NO156129B - PROCEDURE FOR PREPARING MIXTURES OF SULPHATED POLYSACCARIDES WITH A GENERAL HEPARIN POLYSACCARID STRUCTURE. - Google Patents

Description

The present invention relates to the production of mixtures of sulfated polysaccharides with a general heparin polysaccharide structure with a lower average molecular weight than heparin, and which can be used as anticoagulant and antithrombotic agents to prevent or treat thrombosis and as hypolipemic agents.

Heparin is a mixture of sulfated mucopolysaccharides

of animal origin, and which is essentially used because of its anticoagulant and antithrombotic properties, especially to prevent postoperative vein thrombosis, and because of its hypolipemic properties. It is known that heparin works during the coagulation process by activating a natural coagulation inhibitor found in the blood, i.e. antithrombin III. The activation of this protein has the effect of preventing the action of two proteases, i.e. X-activated factor (factor Xa) on the one hand and thrombin on the other.

The anticoagulant activity of heparin in vitro is usually measured by the methods described in various pharmacopoeias, especially the American, English or French pharmacopoeia, as an international standard is used here. However, it is now possible to measure its specific activity in vitro on the one hand with respect to factor Xa and on the other hand with respect to thrombin (see, for example, A. Teien et al., "Thrombosis Research", 11,

107, 1977).

Commercial heparins which are a mixture of polysaccharides where the average molecular weight is above 10,000 daltons and where the molecular weight distribution goes from 4,000 to approx.

45,000 daltons, has the following activity in vitro:

Anticoagulant activity, measured by the method described in the French Pharmacopoeia, 8th edition, under heparin, by reference to the 3rd International Standard and "Codex": 140 to 200 I.U./mg.

Anti-Xa activity measured by Teien's method and by reference to the 3rd international standard 16 to 180 i.e./mg.

APTT activity (Activated partial thromboplastin time) measured by Teien's method, with references to the 3rd international standard: 150 to 170 i.e./mg.

Heparin is always administered parenterally (usually subcutaneously)

and the effect is relatively short-lived, which means that one has the following two disadvantages: it is necessary to add the heparin at least three times per day, and that there is a relatively high frequency of post-operative bleeding.

It is known (cf. Johnson et al, "Thrombos. Haemostas", Stuttgart, 1976, 35, 586-591; Lane et al, "Thrombosis Research" 16, 651-662, Pergamon Press Ltd. 1979; Lasker, Chiu, "Annals N.Y. Acad. Sei., 222, 1973, 971-977; Lasker, "Adv. Exp. Mod. Biol." 52, 1975, 119-130) that when fractionating heparin, e.g. by filtering over "Sephadex" gel, obtain fractions with an average molecular weight that is smaller than that of heparin, and where the molecular weight distribution is narrower than that of heparin. The tests carried out in vitro and in vivo with such fractions show on the one side a relatively more active X-activated factor than on thrombin (ie that they have a ratio of anti-Xa activity

anti-thrombin activity

which is significantly higher than 1), and that said fractions, on the other hand, are more easily absorbed into the blood by subcutaneous injection than for heparin, whereby a higher and longer-lasting activity in plasma is obtained than for the heparin itself.

It is also known (cf. Lasker and Chiu as cited above, Lasker as cited above; US-PS 3,766,167; Perlin et al., "Carbohydrate Research", 18, 1971, 185-194), that by enzymatic hydrolysis of heparin can produce depolymerization products with a low average molecular weight (from 5,300 to 4,500 daltons, as determined by ultracentrifugation) and with an anticoagulant activity, as determined by method USP nr# XVII,

this method is described in the American Pharmacopée, pages 609-611, article "Sodium heparin") of approx. 70 i.e./mg. These depolymerization products can be fractionated by filtration over "Sephadex" gel into fractions with an average molecular weight, as determined by ultracentrifugation, of from 3,200 to 5,900 daltons and with an anticoagulant activity from 45 to 95 i.e./mg (USP method ). These depolymerization products and fractions are active, both orally and parenterally.

It is also known (cf. Lasker as cited above) that by depolymerizing heparin using ascorbic acid and hydrogen peroxide, products with a low average molecular weight can be produced. Depolymerization using ascorbic acid and hydrogen peroxide will, after fractionation in alcohol, give fractions with an average molecular weight of from 4,000 to 7,100 daltons and an anticoagulant activity of from 12 to 100 i.e./mg (uSP method).

Finally, it is also known (GB-PS 2.002.406) that one knows

a resulphation of depolymerization products of heparin without anticoagulant activity can produce oligo-polysaccharides with an average molecular weight, as determined by the Somogy method, (this method is described in GB-PS 2,002,406 and in "Clinical Chemistry", 1960, vol.6, p. 21) of from 2,600 to 5,500 daltons, a specific rotation, measured in an aqueous solution at 20° of from +40°C to +50°C, and an anticoagulant activity of less than 50 i.e./mg ( USP method), and which can be taken either orally or parenterally to prevent thrombosis.

