RU2161489C2 - Improved copolymer-1 in copolymer composition - Google Patents

Abstract

FIELD: organic chemistry, chemistry of proteins. SUBSTANCE: fraction of copolymer-1 is a mixture of polypeptides consisting of alanine, glutamic acid, lysine and tyrosine. Copolymer-1 fraction has more 75% of molecules with molecular mass about from 2 to 20 kDa and less 5% of molecules with molecular mass above 40 kDa. Composition for treatment of patients with cerebrospinal sclerosis has pharmaceutically acceptable carrier and pharmaceutically effective amount of copolymer-1 fraction. EFFECT: use of improved copolymer-1 as therapeutic agent against cerebrospinal sclerosis. 8 cl, 2 dwg, 1 tbl, 4 ex

Description

State of the art
Copolymer-1 is a synthetic polypeptide analogue of the myelin basic protein (MBP), a natural component of the myelin sheath. It has been proposed to be used as a potential therapeutic agent for multiple sclerosis (Eur. J. lmmunol. [1971] 1: 242; and J. Neurol. Sci. [1977] 31: 433). All references cited herein are fully incorporated in this application. Interest in copolymer-1 as an immunotherapeutic agent for multiple sclerosis arose in the 1950s when it was discovered that components of myelin, such as MBP, prevent or delay experimental autoimmune encephalomyelitis (EAI). EAI is a disease similar to multiple sclerosis that can be caused in animals affected by this disease.

 Copolymer-1 was studied at the Weisman Institute (Rehovot, Israel) by Dr. Sela, Arnon and colleagues who have found that it suppresses EAI (Eur. J. lmmunol. [1971] 1: 242: US patent 3849550). Recently it became known that copolymer-1 has a beneficial effect on patients with an exacerbating and weakening form of multiple sclerosis (N. Engl. J. Med. [1987] 317: 408). Patients who were daily injected with copolymer-1 showed fewer cases of exacerbation and a slight development of disability in comparison with control patients.

 Copolymer-1 is a mixture of polypeptides consisting of alanine, glutamic acid, lysine and tyrosine in a molar ratio of about 6: 2: 5: 2, respectively. It is synthesized by polymerizing these four amino acids to form products with an average molecular weight of 23,000 Da (US Patent 3849550).

 The purpose of the invention is to obtain an improved composition of the copolymer-1.

SUMMARY OF THE INVENTION
The invention relates to a composition of copolymer-1, essentially free of copolymer-1 with a molecular weight of more than 40 kDa.

 In addition, the invention relates to copolymer-1, in which a molar fraction with a molecular weight of from about 2 kDa to about 20 kDa exceeds 75%.

 Additionally, the invention relates to copolymer-1 with an average molecular weight of from about 4 to about 8.6 kDa.

 The invention also relates to a pharmaceutical composition and to a method for treating multiple sclerosis using the described copolymer-1.

Brief Description of the Drawings
Figure 1 shows the molecular weight distribution of three doses of copolymer-1 and the proportion of species with a molecular weight above 40 kDa. In FIG. 2 shows the same data related to the molar fraction.

DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a composition of copolymer-1, essentially free of species of copolymer-1 with a molecular weight of more than 40 kDa. This composition preferably contains less than 5%, and more preferably less than 2.5%, of a copolymer-1 with a molecular weight of 40 kDa or more.

 The invention also relates to copolymer-1, in which a molar fraction with a molecular weight of from about 2 kDa to about 20 kDa exceeds 75%.

 Additionally, the invention relates to copolymer-1 with an average molecular weight of from about 4 to about 8.6 kDa. In particular, the invention relates to copolymer-1 with an average molecular weight of from about 4 to about 8.6 kDa and to copolymer-1 with an average molecular weight of from about 6.25 to about 8.4 kDa.

The copolymer-1 according to the invention can be obtained by known methods, for example, the method according to US patent N 3849550, according to which the N-carboxyanhydrides of tyrosine, alanine, γ-benzylglutamate and EN-trifluoroacetylisine are polymerized at ambient temperature in anhydrous dioxane with diethylamine as an initiator. Next, the γ-carboxyl group of glutamic acid is deprotected with hydrogen bromide in glacial acetic acid, followed by removal of trifluoroacetyl groups from lysine residues with 1M piperidine. In the application, the terms "ambient temperature" and "room temperature" should be understood as a temperature of from about 20 to about 26 ^o C.

 Copolymer-1 with the desired molecular weight profile can be obtained essentially by any known method. Such methods include chromatography of copolymer-1 containing high molecular weight species, and collecting fractions without undesirable species or partial acidic or enzymatic hydrolysis to remove high molecular weight species, followed by purification by dialysis or ultrafiltration. Another way to obtain copolymer-1 with the desired molecular weight profile is to obtain the desired species with protected amino acids and then obtain the desired species immediately after deprotection. The formulation of the compositions according to the invention can be formulated by well-known methods. Preferably, the composition is lyophilized or converted into an aqueous solution suitable for subcutaneous injection. The copolymer-1 can also be given any of the known forms suitable for the preparation of peptide drugs for oral, nasal, buccal or rectal administration.

