IE51283B1 - Sulphated polysaccharides,processes for their preparation and their use as medicaments - Google Patents

Abstract

1. Mixtures of sulphated polysaccharides having the general structure of the polysaccharides present in heparin, whose acid groups are in the free form or converted into a salt form, characterised in that the polysaccharides present in the said mixtures have an ethylenic double bond at one of the ends of their chain and in that, in the form of the sodium salt, the mixtures have the following characteristics : - percentage of sulphur : 9% to 13.5% - percentage of nitrogen : 1.8% to 2.5% - percentage of uronic acids : 20% to 30% - weight-average molecular weight : 2000 to 10 000 daltons - specific rotatory power in aqueous solution at 20 degrees C : [alpha]D-20 = + 25 degrees to + 55 degrees

Description

The present invention relates to mixtures of sulphated polysaccharides of weight mean molecular weight less than that of heparin, which can be used as anti-coagulant and anti-thrombotic agents in the prevention and treatment of thromboses and as hypolipemic agents, and to processes for their preparation.

Heparin is a mixture of sulphated mucopolysaccharides of animal origin, largely used for its anti-coagulant and anti-thrombotic properties, notably in the prevention of post-operation venous thromboses, and for its hypolipemiant properties. It is known that it acts in the coagulation process by activating a natural inhibitor, antithrombin III of coagulation which is contained in blood. The activation of this protein has the effect of inhibiting the action of two proteases, the X-activated factor (factor Xa) on the one hand and thrombin on the other.

The anti-coagulant activity in vitro of the heparin is generally measured by the official methods of the Pharmacopeia, especially of the American, English or French Pharmacopeia, by referring to an international standard.

But it is now possible to measure its specific activities 51383 in vitro, on the one hand with respect to the factor Za and on the other hand with respect to thrombin (cf. for exanple A. TBIEN et al., Thrombosis Research, 11, 107, 1977).

The commercial heparins, which are mixtures of polysaccharides whose weight mean molecular weight is greater than 10,000 daltons and of which the spread of molecular weight is from about 4»000 to about 45»000 daltons, show the following activities in vitro:Anti-coagulant activity measured by the method of the French Pharmacopeia, 8th edition, heparin monograph, by referring to the 3rd international standard (or Codex anti-coagulant activity): 140 to 200 l.u./ng.

Anti-Xa activity measured by the method of TBIEN et al. (op. cit.), by referring to the 3rd international standard : 160 to 180 i.u./mg.

A.P.T.T. activity (Activated Partial Thromboplastin Time) measured by the method of TEIEN et al.( op. cit.), by referring to the 3rd international standard: 150 to 170 i.u./mg.

The heparin is obligatorily administered parenterally (in practice subcutaneously) and the action is relatively short, as a result of which there are the two following important disadvantages: the need to carry out three administrations per day and the relatively 512 8 3 high frequency of post—operative haemorrhagic accidents It is knovm (cf. JOHNSON et al, Thromhos, Haemostss. Stuttg. 1976» 35» 586-591» LANE et al, Thrombosis Research 16, 651-662, Pergamon Eress Ltd. 1979 LASKER, CHIU, Anmals N.Y. Acad., Sci., 222, 1973, 971-977} LASKER, Adv. Exp. Med.Biol. 52, 1975, 119-130) that, by fractionation of heparin, for example by filtration over Sephadex gel, fractions of mean molecular weight less than that of heparin and having a spread of molecular weights smaller than that of heparin can be obtained. The .tests made in vitro and in vivo show that such fractions on the one hand are relatively more active on the X-activated factor than on the thrombin (that is that they show a anti-Xa activity__ ratio distinctly greater than anti-thrombinic activity 1), and on the other hand are more easily absorbed in the circulation from a subcutaneous injection than heparin itself, hence they have a higher and longer20 lasting plasmatic activity than that of heparin.

It is also known (cf. LASKER and CHIU op. cit.; LASKER og. cit.; U.S. Patent No. 3,766,167; PERUN et al., Carboyhdrate Research, 18, 1971, 185-194) to prepare, by enzymatic hydrolysis of heparin, depolymer25 isation products having a low mean molecular weight (practically 5,300 to 4,500 daltons, determined by ultracentrifugation) and an anti-coagulant activity, determined by the method U.S. Pharmacopeia Ho. XVII, of about 70 i.u./mg. These products of depolymerisation can be fractionated, by filtration over Sephadex gel, into fractions whose mean molecular weight, determined by ultracentrifugation is from 3200 to 5900 daltons and with anti-coagulant activities from 45 to 95 i.u./mg (U.S.P, method). These depolymerisation products and the fractions which a re obtained are active, taken orally ae well as parenterally.

It is also known (cf, LASKER op. cit.) to prepare, by depolymerisation of heparin by means of ascorbic acid and hydrogen peroxide, products of low mean molecular weight. The depolymerisation by means of ascorbic acid and hydrogen peroxide leads, after fractionation in alcohol, to fractions having a mean molecular weight from 4000 to 7100 daltons and an anti-coagulant activity of 12 to 100 i.u./mg (U.S. Pharmacopeia method).

Finally, it is known (cf. British Patent Application No. 2,002,406 A published on 21.02.1979) to prepare, by resulphating of depolymerisation products of heparin ifevoid of anti-coagulant activity, oligopolysaccharides having a mean molecular weight, determined by the Somogy method, of 2600 to 5500 daltons, a specific rotatory power, measured in aqueous solution at. 20°C, of +40° to +50°C, and an anti-coagulant activity less than 50 i.u./mg (U.S Pharmacopeia method), which would be as active taken orally as parenterally for the prevention of thromboses.

The mixtures of sulphated polysaccharides accord5 ing to the invention are mixtures of sulphated polysaccharides having the general structure of the polysaccharides that constitute heparin, that is showing the same elementary linkages as the polysaccharides that constitute heparin with the elementary linkages being linked in the same way as in the latter0 However, contrary to the polysaccharides that constitute heparin, the polysaccharides that constitute the mixtures according to the invention comprise an ethylenic double bond at one of the ends of their chain. In the mixtures according to the invention, the acid groups of the sulphated polysaccharides may be in free form or in the form of a salt, in particular, in the form of a sodium, calcium or magnesium salt. The mixtures according to the invention have, in the fora of the sodium salt, the following characteristics: - percentage of sulphur : to 13.55» - percentage of nitrogen : 1.80 to 2.5% - percentage of uronic acids : 200 to 30$ - weight mean molecular weight : 2000 to 10,000 daltons - specific rotatory power in aqueous solution at 20°C: D ί +25° to +55' The sulphur and nitrogen contents and the rot5 atory power indicated above have been determined by the methods of the French Pharmacopeia, 8th edition, heparin monograph. The content of uronic acids has been determined by the method of K. BIUMENKRAIITZ et al. (Analytical Biochemistry, 54, 484, 1975). The weight mean molecular weight has been determined by gel permeation chromatography on a polyacrylamide-agarose gel by referring to the standard constituted by heparins of known mean molecular weight, according to the method of AHDERSON et al. (Thrombosis Research, 9, 575, 1976).

