US20030229010A1 - Oral insulin-oligomer conjugates - Google Patents
Oral insulin-oligomer conjugates Download PDFInfo
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- US20030229010A1 US20030229010A1 US10/448,535 US44853503A US2003229010A1 US 20030229010 A1 US20030229010 A1 US 20030229010A1 US 44853503 A US44853503 A US 44853503A US 2003229010 A1 US2003229010 A1 US 2003229010A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/4841—Filling excipients; Inactive ingredients
- A61K9/4858—Organic compounds
Definitions
- the present invention relates to microemulsion formulations of free-form and/or conjugation-stabilized therapeutic agents, and to methods of making and using same.
- the compositions of the invention may comprise therapeutic agents such as proteins, peptides, nucleosides, nucleotides, antiviral agents, antineoplastic agents, antibiotics, antiarrhythmics, anti-coagulants, etc., and prodrugs, precursors, derivatives, and intermediates thereof.
- polypeptides and proteins for the systemic treatment of specific diseases is now well accepted in medical practice.
- the role that the peptides play in replacement therapy is so important that many research activities are being directed towards the synthesis of large quantities by recombinant DNA technology.
- Many of these polypeptides are endogenous molecules which are very potent and specific in eliciting their biological actions.
- Other non-(poly)peptidyl therapeutic agents are equally important and pharmaceutically efficacious.
- a major factor limiting the usefulness of these therapeutic substances for their intended application is that they are easily metabolized by plasma proteases when given parenterally.
- the oral route of administration of these substances is even more problematic because in addition to proteolysis in the stomach, the high acidity of the stomach destroys them before they reach their intended target tissue.
- polypeptides and protein fragments produced by the action of gastric and pancreatic enzymes, are cleaved by exo and endopeptidases in the intestinal brush border membrane to yield di- and tripeptides, and even if proteolysis by pancreatic enzymes is avoided, polypeptides are subject to degradation by brush border peptidases. Any of the therapeutic agent that survives passage through the stomach is further subjected to metabolism in the intestinal mucosa where a penetration barrier prevents entry into the cells.
- protease inhibitors such as aprotinin, soybean trypsin inhibitor, and amastatin
- aprotinin such as aprotinin
- soybean trypsin inhibitor such as soybean trypsin inhibitor
- amastatin aprotin inhibitor
- these protease inhibitors are not selective, and endogenous proteins are also inhibited. This effect is undesirable.
- Boccu et al. disclosed that polyethylene glycol could be linked to a protein such as superoxide dismutase (“SOD”).
- SOD superoxide dismutase
- the resulting conjugated product showed increased stability against denaturation and enzymatic digestion.
- the polymers did not contain moieties that are necessary for membrane interaction and thus suffer from the same problems as noted above in that they are not suitable for oral administration.
- the modifying compounds are not polymers and accordingly do not contain moieties necessary to impart both the solubility and membrane affinity necessary for bioavailability following oral as well as parenteral administration. Many of these preparations lack oral bioavailability.
- the insulin molecule consists of two chains of amino acids linked by disulfide bonds; the molecular weight of insulin is around 6,000.
- the ⁇ -cells of the pancreatic islets secrete a single chain precursor of insulin, known as proinsulin. Proteolysis of proinsulin results in removal of four basic amino acids (numbers 31, 32, 64 and 65 in the proinsulin chain: Arg, Arg, Lys, Arg respectively) and the connecting (“C”) peptide.
- the A chain has glycine at the amino terminus
- the B chain has phenylalanine at the amino terminus.
- Insulin may exist as a monomer, dimer or a hexamer formed from three of the dimers. The hexamer is coordinated with two Zn 2+ atoms. Biological activity resides in the monomer. Although until recently bovine and porcine insulin were used almost exclusively to treat diabetes in humans, numerous variations in insulin between species are known. Porcine insulin is most similar to human insulin, from which it differs only in having an alanine rather than threonine residue at the B-chain C-terminus. Despite these differences most mammalian insulin has comparable specific activity. Until recently animal extracts provided all insulin used for treatment of the disease. The advent of recombinant technology allows commercial scale manufacture of human insulin (e.g., HumulinTM insulin, commercially available from Eli Lilly and Company, Indianapolis, Ind.).
- human insulin e.g., HumulinTM insulin, commercially available from Eli Lilly and Company, Indianapolis, Ind.
- Formulated insulin is prone to numerous types of degradation.
- Nonenzymatic deamidation occurs when a side-chain amide group from a glutaminyl or asparaginyl residue is hydrolyzed to a free carboxylic acid.
- Published reports suggest that the three Asn residues are most susceptible to such reactions.
- Such agents In addition to the in vivo usage of therapeutic agents including polypeptides, proteins. nucleosides, and other biologically active molecules, such agents also find substantial and increasing use in diagnostic reagent applications. In many such applications, these agents are utilized in solution environments wherein they are susceptible to thermal and enzymic degradation.
- diagnostic agents include enzymes, peptide and protein hormones, antibodies, enzyme-protein conjugates used for immunoassay, antibody-hapten conjugates, viral proteins such as those used in a large number of assay methodologies for the diagnosis or screening of diseases such as AIDS, hepatitis, and rubella, peptide and protein growth factors used for example in tissue culture, enzymes used in clinical chemistry, and insoluble enzymes such as those used in the food industry.
- alkaline phosphatase is widely utilized as a reagent in kits used for the colorimetric detection of antibody or antigen in biological fluids.
- enzyme is commercially available in various forms, including free enzyme and antibody conjugates, its storage stability in solution often is limited.
- alkaline phosphatase conjugates are frequently freeze-dried, and additives such as bovine serum albumin and Tween 20 are used to extend the stability of the enzyme preparations.
- additives such as bovine serum albumin and Tween 20 are used to extend the stability of the enzyme preparations.
- a recent approach involves formulation of insulin in a liquid medium, using absorption enhancers.
- U.S. Pat. No. 5,653,987 to Modi and Chanonga teaches that insulin can be formulated for oral or nasal delivery using at least two different absorption enhancers.
- a close examination of the enhancers described in this reference reveals that these enhancers are not pharmaceutically acceptable.
- the formulation and synthesis method of U.S. Pat. No. 5,653,987 differ fundamentally from the concept and method of the present invention. Additionally, unlike the present invention, the formulation approach of U.S. Pat. No. 5,653,987 does not address enzymatically stabilized insulin conjugates.
- Desai, A. J., U.S. Pat. No. 5,206,219 describes another microemulsion formulation for oral administration, in which a liquid polyol solvent and lipid cosolvent containing a proteinaceous medicament, e.g., insulin, is treated to form a microemulsion in the gastrointestinal tract at sites of absorption.
- a vital ingredient in the formulation is a protease inhibitor. The purpose of using the inhibitor is to prevent the degradation of the proteinaceous medicament. Problems that arise from using protease inhibitors to aid in oral delivery of proteinaceous drugs have been enumerated hereinabove.
- the present invention relates to microemulsion formulations of free-form and/or conjugation-stabilized therapeutic agents, and to methods of making and using same.
- the compositions of the invention may comprise therapeutic agents such as proteins, peptides, nucleosides, nucleotides, antiviral agents, antineoplastic agents, antibiotics, antiarrhythmics, anti-coagulants, etc., and prodrugs, precursors, derivatives, and intermediates thereof.
- the formulations of the present invention may utilize conjugation-stabilized therapeutic and/or diagnostic agent compositions, which are conjugatively stabilized as more fully described in U.S. Pat. No. 5,681,811 issued Oct. 28, 1997, U.S. Pat. No. 5,438,040 issued Aug. 1, 1995 and U.S. Pat. No. 5,259,030 issued Oct. 25, 1994, all in the name of Nnochiri Nkem Ekwuribe, the disclosures of which are hereby incorporated herein in their entirety.
- the formulations of the present invention may utilize covalently conjugated therapeutic and/or diagnostic complexes wherein the therapeutic and/or diagnostic agent peptide is covalently bonded to one or more molecules of a polymer incorporating as an integral part thereof a hydrophilic moiety, e.g., a linear polyalkylene glycol, and wherein said polymer incorporates a lipophilic moiety as an integral part thereof.
- a polymer incorporating as an integral part thereof a hydrophilic moiety, e.g., a linear polyalkylene glycol, and wherein said polymer incorporates a lipophilic moiety as an integral part thereof.
