`
`111111 11111En1111 11111 1m101109151191091111 mil mil
`
`
`
`
`
`United States Patent [19]
`Ekwuribe
`
`5,359,030
`[11] Patent Number:
`[45] Date of Patent: Oct. 25, 1994
`
`[54] CONJUGATION-STABILIZED
`POLYPEPTIDE COMPOSITIONS,
`THERAPEUTIC DELIVERY AND
`DIAGNOSTIC FORMULATIONS
`COMPRISING SAME, AND METHOD OF
`MAKING AND USING THE SAME
`[75] Inventor: Nnochiri N. Ekwuribe, Southfield,
`Mich.
`
`[73] Assignee: Protein Delivery, Inc., Durham, N.C.
`
`[22] Filed:
`
`[51] Int. Cl .5
`
`[21] Appl. No.: 59,701
`May 10, 1993
`C07K 7/40; C07K 7/36;
`C07K 17/08; C08H 1/00
` 530/303; 530/307;
`[52] U.S. Cl.
`530/309; 530/322; 530/345; 530/402; 530/351;
`530/409; 530/410; 530/411; 435/188; 424/85.1;
`424/85.4; 424/94.3
` 435/188; 514/3, 4, 12;
`[58] Field of Search
`530/303, 307, 324, 309, 345, 322, 402, 326, 409,
`410, 411, 325, 351; 424/85.1, 85.2, 85.4, 85.5,
`85.6, 85.9, 94.3
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`4,003,792 1/1977 Mill et al.
`4,094,196 8/1977 Hilper et al.
`4,179,337 12/1979 Davis et al.
`4,585,754 4/1986 Meisner et al.
`4,849,405 7/1989 Ecanow
`4,963,367 10/1990 Ecanow
`5,013,556 5/1991 Woodle et al.
`
` 530/303
` 526/271
` 435/181
` 514/8
` 514/3
` 424/485
` 424/450
`
`OTHER PUBLICATIONS
`Nucci, et al. "The Therapeutic Value of Poly(ethylen
`Glycol)—Modified Proteins" Ac. Drug. Del. Rev. 6:
`133-151 1991.
`Conradi, R. A., et al., "The Influence of Peptide Struc-
`ture on Transport Across Caco-2 Cells," Pharm. Res.,
`1991, 8 (12): 1453-1459.
`Abuchowski, A. and F. F. Davis, "Soluble Polymer--
`Enzyme Adducts," pp. 368-383, Enzymes as Drugs, J.
`S. Holcenberg, John Wiley, 1981.
`Boccu, E. et al., "Pharmacokinetic Properties of Poly-
`
`ethylene Glycol Derivatized Superoxide Dismutase,"
`Pharm. Res. Comm., 1982 14: 11-120.
`(List continued on next page.)
`
`Primary Examiner—Jeffrey E. Russel
`Assistant Examiner—Nancy J. Gromet
`Attorney, Agent, or Firm—Steven J. Hultquist; Fran S.
`Wasserman
`[57]
`ABSTRACT
`A stabilized conjugated peptide complex comprising a
`peptide conjugatively coupled to a polymer including
`lipophilic and hydrophilic moieties, wherein the peptide
`may for example be selected from the group consisting
`of insulin, calcitonin, ACTH, glucagon, somatostatin,
`somatotropin, somatomedin, parathyroid hormone,
`erythropoietin, hypothalamic releasing factors, prolac-
`tin, thyroid stimulating hormones, endorphins, enke-
`phalins, vasopressin, non-naturally occurring opioids,
`superoxide dismutase, interferon, asparaginase, argi-
`nase, arginine deaminease, adenosine deaminase, ribo-
`nuclease, trypsin, chymotrypsin, and papain. In a partic-
`ular aspect, the invention comprises an insulin composi-
`tion suitable for parenteral as well as non-parenteral
`administration, preferably oral or parenteral administra-
`tion, comprising insulin covalently coupled with a poly-
`mer including (i) a linear polyalkylene glycol moiety
`and (ii) a lipophilic moiety, wherein the insulin, the
`linear polyalkylene glycol moiety and the lipophilic
`moiety are conformationally arranged in relation to one
`another such that the insulin in the composition has an
`enhanced in vivo resistance to enzymatic degradation,
`relative to insulin alone. One, two, or three polymer
`constituents may be covalently attached to the insulin
`molecule, with one polymer constituent being pre-
`ferred. The conjugates of the invention are usefully
`employed in therapeutic as well as non-therapeutic, e.g.,
`diagnostic, applications, and the peptide and polymer
`may be covalently coupled to one another, or alterna-
`tively may be associatively coupled to one another, e.g.,
`by hydrogen bonding or other associative bonding rela-
`tionship.
