EP0670728A1 - Lipophilic peptide-based carriers for targeted drug delivery using rational drug-binding design - Google Patents

Lipophilic peptide-based carriers for targeted drug delivery using rational drug-binding design

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Publication number
EP0670728A1
EP0670728A1 EP94901482A EP94901482A EP0670728A1 EP 0670728 A1 EP0670728 A1 EP 0670728A1 EP 94901482 A EP94901482 A EP 94901482A EP 94901482 A EP94901482 A EP 94901482A EP 0670728 A1 EP0670728 A1 EP 0670728A1
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EP
European Patent Office
Prior art keywords
group
moiety
drug
combinations
pharmaceutical agent
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EP94901482A
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German (de)
French (fr)
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EP0670728A4 (en
Inventor
David Schlesinger
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Integenx Acquisition Corp
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Molecular Dynamics Inc
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Publication of EP0670728A1 publication Critical patent/EP0670728A1/en
Publication of EP0670728A4 publication Critical patent/EP0670728A4/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3526—Organic compounds containing nitrogen
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q15/00—Anti-perspirants or body deodorants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/57—Compounds covalently linked to a(n inert) carrier molecule, e.g. conjugates, pro-fragrances

Definitions

  • the present invention relates to the field of drug delivery and to the composition, methods of synthesis and uses of pH-dependent, peptide-based lipophilic drug carriers. More specifically, this invention relates to carriers that: (a) insulate the drug during transport through the lumen of the gastrointestinal ("GI") tract and cardiovascular circulation and during presentation to physiological barriers including, e.g., the mucosal linings of the GI tract and endothelial linings of the cardiovascular system including the blood brain barrier (“BBB”), and (b) enhance the drug's transport across such physiological barriers, such that during such transport, the drug remains structurally, chemically and biologically unmodified, thereby rendering the carrier efficacious for targeted xenobiotic and endogenous drug delivery.
  • GI gastrointestinal
  • BBB blood brain barrier
  • labile pharmaceutical agents comprising biologically active peptide or protein compositions, such as hormones and releasing factors, mucopolysaccharides (e.g., heparin) , polynucleotides, antibiotics, antivirals, and antifungals are administered via the parenteral route to avoid denaturation or degradation of the drug by stomach acid (HC1) or enzymes present in the GI tract (pepsin, trypsin, chymotrypsin and other endopeptidases and exopeptidases) .
  • HC1 stomach acid
  • pepsin, trypsin, chymotrypsin and other endopeptidases and exopeptidases enzymes present in the GI tract
  • drugs examples include insulin, growth hormone, releasing factors (e.g., corticotropin releasing factor, luteotropin-releasing hormone (“LHRH”), growth hormone releasing factor) and protein and glycoprotein immuno-stimulatory drugs (e.g., lymphokines, cytokines, poly I:poly C 12 U, interleukines, interferon, and macrophage granulocyte stimulating factor) .
  • releasing factors e.g., corticotropin releasing factor, luteotropin-releasing hormone (“LHRH”), growth hormone releasing factor
  • protein and glycoprotein immuno-stimulatory drugs e.g., lymphokines, cytokines, poly I:poly C 12 U, interleukines, interferon, and macrophage granulocyte stimulating factor.
  • xenobiotics are drugs that are not naturally produced or occurring in the body of the human or animal, but are synthesized or otherwise created externally for delivery internally.
  • the primary site of action of insulin is the liver hepatocyte so that an oral route of delivery of insulin would direct the drug via the hepatic portal vein to the liver.
  • the drug In order to achieve delivery, however, the drug must be rendered impervious to degradation in the GI tract, while simultaneously rendered permeable across the GI tract's epithelial linings.
  • gastrointestinal tract long considered a major lymphoid organ because of its lining with lymphoid tissues (such as Payer's patches and abundant lymphoid cells) , is an appropriate target for absorption of the vaccine, since absorption at this target would result in a substantial stimulation of an immunological response.
  • ectopic and thus non- parenteral method utilizes the co-administration of adjuvants to facilitate absorption.
  • adjuvants include resorcinols, surfactants such as polyoxyethyleneoleyl ether and n-hexadecyl polyethylene ether, and muramyl dipeptide (Vaccine S.A.).
  • ectopic methods limit the types of pharmaceutical agents that can be administered, as well as the medical conditions to be treated. Indeed, chronic use of ectopics is not medically recommended because of limited absorption of the drug.
  • Another approach involves the co-administration of enzyme inhibitors, such as trasylol, diisopropylfluorophosphate (DFP) and pancreatic trypsin inhibitor.
  • enzyme inhibitors such as trasylol, diisopropylfluorophosphate (DFP) and pancreatic trypsin inhibitor.
  • DFP diisopropylfluorophosphate
  • pancreatic trypsin inhibitor Such inhibitors are typically administered parenterally together with the pharmaceutical agent to inhibit enzymatic degradation of the agent.
  • Enzyme inhibitors do not, however, protect against the acidic conditions and resultant degradation of the pharmaceutical agent in the stomach and do not facilitate intestinal permeation. Consequently, this approach is not well-suited for exogenous, or more specifically, oral drug delivery.
  • liposomes i.e., non-protein based water-in-oil-water emulsions that are non-selective, non-specific binders of the pharmaceutical agent and are thought to be suitable microreserves for controlled release of the bound agent.
  • Liposomes containing heparin have been disclosed in U.S. Patent No. 4,239,734.
  • Several publications have also described the administration of insulin via liposomes (Patel, et al., FEBS Letters 62:60 (1976) and Hashimoto, et al. Endocrinol. Japan 26:337 (1979)).
  • Liposomes are, however, non-selective, i.e., not specifically developed and modified for binding with specific drugs, and are rather a class of emulsions.
  • liposomes are not rapidly degraded, and once degraded, have potentially deleterious effects as a consequence of their fatty acid-based composition.
  • liposome technology has not yet been proven efficacious.
  • liposome A more specific form of liposome has been disclosed by Yessair, "Phosphatidyl Choline and Pysophosphatidyl Choline in Mixed Lipid Micelles as Novel Drug Delivery Systems," PhosDholipids, 1990, pp. 83-106, and U.S. Patent Nos.
  • Yessair recognizes the need to create an oral form of drug delivery, he solves the need by developing a non-selective carrier. Like liposome technology, though Yessair's carrier increases permeation across the epithelial lining of the GI tract, the base of the carrier is fatty acid in composition, and thus may have deleterious physiological consequences upon degradation. This form of delivery is, as well, not conceived using a rational drug design.
  • microspheres Oral administration of heparin and insulin purportedly "encapsulated” in solid phase hollow particles called “microspheres,” has been disclosed in U.S. Patent No. 4,925,673. Such microspheres are allegedly created from linear peptides that "self-assemble” into solid phase particles by thermal condensation. See, e.g., Fox, S. . and Harada, K. (1960) J. Amer. Chem. Soc. 82:3745. There has, however, been no rigorous proof of their true existence, and thus the concept is at best hypothetical. The production of such "microspheres” involves the use of a number of different L-amino acids dispersed in a medium which is then thermally co-polymerized.
  • Acrylics of proprietary composition have also been suggested as oral drug forms for administration of insulin by Cortecs, with inconclusive results.
  • non-toxicity has not been established, and bio-reactivity has not been considered in sufficient detail to render these oral formulations more than hypothetical.
  • bio-reactivity has not been considered in sufficient detail to render these oral formulations more than hypothetical.
  • There is also a potential that such materials will be deposited in the liver and thus increase toxic effects. Again, such materials are non-specific, and do not incorporate rational drug design in their formulation.
  • Bile salts have been combined with proteins which then can be administered intra-nasally with drugs to improve bioavailability (California Biotechnology) .
  • this technique is not a rationally created drug design, is non ⁇ specific and non-oral, and is limited to only those drugs that are viable for intranasal delivery.
  • Bodor, et al. report the use of "packaged" enkephalins which enable the delivery of analgesics across the BBB.
  • Bodor, et al. "A Strategy for Delivering Peptides into the Central Nervous System by Sequential Metabolism," Science, Vol. 257, September 18, 1992, pp. 1698-1700.
  • His package comprises the attachment of a steroid nucleus to a polypeptide base (YAGFL) to render the thereby-created conjugate lipophilic in nature, and thus less susceptible to enzymatic degradation, while increasing permeability.
  • YAGFL polypeptide base
  • this conjugate is specialized only for the BBB and leaves a large, bulky steroid moiety in the CNS.
