AU741590B2 - Copolymer-1 improvements in compositions of copolymers - Google Patents

Description

*K

S F Ref: 359455D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

o oo Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: Yeda Research and Development Co. Ltd.

Rehovot P.O. Box 76100

ISRAEL

Ellezer Konfino, Michael Sela, Dvora Teltelbaum, Ruth Arnon Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Copolymer-1 Improvements in Compositions of Copolymers r The following statement is a full description best method of performing it known to me/us:of this invention, including the 5845 COPOL MER-1 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 :n 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 S' a disease resembling multiple sclerosis that can be 20 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) 25 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-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 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 There is herein disclosed a composition of copolymer-1 substantially free of species of copolymer-1 having a molecular weight of over 40 kilodaltons (KDa).

There is herein disclosed 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, there is disclosed a copolymer-1 having an average molecular weight of about 4 to about 8.6 KDa.

Moreover, there is disclosed to a pharmaceutical composition and a method for the treatment of multiple sclerosis, using the above-discussed copolymer-1.

i5 According to a first embodiment of the invention, there is provided a copolymer-1 having a molecular weight of about 5 to 9 kilodaltons, made by a process comprising the steps of: reacting protected copolymer-1 with hydrobromic acid to form trifluoroacetyl copolymer-1, 20 treating said trifluoroacetyl copolymer- with aqueous piperidine solution to form copolymer-1, and purifying said copolymer-1, to result in copolymer-1 having a molecular weight of about 5 to 9 kilodaltons.

According to a second embodiment of the invention, there is provided a composition for the treatment of multiple sclerosis comprising a pharmaceutically effective amount of a copolymer-1 fraction, wherein said fraction contains less than of species of copolymer-1 having a molecular weight of over 40 kilodaltons; 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 copolymer-1 having over 75% of its molar fraction within the molecular weight range Sfrom about 2kDa to about 20kDa, wherein said reaction takes place for a time and at a S"temperature predetermined by test reaction, and [I:\DayLib\LIBFF]09905spec.doc:gcc 2a treating said trifluoroacetyl copolymer-1 having over 75% of its molar fraction within the molecular weight range from about 2kDa to about 20kDa with aqueous piperidine solution to form copolymer-1 having over 75% of its molar fraction within the molecular weight range from about 2kDa to about According to a third embodiment of the invention, there is provided a method for the treatment of multiple sclerosis comprising administering a therapeutically effective amount of the copolymer-1 in accordance with the first embodiment of the present invention, or the composition in accordance with the second embodiment of the present invention.

According to a fourth embodiment of the invention, there is provided the copolymer-1 in accordance with the first embodiment of the present invention, or the composition in accordance with the second embodiment of the present invention, when used in the treatment of multiple sclerosis.

According to a fifth embodiment of the invention, there is provided the use of the 15 copolymer-1 in accordance with the first embodiment of the present invention, in the preparation of a medicament for the treatment of multiple sclerosis.

According to a sixth embodiment of the invention, there is provided a method of manufacturing copolymer-1, comprising reacting protected copolymer-1 with hydrobromic acid to form trifluoroacetyl copolymer-1, treating said trifluoroacetyl 999 20 copolymer- 1 with aqueous piperidine solution to form copolymer-1, and purifying said copolymer-1, to result in copolymer-1 having a molecular weight of about 5 to 9 kilodaltons.

According to a seventh embodiment of the invention, there is provided a copolymer-I having over 75% of its molar fraction within the molecular weight range from about 2kDa to about 20kDa, prepared by a process comprising the steps of: reacting protected copolymer-1 with hydrobromic acid to form trifluoroacetyl copolymer- having over 75% of its molar fraction within the molecular weight range from about 2kDa to about 20kDa, 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 2kDa to about 20kDa with aqueous piperidine solution to form copolymer-1 having over 75% of its molar fraction within the molecular weight range from about 2kDa to about F According to an eighth embodiment of the invention, there is provided a Strifluoroacetyl copolymer-1 having over 75% of its molar fraction within the molecular [I:\DayLib\LIBFF]09905spec.doc:gcc 2b weight range from about 2kDa to about 20kDa, produced by a process comprising the steps of reacting protected copolymer-1 with hydrobromic acid for a time and at a temperature predetermined by test reaction.

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 1 0 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 *i [I:\DAYLIB\LBFFO9905spec I.doc:ANB 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-i 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-i having an average molecular weight of about 4 to about 8.6 KDa. In particular, the invention relates to a copolymer-i having an average molecular weight of about 4 to about 8 KDa and a copolymer-l 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 25 by the removal of the trifluoroacetyl groups from the lysine residues by lM 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 OC.

The copolymer-l 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 15 suffering from multiple sclerosis at a dosage of

*T

The invention will be exemplified but not necessarily limited by the following Examples.

20 EXAMPLE 1 ChromatoQraDhic method of Drearation of low-toxicity coDolvmer-1 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-l 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-i sDecies present in this batch had a molecular weight of over 15 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-l species in this batch had a molecular weight of over 40 KDa.

S* EXAMPLE 2 Toxicity Analysis 25 A: In Vivo Three batches of copolymer-l having an average molecular weight of 7.3 and 8.4 KDa (less than 2.5% copolymer-i 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-l 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".

15 B: In Vitro .RBL Deqranulation 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 Sphysiological, 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-l 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-2H 3 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-1, with average molecular 15 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 Trifluoroacetyl-CoDolymer-l 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.

15 EXAMPLE 4 PreDaration of Trifluoroacetvl-Copolymer-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.

O..