`
`33 Claims, 2 Drawing Sheets
`
`700
`
`600
`
`E
`
`500 r
`
`8
`co
`=
`„,
`
`400 F.
`
`300
`
`200 ;-
`
`100
`
`• BASEUNE
`• GLUCOSE 055 (5 g/kg) P.O.
`(100 Ag/kg) SC.; GLUCOSE (5 g/kg) P.O.
`3
`• MINN (1.5 mg/kg) P.O.; GLUCOSE (5 9/kg) P.O.
`o NUN COMPLEX (100 Ag/kg) S.C.; GLUCOSE (5 g/kg) P.O.
`• INSULIN COMPLEX (250 µg/kg) S.C.; GLUCOSE (5 g/kg) P.O.
`INSULIN COMPLEX (1.5 mg/kg) P.O.; GLUCOSE (5 g/kg) P.O.
`A INSULIN COUPLES (100 Ag/kg) P.O.: GLUCOSE (5 9/kg) P.O.
`
`J
`
`.. .... ......
`
`.
`
`.
`
`0
`
`30
`
`60
`lIME (MINUTES)
`
`90
`
`120
`
`MYLAN EXHIBIT - 1009
`Mylan Pharmaceuticals, Inc. v. Bausch Health Ireland, Ltd. - IPR2022-00722
`
`

`

`5,359,030
`
`Page 2
`
`OTHER PUBLICATIONS
`Igarashi, R. et al, "Biologically Active Peptides Conju-
`gated with Lecithin for DDS" Proceed. Intern. Symp.
`Cont. Rel. Bioactiv. Mater. 1990, 17 367-368.
`Taniguchi, T. et al, "Synthesis of Acyloyl Lysozyme
`and Improvement of its Lymphatic Transport Follow-
`ing Small Intestinal Administration in Rats" Proceed.
`Intern. Symp. Control. Rel. Bioactiv. Mater., 1992, 19:
`104-105.
`Russell-Jones, G. J. "Vitamin B12 Drug Delivery",
`Proceed. Intern. Symp. Control. Rel. Bioactive. Mater.,
`1992, 19: 102-103.
`Baudys, M. et al, "Synthesis and Characteristics of Dif-
`ferent Glycosylated. Derivatives of Insulin" Proceed.
`Intern. Symp. Cont. Rel. Bioactive. Mater., 1992, 19:
`210-211.
`Chien, Y. W., Novel Drug Delivery Systems, pp.
`678-679, Marcell Deffer, Inc., New York, N.Y., 1992.
`Santiago, N. et al, "Oral Immunization of Rats with
`Influenza Virus M Protein (M1) Microspheres," Pro-
`ceed. Intern. Symp. Cont. Rel. Bioactive. Mater., 1992,
`19: 116-117.
`Banting, R. G., et al, "Pancreatic Extracts in the Treat-
`ment of Diabetes Mellitus," The Canadian Med. Assoc.
`J. 1922, 12: 141-146.
`
`Brange, J. et al, "Chemical Stability of Insulin. 1. Hy-
`drolytic Degradation During Storage of Pharmaceuti-
`cal Preparations," Pharm. Res., 1992, 9 (6): 715-726.
`Brange, J. et al, "Chemical Stability of Insulin. 2. For-
`mation of Higher Molecular Weight Transformation
`Products During Storage of Pharmaceutical Prepara-
`tions," Pharm. Res., 1992, 9 (6) 727-734.
`Robbins, D. C. et al, "Antibodies to Covalent Aggre-
`gates of Insulin in Blood of Insulin-Using Diabetic
`Patients" Diabetes, 1987, 36: 838-841.