The polysaccharides that constitute the mixtures according to the invention particularly correspond to the following formulas wherein R is hydrogen atom or a carboxylic group, in (I) the free acid state or in the form of a salt, R’ is an OH group or a sulphate group, in the free acid form or in the form of a salt, R^ is an OH group or a sulphate group, in the free acid form or in the form of a salt, R£ is a sulphonate group, in the free acid form or in the form of a salt, or an acetyl group, -0is an oxygen bridge, the G linkages are linkages of the glucosamine type present in the structure of heparin, the U linkages are linkages of the uronic acid type (D-glucuronic acid, D-iduronic acid and sulphated 1-iduronic acid) present in the structure of heparin, and n is a whole number from 3 to 20.

In the above formula (I), the acid groups of the polysaccharides may be in free form or in the form of a salt, particularly a sodium, calcium or magnesium salt.

Although the present invention relates to all the mixtures previously defined it is more particularly concerned with those mixtures which, in the form of the sodium salt, show the additional characteristics indicated for categories I, II, III in the following Table A.

TABER A Category Category II Category III Weight mean molecular weight (in daltons) 8000 to 10,000 3000 to 8000 2000 to 7000 In vitro Codex anticoagulant activity 130 to 160 80 to 140,οεδ° In vitro anti-Xa activity o 40 o O 00 K\T- 120 to 250 80 to 250 In vitro A.P.T.T. activity 100 to 150 80 to 120 10 to 80 Ratio In vitro anti-Xa activity In vitro A.P.T.T. activity ------- -- -------« 1 to 1.5 1.4 to 3 2 to 10 jL5 The Codex anti-coagulant activity indicated in the Table above has been determined by the method of the Stench Pharmacopeia, 8th edition, heparin monograph. The anti-Xa and A.P.T.T activities indicated in Table A as well as those indicated in Tables B and C below have been determined by the method of TSIEK et al. ( op. cit.), according to the following procedures: a) Determination of the anti-Xa activity; This activity is determined on bovine plasma, free of platelets, by means of the chromogenic substrate S 2222 £chromogenic peptide having the structureί (N-benzoyl)Ile-Glu-Gly-Arg-(p-nitro)anilideJ , referring to the 3rd international standard. 1283 ίο 100 μΐ of citrated bovine plasma, diluted from to 5 with an aqueous buffer tris/EDTA pH 8.4 i.e., two parts by volume of the bovine plasma are diluted with the aqueous buffer so as to obtain 5 parts by volume of the resulting solution are added to 100 pi of a solution of the product to be tested or of the standard in an aqueous buffer tris/EDTA pH 8.4, the 100 pi corresponding to 0.02 to 0.08pg of product of standard. After 3 minutes of incubation at 37°C, 100 μΐ of an aqueous solution of bovine Xa factor, which correspond to 7 n Kat of factor Xa, are added. The expression n Kat is an abbreviated form of nanokatal which is a unit for measuring the quantity of factor Xa. After a 30 seconds incubation period,200 pi of an aqueous solution of S 2222, which correspond to 0.2 pmole of S 2222, are added. After a three minutes incubation period, 300 pi of acetic acid are added and the optical density of the solution is measured at 405 nm by comparison with distilled water.

By plotting the optical density as a function of the concentration of product or standard, one obtains two straight lines, one relating to the product to be tested, the other relating to the standard. The activity of the product, expressed in international units per mg, is given by the formula: 173 slope of the straight line relating to the product to be tested slope of the straight line relating to the standard the number 173 corresponding to the value of the activity for the third international standard. b) Determination of the A.P.T.T. activity The product to be tested and the third inter51283 national standard are dissolved in a 0.15 H aqueous solution of sodium chloride, then diluted with a citrated bovine plasma, free of platelets, so as to obtain concentrations of product to be tested (or of standard) from 0 to 4 pg/ml. 100 μΐ of the reagent automated APTT Precibio (reagent based on phospholipids of rabbit brain and micronized silica) are added to 100 pi of the solution so obtained. After a five minutes incubation period at 57°°» 1°° pi of a 0.025 K aqueous solution of calcium chloride are added. The clotting time is measured by means of a Bio-IIerieux fibrometer.

Py plotting the logarithm of the clotting time as a function of the concentration of the product or of the standard, one obtains two straight lines, one relating to the product to be tested, the other relating to the standard. The activity of the product, expressed in international units per mg, is given by the formula: 173 slope of the straight line relating to the product .to be tested slope of the straight line relating to the standard the number 173 corresponding to the value of the activity for the third international standard.

All the activities appearing in Tables A,B and 0 are expressed in international units per mg (i.u./mg), referring to the 3rd international standard.

The mixture according to the invention may be prepared for example by the action of an inorganic or organic base on a heparin ester resulting from the partial or total esterification of the carboxylic acid groups of the heparin. In this ester the acid groups of heparin which are not esterified (that is, the acid sulphate groups and possibly a part of the carboxylic acid groups) may be in the free state or in the form of salts, in particular an alkali metal salt such as for 2o example the sodium salt, an alkaline earth salt such as for example the calcium salt, a magnesium salt or quaternary ammonium salt with a long chain such as for example the benzethonium salt - long chain in this regard means a quaternary alkyl or aralkyl ammonium salt wherein at least one of the alkyl or aralkyl chains has to 20 carbon atoms.

In the case where the heparin ester is soluble in water (for example where the acid groups which are not esterified are in the form of the sodium salt), the 2o reaction between the ester and the base may be effected in water, at a temperature of 20°C to 80°C, the molar concentration of the base in the medium being preferably between 0.1 and 0.6. Bases which may be used are those which are water-soluble and in particular sodium hydroxide, potassium hydroxide, alkali metal carbonates, triethylamine, tri ethylene-diamine, quinuclidine, 1,5-diazabicyclo £-4.3.cD 5-nonene and 1,5-diaza-bicyclo {_5.4.o] 5-undecene.

Once the reaction has finished, the product of depolymerisation formed is isolated, for example by precipitation by the addition of sodium chloride and then methanol.

The reaction between the ester and the base may also be effected, particularly when the acid groups of the ester which are not esterified are in the form of a quaternary ammonium salt with a long chain, in an organic solvent unreactive towards the ester, such as for example dichloromethane, dimethylformamide, formamide or tetrahydrofuran, at a temperature preferably of 20°C to 80°C. Bases which may be used are those which are soluble in the solvent used and in particular 1,5-diaza-bicyclo [4.3.0] 5-nonene, quinuclidine and 1,5-diaza-bicyclo [5·4·θ| 5-undecene.

