United States Patent (19)
`
`Ekwuribe
`
`||||||||||||||||
`USOO535903OA
`11
`Patent Number:
`5,359,030
`45
`Date of Patent:
`Oct. 25, 1994
`
`54 CONJUGATION-STABILIZED
`POLYPEPTIDE COMPOSITIONS,
`THERAPEUTIC DELVERY AND
`DIAGNOSTIC FORMULATIONS
`COMPRISING SAME, AND METHOD OF
`MAKING AND USING THE SAME
`(75) Inventor:
`Ninochiri N. Ekwuribe, Southfield, .
`Mich.
`Assignee: Protein Delivery, Inc., Durham, N.C.
`Appl. No.: 59,701
`Filed:
`May 10, 1993
`Int. Cl. .......................... C07K 7/40; C07K 7/36;
`C07K 17/08; C08H 1/00
`U.S. Cl. .................................... 530/303; 530/307;
`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
`Field of Search ................. 435/188: 514/3, 4, 12;
`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
`
`(73)
`(21)
`22
`(51)
`(52)
`
`58)
`
`56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`4,003,792 1/1977 Mill et al. ............................ 530/303
`4,094,196 8/1977 Hiper et al...
`... 526/271
`4,179,337 12/1979 Davis et al....................
`... 435/181
`4,585,754 4/1986 Meisner et al. ......................... 514/8
`4,849,405 7/1989 Ecanow .................................. 514/3
`4,963,367 10/1990 Ecanow ........ ........
`... 424/485
`5,013,556 5/1991 Woodle et al. ..................... 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
`ABSTRACT
`57
`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,
`Sonatotropin, 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
`
`t
`S.
`E 500 :
`3 400
`g"
`S 30
`
`eve
`
`200 -
`
`
`
`100
`
`o BASELNE
`o GUCOSE ONLY (5 g/kg) P0.
`v NSUUN (100 g/kg) S.C.; GUCOSE (5 g/kg) P0.
`v NSUN (1.5 mg/kg) P0; GUCOSE (5 g/kg) P0.
`O NSAIN COMPEX (100 g/kg SC; GLUGOSE (5 g/kg) P0.
`INSULIN COPLEX (250 ug/kg) S.C.; GLUCOSE (5 g/kg) P0.
`a NSYLIN COAPEX (15 mg/kg) P0; G:0C0SE (5 g/kg) P0.
`via ISUNCIPLE (10 ug/kg) P0; GLUGOSE (5 g/kg) P0.
`r
`
`-
`
`i
`
`-
`
`90
`
`20
`
`TIME (MINUIES)
`
`MPI EXHIBIT 1019 PAGE 1
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`MPI EXHIBIT 1019 PAGE 1
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`

`

`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, 17367-368.
`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. etal, "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. Maislosetal, “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.
`
`MPI EXHIBIT 1019 PAGE 2
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`MPI EXHIBIT 1019 PAGE 2
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`

`

`U.S. Patent
`
`Oct. 25, 1994
`
`Sheet 1 of 2
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`5,359,030
`
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`SHQOH ‘BW||
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`MPI EXHIBIT 1019 PAGE 3
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`MPI EXHIBIT 1019 PAGE 3
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`

`

`U.S. Patent
`
`Oct. 25, 1994
`
`Sheet 2 of 2
`
`3,359,030
`
`
`
`
`
`
`
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`MPI EXHIBIT 1019 PAGE 4
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`MPI EXHIBIT 1019 PAGE 4
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`MPI EXHIBIT 1019 PAGE 4
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`