`M. Maislos et al, "The Source of the Circulating Aggre-
`gate of Insulin in Type I Diabetic Patients is Therapeu-
`tic Insulin" J. Clin. Invest., 1986, 77: 717-723
`Ratner, R. E. et al, "Persistent Cutaneous Insulin Al-
`lergy Resulting from High-Molecular Weight Insulin
`Aggregates," Diabetes, 1990, 39: 728-733.
`Oka, K. et al, "Enhanced Intestinal Absorption of a
`Hydrophobic Polymer-conjugated Protein Drug,
`Smancs, in an Oily Formulation" Pharm. Res., 1990, 7
`(8): 852-855.
`Saffran, M. et al, "A New Approach to the Oral Admin-
`istration of Insulin and Other Peptide Drugs," Science,
`1986, 233: 1081-1084.
`
`

`

`lualua *S11
`
`t66I `SZ 'PO
`
`Z Jo I PatIS
`
`FIG.1
`TIME, HOURS
`
`8
`I
`
`7
`I
`
`6
`I
`
`5
`I
`
`4
`I
`
`3
`I
`
`2
`I
`
`1
`I
`
`0
`50
`
`
`
`100
`
`4
`
`150 --
`
`x
`
`GLUCOSE 5g/Kg P.S. (NO INSULIN) GROUP 5
`CONJUGATE 1 100 µµg/KgKg S.C.: GROUP 4
`CONJUGATE 1 100 g/
`3
`INSULIN 1.5 mg/Kg P.O.: GROUP 2
`INSULIN 100 g/Kg S.C.: GROUP 1
`
`P.O.:
`
`2
`00
`0 c.)
`E Lunt)
`300
`
`C.,
`
`350 -
`
`400
`
`450
`
`

`

`lualud 'S'il
`
`t661 `SZ 'PO
`
`Z Jo Z lamIS
`
`FIG.2
`
`120
`
`90
`
`60
`
`30
`
`TIME (MINUTES)
`
`............. ....... ............
`
`....................... ............. ..............
`
`„,,s,,,,,
`
`\ •
`
`‘‘• A INSULIN COMPLEX (100 µg/kg) P.O.; GLUCOSE (5 g/kg) P.O.
`A INSULIN COMPLEX (1.5 mg/kg) P.O.; GLUCOSE (5 g/kg) P.O.
`• INSULIN COMPLEX (250 µg/kg) S.C.; GLUCOSE (5 g/kg) P.O.
`INSULIN COMPLEX (100 µg/kg) S.C.; GLUCOSE (5 g/kg) P.O.
`O
`3 INSULIN (1.5 mg/kg) P.O.; GLUCOSE (5 g/kg) P.O.
`3 INSULIN (100 µg/kg) S.C.; GLUCOSE (5 g/kg) P.O.
`• GLUCOSE ONLY (5 g/kg) P.O.
`O BASELINE
`
`
`
`\
`\\
`'•.,,
`..,
`\
`.
`,,
`•,
`‘,
`\\,
`\
`,
`,
`,
`,
`,
`
`//
`II I'Y
`
`,
`,, , ,
`, , /
`
`/1/
`/
`
`0
`
`0
`
`100
`
`200
`
`300
`
`ffiU,
`
`c.D
`c) 400
`
` 500
`
`gn
`
`600
`
`700
`
`

`

`CONJUGATION-STABILIZED POLYPEPTIDE
`COMPOSITIONS, THERAPEUTIC DELIVERY
`AND DIAGNOSTIC FORMULATIONS
`COMPRISING SAME, AND METHOD OF MAKING 5
`AND USING THE SAME
`
`10
`
`25
`
`30
`
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`The present invention relates to conjugation-stabil-
`ized (poly)peptide and protein compositions and formu-
`lations, and to methods of making and using same.
`2. Description of the Related Art
`The use of polypeptides and proteins for the systemic
`treatment of certain diseases is now well accepted in 15
`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 20
`which are very potent and specific in eliciting their
`biological actions.
`A major factor limiting the usefulness of these sub-
`stances 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, pro-
`duced 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 35
`peptidases. Any of the given peptides that survive pas-
`sage through the stomach are further subjected to me-
`tabolism in the intestinal mucosa where a penetration
`barrier prevents entry into the cells.
