NZ537411A - Heparin-derived polysaccharide mixtures and preparation method - Google Patents

Abstract

Disclosed is a method of preparing a mixture of sulphated polysaccharides possessing the general structure of the constituent polysaccharides of heparin and exhibiting characteristics as described in the form of an alkali or alkaline-earth metal salt wherein the method comprises: a) depolymerising a quaternary ammonium salt of the benzyl ester of heparin in an organic medium by means of a strong organic base with a pKa greater than 20, wherein said strong organic is of formula (I); wherein R1 to R5 each independently represent a straight of branched chain alkyl radical having from 1 to 6 carbon atoms, with the proviso that the strong organic base is not 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine; b) Converting the quaternary ammonium salt of the benzyl ester of the depolymerised heparin to a sodium salt; c) Saponifying the ester; and optionally d) Purifying the product. (61) Addition to 524028

Description

New Zealand Paient Spedficaiion for Paient Number 537411 53 1 U NEW ZEALAND PATENTS ACT, 1953 No: Patent of Addition to NZ 524028 Date: Dated 18 July 2001 complete specification HEPARIN-DERIVED POLYSACCHARIDE MIXTURES AND PREPARATION METHOD We, AVENTIS PHARMA S.A., of 20 Avenue Raymond Aron, F-92160 ANTONY, France, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: - 1 2 r\ ^ uC. jivju'I R E C F ! V F H 9 Ji 307014-1 HEPARIN-DERIVED POLYSACCHARIDE MIXTURES AND PREPARATION METHOD This application is a patent of addition to New Zealand specification No. 524028 (NZ 524028) filed on 18 July 2001.

TECHNICAL FIELD The present invention relates to mixtures of polysaccharides derived from heparin and, more particularly, to their method of preparation which comprises a modification of the invention of NZ 524028.

BACKGROUND ART Heparin is a heterogeneous group of sulphated mucopolysaccharides of animal origin. As used herein, "heparin" refers to a composition comprising one or more individual sulphated mucopolysaccharides in this group.

Heparin is used for its anticoagulant and antithrombotic properties.

Heparin nevertheless has disadvantages which limit the conditions for its use. In particular, its high anticoagulant activity (anti-IIa activity) can cause haemorrhages.

Low molecular weight heparins obtained by basic depolymerization of heparin esters have been proposed in EP 00 40144; however, these low molecular weight heparins still have a high anti-II anticoagulant activity.

Accordingly, it is an object of the present invention to provide a method of preparing a mixture of sulphated polysaccharides possessing a more selective activity towards activated factor X (factor Xa) and activated factor II 307014-1 (factor Ila) than heparin, or to at least provide the public with a useful choice.

Other objects of the invention may become apparent from the following description which is given by way of example only.

Any discussion of documents, acts, materials, devices, articles or the like which has.been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date.

SUMMARY OF THE INVENTION Accordingly, in one aspect, the present invention provides a method of preparing a mixture of sulphated polysaccharides possessing the general structure of the constituent polysaccharides of heparin and exhibiting the following characteristics: - having a mean molecular weight of 1500 to 3000 daltons, an anti-Xa activity of 100 to 150 IU/mg, an anti-Ila activity of 0 to 10 IU/mg and an anti-Xa activity/anti-Ila activity ratio greater than 10, - the constituent polysaccharides of the mixture containing 2 to 26 saccharide units and having a 4,5 unsaturated glucuronic acid 2-O-sulphate unit at one of their ends, in the form of an alkali or alkaline-earth metal salt, wherein the method comprises: 307014-1 (a) depolymerising a quaternary ammonium salt of the benzyl ester of heparin in an organic medium by means of a strong organic base with a pKa greater than 20, wherein said strong organic base has the formula: wherein R1 to R5 each independently represent a straight or branched chain alkyl radical having from 1 to 6 carbon atoms, with the proviso that the strong organic base is not 2-tert-butylimino-2-diethylamino-l,3-dimethylperhydro-l,3,2-diazaphosphorine; (b) converting the quaternary ammonium salt of the benzyl ester of the depolymerized heparin to a sodium salt; (c) saponifying the ester; and optionally (d) purifying the product.