  • Bodor's conjugate is an injectable; he does not discuss oral forms of delivery in any manner, nor the specialized nature of a CNS oral drug delivery vehicle, i.e., the need to first facilitate absorption across the intestinal barrier, followed by delivery without degradation to the BBB, and finally absorption across the BBB.
  • His injectable vehicle provides merely for the step across the BBB, and thus is an unlikely candidate for oral delivery.
  • the ester bond between the steroid moiety and analgesic peptide would be a target for cleavage by esterases in the lower GI tract. Such susceptibility to cleavage renders his vehicle inappropriate as an oral drug form.
  • Alkermes Other methods for targeting and crossing the BBB have been suggested by Alkermes.
  • the Alkermes method incorporates a re-engineered bradykinin carrier to dilate the endothelial linings of the brain capillaries.
  • other potentially deleterious compounds can be transported across the BBB as a consequence of its dilation.
  • the process is non-specific, and is not of a rational drug design.
  • Recent publications also disclose the abundant need for an oral form of drug delivery. For example, Kleinert, et al. , Discovery of a Peptide-Based Renin Inhibitor with Oral Bioavailability and Efficacy, Science, Vol. 257, 9/25/92, pp.
  • Rational drug design involves creating a pre-determined composition whose constituent moieties are selected for their specific chemical interactions and binding with the drug to be delivered, as well as for the physiological qualities of the environment in which the drug and its carrier will be placed.
  • Rational drug design the most potent technology for understanding the relationships between structure and function, requires a knowledge of primary, secondary and tertiary structure of peptides and proteins, and involves a host of computer-based tools that will allow three-dimensional viewing of both the chemical construct of the carrier, as well as the drug, and the final complex of the construct and the drug formed, with the goal of maximizing drug binding and intestinal permeation.
  • the tools also involve a combination of crystallography, spectroscopy, and computational methods to avoid the typically employed high-volume randomized screening of naturally occurring and/or synthesized drugs and their analogues and the types of carriers required to transport them across physiological barriers.
  • X is an amino acid blocking group
  • A is selected from the group consisting of at least one amino acid
  • B is selected from the group consisting of lysine, ornithine and combinations thereof
  • C is selected from the group consisting of a free carboxyl group, or a protected carboxamide (CONH 2 )
  • n is the number of cycles of addition of B.
  • the invention further comprises creating a complex of the carrier and a xenobiotic or endogenous drug.
  • Branched chain ligands defined herein as “kepitopes, " are constructed at the molecular level of specific amino acids such that the resultant kepitope behaves chemically at low pH as if it were an aqueous-immiscible oil. These ligands bind to active pharmaceutical agents in molecular form without altering the structure and biological functionality of the agent. These kepitope constructs are capable of transporting drugs across epithelial linings of the lower gastrointestinal tract, and the process primarily, but not exclusively, involves facilitated passive diffusional mechanisms.
  • these constructs are homogeneous molecular species, typically between 1000 to 5000 Daltons, prepared by solid phase peptide synthesis (though solution phase synthesis is feasible, as well) and consist of multiple copies of one or more surface epitopes constructed on a polylysine, polyornithine or polylysine-polyornithine branched chain peptide matrix (preferably polylysine) .
  • the surface epitopes contain acidic (dicarboxylic) amino acids for oral delivery formulations (so that residues are uncharged at low pH) but are predominantly hydrophobic in nature, favoring oil droplets (aqueous phase immiscibility) and contain sufficient ionic and/or hydrophilic character to allow the drug to be bound non-covalently via salt bridges (ionic) and/or by hydrophobic interaction, and thus provide reversible, pH dependent partitioning of the drug carrier complex between aqueous and organic phases.
  • acidic (dicarboxylic) amino acids for oral delivery formulations (so that residues are uncharged at low pH) but are predominantly hydrophobic in nature, favoring oil droplets (aqueous phase immiscibility) and contain sufficient ionic and/or hydrophilic character to allow the drug to be bound non-covalently via salt bridges (ionic) and/or by hydrophobic interaction, and thus provide reversible, pH dependent partitioning of the drug carrier complex between aqueous and organic phases
  • Fig. 1 is a diagrammatical representation of the complex of kepitope and drug in accordance with the invention.
  • Solid phase synthesis is performed according to a modified method (Schlesinger, D.H., Conchrane, A.H., Gwadz, R.W. , Godson, G.N., Melton, R. , Nussenzweig, R. and Nussenzweig, V., Biochemistry 23:5665-5670, 1984) of the solid phase peptide synthesis protocol (Merrifield, R.B. J. Amer. Chem. Soc, 85:2149-2154, 1963).
  • the K constructs are synthesized by a modified protocol of Tarn (1988) on a benzhydryl amine resin with a substitution of 0.65 meq/gm.
  • the resin is washed with multiple additions of ethanol, dicloromethane (DCM) , and dimethyl formamide. It is then treated with 50% (in DCM) trifluoroacetic acid (TFA) for 30 min. and then washed with DCM.
  • DCM dicloromethane
  • TFA trifluoroacetic acid
  • the amino function on the resin is neutralized with diisopropyl ethyl amine (DIPEA) (10%in DCM) and then washed with DCM.
  • DIPEA diisopropyl ethyl amine
  • a three-fold molar excess of bis-boc-lysine (or bis-boc-ornithine) and hydroxybenzotriazole (HOBT) is added to the resin and the reaction allowed to proceed for 2 hours followed by test for completion of coupling with ninhydrin.
  • lysine or ornithine, as the case may be
  • the selection is based on rational drug carrier design parameters, including the properties of the drug and the environment in which delivery is anticipated.
  • the pre-selected series of polypeptide surface epitopes are then cyclically added to each of the molecular weight constructs using the identical protocol for the K constructs themselves (there being three in number under the prior embodiment): the lysine- (or ornithine-) resins are first washed, then the multiple N-terminal boc groups are removed with TFA and the lysine- (or ornithine-) resin then neutralized with DIPEA. A three-fold molar excess of each amino acid along with a three-fold excess of HOBT is then added to the lysine- (or ornithine-) resin (to inhibit racimization) and allowed to react for 2 hours followed by ninhydrin test for completion of coupling.
  • the N-terminus of the completed surface epitope-K- linked construct can be blocked to remove the additional positive charge on the epitope with pyroglutamate, or can simply be untreated with TFA to preserve the Boc or Fmoc group.
  • the completed construct is then treated with anhydrous HF (0° C, 1 hr.) to remove side-chain protecting groups and to cleave the lysine- (or ornithine-) branched peptide Kepitope ligand from the benzhydryl amine resin. This is accomplished by the addition of anhydrous HF (10 ml/gm peptide resin) .
  • the HF is removed by a stream of N 2 for 30 min., followed by water aspiration.
  • the mixture is then washed with cold anhydrous ether to remove excess anisole.
  • the peptide is then extracted with alternate washes of water and acetic acid and then lophilized.
  • the peptide constructs can first be purified by gel permeation chromatography for desalting and then by reversed phase HPLC as the final purification step.
  • the kepitope can also be synthesized in solution phase.
  • Solution phase synthesis of the K-construct and carrier may be a long-term preferred method in light of the fact that it is more cost-effective for synthesizing larger quantities. In this methodology, there is no inert support, but the protecting groups are very similar.
  • One of reasonable skill in the art easily understands, when using the teachings contained herein, how to synthesize the construct and carriers using solution phase techniques.
  • Kepitopes 1. Amino acid analysis. The constructs are hydrolized back to their constituent amino acids with 6NHCL at 110°C for 24 hours and then evaporated to dryness, reconstituted and subjected to reverse phase HPLC for amino acid identification and quantification. 2. Test for homogeneity by HPLC and sequence analysis.
  • Homogeneity of the carrier is performed by reverse-phase HPLC on a C18 ODS column as the stationary phase and a linear gradient of water against acetonitrile as the mobile phase, with an increasing percentage of acetonitrile to develop the gradient.
  • Automated sequencing of the carrier is performed via gas phase sequencing using a modified procedure of Edman, P. and Begg, E.G. (Europ, J. Biochem 1:80-90, 1967). Binding of drug to Kepitope
  • the epitope is designed with the ionic and hydrophobic characteristics favored to bind reversibly according to the drug's physical and chemical parameters (Table I, below) .
  • hydrophobicity of the drug requires a hydrophobic character of the surface epitope.
  • Anionic drugs such as heparin
  • the ligand have a net positive charge for heparin binding, as well as enough hydrophobic character to penetrate the intestinal mucosa or the BBB.
