(12) United States Patent
`US 6,342,476 B1
`(10) Patent N0.:
`Konfino et al.
`
`(45) Date of Patent: Jan. 29, 2002
`
`US006342476B1
`
`(54) COPOLYMER-l IMPROVEMENTS IN
`COMPOSITIONS OF COPOLYMERS
`
`(75)
`
`Inventors: Eliezer Konfino, Ramat Gan; Michael
`Sela, Rehovot; Dvora Teitelbaum,
`Rehovot; Ruth Arnon, Rehovot, all of
`(IL)
`
`(73) Assignee: Yeda Research & Development
`Company Limited, Rehovot (IL)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.: 09/510,141
`
`(22)
`
`Filed:
`
`Feb. 22, 2000
`
`Related US. Application Data
`
`(63) Continuation of application No. 09/032,334, filed on Feb.
`27, 1998, now Pat. No. 6,048,898, which is a continuation
`of application No. 08/447,146, filed on May 22, 1995, now
`Pat. No. 5,800,808, which is a continuation—in—part of appli—
`cation No. 08/344,248, filed on NOV. 23, 1994, now aban—
`doned, which is a continuation of application No. 08/248,
`037, filed on May 24, 1994, now abandoned.
`
`(51)
`
`Int. Cl.7 ......................... A01N 43/16; A61K 31/35
`
`(52) US. Cl.
`
`............................. 514/2; 514/12; 514/903;
`424/78.37
`
`(58) Field of Search .............................. 514/2, 12, 903;
`424/78.37
`
`(56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`........... 424/78
`3,849,550 A * 11/1974 Teitlebaum et al.
`4,594,409 A
`6/1986 Hayashii et al.
`............ 525/420
`FOREIGN PATENT DOCUMENTS
`
`DE
`EP
`EP
`EP
`SU
`SU
`
`39 30733
`03 78246
`0 383 620
`9 83020
`1882051
`1664845
`
`*
`
`3/1991
`7/1990
`8/1990
`8/1990
`9/1985
`7/1991
`
`OTHER PUBLICATIONS
`
`D. Teitelbaum et al., “Dose—Response Studies on Experi-
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`D. Teitelbaum et al., “Suppression of Experimental Allergic
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`1972, 240, pp.
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`
`C. Webb et al., “Further Studies on the Suppression of
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`
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`279—286 (1973).
`D. Teitelbaum et al., “Suppression of Experimental Allergic
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`Copolymer”, Clin.
`Immunol.
`Immunopath, 1974, 3, pp.
`256—262.
`
`D. Teitelbaum et al., “Dose—response Studies on Experi-
`mental Allergic Encephalomyelitis Suppression by COP—1”,
`Israel J. Med. Sci., 1974, 10, pp. 1172—1173.
`C. Webb et al., “Suppression of Experimental Allergic
`Encephalomyelitis in Rhesus Monkeys by a Synthetic Basic
`Copolymer”, Isr. J. Med. Sci., 1975, 11, p. 1388 (abstract).
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`pression of EAE by Synthetic Basic Copolymers of Amino
`Acids”, Immunochemistry, 1976, 13, pp. 333—337.
`0. Abramsky et al., “Effect of a Synthetic Polypeptide
`(COP—1) on Patients with Multiple Sclerosis and with Acute
`Disseminated Encephalomyelitis”, J. Neurol. Sci., 1977, 31,
`pp. 433—438.
`D. Teitelbaum et al., “Suppression of Experimental Allergic
`Encephalomyelitis in Baboons by COP—1.” Israel J. Med.
`Sci., 1977, 13, 1038 (abstract).
`M. Sela et al., “Experimental Allergic Encephalomyelitis in
`Menarini Series on Immunopathology, vol. 1, First Sympo-
`sium of Organ Specific Autoimmunity”, Cremona, Italy, Jun.
`1977, Miescher P.A. eds., pp. 9—21, Schwabe Co., Basel,
`(1978).
`R. Arnon et al., “Suppression of EAE in Baboons by a
`Synthetic Polymer of Amino Acids”, Neurology, 1978, 28,
`336 (abstract).
`E.C.Alvord et al., “Myelin Basic Protein Treatment of
`Experimental Allergic Encephalomyelitis in Monkeys”,
`Ann. Neurol, 1979, 6, pp. 469—473.
`A.B. Keith et al., “The Effect of COP—1, a Synthetic
`Polypeptide, on Chronic Relapsing Experimental Allergic
`Encephalomyelitis in Guinea Pigs”, J. Neurol. Sci., 1979,
`42, pp. 267—274.
`Z. Lando et al., “Effect of Cyclophosphamide on Suppressor
`Cell Activity in Mice Unresponsive to EAE”, J. Immunol,
`1979, 123, pp. 2156—2160 (abstract).
