OA12340A - Heparin - derived polysaccharide mixtures, their preparation and pharmaceutical compositions containing them. - Google Patents

Abstract

The invention concerns heparin-derived polysaccharide mixtures, their preparation method and pharmaceutical compositions containing them

Description

012340

The present invention relates to mixtures of heparin-derived polysaccharides, to a process for their preparation and to pharmaceutical compositions containing them. Heparin is a mixture of sulfated mucopolysaccharides of animal origin, used in particular for its anticoagulant and antithrombotic properties. Heparin, however, has drawbacks that limit the conditions of its use. In particular, its anticoagulant activity (anti-IIa activity) importantpeut cause hemorrhages.

Low molecular weight heparins obtained by basic depolymerization of heparin esters have been proposed (EP40144); however, these products have significant anti-IIa activity. The invention relates to mixtures of heparin-derived polysaccharides having a more selective activity with respect to activated factor X (factor Xa) and activated factor II (factor Ha) than heparin.

The present invention more particularly relates to sulfated polysaccharide mixtures having the general structure of the polysaccharides constituting heparin and having the following characteristics: they have an average 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-IIa activity ratio of greater than 10; the constituent polysaccharides of the mixtures contain 2 to 26 and have a 4,5-glucuronic acid unit unsaturated 2-O-sulphate at one end, in the form of an alkali or alkaline earth metal salt.

As the alkali or alkaline earth metal salt, the sodium, potassium, calcium and magnesium salts are preferred. 2 012340

The average molecular weight is determined by liquid chromatography hantepression using two columns in series for example those sold under the name of TSK G3000 XL and TSK G2000 XL. The detection is carried out by refractometry. The eluent used is lithium nitrate and the flow rate is 0.6 ml / min. The system is calibrated with standards prepared by fractionation of enoxaparin (AVENTIS) by agarose-polyacrylamide gel (IBF) chromatography. This preparation is carried out according to the technique described by Barrowcliffe et al., Thromb. Res., 12, 27-36 (1977-78) or D.A. Lane et al., Thromb. Res., 12, 257-271 (1977-78). The results are calculated using the GPC6 software (PerkinElmer). Anti-Xa activity is measured by the amidolytic method on a substratchromogenic described by Teien et al., Thromb. Res., 10, 399-410 (1977), as a standard the first international standard for low molecular weight heparins. Anti-Ha activity is measured by the technique described by Anderson L.O. et al., Thromb. Res., 15, 531-541 (1979), with as standard the first international standard of low molecular weight heparins.

Preferably, the mixtures as described above have an anti-toxic activity between 125 and 150 IU / mg.

In particular, the mixtures as described above have an activity Xa between 140 and 150 IU / mg and have an average molecular weight between 2000 and 3000 daltons.

Preferably, the mixtures according to the invention have an anti-IIa activity of 0 to 5 IU / mg.

Even more preferably, the mixtures have an anti-Xa activity / anti-IIa activity ratio greater than 25.

The oligosaccharide mixtures according to the invention can be prepared by the depolymerization of a quaternary ammonium salt of the benzyl ester of heparin in organic medium, by means of a strong organic base of pka greater than 20 or of imidazolate of sodium, conversion of the quaternary ammonium salt of the depolymerized heparin benzyl ester to the sodium salt, saponification of the ester and optionally purification. 012340

The quaternary ammonium salt of the benzyl ester of heparin is preferably the benzethonium, cetylpyridinium or cetyltrimethylammonium salt.

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

As a strong organic base of pka greater than 20, 1,5,7-triazabicyclo [4.4.0] dec-5-ene, 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1 can be used. , 3,2-diazaphosphorine, the bases of the family of guanidine and phosphazenes. The bases of the family of guanidines are preferably those of formula:

R4 R3 in which R! is hydrogen or alkyl, R 3, R 3, R 4 and R 5, which are identical or different, each represent an alkyl radical.

More particularly, R! is hydrogen and R2, R3, R4 and R5 are methyl radicals. Strong organic bases related to the family of phosphazenes are defined for example according to R. Schwesinger et al., Angew. Chem. Int. Ed. Engl. 26, 1167-1169 (1987), R. Schwesinger et al., Angew. Chem. 105, 1420 (1993).

Among the bases of the phosphazenes family, those of the formula R 3 R 2 -N, R 4, R 1 -N = P-N-R 5 N-R 6 / R 7 in which the radicals R 1 to R 7 are identical or different are preferably used. and represent alkyl radicals. 012340

In the preceding formulas, the alkyl radicals contain 1 to 6 carbon atoms in straight or branched chain.

