SU1639432A3 - Method for depolymerization of heparin, sulfomucopolysaccharide or sodium deoxyribonucleinate - Google Patents

Description

I

(21) 4203197/04

(22) 08/03/87

(31) 304799

(32) 08/05/86

(33) AR

(46) 03/30/91. Bil № 12

(71) AHORKA S.A. (AR)

(72) Victor Batista Diaz, Ricardo Hugo Domanico (AR) and Fernando Fussi (GG)

(53) 547.995.17.07 (088.8)

(56) Application EP I 021067 Al, cl. From 08 to 37/10, 1984.

(54) METHOD OF DEPOLYMERIZATION OF HEPARIN, SULFOMUKOPOLYSACCHARIDE OR SODIUM LEONOXYRY BONUCLEINATE

(57) FIELD: chemistry.

polysaccharides, in particular, to the depolymerization of heparin, sulfomucopolyaccharide or sodium deoxyribonucleate, which have an antithrombotic effect. The purpose of the invention is to simplify the process, the ability to control the degree of destruction while maintaining the original product by the target product. Depolymerization is carried out by treatment with hydrogen peroxide in the presence of catalytic amounts of Fe (2+) sulfate at pH 4-6.3, temperature 50-80 ° C and in an aqueous medium. The process is carried out at a ratio of the initial product (May.): 30% hydrogen peroxide (vol.), Equal to 5: 1, with the termination of the process at the right time by treatment with ethanol at pH 7. 4 table.

SS

(L

The invention relates to the chemistry of polysaccharides, specifically to a method for the depolymerization of heparin, sulfomopolysaccharide or sodium deoxyribonucleate, which have an antithrombotic effect.

The aim of the invention is to simplify the process and provide the ability to control the degree of destruction while maintaining the original product by the target product.

According to the proposed method, hydrogen peroxide is used as a depolymerizing agent in the presence of catalytic amounts of iron (II) sulfate and the process is carried out at pH 4-6.3, temperature 50-80 ° C with a ratio of the starting product (May): 30% peroxide hydrogen (vol.), equal to 5: 1, with the termination of the process at the right time by treatment with ethanol at pH 7.

Example 1. 20 g (1.666 mmol) of heparin sodium (American Pharmaceuticals) are dissolved in 100 ml of water (MV 12000), 4 g of cationic resin (AmberR-f

lite IR - 120, H form) add

to the solution with stirring. As the pH of the solution decreases to 4.0-4.5, the resin is filtered off and the filtrate is collected in a flask with a round bottom. In the process of heating under reflux, it is heated at 80 ° C in a water bath, and then 4.2 ml of the reagent obtained by mixing 4.0 ml of 30% Hg02 and 0.2 ml of solution are added to the solution with stirring

GO

with

СО СО WITH N5

 O4

pa Fe (ll) (solution, 5 g FeS04 7H20 in 100 ml of water).

The solution is maintained at 80 ° C and the pH is fixed. In advance

Specific time points from the flask are taken in 20 ml samples. A few drops of 0.1 M NaOH were quickly added to each sample with simultaneous stirring in order to raise the pH to J 7.0, 5 ml of 0.5 M NaCl solution, 25 ml of ethanol using the drop method, 3.3 ml of 20% - NaCl solution. The precipitate is collected, washed in ethanol, and then in acetone. From evening to morning it is sewn in a vacuum oven at 50-60 ° C and the samples are monitored using viscometry to determine the molecular weight Offi) and concentration (strength) .2

The results are presented in the table.

Example 2 20 g of sodium heparin is dissolved in 100 ml of water with natural 6. The solution is heated to 50 ° C under reflux in a 2 water bath, then 4.6 ml of the reagent is added to it, obtained by mixing 4.0 ml of 30% H202 and 0.6 ml of solution A (see Example 1 ). The reaction mixture is maintained at 3 50 ° C and its pH value is fixed. At predetermined periods of time, 20 ml samples are taken from the flasks. The samples are then subjected to a treatment analogous to example 1.

The results are summarized in table. 2

Example 20 g of the naturally occurring sulfomopolysaccharide mixture of the following composition,%:

Condroitin sulfate (CS) 20

Dermatan sulfate (DS) 50

Heparan sulfate (HS) 30

(, 0 at 80 ° C, 30 ppm Fe-II), having the following characteristics:

Specific optical

rotation, hail-20

Hexuronic acid

after hydrolysis

(met Dische (carbazole),% 30

Hexoamine after hydrolysis (met Elson - Morgan) acetylacetone + + p-dimethylaminobenzaldehyde),% 30

Organic Sulphates

(in terms of S),% 7

Definition:

50

5 0 5

0

five

0

five

Components (acetylcellulose electrophoresis, 0.04 M barium acetate, PH 5.0),%,,. .

Chondroitin Sulfate 20 Dermatan Sulfate50

Heparan sulfate30

subjected to the treatment described in Example 1. Samples are taken and their pH values are recorded, true viscosity and electrophoresis are evaluated.

The results are shown in Table. 3

Example4. 20 g sodium deoxyribonucleate of a mammal is dissolved in 100 ml of water with natural 3. The solution is dissolved, heated under reflux, and the procedure described in Example 1 is then carried out. At predetermined periods of time, samples are taken from the flask. For each sample, the pH, the residual H2O2 and the opalescence point are recorded (adding ethanol dropwise in an amount necessary for permanent opalescence of the solution). Then, two volumes of ethanol are added to the precipitate of the depolymerized nucleins.

The results are presented in table 4.

Thus, the proposed method allows to obtain molecules of exactly the same structure as the original, and differing only in MB, due to which it is possible to study changes in their biological and pharmacological properties depending only on MB; allows to obtain the target product with a rather low content of toxic heavy metals, such as copper, when used, it eliminates the need to treat with toxic chelating agents to remove contaminants. Since the reaction can be easily stopped at 0 at any given point in time, using the proposed method it is possible to obtain a uniform well identifiable product.

According to the proposed method, it is possible to obtain oligomers that are different from the starting molecule only MB, which do not have side toxic effects characteristic of the starting products. In addition, the main side effects caused by the initial molecules, such as bleeding, anaphylactic reactions, inhibition of blood plate aggregation, can be eliminated by decreasing MB c. using the proposed method without

changes with therapeutic and pharmacological properties.

Claims (1)

Invention Formula about The method of depolymerization of heparin, sulfomopolysaccharide or sodium deoxyrybonucleinate by treating depolymerisin with an agent containing hydrogen peroxide and a bivalent metal salt, at elevated temperatures in an aqueous medium, about t and h and roil, and so that, in order to simplify the process and provide the ability to control the degree of destruction while maintaining the original product as the target product the measuring agent is hydrogen peroxide in the presence of catalytic amounts of iron (II) sulphate and the process is carried out at pH 4-6.3, temperature 50-80 ° C with a ratio of the starting product (May): 30% hydrogen peroxide (vol. ), equal to 5: 1, with termination of PRC the jar at the right time by treatment with ethanol at pH 7. Table 1 Time 4i PH T 5.9 6.5 6.5 6.5 6.5 TableA Residual H20Z,% Opalescence point, v / v 0.500 0.550 0.630 0.680 0.680