In a further aspect, the present invention provides a mixture of sulphated polysaccharides possessing the general structure of the constituent polysaccharides of heparin and exhibiting the following characteristics: - having a mean molecular weight of 1500 to 3000 daltons, an anti-Xa activity of 100 to 150 IU/mg, an anti-Ila activity of 0 to 10 IU/mg and an anti-Xa activity/anti-Ila activity ratio greater than 10, - the constituent polysaccharides of the mixture containing 2 to 26 saccharide units and having a 4,5 307014-1 unsaturated glucuronic acid 2-0-sulphate unit at one of their ends, in the form of an alkali or alkaline-earth metal salt, when prepared by a method of the invention.

In a yet further aspect, the present invention provides a pharmaceutical composition comprising a mixture of sulphated polysaccharides prepared by a method of the invention.

In a still further aspect, the present invention provides a use of a mixture of sulphated polysaccharides prepared by a method of the invention in the manufacture of an anti-thrombotic medicament.

This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Although the present invention is broadly as defined above, those persons skilled in the art will appreciate that the invention is not limited thereto and that the invention 307014-1 also includes embodiments of which the following description gives examples.

DETAILED DESCRIPTION OF THE INVENTION The subject of the present invention is more particularly a method of preparing a mixture of sulphated polysaccharides possessing the general structure of the constituent polysaccharides of heparin and exhibiting the following characteristics: - having a mean molecular weight of 1500 to 3000 daltons, an anti-Xa activity of 100 to 150 IU/mg, an anti-Ila activity of 0 to 10 IU/mg, and an anti-Xa activity/anti-Ila activity ratio greater than 10:1, - the constituent polysaccharides of the mixture containing 2 to 26 saccharide units and having a 4,5-unsaturated glucuronic acid 2-O-sulphate unit at one of their ends, in the form of an alkali or alkaline-earth metal salt.

Preferred alkali or alkaline-earth metal salts are the sodium, potassium, calcium and magnesium salts.

The mean molecular weight of compositions of the invention can be determined by high-pressure liquid chromatography. An example of this method uses two columns in series, for example those marketed under the name TSK G3000 XL and TSK G2000 XL. In this method, the detection can be carried out by refractometry using a lithium nitrate eluent and a flow rate of 0.6 ml/min. The system may be calibrated with standards prepared by fractionation of enoxaparin (AVENTIS) by chromatography on agarosepolyacrylamide gel (IBF), according to the technique described by Barrowcliffe et al., Thromb. Res., 12, 27-36 307014-1 (1977-78), or D. A. Lane et al., Thromb. Res., 12, 257-271 (1977-78). The results can be calculated with the GPC6 software (Perkin Elmer).

The anti-Xa activity of a composition of the invention may be measured by the amidolytic method on a chromogenic substrate described by Teien et al., Thromb. Res., 10, 399-410 (1977), with, as standard, the first international standard for low-molecular weight heparins.

The anti-IIa activity of a composition of the invention may be measured by the technique described by Anderson L. 0. et al., Thromb. Res., 15, 531-541 (1979), with, as standard, the first international standard for low-molecular weight heparins.

Preferably, the mixture of sulphated polysaccharides prepared according to the method of the invention exhibits an anti-Xa activity of between 125 and 150 IU/mg.

More preferably, the mixture of sulphated polysaccharides prepared according to the method of the invention exhibits an anti-Xa activity of between 140 and 150 IU/mg and has a mean molecular weight of between 2000 and 3000 daltons.

Preferably, the mixture of sulphated polysaccharides prepared according to the method of the invention has an anti-IIa activity of 0 to 5 IU/mg.

More preferably, the mixture of sulphated polysaccharides prepared according to the method of the invention has an anti-Xa activity/anti-IIa activity ratio greater than 25.

The mixture of sulphated polysaccharides is prepared by depolymerization of a quaternary ammonium salt of the benzyl 307014-1 ester of heparin in an organic medium, by means of a strong organic base with a pKa greater than 20, conversion of the quaternary ammonium salt of the benzyl ester of the depolymerized heparin to a sodium salt, saponification of the ester, and optionally purification.

