`Konfino et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 6,342,476 B1
`Jan. 29, 2002
`
`US006342476B1
`
`(54) COPOLYMER-1 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 U.S. 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) U.S. CI.
`
`........................... .. 514/2; 514/12; 514/903;
`424/7837
`
`(58) Field of Search ............................ .. 514/2, 12, 903;
`424fl8.37
`
`(56)
`
`References Cited
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`
`3,849,550 A " ll/1974 Teitlebaum et al. ......... .. 424/78
`4,594,409 A
`6/1986 I-Iayashii elal.
`5E/420
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`
`DE
`EP
`EP
`EP
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`
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`0 383 620
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`
`"‘
`
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`9/1985
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`
`OTHER PUBLICATIONS
`
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`
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`
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`
`D. Teitelbaum et al., “Dose—response Studies on Experi-
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`C. Webb et al., “Suppression of Experimental Allergic
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`R. Amon et al., “Suppression of EAE in Baboons by a
`Synthetic Polymer of Amino Acids", Neurology, 1978, 28,
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`E.CAlvord 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 Eflect of COP—1, a Synthetic
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`42, pp. 267-274.
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`
`(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-1 comprising Copolymer-1 substantially free of
`species having a molecular weight of over 40 kilodaltons.
`
`1 Claim, 2 Drawing Sheets
`
`AMNEAL
`
`EXHIBIT NO. 1024 Page 1
`
` AMNEAL
`
`
`
`US 6,342,476 B1
`Page 2
`
`OTHER PUBLICATIONS
`
`Z. l..ando et al., “Experimental Allergic Encephalomyelitis
`in Mice—Suppression and Prevention with COP—1”, 1sraelJ.
`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. Amon, “A Synthetic Copolymer of Amino Acids in a
`Clinical Trail for MS Therapy" in Progress in Multiple
`Sclerosis Research, Bauer, Ritter, eds. Springer Verlag N.Y.,
`1980 pp. 416-418.
`M. B. Bomstein et al., “Treatment of Multiple Sclerosis with
`a Synthetic Polypeptide: Preliminary results”, Trans. Am.
`Neurol. Assoc., 1980, 105, pp. 348-350.
`M. B. Bomstein 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-
`ral. Sci., 1980, 46, pp. 137-143.
`R. Amon, “Experimental Allergic EncephalomyelitisnSus-
`ceptibility and Suppression”, Immunological Rev., 1981, 55,
`pp. 5-30.
`M. B. Bomstein et al., “Multiple Sclerosis: Trial of a
`Synthetic Polypeptide”,Ann. Neunal, 1982, 11, pp. 317-319.
`C. G. Brosnan et al., “The Response of Normal Human
`Lymphocytes to Copolymer—1.”J. Neurapath. Exp. Neural.,
`1983, 42, pp. 356 (abstract).
`R. P. Lisak et al., “Effect of Treatment with Copolymer 1
`(COP—1) on the in VlV0 and in Vitro Manifestations of
`Experimental Allergic Encephalomyelitis (EAE)", J. Neu-
`ral. Sci., 1983, 62, 281-293.
`M. B. Bomstein et al., “Clinical Trials of Copolymer 1 in
`Multiple Sclerosis”, Ann. N.l’. Acad. Sci. (USA), 1984, pp.
`366-372.
`
`M. B. Bomstein 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: Efiect 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., “lmmunogenic potentials of copolymer
`1
`in normal
`lymphocytes” Neurology, 1985, 35, pp.
`1754-1759.
`
`M. B. Bomstein 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. Bomstein, “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. Amon et al., “Suppression of Demyelinating Diseases by
`Synthetic Copolymers”, from: A multidisciplinary approach
`to myelin disease G. Serlupi Crescenzi, ed. Plenum Publish-
`ing Corporation, 1988, pp. 243-250.
`M. B. Bomstein 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 lmmune Response to
`Copolymer 1 in Multiple Sclerosis Measured by the Mac-
`rophage Procoagulant Activity Assay”, Int. lmmunol, 1990,
`2, pp. 714-718.
`M. B. Bomstein 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. (Pohmd),
`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. Bomstein 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.
`D. Teitelbaum et al., “Synthetic Copolymer 1 Inhibits
`Human T—Ce1l 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. Amon et al., “On the Existence of Suppressor Cells”, Int.
`Arch. Allergy Immunol., 1993, 100, pp. 2-7.
`M. Sela, “Polymeric Drugs as lmmunomodulatory Vaccines
`Against Multiple Sclerosis”, Makromol. Chem. Macromol.
`Symp., 1993, 70/71, pp. 147-155.
`
`AMNEAL
`
`EXHIBIT NO. 1024 Page2
`
` AMNEAL
`
`
`
`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
`fol1owup.”, 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 [BC 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 Histocompatfloility 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 Histooompatibility Complex
`Molecules On Living Antigen Presenting Cells”, J. Neuro-
`chem, 63, Suppl. 1, S61, 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”,
`Assafliamfelr Medical Center, Sackler Sdiool ofMedicine,
`Tel—Aviv University, University of Marland School of Medi-
`cine, p. 22 (1994).
`R. Milo et al., “Additive Elfects 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”,
`JAMA 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 Dilficulties 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 M.D. et al., “Access to Investigational
`Dmgs for Treatment Purposes”, American Family Physi-
`cian, Sep. 15, 1994, pp. 845-847.
