AU2004202245B2 - Copolymer-1 Improvements in Compositions of Copolymers - Google Patents
Copolymer-1 Improvements in Compositions of Copolymers Download PDFInfo
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- AU2004202245B2 AU2004202245B2 AU2004202245A AU2004202245A AU2004202245B2 AU 2004202245 B2 AU2004202245 B2 AU 2004202245B2 AU 2004202245 A AU2004202245 A AU 2004202245A AU 2004202245 A AU2004202245 A AU 2004202245A AU 2004202245 B2 AU2004202245 B2 AU 2004202245B2
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- 102000004196 processed proteins & peptides Human genes 0.000 claims description 26
- 229920001184 polypeptide Polymers 0.000 claims description 25
- 201000006417 multiple sclerosis Diseases 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 15
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 10
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 10
- 235000004279 alanine Nutrition 0.000 claims description 10
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 10
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 8
- 235000013922 glutamic acid Nutrition 0.000 claims description 8
- 239000004220 glutamic acid Substances 0.000 claims description 8
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 7
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 7
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 7
- 239000004472 Lysine Substances 0.000 claims description 7
- 235000018977 lysine Nutrition 0.000 claims description 7
- 235000002374 tyrosine Nutrition 0.000 claims description 7
- 239000008194 pharmaceutical composition Substances 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 241000894007 species Species 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 10
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 10
- 229940076279 serotonin Drugs 0.000 description 7
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 4
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical compound Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 4
- 229960000583 acetic acid Drugs 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 description 3
- 201000002491 encephalomyelitis Diseases 0.000 description 3
- 239000013628 high molecular weight specie Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- KNCHTBNNSQSLRV-YFKPBYRVSA-N (2s)-6-amino-2-[(2,2,2-trifluoroacetyl)amino]hexanoic acid Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)C(F)(F)F KNCHTBNNSQSLRV-YFKPBYRVSA-N 0.000 description 2
- KISWVXRQTGLFGD-UHFFFAOYSA-N 2-[[2-[[6-amino-2-[[2-[[2-[[5-amino-2-[[2-[[1-[2-[[6-amino-2-[(2,5-diamino-5-oxopentanoyl)amino]hexanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]-5-(diaminomethylideneamino)p Chemical compound C1CCN(C(=O)C(CCCN=C(N)N)NC(=O)C(CCCCN)NC(=O)C(N)CCC(N)=O)C1C(=O)NC(CO)C(=O)NC(CCC(N)=O)C(=O)NC(CCCN=C(N)N)C(=O)NC(CO)C(=O)NC(CCCCN)C(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=C(O)C=C1 KISWVXRQTGLFGD-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 208000016557 Acute basophilic leukemia Diseases 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 2
- 239000005695 Ammonium acetate Substances 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 229940043376 ammonium acetate Drugs 0.000 description 2
- 235000019257 ammonium acetate Nutrition 0.000 description 2
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- 238000002523 gelfiltration Methods 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-L glutamate group Chemical group N[C@@H](CCC(=O)[O-])C(=O)[O-] WHUUTDBJXJRKMK-VKHMYHEASA-L 0.000 description 2
- 229960001340 histamine Drugs 0.000 description 2
- 229910000042 hydrogen bromide Inorganic materials 0.000 description 2
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- 231100000053 low toxicity Toxicity 0.000 description 2
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- 238000006116 polymerization reaction Methods 0.000 description 2
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- 230000001988 toxicity Effects 0.000 description 2
- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 description 1
- 208000032116 Autoimmune Experimental Encephalomyelitis Diseases 0.000 description 1
- 208000001718 Immediate Hypersensitivity Diseases 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 102000047918 Myelin Basic Human genes 0.000 description 1
- 102000006386 Myelin Proteins Human genes 0.000 description 1
- 108010083674 Myelin Proteins Proteins 0.000 description 1
- 101710107068 Myelin basic protein Proteins 0.000 description 1
- 108010039918 Polylysine Proteins 0.000 description 1
- 206010045240 Type I hypersensitivity Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 description 1
- VREFGVBLTWBCJP-UHFFFAOYSA-N alprazolam Chemical compound C12=CC(Cl)=CC=C2N2C(C)=NN=C2CN=C1C1=CC=CC=C1 VREFGVBLTWBCJP-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
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- 208000010216 atopic IgE responsiveness Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
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- 238000007796 conventional method Methods 0.000 description 1
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- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 230000005713 exacerbation Effects 0.000 description 1
- 208000012997 experimental autoimmune encephalomyelitis Diseases 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
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- 239000003999 initiator Substances 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000013365 molecular weight analysis method Methods 0.000 description 1
- 210000005012 myelin Anatomy 0.000 description 1
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- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229940124606 potential therapeutic agent Drugs 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
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- 238000010512 small scale reaction Methods 0.000 description 1
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- 238000010254 subcutaneous injection Methods 0.000 description 1
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- 229920001059 synthetic polymer Polymers 0.000 description 1
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- 239000012085 test solution Substances 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 125000004044 trifluoroacetyl group Chemical group FC(C(=O)*)(F)F 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
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Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
S&F Ref: 359455D3
AUSTRALIA
PATENTS ACT 1990 COMPLETE
SPECIFICATION
FOR A STANDARD
PATENT
Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: Yeda Research and Development Co. Ltd, of P.O Box 76100, Rehovot, Israel Eliezer Konfino Ruth Arnon Dvora Teitelbaum Michael Sela Spruson Ferguson St Martins Tower Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Copolymer-1 Improvements in Compositions of Copolymers The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c COPOLYMER-1 IMPROVEMENTS IN COMPOSITIONS OF COPOLYMERS 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 therapeutic 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 1950's that myelin components such as MBP prevent or arrest experimental autoimmune 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; U.S. Patent 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 injections of copolymer-I 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
I
2 ratio of approximately 6:2:5:1, respectively. It is synthesised by chemically polymerising the four amino acids forming products with average molecular weights of 23,000 daltons Patent No. 3,849,550).