Once the reaction has ended, the product of depolymerisation formed in which the carboxylic acid groups are still esterified, is isolated for example in the form of an alkal metal salt, and is hydrolysed by an aqueous solution of alkali metal hydroxide, especially sodium hydroxide, at least IN, at low temperature (0°C to +5°C). The final product is separated, for exanple by precipitation by the addition of sodium chlorine and then methanol.

Heparin esters which may be used as starting products for preparing the mixtures of polysaccharides according to the invention nay be non-selective esters.

By non-selective esters we mean heparin esters wherein the carboxyl groups of the D-glucuronic acid, unsulphated L-iduronic acid and sulphated L-iduronic acid linkages are indiscriminately esterified. By selective esters we mean heparin esters wherein only the carboxyl groups of the D-glucuronic acid linkages or only the carboxyl groups of the D-glucuronic acid and unsulphated L-iduronic acid linkages, or only the carboxyl groups of the unsulphated L-iduronic acid and sulphated L-iduronic acid linkages, or only the carboxyl groups of the sulphated L-iduronic acid linkages, are esterified, partially or completely.

Esters which may be used as starting products in the processes according to the invention are, in particular, the heparin esters described in French Patent Ko. 2,150,724 and in British Patent No. 1,501,095, as well as the methyl, ethyl, ethoxycarbonylmethyl, cyanomethyl and particularly the benzyl and substituted benzyl (especially 4-chloro-benzyl, 4-nitro-benzyl) esters of heparin. The benzyl esters or substituted benzyl esters of heparin are preferably used as starting products.

By the term substituted benzyl is meant benzyl or phenyl substituted with one or two substituents selected from chlorine, nitro, alkyl with 1 to 4 carbon atoms and alkoxy with 1 to 4 carbon atoms. The heparin esters used as starting substances in the processes according to the invention may be derived from heparin of any origin (for example bovine lung heparin, heparin from pig mucous membranes or heparin from cattle intestines).

The non-selective methyl, ethyl, ethoxycarbonylmethyl, cyanomethyl, benzyl or substituted benzyl ir> e3ter3 of heparin may be obtained,for example, by the action of a neutral quaternary ammonium or amine salt of heparin with a halogenated derivative of formula Hal - CHg - R(II), in whieh Hal represents a chlorine, bromine or iodine atom and R represents a hydrogen atom or a methyl, ethoxyearbonyl, cyano, phenyl or substituted phenyl group. This reaction is effected in solution or in suspension in an inert solvent such asfior examplej dimethylformamide, methylene chloride, dimethylsulphoxide, tetrahydrofuran -or acetone, at a temperature of from -20°C to +60?C.

The methyl, ethyl, ethoxycarbonylmethyl,cyanomethyl, benzyl or substituted benzyl esters of heparin wherein only the carboxyl groups of the D-glucuronic acid linkages or only the carboxyl groups of the D-glucuronie aoid and unsulphated L-iduronic acid linkages are esterified, partially or completely may be obtained for example by reacting a halogen derivative of the above formula (II) with an acid quaternary ammonium salt of heparin in which, besides the sulphate groups, only the carboxyl groups of the D-glucuronio aoid linkages, or only the carboxyl groups of the Dglucuronic aoid and unsulphated L-iduronic acid linkages, are in salt form the other carboxy), groups being in the free acid form. This reaction is carried out under the same conditions as the reaction of the halogen derivative of the formula (II) with a neutral quaternary ammonium salt of heparin.

The acid quaternary ammonium salts of heparin in which, besides the sulphate groups, only the carboxyl groups of the D-glucuronic acid linkages, are in salt form may be prepared by reacting a quaternary ammonium salt with heparin, in an aqueous medium with a pH of from 3 to 4.

The acid quaternary ammonium salts of heparin in which, besides the sulphate groups, only the carboxyl groups of the D-glucuronic acid and unsulphated 1iduronic acid linkages, are in salt form may be obtained by reacting a quaternary ammonium salt with heparin, in an aqueous medium the pH of which is low enough to form the quaternary ammonium salt of heparin wherein only the sulphate groups are salified (in practice the pH is from 2 to 2.5) , then selectively neutralizing the carboxyl groups of the D-glucuronic acid and unsulphated 1-iduronic acid linkages of the product so obtained by the addition of a determined amount of quaternary ammonium hydroxide in a dimethylformamide medium» The amount of quaternary ammonium hydroxide to be added is deduced from the neutralisation curve in a dimethylformamide medium for a sample of the product having a known weight.

The methyl, ethyl, ethoxycarbonylmethyl,cyanomethyl, benzyl and substituted benzyl esters of heparin wherein only the carboxyl groups of the sulphated L-iduronic acid linkages or only the carboxyl groups of the unsulphated L-iduronic acid and sulphated L-iduronic acid linkages are esterified, partially or completely may be prepared by reacting an alcohol of the formula HO-CHj-R (III), in which R is a hydrogen atom or a methyl, ethoxycarbonyl, cyano, phenyl or substituted phenyl group, with heparin, In an agueous medium, in the presence of a watersoluble condensation agent of the earbodiimide type such as, for example, l-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide, the pH of the medium being adjusted to a value in the range 3.5 - 4.5 in the first case and inthe range 2-3 in the second case. By the term substituted phenyl is meant benzyl or phenyl substituted with one or two substituents selected from chlorine, nitro, alkyl with 1 to 4 carbon atoms and alkoxy with 1 to 4 carbon atoms. Preferred alcohols of formula (III) which can be used are methanol and ethanol in which case one obtains respectively a selective methyl ester of heparin or a selective ethyl ester of heparin.

Certain heparin esters mentioned above, in particular the said selective esters are new products which are described and claimed, in addition to processes for their preparation, in Patent Specification No. 107¾) 81.

The processes according to the invention using the action of an inorganic or organic base on a heparin ester, which are processes by β-elimination, permit partial and controlled depolymerisation of the heparin without altering its general structure.

The mixtures of sulphated polysaccharides according to the invention possess anti-coagulant and anti-thrombotic activities and a hypolipemic activity.

For mixtures having a sufficiently low mean molecular weight (in practice less than or equal to 7000 daltons), the anti-thrombotic activity is greater than the anti-coagulant activity. The mixtures according to the invention have little toxicity.

For example, the product of Example 9 below is not toxic at a dose of 300 mg/kg when administered intravenously to rats and mice. When administered subcutaneously, its toxicity is equal to that of heparin.

The mixtures of sulphated polysaccharides according to the invention in which the acid groups of the polysaccharides are in the form of pharmace15 utically acceptable salts, especially in the form of the sodium, calcium or magnesium salt, can be used, as anti-coagulant and anti-thrombotic agents, for the prevention and treatment of thrombosis.

They nay also be used for the treatment of hyperlipemia.