`
`(List continued on next page.)
`
`Primary Examiner—Frederick Krass
`(74) Attorney, Agent, or Firm—Kenyon & Kenyon
`
`(57)
`
`ABSTRACT
`
`The present invention relates to an improved composition of
`copolymer-l comprising copolymer-l substantially free of
`species having a molecular weight of over 40 kilodaltons.
`
`1 Claim, 2 Drawing Sheets
`
`MYLAN INC. EXHIBIT NO. 1024 Page 1
`
`MYLAN INC. EXHIBIT NO. 1024 Page 1
`
`

`

`US 6,342,476 B1
`Page 2
`
`OTHER PUBLICATIONS
`
`Z. Lando et al., “Experimental Allergic Encephalomyelitis
`in Mice—Suppression and Prevention with COP—1”, Israel J.
`Med. Sci., 1979, 15, pp. 868—869 (abstract).
`D. Teitelbaum et al., “Blocking of Sensitization to Encepha-
`litogenic Basic Protein in Vitro by Synthetic Basic Copoly-
`mer (COP—1).” in Cell biology and Immunology of Leuko-
`cyte Function, Academic Press, New York, 1979, pp.
`681—685.
`
`D. Teitelbaum, “Suppression of Experimental Allergic
`Encephalomyelitis with a Synthetic Copolymer—relevance
`to Multiple Sclerosis”, in Humoral Immunity in Neurologi-
`cal Diseases, Karcher D., Lowenthal A. & Strosberg A.D.
`eds. Plenum Publishing Corp., 1979, pp. 609—613.
`R. Arnon et al., “Desensitization of Experimental Allergic
`Encephalomyelitis with Synthetic Peptide Analogues” in
`The Suppression of Experimental Allergic Encephalomyeli-
`tis and Multiple Sclerosis, Academic Press, New York, 1980
`pp. 105—107.
`R. Arnon, “A Synthetic Copolymer of Amino Acids in a
`Clinical Trail for MS Therapy” in Progress in Multiple
`Sclerosis Research, Bauer, Ritter, eds. Springer Verlag NY,
`1980 pp. 416—418.
`M. B. Bornstein et al., “Treatment of Multiple Sclerosis with
`a Synthetic Polypeptide: Preliminary results.”, Trans. Am.
`Neurol. Assoc., 1980, 105, pp. 348—350.
`M. B. Bornstein et al., “Treatment of Multiple Sclerosis with
`a Synthetic Polypeptide: preliminary results”, Ann. Neuro.,
`1980, 8, pp. 117 (abstract).
`J. R. McDermott et al., “Antigen—induced Suppression of
`Experimental Allergic Neuritis in the Guinea Pig.”, J. Neu-
`rol. Sci., 1980, 46, pp. 137—143.
`R. Arnon, “Experimental Allergic Encephalomyelitis—Sus-
`ceptibility and Suppression”, Immunological Rev., 1981, 55,
`pp. 5—30.
`M. B. Bornstein et al., “Multiple Sclerosis: Trial of a
`Synthetic Polypeptide”,Ann. Neurol, 1982, 11, pp. 317—319.
`C. G. Brosnan et al., “The Response of Normal Human
`Lymphocytes to Copolymer—1.”J. Neuropath. Exp. Neurol.,
`1983, 42, pp. 356 (abstract).
`R. P. Lisak et al., “Effect of Treatment with Copolymer 1
`(COP—1) on the in Vivo and in Vitro Manifestations of
`Experimental Allergic Encephalomyelitis (EAE)”, J. Neu-
`rol. Sci., 1983, 62, 281—293.
`M. B. Bornstein et al., “Clinical Trials of Copolymer 1 in
`Multiple Sclerosis”, Ann. NY. Acad. Sci. (USA), 1984, pp.
`366—372.
`
`M. B. Bornstein et al., “Clinical Trials of a Synthetic
`Polypeptide (Copolymer 1) for the Treatment of Multiple
`Sclerosis” in R.E. Gonsett et al., eds. Immunological and
`clinical aspects of multiple sclerosis, 1984, pp. 144—150.
`C. F. Brosnan et al., “Copolymer 1: Effect on Normal
`Human Lymphocytes”, Ann. NY. Acad. Sci. (USA), 1984,
`436, pp. 498—499.
`J. Burns et al., “Human Cellular Immune Response in Vitro
`to Copolymer 1 and Myelin Basic Protein (MBP)”, Neurol-
`ogy, 1985, 35, (suppl 1), 170 (abstract).
`C. F. Brosnan, et al., “Immunogenic potentials of copolymer
`1
`in normal
`lymphocytes” Neurology, 1985, 35, pp.
`1754—1759.