- the present invention relates to a formulation including a physiologically active therapeutic agent covalently coupled with a polymer comprising (i) a linear polyalkylene glycol moiety and (ii) a lipophilic moiety, wherein the therapeutic agent, linear polyalkylene glycol moiety, and the lipophilic moiety are conformationally arranged in relation to one another such that the physiologically active therapeutic agent in the formulation has an enhanced in vivo resistance to enzymatic degradation, relative to the physiologically active therapeutic agent alone (i.e., in an unconjugated form devoid of the polymer coupled thereto).
- the invention in another aspect, relates to a formulation including a physiologically active therapeutic agent composition of three-dimensional conformation comprising a physiologically active therapeutic agent covalently coupled with a polysorbate complex comprising (i) a linear polyalkylene glycol moiety and (ii) a lipophilic moiety, wherein the physiologically active therapeutic agent, the linear polyalkylene glycol moiety and the lipophilic moiety are conformationally arranged in relation to one another such that (a) the lipophilic moiety is exteriorly available in the three-dimensional conformation, and (b) the physiologically active therapeutic agent in the physiologically active therapeutic agent composition has an enhanced in vivo resistance to enzymatic degradation, relative to the physiologically active therapeutic agent alone.
- a physiologically active therapeutic agent composition of three-dimensional conformation comprising a physiologically active therapeutic agent covalently coupled with a polysorbate complex comprising (i) a linear polyalkylene glycol moiety and (ii) a lipophilic moiety, wherein the physiologically active therapeutic agent, the
- the invention relates to formulations including a multiligand conjugated therapeutic agent complex comprising a triglyceride backbone moiety, having:
- a bioactive therapeutic agent covalently coupled with the triglyceride backbone moiety through a polyalkylene glycol spacer group bonded at a carbon atom of the triglyceride backbone moiety;
- At least one fatty acid moiety covalently attached either directly to a carbon atom of the triglyceride backbone moiety or covalently joined through a polyalkylene glycol spacer moiety.
- the ⁇ and ⁇ carbon atoms of the triglyceride bioactive moiety may have fatty acid moieties attached by covalently bonding either directly thereto, or indirectly covalently bonded thereto through polyalkylene glycol spacer moieties.
- a fatty acid moiety may be covalently attached either directly or through a polyalkylene glycol spacer moiety to the a and a′ carbons of the triglyceride backbone moiety, with the bioactive therapeutic agent being covalently coupled with the ⁇ -carbon of the triglyceride backbone moiety, either being directly covalently bonded thereto or indirectly bonded thereto through a polyalkylene spacer moiety.
- the bioactive therapeutic agent may advantageously be covalently coupled with the triglyceride modified backbone moiety through alkyl spacer groups, or alternatively other acceptable spacer groups, within the broad scope of the invention.
- acceptability of the spacer group refers to steric, compositional, and end use application specific acceptability characteristics.
- the invention relates to a formulation including a polysorbate complex comprising a polysorbate moiety including a triglyceride backbone having covalently coupled to a, a′ and ⁇ carbon atoms thereof functionalizing groups including:
- a polyethylene glycol group having a physiologically active moiety covalently bonded thereto e.g., a physiologically active moiety is covalently bonded to an appropriate functionality of the polyethylene glycol group.
- Such covalent bonding may be either direct, e.g., to a hydroxy terminal functionality of the polyethylene glycol group, or alternatively, the covalent bonding may be indirect, e.g., by reactively capping the hydroxy terminus of the polyethylene glycol group with a terminal carboxy functionality spacer group, so that the resulting capped polyethylene glycol group has a terminal carboxy functionality to which the physiologically active moiety may be covalently bonded.
- the invention relates to a further aspect to a formulation including a stable, aqueously soluble, conjugated therapeutic agent complex comprising a physiologically active therapeutic agent covalently coupled to a physiologically compatible polyethylene glycol modified glycolipid moiety.
- the physiologically active therapeutic agent may be covalently coupled to the physiologically compatible polyethylene glycol modified glycolipid moiety by a labile covalent bond at a free amino acid group of the therapeutic agent, wherein the labile covalent bond may be scissionable in vivo by biochemical hydrolysis and/or proteolysis.
- the physiologically compatible polyethylene glycol modified glycolipid moiety may advantageously comprise a polysorbate polymer, e.g., a polysorbate polymer comprising fatty acid ester groups selected from the group consisting of monopalmitate, dipalmitate, monolaurate, dilaurate, trilaurate, monoleate, dioleate, trioleate, monostearate, distearate, and tristearate.
- a polysorbate polymer e.g., a polysorbate polymer comprising fatty acid ester groups selected from the group consisting of monopalmitate, dipalmitate, monolaurate, dilaurate, trilaurate, monoleate, dioleate, trioleate, monostearate, distearate, and tristearate.
- the physiologically compatible polyethylene glycol modified glycolipid moiety may suitably comprise a polymer selected from the group consisting of polyethylene glycol ethers of fatty acids, and polyethylene glycol esters of fatty acids, wherein the fatty acids for example comprise a fatty acid selected from the group consisting of lauric, palmitic, oleic, and stearic acids.
- the physiologically active therapeutic agent may illustratively comprise a peptide, protein, nucleoside, nucleotide, antineoplastic, agent, antibiotic, anticoagulant, antiarrhythmic agent, antiviral agent, or prodrugs, precursors, intermediates, or derivatives thereof.
- the therapeutic agent may comprise peptide selected from the group consisting of insulin, calcitonin, ACTH, glucagon, somatostatin, somatotropin, somatomedin, parathyroid hormone, erythropoietin, hypothalmic releasing factors, prolactin, thyroid stimulating hormones, endorphins, enkephalins, vasopressin, non-naturally occurring opioids, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminease, adenosine deaminase ribonuclease. trypsin, chemotrypsin, and papain.
- peptide selected from the group consisting of insulin, calcitonin, ACTH, glucagon, somatostatin, somatotropin, somatomedin, parathyroid hormone, erythropoietin, hypothalmic releasing factors, prolactin, thyroid stimulating
- the therapeutic agent may comprise.
- An antiviral such as: Ara-A (Arabinofuranosyladenine), Acylguanosine, Nordeoxyguanosine, Azidothymidine, Dideoxyadenosine, or Dideoxycytidine; an anti-cancer agent such as Dideoxyinosine Floxuridine, 6-Mercaptopurine, Doxorubicin, Daunorubicin, or 1-darubicin; and antibiotic such as Erythormycin, Vancomycin, oleandomycin, or Ampicillin; an antiarrhythmic such as Quinidine; or an anticoagulant such as Heparins.
- the present invention relates to an oral administration dosage form for the mediation of insulin deficiency, comprising a formulation including a pharmaceutically acceptable carrier and a stable, aqueously soluble, conjugated insulin complex comprising insulin or proinsulin covalently coupled to a physiologically compatible polyethylene glycol modified glycolipid moiety.
- the invention relates to a method of treating insulin deficiency in a human or non-human mammalian subject exhibiting such deficiency, comprising orally administering to the subject an effective amount of a conjugated insulin composition comprising a stable, aqueously soluble, conjugated insulin complex comprising insulin covalently or proinsulin covalently coupled to a physiologically compatible polyethylene glycol modified glycolipid moiety.
- peptide as used herein is intended to be broadly construed as inclusive of polypeptides per se having molecular weights of up to about 10,000, as well as proteins having molecular weights of greater than about 10,000, wherein the molecular weights are number average molecular weights.
- covalently coupled means that the specified moieties are either directly covalently bonded to one another, or else are indirectly covalently joined to one another through an intervening moiety or moieties, such as a bridge, spacer, or linkage moiety or moieties.
- conjugatively coupled means that the specified moieties are either covalently coupled to one another or they are non-covalently coupled to one another, e.g., by hydrogen bonding, ionic bonding, Van der Waals forces, etc.
- free in reference to a therapeutic agent means that the therapeutic agent is not conjugatively coupled in the specific formulation.
- therapeutic agent means an agent which is therapeutically useful, e.g., an agent for the treatment, remission or attenuation of a disease state, physiological condition, symptoms, or etiological factors, or for the evaluation or diagnosis thereof.
- HLB hydrophilic and lipophilic balance
- the HLB value is an empirical value on an arbitrary scale that is conveniently and widely used in surfactant chemistry to provide a measure of the polarity of a surfactant or mixture of surfactants, as for example is referenced in U.S. Pat. No. 5,646,109.
- HLB values are commonly provided by surfactant suppliers, and HLB values for a mixture of surfactants are calculated on a volumetric basis. See also U.S. Pat. No. 5,206,219 at column 5, lines 3-17, giving illustrative examples of HLB values for various esters of polyethylene glycol, polyethylene fatty acid esters and polyoxyethylated fatty acids.