`In spite of these obstacles, there is substantial evi- 40
`dence in the literature to suggest that nutritional and
`pharmaceutical proteins are absorbed through the intes-
`tinal mucosa. On the other hand, nutritional and drug
`(poly)peptides are absorbed by specific peptide trans-
`porters in the intestinal mucosa cells. These findings 45
`indicate that properly formulated (poly)peptides and
`proteins may be administered by the oral route, with
`retention of sufficient biological activity for their in-
`tended use. If, however, it were possible to modify
`these peptides so that their physiological activities were 50
`maintained totally, or at least to a significant degree,
`and at the same time stabilize them against proteolytic
`enzymes and enhance their penetration capability
`through the intestinal mucosa, then it would be possible
`to utilize them properly for their intended purpose. The 55
`product so obtained would offer advantages in that
`more efficient absorption would result, with the con-
`comitant ability to use lower doses to elicit the optimum
`therapeutic effect.
`The problems associated with oral or parenteral ad- 60
`ministration of proteins are well known in the pharma-
`ceutical industry, and various strategies are being used
`in attempts to solve them. These strategies include in-
`corporation of penetration enhancers, such as the salic-
`ylates, lipid-bile salt-mixed micelles, glycerides, and 65
`acylcarnitines, but these frequently are found to cause
`serious local toxicity problems, such as local irritation
`and toxicity, complete abrasion of the epithelial layer
`
`5,359,030
`
`2
`and inflammation of tissue. These problems arise be-
`cause enhancers are usually coadministered with the
`peptide product and leakages from the dosage form
`often occur. Other strategies to improve oral delivery
`include mixing the peptides with protease inhibitors,
`such as aprotinin, soybean trypsin inhibitor, and amasta-
`tin, in an attempt to limit degradation of the adminis-
`tered therapeutic agent. Unfortunately these protease
`inhibitors are not selective, and endogenous proteins are
`also inhibited. This effect is undesirable.
`Enhanced penetration of peptides across mucosal
`membranes has also been pursued by modifying the
`physicochemical properties of candidate drugs. Results
`indicate that simply raising lipophilicity is not sufficient
`to increase paracellular transport. Indeed it has been
`suggested that cleaving the peptide-water hydrogen
`bonds is the main energy barrier to overcome in obtain-
`ing peptide diffusion across membranes (Conradi, R. A.,
`Hilgers, A. R., Ho, N. F. H., and Burton, P. S., "The
`influence of peptide structure on transport across Caco-
`2 cells", Pharm. Res., 8, 1453-1460, (1991)). Protein
`stabilization has been described by several authors.
`Abuchowski and Davis ("Soluble polymers-Enzyme
`adducts", In: Enzymes as Drugs, Eds. Holcenberg and
`Roberts, J. Wiley and Sons, New York, N.Y., (1981))
`disclosed various methods of derivatization of enzymes
`to provide water soluble, non-immunogenic, in vivo
`stabilized products.
`A great deal of work dealing with protein stabiliza-
`tion has been published. Abuchowski and Davis dis-
`close various ways of conjugating enzymes with poly-
`meric materials (Ibid). More specifically, these poly-
`mers are dextrans, polyvinyl pyrrolidones, glycopep-
`tides, polyethylene glycol and polyamino acids. The
`resulting conjugated polypeptides are reported to retain
`their biological activities and solubility in water for
`parenteral applications. The same authors, in U.S. Pat.
`No. 4,179,337, disclose that polyethylene glycol ren-
`dered proteins soluble and non-immunogenic when
`coupled to such proteins. These polymeric materials,
`however, did not contain fragments suited for intestinal
`mucosa binding, nor did they contain any moieties that
`would facilitate or enhance membrane penetration.
`While these conjugates were water-soluble, they were
`not intended for oral administration.
`Meisner et al., U.S. Pat. No. 4,585,754, teaches that
`proteins may be stabilized by conjugating them with
`chondroitin sulfates. Products of this combination are
`usually polyanionic, very hydrophilic, and lack cell
`penetration capability. They are usually not intended
`for oral administration.