`
`M. B. Bornstein et al., “Multiple Sclerosis: Clinical Trials of
`a Synthetic Polypeptide, Copolymer 1.”, Neurology, 1985,
`35, (suppl 1), p. 103 (abstract).
`
`D. Teitelbaum et al., “Monoclonal antibodies to Myelin
`Basic Protein Cross Road with a Synthetic EAE Suppressive
`Copolymer, COP 1.”, Proc. 7th European Immunology
`Meeting, Jerusalem, 1985 (abstract).
`J. Burns et al., “Human Cellular Immune Response to
`Copolymer 1 and Myelin Basic Protein.” Neurology, 1986,
`36, pp. 92—94.
`M. B. Bornstein, “COP—1 may be Beneficial for Patients
`with Exacerbating—remitting Form of Multiple Sclerosis”,
`Adv. Ther. (USA), 1987, 4, p. 206 (Abstract).
`H. L. Winer , “COP—1 Therapy for Multiple Sclerosis”, New
`England Journal of Medicine, 1987, 317, pp. 442—444.
`R. Arnon et al., “Suppression of Demyelinating Diseases by
`Synthetic Copolymers”, from: A multidisctplinary approach
`to myelin disease G. Serlupi Crescenzi, ed. Plenum Publish-
`ing Corporation, 1988, pp. 243—250.
`M. B. Bornstein et al., “Pilot Trial of COP—1 in Chronic
`Progressive Multiple Sclerosis:
`Preliminary Report”,
`Elsevier Science Publisher, 1989, pp. 225—232; Conference
`“The International Multiple Sclerosis Conference: an update
`on Multiple Sclerosis” Roma (Italy), Sep. 15—17, 1988.
`E. Grgacic et al., “Cell—mediated Immune Response to
`Copolymer 1 in Multiple Sclerosis Measured by the Mac-
`rophage Procoagulant Activity Assay”, Int. Immunol, 1990,
`2, pp. 714—718.
`M. B. Bornstein et al., “Clinical Trials of COP—1 in Multiple
`Sclerosis”, Handbook of Multiple Sclerosis, S. D. Cook
`Marcel Rekker et al., 1990, pp. 469—480.
`M. Wender, Copolymer 1 (COP—1)
`in the Treatment of
`Multiple Sclerosis (letter) Neur. Neurochir. Pol. (Poland),
`1990, 24, pp. 113.
`Z. Meiner, “COP—1 Multicenter Clinical Trial in Exacerbat-
`ing—remitting Multiple—Sclerosis: one year follow—up”,J. of
`Neurol., 1991, supp 1. (abstract).
`D. Teitelbaum et al., “Cross—reactions and Specificities of
`Monoclonal Antibodies Against Myelin Basic Protein and
`Against The synthetic, Copolymer 1.” Proc. Natl. Acad. Sci.,
`(USA) 1991, 88, pp. 9528—9532.
`M Salvetti et al., “Myelin Basic Protein T Cell Epitopes in
`Patients with Multiple Sclerosis”, 72 (Abstract) (1991).
`M. B. Bornstein et al., “Treatment of Multiple Sclerosis:
`Trial design, results and future Perspectives”, Rudick R.K.
`& Goodkin D.E., eds. Springer Verlag, London, New York,
`1992, pp. 173—198.
`Inhibits
`1
`D. Teitelbaum et al., “Synthetic Copolymer
`Human T—Cell Lines Specific for Myelin Basic Protein”,
`Proc. Natl. Acad., Sci, (USA), 1992, 89, 137—141.
`K. P. Johnson, “Clinical Studies in Copolymer 1 Therapy for
`Exacerbating—remitting Multiple Sclerosis”, Comm. pre-
`sented at the Congress for Advances in the Understanding
`and Treatment of Multiple Sclerosis, Boston (USA), Oct.
`28—29, 1992.
`
`R. Milo et al., “Inhibition of Myelin Basic Protein—Specific
`Human T—Cell Lines by COP—1”, Israel J. Med. Sci., 1992,
`28, p. 486 (Abstract).
`R. Arnon et al., “On the Existence of Suppressor Cells”, Int.
`Arch. Allergy Immunol., 1993, 100, pp. 2—7.
`M. Sela, “Polymeric Drugs as Immunomodulatory Vaccines
`Against Multiple Sclerosis”, Makromol. Chem. Macromol.
`Symp., 1993, 70/71, pp. 147—155.
`
`MYLAN INC. EXHIBIT NO. 1024 Page 2
`
`MYLAN INC. EXHIBIT NO. 1024 Page 2
`
`

`

`US 6,342,476 B1
`
`Page 3
`
`Z. Meiner et al., “The Israeli COP—1 Multicenter Clinical
`Trial in Exacerbating—remitting Multiple Sclerosis two—year
`followup.”, 9th Congress of the European Committee for
`Treatment and Research in Multiple Sclerosis, Florence
`(Italy), Oct.—Nov., 1993 Abstract.