The quaternary ammonium salt of the benzyl ester of heparin utilised in the method of the present invention is preferably the benzethonium, cetylpyridinium, or cetyltrimethylammonium salt.

The depolymerization of the method of the invention is generally carried out in an inert organic solvent such as a chlorinated solvent (for example, dichloromethane), tetrahydrofuran, or anisole, and at a temperature ranging from -20°C. to 40°C.

NZ 524028 describes and claims a method of preparing a mixture of sulphated polysaccharides which uses a strong organic base with a pKa greater than 20 which is selected from 1,5,7-triaza-bicyclo-[4.4.0]-dec-5-ene, 2-tert-butylimino-2-diethylamino-l,3-dimethylperhydro-l, 3, 2-diaza-phosphorine, guanidine bases and phosphazene bases.

The guanidine bases useful in the invention of NZ 524028 are preferably of formula: wherein Ri is hydrogen or a Ci~C6 alkyl group, and R2, R3, R4, and R5, which are identical or different, each represent a Ci-C6 alkyl group. r4 r3 307014-1 The strong organic phosphazene bases useful in the invention of NZ 524028 are described in R. Schwesinger et al., Angew. Chem. Int. Ed. Eng. 26, 1167-1169-(1987) , and R. Schwesinger et al., Angew. Chem. 105, 1420 (1993).

Phosphazene bases preferred for use in the invention of NZ 524028 are of the formula: Rs / R2 N R4 \ / R-i N=R N R5 \ N R6 r7 wherein Ri to R7 are identical or different and represent Ci~ C6 alkyl groups.

As used herein, the term "C1-C6 alkyl" includes straight and branched hydrocarbon groups.

The method of the present invention utilises a strong organic base with a pKa greater than 20, wherein said strong organic base has the formula: wherein R1 to R5 each independently represent a straight or branched chain alkyl radical having from 1 to 6 carbon atoms, with the proviso that the strong organic base is not 2-tert-butylimino-2-diethylamino-l,3-dimethylperhydro-l,3,2-diazaphosphorine. 307014-1 Advantageously, the mol ratio of the strong organic base with a pKa greater than 20 to the quaternary ammonium salt of the benzyl ester of heparin is between 0.2 and 5 and preferably between 1 and 4.

Preferably, the quaternary ammonium salt of the benzyl ester of heparin has a degree of esterification between 50% and 100%, more preferably between 70% and 90%. This degree of esterification corresponds to the molar percentage of esterification of the uronic acids of the heparin.

The conversion of the quaternary ammonium salt of the benzyl ester of the depolymerized heparin to a sodium salt is generally carried out by treating the reaction medium with an alcoholic solution of sodium acetate. For example, the conversion may be carried out by treating the reaction medium with a 10% solution of sodium acetate in methanol (weight/volume), at a temperature between 15°C to 25°C. The equivalent by weight of acetate added is preferably 3 times greater than the mass of quaternary ammonium salt of the benzyl ester of heparin used in the depolymerization reaction.

The saponification may be carried out using an alkali metal hydroxide. Suitable alkali metal hydroxides include sodium hydroxide, potassium hydroxide and lithium hydroxide. The saponification is generally carried out in an aqueous medium, at a temperature between 0°C and 20°C, preferably between 0°C and 10°C. Generally, 1 to 5 molar equivalents of alkali metal hydroxide can be used. Preferably, the saponification is carried out in the presence of 2 to 3 molar equivalents of alkali metal hydroxide.

The final product may be optionally purified by any known method of purifying depolymerized heparins, for example according to the method described in EP 0037319. 307014-1 Preferably, the purification is carried out using hydrogen peroxide, in an aqueous medium, at a temperature between 10°C and 50°C. Preferably, this operation is carried out between 20°C and 40°C.

The quaternary ammonium salt of the benzyl ester of heparin may be prepared according to the following reaction scheme: (a) conversion of the heparin in the form of a sodium salt using benzethonium chloride in order to obtain benzethonium heparinate, (b) esterification of the benzethonium salt obtained above using benzyl chloride and treatment with an alcoholic solution of sodium acetate to obtain the sodium salt of the benzyl ester of heparin, and (c) conversion of the sodium salt of the benzyl ester of heparin to a quaternary ammonium salt, preferably a benzethonium, cetylpyridinium, or cetyltrimethylammonium salt.