`Mill et al., U.S. Pat. No. 4,003,792, teaches that cer-
`tain acidic polysaccharides, such as pectin, algesic acid,
`hyaluronic acid and carrageenan, can be coupled to
`proteins to produce both soluble and insoluble products.
`Such polysaccharides are polyanionic, derived from
`food plants. They lack cell penetration capability and
`are usually not intended for oral administration.
`In Pharmacological Research Communication 14,
`11-120 (1982), Boccu et al. disclosed that polyethylene
`glycol could be linked to a protein such as superoxide
`dismutase ("SOD"). 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.
`
`

`

`5,359,030
`
`3
`Other techniques of stabilizing peptide and protein
`drugs in which proteinaceous drug substances are con-
`jugated with relatively low molecular weight com-
`pounds such as aminolethicin, fatty acids, vitamin B12,
`and glycosides, are revealed in the following articles: R.
`Igarishi et al., "Proceed. Intern. Syrup. Control. Rel.
`Bioact. Materials", 17, 366, (1990); T. Taniguchi et al.
`Ibid 19, 104, (1992); G. J. Russel-Jones, Ibid, 19, 102,
`(1992); M. Baudys et al., Ibid, 19, 210, (1992). The modi-
`fying compounds are not polymers and accordingly do
`not contain moieties necessary to impart both the solu-
`bility and membrane affinity necessary for bioavailabil-
`ity following oral as well as parenteral administration.
`Many of these preparations lack oral bioavailability.
`Another approach which has been taken to lengthen
`the in vivo duration of action of proteinaceous sub-
`stances is the technique of encapsulation. M. Safran et
`al., in Science, 223, 1081, (1986) teaches the encapsula-
`tion of proteinaceous drugs in an azopolymer film for
`oral administration. The film is reported to survive
`digestion in the stomach but is degraded by microflora
`in the large intestine, where the encapsulated protein is
`released. The technique utilizes a physical mixture and
`does not facilitate the absorption of released protein
`across the membrane.
`Ecanow, U.S. Pat. No. 4,963,367, teaches that physio-
`logically active compounds, including proteins, can be
`encapsulated by a coacervative-derived film and the
`finished product can be suitable for transmucosal ad-
`ministration. Other formulations of the same invention
`may be administered by inhalation, oral, parenteral and
`transdermal routes. These approaches do not provide
`intact stability against acidity and proteolytic enzymes
`of the gastrointestinal tract, the property as desired for
`oral delivery.
`Another approach taken to stabilize protein drugs for
`oral as well as parenteral administration involves en-
`trapment of the therapeutic agent in liposomes. A re-
`view of this technique is found in Y. W. Chien, "New
`Drug Delivery Systems", Marcel Dekker, New York,
`N.Y., 1992. Liposome-protein complexes are physical
`mixtures; their administration gives erratic and unpre-
`dictable results. Undesirable accumulation of the pro-
`tein component in certain organs has been reported, in
`the use of such liposome-protein complexes. In addition
`to these factors, there are additional drawbacks associ-
`ated with the use of liposomes, such as cost, difficult
`manufacturing processes requiring complex lypophili-
`zation cycles, and solvent incompatibilities. Moreover,
`altered biodistribution and antigenicity issues have been
`raised as limiting factors in the development of clini-
`cally useful liposomal formulations.
`The use of "proteinoids" has been described recently
`(Santiago, N., Milstein, S. J., Rivera, T., Garcia, E.,
`Chang., T. C., Baughman, R. A., and Bucher, D., "Oral
`Immunization of Rats with Influenza Virus M Protein
`(M1) Microspheres", Abstract #A 221, Proc. Int. Symp.
`Control Rel. Bioac. Mater., 19, 116 (1992)). Oral delivery
`of several classes of therapeutics has been reported
`using this system, which encapsulates the drug of inter-
`est in a polymeric sheath composed of highly branched
`amino acids. As is the case with liposomes, the drugs are
`not chemically bound to the proteinoid sphere, and
`leakage of drug out of the dosage form components is
`possible.
`A peptide which has been the focus of much synthesis
`work, and efforts to improve its administration and
`bioassimilation, is insulin.