`R. Milo et al., “Copolymer—1 (COP—1) regulates class II
`MHC Expression and Cytokine Synthesis in the Mono-
`cyte—Macrophage Cell Line”, The IBC Conference on Mul-
`tiple Sclerosis, San Diego, Dec. 10, 1993 (Abstract).
`M. Fridkis—Hareli et al., “Specific and Promiscuous Binding
`of Synthetic Copolymer—1 to Class II Major Histocompati-
`bilty Complex Molecules on Living Antigen Presenting
`Cells”, Israeli Biochemistry Society, 1994, Mar. pp. 21—22
`(Abstract).
`M. Fridkis—Hareli et al., “Synthetic Copolymer—1 and
`Myelin Basic Protein do not Undergo Processing Prior to the
`Binding to Class 11 Major Histocompatibility Complex
`Molecules on Antigen Presenting Cells”, The Israeli Immu-
`nol. Soc., May 3—4, 1994 (Abstract).
`R. Arnon et al., “Immunospecific Drug Design—Prospects
`for Treatment of Autoimmune Diseases”, Therapeutic
`Immunol., 1994, 1, pp. 65—70.
`M. Fridkis—Hareli et al., “Synthetic Copolymer—1 Inhibits
`the Binding of MBP, PLP and MOG Peptides to Class 11
`Major Histocompatibility Complex Molecules on Antigen
`Presenting Cells” Neurochem Meeting, Aug. 14—19, 1994.
`Masha Fridkis—Hareli et al., “Synthetic Copolymer 1 and
`Myelin Basic Protein Do Not Require Processing Prior to
`Binding To Class II Major Histocompatibility Complex
`Molecules On Living Antigen Presenting Cells”, J. Neuro-
`chem, 63, Suppl. I, 561, 1994.
`Kenneth P. Johnson, “Experimental Therapy of Relapsin-
`g—Remitting Multiple Sclerosis With Copolymer—1”,Ameri-
`can Neurological Association, 1994, 36, pp. 115—117.
`E. Kott et al., “COP—1 Increases Suppressor Cells Number
`In Multiple Sclerosis”, Israel Neurological Association,
`1994, p. 17.
`The COP—1 Multicenter Clinical and Research Group Study,
`“COP—1 Multicenter Trial in Relapsing Remitting Multiple
`Sclerosis: 3 Year Follow Up”, Abstracts of Symposia and
`Free Communications, Jun. 25—29, 1994, suppl 1, 241, p. 6.
`Yafit Stark, “Expanded Clinical Trials of Treatments for
`Multiple Sclerosis: Copolymer 1 Treatment Investigational
`New Drug Program”, Ann. Neurol, 1994, 36, pp. 114—115.
`R. Milo et al., “Additive Effect of Copolymer—1 and Inter-
`feron—B on the Immune Response to Myelin Basic Protein”,
`AssafHarofeh Medical Center; Sackler School of Medicine,
`Tel—Aviv University, University of Marlana' School of Medi-
`cine, p. 22 (1994).
`R. Milo et al., “Additive Effects of COP—1 and IFN—Beta on
`Immune Responses to Myelin Basic Protein”, Neurology,
`1994, 44, suppl. 2 A212.
`D. Teitalbaum et al., “Immunological Parameters in a Mul-
`ticenter Clinical Trial of COP1 in Multiple Sclerosis: A
`2—year follow—up”, Neurology, 1994 44, suppl. 2 A358.
`
`M. Fridkis—Hareli et al., “Copolymer 1 Displaces MBP, PLP
`and MOG, but can not be displaced by these antigens from
`the MHC Class II binding Site”(1994).
`Paul Cotton, “Options for Multiple Sclerosis Therapy”,
`JAIVIA Medical News & Perspectives, 1994, 272, No. 18.
`
`Lawrence Jacobs, “Advances in specific therapy for multiple
`sclerosis”, Neurology, 1994, 7, pp. 250—254.
`
`L. Durelli, “Immunotherapeutics of Multiple Sclerosis” pp.
`467—475 (1994).
`
`Lawrence Myers et al., “The Peculiar Difficulties of Thera-
`peutic Trials for Multiple Sclerosis”, Neurologic Clinics,
`Feb. 1990, 8, pp. 119—141.
`
`D. A. Francis, “The Current Therapy of Multiple Sclerosis”,
`Journal of Clinical Pharmacy and Therapeutics, 1993, 18,
`pp. 77—84.
`
`Jonathan L. Carter et al., “Newer Drug Therapies for Mul-
`tiple Sclerosis”, Drug Therapy, Mar. 1990, pp. 31—43.