`

`10
`
`15
`
`1.
`
`CONUGATION-STABILIZED POLYPEPTOE
`COMPOSITIONS, THERAPEUTIC DELIVERY
`AND DAGNOSTIC FORMULATIONS
`COMPRISING SAME, AND METHOD OF MAKING
`5
`AND USING THE SAME
`
`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.
`
`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
`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
`25
`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
`30
`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
`
`MPI EXHIBIT 1019 PAGE 5
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`MPI EXHIBIT 1019 PAGE 5
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`1O
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`15
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`25
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`35
`
`5,359,030
`3
`4
`Other techniques of stabilizing peptide and protein
`The use of insulin as a treatment for diabetes dates
`drugs in which proteinaceous drug substances are con
`back to 1922, when Banting et al. ("Pancreatic Extracts
`jugated with relatively low molecular weight com
`in the Treatment of Diabetes Mellitus,' Can. Med.
`pounds such as aminolethicin, fatty acids, vitamin B12,
`Assoc. J., 12, 141-146 (1922) showed that the active
`extract from the pancreas had therapeutic effects in
`and glycosides, are revealed in the following articles: R.
`Igarishi et al., “Proceed. Intern. Syrup. Control. Rel.
`diabetic dogs. Treatment of a diabetic patient in that
`same year with pancreatic extracts resulted in a dra
`Bioact. Materials', 17, 366, (1990); T. Taniguchi et al.
`matic, life-saving clinical improvement. A course of
`Ibid 19, 104, (1992); G. J. Russel-Jones, Ibid, 19, 102,
`daily injections of insulin is required for extended re
`(1992); M. Baudys et al., Ibid, 19, 210, (1992). The modi
`fying compounds are not polymers and accordingly do
`covery.
`not contain moieties necessary to impart both the solu
`The insulin molecule consists of two chains of amino
`bility and membrane affinity necessary for bioavailabil
`acids linked by disulfide bonds; the molecular weight of
`ity following oral as well as parenteral administration.
`insulin is around 6,000. The 6-cells of the pancreatic
`Many of these preparations lack oral bioavailability.
`islets secrete a single chain precursor of insulin, known
`as proinsulin. Proteolysis of proinsulin results in re
`Another approach which has been taken to lengthen
`the in vivo duration of action of proteinaceous sub
`moval of four basic amino acids (numbers 31, 32, 64 and
`65 in the proinsulin chain: Arg, Arg, Lys, Arg respec
`stances is the technique of encapsulation. M. Safran et
`tively) and the connecting (“C”) peptide. In the result
`al., in Science, 223, 1081, (1986) teaches the encapsula
`tion of proteinaceous drugs in an azopolymer film for
`ing two-chain insulin molecule, the A chain has glycine
`oral administration. The film is reported to survive
`at the amino terminus, and the B chain has phenylala
`digestion in the stomach but is degraded by microflora
`nine at the amino terminus.
`in the large intestine, where the encapsulated protein is
`Insulin may exist as a monomer, dimer or a hexamer
`released. The technique utilizes a physical mixture and
`formed from three of the dimers. The hexamer is coor
`dinated with two Zn2+ atoms. Biological activity re
`does not facilitate the absorption of released protein
`sides in the monomer. Although until recently bovine
`across the membrane.
`and porcine insulin were used almost exclusively to
`Ecanow, U.S. Pat. No. 4,963,367, teaches that physio
`logically active compounds, including proteins, can be
`treat diabetes in humans, numerous variations in insulin
`encapsulated by a coacervative-derived film and the
`between species are known. Porcine insulin is most
`finished product can be suitable for transmucosal ad
`similar to human insulin, from which it differs only in
`having an alanine rather than threonine residue at the
`ministration. Other formulations of the same invention
`may be administered by inhalation, oral, parenteral and
`B-chain C-terminus. Despite these differences most
`mammalian insulin has comparable specific activity.
`transdermal routes. These approaches do not provide
`intact stability against acidity and proteolytic enzymes
`Until recently animal extracts provided all insulin used
`of the gastrointestinal tract, the property as desired for
`for treatment of the disease. The advent of recombinant
`oral delivery.
`technology allows commercial scale manufacture of
`Another approach taken to stabilize protein drugs for
`human insulin (e.g., Humulin TM 0 insulin, commer
`cially available from Eli Lilly and Company, Indianap
`oral as well as parenteral administration involves en
`trapment of the therapeutic agent in liposomes. A re
`olis, Ind.).
`view of this technique is found in Y. W. Chien, "New
`Although insulin has now been used for more than 70
`Drug Delivery Systems', Marcel Dekker, New York,
`years as a treatment for diabetes, few studies of its for
`40
`N.Y., 1992. Liposome-protein complexes are physical
`mulation stability appeared until two recent publica
`tions (Brange, J., Langkjaer, L., Havelund, S., and Vo
`mixtures; their administration gives erratic and unpre
`lund, A., "Chemical stability of insulin. I. Degradation