They can be advantageously substituted for heparin for ouch applications. In fact, when administered subcutaneously, they show a longer-lasting action than heparin, which enables the frequency of the injections to be reduced. Further they provoke loss secondary effects (hamorrhagio effects) than heparin. ' 51383 They can be administered, in admixture vith a pharmaceutically acceptable carrier, intravenously, subcutaneously, via the lungs (inhalation), rectum and, for the mixtures of sufficiently low mean molecular weight (mixtures of category III in particular),orally.

The doses administered depend on the method of administration and on the desired effect (anti-thrombotic or hypolipemic effect).

The invention is illustrated by the following non-limiting Examples.

The neutral benzethonium salt of heparin or benzethonium heparinate used as the starting substance in Examples 1 to 3, 10 and 12 to 15 comes from a heparin fran pig intestines having the following characteristics: - weight mean molecular weight : 16,000 daltons - specific rotatory power in aqueous solution at °C -Codex anti-coaaulant activity : 157 i.u./mg The neutral benzethonium salt of heparin or benzethonium heparinate used as starting product in Examples 4 to 9 and 11 comes from a heparin from cattle intestines having the following characteristics: - weight mean molecular weight : 11,400 daltons D s +37 - Codex anti-coagulant activity : 128 i.u./mg The neutral henzethonium salt of heparin or benzethoniuw heparinate used as the starting product 5 in Example 16 comes from a pig’s mucous membrane heparin having a weight mean molecular weight of 16,000 daltons, a specific rotatory power in aqueous solution at 20°C of +44° and a Codex anti-coagulant activity of 180 i,u./mg.

The sodium salt of heparin used as the starting product in Exanples 17 to 19 corresponds to the above pig’s mucous membrane heparin» EXAMPIiE 1 g of (4-chloro)benzyl chloride are added; to a solution of 30 g of henzethonium. heparinate in 600 ml of dimethylformamide. After solution has taken place the reactants are left in contact for 60 hours at the ambient temperature (about 20°C), then 600 ml of a 10ja solution of sodium acetate in methanol are added. The precipitate formed is separated by filtration, washed with, methanol and dried in vacuo. 10.75 g of the 4-chloro-bensyl ester of heparin, in the form of the sodium salt, are thus obtained.

The ester obtained is contacted, with stirring, with 269 ml of a 0.4N aqueous solution of sodium hydroxide at 25°C. At the end of 2 hours, neutralisation is effected by the addition of a 0.4N aqueous solution of hydrochloric acid and precipitation is effected by the addition of two volumes (that is, double the volume of the aqueous phase) of methanol. 8,46 g of depolymerised heparin in the form of the sodium salt are isolated by filtration.

EXAMPLE 2 g of benzyl chloride are addded to a solution of 50 g of benzethonium heparinate in 600 ml of dimethylformamide. After solution has taken place the reactants are left in contact for 60 hours at the ambient temperature, and precipitation is effected by the addition of 600 ml of a 1Q£ solution of sodium acetate in methanol. The precipitate is isolated by filtration, washed with methanol and dried in vacuo. 11.4 g of the benzyl ester of heparin is thus obtained, in the form of the sodium salt. g of the above ester are left in contact for 2 hours, with stirring, with 75 ml of a 0.4N aqueous solution of sodium hydroxide at 20°C25°C. Then the solution is neutralised by the addition of a 0.4N aqueous solution of hydrochloric acid and precipitation is effected by adding 2 volumes of methanol. 2.23 β of depolymerised heparin are thus isolated, in the form of the sodium salt. EXAMPIE 3 g of benzyl chloride are added to a solution 5 of 10 g of benzethonium heparinate in 250 ml of dichloromethane.

After solution, the mixture is left for 24 hours at the ambient temperature, then the solvent is evaporated in vacuo. The residue is dissolved in 150 ml of n imethylfnrmamin a and precipitation is effected by the addition of 150 ml of a 105» solution of sodium acetate in methanol. 3.67 g of the benzyl ester of heparin are separated by filtration, in the form of the sodium salt. 2 g of the above ester are treated for 2 hours, with stirring, with 50 ml of a 0.1K aqueous solution of sodium hydroxide at 60°C. After cooling, the solution is neutralized by the addition of a 0.1N aqueous solution of hydrochloric acid, then precipitat20 ion is effected by the add.ition of 2 volumes of methanol. ’ 1.54 g of depolymerised heparin are thus obtained, in the form of the sodium salt.

EXAMPLE 4 g of ethyl chloracetate are added to a solution of 5 g of benzethonium heparinate in 125 ml of dichloromethane and, after solution, the substances are left in contact for 3 days at the ambient temperature. The solvent is evaporated under vacuum, the residue i3 taken up with 75 ml of dimethylformamide and precipitation is effected by the addition of 75 ml of a 10% solution of sodium acetate in methanol. The precipitate, separated by filtration, is washed in methanol, then dried in vacuo. 1.72 g of the carbethoxymethyl ester of heparin are thus obtained, in the form of the sodium salt. 1.7 E of the above ester are treated with 45 ml of a 0.1N aqueous solution of sodium hydroxide at 60°C, with stirring, for two hours. After cooling, the solution is neutralised by the addition of a 0.1N aqueous solution of hydrochloric acid and precipitation is effected by the addition of two volumes of methanol. 1.33 g of depolymerised heparin are isolated by filtration, in the form of the sodium salt.

EXAMPEB 5 g of (4-chloro)benzyl chloride are added to a solution of 10 g of benzethonium heparinate in 250 ml of dichloromethane, and dissolved by stirring. The solution is left for 24 hours at the ambient temperature, then the solvent is evaporated in vacuo.

The residue is taken up with 150 ml of dimethylformamide and precipitation is effected by the addition of 150 ml of a 10% solution of sodium acetate in methanol.

After filtration, washing the precipitate with methanol and drying in vacuo, 3.84 g of the (4chloro)-benzyl ester of heparin are isolated in the form of the sodium salt. 2 g of the above ester are treated with 50 ml of a O.UI aqueous solution of sodium hydroxide at 60°C, with stirring, for two hours. After cooling and neutralisation with a O.lli aqueous solution of hydrochloric acid, precipitation is effected by the addition of two volumes of methanol. The precipitate is isolated by filtration, washed with methanol and dried in vacuo. 1.38 g of depolymerised heparin are thus obtained, in the form cf the sodium salt. ΕΧΑΚΠ3 6 g of (4-nitro)benzyl chloride are added to a solution of 5 g of benzethonium heparinate in 125 ml of dichloromethane and dissolved by stirring. The solution is then left fcr 3 days at the ambient temperature, then the solvent is evaporated in vacuo and the residue is dissolved in 75 ml of dimethylformamide. The ester formed is precipitated by the addition of 75 ml of a 10^ solution of sodium acetate in methanol. The precipitate is collected by filtration, washed in methanol and dried in vacuo. 1.89 g of the (4-nitro)-benzyl ester of heparin are thus obtained, in the form of the sodium salt. 1.85 g of the above ester are treated with 46 ml of a 0.1N aqueous solution of sodium hydroxide at 60°C for two hours, with stirring. After cooling, neutralisation is effected by the addition of a 0.1K aqueous soluticn of hydrochloric acid, and precipitation is effected bjr the addition of two volumes of methanol. The precipitate is isolated by filtration, washed with methanol and dried in vacuo. 1.15 g of depolymerised heparin are thus obtained, in the form of the sodium salt.