- the invention thus comprehends various compositions for therapeutic (in vivo) application, wherein the therapeutic agent component of the conjugated therapeutic agent complex is a physiologically active, or bioactive, therapeutic agent.
- the conjugation of the therapeutic agent component to the polymer comprising hydrophilic and lipophilic moieties may be direct covalent bonding or indirect (through appropriate spacer groups) bonding, and the hydrophilic and lipophilic moieties may also be structurally arranged in the polymeric conjugating structure in any suitable manner involving direct or indirect covalent bonding, relative to one another.
- a wide variety of therapeutic agent species may be accommodated in the broad practice of the present invention, as necessary or desirable in a given end use therapeutic application.
- covalently coupled therapeutic agent compositions such as those described above may utilize therapeutic agent components intended for diagnostic or in vitro applications, wherein the therapeutic agent is for example a diagnostic reagent, or a complement of a diagnostic conjugate for immunoassay or other diagnostic or non-in vivo applications.
- the complexes of the invention are highly usefully employed as stabilized compositions which may for example be formulated as hereinafter more fully described with compatible solvents or other solution-based formulations to provide stable compositional forms which are of enhanced resistance to degradation.
- the present invention relates in one broad compositional aspect to formulations including covalently, conjugated therapeutic agent complexes wherein the therapeutic agent is covalently bonded to one or more molecules of a polymer incorporating as an integral part of said polymer a hydrophilic moiety, e.g., a polyalkylene glycol moiety, and a lipophilic moiety, e.g., a fatty acid moiety.
- a hydrophilic moiety e.g., a polyalkylene glycol moiety
- a lipophilic moiety e.g., a fatty acid moiety.
- the therapeutic agent may be covalently conjugated by covalent bonding with one or more molecules of a linear polyalkylene glycol polymer incorporated in which, as an integral part thereof is a lipophilic moiety, e.g., a fatty acid moiety.
- the present invention relates to formulations of non-covalently conjugated therapeutic agent complexes wherein the therapeutic agent is non-covalently associated with one or more molecules of a polymer incorporating as an integral part thereof a hydrophilic moiety, e.g., a polyalkylene glycol moiety, and a lipophilic moiety, e.g., a fatty acid moiety.
- a hydrophilic moiety e.g., a polyalkylene glycol moiety
- a lipophilic moiety e.g., a fatty acid moiety.
- the polymer may be variously structured and arranged analogous to description of the polymer in the covalently conjugated therapeutic agent complexes described above, but wherein the therapeutic agent is not bonded to the polymer molecule(s) in a covalent manner, but is nonetheless associated with the polymer, as for example by associative bonding, such as hydrogen bonding, ionic bonding or complexation, Van der Waals bonding, micellular encapsulation or association (of the specific therapeutic agent), etc., or alternatively wherein the therapeutic agent is in a free form in the formulation, unassociated with any conjugating polymer.
- associative bonding such as hydrogen bonding, ionic bonding or complexation, Van der Waals bonding, micellular encapsulation or association (of the specific therapeutic agent), etc.
- the polymer component may be suitably constructed, modified, or appropriately functionalized to impart the ability for associative conjugation in a selectively manner (for example, to impart hydrogen bonding capability to the polymer viz-a-vis the therapeutic agent), within the skill of the art.
- compositions of therapeutic agent component(s) and polymeric moiety/(ies) within the broad scope of the present invention may for example utilize a therapeutic agent component for therapeutic (e.g., in vivo) applications, as well as non-therapeutic therapeutic agent components, e.g., for diagnostic or other (in vitro ) use.
- the formulations of the present invention advantageously comprise a microemulsion of a therapeutic agent in free and/or conjugatively coupled form, wherein the microemulsion has a hydrophilic and lipophilic balance (HLB) value of between 3 and 7, more preferably from about 5 to about 6.5 and most preferably from about 5 to about 6.
- the microemulsion suitably comprises a water-in-oil (w/o) emulsion of such character having a clear optical character.
- the present invention relates to an oral dosage form of insulin.
- insulin may be in the free state and/or conjugatively coupled with an amphiphilic polymer (the conjugative coupling may be covalent coupling and/or associative (non-covalent) coupling).
- the present invention also relates to a method by which the insulin conjugates and/or free insulin may be prepared in microemulsion formulation.
- the present invention provides a safe and effective oral dosage form applicable to free insulin and/or insulin conjugates, as well as to other therapeutic agents, e.g., proteinaceous medicaments, in free form and/or modified by amphiphilic polymers for the purpose of enhancing their stability for oral administration.
- Unmodified therapeutic agents that are amphiphilic in character can also be usefully employed in the broad practice of the present invention.
- the present invention differs from the composition and method of preparation of other known microemulsions.
- the current state of the art recognizes the instability of protein drugs in the gastrointestinal tract and has endeavored to circumvent such problem by formulating protein drugs with protease inhibitors.
- protease inhibitors by their intrinsic character function to protect harmful proteinaceous material in the tract from degradation, and the added protease inhibitors thereby interfere with natural and desirable physiological digestive processes.
- the presence of protease inhibitors in oral formulations may therefore have quite serious consequences to the health and well-being of the patient
- the present invention contemplates the use of therapeutic formulations in which the therapeutic agent(s) can be usefully and effectively stabilized without protease inhibitors and their attendant problems.
- microemulsion formulation of the present invention provides a dosage form for the safe and effective oral administration of (i) the free form of therapeutic agents such as insulin, (ii) amphiphilic conjugates of such therapeutic agents, or (iii) mixtures of such free form therapeutic agents and their conjugates.
- the method of the invention for preparing microemulsion formulations useful as oral dosage forms of therapeutic agents such as insulin utilizes emulsification techniques in a novel fashion with respect to the selection of oil, water and cosurfactant components, to provide a microemulsion having a specific HLB character useful for oral drug delivery.
- the oil component of the microemulsion formulation of the invention -appropriately comprises a pharmaceutically acceptable oil, preferably of a food grade quality.
- the selection of oil, water and cosurfactant constituents to yield a microemulsion having an HLB value of less than 7 provides a stable formulation for incorporating free form insulin and/or insulin conjugates for effective oral administration of the insulin.
- particularly preferred insulin forms include the so-called hex-insulin mixture hereinafter disclosed, which is amphiphilic and able to distribute well in oil, surfactant and water.
- the hex-insulin mixture is formed from insulin conjugated with a hexyl polymer which forms a mixture of mono-, di- and tri-conjugates of insulin as well as free insulin as the conjugative reaction product (the mono-, di- and tri- prefixes referring to the number of polymer moieties attached to the insulin molecule).
- the hex-insulin mixture is more stable than unconjugated insulin against proteolytic digestion (Table B). In closed loop assay determinations (FIG. 3, 4), the hex-insulin mixture is better absorbed than unconjugated insulin and gives better glucose reduction than insulin (on an insulin weight basis).
- the concentration of the hex-insulin mixture in the formulation is able to be increased above the concentration possible for free zinc insulin alone, and the formulation of both the hex-insulin mixture and free zinc insulin in the microemulsion compositions of the present invention shows improved absorption of the hex-insulin mixture in relation to absorption of zinc insulin (FIG. 6). Accordingly, the hex-insulin mixtures of the invention are highly efficacious in providing superior absorption of the insulin component and concomitant reduction of blood glucose in therapeutic use.
- the insulin when used in free form in the formulations of the present invention, the insulin may be used in any suitable form, such as in salt forms such as zinc insulin, sodium insulin, etc. or other pharmaceutically acceptable forms of insulin.
- formulation of the invention may also be used for parenteral administration or any other suitable mode of administration.
- FIG. 1 is a graph of serum glucose, in mg/dL, as a function of time, in minutes, for administration of insulin per se and in complexed forms.
- FIG. 2 is a graph of serum glucose, in mg/dL, as a function of time, in hours, for administration of insulin in various forms.
- FIG. 3 is a graph of ELISA assay results for native insulin in the NRS pool of various Sprague-Dawley rats, showing insulin concentration as a function of time, prior to and after dosing with insulin, at respective doses of 10 milligrams of insulin per kilogram of body weight, and at 30 milligrams of insulin per kilogram of body weight.
- FIG. 4 is a graph of ELISA assay results for insulin in the NRS pool of various Sprague-Dawley rats, showing insulin concentration as a function of time, prior to and after dosing with insulin, at respective doses of 10 milligrams of insulin per kilogram of body weight, and at 30 milligrams of insulin per kilogram of body weight, wherein the insulin is administered as a conjugate, insulin-hexyl-PEG 7 -OMe.