The present invention aims to improve the known technique and thus relates to a method for the production of

mixtures of sulfated polysaccharides with a general heparin polysaccharide structure with a lower average molecular weight than heparin where the acidic groups are present in free or salted form, the polysaccharides in the mixtures have an ethylenic double bond

at one of the chain ends and where the mixtures in the sodium salt state exhibit the following characteristics:

percentage of sulphur: 9 to 13.5,

percentage of nitrogen; 1.8 to 2.5, percentage uronic acid: 20 to 30,

average molecular weight 20,000 to 10,0000 daltons, specific rotation in aqueous solution: (a)<20> = +25 to +55°,

D

and this method is characterized by the fact that a heparin ester obtained by partial or total esterification of the carboxylic acid groups in heparin is reacted with an alkali or organic

base at a temperature of 20 to 80°C either in an aqueous environment if the heparin ester is soluble in water or in an inert solvent for the ester, the depolymerization product thus obtained is isolated as an alkali salt and the latter is hydrolyzed using an aqueous, at least 1 N sodium hydroxy solution at a temperature of 0 to 5°C.

The sulfur and nitrogen content as well as the above-mentioned rotation are determined by the methods described in the 8th edition of the French pharmacopoeia under heparin. The content of uronic acid was determined by the method described by N. Blumen-'krantz et al. ("Analytical Biochemistry", 54, 484, 1973). The average molecular weight was determined by so-called gel permeation chromatography on a gel of polyacrylamide agarose using a standard of heparin of known molecular weight, according to the method of Andersson et al. ("Thrombosis Research", 9, 575, 1976).

The polysaccharide component in the mixtures prepared according to the present invention has more particularly the following formula:

where R is a hydrogen atom or a carboxylic acid group, as free acid or in the form of a salt, R' is an OH group or sulfate group, in free acid form or in the form of a salt, R^ is an OH group or a sulfate group, in free acid form or in the form of a salt, R2 is a sulfonic acid group in free acid form or in the form of a salt, or an acetyl group, -0- is an oxygen bridge, the G bond is the bond of the glucosamine type present in the structure of heparin , the U bonds are bonds of the uronic acid type

(D-glucuronic acid, L-iduronic acid, sulfated L-iduronic acid)

of the same type as in heparin, and n is an integer from 3 to 20.

In formula I, the acid groups in the polysaccharides can be in free form or in the form of a salt, in particular a sodium, calcium or magnesium salt.

Although the present invention relates to all mixtures as defined above, it relates more particularly to those mixtures which, in the form of the sodium salt, show the additional properties specified for categories I, II and III

in Table A below:

(chromogenic peptide with the following structure: (N-benzoyl)-Ile-Glu-Gly-Arg-(p-nitro)anilide), referring to

3. international standard.

100 ul of citrated bovine plasma, diluted from 2 to

5 times with an aqueous buffer, tris(EDTA, pH 8.4, was added 100 µl of a solution of the product to be tested, or of the standard in aqueous buffer, tris/EDTA,

pH 8.4, and where said 100 µl corresponds to 0.02 to 0.08 µg of product or standard. After 3 minutes of incubation at 37°C, an aqueous solution of factor Xa from ox was added which corresponded to 7 n Kat (where n Kat denotes nanocatal where catal is the unit of enzymes that catalyze the transformation of 1 mol of substrate per sec. under standardized conditions for substrate, temperature and pH value) of factor Xa. After a 30-second incubation period, 200 µl of an aqueous solution of S 2222 was added, which corresponds to 0.2 µmol of S 2222. After a three-minute incubation period, 300 µl of acetic acid was added, after which the optical density of the solution was measured at 405 nm in comparison with distilled water.

By plotting the optical density as a function of the concentration of the product or standard, two straight lines are obtained, one relating to the product under test, the other relating to the standard. The product's activity expressed in international units (i.e.) per mg is then given by the following formula:

as the number 173 corresponds to the value for the activity of the third international standard.

In the third international heparin standard, a heparin whose activity value is 173 international units per mg. This unit is provided by the "National Institute of Biological Standards and Control" in London.

b) Determination of APTT activity

The product to be tested and the third international

standard was dissolved in a 0.15 M aqueous solution of sodium chloride, and then diluted with a citrated bovine plasma, free of platelets, so that a product concentration (or for said standard) of from 0 to 4 µg/ml was obtained.

100 µl of the reagent "automated APTT Precibio" (reagent based on rabbit brain phospholipids and micronized silicon dioxide) was added to 100 µl of the prepared solution. After five minutes of incubation at 37°C, 100 ul were added

of a 0.025 M solution of calcium chloride. The bonding time was then measured using a "Bio-Mérieux" fibrometer.

By plotting the logarithm of the sticking time as a function of the product concentration or of the standard, one obtains two straight lines, one relating the product below

n

testing, and the other which indicates the standard. The product's activity expressed in international units per mg. is then given by the following formula:

where "173" is as indicated above.

All the activities listed in tables A, B and C are expressed in international units per mg. (i.e./mq), referring to the third international standard. The mixture is prepared according to the present invention as mentioned by allowing an alkali or organic base to act on a heparin ester, whereby a partial or total esterification of the carboxylic acid groups in the heparin is obtained.

In this ester, the acid groups of the heparin that are not esterified (i.e. sulfuric acid groups and possibly partially carboxylic acid groups) will either be in the form of free acids or in the form of salts, especially an alkali metal salt such as sodium salt, an alkaline earth metal salt, such such as a calcium salt, a magnesium salt or a quaternary ammonium salt with a long chain such as the benzethonium salt.