  • the pH of the aqueous solvent must exist at or very near the isoelectric point (pKI) of the ligand.
  • the drug 0.2-200mg/ml
  • the aqueous phase can be removed by lyophilization. Alternatively, the two phases are separated by sedimentation in a low speed centrifuge.
  • the drug-ligand complex can cleaned in such a manner as to remove any biologically active drug from the surface of the complex by dialysis against a pH 2 solution (HCl-acetic acid, 0.05 N) containing pepsin (1:100 enzyme to complex ratio) .
  • This procedure enzymatically degrades biologically active drug from the surface of the complex (pepsin being complex-impenetrable at these concentrations and pH) .
  • Pepsin remaining in the resulting dialysate is readily degraded in vivo in the lower G.I. tract by the animal's own endogenous chymotrypsin and trypsin and other endopeptides.
  • FIG. 1 is a diagrammatical representation of the complex of kepitope and drug, in accordance with the invention.
  • the overall circle depicts the organic phase containing the hydrophobic drug-ligand complex in an aqueous solvent.
  • Ligand consists of multiple surface epitopes (SE) 6, covalently attached to the K construct 2, which determines the ligand's hydrophobicity and pKI.
  • Epitopes are covalently linked to the branched chain K construct which determines the number of epitopes and hence the ligands molecular weight.
  • Hatched squares 8 depict drug; K x , K 2/ and K 3 represent serial additions of lysine during synthesis of the K construct.
  • the drug is bound to the ligand through weak forces, such as ionic and hydrophobic bonds only, facilitating release at physiological pH at which the ligand is highly charged and becomes miscible in aqueous solvents such as blood.
  • the N- terminus contains pyroglutamic acid (pE or pGlu) or N-blocking group such as N-acetyl, Boc, Fmoc.
  • the C-terminus 4, as shown in Fig. 1, is CONH 2 , though it is understood that terminus 4 may be in the form of a free carboxylic acid.
  • binding constants are express in terms of the partition coefficients ("K") of the pharmaceutical agent ("A"), in accordance with the following formula:
  • K [A organic phase] / [A aqueous phase] Table I was created in the following manner. First the particular kepitope (surface epitope on a K 7 construct) was synthesized using the techniques set forth, above. Second, the agent (0.2 mg/ml) together with the kepitope (40 mg/ml) were dissolved in water and the pH was raised to pH 8 with NH 4 OH. Next, the pH of the solution was lowered to pH 3.5 to 4.0. At this pH, carboxyl groups are no longer ionized, and two phases are formed. The resulting phases are separated by low speed centrifugation. The concentration of the drug is measured by high performance liquid chromatography ("HPLC") of an aliquot of a sample from each phase.
  • HPLC high performance liquid chromatography
  • the concentration and resulting pH of the kepitope is selected such that the plait point (formation of a single phase) is not reached.
  • the molecular weights of luteinizing hormone-releasing hormone ("LHRH"), vasopressin, substance P and oxytocin are about 1000 Daltons.
  • the lysine (or ornithine) branched chain matrix used in Table I is a K 7 construct (molecular weight of 1000 Daltons) . Since the complete kepitope contains 8 copies of the surface epitopes, the molecular weight of the kepitope is 9000 Daltons. As can be observed in Table I, above, selection of the particular surface epitopes will change the affinity of the
  • peptides and proteins tested for bioavailability by means of RIA include neuropeptide Y (NPY) , insulin, an immunoglobulin (IgG) , substance P, and calcitonin as well as the ligand itself.
  • Adjuvants are typically utilized to increase localized blood flow to aid in the absorption of the vaccine, upon administration via skin-based infusion.
  • the surface epitopes (SE's) attached to the polylysine (or polyornithine) matrix are selected based upon the primary structure and chemical properties of the vaccine. For example, where the vaccine possesses numerous surface polypeptides that are relatively uncharged, e.g., phenylalanine, then the SE's selected are also hydrophobic and are selected from the group comprising tyrosine, valine, isoleucine, leucine, methionine, cysteine, phenylalanine, alanine, proline, and glycine. In this instance, the bonding is achieved through hydrophobic interaction. For the aromatic amino acids, such bonding is ⁇ - ⁇ . It should be appreciated that the synthesized adjuvant-vaccine composition appears as an oil, and is administered as such.
  • Vaccines are prepared in accordance with known technology, and can include, where necessary to prevent enzymatic degradation, D-amino acids, rather than the susceptible, more common and naturally occurring L-form. Since such hydrophobic bonding is weak and therefore reversible, upon interaction with the physiological fluids present upon administration of the vaccine, such bonds will be broken and the vaccine will be released in the same manner as is well known in the art. In this instance, a two-phased condition will result wherein the adjuvant, having oil-like characteristics, will separate in physiological fluid from the vaccine which will slowly dissolve, pass into the aqueous phase and thereby facilitate absorption-based delivery. It should be appreciated that such vaccine delivery can be used for both humans and animals, including pets and livestock. Vaccine Delivery Via Oral Administration
  • the SE's (and vaccines) selected may be the same as those selected for adjuvant-based delivery in order to facilitate bonding of the matrix to the vaccine. It is theorized that this form of vaccine will cause an improved immunological response in that the oil-like characteristics of the construct will render the engulfed vaccine somewhat more impervious to enzymatic degradation and denaturation and hydrolysis by stomach acid than that heretofore known.
  • the pathways for absorption are the same as those explained above in connection with pharmaceutical delivery, wherein the vaccine is released after the construct-vaccine complex is absorbed into the blood stream. Upon absorption, the complex dissociates, and the vaccine migrates into the aqueous phase.
  • D-amino acids in the synthesis of the carrier to prevent enzymatic degradation of the vaccine in the intestine and/or blood stream prior to immuno-stimulatory action.
  • selection and synthesis techniques for the vaccine are well-known in the art. It should be recognized that such vaccine delivery systems are suitable for use in both humans and animals, including pets and livestock.
  • BBB Blood/Brain Barrier
  • the carrier-drug complex must permeate and overcome without dissociating, in order to deliver the drug to the CNS target: intestinal and BBB.
  • the SE attached to the K-construct must be of a nature that they are highly-hydrophobic for absorption at both barriers, while non-susceptible to blood- based enzymatic attack prior to presentation at the BBB.
  • this is a specialized instance of rational drug design, since the SE's must be selected with the nature of the particular drug in mind.
  • microreserve systems are created in order to have a controlled form of drug delivery over time, releasing their drug-bound products within the cardiovascular system. Consequently, the complex must be created with the use of amino acids that do not immediately become charged when the pH rises after passing the intestinal barrier, such that dissociation is slower, and a time continuum is created.
  • SE's in this instance need not be the aspartic and glutamic acids utilized for rapid dissociation, and a more rational drug schemata should employ a constellation of other hydrophobic amino acids which may include a greater percentage of D-amino acids which are known to be less enzymatically degradable, or degrade at a slower rate.
  • a high molecular weight carrier rendering low molecular weight drugs less likely to be filtered by the kidney and with a high degree of non- degradability by the liver and circulating proteases in the blood.
  • Adding more lysines or ornithines to the construct exponentially increases the size and molecular weight of the carrier by a function of 2 n , where n equals the number of cycles of addition of lysine or ornithine during synthesis.
  • Higher molecular weight complexes will be more impervious to kidney filtration, since clearance times are increased and such complexes will linger longer in the blood stream.
  • the molecular weight of the complex can also be increased by using longer chain epitopes.
  • Antiperspirants are known to be relatively inactive until placed in the aqueous environment of perspiration.
  • a complex can be formed using SE's rationally selected to associate with molecular antiperspirant to form oil droplets which can then be applied ectopically and be available "on demand," i.e., upon emersion within the aqueous environment of perspiration. Since perspiration has a pH near neutral (7.0), it will cause ionization of the construct followed by dissociation of the bound active ingredient in anti-perspirant, thereby liberating its effects.
  • perfumes are best if they can be released over time, rendering them long acting.
  • a carrier having perfume molecules bound to it will ionize upon presence of perspiration and thereby dissociate and liberate the perfume molecules, which then volatilize.
  • the carrier can be used to enable the controlled delivery of preservatives and additives, and will, as a consequence of its hydrophobicity, itself act as a preservative.
  • specific SE's can be selected which will enable binding to the preservative and additive, when needed, and will thereby ensure controlled release.
  • a food additive such a complex will preserve the flavor, consistency and surface characteristics of foods over time.