`dictable results. Undesirable accumulation of the pro
`during storage of pharmaceutical preparations, Pharm.
`tein component in certain organs has been reported, in
`the use of such liposome-protein complexes. In addition
`Res., 9, 715-726, (1992); and Brange, J. Havelund, S.,
`and Hougaard, P., "Chemical stability of insulin. 2.
`to these factors, there are additional drawbacks associ
`ated with the use of liposomes, such as cost, difficult
`Formulation of higher molecular weight transformation
`manufacturing processes requiring complex lypophili
`products during storage of pharmaceutical prepara
`zation cycles, and solvent incompatibilities. Moreover,
`tions,” Pharm. Res., 9, 727-734, (1992)). In these publi
`cations, the authors exhaustively describe chemical
`altered biodistribution and antigenicity issues have been
`stability of several insulin preparations under varied
`raised as limiting factors in the development of clini
`cally useful liposomal formulations.
`temperature and pH conditions. Earlier reports focused
`The use of “proteinoids' has been described recently
`almost entirely on biological potency as a measure of
`(Santiago, N., Milstein, S. J., Rivera, T., Garcia, E.,
`insulin formulation stability. However the advent of
`Several new and powerful analytical techniques-disc
`Chang., T. C., Baughman, R. A., and Bucher, D., "Oral
`electrophoresis, size exclusion chromatography, and
`Immunization of Rats with Influenza Virus M Protein
`(M1) Microspheres', Abstract #A 221, Proc. Int. Symp.
`HPLC-allows a detailed examination of insulin's
`chemical stability profile. Early chemical studies on
`Control Rel. Bioac. Mater, 19, 116 (1992)). Oral delivery
`insulin stability were difficult because the recrystallized
`of several classes of therapeutics has been reported
`using this system, which encapsulates the drug of inter
`insulin under examination was found to be no more than
`80-90% pure. More recently monocomponent, high
`est in a polymeric sheath composed of highly branched
`purity insulin has become available. This monocompo
`amino acids. As is the case with liposomes, the drugs are
`not chemically bound to the proteinoid sphere, and
`nent insulin contains impurities at levels undetectable by
`leakage of drug out of the dosage form components is
`current analysis techniques.
`possible.
`Formulated insulin is prone to numerous types of
`A peptide which has been the focus of much synthesis
`degradation. Nonenzymatic deamidiation occurs when
`a side-chain amide group from a glutaminyl or asparagi
`work, and efforts to improve its administration and
`nyl residue is hydrolyzed to a free carboxylic acid.
`bioassimilation, is insulin.
`
`60
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`45
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`50
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`55
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`65
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`MPI EXHIBIT 1019 PAGE 6
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`O
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`15
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`5,359,030
`5
`6
`There are six possible sites for such deamidiation in
`would be a substantial advance in the pharmaceutical
`insulin: Gln-45, Gln-415, Asn118, Asn12, Asn-3, and
`and medical arts, and modifications providing this sta
`Gln B4. Published reports suggest that the three Asn
`bility (and in addition providing the possibility of oral
`residues are most susceptible to such reactions.
`availability of insulin) would make a significant contri
`Brange et al. (ibid) reported that in acidic conditions
`bution to the management of diabetes.
`insulin is rapidly degraded by extensive deamidation at
`In addition to the in vivo usage of polypeptides and
`proteins as therapeutic agents, polypeptides and prote
`Asn121. In contrast, in neutral formulations deamidation
`takes place at Asn-3 at a much slower rate, independent
`ins also find substantial and increasing use in diagnostic
`reagent applications. In many such applications, poly
`of insulin concentration and species of origin of the
`insulin. However, temperature and formulation type
`peptides and proteins are utilized in solution environ
`play an important role in determining the rate of hydro
`ments wherein they are susceptible to thermal and en
`lysis at B3. For example, hydrolysis at B3 is minimal if
`Zymic degradation of (poly)peptides and proteins such a
`the insulin is crystalline as opposed to amorphous. Ap
`enzymes, peptide and protein hormones, antibodies,
`parently the reduced flexibility (tertiary structure) in
`enzyme-protein conjugates used for immunoassay, anti
`the crystalline form slows the reaction rate. Stabilizing
`body-hapten conjugates, viral proteins such as those
`the tertiary structure by incorporating phenol into neu
`used in a large number of assay methodologies for the
`diagnosis or screening of diseases such as AIDS, hepati
`tral formulations results in reduced rates of deamida
`tis, and rubella, peptide and protein growth factors used
`tion.
`In addition to hydrolytic degradation products in
`for example in tissue culture, enzymes used in clinical
`insulin formulations, high molecular weight transforma