`
`Shalini Bansil, M.D. 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 oopolymer 1 to class 1] major histo-
`compatibility complex molecules on living antigen—present-
`ing oelIs—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 I-listooompatibility Complex Mol-
`ecules on Living Antigen Presenting Cells”, Department of
`Chemical Immunology, The Weizmann Institute of Science,
`Rehovot, 76100, Israel (1993).
`
`Rolak, “CopoIymer—I Therapy for Multiple Sclerosis,”
`Dept. of Neurology, Baylor College of Medicine, Clinical
`Neuropharmaoology, pp. 391—396.* (1993).
`
`M. Bodanszky, “Principles of Peptide Synthesis, " Spring-
`er—Verlag, Berlin, Heidelberg, New York, Tokyo, 1984,
`118-2293’
`
`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
`
`AMNEAL
`
`EXHIBIT NO. 1024 Page3
`
` AMNEAL
`
`
`
`U.S. Patent
`
`Jan. 29, 2002
`
`Sheet 1 of 2
`
`US 6,342,476 B1
`
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`
`
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`
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`
`
`
`Em_m>>.m_:om_o_>_
`
`—.G_n_
`
`
`
`om.....--,5.5..............-EIII
`
`m9.:hoox...
`
`
`
`mmmE_m..2
`
`AMNEAL
`
`EXHIBIT NO. 1024 Page4
`
` AMNEAL
`
`
`
`
`
`U.S. Patent
`
`Jan. 29, 2002
`
`Sheet 2 of 2
`
`US 6,342,476 B1
`
`mo<mm><
`
`
`
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`
`AMNEAL
`
`EXHIBIT NO. 1024 Page5
`
` AMNEAL
`
`
`
`US 6,342,476 B1
`
`1
`COPOLYMER-l INIPROVEMENTS IN
`COMPOSITIONS OF COPOLYMERS
`
`This application is a continuation of application Ser. No.
`09/032,334, filed Feb. 27, 1998, U.S. Pat. No. 6,048,898
`which is a continuation of U.S. Ser. No. 08/447,146, filed
`May 22, 1995, now U.S. Pat. No. 5,800,808, which is a
`continuation-in-part of U.S. Ser. No. 08/344,248, filed Nov.
`23, 1994, now abandoned, which is a continuation of U.S.
`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] 31:433). 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
`l950’s that myelin components such as MBP prevent or
`arrest experimental autoinimune encephalomyelitis
`EAE is a disease resembling multiple sclerosis that can be
`induced in susceptible animals.
`Copolymer-1 was developed by Drs. Sela, Amon, and
`their co-workers at the Weizmann Institute (Rehovot, Israel).
`It was shown to suppress EAE (Eur. J. Immunol. [1971]
`1:242; U.S. 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] 3l7:408). 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 6:2:5:1, respectively. It is synthesized by
`chemically polymerizing the four amino acids forming prod-
`ucts with average molecular weights of 23,000 daltons (U.S.
`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-l
`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
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`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 U.S. Pat. No. 3,849,550, wherein the
`N-carboxyanhydrides of tyrosine, alanine, y-benzyl
`glutamate and E-N-trifluoro-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 elfected by hydrogen bromide in
`glacial acetic acid and is followed by the removal of the
`trifluoroacetyl groups fi'om 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
`suflering 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, U.S. Pat. No.
`3,849,550.
`
`One batch was then subjected to chromatographic
`separation, as described below.
`A column for gel filtration, FRACTOGEL TSK HWSS
`(600><26 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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`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. A fraction having
`an average molecular weight of 7-8 lG)a 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% oopolymer-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.
`Isetsky, M. G. Petrino and R. P. Siraganian. Eur. J. lmmunol.
`11, 317 (1981)). The physiological stimulus for histamine
`release involves binding of the antigen to membrane-bound
`lgE molecules, resulting in the latter’s cross-linking and the
`consequent triggering of an intricate biochemical cascade.
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`60
`
`65
`
`4
`
`immunoglobulin-mediated
`Beside these physiological,
`triggers, degranulation can be induced by dilferent 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 etfects.
`
`ll. 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.
`lll. 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
`
`d.5
`d.S
`>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. Imrnun. Vol. 1 p. 242 (1971) from the
`N-carboxyanhydrides of tyrosine (18 g). alanine (50 g),
`y-benizyl glutamate (35 g) and trifluoroaoetyllysine (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
`trifluoroacetyl-copolymer-1.
`
`EXAMPLE 4
`
`Preoaration of Trifluoroacetyl-Copolymer-1
`
`Protected copolymer-1 is prepared as described by Teitel-
`baum et al. Eur. J. lmmun. Vol. 1 p. 242 (1971) from the
`N-carboxyanhydrides of tyrosine (18 g), alanine (50 g),
`1:-benzyl glutamate (35 g) and trifluoroacetyllysine (83 g)
`dissolved in 3.5 liters of dioxane.
`
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`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 S-carboxylate of
`the glutamate residue and cleaves the polymer to smaller
`polypeptides. The time needed for obtaining eopolymer-1 of
`molecular weight 7,000:t: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 dilferent 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,(X)0:2,000 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 trifluoroacetylcopolymer-1.
`
`Preparation of Low-toxicity Copolymer-1
`
`20 g of trifluoroacetyl-copolymer-l 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
`
`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 elfective 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 trifluoroacetyl eopolymer-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 trifluoroacetyl 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.
`*
`It
`It
`50!
`III
`
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