The reaction of step (a) may be carried out in an aqueous medium by the action of excess benzethonium chloride on heparin in the form of a sodium salt, at a temperature between 15°C and 25°C.

Advantageously, the molar ratio of benzethonium chloride/heparin in the form of a sodium salt is between 2 and 3, and preferably 2.5.

The starting heparin in the form of a sodium salt used is preferably a pig heparin, which may be purified beforehand in order to reduce its dermatan sulphate level according to the method described in FR 2663639. 307014-1 The esterification of step (b) is preferably carried out in a chlorinated organic solvent (for example, chloroform or methylene chloride), at a temperature between 25°C and 45°C and preferably between 30°C and 40°C. The sodium salt of the ester may be recovered by precipitation using sodium acetate at 10% by weight/volume in an alcohol, for example methanol. Generally, 1 to 1.2 volumes of alcohol are used per volume of reaction medium. The quantity of benzyl chloride and the reaction time may be adjusted to obtain a degree of esterification between 50% and 100%, preferably between 70% and 90%. Preferably, 0.5 to 1.5 parts by weight benzyl chloride are used for 1 part by weight of benzethonium salt of heparin. The reaction time will typically range from 10 to 35 hours.

The conversion step (c) may be carried out using a quaternary ammonium chloride. Preferably, benzethonium chloride, cetylpyridinium chloride, or cetyltrimethylammonium chloride are used, in an aqueous medium, at a temperature between 10°C and 25°C. Advantageously, the mol ratio of the quaternary ammonium chloride to the sodium salt of the benzyl ester of heparin is between 2 and 3.

The mixture of sulphated polysaccharides prepared according to the method of the invention when obtained in the form of a sodium salt, may be converted to a salt of another alkali or alkaline-earth metal. The conversion from one salt to another is optionally achieved using the method described in US 4,168,377.

The mixture of sulphated polysaccharides prepared according to the method of the invention is not toxic and may be used as a medicament. 307014-1 The mixture of sulphated polysaccharides prepared according to the method of the invention may be used as an antithrombotic agent. Such antithrombotic agents may be useful in the treatment or the prevention of venous thromboses and arterial thrombotic accidents, including myocardial infarction. The mixture of sulphated polysaccharides prepared according to the method of the invention may also be useful in the prevention and treatment of the proliferation of the smooth muscle cells, angiogenesis, and as a neuroprotective agent for atherosclerosis and for arteriosclerosis.

The present invention also relates to a pharmaceutical composition comprising a mixture of sulphated polysaccharides prepared according to the method of the invention a composition according to the invention, optionally in combination with one or more inert excipients.

The pharmaceutical compositions may, for example, be formulated as solutions which can be injected subcutaneously, intramuscularly or intravenously. Alternatively it may be formulated for pulmonary administration (inhalation).

The dosage may vary according to the age, weight and state of health of the patient. For an adult, a suitable dosage may range from 20 to 100 mg per day when administered by subcutaneous, intramuscular or intravenous route.

The following non-limiting examples are provided to illustrate the present invention and in no way limit the scope thereof. 307014-1 EXAMPLES EXAMPLE 1: Preparation of the Benzethonium Salt of the Benzyl Ester of Heparin A. Benzethonium Heparinate A solution of 25 g of benzethonium chloride in 125 ml of water is added to a solution of 10 g of heparin in the form of a sodium salt in 100 ml of water at a temperature in the region of 20°C. The product is filtered, washed with water and dried.

B. Benzyl Ester of Heparin (Sodium Salt) Benzyl chloride (16 ml) is added to a solution of 20 g of benzethonium heparinate in 80 ml of methylene chloride. The solution is heated at a temperature of 30°C for 12 hours. 108 ml of a 10% solution of sodium acetate in methanol are then added, the mixture is filtered, washed with methanol and dried. 7.6 g of benzyl ester of heparin are thus obtained in the form of a sodium salt with a degree of esterification of 77%.