It is an object of the present invention to provide an improved composition of copolymer-1.
Summary of the Invention The present invention relates to a composition of copolymer-I 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 composition and a method for the treatment of multiple sclerosis, using the above-discussed copolymer-1.
According to a first embodiment of the invention, there is provided a composition comprising a mixture of polypeptides composed of glutamic acid, lysine, alanine and tyrosine, wherein the mixture has an average molecular weight of about 4 to about 9 kilodaltons, and wherein the mixture of polypeptides is non-uniform with respect to molecular weight and constitution.
According to a second embodiment of the invention, there is provided a pharmaceutical composition comprising a dose therapeutically effective to treat multiple sclerosis of the composition in accordance with the first embodiment of the present invention.
According to a third embodiment of the invention, there is provided a method for treating a patient suffering from multiple sclerosis comprising administering to a patient in need thereof the composition in accordance with the second embodiment of the present invention.
According to a fourth embodiment of the invention, there is provided a composition comprising a mixture of polypeptides composed of glutamic acid, lysine, alanine and tyrosine, wherein the mixture has an average molecular weight of 4 to 9 kilodaltons, and [R:\PAL Speciflcations\W59455]77527spec.doc:gcc wherein the mixture of polypeptides is non-uniform with respect to molecular weight and constitution.
According to a fifth embodiment of the invention, there is provided a pharmaceutical composition comprising a dose therapeutically effective to treat multiple sclerosis of the composition in accordance with the fourth embodiment of the present invention.
According to a sixth embodiment of the invention, there is provided a method for treating a patient suffering from multiple sclerosis comprising administering to a patient in need thereof the composition in accordance with the fifth embodiment of the present 0o invention.
Brief Description of the Drawings Figure 1 displays the molecular weight distribution of three batches of copolymer-1, showing the proportion of species with molecular weight above 40 KDa. Figure 2 shows similar data relating to the molar fraction.
Detailed Description of the Invention The present invention relates to a composition of copolymer-1 substantially free of species of copolymer-1 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 [R:\PA L Specifications\35945 577527spec.doc:gcc 3 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-l, according to the present invention, may be prepared by methods known in the art, for example, the process disclosed in U.S. Patent 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 diethylamine as initiator. The deblocking of the ycarboxyl 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 piperidine. 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 containing 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 ultrafiltration. 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 protection. 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 The invention will be exemplified but not necessarily limited by the following Examples.
EXAMPLE 1 ChromatocraDhic method of oreDaration of low-toxicity coDolvmer-l Two batches of copolymer-1 were prepared according to the methods known in the art, for example, U.S. Patent No. 3,849,550.
One batch was then subjected to chromatographic separation, as described below.
A column for gel filtration, FRACTOGEL TSK HW55 (600 x 26mm) was prepared in a Superformance 26 Merck cartridge according to the manufacturer's instructions. The column was equilibrated with water and acetone solution was injected for total volume determination. The column was equilibrated with 0.2M ammonium acetate buffer pH 30 ml copolymer-1 samples (20mg/ml, in 0.2M ammonium 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 KDa was isolated between 120-130 minutes (Batch A).
Molecular Weight Analysis UV absorbance at 275 nm was determined in a UVIKON 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 avera:e molecular weight of 7-8 KDa. 2.5% of this batch had a molecular weight above 32 KDa, but no copolymer-1 species present in this batch had a molecular weight of over KDa.