`
`Brian G. Weinshenker et al., “Natural history and treatment
`of multiple sclerosis”, Current Opinion in Neurology and
`Neurosurgery, 1992, 5, pp. 203—211.
`
`Stuart Nightingale MD. et al., “Access to Investigational
`Drugs for Treatment Purposes”, American Family Physi-
`cian, Sep. 15, 1994, pp. 845—847.
`
`Shalini Bansil, MD. et al., “Multiple Sclerosis: Pathogen-
`esis and Treatment”, Seminars in Neurology, 1994, 14, No.
`2, pp. 146—153.
`
`Masha Fridkis—Hareli et al., “Direct binding of myelin basic
`protein and synthetic copolymer 1 to class II major histo-
`compatibility complex molecules on living antigen—present-
`ing cells—specificity and promiscuity”, Proc. Natl. Acad. Sci,
`USA, May 1994, 91, pp. 4872—4876.
`
`E. Gurevich, “Study of the MHC—competition between BP
`and Cop 1 using human cytotoxic T—cell clones” Isr. J. Med.
`Sci, 1993 (Abstract).
`
`Masha Fridkis—Hareli et al., “Synthetic Copolymer 1 and
`Myelin Basic Protein do not require Processing prior to
`binding to class II major Histocompatibility Complex Mol-
`ecules on Living Antigen Presenting Cells”, Department of
`Chemical Immunology, The Weizmann Institute of Science,
`Rehovot, 76100, Israel (1993).
`
`Rolak, “Copolymer—I Therapy for Multiple Sclerosis,”
`Dept. of Neurology, Baylor College of Medicine, Clinical
`Neuropharmacology, pp. 391—396.* (1993).
`
`M. Bodanszky, “Principles of Peptide Synthesis, ” Spring-
`er—Verlag, Berlin, Heidelberg, New York, Tokyo, 1984,
`118—229.*
`
`J. Burns et al., “Failure of Copolymer 1 to Inhibit the Human
`T—cell Response to Myelin Basic Protein”, Neurology, 41,
`pp. 1317—1319, 1991.
`
`Clinical Trial Protocol No. 9001; first patient enrolled Oct.
`23, 1991.
`
`Clinical Trial Protocol No. 9002; first patient enrolled Jun.
`17, 1993.
`
`* cited by examiner
`
`MYLAN INC. EXHIBIT NO. 1024 Page 3
`
`MYLAN INC. EXHIBIT NO. 1024 Page 3
`
`

`

`US. Patent
`
`Jan. 29, 2002
`
`Sheet 1 0f 2
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`MYLAN INC. EXHIBIT NO. 1024 Page 4
`
`MYLAN INC. EXHIBIT NO. 1024 Page 4
`
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`

`US. Patent
`
`Jan. 29, 2002
`
`Sheet 2 0f 2
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`MYLAN INC. EXHIBIT NO. 1024 Page 5
`
`MYLAN INC. EXHIBIT NO. 1024 Page 5
`
`

`

`US 6,342,476 B1
`
`1
`COPOLYMER-l IMPROVEMENTS IN
`COMPOSITIONS OF COPOLYMERS
`
`This application is a continuation of application Ser. No.
`09/032,334, filed Feb. 27, 1998, US. Pat. No. 6,048,898
`which is a continuation of US. Ser. No. 08/447,146, filed
`May 22, 1995, now US. Pat. No. 5,800,808, which is a
`continuation-in-part of US. Ser. No. 08/344,248, filed Nov.
`23, 1994, now abandoned, which is a continuation of US.
`Ser. No. 08/248,037, filed May 24, 1994, now abandoned.
`
`BACKGROUND OF THE INVENTION
`
`Copolymer-1 is a synthetic polypeptide analog of myelin
`basic protein (MBP), which is a natural component of the
`myelin sheath. It has been suggested as a potential thera-
`peutic agent for multiple sclerosis (Eur. J. Immunol. [1971]
`1:242; and J. Neurol. Sci. [1977] 312433). All references
`cited herein are hereby incorporated by reference in their
`entirety. Interest in copolymer-1 as an immunotherapy for
`multiple sclerosis stems from observations first made in the
`1950’s that myelin components such as MBP prevent or
`arrest experimental autoinimune encephalomyelitis (EAE).
`EAE is a disease resembling multiple sclerosis that can be
`induced in susceptible animals.
`Copolymer-1 was developed by Drs. Sela, Arnon, and
`their co-workers at the Weizmann Institute (Rehovot, Israel).
`It was shown to suppress EAE (Eur. J. Immunol. [1971]
`1:242; US. Pat. No. 3,849,550). More recently, copolymer-1
`was shown to be beneficial
`for patients with the
`exacerbating-remitting form of multiple sclerosis (N. Engl.