`chemistry, and insoluble enzymes such as those used in
`20
`tion products are also formed. Brange et al. showed by
`the food industry. As a further specific example, alka
`size exclusion chromatography that the main products
`line phosphatase is widely utilized as a reagent in kits
`formed on storage of insulin formulations between 4
`used for the colorimetric detection of antibody or anti
`gen in biological fluids. Although such enzyme is com
`and 45 C. are covalent insulin dimers. In formulations
`containing protamine, covalent insulin protamine prod
`mercially available in various forms, including free
`25
`enzyme and antibody conjugates, its storage stability
`ucts are also formed. The rate of formulation of insulin
`dimer and insulin-protamine products is affected signifi
`and solution often is limited. As a result, alkaline phos
`cantly by temperature. For human or porcine insulin,
`phatase conjugates are frequently freeze-dried, and
`(regular N1 preparation) time to formation of 1% high
`additives such as bovine serum albumin and Tween 20
`molecular weight products is decreased from 154
`are used to extend the stability of the enzyme prepara
`30
`months to 1.7 months at 37 C. compared to 4 C. For
`tions. Such approaches, while advantageous in some
`zinc suspension preparations of porcine insulin, the
`instances to enhance the resistance to degradation of the
`same transformation would require 357 months at 4 C.
`polypeptide and protein agents, have various shortcom
`ings which limit their general applicability.
`but only 0.6 months at 37 C.
`These types of degradation in insulin may be of great
`35
`SUMMARY OF THE INVENTION
`significance to diabetic subjects. Although the forma
`tion of high molecular weight products is generally
`The present invention relates generally to conjuga
`slower than the formation of hydrolytic (chemical)
`tion-stabilized (poly)peptide and protein compositions
`degradation products described earlier, the implications
`and formulations, and to methods of making and using
`may be more serious. There is significant evidence that
`Sale.
`the incidence of immunological responses to insulin
`More particularly, the present invention relates in
`may result from the presence of covalent aggregates of
`one broad compositional aspect to covalently conju
`insulin (Robbins, D. C. Cooper, S. M. Fineberg, S. E.,
`gated peptide complexes wherein the peptide is cova
`and Mead, P.M., "Antibodies to covalent aggregates of
`lently bonded to one or more molecules of a polymer
`insulin in blood of insulin-using diabetic patients', Dia
`incorporating as an integral part thereof a hydrophilic
`45
`moiety, e.g., a linear polyalkylene glycol, and wherein
`betes, 36, 838–841, (1987); Maislos, M., Mead, P. M.,
`said polymer incorporates a lipophilic moiety as an
`Gaynor, D. H., and Robbins, D.C., "The source of the
`circulating aggregate of insulin in type I diabetic pa
`integral part thereof.
`tients is therapeutic insulin', J. Clin. Invest., 77,
`In one particular aspect, the present invention relates
`to a physiologically active peptide composition com
`717-723. (1986); and Ratner R. E., Phillips, T. M., and
`prising a physiologically active peptide covalently cou
`Steiner, M., "Persistent cutaneous insulin allergy result
`ing from high molecular weight insulin aggregates",
`pled with a polymer comprising (i) a linear polyalkylene
`glycol moiety and (ii) a lipophilic moiety, wherein the
`Diabetes, 39,728-733, (1990)). As many as 30% of dia
`betic subjects receiving insulin show specific antibodies
`peptide, linear polyalkylene glycol moiety, and the
`lipophilic moiety are conformationally arranged in rela
`to covalent insulin dimers. At a level as low as 2% it
`55
`was reported that the presence of covalent insulin di
`tion to one another such that the physiologically active
`mers generated a highly significant response in lympho
`peptide in the physiologically active peptide composi
`cyte stimulation in allergic patients. Responses were not
`tion has an enhanced in vivo resistance to enzymatic
`significant when dimer content was in the range
`degradation, relative to the physiologically active pep
`0.3-0.6%. As a result it is recommended that the level of
`tide alone (i.e., in an unconjugated form devoid of the
`60
`polymer coupled thereto).
`covalent insulin dimers present in formulation be kept
`below 1% to avoid clinical manifestations.
`In another aspect, the invention relates to a physio
`Several insulin formulations are commercially avail
`logically active peptide composition of three-dimen
`able; although stability has been improved to the extent
`sional conformation comprising a physiologically active
`that it is no longer necessary to refrigerate all formula
`peptide covalently coupled with a polysorbate complex
`65
`comprising (i) a linear polyalkylene glycol moiety and
`tions, there remains a need for insulin formulations with
`(ii) a lipophilic moiety, wherein the physiologically
`enhanced stability. A modified insulin which is not
`prone to formation of high molecular weight products
`active peptide, the linear polyalkylene glycol moiety
`
`40
`
`50
`
`MPI EXHIBIT 1019 PAGE 7
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`MPI EXHIBIT 1019 PAGE 7
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`