  • the objective here is to give protection to medicinals against fluid-based inactivation.
  • Some drugs readily hydrolyze or are labile in the presence of water, i.e., may undergo conformational changes or changes in secondary structure.
  • the complex would be formed of a hydrophobic epitope which binds to the medicinal. While not impeding the activity of the medicinal, such a complex, as a consequence of its hydrophobicity, would prevent fluid entry and concomitant inactivation.
  • rational drug design is employed to carefully select SE's to be placed on the polylysine (or polyornithine) matrix which will achieve such results.
  • the kepitopes of the instant invention are well-suited for rational drug design (drug-binding design) .
  • the process involves first ascertaining the properties of the drug sought to be delivered via one of the methods set forth herein, e.g., orally.
  • the mode of delivery must be selected, e.g., injectable, ectopic, oral, intranasal, ocular, buccal, vaginal, rectal, transdermal, since the physiological barriers that must be overcome, while maintaining the complex in tact, will determine the nature of the kepitope.
  • the primary structure (i.e., amino acid sequence) and secondary structure (i.e., how it folds in two dimensions by forming disulfide bridges) of a series of kepitopes are determined via a series of tools which include three-dimensional, computer based modeling. Since the size of surface epitopes utilized herein will likely be small, there is little concern about the tertiary structure, since three-dimensional conformational changes are unlikely. Also, a series of kepitope candidates, rather than a single kepitope, is desirable to allow selection of the candidate that provides the best bioavailability data.

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Abstract

Composition, methods of synthesis and uses of a peptide-based moiety for oral drug delivery, having the general formula: [X-A]2$(1,2)$ - [B]2$(1,2)$-1 - C, wherein X is an amino acid blocking group; A is selected from the group consisting of at least one amino acid; B is selected from the group consisting of lysine, ornithine and combinations thereof; C is selected from the group consisting of a free carboxyl group, or a protected carboxamide (CONH2); and n is the number of cycles of addition of B, including the addition of various xenobiotics and endogenous drugs.

Description

LIPOPHILIC PEPTIDE-BASED CARRIERS FOR TARGETED DRUG DELIVERY USING RATIONAL DRUG-BINDING DESIGN
Field of the Invention
The present invention relates to the field of drug delivery and to the composition, methods of synthesis and uses of pH-dependent, peptide-based lipophilic drug carriers. More specifically, this invention relates to carriers that: (a) insulate the drug during transport through the lumen of the gastrointestinal ("GI") tract and cardiovascular circulation and during presentation to physiological barriers including, e.g., the mucosal linings of the GI tract and endothelial linings of the cardiovascular system including the blood brain barrier ("BBB"), and (b) enhance the drug's transport across such physiological barriers, such that during such transport, the drug remains structurally, chemically and biologically unmodified, thereby rendering the carrier efficacious for targeted xenobiotic and endogenous drug delivery.
Background of the Invention It is well known that a wide variety of labile pharmaceutical agents comprising biologically active peptide or protein compositions, such as hormones and releasing factors, mucopolysaccharides (e.g., heparin) , polynucleotides, antibiotics, antivirals, and antifungals are administered via the parenteral route to avoid denaturation or degradation of the drug by stomach acid (HC1) or enzymes present in the GI tract (pepsin, trypsin, chymotrypsin and other endopeptidases and exopeptidases) . Examples of such drugs include insulin, growth hormone, releasing factors (e.g., corticotropin releasing factor, luteotropin-releasing hormone ("LHRH"), growth hormone releasing factor) and protein and glycoprotein immuno-stimulatory drugs (e.g., lymphokines, cytokines, poly I:poly C12U, interleukines, interferon, and macrophage granulocyte stimulating factor) . Definitionally, "xenobiotics" are drugs that are not naturally produced or occurring in the body of the human or animal, but are synthesized or otherwise created externally for delivery internally.
An oral pathway for delivery of xenobiotics and endogenous drugs, especially suitable for chronic application, has long been sought and desired, though heretofore largely unavailable. Such a pathway would avoid the physically intrusive nature of infusions and the necessity of hospital out-patient or supervised in-home care, as well as catheterization typically associated with regular intravenous infusions. Also, it should be appreciated that the oral route of administration which is then followed by intestinal absorption into the blood stream or lymphatic system, may be more efficacious than intravenous administration, since delivery of the drug into circulation via oral administration occurs slowly over time.
For example, the primary site of action of insulin is the liver hepatocyte so that an oral route of delivery of insulin would direct the drug via the hepatic portal vein to the liver. In order to achieve delivery, however, the drug must be rendered impervious to degradation in the GI tract, while simultaneously rendered permeable across the GI tract's epithelial linings.
Similarly, it would be medically preferred to administer vaccines orally rather than intravenously for all of the foregoing reasons, as well as for the desire to deliver the drug directly to the GI tract. The gastrointestinal tract, long considered a major lymphoid organ because of its lining with lymphoid tissues (such as Payer's patches and abundant lymphoid cells) , is an appropriate target for absorption of the vaccine, since absorption at this target would result in a substantial stimulation of an immunological response.
Much effort has been directed towards circumventing the physiological barriers associated with drug delivery of pharmacologically active agents. One ectopic (and thus non- parenteral) method utilizes the co-administration of adjuvants to facilitate absorption. Examples of non-toxic adjuvants include resorcinols, surfactants such as polyoxyethyleneoleyl ether and n-hexadecyl polyethylene ether, and muramyl dipeptide (Vaccine S.A.). Of course, ectopic methods limit the types of pharmaceutical agents that can be administered, as well as the medical conditions to be treated. Indeed, chronic use of ectopics is not medically recommended because of limited absorption of the drug. Another approach involves the co-administration of enzyme inhibitors, such as trasylol, diisopropylfluorophosphate (DFP) and pancreatic trypsin inhibitor. Such inhibitors are typically administered parenterally together with the pharmaceutical agent to inhibit enzymatic degradation of the agent. Enzyme inhibitors do not, however, protect against the acidic conditions and resultant degradation of the pharmaceutical agent in the stomach and do not facilitate intestinal permeation. Consequently, this approach is not well-suited for exogenous, or more specifically, oral drug delivery.
Other parenteral approaches have included the use of liposomes, i.e., non-protein based water-in-oil-water emulsions that are non-selective, non-specific binders of the pharmaceutical agent and are thought to be suitable microreserves for controlled release of the bound agent.
Liposomes containing heparin have been disclosed in U.S. Patent No. 4,239,734. Several publications have also described the administration of insulin via liposomes (Patel, et al., FEBS Letters 62:60 (1976) and Hashimoto, et al. Endocrinol. Japan 26:337 (1979)). Liposomes are, however, non-selective, i.e., not specifically developed and modified for binding with specific drugs, and are rather a class of emulsions. Also, liposomes are not rapidly degraded, and once degraded, have potentially deleterious effects as a consequence of their fatty acid-based composition. Lastly, liposome technology has not yet been proven efficacious.
A more specific form of liposome has been disclosed by Yessair, "Phosphatidyl Choline and Pysophosphatidyl Choline in Mixed Lipid Micelles as Novel Drug Delivery Systems," PhosDholipids, 1990, pp. 83-106, and U.S. Patent Nos.
4,874,795 and 4,298,594. Although Yessair recognizes the need to create an oral form of drug delivery, he solves the need by developing a non-selective carrier. Like liposome technology, though Yessair's carrier increases permeation across the epithelial lining of the GI tract, the base of the carrier is fatty acid in composition, and thus may have deleterious physiological consequences upon degradation. This form of delivery is, as well, not conceived using a rational drug design.
Oral administration of heparin and insulin purportedly "encapsulated" in solid phase hollow particles called "microspheres," has been disclosed in U.S. Patent No. 4,925,673. Such microspheres are allegedly created from linear peptides that "self-assemble" into solid phase particles by thermal condensation. See, e.g., Fox, S. . and Harada, K. (1960) J. Amer. Chem. Soc. 82:3745. There has, however, been no rigorous proof of their true existence, and thus the concept is at best hypothetical. The production of such "microspheres" involves the use of a number of different L-amino acids dispersed in a medium which is then thermally co-polymerized. The products of this process are extremely heterogenous, representing the many different copolymers that can be formed, such as anhydrides, diketopiperizenes, and linear, branched chain and cyclic peptides of undetermined length and sequence. Likewise, there is no evidence that such "microspheres" are non-degradable by stomach acid or peptidase action, nor that such particles will be permeable in the GI epithelial lining. The mechanisms of absorption in the GI tract, if any, are unknown.