The other batch of copolymer-1 which was not subjected to chromatography, had an average molecular weight of 12 KDa. 2.5% of the batch had a molecular weight above 42KDa 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 molecular weight of 7.3 and 8.4 KDa (less than 2.5% copolymer-1 species over 40KDa) and 22KDa (more than 5% copolymer-1 species over 40KDa) 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 2mg/ml of the active ingredient. Each mouse was injected with 0.5ml 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 designated "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 22KDa, 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 Basophilic Leukemia cell line (RBL-2H 3 was developed and characterized as a highly sensitive, uniform, easy to maintain 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. Beside these physiological, immunoglobulin-mediated triggers, degranulation can be induced by different non-IgEmediated stimuli. Among these are various peptides and synthetic polymers, e.g. polylysine 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 systemic side effects.
II. Principle of the test method Rat Basophilic Leukemia cells (RBL-2H3 are loaded with 3 H]-serotonin, followed by incubation with 100 pg of the copolymer-1 to be tested. Batches of copolymer-1 which induce non-specific degranulation, release 3 H]-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-l, with average molecular weight between 6,250-14,500 were analyzed for both of the species with molecular weight over 40KDa and for degranulation of RBL's. Results are summarized in the following table.
Average of species with Serotonin M.W. (Daltons) M.W. over 40KDa Release 6,250 2.5 12.4 7,300 2.5 21.0 13,000 5 66.9 14,500 5 67.8 As can be seen, when the of high molecular weight species is low the release of serotonin, indicative of toxicity, is low, and vice versa.
EXAMPLE 3 Preparation of Trifluoroacetvl-Copolvmer-1 Protected copolymer-1 is prepared as described by Teitelbaum et al. Eur. J. Immun. Vol. 1 p. 242 (1971) from the N-carboxyanhydrides of tyrosine (18g), alanine (50g), y-benzyl glutamate (35g) and trifluoroacetyllysine (83g) 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% hydrobromic 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 Preparation of Trifluoroacetvl-CoDolymer-1 Protected copolymer-1 is prepared as described by Teitelbaum et al. Eur. J. Immun. Vol. 1 p. 242 (1971) from the N-carboxyanhydrides of tyrosine (18g), alanine (50g), T-benzyl glutamate (35g) and trifluoroacetyllysine (83g) 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.
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 28 0 C a test reaction is performed 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 0 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 copolvmer-1 of trifluoroacetyl-copolymer-1 are dispersed in 1 liter of water to which 100g 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 100-20°C against water until a pH 8 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 solution is then concentrated and lyophilized to dryness.
Claims (27)
1. A composition comprising a mixture of polypeptides composed of glutamic acid, lysine, alanine and tyrosine, wherein the mixture has an average molecular weight of about 4 to about 9 kilodaltons, and wherein the mixture of polypeptides is non-uniform with respect to molecular weight and constitution.
2. The composition of claim 1, wherein over 75% of the polypeptides of the mixture, on a molar fraction basis, have a molecular weight in a range of about 2 kilodaltons to about 20 kilodaltons.
3. The composition of claim 1, wherein less than 5% of the polypeptides of the 0o mixture have a molecular weight of over 40 kilodaltons.
4. The composition of claim 3, wherein over 75% of the polypeptides of the mixture, on a molar fraction basis, have a molecular weight in a range of about 2 kilodaltons to about 20 kilodaltons. The composition of claim 4, wherein the mixture has an average molecular weight of 6.25 to 8.4 kilodaltons.
6. The composition of claim 1, wherein the mixture has an average molecular weight of about 4 to 8.6 kilodaltons.
7. The composition of claim 1, wherein the mixture has an average molecular weight of about 5 to about 9 kilodaltons.
8. The composition of claim 1, wherein less than 2.5% of the polypeptides of the mixture have a molecular weight of over 40 kilodaltons.
9. The composition of claim 8, wherein over 75% of the polypeptides of the mixture, on a molar fraction basis, have a molecular weight in a range of about 2 kilodaltons to about 20 kilodaltons.
10. The composition of claim 9, wherein the mixture has an average molecular weight of 6.25 to 8.4 kilodaltons.
11. The composition of claim 1, wherein the mixture has a molecular weight distribution substantially as depicted in the curves of Figure 1 or Figure 2 in which the average molecular weight is about 7.7 kDa.
12. A pharmaceutical composition comprising a dose therapeutically effective to treat multiple sclerosis of the composition of any one of claims 1-11.