`
`4
`The use of insulin as a treatment for diabetes dates
`back to 1922, when Banting et al. ("Pancreatic Extracts
`in the Treatment of Diabetes Mellitus," Can. Med.
`Assoc. J., 12, 141-146 (1922)) showed that the active
`5 extract from the pancreas had therapeutic effects in
`diabetic dogs. Treatment of a diabetic patient in that
`same year with pancreatic extracts resulted in a dra-
`matic, life-saving clinical improvement. A course of
`daily injections of insulin is required for extended re-
`10 covery.
`The insulin molecule consists of two chains of amino
`acids linked by disulfide bonds; the molecular weight of
`insulin is around 6,000. The 13-cells of the pancreatic
`islets secrete a single chain precursor of insulin, known
`15 as proinsulin. Proteolysis of proinsulin results in re-
`moval of four basic amino acids (numbers 31, 32, 64 and
`65 in the proinsulin chain: Arg, Arg, Lys, Arg respec-
`tively) and the connecting ("C") peptide. In the result-
`ing two-chain insulin molecule, the A chain has glycine
`20 at the amino terminus, and the B chain has phenylala-
`nine at the amino terminus.
`Insulin may exist as a monomer, dimer or a hexamer
`formed from three of the dimers. The hexamer is coor-
`dinated with two Zn2+ atoms. Biological activity re-
`25 sides 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
`30 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
`35 technology allows commercial scale manufacture of
`human insulin (e.g., Humulin TM 0 insulin, commer-
`cially available from Eli Lilly and Company, Indianap-
`olis, Ind.).
`Although insulin has now been used for more than 70
`40 years as a treatment for diabetes, few studies of its for-
`mulation stability appeared until two recent publica-
`tions (Brange, J., Langkjaer, L., Havelund, S., and Vo-
`lund, A., "Chemical stability of insulin. I. Degradation
`during storage of pharmaceutical preparations," Pharm.
`45 Res., 9, 715-726, (1992); and Brange, J. Havelund, S.,
`and Hougaard, P., "Chemical stability of insulin. 2.
`Formulation of higher molecular weight transformation
`products during storage of pharmaceutical prepara-
`tions," Pharm. Res., 9, 727-734, (1992)). In these publi-
`50 cations, the authors exhaustively describe chemical
`stability of several insulin preparations under varied
`temperature and pH conditions. Earlier reports focused
`almost entirely on biological potency as a measure of
`insulin formulation stability. However the advent of
`55 several new and powerful analytical techniques—disc
`electrophoresis, size exclusion chromatography, and
`HPLC—allows a detailed examination of insulin's
`chemical stability profile. Early chemical studies on
`insulin stability were difficult because the recrystallized
`60 insulin under examination was found to be no more than
`80-90% pure. More recently monocomponent, high-
`purity insulin has become available. This monocompo-
`nent insulin contains impurities at levels undetectable by
`current analysis techniques.
`Formulated insulin is prone to numerous types of
`degradation. Nonenzymatic deamidiation occurs when
`a side-chain amide group from a glutaminyl or asparagi-
`nyl residue is hydrolyzed to a free carboxylic acid.
`
`65
`
`

`

`5,359,030
`
`5
`There are six possible sites for such deamidiation in
`insulin: GlnA5, GlnA15, AsnA18, ASnA2l, AsnB3, and
`GlnB4. Published reports suggest that the three Asn
`residues are most susceptible to such reactions.
`Brange et al. (ibid) reported that in acidic conditions
`insulin is rapidly degraded by extensive deamidation at
`AsnA21. In contrast, in neutral formulations deamidation
`takes place at AsnB3 at a much slower rate, independent
`of insulin concentration and species of origin of the
`insulin. However, temperature and formulation type
`play an important role in determining the rate of hydro-
`lysis at B3. For example, hydrolysis at B3 is minimal if
`the insulin is crystalline as opposed to amorphous. Ap-
`parently the reduced flexibility (tertiary structure) in
`the crystalline form slows the reaction rate. Stabilizing
`the tertiary structure by incorporating phenol into neu-
`tral formulations results in reduced rates of deamida-
`tion.