Advantageously, the molar ratio of strong organic base of pka greater than 20 sodium or sodium quaternary ammonium salt of the benzyl ester of heparinis between 0.2 and 5 and preferably between 1 and 4.

More particularly, the esterification level of the quaternary ammonium salt of heparin benzyl ester is between 50 and 100% and preferably between 70 and 90%. This degree of esterification corresponds to the molar percentage of esterification of the uronic acids of heparin.

The conversion of the quaternary ammonium salt of the benzyl ester of the heparin-polymerization to the sodium salt is generally carried out by treating the reaction mixture with an alcoholic solution of sodium acetate and, preferably, with a 10% strength solution. sodium acetate in methanol (w / v), at a temperature between 15 and 25 ° C. The weight equivalent of added acetate ispreferentially 3 times greater than the mass of quaternary ammonium salt of the benzyl ester of heparin engaged in the depolymerization reaction.

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

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

The quaternary ammonium salt of the benzyl ester of heparin can be prepared according to the following reaction scheme: a) transformation of heparin in the sodium salt form with benzenemonium chloride to give benzethonium heparinate, B) esterification of the benzethonium salt obtained previously with benzyl chloride and treatment with an alcoholic solution of sodium acetate to obtain the sodium salt of the benzyl ester of heparin, c) transalification of the sodium salt of the benzyl ester of heparin to quaternary ammonium salt and, preferably, benzethonium salt, decetylpyridinium or cetyltrimethylammonium salt,

The reaction of step a) is carried out in aqueous medium, by the action of excess benzenium chloride, on heparin in the form of sodium salt, at a temperature of about 15 to 25 ° C. Advantageously, the molar ratio of benzenemonium chloride / heparin in the form of sodium salt is between 2 and 3 and more particularly 2.5. The starting sodium salt heparin used is preferably a porcine heparin. This may be preliminarily purified to reduce its dermatosan sulfate level according to the process described in FR2663639. The esterification of step b) is preferably carried out in an organic chlorinated solvent (chloroform, methylene chloride, for example) at a temperature of between 25 and 45 ° C. and preferably between 30 and 40 ° C. . The ester in the form of sodium salt is then recovered by precipitation with 10% w / v sodium acetate in an alcohol such as methanol. Generally 1 to 1.2 volumes of alcohol per volume of reaction medium are employed. The amount of benzyl chloride and the reaction time are adapted to obtain an esterification rate of between 50 and 100% and preferably between 70 and 90%. Preferably, 0.5 to 1.5 parts by weight of benzyl chloride are used per 1 part by weight of the benzenemonium salt of heparin. Likewise, preferably, the reaction time will be between 10 and 35 hours.

The transalification of step c) is carried out by means of a quaternary ammonium chloride and, preferably, by means of benzethonium chloride, cetylpyridinium chloride or cetyltrimethylammonium chloride, in an aqueous medium, at a temperature of between 10.degree. and 25 ° C. Advantageously, the ratio of quaternary ammonium hydroxide / sodium salt of the benzyl ester of heparin is comprised between 2 and 3.

The mixtures according to the invention in the form of sodium salt can be converted into another salt of an alkali metal or alkaline earth metal. One passes optionally from one to the other using the method described in US4168377.

The mixtures according to the invention are not toxic and can thus be used as medicaments.

The mixtures of the present invention can be used as thithrombotic agents. In particular, they are useful for the prevention of thrombosesveins, arterial thrombotic accidents, especially in the case of myocardial infarction. They are also useful in the prevention and treatment of smooth muscle cell proliferation, angiogenesis, and as agents-inhibitors of atherosclerosis and arteriosclerosis.

The present invention also relates to pharmaceutical compositions comprising as active principle a mixture of polysaccharides according to the invention optionally in association with one or more inert excipients.

The pharmaceutical compositions are, for example, injectable solutions that are administered subcutaneously or intravenously. They are also useful by the pulmonary route (inhalation).

The dosage may vary according to the age, weight and state of health of the patient.For an adult, it is generally between 20 and 100 mg per day by intramuscular or subcutaneous route.

The following examples illustrate the invention.

EXAMPLE A PREPARATION OF BENZETHONIUM SALT OF ESTERBENZYLIC HEPARIN

Benzethonium heparinate To a solution of 10 g of heparin in the form of sodium salt in 100 ml of water at a temperature in the region of 20 ° C., a solution of 25 g of benzenemonium chloride in 125 ml of water is added. The product is filtered, washed with water and dried.

Heparin benzyl ester (sodium salt) To a solution of 20 g of benzethonium heparinate in 80 ml of methylene chloride was added 16 ml of benzyl 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, filtered, washed with methanol and dried. 7.6 g of benzyl ester of heparin are thus obtained in the form of sodium salt, the esterification rate of which is 77%.