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-l. 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 coolvmer-1 of trifluoroacetyl-copolymer-l 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 15 distributed into dialysis bags and dialyzed at 100-200o against water until a pH 8 is attained. It is then dialyzed against about 0.3% acetic acid and again water S:until a pH 5.5-6.0 is obtained. This solution is then concentrated and lyophilized to dryness.

*o

Claims (22)

1. Copolymer-1 having a molecular weight of about 5 to 9 kilodaltons, made by a process comprising the steps of: reacting protected copolymer-1 with hydrobromic acid to form trifluoroacetyl s copolymer-1, treating said trifluoroacetyl copolymer-1 with aqueous piperidine solution to form copolymer-1, and purifying said copolymer-1, to result in copolymer-1 having a molecular weight of about 5 to 9 kilodaltons. o0 2. The copolymer-1 of claim 1, wherein said copolymer-1 has an average molecular weight of about 6.25 to about 8.4 kilodaltons.

3. A composition for the treatment of multiple sclerosis comprising a pharmaceutically 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 99 s kilodaltons; 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: :i reacting protected copolymer-1 with hydrobromic acid to form trifluoroacetyl copolymer-1 having over 75% of its molar fraction within the molecular weight range 20 from about 2kDa to about 20kDa, wherein said reaction takes place for a time and at a temperature predetermined by test reaction, and 9 treating said trifluoroacetyl copolymer- having over 75% of its molar fraction within the molecular weight range from about 2kDa to about 20kDa with aqueous piperidine solution to form copolymer-1 having over 75% of its molar fraction within the molecular weight range from about 2kDa to about

4. The composition of claim 3, wherein said copolymer-1 has an average molecular weight of about 4 to about 8.6 kilodaltons. The composition of claim 4, wherein said copolymer-I has an average molecular weight of about 4 to about 8 kilodaltons.

6. The composition of claim 4, wherein said copolymer-1 in said fraction has an average molecular weight of about 6.25 to about 8.4 kilodaltons.

7. A method for the treatment of multiple sclerosis comprising administering a S therapeutically effective amount of the copolymer-1 of any one of claims 1-2, or the Somposition of any one of claims 3-7. 1:\DayLib\L BFF]09905spec.doc:gcc 11

8. The copolymer-1 of any one of claims 1-2, or the composition of any one of claims 3-7 when used in the treatment of multiple sclerosis.

9. Use of the copolymer- of any one of claims 1-2, in the preparation of a medicament for the treatment of multiple sclerosis.

10. A method of malnufacturing copolymer-1, comprising reacting protected copolymer-1 with hydrobromic acid to form trifluoroacetyl copolymer-1, treating said trifluoroacetyl copolymer-1 with aqueous piperidine solution to form copolymer-1, and purifying said copolymer-1, to result in copolymer-1 having a molecular weight of about to 9 kilodaltons.

11. The method of claim 10, wherein said protected copolymer-1 is reacted with hydrobromic acid for about 10-50 hours at a temperature of about 20-28 0 C.

12. The method of claim 10, wherein said protected copolymer-1 is reacted with hydrobromic acid for about 17 hours at a temperature of about 26 0 C.

13. The method of any one of claims 10-12, wherein said protected copolymer-1 s15 has a molecular weight of about 5 to 9 kilodaltons. S

14. A copolymer-1 as prepared in accordance with the method of any one of claims 10-13.

15. Copolymer-1 having over 75% of its molar fraction within the molecular weight range from about 2kDa to about 20kDa, prepared by a process comprising the steps of: reacting protected copolymer-1 with hydrobromic acid to form trifluoroacetyl copolymer-1 having over 75% of its molar fraction within the molecular weight range S.from about 2kDa to about 20kDa, 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 2kDa to about 20kDa with aqueous piperidine solution to form copolymer-1 having over 75% of its molar fraction within the molecular weight range from about 2kDa to about

16. The copolymer-1 of claim 15 wherein said protected copolymer-1 is reacted with hydrobromic acid for about 10-50 hours at a temperature of about 20-28"C.

17. The copolymer-I of claim 15, wherein said protected copolymer-1 is reacted with hydrobromic acid for about 17 hours at a temperature of about 26"C.

18. The copolymer-1 of any one of claims 15-17, wherein said copolymer-1 has a s'F ecular weight of about 5 to about 9 kilodaltons. [I:\DayLib\LI BFF]09905spec.doc:gcc 12

19. Trifluoroacetyl copolymer-1 having over 75% of its molar fraction within the molecular weight range from about 2kDa to about 20kDa, produced by a process comprising the steps of reacting protected copolymer-1 with hydrobromic acid for a time and at a temperature predetermined by test reaction.

20. The trifluoroacetyl copolymer-1 of claim 19 wherein said protected copolymer-1 is reacted with hydrobromic acid for about 10-50 hours at a temperature of about 20-28°C.

21. The trifluoroacetyl copolymer-1 of claim 20 wherein said protected copolymer- 1 is reacted with hydrobromic acid for about 17 hours at a temperature of about 26°C.

22. A copolymer-1 as claimed in claim 1, substantially as hereinbefore described with reference to any one of the examples. i; 23. A method of manufacturing copolymer-1 as claimed in claim 10, substantially as hereinbefore described with reference to any one of Examples 1, 3 or 4.

24. A copolymer-1 as claimed in claim 15 substantially as hereinbefore described 1 s5 with reference to any one of Examples 1, 3 or 4. Trifluoroacetyl copolymer-1 as claimed in claim 19 substantially as hereinbefore described with reference to Example 3 or 4.

26. Copolymer-1 prepared in accordance with the method of claim 23. 20 Dated 22 October, 2001 Yeda Research and Development Co. Ltd Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [I:\DAYLIB\LIBFF]09905spec I.doc:ANB