`In addition to hydrolytic degradation products in
`insulin formulations, high molecular weight transforma-
`tion products are also formed. Brange et al. showed by
`size exclusion chromatography that the main products
`formed on storage of insulin formulations between 4°
`and 45° C. are covalent insulin dimers. In formulations
`containing protamine, covalent insulin protamine prod-
`ucts are also formed. The rate of formulation of insulin-
`dimer and insulin-protamine products is affected signifi-
`cantly by temperature. For human or porcine insulin,
`(regular N1 preparation) time to formation of 1% high
`molecular weight products is decreased from 154
`months to 1.7 months at 37° C. compared to 4° C. For
`zinc suspension preparations of porcine insulin, the
`same transformation would require 357 months at 4° C.
`but only 0.6 months at 37° C.
`These types of degradation in insulin may be of great
`significance to diabetic subjects. Although the forma-
`tion of high molecular weight products is generally
`slower than the formation of hydrolytic (chemical)
`degradation products described earlier, the implications
`may be more serious. There is significant evidence that
`the incidence of immunological responses to insulin
`may result from the presence of covalent aggregates of
`insulin (Robbins, D. C. Cooper, S. M. Fineberg, S. E.,
`and Mead, P. M., "Antibodies to covalent aggregates of
`insulin in blood of insulin-using diabetic patients", Dia-
`betes, 36, 838-841, (1987); Maislos, M., Mead, P. M.,
`Gaynor, D. H., and Robbins, D. C., "The source of the
`circulating aggregate of insulin in type I diabetic pa-
`tients is therapeutic insulin", J. Clin. Invest., 77,
`717-723. (1986); and Ratner R. E., Phillips, T. M., and
`Steiner, M., "Persistent cutaneous insulin allergy result-
`ing from high molecular weight insulin aggregates",
`Diabetes, 39, 728-733, (1990)). As many as 30% of dia-
`betic subjects receiving insulin show specific antibodies
`to covalent insulin dimers. At a level as low as 2% it
`was reported that the presence of covalent insulin di-
`mers generated a highly significant response in lympho-
`cyte stimulation in allergic patients. Responses were not
`significant when dimer content was in the range
`0.3-0.6%. As a result it is recommended that the level of
`covalent insulin dimers present in formulation be kept
`below 1% to avoid clinical manifestations.
`Several insulin formulations are commercially avail-
`able; although stability has been improved to the extent
`that it is no longer necessary to refrigerate all formula-
`tions, there remains a need for insulin formulations with
`enhanced stability. A modified insulin which is not
`prone to formation of high molecular weight products
`
`6
`would be a substantial advance in the pharmaceutical
`and medical arts, and modifications providing this sta-
`bility (and in addition providing the possibility of oral
`availability of insulin) would make a significant contri-
`5 bution to the management of diabetes.
`In addition to the in vivo usage of polypeptides and
`proteins as therapeutic agents, polypeptides and prote-
`ins also find substantial and increasing use in diagnostic
`reagent applications. In many such applications, poly-
`10 peptides and proteins are utilized in solution environ-
`ments wherein they are susceptible to thermal and en-
`zymic degradation of (poly)peptides and proteins such a
`enzymes, peptide and protein hormones, antibodies,
`enzyme-protein conjugates used for immunoassay, anti-
`15 body-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, hepati-
`tis, and rubella, peptide and protein growth factors used
`for example in tissue culture, enzymes used in clinical
`20 chemistry, and insoluble enzymes such as those used in
`the food industry. As a further specific example, alka-
`line phosphatase is widely utilized as a reagent in kits
`used for the colorimetric detection of antibody or anti-
`gen in biological fluids. Although such enzyme is com-
`25 mercially available in various forms, including free
`enzyme and antibody conjugates, its storage stability
`and solution often is limited. As a result, alkaline phos-
`phatase conjugates are frequently freeze-dried, and
`additives such as bovine serum albumin and Tween 20
`30 are used to extend the stability of the enzyme prepara-
`tions. Such approaches, while advantageous in some
`instances to enhance the resistance to degradation of the
`polypeptide and protein agents, have various shortcom-
`ings which limit their general applicability.