Benzyl heparin ester (benzethonium salt)

In a 2 liter Erlenmeyer flask, 36 g (0.0549 mol) of benzyl ester of heparin (sodium salt) and 540 ml of distilled water are introduced. After homogenization at a temperature of about 20 ° C, a pale yellow solution is obtained. With magnetic stirring, a solution of 64.45 g (0.1488 mol) of benzethonium chloride and 450 ml of water is prepared in a 1 liter Erlenmeyer flask B. The solution of Erlenmeyer B is poured in about 35 minutes into the solution of the stirrer as stirring. The formation of an abundant white precipitate is observed. The Erlenmeyer flask is rinsed with 200 ml of distilled water and the washing water is introduced into the Erlenmeyer flask. Stirring is then stopped and the suspension is allowed to settle for 12 hours. The clear portion of the supernatant is removed and discarded. On the sedimented precipitate (slurry appearance), 560 ml of water is added and stirred for 20 minutes. The precipitate is left to sediment in about 30 minutes. The supernatant is removed and discarded (560 ml). On the sedimented precipitate, this wash operation is twice repeated with about 560 ml of distilled water. In the last washing operation, the precipitate is left in suspension and filtered on Sintered Glass 3. The cake is then washed with 4 times 200 ml of distilled water. The wet white solid is drained and then dried under reduced pressure (2.7 kPa), at a temperature in the region of 60 ° C. After 12 hours of drying, 87.5 g of benzyl ester of heparin, benzethonium salt, are obtained. The yield obtained is 94.9%. EXAMPLE 1 Depolymerization and transformation into sodium salt

In a 50 ml Erlenmeyer flask, 28 ml of dichloromethane are introduced. Under stirring, 4 g (0.00238 mol) of benzyl ester of heparin (esterification rate: 77%, benzethonium salt) obtained as described in Example A were slowly charged. After complete dissolution, 1.32 was added. g (0.00948 moles) of 1,5,7,8-triazabicyclo [4.4.0] dec-5-ene. 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 in 120 ml of methanol is prepared at 4 ° C. in an Erlenmeyer flask B. With magnetic stirring, the reaction mixture of the Erlenmeyer flask A is poured into the ethanolic sodium acetate solution. A gelatinous yellow precipitate almost translucent appears. The stirring is then stopped and the suspension is allowed to settle for a time. The clear portion of the supernatant is removed and discarded (62 ml). On the precipitated sediment (yellow slurry appearance), 50 ml of methanol was added and stirred for 20 minutes. The precipitate is left to sediment for approximately 30 minutes. The supernatant is removed and discarded (49 ml). On the sedimented precipitate, 50 ml of methanol are added and the mixture is stirred for 20 minutes. The precipitate in suspension is then filtered on Glass-Filled 4. The gold-yellow cake obtained is then washed with twice 25 ml of methanol. The wet solid is drained and then dried under reduced pressure (2.7 kPa) at a temperature of 60 ° C. After 12 hours of drying, 1.21 g of heparinedepolymerized (benzyl ester, sodium salt) are obtained. The yield obtained is 77.2%.

Saponification:

In a 25-ml Erlenmeyer flask, 1.21 g (0.0018 mol) of the previously obtained heparin-polymerization (benzyl ester, sodium salt) and 11 ml of water are introduced. Under magnetic stirring, 0.18 ml (0.0018 mol) of 30% desolder liquor is introduced. After addition, the mixture is cooled to 4 ° C. and stirred for 2 hours. 1.43 g of NaCl are added and the solution is neutralized by addition of 1 mol / l HCl (14 ml). The mixture is transferred to a 100 ml Erlenmeyer flask and 52 ml of methanols are added. The formation of a yellow precipitate is observed. The stirring is then stopped and the suspension is left to sediment for 12 hours at a temperature in the region of 20 ° C. The supernatant is then removed and discarded (44 ml). On the sedimented precipitate (yellow slurry appearance), 25 ml of methanol are added and stirred for 20 minutes. The precipitate is left to sediment for approximately 30 minutes. The supernatant is removed and discarded (21 ml). On the sedimented precipitate, 25 ml of methanol are added and the mixture is stirred for 20 minutes. The precipitate in suspension is then filtered on Refrigerated Glass 3. The light yellow cake obtained is then washed with twice 10 ml of methanol. The wet solid is drained and then dried under reduced pressure (2.7 kPa) at a temperature of 60 ° C. After 12 hours of drying, 0.66 g of crude heparinedepolymerized (sodium salt) is obtained. The yield obtained is 60%. 9 012340

Purification:

In a 10 ml Erlenmeyer flask was charged with 0.66 g of crude depolymerized heparin obtained previously and 5.9 ml of distilled water. The mixture is heated to 40 ° C. under magnetic stirring. By adding sodium hydroxide at 0.1 mol / l, the pH is brought to between 9 and 10 and 33 microliters of a 30% aqueous solution of hydrogen peroxide are added. After stirring for approximately 2 hours, 0, 65 g of sodium chloride. The mixture is then neutralized by addition of HCl to 0.1 mol / l. The solution is then filtered and transferred into a 25 ml Erlenmeyer flask. 23.3 ml of methanol are poured. The formation of a white precipitate is observed. Stirring is then stopped and the suspension is allowed to feed for 12 hours at a temperature in the region of 20 ° C. The odorant is then removed and discarded (5 ml). On the sedimented precipitate (debossed appearance), 5 ml of methanol and 5 ml of methanol are added. it is stirred for 20 minutes. The precipitate is left to sediment for approximately 30 minutes. The supernatant is removed and discarded (5 ml). On the sedimented precipitate, 5 ml of methanol are added and the mixture is stirred for 20 minutes. The precipitate in suspension is then filtered on Sintered Glass 3. The resulting white cake is then washed with 2 times 5 ml of methanol. The moist solid is drained and dried under reduced pressure (2.7 kPa), at a temperature in the region of 60 ° C. After 12 hours of drying, 0.51 g of a purified mixture of polysaccharides (sodium salt) are obtained. The yield obtained is 77.2%.

The characteristics of this mixture are as follows:

Average molecular weight: 1600 daltons

Anti-Xa activity: 94 IU / mg

Anti-It activity: <0.1 IU / mg

Anti-Xa activity ratio / anti-IIa activity:> 100 EXAMPLE 2 Depolymerization and transformation to sodium salt:

In a 100 ml Erlenmeyer flask, 70 ml of dichloromethane are introduced. Under stirring, 10 g (0.00595 mol) of benzyl ester of heparin (esterification rate: 77%, benzethonium salt) obtained as described in 10 were slowly charged. After complete dissolution, 1.7 ml (0.00587 moles) of 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine are added. At a temperature of about 20 ° C, the reaction is allowed to proceed for about 3 hours and 30 minutes. 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 B. With magnetic stirring, the reaction mixture of the Erlenmeyer flask A is poured into the ethanolic sodium acetate solution. A gelatinous yellow precipitate almost translucent appears. The stirring is then stopped and the suspension is allowed to settle for a time. The clear portion of the supernatant is removed and discarded (204 ml). On the sedimented precipitate (yellow slurry appearance), 125 ml of methanol are added and the mixture is stirred for 20 minutes. The precipitate is left to sediment for approximately 30 minutes. The odorant is removed and discarded (162 ml). On the sedimented precipitate, 125 ml of methanol are added and stirred for 20 minutes. The gelatinous precipitate in suspension is then filtered on Sintered Glass 3. The yellow gelatinous cake obtained is then washed with 2 portions of 63 ml of methanol. The gelatinous solid is drained and then dried under reduced pressure (2.7 kPa) at a temperature in the region of 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:

1.67 g of depolymerized heparin (benzyl ester, sodium salt) obtained above are saponified according to the saponification process described in Example 1. 0.94 g of a light yellow powder are obtained. The yield of crude depolymerized heparin (sodium salt) is 61%.

Purification:

0.94 g of crude depolymerized heparin (sodium salt) previously obtained according to the purification method described in Example 1 are purified. 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:

Average molecular weight: 2500 Dalton

Anti-Xa activity: 146.6 IU / mg 11 012340

Anti-IIa activity: 2.15 μl / mg

Anti-Xa activity / anti-IIa activity ratio: EXAMPLE 3 Depolymerization and conversion to sodium salt

In a 50 ml Erlenmeyer flask, 28 ml of dichloromethane are introduced. Under stirring, 4 g (0.00238 mol) of benzyl ester of heparin (esterification rate: 77%, benzethonium salt) obtained according to Example A were slowly charged. After complete dissolution and cooling to 2 ° C., 0.333 g (0.00239 mol) of 1,5,7-triazabicyclo [4.4.0] dec-5-ene. At a temperature of 20 ° C, the reaction is allowed to continue for about 3 hours and 30 minutes. During this time, a solution of 12 g of sodium acetate in 120 ml of methanol is prepared at 4 ° C. in an Erlenmeyer flask B. With magnetic stirring, the reaction mixture of Erlenmeyer A is poured into the methanolic solution of sodium acetate. A yellow precipitate appears. The agitation is then stopped and the suspension is allowed to settle for one hour. The clear portion of the supernatant is removed and discarded (90 ml). On the sedimented precipitate (yellow slurry appearance), 50 ml of ethanol are added and stirred for 20 minutes. The precipitate is left to sediment for approximately 30 minutes. The supernatant is removed and discarded (61 ml). On the precipitate, 50 ml of methanol are added and stirred for 20 minutes. The precipitate in suspension is then filtered on Sintered Glass 4. The cake obtained is then washed with 2 times 25 ml of methanol. The wet solid is drained and then dried under reduced pressure (2.7 kPa), at a temperature in the region of 60 ° C. After 12 hours of drying, 1.19 g of depolymerized heparin (benzyl ester, sodium salt) are obtained. The solid is intense yellow. The yield obtained is 75.9%.