Acrylics of proprietary composition have also been suggested as oral drug forms for administration of insulin by Cortecs, with inconclusive results. There are also significant medical concerns associated with the use of such polymers. In particular, non-toxicity has not been established, and bio-reactivity has not been considered in sufficient detail to render these oral formulations more than hypothetical. There is also a potential that such materials will be deposited in the liver and thus increase toxic effects. Again, such materials are non-specific, and do not incorporate rational drug design in their formulation.
Bile salts have been combined with proteins which then can be administered intra-nasally with drugs to improve bioavailability (California Biotechnology) . However, this technique is not a rationally created drug design, is non¬ specific and non-oral, and is limited to only those drugs that are viable for intranasal delivery. With respect to rational drug design, Bodor, et al. report the use of "packaged" enkephalins which enable the delivery of analgesics across the BBB. Bodor, et al., "A Strategy for Delivering Peptides into the Central Nervous System by Sequential Metabolism," Science, Vol. 257, September 18, 1992, pp. 1698-1700. His package comprises the attachment of a steroid nucleus to a polypeptide base (YAGFL) to render the thereby-created conjugate lipophilic in nature, and thus less susceptible to enzymatic degradation, while increasing permeability. However, this conjugate is specialized only for the BBB and leaves a large, bulky steroid moiety in the CNS. Likewise, Bodor's conjugate is an injectable; he does not discuss oral forms of delivery in any manner, nor the specialized nature of a CNS oral drug delivery vehicle, i.e., the need to first facilitate absorption across the intestinal barrier, followed by delivery without degradation to the BBB, and finally absorption across the BBB. His injectable vehicle provides merely for the step across the BBB, and thus is an unlikely candidate for oral delivery. In particular, the ester bond between the steroid moiety and analgesic peptide would be a target for cleavage by esterases in the lower GI tract. Such susceptibility to cleavage renders his vehicle inappropriate as an oral drug form.
Other methods for targeting and crossing the BBB have been suggested by Alkermes. In particular the Alkermes method incorporates a re-engineered bradykinin carrier to dilate the endothelial linings of the brain capillaries. However, other potentially deleterious compounds can be transported across the BBB as a consequence of its dilation. Thus, the process is non-specific, and is not of a rational drug design. Recent publications also disclose the abundant need for an oral form of drug delivery. For example, Kleinert, et al. , Discovery of a Peptide-Based Renin Inhibitor with Oral Bioavailability and Efficacy, Science, Vol. 257, 9/25/92, pp. 1940-1943, teaches the use of an analogue of a peptide-based renin inhibitor which is orally active, and characterized as lipophilic. However, the molecular species is the drug, not a carrier for the drug. In other words, there is no carrier, and the fact that the drug is lipophilic is merely accidental. Such an accidental finding supports the theory that increased lipophilicity increases intestinal absorption. This theory of lipophilicity is, as shown below, supported and proven by the present invention which does not accidentally achieve lipophilicity of the drug, but sets it as an important goal of the carrier's rational design.
Thus, it can be observed that the prior art does not, in typical fashion, teach rational drug design for oral delivery. "Rational drug design" (or drug binding design) for oral delivery involves creating a pre-determined composition whose constituent moieties are selected for their specific chemical interactions and binding with the drug to be delivered, as well as for the physiological qualities of the environment in which the drug and its carrier will be placed. Rational drug design, the most potent technology for understanding the relationships between structure and function, requires a knowledge of primary, secondary and tertiary structure of peptides and proteins, and involves a host of computer-based tools that will allow three-dimensional viewing of both the chemical construct of the carrier, as well as the drug, and the final complex of the construct and the drug formed, with the goal of maximizing drug binding and intestinal permeation. The tools also involve a combination of crystallography, spectroscopy, and computational methods to avoid the typically employed high-volume randomized screening of naturally occurring and/or synthesized drugs and their analogues and the types of carriers required to transport them across physiological barriers.
It is clear that such methods of rational drug design have heretofore been unknown. For example, Kuntz, Irwin D., "Structure-Based Strategies for Drug Design and Discovery, "
Science, Vol. 257, August 21, 1992 pp. 1078-1082 teaches away from the invention by suggesting that while it is desirable to have such rational methods of examining drugs, carriers, and complexes structurally and functionally, such methods of design are not yet available.
It is therefore an object of the present invention to create a carrier that is variable in chemical construction to enable binding with various xenobiotics as well as endogenous drugs to create a complex.
It is another object of the present invention to create a complex of a xenobiotic (or endogenous drug) and carrier that possesses chemical characteristics suitable for targeted delivery of the drug.
It is a further object of the present invention to provide a rational form of drug delivery that is non- degradable until it reaches its targeted location for delivery. It is a still further object of the present invention to provide a rational form of drug delivery having constituent amino acids that are predetermined to enhance affinity for particular drugs as well as to insulate the drug during transport to, and presentation at physiological barriers, and to facilitate permeation of said barriers and delivery of a structurally, chemically and biologically unmodified drug to its target.
It is yet a still further object of the present invention to provide a carrier that it is easily degraded after delivery of its drug cargo without any potentially deleterious or toxic effects.
Summary of the Invention
The foregoing and other objects are achieved by the present invention, which is directed to the composition, methods of synthesis and uses of a peptide-based moiety for drug delivery, having the general formula:
[X-A] 2n :B] 2r--l wherein X is an amino acid blocking group; A is selected from the group consisting of at least one amino acid; B is selected from the group consisting of lysine, ornithine and combinations thereof; C is selected from the group consisting of a free carboxyl group, or a protected carboxamide (CONH2) ; and n is the number of cycles of addition of B. The invention further comprises creating a complex of the carrier and a xenobiotic or endogenous drug.
Branched chain ligands, defined herein as "kepitopes, " are constructed at the molecular level of specific amino acids such that the resultant kepitope behaves chemically at low pH as if it were an aqueous-immiscible oil. These ligands bind to active pharmaceutical agents in molecular form without altering the structure and biological functionality of the agent. These kepitope constructs are capable of transporting drugs across epithelial linings of the lower gastrointestinal tract, and the process primarily, but not exclusively, involves facilitated passive diffusional mechanisms. Chemically, these constructs are homogeneous molecular species, typically between 1000 to 5000 Daltons, prepared by solid phase peptide synthesis (though solution phase synthesis is feasible, as well) and consist of multiple copies of one or more surface epitopes constructed on a polylysine, polyornithine or polylysine-polyornithine branched chain peptide matrix (preferably polylysine) . The surface epitopes contain acidic (dicarboxylic) amino acids for oral delivery formulations (so that residues are uncharged at low pH) but are predominantly hydrophobic in nature, favoring oil droplets (aqueous phase immiscibility) and contain sufficient ionic and/or hydrophilic character to allow the drug to be bound non-covalently via salt bridges (ionic) and/or by hydrophobic interaction, and thus provide reversible, pH dependent partitioning of the drug carrier complex between aqueous and organic phases. These hydrophobic and ionic interactions render otherwise labile drugs resistant to stomach acid and digestive enzymes of the gastrointestinal tract and facilitate permeation across mucosal (epithelial) barriers of the GI and endothelial linings of the BBB. As such, these types of peptide-based ligands are excellent carriers and thus operate effectively as an oral drug delivery system. It is thus a feature of the invention that provides a rational form of drug delivery having amino acids that are predetermined to enhance affinity for particular drugs, which insulate the drug during transport to, and presentation at physiological barriers, and which facilitate permeation of said barriers and delivery of a structurally, chemically and biologically unmodified drug to its target.
It is another feature of the present invention that provides a carrier that it is easily degraded after delivery of its drug cargo without any potentially deleterious or toxic effects.
It is still another feature of the present invention that provides a composition, methods of synthesis and use of a drug carrier that is variable in chemical construction to enable binding with various xenobiotics and endogenous drugs to create a complex that promotes drug delivery to its targeted environment.
It is yet another feature of the present invention that, depending upon the surface epitopes employed, provides a carrier that permeates physiological barriers, and thus is suitable for, among other things, vaccine delivery via non- toxic adjuvants, vaccine delivery via oral administration, drug passage across the BBB, drug-delivery via circulating microreserve systems, antiperspirant delivery, perfume delivery, as a food preservative and additive, and as an additive in medicinals.
Brief Description of the Drawing
Fig. 1 is a diagrammatical representation of the complex of kepitope and drug in accordance with the invention.