- FIG. 5 is a graph of percent of pre-dose blood glucose level as a function of time, for free insulin, and for conjugated insulin at respective doses of 1, 3 and 10 milligrams of insulin per kilogram of body weight, against a control of phosphate buffered solution (PBS) and bovine serum albumen (BSA) in various Sprague Dawley rats.
- PBS phosphate buffered solution
- BSA bovine serum albumen
- FIG. 6 is a graph of insulin and blood glucose levels in pancreatomized dogs as a function of time, after dosing with a microemulsion insulin formulation representative of the present invention.
- Modification of therapeutic agents with non-toxic, non-immunogenic polymers affords certain advantages. If modifications are made in such a way that the products (polymer-therapeutic agent conjugates) retain all or most of their biological activities the following properties may result: epithelial penetration capability may be enhanced; the modified therapeutic agent may be protected from proteolytic digestion and subsequent abolition of activity; affinity for endogenous transport systems may be improved; chemical stability against stomach acidity may be imparted; and the balance between lipophilicity and hydrophobicity of the polymers may be optimized. Proteinaceous substances endowed with the improved properties described above can be effective as replacement therapy following either oral or parenteral administration. Other routes of administration, such as nasal and transdermal, are potentially advantageously employed with the modified therapeutic agent.
- conjugation-stabilization of diagnostic and/or reagent species of peptides, nucleosides, or other therapeutic agents, including precursors and intermediates of end-use nucleosides, peptides or other products provides corresponding advantages, when the conjugation component is covalently bonded to a polymer as hereinafter disclosed.
- the resultingly covalently conjugated agent is resistant to environmental degradative factors, including solvent- or solution-mediated degradation processes.
- the shelf life of the active ingredient is able to be significantly increased, with concomitant reliability of the therapeutic agent-containing composition in the specific end use for which same is employed.
- Analogous benefits are realized when therapeutic, diagnostic, or reagent species are non-covalently, associatively conjugated with polymer molecule(s) in the manner of the present invention.
- the formulation of the present invention may utilize the therapeutic agent in a free form, as previously described.
- compositions of the invention may utilize the therapeutic agent in any suitable combination or permutation of the foregoing forms (covalently conjugated, associatively conjugated, and/or free form (unconjugated)).
- a peptide covalently bonded to the polymer component as an illustrative embodiment of the form of the therapeutic agent used in the practice of the invention, the nature of the conjugation, involving cleavable covalent chemical bonds, allows for control in terms of the time course over which the polymer may be cleaved from the peptide (insulin). This cleavage may occur by enzymatic or chemical mechanisms.
- the conjugated polymer-peptide complex will be intrinsically active. Full activity will be realized following enzymatic cleavage of the polymer from the peptide. Further, the chemical modification will allow penetration of the attached peptide, e.g., insulin, through cell membranes.
- suitable polymers for conjugation with the therapeutic agents are employed so as to obtain the desirable characteristics enumerated above.
- modified therapeutic agents may be employed for sustained In vivo delivery of the therapeutic agent.
- the present invention provides microemulsion formulations for delivery of therapeutic agents orally in their active form.
- the invention may also be practiced with amphiphilic prodrugs that are therapeutically effective by oral or parenteral administration.
- microemulsion formulations are provided including agents useful for therapeutic applications, as well as immunoassay, diagnostic, and other non-therapeutic (e.g., in vitro ) applications.
- the formulations of the present invention may be employed to provide stabilized peptide and nucleoside compositions variously suitable for in vivo as well as non-in vivo applications, wherein the peptide and nucleoside agents may be conjugated and/or unconjugated in character.
- the therapeutic agent employed in the formulation when the therapeutic agent employed in the formulation is conjugated, a single polymer molecule may be employed for conjugation with a plurality of therapeutic agent species, and it may also be advantageous in the broad practice of the invention to utilize a variety of polymers as conjugating agents for a given therapeutic agent; combinations of such approaches may also be employed.
- the conjugating polymer(s) may utilize any other groups, moieties, or other conjugated species, as appropriate to the end use application By way of example, it may be useful in some applications to covalently bond to the polymer a functional moiety imparting UV-degradation resistance, or antioxidant character, or other properties or characteristics to the polymer.
- the polymer may contain any functionality, repeating groups, linkages, or other constituent structures which do not preclude the efficacy of the conjugated composition for its intended purpose.
- polymers that may usefully be employed achieve these desirable characteristics are described herein below in an exemplary reaction scheme.
- the polymers may be functionalized and then coupled to free amino acid(s) of the peptide(s) to form labile bonds which permit retention of activity with the labile bonds intact. Removal of the bond by chemical hydrolysis and proteolysis then enhances the peptidal activity.
- the polymers utilized in the invention when the therapeutic agent employed in the formulation is conjugated, may suitably incorporate in their molecules constituents such as edible fatty acids (lipophilic end), polyethylene glycols (water soluble end), acceptable sugar moieties (receptor interacting end), and spacers for therapeutic agent attachment.
- polysorbates are particularly preferred and are chosen to illustrate various embodiments of the invention in the ensuing discussion herein, The scope of this invention is of course not limited to polysorbates, and various other polymers incorporating above-described moieties may usefully be employed in the broad practice of this invention.
- it may be desirable to eliminate one of such moieties and to retain others in the polymer structure, without loss of objectives. When it is desirable to do so, the preferred moieties to eliminate without losing the objectives and benefits of the invention are the sugar and/or the spacer moieties.
- polyalkylene glycol residues of C 2 -C 4 alkyl polyalkylene glycols preferably polyethylene glycol (PEG) are advantageously incorporated in the polymer systems of interest.
- PEG polyethylene glycol
- the fatty acid portion of the polymer is provided to associate with the hydrophobic domain of the peptide and thus prevent aggregation in solution.
- the resulting polymer-peptide conjugates thus will be: stabilized (to chemical and enzymatic hydrolysis); water-soluble, due to the PEG residue; and, by virtue of the fatty acid-hydrophobic domain interactions, not prone to aggregation.
- Polyalkylene glycol derivatization has a number of advantageous properties in the formulation of polymer-therapeutic agent conjugates in the practice of the present invention, as associated with the following properties of polyalkylene glycol derivatives: improvement of aqueous solubility, while at the same time eliciting no antigenic or immunogenic response; high degrees of biocompatibility; absence of in vivo biodegradation of the polyalkylene glycol derivatives; and ease of excretion by living organisms.
- the polymers employed in the practice of the present invention as conjugating components of the inventive formulation thus comprise lipophilic and hydrophilic moieties, rendering the resulting polymer-drug conjugate highly effective (bioactive) in oral as well as parenteral and other modes of physiological administration.
- drug and “therapeutic agent” are used interchangeably.
- polymer-therapeutic agent conjugates of the present invention are the formulae of covalently bonded conjugates denoted for ease of subsequent reference as Conjugate 1, Conjugate 2, and Conjugate 3, wherein “drug” is insulin or other therapeutic agent, and specific values of m, n, w, x, and y will be described in the ensuing discussion.
- m and n each are independently from 1 to 125.
- m and n each are independently from 1 to 125.
- Conjugate 1 features commercially available polysorbate monooleate at the center of the polymeric system, a sugar derivative used in many pharmaceutical applications. Lipophilic and absorption enhancing properties are imparted by the fatty acid chain, while the polyethylene glycol (PEG) residues provide a hydrophilic (hydrogen bond accepting) environment. Drug is attached through a carbamate linkage adjacent to the PEG region of the polymer.
- PEG polyethylene glycol
- Conjugate 2 the sugar residue is excluded, but drug is once again attached to the polymer through a carbamate bond adjacent to the hydrophilic PEG region of the polymer.
- the lipophilic fatty acid region of the polymer is thus some distance from the point of attachment to drug, e.g., insulin.
- the polymers utilized in therapeutic agent conjugation in accordance with the invention are designed to incorporate good physical characteristics that enable them to achieve the desired objectives.
- Absorption enhancers while enabling penetration of the drug through the cell membrane, do not improve the stability characteristics of the drug, and in vivo applications may therefore utilize the polymer-drug conjugates of the invention in formulations devoid of such penetration enhancers.
- One aspect of the present invention therefore relates to the incorporation of fatty moiety within the polymer, to mimic penetration enhancers.
- the microemulsion formulations of the invention are preferably devoid of any protease inhibitors. Protease inhibitors have been used in the prior art to enhance the stability of proteinaceous therapeutic agents, as hereinearlier discussed, but are desirably absent in the microemulsion formulations of the present invention.
- the drug may be covalently attached to the water-soluble polymer by means of a labile chemical bond.
- This covalent bond between the drug and the polymer may be cleaved by chemical or enzymatic reaction.