In cases where the heparin ester is soluble in water (e.g. when the non-esterified acid groups are in the form of the sodium salt), then the reaction between the ester and the base can be carried out in water at temperatures between 20 and 80°C, as the molar concentration of the base in the medium is preferably between 0.1 and 0.6. Bases that can be used are those that are soluble in water and in particular sodium hydroxide, potassium hydroxide, alkali metal carbonates, triethylamine, and 1,5-diazabicyclo (4,3,0)5-nonene. as soon as the reaction is over, the depolymerization product can be isolated, e.g. by precipitation by adding sodium chloride and then methanol.

The reaction between the ester and the base can also be carried out, especially when the non-esterified acid groups in the ester are in the form of a quaternary ammonium salt with a long chain in an inert organic solvent for said ester, e.g. dichloromethane, dimethylformamide, formamide or tetrahydrofuran, at temperatures between 20 and 80°C. Bases that can be used are bases that are soluble in the solvent used, and in particular 1,5-diaza-bicyclo(4,3,0)-5-nonene.

As soon as the reaction is over, the depolymerization product, where the carboxylic acid groups are still esterified, can be isolated in the form of an alkaline salt, and can then be hydrolyzed with the help of an aqueous solution of an alkali metal hydroxide, especially sodium hydroxide with a strength of at least 1 N, at a low temperature from 0°C to 5°C.

The final product can be separated, e.g. by precipitation, by adding sodium chloride and then methanol.

Heparin esters that can be used as starting products for the preparation according to the present invention of mixtures of polysaccharides can be non-selective esters or selective esters. Non-selective esters include heparin esters where the carboxyl groups in the D-glucuronic acid, the unsulphated L-iduronic acid and the sulphated L-iduronic acid bonds are arbitrarily esterified. Selective esters are heparin esters that are fully or partially esterified, either only on the carboxyl groups in the D-glucuronic acid bonds or only the carboxyl groups in the D-glucuronic acid and the unsulfated L-iduronic acid bonds, or only the carboxyl groups in the unsulfated L-iduronic acid and the sulfated L- the iduronic acid bonds, or just the carboxyl groups in sulfated L-iduronic acid bonds.

Heparin esters which can be used as starting products for the present processes are in particular the heparin esters described in FR-PS 2,150,724 and GB-PS 1,501,095, as well as methyl, ethyl, ethoxycarbonylmethyl, cyanomethyl, benzyl and substituted benzyl (especially 4-chloro-benzyl and 4-nitrobenzyl) esters of heparin. Those which are preferably used as starting products are benzyl or substituted benzyl esters of heparin. The heparin esters used as starting compounds in the present method can come from heparin of any origin (either lung heparin, heparin from pig membranes, heparin from the intestinal tract of cattle, etc.).

The non-selective methyl, ethyl, ethoxycarbonylmethyl, cyanomethyl, benzyl and substituted benzyl esters of heparin can e.g. is produced by reacting a neutral quaternary ammonium or amino salt of heparin with a halogenated derivative of formula

where Hal represents a chlorine, bromine or iodine atom and R represents a hydrogen atom or a methyl, ethoxycarbonyl, cyano, phenyl or substituted phenyl group. This reaction is carried out in a solution or a suspension in an inert solvent, such as dimethylformamide, methylene chloride, dimethylsulfoxide, tetrahydrofuran or acetone, at temperatures between -20°C and +60°C.

Methyl, ethyl, ethoxycarbonylmethyl, cyanomethyl, benzyl or substituted benzyl esters of heparin which are either fully or partially esterified, either only with the carboxyl groups in the D-glucuronic acid linkages or only with the carboxyl groups in the D-glucuronic acid or unsulfated L-iduronic acid linkages, can be prepared by reacting a halogen derivative with the above-mentioned formula (II) with an acidic, quaternary ammonium salt of heparin in which, in addition to the sulfate groups, either only the carboxyl groups of the D-glucuronic acid bonds, or only the carboxyl groups of D-glucuronic acid1 and unsulfated L-iduronic acid bonds are salted, while the other carboxyl groups are in the form of free acids. This reaction is carried out under the same conditions as for the reaction between halogen derivatives of formula (II) and said neutral, quaternary ammonium salt of heparin.

The acidic, quaternary ammonium salts of heparin where, apart from the sulphate groups, only the carboxyl groups in the D-glucuronic acid bonds are salt-formed, are produced by reacting a quaternary ammonium salt with heparin in an aqueous medium at a pH value between 3 and 4.

The acidic, quaternary' ammonium salts of heparin, in which, in addition to the sulfate groups, only the carboxyl groups in the D-glucuronic acid and the unsulfated L-iduronic acid bonds are salt-formed, are produced by reacting a quaternary ammonium salt with heparin in an aqueous medium with a sufficiently low pH value that a quaternary ammonium salt of heparin is formed where only the sulfate groups are salt-formed (in practice a pHr<y>ecdifra from 2 to 2.5 is used), after which the carboxyl groups in the D-glucuronic acid and the unsulfated L-iduronic acid bonds in the product are selectively neutralized by adding a predetermined amount of a quaternary ammonium hydroxide, in a dimethylformamide medium.