BXAffPEE 7 g of benzyl chloride are added to a solution of 50 g of benzethonium heparinate in 500 ml of dichloromethane and dissolved by stirring. The solution is then left at ambient temperature for 24 hours, then the solvent is evaporated in vacuo and the residue is taken up with 400 ml of ether. The insoluble material is separated by filtration. 50 g of the benzyl ester of heparin are thus obtained, in the form of the benzethonium salt. This ester is dissolved in 200 ml of dichloro-rcethane containing 8 ml of 1,5-diaza-bicyclo [4.3.θ] 5-nonene. The solution is refluxed for 3h 30, then tlie solvent is evaporated in vacuo. The residue is dissolved in 450 ml of dimethylformamide and an equal volume of a 10?« solution of sodium acetate in methanol is added. The precipitate is collected by filtration and washed in methanol. Then it is .treated at 0°C for an hour by a IN aqueous solution of sodium hydroxide. After neutralisation, precipitation is effected by the addition of two volumes of methanol. The precipitate is isolated hy filtration, washed with methanol and dried in vacuo. 6.6 g of depolymerised heparin are obtained, in the form of the sodium salt.

EXAMPLE 8 g of (4-chloro)benzyl chloride are added to a solution of 10 g of benzethonium heparinate in 250 ml of dichloromethane and dissolved by stirring.

The solution is left at ambient temperature for 24 hours, then the solvent is evaporated in vacuo.

The residue is taken up with 200 ml of ether and the precipitate formed is isolated by filtration. 10 g of the (4-chloro)-benzyl ester oi heparin are thus obtained, in the form of the benzethonium salt. g of this product are dissolved in 100 ml of dichloro-methane containing 1o5 ml of 1,5-diazabicyclo [4.3 «θ} 5-nonene and refluxed for 4 hours.

Then the solvent is evaporated in vacuo, the residue is taken up with 50 ml of dimethylformamide and 100 ml of a 100 solution of sodium acetate in methanol are added. The precipitate formed is isolated by filtration, washed with methanol, then treated by 24 ml of a IN aqueous solution of sodium hydroxide for an hour at 0°C. The solution is neutralised by the addition of a IN aqueous solution of hydrochloric acid and precipitation is effected by the addition of two volumes of methanol. The precipitate is separated by filtration. After washing with methanol and drying in vacuo, 1 g of depolymerised heparin is obtained, in the form of the sodium salt.

Exnmple 9 ε oT benzyl chloride are added to a solution of 30 ε of benzethonium heparinate in 600 ml of dimethylformamide. After solution has taken place, the reactants are left in contact for 60 hours at the ambient temperature, then the ester formed is precipitated by the addition of 1200 ml of a 10% solution of sodium acetate in methanol. The precipitate is isolated by filtration, washed with methanol and dried in vacuo. io 11.4 g of the benzyl ester of heparin are thus obtained, in the form of the sodium salt. g of the above ester are treated with 250 ml of a 0.1 N aqueous solution of sodium hydroxide at 60°C, for two hours with stirring. After cooling, the solution is neutralised by a 0.1 N aqueous solution of hydrochloric acid and precipitation is effected by the addition of two volumes of methanol. The precipitate is filtered, washed with methanol and dried in vacuo. 6.65 6 of depolymerised heparin are thus obtained, in 2o the form of the sodium salt.

Example 10 120 g of (4-chloro)benzyl chloride are added to a solution of 120 g of benzethonium heparinate in 2.4 1 of dimethylformamide and dissolved by stirring. The solution is then left at ambient temperature for 60 hours, then 2.4 1 of a 10% solution of sodium acetate in methanol are added. The precipitate formed is separated by filtration, washed with methanol and dried in vacuo. 46 g of the (4-chloro)-benzyl ester of heparin are thus obtained, in the form of the sodium salt. g of the above ester are treated with 500 ml of a 0.1 N aqueous solution of sodium hydroxide at 60°C, for two hours, with stirring. After cooling and neutralisation, precipitation is effected by the addition of two volumes of methanol. 11.7 g of depolymerised heparin are isolated by filtration in the form of the sodium salt.

Example 11 g of methyl iodide are added to a solution of 30 g of benzethonium heparinate in 750 ml of dichloromethane and dissolved by stirring. The solution is left for 46 hours at the ambient temperature, then the solvent is evaporated in vacuo. The residue is taken up with 450 ml of dimethylformamide and precipitation is effected by the addition of 450 ml of a 10% solution of sodium acetate in methanol. After filtration, the precipitate is washed with methanol and dried in vacuo. 10.5 g of the methyl ester of heparin are thus isolated in the form of the sodium salt, g of the above ester are treated with 50 ml of a 0.1 N aqueous solution of sodium hydroxide at 60°C, with stirring for two hours. After cooling, the pH of the solution is brought to about 4.5 by stirring with a carboxylic ion exchange resin in the H+ form. The . 51283 resin is then separated by filtration and washed with water. The collected aqueous phases are neutralised, by the addition of a dilute aqueous solution of sodium hydroxide, and are then lyophilised. 2 g of depoly5 merised heparin are thus obtained, in the form of the sodium salt.

Example 12. g of the (h-chloro)-benzyl ester of heparin obtained in Example 10 are treated with 120 ml of a 10% aqueous solution of sodium carbonate at 60°C, for two hours, with stirring. After cooling, the solution is neutralised by a O.AN aqueous solution of hydrochloric acid and precipitation is effected by the addition of two volumes of methanol. 1-57 B of depolymerised heparin are isolated by filtration, in the form of the sodium salt.

Example 13 JO g of ethyl chloracetate are added to a solution of JO g of henzethonium heparinate in 600 ml of dimethyl20 formamide. After solution, the substances are left in contact for 60 hours at the ambient temperature, then 600 ml of a 10% solution of sodium acetate in methanol are added. The precipitate formed is separated hy filtration, washed with methanol and dried in vacuo. .78 g of the carbethoxymethyl ester of heparin are thus obtained, in the form of the sodium salt. g of the above ester are put in contact with 100 ml of a 3% aqueous solution of triethylamine at a temperature of 60°C. At the end of 5 hours, the solution is neutralised hy the addition of an aqueous solution of hydrochloric acid, then precipitation is effected by the addition of 2 volumes of methanol. 2.5 g of depolymerised heparin are isolated by filtration, in the form of the sodium salt.