- the polymer-drug product retains an acceptable amount of activity; full activity of the component drug is realized when the polymer is completely cleaved from the drug.
- portions of polyethylene glycol are present in the conjugating polymer to endow the polymer-drug conjugate with high aqueous solubility and prolonged blood circulation capability.
- the modifications described above confer improved solubility, stability, and membrane affinity properties on the drug. As a result of these improved characteristics the invention contemplates parenteral and oral delivery of both the active polymer-drug species and, following hydrolytic cleavage, bioavailability of the drug per se, in in vivo applications.
- the polymers used in the embodiment described below can be classified as polyethylene glycol modified lipids and polyethylene glycol modified fatty acids.
- preferred conjugating polymers may be mentioned polysorbates comprising monopalmitate, dipalmitate, tripalmitate, monolaurate, dilaurate, trialaurate, monooleate, dioleate, trioleate, monostearate, distearate, and tristearate.
- Other lower fatty acids can be utilized.
- the number average molecular weight of polymer resulting from each combination is preferred to be in the range of from about 750 to about 5,000 daltons.
- Alternative polymers of preference are polyethylene glycol ethers or esters of fatty acids, such fatty acids being lauric, palmitic, oleic, and stearic acids, and other lower fatty acids can be utilized, with the polymers ranging from 250 to 5,000 daltons in number average molecular weight. It is preferred to have a derivatizable group in the polymer, where such group can be at the end terminating with polyethylene glycol or at the end terminating with fatty moiety. The derivatizable group may also be situated within the polymer and thus may serve as a spacer between the peptide (or other therapeutic agent) and the polymer.
- R 1 , R 2 and R 3 are each independently selected from the group consisting of lauric, oleic, palmitic and stearic acid radicals, or R 1 and R 2 are each hydroxyl while R 3 is lauric, palmitic, oleic or stearic acid radical, or lower fatty acid.
- These polymers are commercially available and are used in pharmaceutical formulations. Where a higher molecular weight polymer is desired, it may be synthesized from glycolipids such as sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate or sorbitan monostearate, and an appropriate polyethylene glycol. Structures of glycolipids which may be used as starting reagents are depicted below.
- glycolipid polymers substituted in three positions with polyethylene glycol a desired polyethylene glycol having two free hydroxyls at the termini is protected at one terminus with a trityl group in pyridine using one mole of trityl chloride. The remaining free hydroxyl group of the polyethylene glycol is converted to either tosylate or bromide.
- the desired glycolipid is dissolved in a suitable inert solvent and treated with sodium hydride.
- the tosylate or bromide of the protected polyethylene glycol is dissolved in inert solvent and added in excess to the solution of glycolipid.
- the product is treated with a solution of para-toluenesulfonic acid in anhydrous inert solvent at room temperature and purified by column chromatography. The structures of the transformation are depicted below.
- each n and m may vary independently, and have any suitable value appropriate to the specific drug being stabilized, e.g., from 1 to 16.
- the primary alcohol is first esterified or esterified with a fatty acid moiety such as lauric, oleic, palmitic or stearic; the amino group is derivatized with fatty acids to form amides or secondary amino groups, as shown below.
- a fatty acid moiety such as lauric, oleic, palmitic or stearic
- the amino group is derivatized with fatty acids to form amides or secondary amino groups, as shown below.
- m may have any suitable value, e.g., from 10 to 16.
- fatty acid derivatives in different parts of the polyethylene glycol chain to achieve certain physicochemical properties similar to polysorbates that have been substituted with two/three molecules of fatty acids, e.g., polysorbate trioleate.
- polymer A it is desirable to protect the hydroxyl moieties on the first and second carbon of glycerol, e.g. solketal.
- the remaining hydroxyl group is converted to the sodium salt in an inert solvent and reacted with halogenated or tosylated polyethylene glycol in which one end of the polyethylene glycol has been protected as an ester.
- the glycerol protection is removed and the resulting two free hydroxyl groups are converted to the corresponding sodium salts.
- These salts are reacted in inert solvent with polyethylene glycol which has been partially derivatized with fatty acids. Reaction takes place after the free hydroxyl is converted to the tosylate or bromide.
- Polymer G is synthesized in the same manner except that the protected glycerol is first reacted with esters of fatty acids which have been halogenated at the terminal carbon of the acid.
- polymer C In the synthesis of polymer C, it is preferable to start with 1, 3-dihalo-2-propanol.
- the dihalo compound is dissolved in inert solvent and treated with the sodium salt of two moles of polyethylene glycol which has been previously derivatized with one mole of a fatty acid moiety.
- the product is purified by chromatography or dialysis.
- the resulting dry product is treated, in inert solvent, with sodium hydride.
- the sodium salt thus formed is reacted with a halo derivative of partially protected polyethylene glycol.
- the resulting polymer still contains a polyethylene glycol fragment.
- the membrane affinity properties of the fatty acid moiety may be retained by substituting a fatty acid proper with a lipophilic long chain alkane; biocompatibility is thus preserved.
- the polyethylene glycol with two terminal free hydroxyl groups is treated with sodium hydride in inert solvent.
- One equivalent weight of a primary bromide derivative of a fatty acid-like moiety is added to the polyethylene glycol solvent mixture.
- the desired product is extracted in inert solvent and purified by column chromatography if necessary.
- the acid chloride of the acid is treated with excess of desired polyethylene glycol in suitable inert solvent.
- the polymer is extracted in inert solvent and further purified by chromatography if necessary.
- the polymer is synthesized with the derivatizable function placed on the fatty acid moiety.
- a solution of mono-methoxypolyethylene glycol of appropriate molecular weight in inert solvent is treated with sodium hydride followed by the addition of solution containing the ethyl ester of a fatty acid bearing a leaving group at the terminal carbon of the acid.
- the product is purified after solvent extraction and if necessary, by column chromatography.
- ester protection is removed by treating with dilute acid or base.
- the carboxyl or ester is converted to a hydroxyl group by a chemical reduction method known in the art.
- the functional groups that are used in the drug conjugation are usually at a terminal end of the polymer, but in some cases, it is preferred that the functional group is positioned within the polymer. In this situation, the derivatizing groups serve as spacers.
- a fatty acid moiety may be brominated at the carbon alpha to the carboxylic group and the acid moiety is esterified. The experimental procedure for such type of compound is similar to the one outlined above, resulting in the product shown below.
- a polyethylene glycol monoether may be converted to an amino group and treated with succinic anhydride that has been derivatized with a fatty acid moiety.
- a desired polyethylene glycol bearing primary amine is dissolved in sodium phosphate buffer at pH 8.8 and treated with a substituted succinic anhydride fatty acid moiety as shown in the scheme below.
- the product is isolated by solvent extraction and purified by column chromatography if necessary.
- the reaction of the polymer with the drug to obtain covalently conjugated products is readily carried out
- the polymer is referred to as (P).
- the polymer contains a hydroxyl group, it is first converted to an active carbonate derivative such as para-nitrophenyl carbonate. The activated derivative then is reacted with the amino residue of the drug in a short period of time under mild conditions producing carbamate derivatives.
- reaction and reagent only serve as illustration and are not exclusive; other activating reagents resulting in formation of urethane, or other, linkages can be employed.
- the hydroxyl group can be converted to an amino group using reagents known in the art. Subsequent coupling with drug through their carboxyl groups results in amide formation.
- the polymer contains a carboxyl group
- it can be converted to a mixed anhydride and reacted with the amino group of the drug to create a conjugate containing an amide bond.
- the carboxyl group can be treated with water-soluble carbodimide and reacted with the drug to produce conjugates containing amide bonds.
- the activity and stability of the drug conjugates can be varied in several ways, by using a polymer of different molecular size. Solubilities of the conjugates can be varied by changing the proportion and size of the polyethylene glycol fragment incorporated in the polymer composition. Hydrophilic and hydrophobic characteristics can be balanced by careful combination of fatty acid and polyethylene glycol moieties.
- various techniques may be advantageously employed to improve the stability characteristics of the polymer conjugates of the present invention, including: the functionalization of the polymer with groups of superior hydrolysis resistance, e.g., the previously illustrated conversion of ester groups to ether groups; modifying the lipophilic/hydrophilic balance of the conjugating polymer, as appropriate to the drug being stabilized by the polymer; and tailoring the molecular weight of the polymer to the appropriate level for the molecular weight of the drug being stabilized by the polymer.
- polyalkylene glycol-derived polymers of value for therapeutic applications of the present invention is general biocompatibility.