`J. Med. [1987] 317:408). Patients treated with daily injec-
`tions of copolymer-1 had fewer exacerbations and smaller
`increases in their disability status than the control patients.
`Copolymer-1 is a mixture of polypeptides composed of
`alanine, glutamic acid, lysine, and tyrosine in a molar ratio
`of approximately 6:2:521, respectively. It is synthesized by
`chemically polymerizing the four amino acids forming prod-
`ucts with average molecular weights of 23,000 daltons (US.
`Pat. No. 3,849,550).
`It is an object of the present invention to provide an
`improved composition of copolymer-1.
`
`SUMMARY OF THE INVENTION
`
`invention relates to a composition of
`The present
`copolymer-1 substantially free of species of copolymer-1
`having a molecular weight of over 40 kilodaltons (KDa).
`The invention further relates to a copolymer-1 having
`over 75% of its molar fraction within the molecular weight
`range from about 2 KDa to about 20 KDa.
`In addition, the invention relates to a copolymer-1 having
`an average molecular weight of about 4 to about 8.6 KDa.
`Moreover, the invention relates to a pharmaceutical com-
`position and a method for the treatment of multiple sclerosis,
`using the above-discussed copolymer-1.
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 displays the molecular weight distribution of three
`batches of copolymer-1, showing the proportion of species
`with molecular weight above 40 KDa. FIG. 2 shows similar
`data relating to the molar fraction.
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`invention relates to a composition of
`The present
`copolymer-1 substantially free of species of copolymer-1
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`2
`
`having a molecular weight of over 40 kilodaltons (KDa).
`Preferably, the composition contains less than 5% of species
`of copolymer-1 having a molecular weight of 40 KDa or
`more. More preferably, the composition contains less than
`2.5% of species of copolymer-1 having a molecular weight
`of 40 KDa, or more.
`The invention further relates to a copolymer-1 having
`over 75% of its molar fraction within the molecular weight
`range from about 2 KDa to about 20 KDa.
`In addition, the invention relates to a copolymer-1 having
`an average molecular weight of about 4 to about 8.6 KDa.
`In particular, the invention relates to a copolymer-1 having
`an average molecular weight of about 4 to about 8 KDa and
`a copolymer-1 having an average molecular weight of about
`6.25 to about 8.4 KDa.
`
`Copolymer-1, according to the present invention, may be
`prepared by methods known in the art, for example, the
`process disclosed in US. Pat. No. 3,849,550, wherein the
`N-carboxyanhydrides of tyrosine, alanine, y-benzyl
`glutamate and E-N-trifiuoro-acetyllysine are polymerised at
`ambient
`temperature in anhydrous dioxane with diethy-
`lamine as initiator. The deblocking of the y-carboxyl group
`of the glutamic acid is effected by hydrogen bromide in
`glacial acetic acid and is followed by the removal of the
`trifluoroacetyl groups from the lysine residues by 1M pip-
`eridine. For the purposes of the application,
`the terms
`“ambient temperature” and “room temperature” should be
`understood to mean a temperature ranging from about 20 to
`about 26° C.
`
`The copolymer-1 with the required molecular weight
`profile can be obtained either by methods known per se.
`Such methods include chromatography of copolymer-1 con-
`taining high molecular weight species and collecting the
`fractions without the undesired species or by partial acid or
`enzymatic hydrolysis to remove the high molecular weight
`species with subsequent purification by dialysis or ultrafil-
`tration. A further method to obtain copolymer-1 with the
`desired molecular weight profile is by preparing the desired
`species while the amino acids are still protected and then
`obtain the correct species directly upon removing the pro-
`tection. The compositions of the present invention may be
`formulated by conventional methods known in the art.
`Preferably, the composition is lyophilized and formed into
`an aqueous solution suitable for sub-cutaneous injection.
`Alternatively, copolymer-1 may be formulated in any of the
`forms known in the art for preparing oral, nasal, buccal, or
`rectal formulations of peptide drugs.
`Typically, copolymer-1 is administered daily to patients
`suffering from multiple sclerosis at a dosage of 20 mg.
`The invention will be exemplified but not necessarily
`limited by the following Examples.
`
`EXAMPLE 1
`
`Chromatographic Method of Preparation of Low-
`toxicity Cooolymer-1
`
`Two batches of copolymer-1 were prepared according to
`the methods known in the art, for example, US. Pat. No.
`3,849,550.
`One batch was then subjected to chromatographic
`separation, as described below.
`A column for gel filtration, FRACTOGEL TSK HW55
`(600x26 mm) was prepared in a Superformance 26 Merck
`cartridge according to the manufacturer’s instructions. The
`column was equilibrated with water and acetone solution
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`US 6,342,476 B1
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`3
`was injected for total volume determination. The column
`was equilibrated with 0.2M ammonium acetate buffer pH
`5.0. 30 ml copolymer-1 samples (20 mg/ml, in 0.2M ammo-
`nium acetate pH 5.0) were loaded on the column and
`fractions were collected every 10 minutes. Afraction having
`an average molecular weight of 7—8 KDa was isolated
`between 120—130 minutes (Batch A).