 Typically, patients suffering from multiple sclerosis, copolymer-1 is administered daily in doses of 20 mg.

 The invention is described in the examples, not limiting its scope.

Example 1
Chromatographic method for producing a low toxicity copolymer-1
Two doses of copolymer-1 were obtained in a known manner, for example, according to US patent N 3849550.

 Then, a single dose was subjected to chromatographic separation, as described below.

 The gel filtration column FRACTOGEL TSK HWSS (600 x 26 mm) was assembled in a Superformance 26 Merck cartridge according to the manufacturer's instructions. The column was equilibrated with water and an acetone solution was injected to determine the total volume. The column was equilibrated with 0.2 M pH 5.0 ammonium acetate buffer, loaded with 30 ml of copolymer-1 samples (20 mg / ml in 0.2 M ammonium acetate buffer with pH 5.0), and fractions were taken every 10 min. A fraction with an average molecular weight of 7-8 kDa was isolated between 120-130 minutes (Dose A).

Molecular weight analysis
Absorption of ultraviolet light (CEM) at 275 nm was determined by a UVIKON 810 spectrophotometer. Samples were diluted to achieve a CEM level below 1 absorption unit. The molecular distribution of two doses was determined on a calibrated gel filtration column (Superose 12).

 It was found that dose A of copolymer-1 had an average molecular weight of 7-8 kDa. In this dose, of all types of copolymer-1, 2.5% had a molecular weight above 42 kDa, and 5% had more than 40 kDa.

 The average molecular weight of another, not subjected to chromatographic separation, dose of copolymer-1 was 12 kDa. In this dose, of all types of copolymer-1, 2.5% also had a molecular weight above 42 kDa, and 5% had more than 40 kDa.

Example 2 Toxicity Test
A: In Vivo
Three doses of copolymer-1 with an average molecular weight of 7.3 kDa, 8.4 kDa (less than 2.5% of copolymer-1 has a molecular weight of more than 40 kDa) and 22 kDa (more than 5% of copolymer-1 has a molecular weight of more than 40 kDa ) tested for toxicity as described below. In each case, 5 mice were used in each experimental group.

Methodology
Copolymer-1 was dissolved in distilled water to obtain a solution with a concentration of the active ingredient of 2 mg / ml. Each mouse was injected with 0.5 ml of the test solution into the tail vein. Mice were observed for more than 48 hours to determine mortality and related clinical signs. Observation results were recorded 10 minutes, 24 hours and 48 hours after injection. If by the end of 48 hours all the animals were alive and no adverse signs were detected, then such a dose was considered "Non-toxic." If one or more mice died or showed adverse signs, then such a dose was considered "toxic."

 Doses with an average molecular weight of 7.3. and 8.4 kDa were classified as "non-toxic", and in experiments with a dose with an average molecular weight of 22 kDa, 3 out of 5 mice died by the end of 48 hours and therefore it was recognized as "toxic".

In: In Vitro
BLK degranulation test
1. Introduction. Histamine (or serotonin) isolated from basophilic cells serves as an in vitro model of an immediate allergic reaction.

A rat basophilic leukemia cell line (BLK-2H ₃ ) was obtained, which was a reproducible homogeneous easily stored system with high sensitivity (EL Basumian, C. lsersky, MG Petrino and RP Siraganian. Eur. J. lmmunol. 11, 317, 1981 ) The physiological stimulus for histamine release involves the binding of antigen to membrane-bound IgE molecules, resulting in a crosslinking of molecules and the subsequent launch of a complex biochemical cascade. In addition to these physiological immunoglobulin-mediated triggering mechanisms, degranulation can be triggered by various He-IgE-mediated stimuli. Among them are various peptides and synthetic polymers, for example polylysine (RP Siraganian. Trends in Pharmacological Sciences, October 432, 1983). Therefore, the test for BLK-degranulation is used to select those doses of copolymer-1 that cause significant degranulation and, thus, can reveal undesirable local and / or systemic side effects.

II. Fundamentals of the test procedure. [ ³ H] -serotonin was added to rat basophilic leukemia cells (BLK-2H ₃ ) and the mixture was incubated with 100 μg of copolymer-1. Doses of copolymer-1 causing non-specific degranulation released [ ³ H] -serotonin into the medium. Its radioactivity was determined by a scintillation counter, and the total amount of serotonin entering the cells was determined in centrifuged cells. The degree of degradation was calculated as a percentage of serotonin isolated from cells from its total amount.

 III. Results. Four doses of copolymer-1 with an average molecular weight in the range of 6250-14500 were investigated to determine the percentage of species with a molecular weight of more than 40 kDa and for BLK degranulation. The results are summarized in the table.

