Advanced copolymer 1 copolymer compositions

 

(57) Abstract:

The fraction of copolymer-1 is a mixture of polypeptides. Polypeptides are composed of alanine, glutamic acid, lysine and tyrosine. The fraction of copolymer-1 contains more than 75% of molecules with molecular weight of approximately from 2 to 20 kDa and less than 5% of molecules with molecular weight of more than 40 kDa. Composition for treatment of multiple sclerosis contains a pharmaceutically acceptable carrier and a pharmaceutically effective amount of the fraction of copolymer-1. The technical result of the invention consists in the application of advanced copolymer-1 as a therapeutic agent against multiple sclerosis. 6 C. and 2 h.p. f-crystals, 1 tab., 2 Il.

Art

Copolymer-1 is a synthetic polypeptide analog of the basic protein of myelin (exchange rate) is a natural component of the myelin sheath. It was proposed to apply as a potential therapeutic agent against multiple sclerosis (Eur. J. lmmunol. [1971] 1:242; and J. Neurol. Sci. [1977] 31: 433). All these references are fully incorporated in this application. Interest in the copolymer-1 as immunotherapy to cure multiple sclerosis occurred in the 1950's, when he discovered that such components Mali is similar to multiple sclerosis, which can be caused by susceptible animals.

Copolymer-1 was investigated at the Institute of Weisman (Rehovot, Israel) Village doctors, Arnon with staff revealed that it inhibits EAE (Eur. J. lmmunol. [1971] 1:242: U.S. patent 3849550). Recently it became known that the copolymer-1 has a beneficial effect on patients with growing and declining form of multiple sclerosis (N. Engl. J. Med. [1987] 317:408). Of the patients who were daily injected with a copolymer-1, has been less aggravation and a small disability compared 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. It is synthesized, polymerize these four amino acids with the formation of products with average molecular weight of 23000 Yes (U.S. Patent 3849550).

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

The invention

The invention relates to the composition of the copolymer-1, essentially free of copolymer-1 with a molecular mass of more than 40 kDa.

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

The invention also relates to pharmaceutical compositions and to a method of treating multiple sclerosis with the use of the described copolymer-1.

Brief description of drawings

In Fig.1 shows the distribution of the molecular masses of the three doses of copolymer-1 and the proportion of species with molecular weight greater than 40 kDa. In Fig. 2 shows the same data related to the molar fraction.

Detailed description of the invention

The present invention relates to the composition of the copolymer-1, essentially free of species of copolymer-1 with a molecular mass of more than 40 kDa. This composition preferably contains less than 5%, and more preferably less than 2.5% of copolymer-1 with a molecular mass of 40 kDa or more.

The invention also relates to copolymer-1 in which the 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 of 8.6 kDa. In particular, the invention relates to copolymer-1 with an average molecular weight of from about 4 to about an 8.6 kDa and FOSS is retenu can be obtained by known methods, for example, the method according to U.S. patent N 3849550, according to which N-carboxyanhydride tyrosine, alanine, -benzylguanine and E-N-triptorelin will polimerizuet at ambient temperature in anhydrous dioxane with diethylamine as initiator. Next-carboxyl group of glutamic acid will unlock hydrogen bromide in glacial acetic acid, followed by removal trifluoracetyl group of lysine residues 1M piperidine. In the application, the terms "ambient temperature" and "room temperature" should be understood as a temperature of from about 20 to about 26oC.

Copolymer-1 with the desired profile of molecular weight can be obtained essentially by any known method. Such methods include chromatography of the copolymer-1, containing high molecular weight species, and collecting fractions without undesirable species, or partial acid or enzymatic hydrolysis to remove high molecular weight species, followed by purification by dialysis or ultrafiltration. Another method of producing copolymer-1 with the desired profile of molecular weight is in getting the desired species with protected amino acids and the subsequent receipt of the appropriate type directly after removing zacosta lyophilizer or turn in an aqueous solution, suitable for subcutaneous injection. The copolymer-1 can also be formed in any known form, convenient for the preparation of peptide drugs for oral, nasal, cheek or rectal administration.

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

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

Example 1

Chromatographic method of obtaining non-lethal copolymer-1

Two doses of copolymer-1 were obtained in a known manner, for example, U.S. patent N 3849550.

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

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

Analysis of molecular weight

The absorption of ultraviolet light is absorption. The molecular distribution of the two doses was determined on a calibrated gelfiltration column (Superose 12).

It was found that the dose And copolymer-1 had an average molecular weight of 7-8 kDa. At this dose from all kinds of copolymer-1 and 2.5% had a molecular weight of above 42 kDa, and 5% more than 40 kDa.

The average molecular mass of the other, is not subjected to chromatographic separation, the dose of copolymer-1 was 12 kDa. At this dose from all kinds of copolymer-1 and 2.5% had a molecular weight of above 42 kDa, and 5% more than 40 kDa.

Example 2 analysis of the toxicity

A: In Vivo

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

Technique

Copolymer-1 was dissolved in distilled water to obtain a solution with a concentration of active ingredient of 2 mg/ml Each mouse was injected 0.5 ml of the test solution into the vein of the tail. To determine mortality and clinical signs of mice was observed over 48 hours. The results of the s and no adverse signs were not detected, this dose was considered to be "non-Toxic". If one or more of the mice died or showed negative signs, this dose was considered to be "toxic".