Example 145 g of chloracetonitrile are added to a solution of 5 g of benzeuhonium heparinate in 125 ml of dichloromethane and dissolved hy stirring. The solution is left for 48 hours at the ambient temperature, then the solvent is evaporated in vacuo. The residue is dissolved in 75 “1 of dimethylformamide and precipitation is effected hy the addition of 75 ml of a 10% solution of sodium acetate in methanol. The precipitate is separated by filtration, washed with methanol and dried in vacuo. 1.63 g of the cyanomethyl ester of heparin are thus obtained, in the form of the sodium salt.

The ester obtained is treated with 40 ml of a 0.1N aqueous solution of sodium hydroxide at 60°C, with stirring, for two hours. After cooling, the solution is neutralised by the addition of a 0.1N aqueous solution of hydrochloric acid, then precipitation is effected by the addition of two volumes of methanol. 1.33 g of depolymerised heparin are isolated by filtration, in the form of the sodium salt.

Example 15 g of the (4-chloro)-benzyl ester of heparin obtained in Example 10 are dissolved, with stirring, in 120 ml of a 10% aqueous solution of sodium carbonate.

After two hours stirring at a temperature from 20°C to 25°C, the pH of the solution is brought to 6 by the addition of a N aqueous solution of hydrochloric acid, then a volume of methanol equal to twice the volume of the aqueous solution is added. The precipitate formed TO is isolated by filtration and 2.1 g of the (4-chloro)benzyl ester of heparin are thus obtained. g of the above ester are treated, with stirring, for two hours with 50 ml of a 0.1H aqueous solution of sodium hydroxide at 60°C. After cooling, the solution T5 is neutralised by the addition of a 0.1N aqueous solution of hydrochloric acid, then precipitation is effected by the addition of two volumes of methanol. 1.4 g of depolymerised heparin are thus obtained, in the form of the sodium salt. in the preceding Examples 1 to 10 and 12 to 15, before precipitating the product formed by the addition of the two volumes of methanol, the concentration of NaCl in the aqueous phase was adjusted to 10% by addition of sodium chloride.

Example Ιό g of (4-chloro)benzyl chloride are dissolved with stirring in a soluticn of 10 g of benzethonium heparinate in 250 ml of dichloromethane. The solution is left for 24 hours at the ambient temperature, then a 10% solution of sodium acetate in methanol is added.

The precipitate formed, is filtered, washed with methanol and dried in vacuo. 5.72 g of (4-chloro)benzyl ester of heparin are thus obtained, in the form of the sodium salt.

A solution of 0.500 g of the above ester in 10 ml 40 of formamide is treated at 60°C, for 5 hours, with 0.5 ml of 1,5-diaza-bicyclo[4,5,oj5-nonene. After cooling, ml of acetone are added. 0.364 g of a precipitate are collected by filtration. This precipitate is treated at 0°C, for two hours, with 6 ml of a N aqueous solution of sodium hydroxide. The aqueous phase is neutralised by the addition of a N aqueous solution of hydrochloric acid and the concentration of NaCl in the medium is adjusted to 10% hy the addition of sodium chloride. Precipitation is effected hy adding two volumes of methanol. 0.263 g of depolymerised heparin are thus obtained, in the form of the sodium salt.

Example 17 2.5 ml of acetic acid then, slowly and with stirring, 150 ml of a 10% aqueous solution of benzethonium chloride are added to a solution of 10 g of heparin (sodium salt) in 40 ml of water. The precipitate formed is collected hy centrifuging, washed with water and dried. 19.67 E of ‘benzethonium acid heparinate are obtained. g of the above product are dissolved in 110 ml of dimethylformamide and 11 g of (4-chloro)-benzyl chloride are added. The reactants are left in contact for 48 hours at the ambient temperature, then 220 ml of a 10% solution of sodium acetate in methanol are added.

The precipitate formed is isolated by filtration, washed with methanol and dried under vacuum. 4.70 g of (4-chloro) -benzyl ester of heparin are thus obtained in the form of the sodium salt. 4g of the above ester are dissolved in 20 ml of water and 40 ml of a 20% aqueous solution of benzethonium chloride are slowly added with stirring. The precipitate formed is collected by centrifuging, washed vzith water and dried under vacuum. The (4-chloro)-benzyl ester of heparin is thus obtained, in the form of the benzethonium salt. g of the above ester (benzethonium salt) is dissolved in 20 ml of dimethylformamide and is treated with 1 ml of 1,5-diaza-bicyclo Qf-,3,cJ 5-nonene at 60°C for five hours. After cooling, 5θ ml of a 10% solution of sodium .acetate in methanol are added. The precipitate formed (0.346g) is collected and treated at 0°C, for two hours, with 5·θ ml of a K aqueous solution of sodium hydroxide. The aqueous phase is neutralised by the addition of a N aqueous solution of hydrochloric acid and the concentration of NaCl in the medium is adjusted to 10% hy the addition of sodium chloride. Precipitation is effected by adding two volumes of methanol. The precipitate is filtered and washed with methanol. 0.253 g of depolymerised heparin are thus obtained in the form of the sodium salt.

Example 18 2.5 nl of formic acid, then, slowly and with stirring, 150 ml of a 10% aqueous solution of benzethonium chloride are added to a solution of 10 g of heparin (sodium salt) in 40 ml of water. The precipitate is collected by centrifuging, washed with water and dried under vacuum. 20.5 g of benzethonium acid heparinate are thus obtained. 2.95 g of the above product are dissolved in 60 ml of dimethylformamide, then 5.9 b1 of a 0.1 N solution of tetrabutylammonium hydroxide in a n-propanol/methanol mixture are added. After the addition of 2.95 g of (4-chloro)-benzyl chloride, the solution is left for five days at the ambient temperature. 74 ml of a 10% solution of sodium acetate in methanol are added. 1.32g of (4-chloro)-benzyl ester of heparin are isolated by filtration, in the form of the sodium salt.