`
`Molecular Weight Analysis
`UV absorbance at 275 nm was determined in a UVIKON
`
`10
`
`810 spectrophotometer. Samples were diluted to obtain a UV
`absorbance lower than 1 Absorption Unit. The molecular
`distribution of the 2 batches was determined on a calibrated
`
`gel filtration column (Superose 12).
`Copolymer-1 batch A was found to have an average
`molecular weight of 7—8 KDa. 2.5% of this batch had a
`molecular weight above 32 KDa, but no copolymer-1 spe-
`cies present in this batch had a molecular weight of over 40
`KDa.
`
`The other batch of copolymer-1 which was not subjected
`to chromatography, had an average molecular weight of 12
`KDa. 2.56 of the batch had a molecular weight above 42
`KDa and 5% of the total copolymer-1 species in this batch
`had a molecular weight of over 40 KDa.
`
`EXAMPLE 2
`
`Toxicity Analysis
`
`A: In Vivo
`
`Three batches of copolymer-1 having an average molecu-
`lar weight of 7.3 and 8.4 KDa (less than 2.5% copolymer-1
`species over 40 KDa) and 22 KDa (more than 5%
`copolymer-1 species over 40 KDa) were subjected to the
`toxicity test described below. In each case 5 mice were used
`in each experimental group.
`
`Method
`
`Copolymer-1 was dissolved in distilled water to yield a
`solution of 2 mg/ml of the active ingredient. Each mouse
`was injected with 0.5 ml of the test solution into the lateral
`tail vein. Mice were observed for mortality and relevant
`clinical signs over a 48 hour period. Observations were
`recorded 10 minutes, 24 hours and 48 hours post-injection.
`If, at the end of 48 hours, all the animals were alive and no
`adverse signs had been observed, then the batch was des-
`ignated “non-toxic”. If, however, one or more of the mice
`had died or had shown adverse signs, then the batch was
`designated “toxic”.
`The batches with the average molecular weight of 7.3 and
`8.4 KDa were both designated “non-toxic”, whereas in the
`batch with the average molecular weight of 22 KDa, 3 out
`of 5 mice had died at the end of 48 hours, and it was
`consequently designated “toxic”.
`B: In Vitro
`
`RBL—Degranulation test
`I. Introduction
`
`Histamine (or serotonin) release from basophile is an in
`vitro model for immediate hypersensitivity. The Rat Baso-
`philic Leukemia cell line (RBL-2H3) was developed and
`characterized as a highly sensitive, uniform, easy to main-
`tain in culture and reproducible system (E. L. Basumian, C.
`Isersky, M. G. Petrino and R. P. Siraganian. Eur. J. Immunol.
`11, 317 (1981)). The physiological stimulus for histamine
`release involves binding of the antigen to membrane-bound
`IgE molecules, resulting in the latter’s cross-linking and the
`consequent triggering of an intricate biochemical cascade.
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`4
`immunoglobulin-mediated
`Beside these physiological,
`triggers, degranulation can be induced by different non-IgE-
`mediated stimuli. Among these are various peptides and
`synthetic polymers, e.g. polylysine (R. P. Siraganian. Trends
`in Pharmacological Sciences, October 432 (1983)). The
`RBL degranulation test is, therefore, used in order to screen
`out those batches of copolymer-1 which evoke substantial
`degranulation and thus might elicit undesirable local and/or
`svstemtc side effects.
`
`II. Principle of the test method
`Rat Basophilic Leukemia cells (RBL-2H3), are loaded
`with [3H] -serotonin, followed by incubation with 100 pg of
`the copolymer-1 to be tested. Batches of copolymer-1 which
`induce non-specific degranulation, release [3H] -serotonin
`into the medium. The radioactivity in the medium is counted
`by a scintillation counter and the total radiolabeled serotonin
`incorporated into the cells is determined in the pelleted cells.
`Percent degranulation is calculated as the percentage of
`serotonin released out of the total incorporated.
`III. Results
`
`Four batches of copolymer-1, with average molecular
`weight between 6,250—14,500 were analyzed for both % of
`the species with molecular weight over 40 KDa and for
`degranulation of RBL’s. Results are summarized in the
`following table.
`
`Average
`M.W. (Daltons)
`
`% of species with
`M.W. over 4OKDa
`
`% Serotonin
`Release
`
`6,250
`7,300
`13,000
`14,500
`
`<2.5
`<2.5
`>5
`>5
`
`12.4
`21.0
`66.9
`67.8
`
`As can be seen, when the % of high molecular weight
`species is low (<2.5), the % release of serotonin, indicative
`of toxicity, is low, and vice versa.