 As can be seen from the table, with a small (<2.5%) proportion of copolymer-1 with a high molecular weight, it is low (<2.5), the percentage allocated as an indicator of toxicity of serotonin is low and vice versa.

Example 3. Obtaining trifluoroacetylsopolymea-1
Protected copolymer-1 was prepared as described by Teitelbaum et al. (Eur. J. lmmun. Vol. 1 P. 242, 1971) from 18 g of tyrosine N-carboxyanhydrides dissolved in 3.5 l of dioxane, 50 g of alanine, 35 g γ-benzyl glutamate and 83 g of trifluoroacetyl lysine.

 The polymerization was initiated by the addition of 0.01-0.02% diethylamine. The reaction mixture was stirred at room temperature for 24 hours, then poured into 10 L of water. The product (protected copolymer-1) was filtered off, washed with water and dried. Blocking γ-benzyl groups were removed from glutamate residues by treatment with 33% hydrobromic acid at room temperature for 6-12 hours with stirring. The product was poured into excess water, filtered off, washed and dried, yielding trifluoroacetyl copolymer-1 at the outlet.

 Example 4

Obtaining trifluoroacetyl copolymer-1
The protected copolymer-1 was prepared as described by Teitelbaum et al. (Eur. J. lmmun. Vol. 1 p. 242, 1971) from 18 g of tyrosine N-carboxyanhydrides dissolved in 3.5 l of dioxane, 50 g of alanine, 35 g τ-benzyl glutamate and 83 g of trifluoroacetyl lysine.

 The polymerization was initiated by the addition of 0.01-0.02% diethylamine. The reaction mixture was stirred at room temperature for 24 hours, then poured into 10 L of water. The product (protected copolymer-1) was filtered off, washed with water and dried.

The protected copolymer-1 was treated with 33% HBr in acetic acid, removing the protecting omega-benzyl group from the 5-carboxylate glutamate residue, and the polymer was cleaved into shorter polypeptides. The time required to obtain copolymer-1 with a molecular weight of 7000 ± 2000 Da depends on the reaction temperature and the size of the protected copolymer-1. Each dose was introduced into the test reaction at a temperature of 20-28 ^o C and for different times, for example from 10 to 50 hours.

The results regarding molecular weights in these small-scale reactions were calculated and plotted against molecular weight over time. The time required to obtain a molecular weight of 7000 ± 2000 Da was calculated according to the schedule, and this indicator was used for large-scale reactions. On average at 26 ^o C the time is 17 hours. The product was poured into excess water, filtered off, washed and dried, yielding trifluoroacetyl copolymer-1 at the outlet.

Obtaining a low toxicity copolymer-1
20 g of trifluoroacetyl copolymer-1 was dispersed in 1 L of water and 100 g of piperidine was added. The mixture was stirred for 24 hours at room temperature and filtered. The crude copolymer-1 solution was distributed into dialysis chambers and dialyzed at 10 - 20 ^° C against water until a pH of 8 was reached. Then dialysis was performed against 0.3% acetic acid and again against water to a pH of 5.5 to 6.0. Then the solution was concentrated and subjected to freeze drying.

Claims (8)

 1. The fraction of copolymer-1, which is a mixture of polypeptides consisting of alanine, glutamic acid, lysine and tyrosine, which contains more than 75% of molecules with a molecular weight of from about 2 to 20 kDa and less than 5% of molecules with a molecular weight of more than 40 kDa.  2. The copolymer-1 fraction according to claim 1, characterized in that it contains less than 2.5% of molecules with a molecular weight of more than 40 kDa.  3. The fraction of copolymer-1, which is a mixture of polypeptides consisting of alanine, glutamic acid, lysine and tyrosine, having an average molecular weight of from about 4 to 8.6 kDa.  4. The fraction of copolymer-1, which is a mixture of polypeptides consisting of alanine, glutamic acid, lysine and tyrosine, having an average molecular weight of about 6.25 to 8.4 kDa.  5. Composition for the treatment of multiple sclerosis containing a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a fraction of copolymer-1, which is a mixture of polypeptides consisting of alanine, glutamic acid, lysine and tyrosine containing more than 75% of molecules with a molecular weight of from about 2 to 20 kDa and less than 5% of molecules with a molecular weight of more than 40 kDa.  6. The composition according to claim 5, characterized in that it contains a fraction of copolymer-1 having less than 2.5% of molecules with a molecular weight of more than 40 kDa.  7. A composition for treating multiple sclerosis containing a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a fraction of copolymer-1, which is a mixture of polypeptides consisting of alanine, glutamic acid, lysine and tyrosine, having an average molecular weight of from about 4 to 8.6 kDa.  8. A composition for treating multiple sclerosis containing a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a fraction of copolymer-1, which is a mixture of polypeptides consisting of alanine, glutamic acid, lysine and tyrosine, having an average molecular weight of about 6.25 to 8.4 kDa

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