Dose with an average molecular weight of 7.3. and 8.4 kDa were classified as "non-toxic", and in experiments with 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

Test BLK-degranulation

1. Introduction. Selected from basophilic cells histamine or serotonin) is an in vitro model of allergic reactions of immediate type.

Was received line basophilic leukemia cells in rats (BLK-2H3), which represented a reproducible homogeneous easily maintain a system with high sensitivity (E. L. Basumian, C. lsersky, M. G. Petrino and R. P. Siraganian. Eur. J. lmmunol. 11, 317, 1981). The physiological stimulus for the selection of histamine involves the binding of antigen to membrane-bound IgE molecules, resulting in cross-linking molecules and the subsequent launch complex biochemical cascade. In addition to these physiological immunoglobulin-mediated mechanisms run, degranulation can be caused by various He-IgE-mediated stimuli. Among them are various pept is and BLK-degranulation used for selection of those doses of copolymer-1, which cause significant degranulation and, thus, can identify unwanted local and/or systemic side effects.

II. The basics of testing methods. To basophilic leukemia cells in rats (BLK-2H3) was added [3H]-serotonin and the mixture incubated with 100 g of copolymer-1. Dose of copolymer-1, causing non-specific degranulation, release of [3H] -serotonin in the environment. Its radioactivity was determined by scintillation counter, and the total number of infiltrating cells serotonin was determined in the centrifuged cells. The degree of degradation was calculated in percent was isolated from the cells of serotonin from its total number.

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

As the table shows, at low (<2.5 per cent) proportion of copolymer-1 with high molecular weight low (<2,5), the percentage allocated as an indicator of toxicity serotonin is low and Vice versa.

Example 3. Getting cryptanalytically-1

Protected copolymer-1 was obtained as described Tate is on, 35 g-benzylguanine and 83 g triptorelin.

Polymerization was initiated by addition of 0.01-0.02 % diethylamine. The reaction mixture was stirred at room temperature for 24 hours, then was poured into 10 l of water. Product (protected copolymer-1) was filtered, washed with water and dried. Blocking-benzyl group was removed from glutamate residues, processing 33% Hydrobromic acid at room temperature for 6-12 hours under stirring. The product was poured into excess water, was filtered, washed and dried them into triftoratsetilatsetonom-1.

Example 4.

Getting cryptanalytically-1

Protected copolymer-1 was obtained as described by their Teitelbaum and other (Eur. J. lmmun. Vol. 1 R. 242, 1971), dissolved in 3.5 liters of dioxane 18 g of N-carboxyanhydrides tyrosine, 50 g of alanine, 35 g-benzylguanine and 83 g triptorelin.

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

Protected copolymer-1 was treated with 33 % HBr in Walker shorter polypeptides. The time required to obtain a copolymer-1 with a molecular weight of 7000 2000 Yes, depends on the reaction temperature and the size of the protected copolymer-1. Each dose was administered in the test reaction at a temperature of 20-28oC for different times, for example from 10 to 50 hours.

The results of the molecular masses of these small-scale reactions were calculated and built a graph of the dependence of molecular weight from time to time. The time required to obtain the molecular weight of 7000 2000 Yes, was calculated according to the schedule and this figure was used for large-scale reactions. On average 26oC time is 17 hours. The product was poured into excess water, was filtered, washed and dried them into triftoratsetilatsetonom-1.

Obtaining non-lethal copolymer-1

20 g of cryptanalytically-1 was dispersible in 1 l of water was added 100 g of piperidine. The mixture was stirred for 24 hours at room temperature and was filtered. A solution of crude copolymer-1 raspatelli cameras for dialysis and deliberately at 10 - 20oC against water to achieve a pH=8. Then there was dialyzed against 0.3% of acetic acid and again against the water to pH = 5.5 to 6.0. Then a solution of concentation, consisting of alanine, glutamic acid, lysine and tyrosine, which contains more than 75% of molecules with molecular weight of approximately from 2 to 20 kDa and less than 5% of molecules with molecular weight of more than 40 kDa.

2. The fraction of copolymer-1 p. 1, characterized in that it contains less than 2.5% of molecules with 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, with 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, with an average molecular weight of about from 6.25 to 8.4 kDa.

5. Composition for treatment of multiple sclerosis, containing a pharmaceutically acceptable carrier and a pharmaceutically effective amount of the 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 molecular weight of approximately from 2 to 20 kDa and less than 5% of molecules with molecular weight of more than 40 kDa.

6. The composition according to p. 5, characterized in that it contains a fraction of sprinklers is permanent sclerosis, containing a pharmaceutically acceptable carrier and a pharmaceutically effective amount of the fraction of copolymer-1, which is a mixture of polypeptides consisting of alanine, glutamic acid, lysine and tyrosine, with an average molecular weight of from about 4 to 8.6 kDa.

8. Composition for treatment of multiple sclerosis, containing a pharmaceutically acceptable carrier and a pharmaceutically effective amount of the fraction of copolymer-1, which is a mixture of polypeptides consisting of alanine, glutamic acid, lysine and tyrosine, with an average molecular weight of about from 6.25 to 8.4 kDa.

 

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