C. Benzyl Ester of Heparin (Benzethonium Salt) 36 g (0.0549 mol) of benzyl ester of heparin (sodium salt) and 540 ml of distilled water are introduced into a 2-liter Erlenmeyer flask A. After homogenization at a temperature of about 20°C, a pale yellow solution is obtained. A solution of 64.45 g (0.1438 mol) of benzethonium chloride and 450 ml of water is prepared, with magnetic stirring, in a 1-liter Erlenmeyer flask B. Over a period of about 35 minutes, the solution in Erlenmeyer flask B is poured into the solution in Erlenmeyer flask A, with stirring. The formation of an abundant white precipitate is observed. The Erlenmeyer flask B is rinsed with 200 ml of 307014-1 distilled water, and the wash water is introduced into the Erlenmeyer flask A. Stirring is then stopped, and the suspension is allowed to settle for 12 hours. The clear portion of the supernatant is removed and discarded. 560 ml of water are added to the sedimented precipitate (slurry appearance) and the mixture is stirred for 20 minutes. The precipitate is allowed to resediment for about 30 minutes. The supernatant is removed and discarded (560 ml). The sedimented precipitate is washed twice with about 560 ml of distilled water in this manner. In the last washing operation, the precipitate is left in suspension and filtered on a #3 sintered glass. The resulting precipitate cake is then washed 4 times with 200 ml of distilled water. The wet white precipitate solid is drained, then dried under reduced pressure (2.7 kPa) at approximately 60°C. After drying for 12 hours, 87.5 g of benzyl ester of heparin, benzethonium salt, are obtained. The yield obtained is 94.9%.

EXAMPLE 2: Depolymerization and Conversion -to a Sodium Salt Dichloromethane (28 ml) is introduced into a 50 ml Erlenmeyer flask C. 4 g (0.00238 mol) of benzyl ester of heparin (degree of esterification: 77%, benzethonium salt) obtained as described in Example 1 are slowly added, with stirring. After complete dissolution, 1.32 g (0.00948 mol) of 1,5,7-triaza-bicyclo-[4.4.0]-dec-5-ene are added. The mixture is stirred at a temperature in the region of 20°C for 3 hours and 30 minutes. During this time, a solution of 12 g of sodium acetate is prepared at 4°C in an Erlenmeyer flask D in 120 ml of methanol. The reaction mixture in Erlenmeyer flask C is poured into the methanolic solution in Erlenmeyer flask D, with magnetic stirring. A practically translucent gelatinous yellow suspension appears. The stirring is stopped, and the suspension is allowed to 307014-1 separate by settling for one hour. The clear portion of the supernatant is removed and discarded (62 ml). Methanol (50 ml) is added to the sedimented precipitate (yellow slurry appearance) and the mixture is stirred for 20 minutes. The precipitate is allowed to resediment for about 30 minutes. The supernatant is removed and discarded (49 ml). Methanol (50 ml) is added to the sedimented precipitate, and the mixture is stirred for 20 minutes. The suspended precipitate is filtered on a #4 sintered glass. The golden yellow precipitate cake obtained is washed twice with 25 ml of methanol. The wet precipitate solid is drained and then dried under reduced pressure (2.7 kPa), at approximately 60°C After drying for 12 hours, 1.21 g of depolymerized heparin are obtained (benzyl ester, sodium salt). The yield obtained is 77.2%.

Saponification Depolymerized heparin (benzyl ester, sodium salt) obtained above (1.21 g (0.0018 mol)) and 11 ml of water are introduced into a 25 ml Erlenmeyer flask E. 0.18 ml (0.0018 mol) of 30% caustic soda is introduced, with magnetic stirring. After addition, the mixture is cooled to 4°C and stirred for 2 hours. NaCl (1.43 g) is added and the solution is neutralized by addition of 1 mol/1 HCl (14 ml). The mixture is transferred to a 100 ml Erlenmeyer flask F and 52 ml of methanol are added. A yellow precipitate formed. The stirring is stopped, and the suspension is allowed to sediment for 12 hours at approximately 20°C. The supernatant is then removed and then discarded (44 ml). 25 ml of methanol are added to the sedimented precipitate (yellow slurry appearance), and the mixture is stirred for 20 minutes. The precipitate is allowed to resediment for about 30 minutes. The supernatant is removed and discarded (21 ml). Methanol (25 ml) is added to the sedimented precipitate 307014-1 and the mixture is stirred for 20 minutes. The precipitate in suspension is then filtered on a #3 sintered glass. The light yellow precipitate cake obtained is then washed with twice 10 ml of methanol. The wet precipitate solid is drained and then dried under reduced pressure (2.7 kPa), at approximately 60°C. After drying for 12 hours, 0.66 g of crude depolymerized heparin is obtained (sodium salt) . The yield obtained is 60%.