Detailed Description of the Invention
Synthesis of Kepitooes
Solid phase synthesis is performed according to a modified method (Schlesinger, D.H., Conchrane, A.H., Gwadz, R.W. , Godson, G.N., Melton, R. , Nussenzweig, R. and Nussenzweig, V., Biochemistry 23:5665-5670, 1984) of the solid phase peptide synthesis protocol (Merrifield, R.B. J. Amer. Chem. Soc, 85:2149-2154, 1963). The K constructs are synthesized by a modified protocol of Tarn (1988) on a benzhydryl amine resin with a substitution of 0.65 meq/gm. The resin is washed with multiple additions of ethanol, dicloromethane (DCM) , and dimethyl formamide. It is then treated with 50% (in DCM) trifluoroacetic acid (TFA) for 30 min. and then washed with DCM. The amino function on the resin is neutralized with diisopropyl ethyl amine (DIPEA) (10%in DCM) and then washed with DCM. A three-fold molar excess of bis-boc-lysine (or bis-boc-ornithine) and hydroxybenzotriazole (HOBT) is added to the resin and the reaction allowed to proceed for 2 hours followed by test for completion of coupling with ninhydrin. This completes the ¥.__ construct (see Fig. 1) and an aliquot of the bis-boc-lysine (or bis-boc-ornithine) resin is removed from the reaction chamber. This protocol is repeated 2 times more for the synthesis of K3 construct as shown in Fig. 1, after the first repetition from which an aliquot is again removed. A K7 construct is completed following the last of 3 additions of bis-boc-lysine (or bis-boc-ornithine) according to the procedure described above. Thus, it can be observed that 2n lysines can be added in formulating the K construct, where "n" equals the number of cycles of addition of lysine or ornithine. There is no single limit to the number of additions of lysine (or ornithine, as the case may be) that may be added in building the construct. The selection is based on rational drug carrier design parameters, including the properties of the drug and the environment in which delivery is anticipated.
It should be appreciated that the only difference between the use of lysine and the use of ornithine resides in the differences associated with the additional methylene group present in lysine, which may be advantageous in connection with the particular drugs selected for delivery. However, the synthesis methods are the same for both. These two amino acids are unique in their having two reactive amino groups, and thus are essential to the invention.
The pre-selected series of polypeptide surface epitopes are then cyclically added to each of the molecular weight constructs using the identical protocol for the K constructs themselves (there being three in number under the prior embodiment): the lysine- (or ornithine-) resins are first washed, then the multiple N-terminal boc groups are removed with TFA and the lysine- (or ornithine-) resin then neutralized with DIPEA. A three-fold molar excess of each amino acid along with a three-fold excess of HOBT is then added to the lysine- (or ornithine-) resin (to inhibit racimization) and allowed to react for 2 hours followed by ninhydrin test for completion of coupling. Following the completion of addition of each amino acid in the surface epitope, the N-terminus of the completed surface epitope-K- linked construct can be blocked to remove the additional positive charge on the epitope with pyroglutamate, or can simply be untreated with TFA to preserve the Boc or Fmoc group.
The completed construct is then treated with anhydrous HF (0° C, 1 hr.) to remove side-chain protecting groups and to cleave the lysine- (or ornithine-) branched peptide Kepitope ligand from the benzhydryl amine resin. This is accomplished by the addition of anhydrous HF (10 ml/gm peptide resin) .
Following cleavage and deprotection, the HF is removed by a stream of N2 for 30 min., followed by water aspiration. The mixture is then washed with cold anhydrous ether to remove excess anisole. The peptide is then extracted with alternate washes of water and acetic acid and then lophilized.
The peptide constructs can first be purified by gel permeation chromatography for desalting and then by reversed phase HPLC as the final purification step.
The kepitope can also be synthesized in solution phase. Solution phase synthesis of the K-construct and carrier may be a long-term preferred method in light of the fact that it is more cost-effective for synthesizing larger quantities. In this methodology, there is no inert support, but the protecting groups are very similar. One of reasonable skill in the art easily understands, when using the teachings contained herein, how to synthesize the construct and carriers using solution phase techniques.
It should be appreciated that rational drug design plays a heavy role in determining not only the number of lysine or ornithine constituents, but also in the particular surface epitopes to be selected. In particular, the conformation, physical chemical and electrical properties of the drug to be borne by the carrier must first be determined, using any of a number of available techniques, including computer modeling. Thereafter, a number of different surface epitopes are selected as likely candidates for components in the carriers for that specific drug and for the targeted environment. Once the kepitopes (K construct plus surface epitopes) are synthesized and purified, the drug-ligand (kepitope complex) is prepared and subjected to bioavailability measurements. These complexes are compared for the best bio-availability data for the target environment. Characterization of Kepitopes 1. Amino acid analysis. The constructs are hydrolized back to their constituent amino acids with 6NHCL at 110°C for 24 hours and then evaporated to dryness, reconstituted and subjected to reverse phase HPLC for amino acid identification and quantification. 2. Test for homogeneity by HPLC and sequence analysis.
Homogeneity of the carrier is performed by reverse-phase HPLC on a C18 ODS column as the stationary phase and a linear gradient of water against acetonitrile as the mobile phase, with an increasing percentage of acetonitrile to develop the gradient. Automated sequencing of the carrier is performed via gas phase sequencing using a modified procedure of Edman, P. and Begg, E.G. (Europ, J. Biochem 1:80-90, 1967). Binding of drug to Kepitope
Depending on the pharmacological agent to be ionically or hydrophobically bound to the carrier (surface epitope-K- construct) , the epitope is designed with the ionic and hydrophobic characteristics favored to bind reversibly according to the drug's physical and chemical parameters (Table I, below) . For example, hydrophobicity of the drug requires a hydrophobic character of the surface epitope.
Anionic drugs, such as heparin, require that the ligand have a net positive charge for heparin binding, as well as enough hydrophobic character to penetrate the intestinal mucosa or the BBB. It is essential that for the carrier to partition itself in an oil phase immiscible with the aqueous phase, the pH of the aqueous solvent must exist at or very near the isoelectric point (pKI) of the ligand. In a preferred embodiment, the drug (0.2-200mg/ml) is dissolved in a volatile buffer with the pH close to the pKI of the ligand. The construct is dissolved in H20 (200mg/ml) and the two solutions mixed. After the spontaneous formation of oil in the aqueous phase, the aqueous phase can be removed by lyophilization. Alternatively, the two phases are separated by sedimentation in a low speed centrifuge.
Alternatively, the drug-ligand complex can cleaned in such a manner as to remove any biologically active drug from the surface of the complex by dialysis against a pH 2 solution (HCl-acetic acid, 0.05 N) containing pepsin (1:100 enzyme to complex ratio) . This procedure enzymatically degrades biologically active drug from the surface of the complex (pepsin being complex-impenetrable at these concentrations and pH) . Pepsin remaining in the resulting dialysate is readily degraded in vivo in the lower G.I. tract by the animal's own endogenous chymotrypsin and trypsin and other endopeptides.
Figure 1 is a diagrammatical representation of the complex of kepitope and drug, in accordance with the invention. The overall circle depicts the organic phase containing the hydrophobic drug-ligand complex in an aqueous solvent. Ligand consists of multiple surface epitopes (SE) 6, covalently attached to the K construct 2, which determines the ligand's hydrophobicity and pKI. Epitopes are covalently linked to the branched chain K construct which determines the number of epitopes and hence the ligands molecular weight. Hatched squares 8 depict drug; Kx, K2/and K3 represent serial additions of lysine during synthesis of the K construct. The drug is bound to the ligand through weak forces, such as ionic and hydrophobic bonds only, facilitating release at physiological pH at which the ligand is highly charged and becomes miscible in aqueous solvents such as blood. The N- terminus contains pyroglutamic acid (pE or pGlu) or N-blocking group such as N-acetyl, Boc, Fmoc. The C-terminus 4, as shown in Fig. 1, is CONH2, though it is understood that terminus 4 may be in the form of a free carboxylic acid.