`

`15
`
`20
`
`5,359,030
`8
`7
`pled to a physiologically compatible polyethylene gly
`and the lipophilic moiety are conformationally arranged
`col modified glycolipid moiety. In such complex, the
`in relation to one another such that (a) the lipophilic
`physiologically active peptide may be covalently cou
`moiety is exteriorly available in the three-dimensional
`pled to the physiologically compatible polyethylene
`conformation, and (b) the physiologically active pep
`tide in the physiologically active peptide composition
`glycol modified glycolipid moiety by a labile covalent
`bond at a free amino acid group of the polypeptide,
`has an enhanced in vivo resistance to enzymatic degra
`dation, relative to the physiologically active peptide
`wherein the liable covalent bond is scissionable in vivo
`by biochemical hydrolysis and/or proteolysis. The
`alone.
`physiologically compatible polyethylene glycol modi
`In a further aspect, the invention relates to a multili
`gand conjugated peptide complex comprising a triglyc
`fied glycolipid moiety may advantageously comprise a
`O
`polysorbate polymer, e.g., a polysorbate polymer com
`eride backbone moiety, having:
`prising fatty acid ester groups selected from the group
`a bioactive peptide covalently coupled with the tri
`glyceride backbone moiety through a polyalkylene
`consisting of monopalmitate, dipalmitate, monolaurate,
`glycol spacer group bonded at a carbon atom of the
`dilaurate, trilaurate, monoleate, dioleate, trioleate, mon
`triglyceride backbone moiety; and
`Ostearate, distearate, and tristearate. In such complex,
`the physiologically compatible polyethylene glycol
`at least one fatty acid moiety covalently attached
`modified glycolipid moiety may suitably comprise a
`either directly to a carbon atom of the triglyceride
`backbone moiety or covalently joined through a polyal
`polymer selected from the group consisting of polyeth
`kylene glycol spacer moiety.
`ylene glycol ethers of fatty acids, and polyethylene
`In such multiligand conjugated peptide complex, the
`glycol esters of fatty acids, wherein the fatty acids for
`example comprise a fatty acid selected from the group
`o' and 3 carbon atoms of the triglyceride bioactive
`consisting of lauric, palmitic, oleic, and stearic acids.
`moiety may have fatty acid moieties attached by cova
`lently bonding either directly thereto, or indirectly
`In the above complex, the physiologically active
`peptide may by way of illustration comprise a peptide
`covalently bonded thereto through polyalkylene glycol
`spacer moieties. Alternatively, a fatty acid moiety may
`selected from the group consisting of insulin, calcitonin,
`25
`be covalently attached either directly or through a
`ACTH, glucagon, somatostatin, somatotropin, soma
`polyalkylene glycol spacer moiety to the a. and a' car
`tomedin, parathyroid hormone, erythropoietin, hypo
`thalmic releasing factors, prolactin, thyroid stimulating
`bons of the triglyceride backbone moiety, with the bi
`oactive peptide being covalently coupled with the f8
`hormones, endorphins, enkephalins, vasopressin, non
`naturally occurring opiods, superoxide dismutase, inter
`carbon of the triglyceride backbone moiety, either
`30
`being directly covalently bonded thereto or indirectly
`feron, asparaginase, arginase, arginine deaminease,
`bonded thereto through a polyalkylene spacer moiety.
`adenosine deaminase ribonuclease, trypsin, chemotryp
`sin, and papain.
`It will be recognized that a wide variety of structural,
`compositional, and conformational forms are possible
`In another aspect, the present invention rela