`
`EXAMPLE 3
`
`Preparation of Trifluoroacetyl-Copolyrmer-1
`
`Protected copolymer-1 is prepared as described by Teitel-
`baum et al. Eur. J. Immun. Vol. 1 p. 242 (1971) from the
`N—carboxyanhydrides of tyrosine (18 g), alanine (50 g),
`y-benizyl glutamate (35 g) and trifluoroacetyllysine (83 g)
`dissolved in 3.5 liters of dioxane.
`
`The polymerization process is initiated by the addition of
`0.01—0.02% diethylamine. The reaction mixture is stirred at
`room temperature for 24 hours and then poured into 10 liters
`water. The product (protected copolymer-1)
`is filtered,
`washed with water and dried. The removal of the gamma-
`benzyl blocking groups from the glutamate residue is carried
`out by treating the protected copolymer-1 with 33% hydro-
`bromic acid in glacial acetic acid at room temperature for
`6—12 hours with stirring. The product is poured into excess
`water,
`filtered, washed and dried, yielding the
`trifluoro acetyl-copolymer-1.
`EXAMPLE 4
`
`Preoaration of Trifiuoroacetyl-Copolymer-1
`
`Protected copolymer-1 is prepared as described by Teitel-
`baum et al. Eur. J. Immun. Vol. 1 p. 242 (1971) from the
`N—carboxyanhydrides of tyrosine (18 g), alanine (50 g),
`t-benzyl glutamate (35 g) and trifiuoroacetyllysine (83 g)
`dissolved in 3.5 liters of dioxane.
`
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`US 6,342,476 B1
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`5
`The polymerization process is initiated by the addition of
`0.01—0.02% diethylamine. The reaction mixture is stirred at
`room temperature for 24 hours and then poured into 10 liters
`water. The product (protected copolymer-1)
`is filtered,
`washed with water and dried. Protected copolymer-1 is
`treated with 33% HBr in acetic acid which removes the
`omega benzyl protecting group from the 5-carboxylate of
`the glutamate residue and cleaves the polymer to smaller
`polypeptides. The time needed for obtaining copolymer-1 of
`molecular weight 7,000:2,000 Da depends on the reaction
`temperature and the size of protected copolymer-1. At
`temperatures of between 20—28° C. a test reaction is per-
`formed on every batch at different time periods for example,
`from 10—50 hours.
`
`The results concerning the molecular weights of these
`small scale reactions are calculated and a curve of molecular
`
`weight against time is drawn. The time needed for obtaining
`molecular weight 7,000:2,000 Da is calculated from the
`curve and performed on larger scale reaction. On average,
`working at 26° C. the time period is 17 hours. The product
`is poured into excess water, filtered, washed and dried,
`yielding the trifiuoroacetylcopolymer-1.
`
`Preparation of Low-toxicity Copolymer-1
`
`20 g of trifluoroacetyl-copolymer-1 are dispersed in 1 liter
`of water to which 100 g piperidine are added. The mixture
`is stirred for 24 hours at room temperature and filtered. The
`solution of crude copolymer-1 is distributed into dialysis
`bags and dialyzed at 10°—20° C. against water until a pH=8
`
`10
`
`15
`
`20
`
`25
`
`6
`is attained. It is then dialyzed against about 0.3% acetic acid
`and again water until a pH=5.5—6.0 is obtained. This solu-
`tion is then concentrated and lyophilized to dryness.
`What is claimed is:
`
`1. A method for treating multiple sclerosis, comprising
`administering to a subject in need thereof, a pharmaceuti-
`cally effective amount of a copolymer-1 fraction wherein
`said fraction contains less than 5% of species of
`copolymer-1 having a molecular weight of over 40 kilodal-
`tons; and wherein over 75% of said copolymer-1 in said
`fraction is within a molecular weight range of about 2
`kilodaltons to about 20 kilodaltons, wherein said
`copolymer-1 fraction is prepared by a process comprising
`the steps of:
`
`reacting protected copolymer-1 with hydrobromic acid to
`form trifiuoroacetyl copolymer-1 having over 75% of
`its molar fraction within the molecular weight range
`from about 2 kDa to about 20 kDa, wherein said
`reaction takes place for a time and at a temperature
`predetermined by test reaction, and
`treating said trifiuoroacetyl copolymer-1 having over 75 %
`of its molar fraction within the molecular weight range
`from about 2 kDa to about 20 kDa with aqueous
`piperidine solution to form copolymer-1 having over
`75% of its molar fraction within the molecular weight
`range from about 2 kDa to about 20 kDa.
`*
`*
`*
`*
`*
`
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