The amount of quaternary ammonium hydroxide that is added is calculated from the neutralization curve in a dimethylformamide medium for a sample of said product with a known weight.

Methyl, ethyl, ethoxycarbonylmethyl, cyanomethyl, benzyl and substituted benzyl esters of heparin in which either only the carboxyl groups in the sulfated L-iduronic acid bonds or only the carboxyl groups in the unsulfated L-iduronic acid and sulfated L-iduronic acid bonds are fully or partially esterified, is prepared by reacting an alcohol of the formula HO-CI^-R (III), where R is a hydrogen atom or a methyl, ethoxycarbonyl, cyano, phenyl or substituted phenyl group, with heparin in an aqueous medium, and in the presence of a water-soluble condensing agent of the carbodiimide type, e.g. 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide, and where the medium's pH value is adjusted to 3.5 - 4.5 in the first case and in the range from 2 - 3 in the second case. As an alcohol with formula (III), one can e.g. use methanol or ethanol, and in these cases a selective methyl ester of heparin and a selective ethyl ester of heparin will be obtained respectively.

Method according to the present invention where one

uses a mineral or organic base on a heparin ester, and where a so-called 6-elimination is obtained, will give a partial and regulated depolymerization of heparin without changing its general structure.

The mixture of sulfated polysaccharides obtained according to the present invention has anticoagulant and antithrombotic effects, and hypolipemic effects. For mixtures with a sufficiently low average molecular weight (in practice less than or equal to 7000 daltons), the antithrombotic effect will be lower than the anticoagulant effect. Mixtures prepared according to the present invention have little toxicity. For example, the product from example 9 was not toxic at a dose of 300 mg/kg body weight when administered intravenously to rats and mice. When the mixtures were administered subcutaneously, the toxicity was the same as for heparin.

Mixtures of sulfated polysaccharides produced according to the present invention where the acid groups in the polysaccharides are in the form of pharmaceutically acceptable salts, especially in the form of sodium, calcium or magnesium salts, can be used as anticoagulant and antithrombotic agents, to prevent or treat thrombosis. They can also be used for the treatment of hypolipaemia. They can advantageously be used instead of heparin in such compounds. When administered subcutaneously, they show a longer-lasting effect than heparin, which means that the frequency of injections can be lowered. Furthermore, they produce less secondary effects (bleeding effects) than heparin.

They can be administered in admixture with pharmaceutically acceptable diluents or carriers, be it intravenously, subcutaneously, via the lungs (inhalation) or by the rectum, and for sufficiently low average molecular weight mixtures (category III mixtures) also orally. The doses administered will depend on the route of administration and the desired effect (antithrombotic or hypolipemic effect).

The following examples illustrate the invention, the anticoagulant effect being determined by the method according to the French Pharmacopoeia, 8th edition, article "Heparin" (see page 6, section after the table) "Codex".

The neutral benzethonium salt of heparin or benzethonium heparinate used as starting compound in Examples 1 to 3, 10, 12 to 15 was prepared from porcine intestinal heparin with the following properties:

- average molecular weight: 16,000 daltons

- specific rotation in aqueous solution

at 20°C: (a)<20>: +41°C

- anticoagulant activity, "Codex": 157 i.e./mg.

The neutral benzethonium salt of heparin or benzethonium heparinate used as starting product in Examples 4 to 9 and 11 came from a heparin from bovine viscera with the following properties:

- average molecular weight: 11,400 daltons

(a)<2>°s +37°C

- anticoagulant activity "Codex": 128 i.e./mg.

The neutral benzethonium salt of heparin or benzethonium heparinate used as starting product in Example 16 came from a porcine mucosal heparin with the following properties:

- average molecular weight of 16,000 daltons

(a)<20> : +44°C - anticoagulant activity, "Codex": 180 i.e./mg.

The sodium salt of heparin used as starting product in Examples 17 to 19 corresponds to the above-mentioned porcine mucosal heparin.

EXAMPLE 1

30 g of (4-chloro)benzyl chloride was added to a solution

of 30 g of benzethonium heparinate in 600 ml of dimethylformamide. After the solution had been left for 60 hours at room temperature (approx. 20°C), 600 ml of a 10% solution of sodium acetate in methanol was added. The precipitate was filtered off, washed with methanol and dried in vacuum. 10.75 g of the 4-chloro-benzyl ester of heparin was produced in the form of the sodium salt.

The produced ester was added with stirring to 269 ml

0.4N aqueous solution of sodium hydroxide at 25°C.

After 2 hours, the mixture was neutralized by adding 0.4N aqueous hydrochloric acid, after which precipitation was carried out by adding two volumes (double the volume of the aqueous phase) of methanol. By filtration, 8.46 g of depolymerized sodium heparinate are isolated.

EXAMPLE 2

30 g of benzyl chloride was added to a solution of 30 g of benzethonium heparinate in 600 ml of dimethylformamide.

After standing for 60 hours at room temperature, precipitation was carried out by adding 600 ml of a 10% solution of sodium acetate in methanol. The precipitate was filtered off, washed with methanol and dried in vacuum. 11.4 g of the benzyl ester of heparin were produced in the form of the sodium salt.