13. A method for treating a patient suffering from multiple sclerosis comprising administering to a patient in need thereof the composition of claim 12. [R:\IIBF 1 2272spec.doc:gcc
14. A composition comprising a mixture of polypeptides composed of glutamic acid, lysine, alanine and tyrosine, wherein the mixture has an average molecular weight of about 4 to about 9 kilodaltons, and wherein the mixture of polypeptides is non-uniform with respect to molecular weight and constitution, substantially as hereinbefore described with reference to any one of the examples. A composition comprising a mixture of polypeptides composed of glutamic acid, lysine, alanine and tyrosine, wherein the mixture has an average molecular weight of 4 to 9 kilodaltons, and wherein the mixture of polypeptides is non-uniform with respect to molecular weight and constitution.
16. The composition of claim 15, wherein over 75% of the polypeptides of the mixture, on a molar fraction basis, have a molecular weight in a range of 2 kilodaltons to kilodaltons.
17. The composition of claim 15, wherein less than 5% of the polypeptides of the mixture have a molecular weight of over 40 kilodaltons.
18. The composition of claim 17, wherein over 75% of the polypeptides of the mixture, on a molar fraction basis, have a molecular weight in a range of 2 kilodaltons to kilodaltons.
19. The composition of claim 18, wherein the mixture has an average molecular weight of 6.25 to 8.4 kilodaltons.
20. The composition of claim 15, wherein the mixture has an average molecular weight of 4 to 8.6 kilodaltons.
21. The composition of claim 15, wherein the mixture has an average molecular weight of 5 to 9 kilodaltons.
22. The composition of claim 15, wherein less than 2.5% of the polypeptides of the mixture have a molecular weight of over 40 kilodaltons.
23. The composition of claim 22, wherein over 75% of the polypeptides of the mixture, on a molar fraction basis, have a molecular weight in a range of 2 kilodaltons to kilodaltons.
24. The composition of claim 23, wherein the mixture has an average molecular weight of 6.25 to 8.4 kilodaltons. The composition of claim 15, wherein the mixture has a molecular weight distribution substantially as depicted in the curves of Figure 1 or Figure 2 in which the average molecular weight is 7.7 kDa. I R:\PA L Speci fications\359455]77527spec.doc:gcc
26. A pharmaceutical composition comprising a dose therapeutically effective to treat multiple sclerosis of the composition of any one of claims 15-25.
27. A method for treating a patient suffering from multiple sclerosis comprising administering to a patient in need thereof the composition of claim 26.
28. A composition comprising a mixture of polypeptides composed of glutamic acid, lysine, alanine and tyrosine, wherein the mixture has an average molecular weight of 4 to 9 kilodaltons, and wherein the mixture of polypeptides is non-uniform with respect to molecular weight and constitution, substantially as hereinbefore described with reference to any one of the examples.
29. A pharmaceutical composition comprising a dose therapeutically effective to treat multiple sclerosis of the composition of claim 14 and 28. A method for treating a patient suffering from multiple sclerosis comprising administering to a patient in need thereof the composition of claim 29.
31. Use of the composition of any one of claims 1-12 or 14 and 15-26 or 28, for the manufacture of a medicament for the treatment of multiple sclerosis. Dated 30 May, 2006 Yeda Research and Development Co. Ltd Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [R:\PA L Spec fications\359455]77527spec.doc:gcc
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2004202245A AU2004202245B2 (en) | 1994-05-24 | 2004-05-25 | Copolymer-1 Improvements in Compositions of Copolymers |
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US248037 | 1994-05-24 | ||
| US344248 | 1994-11-23 | ||
| AU10161/02A AU775214B2 (en) | 1994-05-24 | 2002-01-14 | Copolymer-1 improvements in compositions of copolymers |
| AU2004202245A AU2004202245B2 (en) | 1994-05-24 | 2004-05-25 | Copolymer-1 Improvements in Compositions of Copolymers |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU10161/02A Division AU775214B2 (en) | 1994-05-24 | 2002-01-14 | Copolymer-1 improvements in compositions of copolymers |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU2004202245A1 AU2004202245A1 (en) | 2004-06-17 |
| AU2004202245B2 true AU2004202245B2 (en) | 2006-06-15 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2004202245A Expired AU2004202245B2 (en) | 1994-05-24 | 2004-05-25 | Copolymer-1 Improvements in Compositions of Copolymers |
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| Country | Link |
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| AU (1) | AU2004202245B2 (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0383620A2 (en) * | 1989-02-17 | 1990-08-22 | Repligen Corporation | Process for making genes encoding random polymers of amino acids |
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2004
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0383620A2 (en) * | 1989-02-17 | 1990-08-22 | Repligen Corporation | Process for making genes encoding random polymers of amino acids |
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| AU2004202245A1 (en) | 2004-06-17 |
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