Saponification:

1.19 g of depolymerized heparin (benzyl ester, sodium salt) obtained above are saponified according to the saponification process described in Example 1. 0.78 g of a light yellow powder is obtained. The yield of crude depolymerized heparin (sodium salt) is 71.5%. 12 012340

Purification:

0.78 g of crude depolymerized heparin (sodium salt) previously obtained according to the purification method described in Example 1 are purified. 0.58 g of a white powder is obtained. The yield is 72.5%.

The purified mixture of polysaccharides (sodium salt) obtained has the following characteristics:

Average molecular weight: 2700 Daltons

Anti-Xa activity: 100.1 IU / mg

Anti-Iia activity: 3.3 IU / mg

Anti-Xa activity ratio / anti-Ha activity: 27.3 EXAMPLE 4 Depolymerization and transformation into sodium salt

In a 50 ml Erlenmeyer flask, 28 ml of dichloromethane are introduced. Under stirring, 4 g (0.00238 mol) of benzyl ester of heparin (esterification level: 77%, benzethonium salt) were slowly charged as described in Example A. After complete dissolution and at 2 ° C. 0.6 ml (0.00222 moles) of 2-tert-butylimino-tris (dimethylamino) phosphorane are added. At a temperature of about 0 ° C., the reaction is allowed to proceed for about 3 hours and 30 minutes. During this time, a solution of 12 g of sodium acetate in 120 ml of sodium acetate is prepared at 4 ° C. in an Erlenmeyer flask B. methanol. With magnetic stirring, the reaction mixture of the Erlenmeyer flask A is poured into the methanolic solution of sodium acetate. An almost translucent gelatinous yellow precipitate appears. Stirring is then stopped and the suspension is allowed to settle for one hour. The clear portion of the cloud is removed and discarded (108 ml). On the sedimented precipitate (yellowish-yellowish appearance), 50 ml of methanol are added and the mixture is stirred for 20 minutes. The precipitate is re-sedimented for about 30 minutes. The supernatant is removed and clear (60 ml). On the sedimented precipitate, 50 ml of methanol are added and stirred for 20 minutes. The yellowish white precipitate in suspension is then filtered on Sintered Glass 4. The cake obtained is then washed with twice 25 ml of methanol. The solid solid is drained and then dried under reduced pressure (2.7 kPa) at a temperature in the region of 60 ° C. After 12 hours of drying, 1.22 g of depolymerized heparin (benzyl ester, sodium salt) are obtained. The yield obtained is 77.8%.

Saponification:

1.22 g of depolymerized heparin (benzyl ester, sodium salt) obtained above are saponified according to the saponification protocol described in Example 1. 0.69 g of a very light yellow powder is obtained. The yield of crude heparinedepolymerized (sodium salt) is 61.6%.

Purification:

0.69 g of crude depolymerized heparin (sodium salt) previously obtained according to the purification procedure described in Example 1 are purified. 0.67 g of a white powder is obtained. The yield is 97.1%.

The purified mixture of polysaccharides (sodium salt) obtained has the following characteristics:

Average molecular weight: 2900 Daltons

Anti-Xa activity: 145.2 IU / mg

Anti-Ha activity: 4.5 IU / mg

Anti-Xa activity / anti-IIa activity ratio: 32.6 EXAMPLE 5 Depolymerization and transformation to sodium salt