3 g of this ester was mixed with 75 ml of a 0.4N aq

solution of sodium hydroxide at 20 to 25°C, and the mixture was allowed to stand for two hours. It was then neutralized by adding 0.4N aqueous hydrochloric acid and precipitation was carried out by adding two volumes of methanol. 2.23 g of depolymerized heparin in the form of the sodium salt were produced.

EXAMPLE 3

10 g of benzyl chloride was added to a solution of 10 g of benzethonium heparinate in 250 ml of dichloromethane.

The mixture was left for 24 hours at room temperature, after which the solvent was removed in vacuo. The residue was dissolved in 150 ml of dimethylformamide, and precipitation carried out by adding 150 ml of a 10% solution of sodium acetate in methanol. 3.67 g of the benzyl ester of heparin in the form of the sodium salt was filtered off. 2 g of this ester were treated for 2 hours with 50 ml of a 0.1N aqueous solution of sodium hydroxide at 60°C. After cooling, the solution was neutralized by adding a 0.1N aqueous solution of hydrochloric acid, after which precipitation was carried out by adding 2 volumes of methanol.

1.54 g of depolymerized heparin in the form of sodium salt was produced.

EXAMPLE 4

5 g of ethyl chloroacetate was added to a solution of 5 g of benzethonium heparinate in 125 ml of dichloromethane, after which the mixture was left for 3 days at room temperature. The solvent was removed in vacuo, after which the residue was dissolved in 75 ml of dimethylformamide, and precipitation carried out by adding 75 ml of a 10% solution of sodium acetate in methanol. The precipitate was filtered off, washed with methanol and dried in vacuum. 1.72 g of the carbethoxymethyl ester of heparin in the form of the sodium salt was produced.

1.7 g of this ester was treated with 43 ml of a 0.1N aqueous solution of sodium hydroxide at 60°C for two hours. After cooling, the solution was neutralized by

put a 0.1N, aqueous solution of hydrochloric acid, after which precipitation was carried out by adding 2 volumes of methanol.

By filtration, 1.33 g of depolymerized heparin was isolated in the form of the sodium salt.

EXAMPLE 5

10 g of (4-chloro)benzyl chloride was added to a solution of 10 g of benzethonium heparinate in 250 ml of dichloromethane. The solution was left for 24 hours at room temperature, where. after the solvent was removed in vacuo. The residue was dissolved in 150 ml of dimethylformamide, and the product precipitated by adding 150 ml of a 10% solution of sodium acetate in 1 methanol. The precipitate was filtered off, washed with methanol and dried in a vacuum, thereby obtaining 3.84 g of the (4-chloro)-benzyl ester of heparin in the form of the sodium salt. 2 of this ester was treated with 50 ml of a 0.1N aqueous solution of sodium hydroxide at 60°C for two hours. After cooling and neutralizing with a 0.IN r aqueous solution of hydrochloric acid, the product was precipitated by adding 2 volumes of methanol. The precipitate was filtered off, washed with methanol and dried in vacuum. 1.38 g of depolymerized heparin was produced in the form of the sodium salt.

EXAMPLE 6

5 g of (4-nitro)benzyl chloride was added to a solution of 5 g of benzethonium heparinate in 12 5 ml of dichloromethane and dissolved with stirring. The solution was left for 3 days at room temperature, after which the solvent was removed in vacuo, and the residue dissolved in 75 ml of dimethylformamide.

The ester formed was precipitated by adding 75 ml of a 10% solution of sodium acetate in methanol. The sediment

was filtered off, washed in methanol and dried in vacuo.

1.89 g of the (4-nitro)benzyl ester i was produced

form of the sodium salt.

1.85 g of this ester was treated with 46 ml of a 0.1N aqueous solution of sodium hydroxide at 60°C for two hours with stirring. After cooling, the solution was neutralized by adding a 0.1N aqueous solution of hydrochloric acid, and the product was precipitated by adding two volumes of methanol. The product was isolated by filtration, washed with methanol and dried in vacuo. 1.13 g of depolymerized heparin was produced in the form of the sodium salt.

EXAMPLE 7

30 g of benzyl chloride was added to a solution of 30 g of benzethonium heparinate in 500 ml of dichloromethane and dissolved by stirring. The solution was left at room temperature for 14 hours, after which the solvent was evaporated in vacuo and the residue dissolved in 400 ml of ether. Insoluble precipitate was filtered off. 30 g of the benzyl ester of heparin was produced in the form of the benzethonium salt.

This ester was dissolved in 200 ml of dichloromethane containing 8 ml of 1,5-diaza-bicyclo(4,3,0)5-nonene. The solution was refluxed for 34 hours, after which the solvent was evaporated in vacuo. The residue was dissolved in 450 ml of dimethylformamide and a corresponding volume of a 10% solution of sodium acetate in methanol was added. The precipitate was filtered off and washed in methanol. It was then treated for 1 hour at 0°C with an IN aqueous solution of sodium hydroxide. After neutralization, the product was precipitated by adding two volumes of methanol. The product was filtered off, washed with methanol and dried in vacuum. 6.6 g of depolymerized heparin was produced in the form of the sodium salt.