`SUMMARY OF THE INVENTION
`The present invention relates generally to conjuga-
`tion-stabilized (poly)peptide and protein compositions
`and formulations, and to methods of making and using
`40 same.
`More particularly, the present invention relates in
`one broad compositional aspect to covalently conju-
`gated peptide complexes wherein the peptide is cova-
`lently bonded to one or more molecules of a polymer
`45 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.
`In one particular aspect, the present invention relates
`50 to a physiologically active peptide composition com-
`prising a physiologically active peptide covalently cou-
`pled with a polymer comprising (i) a linear polyalkylene
`glycol moiety and (ii) a lipophilic moiety, wherein the
`peptide, linear polyalkylene glycol moiety, and the
`55 lipophilic moiety are conformationally arranged in rela-
`tion to one another such that the physiologically active
`peptide in the physiologically active peptide composi-
`tion has an enhanced in vivo resistance to enzymatic
`degradation, relative to the physiologically active pep-
`60 tide alone (i.e., in an unconjugated form devoid of the
`polymer coupled thereto).
`In another aspect, the invention relates to a physio-
`logically active peptide composition of three-dimen-
`sional conformation comprising a physiologically active
`65 peptide covalently coupled with a polysorbate complex
`comprising (i) a linear polyalkylene glycol moiety and
`(ii) a lipophilic moiety, wherein the physiologically
`active peptide, the linear polyalkylene glycol moiety
`
`35
`
`

`

`5,359,030
`
`7
`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 pep-
`tide in the physiologically active peptide composition
`has an enhanced in vivo resistance to enzymatic degra-
`dation, relative to the physiologically active peptide
`alone.
`In a further aspect, the invention relates to a multili-
`gand conjugated peptide complex comprising a triglyc-
`eride backbone moiety, having:
`a bioactive peptide covalently coupled with the tri-
`glyceride backbone moiety through a polyalkylene
`glycol spacer group bonded at a carbon atom of the
`triglyceride backbone moiety; and
`at least one fatty acid moiety covalently attached
`either directly to a carbon atom of the triglyceride
`backbone moiety or covalently joined through a polyal-
`kylene glycol spacer moiety.
`In such multiligand conjugated peptide complex, the
`a' and /3 carbon atoms of the triglyceride bioactive
`moiety may have fatty acid moieties attached by cova-
`lently bonding either directly thereto, or indirectly
`covalently bonded thereto through polyalkylene glycol
`spacer moieties. Alternatively, a fatty acid moiety may
`be covalently attached either directly or through a
`polyalkylene glycol spacer moiety to the a and a' car-
`bons of the triglyceride backbone moiety, with the bi-
`oactive peptide being covalently coupled with the /3-
`carbon of the triglyceride backbone moiety, either
`being directly covalently bonded thereto or indirectly
`bonded thereto through a polyalkylene spacer moiety.
`It will be recognized that a wide variety of structural,
`compositional, and conformational forms are possible
`for the multiligand conjugated peptide complex com-
`prising the triglyceride backbone moiety, within the
`scope of the foregoing discussion.
`In such a multiligand conjugated peptide complex,
`the bioactive peptide may advantageously be covalently
`coupled with the triglyceride modified backbone moi-
`ety through alkyl spacer groups, or alternatively other
`acceptable spacer groups, within the broad scope of the
`invention. As used in such context, acceptability of the
`spacer group refers to steric, compositional, and end use
`application specific acceptability characteristics.
`In yet another aspect, the invention relates to a poly-
`sorbate complex comprising a polysorbate moiety in-
`cluding a triglyceride backbone having covalently cou-
`pled to a,a' and /3 carbon atoms thereof functionalizing
`groups including:
`(i) a fatty acid group; and
`(ii) a polyethylene glycol group having a physiologi-
`cally 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 gly-
`col group, or alternatively, the covalent bonding may
`be indirect, e.g., by reactively capping the hydroxy
`terminus of the polyethylene glycol group with a termi-
`nal carboxy functionality spacer group, so that the re-
`sulting capped polyethylene glycol group has a terminal
`carboxy functionality to which the physiologically ac-
`tive moiety may be covalently bonded.
`The invention relates to a further aspect to a stable,
`aqueously soluble, conjugated peptide complex com-
`prising