As shown in Table I, below, binding constants are express in terms of the partition coefficients ("K") of the pharmaceutical agent ("A"), in accordance with the following formula:
K = [A organic phase] / [A aqueous phase] Table I was created in the following manner. First the particular kepitope (surface epitope on a K7 construct) was synthesized using the techniques set forth, above. Second, the agent (0.2 mg/ml) together with the kepitope (40 mg/ml) were dissolved in water and the pH was raised to pH 8 with NH4OH. Next, the pH of the solution was lowered to pH 3.5 to 4.0. At this pH, carboxyl groups are no longer ionized, and two phases are formed. The resulting phases are separated by low speed centrifugation. The concentration of the drug is measured by high performance liquid chromatography ("HPLC") of an aliquot of a sample from each phase. The higher the number, the greater the affinity of the drug for the kepitope. The concentration and resulting pH of the kepitope is selected such that the plait point (formation of a single phase) is not reached. The molecular weights of luteinizing hormone-releasing hormone ("LHRH"), vasopressin, substance P and oxytocin are about 1000 Daltons. The lysine (or ornithine) branched chain matrix used in Table I is a K7 construct (molecular weight of 1000 Daltons) . Since the complete kepitope contains 8 copies of the surface epitopes, the molecular weight of the kepitope is 9000 Daltons. As can be observed in Table I, above, selection of the particular surface epitopes will change the affinity of the
drug thereto. In terms of rational drug design, it should be appreciated that the surface epitopes must be selected for hydrophobicity of the complex, improved affinity for the drug, and release under appropriate conditions at the target location. Though the goal, in the long run, is to minimize the need for randomized laboratory synthesis and analysis, the selection must nonetheless be tested under in vivo and in vitro conditions . Bioavailability by Radioimmuno assay (RIA, ELISA, IRMA) . The efficacy of this oral drug delivery system can be measured directly by radioimmunoassay rather than indirectly by the biological endpoint or biological action of the pharmaceutical agent in order to eliminate possible feedback loops which may alter the estimated amounts of drug delivered across the epithelial lining of the gut. Direct measurement of blood levels of drug is most easily and accurately performed by radioimmunoassay. Examples of peptides and proteins tested for bioavailability by means of RIA include neuropeptide Y (NPY) , insulin, an immunoglobulin (IgG) , substance P, and calcitonin as well as the ligand itself.
Bioavailability by Intestinal Segmentation and Mechanisms of Intestinal Permeation
There are four basic areas of study for determining the mechanisms of intestinal permeation, and resultant bioavailability of the drug.
(1) Site of absorption.
(2) Cells responsible for permeation. Payer's patches, enterocyte, or columnar epithelial
(3) Pharmacokinetics of absorption. (4) Mechanisms of absorption. Active transport, passive diffusion, facilitated diffusion, carrier mediated. Additional Embodiments Vaccine Delivery Via Non-toxic Adjuvants
Adjuvants are typically utilized to increase localized blood flow to aid in the absorption of the vaccine, upon administration via skin-based infusion. In this embodiment, the surface epitopes (SE's) attached to the polylysine (or polyornithine) matrix are selected based upon the primary structure and chemical properties of the vaccine. For example, where the vaccine possesses numerous surface polypeptides that are relatively uncharged, e.g., phenylalanine, then the SE's selected are also hydrophobic and are selected from the group comprising tyrosine, valine, isoleucine, leucine, methionine, cysteine, phenylalanine, alanine, proline, and glycine. In this instance, the bonding is achieved through hydrophobic interaction. For the aromatic amino acids, such bonding is π-π. It should be appreciated that the synthesized adjuvant-vaccine composition appears as an oil, and is administered as such.
Vaccines are prepared in accordance with known technology, and can include, where necessary to prevent enzymatic degradation, D-amino acids, rather than the susceptible, more common and naturally occurring L-form. Since such hydrophobic bonding is weak and therefore reversible, upon interaction with the physiological fluids present upon administration of the vaccine, such bonds will be broken and the vaccine will be released in the same manner as is well known in the art. In this instance, a two-phased condition will result wherein the adjuvant, having oil-like characteristics, will separate in physiological fluid from the vaccine which will slowly dissolve, pass into the aqueous phase and thereby facilitate absorption-based delivery. It should be appreciated that such vaccine delivery can be used for both humans and animals, including pets and livestock. Vaccine Delivery Via Oral Administration
For many vaccines oral delivery is desirable to boost the immune response by lymphoid tissue in the gut. Consequently, the SE's (and vaccines) selected may be the same as those selected for adjuvant-based delivery in order to facilitate bonding of the matrix to the vaccine. It is theorized that this form of vaccine will cause an improved immunological response in that the oil-like characteristics of the construct will render the engulfed vaccine somewhat more impervious to enzymatic degradation and denaturation and hydrolysis by stomach acid than that heretofore known. The pathways for absorption are the same as those explained above in connection with pharmaceutical delivery, wherein the vaccine is released after the construct-vaccine complex is absorbed into the blood stream. Upon absorption, the complex dissociates, and the vaccine migrates into the aqueous phase.
In the oral route of delivery of the construct-vaccine complex, it may be preferred to use D-amino acids in the synthesis of the carrier to prevent enzymatic degradation of the vaccine in the intestine and/or blood stream prior to immuno-stimulatory action. Such selection and synthesis techniques for the vaccine are well-known in the art. It should be recognized that such vaccine delivery systems are suitable for use in both humans and animals, including pets and livestock.
Drug-deliverv Via Oral Form Across Blood/Brain Barrier (BBB)
As reported by Bodor, et al. in "A Strategy for Delivering Peptides into the Central Nervous System by Sequential Metabolism," Science, Vol. 257, September 18, 1992, pp. 1698-1700, most peptides fail to enter the central nervous system as a consequence of their hydrophilic nature, which also renders them subject to peptidolytic enzymatic attack in the lipoidal blood-brain barrier. Consequently, the complex of the present invention is a perfectly suitable carrier for drug delivery across the BBB.
However, there are two separate barriers that the carrier-drug complex must permeate and overcome without dissociating, in order to deliver the drug to the CNS target: intestinal and BBB. Thus, the SE attached to the K-construct must be of a nature that they are highly-hydrophobic for absorption at both barriers, while non-susceptible to blood- based enzymatic attack prior to presentation at the BBB. Thus, this is a specialized instance of rational drug design, since the SE's must be selected with the nature of the particular drug in mind. In this instance, it is likely that the use of aspartic and glutamic acid as constituent SE's will not be as necessary as that required by the non-BBB drug formulations, because such amino acids are charged in the blood stream and cause rapid dissociation, an outcome not desired for achieving delivery across the BBB. It is anticipated that the hydrophobicity of the complex in accordance with the invention will result in passage of the complex across both barriers, and into the CNS. At this point, upon dissociation, which will inevitably occur over time, the drug will be released from the complex, and will find its way to its particular binding sites. Drug-Delivery Via Circulating Microreserve Systems
Like the preferred embodiment described above for creating a complex that serves as a form of administering a drug across the BBB, microreserve systems are created in order to have a controlled form of drug delivery over time, releasing their drug-bound products within the cardiovascular system. Consequently, the complex must be created with the use of amino acids that do not immediately become charged when the pH rises after passing the intestinal barrier, such that dissociation is slower, and a time continuum is created. SE's in this instance need not be the aspartic and glutamic acids utilized for rapid dissociation, and a more rational drug schemata should employ a constellation of other hydrophobic amino acids which may include a greater percentage of D-amino acids which are known to be less enzymatically degradable, or degrade at a slower rate.
It is also a goal to manufacture a high molecular weight carrier rendering low molecular weight drugs less likely to be filtered by the kidney and with a high degree of non- degradability by the liver and circulating proteases in the blood. Adding more lysines or ornithines to the construct exponentially increases the size and molecular weight of the carrier by a function of 2n, where n equals the number of cycles of addition of lysine or ornithine during synthesis. Higher molecular weight complexes will be more impervious to kidney filtration, since clearance times are increased and such complexes will linger longer in the blood stream. The molecular weight of the complex can also be increased by using longer chain epitopes. Lastly, larger-sized drug moieties will also increase the clearance time of the complex, and thus improve the microreserve nature of the complex. The goals to be achieved are to decrease the likelihood of kidney filtration, liver metabolism, and protease degradation in the blood prior to the drug reaching its target or receptor site. One skilled in the art, when given the teachings contained herein will be able to design a carrier that will achieve these goals. Antiperspirant Delivery Via Complex
Antiperspirants are known to be relatively inactive until placed in the aqueous environment of perspiration. A complex can be formed using SE's rationally selected to associate with molecular antiperspirant to form oil droplets which can then be applied ectopically and be available "on demand," i.e., upon emersion within the aqueous environment of perspiration. Since perspiration has a pH near neutral (7.0), it will cause ionization of the construct followed by dissociation of the bound active ingredient in anti-perspirant, thereby liberating its effects.