EXAMPLE 8

10 g of (4-chloro)benzyl chloride was added to a solution of 10 g of benzethonium heparinate in 250 ml of dichloromethane with stirring. The solution was left at room temperature for 24 hours, after which the solvent was evaporated in vacuo. The residue was dissolved in 200 ml of ether and the precipitate formed was isolated by filtration. 10 g of the (4-chloro)benzyl ester of heparin in the form of the benzethonium salt were produced. 5 g of this product was dissolved in 100 ml of dichloromethane containing 1.5 ml of 1,5-diaza-bicyclo(4,3,0)5-nonene and then refluxed for 4 hours. The solvent was then removed in vacuo, the residue dissolved in 30 ml of dimethylformamide, after which 100 ml of a 10% solution of sodium acetate in methanol was added. The precipitate was filtered off, washed with methanol and then treated with 24 ml of a 1N aqueous solution of sodium hydroxide for 1 hour at 0°C. The solution was neutralized by adding a 1N solution of hydrochloric acid and the product precipitated by adding two volumes of methanol. The product was filtered off and after washing with methanol and drying in vacuum, 1 g of depolymerized heparin was obtained in the form of the sodium salt.

EXAMPLE 9

30 g of benzyl chloride was added to a solution of 30 g of benzethonium heparinate in 600 ml of dimethylformamide. After dissolution, the reactants were left for 60 hours at room temperature, after which the produced ester was precipitated by adding 1200 ml of a 10% solution of sodium acetate in methanol. The precipitate was filtered off, washed with methanol and dried in vacuum. 11.4 g of the benzyl ester of heparin were produced in the form of the sodium salt. 10 g of this ester was treated with 250 ml of a 0.1N aqueous solution of sodium hydroxide at 60°C for two hours with stirring. After cooling, the solution was neutralized with a 0.1N aqueous solution of hydrochloric acid and the product precipitated by adding two volumes of methanol. The precipitate was filtered off, washed with methanol and dried in vacuum. 6.65 g of depolymerized heparin was produced in the form of the sodium salt.

EXAMPLE 10

120 g of (4-chloro)benzyl chloride was added to a solution of 120 g of benzethonium heparinate in 2.4 L of dimethylformamide with stirring. The solution was then left at room temperature for 60 hours, after which 2.4 1 of a 10% solution of sodium acetate in methanol was added. The precipitate formed was filtered off, washed with methanol and dried in vacuo. 46 g of the (4-chloro)-benzyl ester of heparin in the form of the sodium salt was produced. 20 g of this ester was treated with 500 ml of a 0.1N aqueous solution of sodium hydroxide at 60°C for two hours with stirring. After cooling and neutralization, precipitation was carried out by adding 2 volumes of methanol. 11.7 g of depolymerized heparin was isolated by filtration in the form of the sodium salt.

EXAMPLE 11

30 g of methyl iodide was added to a solution of 30 g of benzethonium heparinate in 750 ml of dichloromethane with stirring. The solution was left for 48 hours at room temperature, after which the solvent was removed in vacuo. The residue was dissolved in 450 ml of dimethylformamide and precipitation carried out by adding 450 ml of a 10% solution of sodium acetate in methanol. After filtration, the precipitate was washed with methanol and dried in vacuum. 10.5 g of the methyl ester of heparin was produced in the form of the sodium salt. 2 g of this ester were treated with 50 ml of a 0.1N aqueous solution of sodium hydroxide at 60°C with stirring for two hours. After cooling, the pH of the solution was adjusted to 4.5 by stirring with a carboxyl ion exchange resin in the H+<-> form. The resin was then separated by filtration and washed with water. The combined aqueous phases were neutralized by adding a dilute aqueous solution of sodium hydroxide, after which it was lyophilized. 2 g of depolymerized heparin were produced in the form of the sodium salt.

EXAMPLE 12

3 g of the (4-chloro)benzyl ester of heparin prepared as described in Example 10 was treated with 120 ml of a 10% aqueous solution of sodium bicarbonate for two hours at 60°C with stirring. After cooling, the solution was neutralized with a 0.4N aqueous solution of hydrochloric acid, and the product precipitated by adding 2 volumes of methanol. 1.57 g of depolymerized heparin was produced in the form of the sodium salt.

EXAMPLE 13

30 g of ethyl chloroacetate was added to a solution of 30 g of benzethonium heparinate in 600 ml of dimethylformamide. After dissolution, the two compounds were left in contact for 60 hours at room temperature, after which 600 ml of a 10% solution of sodium acetate in methanol was added. The precipitate formed was filtered off, washed with methanol and dried in vacuum.

10.78 g of the carbethoxymethyl ester of heparin in the form of the sodium salt was produced. 3 g of this ester was contacted with 100 ml of a 3% aqueous solution of triethylamine at 60°C. After 5 hours, the solution was neutralized by adding an aqueous hydrochloric acid solution, after which the product was precipitated by adding 2 volumes of methanol. 2.5 g of depolymerized heparin in the form of the sodium salt was isolated by filtration.