The above ester is dissolved in 6.4 ml of water and 12.8 ml of a 20% aqueous solution of benzethonium chloride are slowly added with stirring. The precipitate formed is collected by centrifuging, washed with water and dried under vacuum. The (4—chloro)-benzyl ester of heparin is thus obtained, in the form of the benzethonium salt. lg of the above ester (benzethonium salt) is dissolved in 20ml of dichloromethane and 1ml of 1,5diaza-hicyclo[4,3,0] 5-nonene is added. The solution is heated with reflux for 5 hours, then the solvent is evaporated under vacuum, the residue is taken up with ml of dimethylformamide and 20 ml of a 10% solution of sodium acetate in methanol are added. The precipitate formed is separated hy filtration and washed with methanol. 0.405g are thus ohtained of a product which is treated at 0°C, for two hours, with 6ml of a H aqueous solution of sodium hydroxide. The aqueous phase is neutralised by the addition of a H aqueous solution of hydrochloric acid and the concentration of HaCl in the medium is adjusted to 10% by the addition of sodium chloride. The precipitation is effected hy adding two volumes of methanol. The precipitate is separated by filtration and washed with methanol. 0.355 g of depolymerised heparin are thus obtained, in the form of the sodium salt. Example 19 0.600 g of heparin (sodium salt) are dissolved in ?ml of water and the pH of the solution is adjusted to 3.5 by the addition of a H aqueous solution of hydro37 chloric acid. 0.300g of l~ethyl-3-(3-dimethylaminopropyl)-carbodiimide are added and, after dissolution, the solution is left for one hour at the ambient temperature. 2.5 ml of an aqueous solution of sodium chloride containing 280g per litre are added, then 15 ml of methanol. The precipitate formed is isolated by filtration, washed with methanol and dried under vacuum. 0.527 g of methyl ester of heparin, the esterification percentage of the carboxyl groups of which is 5θ%> thus obtained in the form of the sodium salt.

O.JOOg of the above ester are dissolved in 7-5 ml of a 0.1 N aqueous solution of sodium hydroxide and the solution is heated at 60°C for 2 hours. After cooling, the solution is neutralised by the addition of a 0.1 N aqueous solution of hydrochloric aoid, the concentration of NaCl in the medium is adjusted to 10% by adding sodium chloride and the precipitation is effected hy adding two volumes of methanol. 0.200 g of depolymerised heparin are thus obtained in the form of the sodium salt.

The following Tables B and.C give the characteristics of the products (depolymerised heparins in the form of the sodium salt) prepared in Examples 1 to 19. The percentages of sulphur, nitrogen and uronic acids, the specific rotatory powers in aqueous solution at 20°C, the mean molecular weights and the activities 3S indicatfed in these Tables have heen determined hy the methods previously described. The column spread of molecular weights gives the approximate extreme values of the molecular weights of the polysaccharides constituting the mixtures, such as determined by gel permeation chromatography on gel of polyacrylamideagarose. In the viscosity column, the viscosities at 25°C of a 10% aqueous solution of the products are shown. lo The OT absorption column gives the absorptions by a cm thickness of the 1% solutions of the products in 0.01 N HCl, the absorptions being measured at the wave-length of the absorption maximum appearing in the range 220 - 232 nm.

TABLE B.

Exam- ple % uronic acids % S % N Moan molecular woight (ln daltons) spread of molacular weights 1 23.0 11.5 2.1 + 44° 7300 1600 - 12,000 2 26.0 11.6 2.0 + 44· 6500 1800 - 13,000 3 25.4 12.0 2.2 + 44· 6400 3000 - 11,000 25 4 23.9 10.9 2.1 + 35° 5500 •2200 - 13,000 5 22 .8 12.2 2.2 + 39° 5000 2000 - 13,000 6 24.5 10.8 2.3 + 33® 4200 1000 - 10,000 7 25.2 11.3 2.0 + -33® 4100 1700 - 10,000 30 Θ 23.7 11.1 2.2 + 35® 4500 1000 - 10,000 9 26.0 11.8 2.2 + 38° 4000 2C00 - 10,000 10 25.2 10.9 2.1 + 41® 3800 1000 - 9000 11 23.8 11.0 2.3 + 27® 2000 1000 - BOJO 35 12 23.9 11.5 2.0 + 40® 4000 2000 - 9000 15 24.3 11.8 2.2 + 45° 9000 3000 - 20,000 14 25,6 12.0 2.1 + 44® 8500 3000 - 20,000 15 25.3 11.8 2.3 + 45® 4000 2000 - 20,000 TABLE B (continued) Exam- ple Vlacoalty (In centipoises) Absorption UV — Codex anticoagulant activity in vitro (i.u./mg) Antl-Xa activity In vitro (i.u./ me) A.P.T.T. activity In vitro fi.57 me) 1 2.87 7.6 127 180 105 2 2.25 7.8 116 148 100 5 2.09 7.9 112 163 89 4 1.95 8.2 83 130 52 δ 1.91 9.7 75 118 43 6 1-.74 12.5 60 140 33 7 1.64 15.0 50 130 50 8 1.61 16.4 54 110 53 9 1.C2 14.0 80 159 45 10 1.67 15.4 62 159 40 11 1.50 22.8 40 90 20 12 1.60 14.4 75 140 35 IS 2.60 5.9 155 140 130 14 2.50 6.1 158 130 130 IS 1.65 10.64 110 170 86 TABLE C.

Exam- ple Mean molecular weight (in daltons) Absorp- tion UV Anticoagulant Activity anti-Xa in vitro Α.Ε.Ϊ'.Ϊ'. in vitro Codex in vitro 16 4200 6 140 63 60 17 5500 7.96 165 80 70 18 4400 6.5 110 60 40 19 5000 10.9 150 45 50 41) Example 20 The product of Example 1 on the one hand and a commercial heparin on the other hand have heen administered subcutaneously at different times, to five healty volunteers, at a dose of 5000 i.u. Codex.

On the blood samples taken 1 hour, 3 hours, 5 hours and 7 hours after the administration, there was measured the plasmatic anti-coagulant activity hy means of the anti-Xa and A.P.T.T. tests previously defined. The results ohtained have been expressed in international units per ml of plasma, by referring to a standard curve traced from tests effected on a control plasma to which has been added known quantities of reference heparin (3rd international standard).

The average results obtained are set out in the following Table D; TABLE D.

Time elapsing bot^oen administration and sampling Anti-Xa activity A.P.T.T, activity Product of Example 1 Commercial Heparin Product of Example 1 Commercial Heparin 1 hour 0.20 0.04 0.04 0.05 3 hours 0.27 0.03 0.05 0.04 5 hours 0.28 0.06 0.03 0.02 7 hours 0.22 0.05 0.02 0.01 It is seen that the product of Example 1 exerte an anti-Xa effect which is much more intense than that of the commercial heparin.