- the polymers have various water solubility properties and are not toxic. They are non-antigenic, non-immunogenic and do not interfere with biological activities of enzymes. They have long circulation in the blood and are easily excreted from living organisms.
- the products of the present invention have been found useful in sustaining the biological activity of therapeutic nucleosides, peptides, and other therapeutic agents, and may be prepared for therapeutic administration by microemulsion formulation as hereinafter more fully described. Administration preferably is by either the parenteral or oral route.
- the drug-polymer conjugates of the present invention In the dry, lyophilized state, the drug-polymer conjugates of the present invention have good storage stability; and solution formulations of the conjugates of the present invention are likewise characterized by good storage stability.
- microemulsion formulations of the present invention may be employed for the prophylaxis or treatment of any condition or disease state for which the drug constituent thereof is efficacious.
- microemulsion formulations of the invention may be employed for the diagnosis of constituents, conditions, or disease states in biological systems or specimens, as well as for diagnosis purposes in non-physiological systems.
- microemulsion formulations of the invention may have application in prophylaxis or treatment of condition(s) or disease state(s) in plant systems.
- the active component of the formulation may have insecticidal, herbicidal, fungicidal, and/or pesticidal efficacy amenable to usage in various plant systems.
- the present invention contemplates a method of treating an animal subject having or latently susceptible to such condition(s) or disease state(s) and in need of such treatment, comprising administering to such animal an effective amount of a microemulsion formulation of the present invention which is therapeutically effective for said condition or disease state.
- Subjects to be treated by the polymer conjugates of the present invention include both human and non-human animal (e.g., bird, dog, cat, cow, horse) subjects, and preferably are mammalian subjects, and most preferably human subjects.
- non-human animal e.g., bird, dog, cat, cow, horse
- animal subjects may be administered the microemulsion formulation of the invention at any suitable therapeutically effective and safe dosage, as may readily be determined within the skill of the art, and without undue experimentation.
- microemulsion formulations of the invention may comprise the drug component per se as well as, or alternatively, such drug component in the form of pharmaceutically acceptable esters, salts, or other physiologically functional derivatives thereof.
- the present invention also contemplates pharmaceutical formulations, both for veterinary and for human medical use, which comprise as the active agent one or more therapeutic agent(s).
- the active agent preferably may be utilized together with one or more pharmaceutically acceptable carrier(s) therefor and optionally any other therapeutic ingredients.
- the carrier(s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not unduly deleterious to the recipient thereof.
- the active agent is provided in an amount effective to achieve the desired pharmacological effect, as described above, and in a quantity appropriate to achieve the desired daily dose.
- the formulations include those suitable for parenteral as well as non-parenteral administration, and specific administration modalities include oral, rectal, buccal, topical, nasal, ophthalmic, subcutaneous, intramuscular, intravenous, transdermal, intrathecal, intra-articular, intra-arterial, sub-arachnoid, bronchial, lymphatic, vaginal, and intra-uterine administration.
- specific administration modalities include oral, rectal, buccal, topical, nasal, ophthalmic, subcutaneous, intramuscular, intravenous, transdermal, intrathecal, intra-articular, intra-arterial, sub-arachnoid, bronchial, lymphatic, vaginal, and intra-uterine administration.
- Formulations suitable for oral and parenteral administration are preferred.
- Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules or other containment structures, each containing a predetermined amount of the formulation; or in the emulsified form per se, for ingestion in a predetermined amount.
- a syrup may be made by adding a sugar, for example sucrose, to the microemulsion, together with any accessory ingredient(s).
- a sugar for example sucrose
- Such accessory ingredient(s) may include flavorings, suitable preservative, agents to retard crystallization of the sugar, and agents to increase the solubility of any other ingredient, such as a polyhydroxy alcohol, for example glycerol or sorbitol.
- Formulations suitable for parenteral administration conveniently comprise the microemulsion in a form which preferably is isotonic with the blood of the recipient (e.g., physiological saline solution).
- formulations may be presented in unit-dose or multi-dose form.
- Nasal spray formulations comprise the purified emulsion with preservative agents and isotonic agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal mucus membranes.
- Formulations for rectal administration may be presented as an enema formulation.
- Ophthalmic formulations are prepared by a similar method to the nasal spray, except that the pH and isotonic factors are preferably adjusted to match that of the eye.
- Topical formulations may comprise the emulsion in one or more media, such as mineral oil, petroleum, polyhydroxy alcohols, or other bases used for topical pharmaceutical formulations.
- the formulations of this invention may further include one or more accessory ingredient(s) selected from diluents, buffers, flavoring agents, disintegrants, surface active agents, thickeners, lubricants, preservatives (including antioxidants), and the like.
- the conjugating polymers described herein may be employed for in vitro stabilization of drugs in therapeutic as well as non-therapeutic applications.
- the polymers may be employed for example to increase the thermal stability and enzymic degradation resistance of the drug. Enhancement of the thermal stability characteristic of the drug via conjugation in the manner of the present invention provides a means of improving shelf life, room temperature stability, and robustness of research reagents and kits.
- the present invention provides pharmaceutically acceptable compositions in the form of a microemulsion whose HLB value is between 3 and 7.
- the formulation may be prepared with surfactant components whose combined HLB strength in the dosage form is less than 7.
- the formulation may for example contain a mixture of surfactants in the lipid phase, and an aqueous phase containing a high HLB surfactant.
- the presence of additional triglycerides, other than the low HLB surfactant, is optional, but not essential.
- the lipid phase of the microemulsion of the invention can contain oil-soluble drugs, short chain, medium chain and long chain fatty acid triglycerides, monoglycerides of fatty acids, diglycerides of fatty acids, and propylene glycol esters of fatty acids, such as propylene glycol alginate.
- the low HLB surfactant of the lipid phase can be any suitable surfactant.
- suitable surfactant examples include Sorbitan oleate (Arlacel 80), monoolein/propylene glycol (Arlacel 186), C 8 /C 10 fatty acid mono- and diglycerides from coconut oil, soy lecithin, egg phosphatides, citric acid esters of monoglycerides, lactic acid esters of monoglycerides, diacetyl tartaric acid esters of monoglycerides, succinic acid esters of monoglycerides, sucrose fatty acid esters, polyglycolyzed glycerides of oleic acids (e.g., Labrafil M1944), polyglycolyzed glycerides of lineleic acid (e.g., Labrafil M 2125), polyglycerol esters of fatty acids, including both long chain and medium chain fatty acids, e.g., polyglyceryl decao
- the hydrophilic phase may contain in addition to water and water-soluble drugs, one or more polyhydroxy compounds such as carbohydrates, polyethylene glycols, and propylene glycol, in any ratio from 0-100% w/w of the aqueous phase.
- the hydrophilic phase may ether contain polyoxyethylene(2) sorbitan oleate (Tween 80), polyoxyethylene(20 glycerol trioleate, C 8 /C 10 polyglycolyzed glycerides from coconut oil (e.g., Labrafac CM 10, and Labrasol), Tween 60, Cremophor EL (ethoxylated castor oil), Cremophor RH 20 (ethoxylated castor oil), and sucrose fatty esters such as sucrose stearate.
- Teween 80 polyoxyethylene(2) sorbitan oleate
- C 8 /C 10 polyglycolyzed glycerides from coconut oil (e.g., Labrafac CM 10, and Labrasol)
- Tween 60 Tween 60
- Cremophor EL ethoxylated castor oil
- Cremophor RH 20 ethoxylated castor oil
- sucrose fatty esters such as sucrose ste
- the broad range for the amount of high HLB surfactant that can be present in the composition can be 0.2%-50% w/w.
- the preferred range for such high HLB surfactant is 0.2-10% w/w.
- the broad range for the lipid phase containing surfactants is 20-90% w/w.
- a preferred concentration is around 70% w/w and the water content of the aqueous phase can be 2-30% w/w with the preferred range being 10-20% w/w.
- the preferred range of polyhydroxy solvents of the hydrophilic phase is 0-20% w/w.
- Optional ingradients for the preparation of these emulsions include stabilizers such hydroxy propyl methyl cellulose, hydroxy propyl cellulose, carboxymethyl cellulose, and carboxy cellulose, and organic acids such as polyacrylic acids and their partial esters can be beneficially used in combination.
- antioxidants and antimicrobial agents can be used in the formulation.
- the presence of unsaturated fatty acids in the lipid may prone them susceptible to oxidation.
- Oil soluble butylated hydroxy anisole, Vitamin-E, and propyl gallate can be used in the formulation in a concentration of 0.1 to 1.2% w/w.
- Antimicrobial preservatives, such as parabens and benzalkonium chloride, may also be used to preserve the microemulsion, e.g., in a concentration of 0.1-0.5% w/w.