EXAMPLE 14

5 g of chloroacetonitrile was added to a solution of 5 g of benzethonium heparinate in 12 5 ml of dichloromethane and dissolved with stirring. The solution was left for 48 hours at room temperature, after which the solvent was removed in vacuo. The residue was dissolved in 75 ml of dimethylformamide and the product precipitated by adding 75 ml of a 10% solution of sodium acetate in methanol. The precipitate was filtered off,

washed with methanol and dried. 1.63 g of the cyanomethylester of heparin in the form of the sodium salt was produced.

The ester produced was treated with 40 ml of a

0.1N, aqueous solution of sodium hydroxide at 60°C with stirring for 2 hours. After cooling, the solution was neutralized by adding a 0.1N aqueous solution of hydrochloric acid, after which precipitation was carried out by adding two volumes of methanol. 1.33 g of depolymerized heparin was produced in the form of the sodium salt.

EXAMPLE 15

3 g of the (4-chloro)benzyl ester of heparin prepared as

described in example 10, was dissolved with stirring in 120 ml of 10% aqueous solution of sodium bicarbonate. After two hours of stirring at 20 to 25°C, the pH of the solution was adjusted

to 6 by adding a 1N aqueous solution of hydrochloric acid, followed by the addition of twice the volume of methanol. The sediment was filtered off, and 2.1 g of (4-chloro)benzyl ester of heparin was produced.

2 g of this was treated with stirring for two hours

50 ml of a 0.1N, aqueous solution of sodium hydroxide at 60°C. After cooling, the solution was neutralized by adding a 0.1N aqueous solution of hydrochloric acid, after which the product was precipitated by adding twice the volume of methanol. 1.4 g of depolymerized heparin was produced in the form of the sodium salt.

In the preceding examples 1 to 10 and 12 to 15, the concentration of NaCl in the aqueous phase was adjusted to 10% by adding sodium chloride before precipitating the product formed by adding the two volumes of methanol.

EXAMPLE 16

10 g of (4-chloro)benzyl chloride was dissolved with stirring in a solution of 10 g of benzethonium heparinate in 250 ml of dichloromethane. The solution was left for 24 hours at room temperature, after which a 10% solution of sodium acetate in methanol was added. The formed precipitate was filtered off, washed with methanol, whereby 3.72 g of (4-chloro)benzyl ester of heparin was produced in the form of the sodium salt.

A solution of 0.500 g of this ester in 10 ml of formamide was treated at 60°C for 5 hours with 0.5 ml of 1,5-diaza-bicyclo(4,3,0)5-nonene. After cooling, 70 ml of acetone was added. 0.364 g of precipitate was filtered off, then at 0°C for two hours, treated with 6 ml of a 1N aqueous solution of sodium hydroxide. The aqueous phase was neutralized by the addition of a 1N hydrochloric acid solution and the concentration of NaCl in the medium was adjusted to 10% by the addition of sodium chloride. The precipitation was carried out using the double volume of methanol. 0.263 g of depolymerized heparin was produced in the form of the sodium salt.

EXAMPLE 17

2.5 ml of acetic acid and then 150 ml of a 10% aqueous solution of benzethonium chloride were added with stirring to

added to a solution of 10 g of heparin (sodium salt) in 40 ml of water. The precipitate was centrifuged off, washed with water and dried. 19.67 g of acidic benzethonium heparinate were produced.

11 g of this product was dissolved in 110 ml of dimethylformamide, after which 11 g of (4-chloro)benzyl chloride was added. The mixture was allowed to stand for 48 hours at room temperature, after which 220 ml of 10% sodium acetate in methanol was added. The precipitate was filtered off, washed with methanol and dried in vacuum. 4.70 g of (4-chloro)benzyl ester of heparin was produced in the form of the sodium salt. 4 g of this ester were dissolved in 20 ml of water, after which 40 ml of a 20% aqueous solution of benzethonium chloride was slowly added while stirring. The precipitate formed was filtered off, washed with water and dried in vacuum. The (4-chloro)benzyl ester of heparin was obtained in the form of the benzethonium salt. 1 g of this ester (benzetonium salt) was dissolved in 20 ml of dimethylformamide and at 60°C and during five hours treated with 1 ml of 1,5-diaza-bicyclo-(4,3,0)5-nonene. After cooling, 50 ml of a 10% solution of sodium acetate in methanol was added. 0.346 g of precipitate was filtered off and treated at 0°C for 2 hours with 5 ml of a 1N aqueous solution of sodium hydroxide. The aqueous phase was neutralized by the addition of an IN, aqueous solution of hydrochloric acid, and the concentration of NaCl in the medium was adjusted to 10% by the addition of sodium chloride. Precipitation was filtered off, washed with methanol, and 0.253 g of depolymerized heparin was produced in the form of the sodium salt.

EXAMPLE 18

2.5 ml of formic acid and 150 ml of a 10% aqueous solution of benzethonium chloride were added to a solution of 10 g of heparin (sodium salt) in 40 ml of water. The precipitate was centrifuged off, washed with water and dried under vacuum.

20.5 g of acidic benzethonium heparinate was produced.

2.95 g of this product was dissolved in 60 ml of dimethylformamide, after which 5.9 ml of a 0.1N solution of tetrabutylammonium hydroxide in n-propanol/methanol was added.