Claims (21)

1. , Λ mixture of sulphated polysaccharides having the general structure of the polysaccharides that constitute heparin and whose acid groups are in the free form or in a salt form, wherein the polysaccharides that constitute said mixture have an ethylenic double bond at one of the ends of their chain, the mixture having, in the form of the sodium salt, the following characteristics: -percentage of sulphur : 9% to 13·5% -percentage of nitrogen : 1.8% to 2.5% -percentage of uronic acids : 20% to 30% -weight mean molecular weight : 2000 to 10,000 daltons -specific rotatory power in aqueous solution at 20°G [oi] d° : +25 ° t0 + 55 °

2. A mixture according to claim 1, in which the polysaccharides that constitute the mixture have the fonnula: wherein R Ϊ3 a hydrogen atom or a carboxylic group, in the free acid form or in the form of a salt, R’ is an OH group or a sulphate group, in the free acid form or in the form of a salt, is an OH group or a sulphate group, in the free acid form or in the form of a salt, R 2 is a sulphonate group, in the free acid form or in the form of a salt, or an acetyl group, -0- is an oxygen bridge, the G 5 linkages are linkages of the glucosamine type present in the structure of heparin, the U linkages are linkages of the uronic acid type (D-glucuronic acid, L-iduronic acid and sulphated L-iduronic acid) present in the structure of heparin, and n is a whole number from 3 to 20, the acid 10 groups of the polysaccharides being in the free form or in the form of a salt.

3. A mixture according to claim 1 or 2 which, in the form of the sodium salt, has the following characteristics: -weight mean molecular weight : 8000 to 10,000 daltons 15 -in vitro Codex anti-coagulant activity : 130 to 160 i.u./mg -in vitro anti-Xa activity : 130 to 180 i.u./mg -in vitro A.P.T.T. (as hereinbefore defined) activity : 100 to 150 i.u./mg 20 _ ratio in vitro anti-Xa activity . χ to 1>5 in vitro A.P.T.T. activity

4. A mixture according to claim 1 to 2 which, in the form of the sodium salt, has the following characteristics: -weight mean molecular weight : 3000 to 8000 daltons 512 8 3 -in vitro Codex -anti-coagulant activity - 80 to 140 i.u./mg -in vitro anti-Xa activity : 120 to 250 i.u./mg -in vitro A.P.T.T. activity ; 80 to 120 i.u./mg ifl vi tro anti-Xa activity -ratio in vitro A.P.T.T. activity : 1.4 to 3

5. A mixture according to claim 1 or 2, which, in the form of the sodium salt, has the following characteristics: -weight mean molecular weight : 2000 to 7000 daltons -in vitro Codex anti-coagulant activity: 10 to 80 i.u./mg -in vitro Anti-Xa activity ; 80 to 250 i.u./mg -in vitro A.P.T.T. activity ; 10 to 80 i.u./mg , . ia vitro anti-Xa activity „ , -ratio ... r-..7-r ...,.1. : 2 to 10 in vitro A.P.T.T.activity

6. A mixture according to claim 5, vzhich, in the form of the sodium salt, has the following characteristics -weight mean molecular weight : 4200 daltons -rotatory power [/J = + 33 -in vitro codex anti-coagulant activity ; 60 i.u./mg -in vitro anti-Xa activity ; 140 i.ii./mg -in vitro A.P.T.T. activity ; 33 i.u./mg

7. A mixture according to Claim 5, which, in the form of the sodium salt, has the following characteristics -weight mean molecular weight : 4100 daltons -rotatory power [c/J ρθ =+ 33° 5X383 -In vitro Codex anti-coagulant activity : 50 i.u./mg -in vitro anti-Xa activity : 130 i.u./mg -in vitro A.P.T.T. activity : 30 i.u./mg

8. A mixture according to claim 5, which, in the form of the sodium salt, has the following characteristics: -weight mean molecular* weight : 4500 daltons -rotatory power jcZJ = + 55° -in vitro Codex anti-coagulant activity : 54 i.u./mg -in vitro anti-Xa activity : 110 i.u./mg -in vitro A.P.T.T. activity : 33 i.u./mg

9. A mixture according to claim 5, which, in the form of the sodium salt, has the following characteristics: -weight mean molecular weight : 4000 daltons -rotatory power [©fj ρθ = + 38° -in vitro Codex anti-coagulant activity · 80 i.u./mg -in vitro anti-Xa activity : 159 i.u./mg -in vitro A.P.T.T. activity ; 45 i.u./mg

10. A mixture according to claim 5, which, in the form of the sodium salt, has the following characteristics: -weight mean molecular weight : 3800 daltons -rotatory power jc/J ” + ^ 1 ° -in vitro Codex anti-coagulant activity : 62 i.u./mg -in vitro anti-Xa activity · 159 i.u./mg -in vitro A.P.T.T. activity : 40 i.u./mg

11. A mixture according to claim 5, which, in the form of the sodium salt, has the following characteristics; -weight mean molecular weight : 2800 daltons -rotatory power d° = + ^7° -in vitro Codex anti-coagulant activity ; 40 i.u./mg 5 -in vitro anti-Xa activity ; 90 i.u./mg -in vitro A.P.T.T. activity ; 20 i.u./mg

12. A mixture according to Claim 5, which, in the form of the sodium salt, has the following characteristics: -weight mean molecular weight ; 4000 daltons 10 -rotatory power M 2 D° -in vitro Codex anti-coagulant activity ; 75 i.u./mg -in vitro anti-Xa activity . 340 i.u./mg -in vitro A.P.T.T. activity · 35 i.u./mg

13. A mixture according to claim 4, which, in the 15 form of the sodium salt, has the following characteristics: -weight mean molecular weight : 4000 daltons -rotatory power = + 45° -in vitro Codex anti-coagulant activity · 110 i.u./mg -in vitro anti-Xa activity · 170 i.u./mg 20 -in vitro A.P.T.T. activity · 86 i.u./mg

14. A mixture of sulphated polysaccharides according to claim 1, substantially as herein described with reference to and as illustrated in any of Examples 1 to 19.

15. A process for the preparation of a mixture 25 according to claim 1, which comprises reacting a water-soluble heparin ester, resulting from the partial or complete esterification of the carboxylic acid groups of heparin, with a water-soluble inorganic or organic base in an aqueous medium and at a temperature from 20°C to 80°C, and isolating the product of depolymerisation thus formed.

16. A process according to claim 15, in which the molar concentration of the base in the medium is 0.1 to 0.6.

17. A process for the preparation of a mixture according to claim 1, which comprises reacting a heparin ester resulting from the partial or complete esterification of the carboxylic acid groups of heparin with a base, in an organic solvent unreactive towards said ester, isolating the product of depolymerisation thus formed as an alkali metal salt and hydrolysing said salt at a low temperature with an aqueous solution of sodium hydroxide whose normality is at least IN.

18. A process according to claim 17, in which the reaction of the heparin ester with the base is effected at a temperature from 20°C to 80°C.

19. A process for the preparation of a mixture of sulphated polysaccharides according to claim 1, substantially as herein described with reference to and as illustrated in any of Examples 1 to 19.

20. A medicament which contains as active ingredient a mixture of sulphated polysaccharides as claimed in any one of claims 1 to 14, the add groups of the polysaccharides being in the form of pharmaceutically acceptable salts.

21. A medicament according to claim 20, in which 5 the acid groups of the sulphated polysaccharides are in the form of the sodium salt, calcium salt or magnesium salt.