Purification Crude depolymerized heparin obtained above (0.66 g) and 5.9 ml of distilled water are introduced into a 10 ml Erlenmeyer flask G. The mixture is heated to 40°C, with magnetic stirring. The pH is brought to between 9 and 10 by addition of 0.1 mol/1 sodium hydroxide, and 33 |il of 30% aqueous hydrogen peroxide are added. After stirring for about 2 hours, 0.65 g of sodium chloride is added. The mixture is then neutralized by addition of 0.1 mol/1 HCl. The solution is then filtered and transferred to a 25 ml Erlenmeyer flask H. 23.3 ml of methanol is added. A white precipitate forms. The stirring is then stopped, and the suspension is allowed to sediment for 12 hours at approximately 20°C. The supernatant is removed and then discarded (5 ml). Methanol (5 ml) is added to the sedimented precipitate (slurry appearance), and the mixture is stirred for 20 minutes. The precipitate is allowed to resediment for about 30 minutes. The supernatant is removed and discarded (5 ml). Methanol (5 ml) is added to the sedimented precipitate and the mixture is stirred for 20 minutes. The precipitate in suspension is then filtered on a #3 sintered glass. The white precipitate cake obtained is then washed twice with 5 ml of methanol. The wet precipitate solid is drained and then dried under reduced pressure (2.7 kPa), at approximately 60°C. After drying for 12 hours, 0.51 g of a 307014-1 purified mixture of polysaccharides (sodium salt) is obtained. The yield obtained is 77.2%.

The characteristics of this mixture are as follows: Mean molecular weight: 1600 daltons Anti-Xa activity: 94 IU/mg Anti-IIa activity: <0.1 IU/mg Anti-Xa activity/anti-IIa activity ratio: >100 EXAMPLE 3: Depolymerization and Conversion to a Sodium Salt Dichloromethane (70 ml) is introduced into a 100 ml Erlenmeyer flask I. 10 g (0.00595 mol) of benzyl ester of heparin (degree of esterification: 77%, benzethonium salt) obtained as described in Example 1 are slowly loaded, with stirring. After complete dissolution, 1.7 ml (0.00587 mol) of 2-tert-butylimino-2-diethylamino-l,3-dimethylperhydro-l,-3,2-diaza-phosphorine are added. The reaction is allowed to continue for about 3 hours and 30 minutes at approximately 20°C. During this time, a solution of 30 g of sodium acetate in 300 ml of methanol is prepared at 4°C in an Erlenmeyer flask J. The reaction mixture in Erlenmeyer I is poured into the methanolic solution of sodium acetate, with magnetic stirring. A practically translucent gelatinous yellow suspension forms. The stirring is then stopped and the suspension is allowed to settle for one hour. The clear portion of the supernatant is removed and discarded (204 ml). Methanol (125 ml) is added to the sedimented precipitate (yellow slurry appearance), and the mixture is stirred for 20 minutes. The precipitate is allowed to resediment for about 30 minutes. The supernatant is then removed and discarded (162 ml). Methanol (125 ml) is added to the sedimented precipitate and the mixture is stirred for 307014-1 minutes. The suspended is then filtered on a #3 sintered glass. The yellow gelatinous precipitate cake obtained is then washed twice with 63 ml of methanol. The gelatinous precipitate solid is drained and dried under reduced pressure (2.7 kPa), at approximately 60°C. After drying for 12 hours, 3.34 g of depolymerized heparin (benzyl ester, sodium salt) are obtained. The yield obtained is 85.3%.

Saponification Depolymerized heparin (benzyl ester, sodium salt) obtained above (1.67 g) is saponified according to the saponification method described in Example 2. 0.94 g of a light yellow powder is obtained. The yield of crude depolymerized heparin (sodium salt) is 61%.