After adding 2.95 g of (4-chloro)benzyl chloride, the solution was left for 5 days at room temperature. 74 ml of a 10% solution of sodium acetate in methanol was then added. 1.32 g of (4-chloro)-benzyl ester of heparin in the form of the sodium salt was filtered off and isolated.

This ester was dissolved in 6.4 ml of water and 12.8 ml of a 20% aqueous solution of benzethonium chloride was slowly added with stirring. The precipitate was separated, washed with water and dried in vacuum. The prepared (4-chloro)-benzyl ester of heparin was obtained in the form of the benzethonium salt.

1 g of this ester was dissolved in 20 ml of dichloromethane, and 1 ml of 1,5-diaza-bicyclo(4,3,0)5-nonene was added. The solution was refluxed for 5 hours, after which the solvent was evaporated under vacuum, and the residue dissolved in 15 ml of dimethylformamide and 20 ml of a 10% solution of sodium acetate in methanol was added. The precipitate was filtered off and washed with methanol. 0.405 g of product was produced, which was treated at 0°C for 2 hours with 6 ml of an IN, aqueous solution of sodium hydroxide. The aqueous phase was neutralized by adding an IN, aqueous solution of hydrochloric acid, and the concentration of sodium chloride. The product was precipitated by adding 2 volumes of methanol. It was filtered out and. washed with methanol. 0.355 g of depolymerized heparin was produced in the form of the sodium salt.

EXAMPLE 19

0.600 g of heparin (sodium salt) was dissolved in 7 ml of water,

and the pH was adjusted to 3.5 by adding N-hydrochloric acid. 0.300 g of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide was added and the solution left for 1 hour at room temperature. 2.5 ml of an aqueous solution of sodium chloride containing 280 g per liter, after which 15 ml of methanol was added. The precipitate formed was filtered off, washed with methanol and dried under vacuum. 0.527 g of methyl ester of heparin was produced, where the percentage of esterification of the carboxyl groups is 50%, and the product was obtained in the form of the sodium salt.

0.300 g of this ester was dissolved in 7.5 ml of 0.1N aqueous sodium hydroxide solution and the solution was kept at 60°C for 2 hours. After cooling, the solution was neutralized by adding a 0.1N aqueous solution of hydrochloric acid, and the concentration of NaCl in the medium was adjusted to 10%, after which precipitation was carried out by adding two volumes of methanol. 0.200 was produced. g of depolymerized heparin in the form of the sodium salt.

Tables B and C indicate properties of the products (depolymerised heparin in the form of the sodium salt) prepared as described in examples 1 to 19. The percentage of sulphur, nitrogen and uronic acids, as well as the specific rotation in aqueous solution at 20°C, in addition to the average molecular weight and the activities indicated in the tables were determined using the methods described previously. In the column for the molecular weight distribution, approximate extreme values are given for the molecular weights of the polysaccharide component in the mixture, as this could be determined by gel penetration chromatography on polyacrylamide-agarose gel. The viscosities at 25°C of a 10% aqueous solution of the products are shown in the viscosity column. In the UV absorption column, the absorption is shown for a layer of 1 cm of a 1% solution of the product in 0.01 N HC1, and the absorption is measured in the wavelength range where maximum absorption was obtained, i.e. in the range from 220 - 232 nm.

EXAMPLE 20

The product from example 1 on the one hand, and a commercial sample of heparin on the other hand, were administered subcutaneously at different times to 5 healthy volunteers at a dose of 5000 i.e. "codex". A blood sample was taken 1 hour, 3 hours, 5 hours and 7 hours after the administration, after which the anticoagulant activity in plasma was measured using the anti-Xa and A.P.T.T.-r tests described earlier. The results achieved are expressed in terms of international units per ml of plasma, referring to a standard curve that was set up from samples carried out on control plasma to which known amounts of reference heparin (3rd international standard) had been added.

The mean results are shown in Table D.

It appears that the product from example 1 has a far stronger anti-Xa effect than commercial heparin.

Claims (2)

1. Process for the production of mixtures of sulfated polysaccharides with a general heparin polysaccharide structure with a lower average molecular weight than heparin where the acidic groups are present in free or salted form, where the polysaccharides in the mixtures have an ethylenic double bond at one of the chain ends and where the mixtures in the sodium salt state exhibit the following characteristics: percent sulfur: 9 to 13.5, percent nitrogen: 1.8 to 2.5, percent uronic acid; 20 to 30, average molecular weight 2000 to 10,000 daltons, specific rotation in aqueous solution: (a)<20> = +25 to +55°, characterized in that a heparin ester obtained by partial or total esterification of the carboxylic acid groups in heparin is reacted with an alkali or organic base at a temperature of 20 to 80°C either in an aqueous environment if the heparin ester is soluble in water or in an inert solvent for the ester, the thus obtained depolymerisation product is isolated as an alkali salt and the latter is hydrolysed by means of an aqueous, at least 1 N sodium hydroxide solution at a temperature of 0 to 5°C. 2. Method according to claim 1, characterized in that a base is used with a molar concentration in the aqueous environment of 0.1 to 0.6. 3- Process according to claim 1, characterized in that alkali hydroxides, alkali carbonates, triethylamine or 1,5-diaza(4,3,0)bicyclo-5-nonene are used as bases.