- the microemulsion of the present invention can be prepared by simply mixing by hand or by mild vortexing of the formulation ingredients.
- the order of addition is not crucial, but in cases where the components are viscous the dilution of viscous ingradients with another oil or surfactant may be helpful.
- Preparation of protein stock solutions containing high concentrations of proteins are convenient to prepare the formulations as compared to dissolving protein powders directly in the emulsion formulation volume.
- highly accurate determinations of protein loading in the emulsion can be made by HPLC trace of the stock solution.
- an aqueous solution containing lipid and lipid-soluble low HLB surfactant is prepared.
- the high HLB surfactant is mixed with the lipid phase.
- Slow addition of appropriate quantities of the aqueous solution to the lipid/surfactant mixture results in a clear transparent solution. Occasional gentle shaking may be desirable during the addition.
- the resulting formulation then can be processed for packaging and end use. For example, formulations containing less than 20% w/w of the hydrophilic phase can be made, and the formulation introduced to soft gel capsules, such as enteric-coated soft gel capsules, as unit dosage forms. Alternatively, the formulation can be given as a liquid in the prescribed or desired dose.
- the polymer content is determined by trinitrobenzenesulfonic acid (TNBS) assay and mass spectrometry, and the protein concentration by Biuret Method. A molar ratio of polymer to insulin is determined to be 1:1. Conjugate I is also obtained by using pure organic solvent (e.g., DMSO, DMF).
- pure organic solvent e.g., DMSO, DMF.
- the terminal hydroxyl group of polyethylene glycol monostearate is activated by reaction with p-nitrophenyl chloroformate as described above.
- bovine insulin 80 mg
- 0.1M phosphate buffer pH 8.8.
- the pH is maintained by careful adjustment with 1N NaOH.
- the reaction mixture is quenched with excess glycine and subjected to gel filtration chromatography using Sephadex G-75. Insulin/polymer conjugate is collected and lyophilized. Protein content is determined by Biuret assay, giving a quantitative yield.
- Each test animal received a single dose of either native insulin (Group 1, 100 ⁇ g/kg, subcutaneously); native insulin (Group 2, 1.5 mg/kg, orally by gavage); Conjugate 1) Group 3, 100 ⁇ g/kg, orally); or Conjugate 1 (Group 4, 100 ⁇ g/kg, subcutaneously) at time 0.
- An additional group (Group 5) received no insulin of any kind but was challenged with glucose 30 minutes before scheduled sampling times. Animals were fasted overnight before treatment and for the duration of the study. All test materials were prepared in phosphate buffered saline, pH 7.4.
- Blood glucose levels for Group 1 animals were approximately 30% of control (Group 5, untreated) animals at the 30 minute time point. This hypoglycemic effect lasted only 3.5 hours in Group 1 animals.
- Native insulin administered orally (Group 2) lowered blood glucose levels to a maximum of 60% of control, this maximum response occurring 30 minutes after treatment with the insulin.
- glucose levels in animals in Group 3 (Conjugate 1, 100 ⁇ g/kg, p.o.) were lowered with an apparent delayed onset of hypoglycemic activity.
- the hypoglycemic activity in Group 3 animals was greater than that in Group 2 animals even though the dose of insulin administered to Group 3 was only one fifteenth of that given to Group 2.
- Baseline blood samples were obtained for serum glucose analysis from 10 fasted untreated albino mice (5 males and 5 females); baseline values in FIG. 2 are denoted by the symbol “O”.
- Three additional groups (five males and five females each) were fasted overnight and loaded with glucose alone orally by gavage (5 g/kg body weight).
- Ten animals were sacrificed at each of three time periods to obtain blood samples for glucose analysis: 30, 60 and 120 minutes after dosing.
- a commercial insulin and Conjugate 1 were each administered both orally (p.o) and parenterally (s.c.) to groups of fasted mice (five males and five females, for sacrifice and blood analysis at each of the three time periods), to provide different treatment regimens.
- Glucose was administered orally by gavage to all but the baseline group of animals at a dose of 5 g/kg (10 mg/kg of a 50% w/v solution in normal saline). When insulin was administered orally by gavage, it was given at a dose of 1.5 mg/kg (18.85 mL/kg of a 0.008% w/v solution in normal saline). When insulin was administered subcutaneously, it was given at a dose of 100 ⁇ g/kg (2.5 mL/kg of a 0.004% w/v solution in normal saline).
- the Conjugate 1 polymer-insulin complex When the Conjugate 1 polymer-insulin complex was administered orally by gavage, it was given at a dose of 1.56 mg/kg (2.0 mL/kg of the undiluted test material). When the Conjugate 1 polymer-insulin complex was administered subcutaneously, it was given at a dose of 100 ⁇ g/kg (1.28 mL/kg of a 1:10 dilution of the 0.78 mg/mL solution received) or 250 ⁇ g/kg (3.20 mL/kg of a 1:10 dilution of the solution received). The modified Conjugate 1 contained 0.1 mL insulin/mL and was dosed at a rate of 1.0 mL/kg to obtain a 100 ⁇ g/kg dose.
- Glucose was measured using the Gemini Centrifugal Analyzer and purchased glucose reagent kits.
- the assay was a coupled enzymatic assay based on the reaction of glucose and ATP catalyzed by hexokinase, coupled with the glucose-6-phosphate dehydrogenase reaction, yielding NADH.
- Duplicate samples were analyzed and the mean value reported. Dilution (1:2 or 1:4) of some serum samples was necessary in order to determine the very high glucose concentration present in certain samples.
- the modified Conjugate 1 administered at 100 ⁇ g/kg produced a significant reduction in blood glucose at 30 minutes.
- Polysorbate monopalmitate is first dried by the azeotropic method using dry benzene.
- the phophatase assay was performed according to the method of A. Voller et al, Bulletin WHO, 53, 55 (1976). An aliquot (50 microliter) was added to microwells and mixed with 200 microliter of substrate solution (10 g/L, 4-nitrophenylphosphate in 20% ethanolamine buffer, pH 9.3) and incubated at room temperature for 45 minutes. The reaction was stopped by 50 microliter of 3M NaOH. The absorbence was measured at 405 nm in a micro plate reader.
- TPSCI (1.3 mmole) was dissolved in 6 ml of anhydrous chloroform and added to tributylAraCMP and stirred for 40 minutes at room temperature.
- the resulting TPS activated tributylAraCMP was added to 876 mg of OT 10 (1.2 mmole) in 2.1 mL of pyridine and stirred at room temperature for 41 ⁇ 2 hours.
- TLC of the reaction mixture in THF: methanol (10:0.75 v/v) showed complete disappearance of tributylated AraCMP. The solvent was evaporated and the product was suspended in 6 ml water and extracted into 2 ⁇ 6 ml of chloroform.
- a microemulsion formulation according to the invention was prepared with the composition shown below. Quantity, % Component Added HLB Value HLB Contribution Capmul MCM 53.0 5.5 4.86 Centrophase 31 5.7 3.4 0.38 Propylene glycol 19.9 Tween 80 1.4 15.0 0.35 Hexyl insulin in 15 mg/mL NaP buffer q.s Total 100 5.59
- the propylene glycol content of the above emulsion can be lowered by reducing combined HLB strength of the emulsion.
- the drug loading in the formulation can be increased, as shown in the following Example.
- a microemulsion formulation was prepared with the composition identified in the following table: Quantity, % Component Added HLB Value HLB Contribution Labrafil M 1944 19.2 3.5 0.855 Capmul MCM 46.1 5.5 3.23 Centrophase 31 11.0 3.4 0.489 Propylene glycol 1.9 Tween 80 2.3 15.0 0.64 Hexyl insulin in q.s., 30 mg NaP buffer protein in buffer Total 100 5.0
- the aqueous phase of the above microemulsion can have polyhydroxylic alcohols such as manitol, which will reduce the amount of propylene glycol and the high HLB surfactant essential to form a similar water in oil (w/o) microemulsion.
- polyhydroxylic alcohols such as manitol
- a microemulsion formulation according to the invention was prepared with the composition shown below. Quantity, % Component Added HLB Value HLB Contribution Capmul MCM 45.3 5.5 4.72 Centrophase 31 6.4 3.4 0.48 Propylene Glycol 14.5 Tween 80 1.0 15.0 0.29 Mannitol 16.8 mg/mL of final formulation Hexyl insulin in q.s., (16.6 NaP buffer mg protein/mL of final emulsion) Total 100 5.49
- the aqueous phase of this type microemulsion can accommodate a mixture of polyethylene glycol 300 and polyethylene glycol 400 in addition to propylene glycol in the aqueous phase. as in the following formulation of Example XIV.