Purification Crude depolymerized heparin (sodium salt) obtained above (0.94 g) is purified according to the method of purification described in Example 2. 0.71 g of a white powder is obtained. The yield is 75.5%.

The purified mixture of polysaccharides (sodium salt) obtained has the following characteristics: Mean molecular weight: 2500 daltons Anti-Xa activity: 146.6 IU/mg Anti-IIa activity: 2.15 IU/mg Anti-Xa activity/anti-IIa activity ratio: 68 EXAMPLE 4: Depolymerization and Conversion to a Sodium Salt Dichloromethane (140 ml) is introduced into a 400 ml reactor A. Benzyl ester of heparin (degree of esterification: 77%, benzethonium salt) obtained as 307014-1 described in Example 1 (20 g (0.0119 mol)) is slowly loaded, with stirring. After complete dissolution, the water content of the reaction medium is measured by the Karl Fisher method. The value obtained is 0.1% water. 3.5 ml (0.0121 mol) of 2-tert-butylimino-2-diethy- 1-amino-l,3-dimethylperhydro-1,3,2-diazaphosphorine are then added. The reaction is allowed to proceed for about 24 hours at approximately 25°C. During this time, a solution of 30 g of sodium acetate in 300 ml of methanol is prepared at 4°C in an Erlenmeyer flask K. Half of the reaction mixture of reactor A is poured into the methanolic solution of sodium acetate, with magnetic stirring. A practically translucent gelatinous yellow precipitate appears. The stirring is maintained for one hour and the suspension is allowed to separate by settling for about 12 hours at 4°C. The clear portion of the supernatant is removed and discarded (220 ml). Methanol (220 ml) is added to the sedimented precipitate (yellow slurry appearance) and the solution is stirred for 50 minutes. The precipitate is allowed to resediment for about 40 minutes. The supernatant is removed and discarded (204 ml). Methanol (204 ml) is added to the sedimented precipitate and the solution is stirred for 40 minutes. The gelatinous precipitate in suspension is then filtered on a #3 sintered glass. The yellow gelatinous precipitate cake obtained is then washed twice with 100 ml of methanol. The gelatinous precipitate solid is drained and then dried under reduced pressure (2.7 kPa), at approximately 60°C. After drying for about 12 hours, 2.6 g of depolymerized heparin (benzyl ester, sodium salt) are obtained. The yield obtained is 70.6% (calculated on the basis of half of the reaction medium treated). 307014-1 Saponification Depolymerized heparin (benzyl ester, sodium salt) obtained above (2.6 g) is saponified according to the saponification method described in Example 2. 1.48 g of a light yellow powder are obtained. The yield of crude depolymerized heparin (sodium salt) is 62.9%.

Purification Crude depolymerized heparin obtained above (1.48 g) and 15 ml of distilled water are introduced into a 50 ml Erlenmeyer flask L. The mixture is heated to 40°C, with magnetic stirring. The pH is brought to between 9 and 10 by addition of 1 mol/1 sodium hydroxide. The solution is filtered on a filter membrane having a porosity of 1 pm. 76 ial of 30% aqueous hydrogen peroxide are then added. After stirring for about 2 hours, 1.5 g of sodium chloride are added. The mixture is then neutralized by addition of 1 mol/1 HCl. The solution is filtered on a filter membrane having a porosity of 1 jam. Methanol (38 ml) is poured into the solution. The formation of a white precipitate is observed. The stirring is then stopped and the suspension is allowed to sediment for 1 hour at approximately 20°C. The supernatant is then removed and then discarded (37 ml). 37 ml of methanol are added to the precipitate and the mixture is stirred for 45 minutes. The precipitate is allowed to resediment for about 45 minutes. The supernatant is removed and discarded (34 ml). Methanol (34 ml) is added to the sedimented precipitate and the mixture is stirred for 15 minutes. The precipitate in suspension is then filtered on a #3 sintered glass. The white precipitate cake obtained is then washed twice with 25 ml of methanol. The wet precipitate solid is drained and then dried under reduced pressure (2.7 kPa), at approximately 60°C. After drying for 12 hours, 1.29 g of 307014-1 pure depolymerized heparin (sodium salt) are obtained. The yield obtained is 87.2%.