Perfume Delivery Via Complex
Like antiperspirants, perfumes are best if they can be released over time, rendering them long acting. A carrier having perfume molecules bound to it will ionize upon presence of perspiration and thereby dissociate and liberate the perfume molecules, which then volatilize. Food Preservatives and Additives Via Complex
The carrier can be used to enable the controlled delivery of preservatives and additives, and will, as a consequence of its hydrophobicity, itself act as a preservative. Using rational drug design, specific SE's can be selected which will enable binding to the preservative and additive, when needed, and will thereby ensure controlled release. Thus, as a food additive such a complex will preserve the flavor, consistency and surface characteristics of foods over time. Additives in Medicinals Via Complex
The objective here is to give protection to medicinals against fluid-based inactivation. Some drugs readily hydrolyze or are labile in the presence of water, i.e., may undergo conformational changes or changes in secondary structure. Thus, the complex would be formed of a hydrophobic epitope which binds to the medicinal. While not impeding the activity of the medicinal, such a complex, as a consequence of its hydrophobicity, would prevent fluid entry and concomitant inactivation. In this instance, as before, rational drug design is employed to carefully select SE's to be placed on the polylysine (or polyornithine) matrix which will achieve such results. Rational Drug Design In view of the foregoing, it should appreciated that the kepitopes of the instant invention are well-suited for rational drug design (drug-binding design) . In particular, the process involves first ascertaining the properties of the drug sought to be delivered via one of the methods set forth herein, e.g., orally. Second, the mode of delivery must be selected, e.g., injectable, ectopic, oral, intranasal, ocular, buccal, vaginal, rectal, transdermal, since the physiological barriers that must be overcome, while maintaining the complex in tact, will determine the nature of the kepitope. Next, the primary structure (i.e., amino acid sequence) and secondary structure (i.e., how it folds in two dimensions by forming disulfide bridges) of a series of kepitopes are determined via a series of tools which include three-dimensional, computer based modeling. Since the size of surface epitopes utilized herein will likely be small, there is little concern about the tertiary structure, since three-dimensional conformational changes are unlikely. Also, a series of kepitope candidates, rather than a single kepitope, is desirable to allow selection of the candidate that provides the best bioavailability data. Similarly, the series of kepitopes can lead to development of a library and thereby narrow the extent of bioavailability testing needed for each subsequent pharmaceutical agent. Next, the carrier is constructed, and the drug bound to it. Lastly, pK and bioavailability studies (in vivo and in vitro) are performed, the results examined and the most suitable candidate selected. This rational method of drug-carrier synthesis is far and away different from the usual mechanisms for developing new drugs andynew carriers which involves determining structure only after functionality is shown. Also, the typical format involves the use of a large number of related compositions and their analogues, and thus selection is merely hit-or-miss. Equivalents
The above description, figure and preferred embodiments are provided not to limit the invention but to assist one skilled in the art in better understanding the invention contained herein. The inventor is not thereby limited to the preferred embodiments, but is only limited by the scope of the claims below. One of reasonable skill in the art can also practice the invention through other and equivalent methods, compositions and techniques which are, as well, included within the scope of the invention, to the extent set forth in the appended claims.

Claims

ClaimsI claim:
1. A peptide-based moiety for drug delivery, having the general formula: [X-A] 2n - [B] 2n_f C wherein X is an amino blocking group; A is selected from the group consisting of at least one amino acid; B is selected from the group consisting of lysine, ornithine and combinations thereof; C is selected from the group consisting of a free carboxyl group, or a protected carboxamide (C0NH2) , and n is the number of cycles of addition of B.
2. The moiety of claim 1, wherein said amino blocking group is selected from the group consisting of pE (pyroglutamate) , Fmoc (9-fluoroenyl methyloxycarbonyl) , T-Boc (tertiary butyloxycarbonyl) and acetyl.
3. The moiety of claim 1, further comprising a pharmaceutical agent.
4. The moiety of claim 3, wherein said pharmaceutical agent is selected from the group consisting of a biologically active peptide composition, protein composition, and combinations thereof.
5. The moiety of claim 4, wherein said biologically active peptide composition and protein composition is selected from the group consisting of a hormone, releasing factor, and combinations thereof.
6. The moiety of claim 5, wherein said releasing factor is selected from the group consisting of corticotropin releasing factor, LHRH, and growth hormone releasing factor.
7. The moiety of claim 3, wherein said pharmaceutical agent is selected from the group consisting of a mucopolysaccharide, polynucleotide, antibiotic, antiviral, antifungal, and combinations thereof.
8. The moiety of claim 7, wherein said mucopolysaccharide is heparin.
9. The moiety of claim 3, wherein said pharmaceutical agent is insulin.
10. The moiety of claim 3, wherein said pharmaceutical agent is selected from the group consisting of a protein and glycoprotein immunostimulatory drug, and combinations thereof.
11. The moiety of claim 10, wherein said protein and glycoprotein immunostimulatory drug is selected from the group consisting of a lymphokine, cytokine, poly I: oly C12U, interleukine, interferon, macrophage granulocyte stimulating factor, and combinations thereof.
12. The moiety of claim 1, further comprising a xenobiotic.
13. The moiety of claim 1, further comprising an endogenous drug.
14. The moiety of claim 1, wherein said at least one amino acid is predetermined to render said moiety hydrophobic.
15. The moiety of claim 1, wherein said at least one amino acid is predetermined to render said moiety hydrophilic.
16. The moiety of claim 1, wherein said at least one amino acid is predetermined to render said moiety lipophilic.
17. A method of synthesizing a drug carrier, comprising the steps of building a peptide matrix by cyclic additions of
(2n-l) amino acid constituents selected from the group consisting of lysine, ornithine and combinations thereof, and attaching 2n surface epitopes to said matrix, where n represents the total number of said cyclic additions.
18. The method of claim 17, further comprising the step of adding a pharmaceutical agent.
19. The method of claim 17, further comprising the step of adding at least one amino blocking group to said peptide matrix.
20. The method of claim 17, wherein at least one of said surface epitopes is an amino acid.
21. The method of claim 19, wherein said amino blocking group is selected from the group consisting of pE (pyroglutamate) , Fmoc (9-fluoroenyl methyloxycarbonyl) , T-Boc (tertiary butyloxycarbonyl) and acetyl.
22. The method of claim 18, wherein said pharmaceutical agent is selected from the group consisting of a biologically active peptide composition, protein composition, and combinations thereof.
23. The method of claim 22, wherein said biologically active peptide composition and protein composition is selected from the group consisting of a hormone, releasing factor, and combinations thereof.
24. The method of claim 23, wherein said releasing factor is selected from the group consisting of corticotropin releasing factor, luteotropin releasing factor, and growth hormone releasing factor.
25. The method of claim 18, wherein said pharmaceutical agent is selected from the group consisting of a mucopolysaccharide, polynucleotide, antibiotic, antiviral, antifungal, and combinations thereof.
26. The method of claim 25, wherein said mucopolysaccharide is heparin.
27. The method of claim 18, wherein said pharmaceutical agent is insulin.
28. The method of claim 18, wherein said pharmaceutical agent is selected from the group consisting of a protein and glycoprotein immunostimulatory drug, and combinations thereof.
29. The method of claim 28, wherein said protein and glycoprotein immunostimulatory drug is selected from the group consisting of a lymphokine, cytokine, poly I: oly C12U, interleukine, interferon, macrophage granulocyte stimulating factor, and combinations thereof.
30. The method of claim 17, further comprising the step of adding a xenobiotic.
31. The method of claim 17, further comprising the step of adding an endogenous drug.
32. The method of claim 17, wherein said surface epitopes are predetermined to render said drug carrier hydrophobic.
33. The method of claim 17, wherein said surface epitopes are predetermined to render said drug carrier hydrophilic.
34. The method of claim 17, wherein said surface epitopes are predetermined to render said drug carrier lipophilic.
35. The moiety of claim 1, further comprising a constituent selected from the group consisting of a vaccine, an antiperspirant, a perfume, a food preservative, a food additive, a medicinal additive, and combinations thereof.
36. The method of claim 17, further comprising the step of adding a constituent selected from the group consisting of a vaccine, an antiperspirant, a perfume, a food preservative, a food additive, a medicinal additive, and combinations thereof.
EP94901482A 1992-11-12 1993-11-12 Lipophilic peptide-based carriers for targeted drug delivery using rational drug-binding design. Withdrawn EP0670728A4 (en)

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EP0884327B1 (en) 1997-06-11 2001-03-14 The School Of Pharmacy, University Of London Dendritic lysine-based polypeptides for targeted drug delivery
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