In a 50 ml flask A, 28 ml of dichloromethane are introduced. Under stirring, 4 g (0.00238 mol) of benzyl heparinate are slowly added (esterification level: 77%, benzethonium salt). After complete dissolution and at 40 ° C, 0.95 g (0.00949 mol) of sodium imidazolate is added. Refluxing the dichloromethane, the reaction is allowed to proceed for about 4 hours. During this time, a solution of 12 g of sodium acetate in 120 ml of ethanol is prepared at 4 ° C. in an Erlenmeyer flask B. Under magnetic stirring, the reaction mixture of Erlenmeyer flask A is 14. 012340 poured into the methanolic solution of sodium acetate. A quasi-translucent yellow gelatinous precipitate appears. Stirring is then stopped and the suspension is allowed to settle for one hour. The clear portion of the orange supernatant is removed and discarded (88 ml). On the sedimented precipitate (orange slurry appearance), 50 ml of methanol are added and stirred for 20 minutes. The precipitate is left to settle for about 30 minutes. The supernatant is removed and discarded (51 ml). On the sedimented precipitate, 50 ml of methanol are added and stirred for 20 minutes. The orange precipitate in suspension is then filtered on Sintered Glass 4. The cake obtained is then washed with twice 25 ml of methanol. The solid is drained and then dried under reduced pressure (2.7 kPa) at a temperature in the region of 60 ° C. After drying for 12 hours, 1.34 g of depolymerized heparin (benzyl ester, sodium salt) are obtained. The yield obtained is 76.6%.

Saponification:

1.2 g of depolymerized heparin (benzyl ester, sodium salt) obtained above are saponified according to the saponification protocol described in Example 1. 0.63 g of a beige powder is obtained. The yield of crude depolymerized heparin (sodium salt) is 52.5%.

Purification:

0.63 g of crude depolymerized heparin (sodium salt) previously obtained according to the purification method described in Example 1 are purified. 0.42 g of a beige-white powder is obtained. The yield is 66.7%.

The purified mixture of polysaccharides (sodium salt) obtained has the following characteristics:

Average Molecular Weight: 2250 Daltons

Anti-Xa activity: 134.5 IU / mg

Anti-IIa activity: 1.5 IU / mg

Anti-Xa activity / anti-IIa activity ratio: 90.5 EXAMPLE 6 Depolymerization and transformation to sodium salt:

In a 50 ml Erlenmeyer flask, 28 ml of dichloromethane are introduced. Under stirring, 4 g (0.00238 mol) of benzyl ester of heparin (esterification rate: 77%, benzethonium salt) obtained as described in Example A were slowly charged. After complete dissolution, 1.33 was added. g (0.00956 mol) of 1,5,7-triazabicyclo [4.4.0] dec-5ene. Stirred at a temperature of 20 ° C for 3 hours and 30 minutes. During this time, a solution of 12 g of sodium acetate in 120 ml of methanol is prepared at 4 ° C. in an Erlenmeyer flask B. With magnetic stirring, the reaction mixture of the Erlenmeyer flask A is poured into the ethanolic sodium acetate solution. A gelatinous yellow precipitate almost translucent appears. The stirring is then stopped and the suspension is allowed to settle for a time. The clear portion of the supernatant is removed and discarded (56 ml). On the precipitated sediment (yellow slurry appearance), 60 ml of methanol was added and stirred for 20 minutes. The precipitate is left to sediment for approximately 30 minutes. The supernatant is removed and discarded (70 ml). On the sedimented precipitate, 50 ml of methanol are added and the mixture is stirred for 15 minutes. The precipitate in suspension is then filtered on Glass-Filled 4. The gold-yellow cake obtained is then washed with twice 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (2.7 kPa) at a temperature of 60 ° C. After 12 hours of drying, 0.92 g of heparinedepolymerized (benzyl ester, sodium salt) are obtained. The yield obtained is 64%.

Saponification:

In a 25 ml Erlenmeyer flask, 0.92 g (0.0014 mol) of heparin-polymerized (benzyl ester, sodium salt) obtained previously and 17.5 ml of water are introduced. With magnetic stirring, 0.38 ml (0.00379 mol) of 30% desolder liquor is introduced. After addition, the mixture is maintained at a temperature of 20 ° C and stirred for 5 hours. 1.8 g of NaCl are added and the solution is neutralized by addition of concentrated HCl (final volume of the solution approximately 18 ml). The mixture is transferred to a 100 ml Erlenmeyer flask and 46 ml of methanol are added. The formation of a yellow precipitate is observed. Stirring is then stopped and the suspension is allowed to feed for 12 hours at a temperature in the region of 20 ° C. The odorant is then removed and discarded (52 ml). On the sedimented precipitate (yellow slurry aspect), 25 ml of methanol are added and stirred for 20 minutes. After 23 hours, the precipitate is re-sedimented for approximately one hour. The supernatant is removed and discarded (27 ml). On the sedimented precipitate, 25 ml of methanol are added and stirred for about 1 hour. The precipitate in suspension is then filtered on Sintered Glass 4. The resulting cake is then washed twice with 10 ml of methanol. The wet solid is stripped and then dried under reduced pressure (2.7 kPa), at a temperature in the region of 60 ° C. After drying for 3 hours, 0.42 g of crude depolymerized heparin (sodium salt) is obtained. The yield obtained is 45.6%.