The purified depolymerized heparin (sodium salt) obtained has the following characteristics: Mean molecular weight: 2250 daltons Anti-Xa activity: 149.6 IU/mg Anti-IIa activity: <0.85 IU/mg Anti-Xa activity/anti-IIa activity ratio: 176 It is not the intention to limit the scope of the invention to the abovementioned examples only. As would be appreciated by a skilled person in the art, many variations are possible without departing from the scope of the invention (as set out in the accompanying claims). 307014-1

Claims (15)

WHAT WE CLAIM IS:

1. A method of preparing a mixture of sulphated polysaccharides possessing the general structure of the constituent polysaccharides of heparin and exhibiting the following characteristics: - having a mean molecular weight of 1500 to 3000 daltons, an anti-Xa activity of 100 to 150 IU/mg, an anti-IIa activity of 0 to 10 IU/mg and an anti-Xa activity/anti-Ila activity ratio greater than 10, - the constituent polysaccharides of the mixture containing 2 to 26 saccharide units and having a 4,5 unsaturated glucuronic acid 2-0-sulphate unit at one of their ends, in the form of an alkali or alkaline-earth metal salt, wherein the method comprises: (a) depolymerising a quaternary ammonium salt of the benzyl ester of heparin in an organic medium by means of a strong organic base with a pKa greater than 20, wherein said strong organic base has the formula: wherein R1 to R5 each independently represent a straight or branched chain alkyl radical having from 1 to 6 carbon atoms, with the proviso that the strong organic base is not 2-tert-butylimino-2-diethylamino-l,3-dimethylperhydro-l,3,2-diazaphosphorine; 307014-1 -24- (b) converting the quaternary ammonium salt of the benzyl ester of the depolymerized heparin to a sodium salt; (c) saponifying the ester; and optionally (d) purifying the product.

2. A method according to claim 1, wherein the quaternary ammonium salt of the benzyl ester of heparin is the benzethonium, cetylpyridinium or cetyltrimethylammonium salt.

3. A method according to claim 1 or claim 2 wherein the mole ratio of the strong organic base to the quaternary ammonium salt of the benzyl ester of heparin is between 0.2 and 5.

4. A method according to any preceding claim wherein the quaternary ammonium salt of the benzyl ester of heparin has a degree of esterification of between 50 and 100%.

5. A method according to any preceding claim wherein the conversion of the quaternary ammonium salt of the benzyl ester of depolymerized heparin to a sodium salt is carried out by treating the reaction medium with an alcoholic solution of sodium acetate.

6. A method according to any preceding claim wherein the saponification is carried out by means of an alkali metal hydroxide.

7. A method according to any preceding claim wherein the purification is carried out by means of hydrogen peroxide.

8. A mixture of sulphated polysaccharides possessing the general structure of the constituent polysaccharides of heparin and exhibiting the following characteristics: 307014-1 -25- - having a mean molecular weight of 1500 to 3000 daltons, an anti-Xa activity of 100 to 150 IU/mg, an anti-IIa activity of 0 to 10 IU/mg and an anti-Xa activity/anti-Ila activity ratio greater than 10, - the constituent polysaccharides of the mixture containing 2 to 26 saccharide units and having a 4,5 unsaturated glucuronic acid 2-0-sulphate unit at one of their ends, in the form of an alkali or alkaline-earth metal salt, when prepared by a method according to any one of claims 1 to 7.

9. A pharmaceutical composition comprising a mixture of sulphated polysaccharides according to claim 8.

10. A pharmaceutical composition according to claim 9 further comprising one or more inert excipients.

11. A use of a mixture of sulphated polysaccharides according to claim 8 in the manufacture of an antithrombotic medicament.

12. A method as defined in claim 1 substantially as herein described with reference to any example thereof.

13. A mixture of sulphated polysaccharides as claimed in claim 8 substantially as herein described with reference to any example thereof.

14. A pharmaceutical composition as defined in claim 9 substantially as herein described with reference to any example thereof.

15. A use as claimed in claim 11 substantially as herein described with reference to any example thereof. 307014-1