- a microemulsion formulation according to the invention was prepared with the composition shown below. Quantity, % Component Added HLB Value HLB Contribution Capmul MCM 54.1 5.5 4.80 Centrophase 31 6.45 3.4 0.42 Propylene glycol 14.5 Tween 80 1.5 15.0 0.36 Polyethylene glycol 4.9 400 Polyethylene glycol 3.9 300 Hexyl insulin in q.s., (18.8 NaP buffer mg protein/mL of final emulsion) Total 100 5.58
- an alcohol free w/o microemulsion of similar combined HBL strength can be formed using propylene glycol mono and diesters of medium chain fatty acids (Captex 200) and sodium octanoate (anionic surfactant).
- the high protein load in the microemulsion formulation is an added advantage, as shown in the following Example XV.
- a microemulsion formulation according to the invention was prepared with the composition shown below. Quantity, % Component Added HLB Value HLB Contribution Capmul MCM 20.0 5.5 3.12 Centrophase 31 13.3 3.4 1.51 Captex 200 40.0 Sodium Octanoate 1.86 20-23.0 1.22 Hexyl insulin in q.s., (46.6 NaP buffer mg protein/mL of final emulsion) Total 100 5.85
- HLB HLB strength microemulsions containing high protein load
- a microemulsion formulation according to the invention was prepared with the composition shown below. Quantity, % Component Added HLB Value HLB Contribution Capmul MCM 63.0 5.5 4.4 Centrophase 31 12.1 3.4 0.61 Caprol 860 4.1 11 0.57 Hexyl insulin in q.s., (40 mg NaP buffer protein/mL of final emulsion) Total 100 5.58
- a solid or semi-solid microemulsion can be prepared.
- excipients which are solids at room temperature
- a solid or semi-solid microemulsion can be prepared.
- Whitepsol H15 solid C 8 /C 10 triglyceride
- Imwitor 308 C 10 mono and diglycerides
- Egg lecithin solid at RT
- Gelucire 44/14 a solid dosage form of desired HLB can be obtained.
- the liquid microemulsions of the above examples can be encapsulated in a suitable solid hard fat and delivered in a capsule as a unit dosage form.
- the capsules can be enteric-coated and targeted for absorption in the intestine.
- the liquid emulsions can be filled in a soft gel capsule and delivered for absorption in the intestine.
- These soft gel capsules can also be enteric-coated for absorption in specific areas of the intestine.
- the protein or other therapeutic agent can be dispersed in a mixture of lipid and surfactant in accordance with the present invention and filled in a soft gel capsule as a pre-emulsion concentrate.
- These concentrates can self-emulsify under biological environment exposure conditions to form the desired final emulsion.
- Insulin was dissolved in dimethyl sulfoxide at 25° C. and reacted with 1.2 molar equivalent of activated methyl(ethyleneglycol) 7 -O-hexanoic acid for 45 minutes at 25° C.
- the product mixture (see Table B) containing monoconjugate (42 ⁇ 2.5%, aveg. M.W 6200), diconjugate (39 ⁇ 2.5%, Ave. M.W 6334), triconjugate (4 ⁇ 2.5%, Avge. M.W 7074) and unreacted insulin (12.5 ⁇ 2.5%, Avge. M.W 5734) was purified by dialysis on a 3500 MW cut off membrane at 5° C.
- the solution was lyophilized and protein content, product distribution and purity was determined by reverse phase HPLC.
- the molecular weights of products were determined by MALDI(TOF)/MS.
- the product mixture was analyzed for moisture, acetic acid and traces of bromo impurity.
- the hex-insulin mixture contains mainly mono and disubstituted insulin conjugates. Triconjugate and free insulin are present as the minor components.
- the specification of the components present in the product is given in Table B below and the stability characteristics of the hex-insulin mixture are shown in Table C below, in comparison with native insulin.
- TABLE B Specification for HEX-Ins Mixture Composition Fraction Composition Free insulin 12.5 ⁇ 2.5% monoconjugate (pK1) 42.0 ⁇ 2.5% diconjugate (pK2) 39.0 ⁇ 2.5% triconjugate (pK3) 4.0 ⁇ 2.5%
- FIG. 3 is a graph of ELISA assay results for native insulin in the NRS pool of various Sprague-Dawley rats, showing insulin concentration as a function of time, prior to and after dosing with insulin, at respective doses of 10 milligrams of insulin per kilogram of body weight, and at 30 milligrams of insulin per kilogram of body weight.
- Two rats were dosed at 10 milligrams insulin per kilogram of body weight (Rats C and I) and two rats were dosed at 30 milligrams insulin per kilogram of body weight (Rats K and L).
- the data show that the baseline level of the NRS pool is about 3.5 nanograms of insulin per milliliter.
- FIG. 4 is a graph of ELISA assay results for insulin in the NRS pool of various Sprague-Dawley rats, showing insulin concentration as a function of time, prior to and after dosing with insulin, at respective doses of 10 milligrams of insulin per kilogram of body weight, and at 30 milligrams of insulin per kilogram of body weight, wherein the insulin is administered as a conjugate, insulin-hexyl-PEG 7 -OMe.
- FIG. 5 is a graph of percent of pre-dose blood glucose level as a function of tine, for free insulin, and for conjugated insulin at respective doses of 1, 3 and 10 milligrams of insulin per kilogram of body weight, against a control of phosphate buffered solution (PBS) and bovine serum albumen (BSA).
- PBS phosphate buffered solution
- BSA bovine serum albumen
- FIG. 6 is a graph of insulin and blood glucose levels as a function of time, after dosing of canine subjects with a microemulsion insulin formulation representative of the present invention.
- the respective curves shown in the graph include native Zinc insulin; glucose ( ), hex-insulin; glucose ( ⁇ ); native Zinc insulin; insulin ( ⁇ ); and hex-insulin; insulin ( ⁇ ).
- Glucose data were normalized to the baseline glucose levels (0 minutes). Dogs were dosed orally with one of two emulsion formulations (native Zinc insulin or Hex-insulin mixture). The dose was adjusted to 1.0 mg/kg. Samples were collected at 30 minute intervals. The data were corrected for Hex-insulin reactivity in the BMI ELISA and Pharmacia RIA tests.
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US08/059,701 US5359030A (en) | 1993-05-10 | 1993-05-10 | Conjugation-stabilized polypeptide compositions, therapeutic delivery and diagnostic formulations comprising same, and method of making and using the same |
US08/276,890 US5438040A (en) | 1993-05-10 | 1994-07-19 | Conjugation-stabilized polypeptide compositions, therapeutic delivery and diagnostic formulations comprising same, and method of making and using the same |
US08/509,422 US5681811A (en) | 1993-05-10 | 1995-07-31 | Conjugation-stabilized therapeutic agent compositions, delivery and diagnostic formulations comprising same, and method of making and using the same |
US08/958,383 US6191105B1 (en) | 1993-05-10 | 1997-10-27 | Hydrophilic and lipophilic balanced microemulsion formulations of free-form and/or conjugation-stabilized therapeutic agents such as insulin |
US61420300A | 2000-07-12 | 2000-07-12 | |
US10/448,535 US20030229010A1 (en) | 1993-05-10 | 2003-06-02 | Oral insulin-oligomer conjugates |
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US10/448,524 Abandoned US20030229006A1 (en) | 1993-05-10 | 2003-05-30 | Amphiphilic oligomers |
US10/448,535 Abandoned US20030229010A1 (en) | 1993-05-10 | 2003-06-02 | Oral insulin-oligomer conjugates |
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US10/448,524 Abandoned US20030229006A1 (en) | 1993-05-10 | 2003-05-30 | Amphiphilic oligomers |
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US20090036353A1 (en) * | 2005-01-27 | 2009-02-05 | Novo Nordisk A/S | Insulin Derivatives Conjugated with Structurally Well Defined Branched Polymers |
US9260501B2 (en) | 2006-10-27 | 2016-02-16 | Novo Nordisk A/S | Peptide extended insulins |
US20080139392A1 (en) * | 2006-12-12 | 2008-06-12 | Edgar Joel Acosta-Zara | Linker-Based Lecithin Microemulsion Delivery Vehicles |
US9918934B2 (en) * | 2006-12-12 | 2018-03-20 | Edgar Joel Acosta-Zara | Linker-based lecithin microemulsion delivery vehicles |
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Also Published As
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US20030229006A1 (en) | 2003-12-11 |
US6191105B1 (en) | 2001-02-20 |
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