Purification:

In a 10 ml Erlenmeyer flask, 0.42 g of crude depolymerized heparinobtained previously and 3.8 ml of distilled water are introduced. The mixture is brought to 38 ° C. under magnetic stirring. By addition of sodium hydroxide at 0.1 mol / l, the pH is brought to between 9 and 10, and 25 microliters of a 30% aqueous solution of hydrogen peroxide are added. After about 2 hours 30 minutes of stirring, the mixture is added. 0.5 g of sodium chloride. The mixture is then neutralized by addition of HCl to 0.1 mol / l. The solution is then filtered and transferred into a 25 ml Erlenmeyer flask. 11.3 ml of methanol are poured. The formation of a white precipitate is observed. Stirring is then stopped and the suspension is allowed to feed for 12 hours at a temperature in the region of 20 ° C. The odorant is then removed and discarded (9.8 ml). On the sedimented precipitate (slurry aspect), 5 ml of methanol are added and stirred for 15 minutes. The precipitate is left to sediment for approximately 3 hours. The supernatant is removed and discarded (6.2 ml). On the sedimented precipitate, 5 ml of methanol are added and the mixture is stirred for 20 minutes. The precipitate in suspension is then filtered on Sintered Glass 3. The resulting white cake is then washed with 2 times 5 ml of methanol. The moist solid is drained and dried under reduced pressure (2.7 kPa), at a temperature in the region of 60 ° C. After about 2 hours of drying, 0.39 g of depolymerized heparin (sodium salt) are obtained. The yield obtained is 92.8%.

The characteristics of the depolymerized heparin thus obtained are as follows:

Average molecular weight: 1950 daltons

Anti-Xa activity: 115.6 IU / mg

Anti-IIa activity: <2 IU / mg

Anti-Xa activity / anti-IIa activity ratio: EXAMPLE 7 Depolymerization and transformation to sodium salt

In a 400 ml reactor A, 140 ml of dichloromethane are introduced. Under stirring, 20 g (0.0119 mol) of benzyl ester of heparin (esterification rate: 77%, benzethonium salt) obtained as described in Example A were slowly charged. After complete dissolution, the water of the reaction medium by the Karl Fisher method. The value obtained is 0.1% water. 3.5 ml (0.0121 mol) of 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine are then added. At a temperature in the region of 25 ° C., the reaction is allowed to proceed for about 24 hours. During this time, a solution of 30 g of sodium acetate in 300 ml of methanol is prepared at 4 ° C. in a B-flask. Under magnetic stirring, half of the reaction mixture of reactor A is introduced into the methanolic acetate solution. sodium. A yellow gelatinous quasi- translucent precipitate appears. Stirring is maintained for one hour and the suspension is allowed to decant for about 12 hours at 4 ° C. The clear portion of the cloud is removed and discarded (220 ml). On the sedimented precipitate (yellow-colored appearance), 220 ml of methanol are added and the mixture is stirred for 50 minutes. The precipitate is re-sedimented for about 40 minutes. The supernatant is removed and clear (204 ml). On the sedimented precipitate, 204 ml of methanol are added and stirred for 40 minutes. The gelatinous precipitate in suspension is then filtered on GlassFruit 3. The yellow gelatinous cake obtained is then washed with 2 portions of 100 ml of methanol. The gelatinous solid is drained and then dried under reduced pressure (2.7 kPa) at a temperature in the region of 60 ° C. After drying for approximately 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).

Saponification:

2.6 g of depolymerized heparin (benzyl ester, sodium salt) previously obtained according to the saponification process described in Example 1 are saponified. 1.48 g of a light yellow powder are obtained. The yield of crude depolymerized heparin (sodium salt) is 62.9%.

Purification: 18

0123AU

In a 50 ml Erlenmeyer flask, 1.48 g of crude depolymerized heparin obtained previously and 15 ml of distilled water are introduced. The mixture is heated to 40 ° C. under magnetic stirring. By addition of sodium hydroxide at 1 mol / l, the pH is brought to between 9 and 10. The solution is filtered on a melting membrane of porosity 1 μm. 76 microliters of a 30% aqueous solution of hydrogen peroxide are then added. After stirring for approximately 2 hours, 1.5 g of sodium chloride are added. The mixture is then neutralized by addition of HCl to 1 mol / l. The solution is filtered on a membrane with a porosity of 1 μm. 38 ml of methanol are poured. The formation of white precipitate is observed. The stirring is then stopped and the suspension is allowed to settle for 1 hour at a temperature of 20 ° C. The supernatant is then removed and discarded (37 ml). On the precipitate, 37 ml of methanol are added and stirred for 45 minutes. The precipitate is left to sediment for approximately 45 minutes. The odor is removed and discarded (34 ml). On the sedimented precipitate, 34 ml of methanol are added and stirred for 15 minutes. The precipitate in suspension is then filtered on Sintered Glass 3. The white cake obtained is then washed with twice 25 ml of ethanol. The moist solid is drained and then dried under reduced pressure (2.7 kPa), at a temperature in the region of 60 ° C. After 12 hours of drying, 1.29 g of pure depolymerized heparin (sodium salt) are obtained. The yield obtained is 87.2%. The purified depolymerized heparin (sodium salt) obtained has the following characteristics:

Average 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

Claims (20)

19 012340 CLAIMS 1 - Sulphated polysaccharide mixtures having the general structure of the polysaccharides constitutive of heparin and having the following characteristics: they have an average molecular weight of 1500 to 3000 daltons, an anti-Xa activity of 100 to 150 IU / mg, an anti-inflammatory activity. -Ha from 0 to 10 IU / mg and an anti-Xa activity / anti-IIa activity ratio greater than 10; -the constituent polysaccharides of the mixtures contain 2 to 26 -saccharide moieties and have an unsaturated 4,5-glucuronic acid unit; O-sulphate at one end thereof, in the form of an alkali metal or alkaline earth salt. 2- mixtures according to claim 1 characterized in that the anti Xa activity iscomprise between 125 and 150 IU / mg 3- mixtures according to claim 1 or 2 characterized in that the anti'anti Xa activity iscomprise between 140 and 150 IU / mg and the average molecular weight is between 2000 and 3000 dalton 4 - Mixtures according to any one of claims 1 to 3 in the form of sodium salt, potassium, calcium or magnesium. 5 - Blends according to any one of claims 1 to 4 having an anti-Ha activity of 0 to 5 IU / mg. 6 - Blends according to any one of claims 1 to 5 having an anti-Xa ratio / anti-IIa activity greater than 25. 7 - Process for the preparation of the mixtures of claim 1 characterized in that one depolymerizes a quaternary ammonium salt of the benzyl ester of heparin in organic medium, with a strong organic base of pka greater than 20 w. Sodium imidazolate converts the quaternary ammonium salt of the depolymerized heparin benzyl ester to the sodium salt, saponifies the ester and optionally purifies the product. 012340 20 8 - Process according to claim 7 wherein the quaternary ammonium salt of benzyl ester of heparin is the benzethonium salt, cetylpyridinium or decetyltrimethylammonium. 9 - Process according to claim 7 wherein the strong organic base of pka5 greater than 20 is chosen from 1,5,7-triazabicyclo [4.4.0] dec-5-ene, 2-tert-butylimino-2-diethylamino 1,3-dimethylperhydro-1,3,2-diazaphosphorin, the bases of the family of guanidine and phosphazenes, 10 - Process according to claim 9 for which the bases of the family desguanidines are those of formula: R 4 R 3 wherein R 1 is hydrogen or alkyl, R 2, R 3, R 4 and R 5, which are identical or different, each represent an alkyl radical, the alkyl radicals having 1 to 6 carbon atoms in a straight or branched chain. 11 - Process according to claim 10 wherein R1 is hydrogen and R2, R3, R4 and R5 are methyl radicals. 12 - Process according to claim 9 wherein the bases of the family of phosphazenes are those of formula: R3 R4 R2-N \ / R1-N-P-N-R5 N-R6 / R7 in which the radicals Rj to R7 are identical or different and represent alkyl radicals containing 1 to 6 carbon atoms, straight or branched chain. 13 - Process according to one of claims 7 to 12 wherein the molar organic mole ratio of pka greater than 20 or sodium imidazolate / quaternary ammonium salt of the benzyl ester of heparin is between 0.2 and 5. 012340 14 - Process according to one of claims 7 to 13 wherein the quaternary ammonium salt of the benzyl ester of heparin has a degree of esterification between 50 and 100%. 15 - Process according to one of claims 7 to 14 for which the transformation of the quaternary ammonium salt of the benzyl ester of depolymerized heparin into the sodium salt is carried out by treatment of the reaction medium with an alcoholic solution of sodium acetate. sodium. 16 - Process according to one of claims 7 to 15 for which the saponification'effectue by means of an alkali metal hydroxide. 17 - Method according to one of claims 7 to 16 for which the purification is effected by means of hydrogen peroxide. 18 - Blends as defined in any one of claims 1 to 6 as medicaments. 19 - Mixtures as defined in any one of claims 1 to 6 as antithrombotic drugs 20 - Pharmaceutical compositions comprising as active ingredient a mixture according to one of claims 1 to 6.