NZ197081A

NZ197081A - Mixtures of sulphated polysaccharides

Info

Publication number: NZ197081A
Application number: NZ197081A
Authority: NZ
Inventors: J Mardiguian
Original assignee: Pharmindustrie
Priority date: 1980-05-14
Filing date: 1981-05-13
Publication date: 1984-07-31
Also published as: FR2482611B1; HU188667B; AT384428B; AU7051981A; ATA213681A; EP0040144B1; HUT34769A; MA19143A1; KR840001753B1; ZA813176B; DK211981A; NO811632L; DZ294A1; IE51283B1; GR82283B; AR229510A1; PT73024A; ES502199A0; DE3165361D1; CA1181744A

Description

<div class="application article clearfix" id="description"> New Zealand Paient Spedficaiion for Paient Number 1 97081 19708 1 Priority Dat©(s): . .*.^9 LZ. PATENT OFFICE 13 MAY 1981 RECEIVED Patents Form No. 5 Patents Act 1953 COMPLETE SPECIFICATION SULPHATED POLYSACCARIDES AND PROCESSES FOR THEIR PKEPARATION We, PHAEMINDUSTRIE a company organised and existing under the laws of France, of 35 Quai du Moulin de Cage, 92231 Gennevilliers, France, hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: 1 97 0 8 1 i The present invention relates to new mixtures of sulphated polysaccharides of mean molecular weight inferior to that of heparin, which can be used as anticoagulant and antithrombotic agents in the prevention and treatment of thrombosis and as hypolipemiant agents. Heparin is a mixture of sulphated mucopolysaccharides of animal origin, largely used for its anticoagulant and antithrombotic properties, notably in the prevention of postoperative venous thrombosis, and of its hypolipemiant properties. It is known that it acts in the coagulation process by activating a natural inhibitor of the coagulation contained in the blood, the antithrobin III. 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 the thrombin on the other. The anticoagulant 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 in vitro, on the one hand with respect to the factor Xa and on the other hand with respect to thrombin (cf. for example A. TEIEN et. al., Thrombosis Research 11, 107, 1977). The commercial heparins, which are mixtures of polysaccharides whose mean molecular weight is greater than 10,000 daltons and of which the dispersion of molecular weight goes from 4,000 to about 45,000 daltons, show the following activities in vitro: - 2 - i 97031 /. L. 6. 8i A. perJ~iJL_. p. Anticoagulant activity ^measured by the method of the /u,l? /ja fn<*-«"K»vaLCop«iA- u-S. ' ^ B'r*p>-nr»'h phn^mifinpni n ; ft-t-h prii-M nn.^ hopQPin 'monograph, by P. L. B. & A. referring to the 3rd international standard (or anticoagulant U5P F activity oodsx): 140 to 200 u.i/mg. f V / 5 l&°( Anti-Xa activity measured by the method of TEIEN et al (already cited), by referring to the 3rd international standard: 160 to 180 u.i./mg. A.P.T.T. activity (Activated Partial Thromboplastin Time) measured by the method of TEIEN et al (already cited), by referring to the 3rd international standard: 150 to 170 u.i./mg. The heparin is administered obligatorily parenterally (in practice subcutaneously) and the action is relatively short, from which there are the two following important disadvantages: the need to carry out three administrations per day and the relatively high frequency of post-operative hemorr-^hagic accidents. It is known (cf. JOHNSON et al, Thrombos. Eaemostas.Stuttg. 1976, 35, 586-591; LANE et al, Thrombosis Research 16, 651-662 Pergamon Press Ltd. 1979; LASKER, CHIU, Annals 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 Sephadox gel, fractions of mean molecular weight smaller than that of the heparin and having a dispersion 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 superior to 1), anti-thrombinic activity - 3 - O"? r\ n 4 * « j 1 and on the other hand are more easily absorbed in the circulation from a subcutaneous injection than heparin itself, hence a higher and longer-lasting plasmatic activity than that of the heparin. It is also known (cf. LASKER and CHIU already cited, LASKER already cited; U.S.Patent No.3,766,167; PERLIN et al; Carbohydrate Research, 18, 1971,185-194) to prepare, by enzymatic hydrolysis of heparin, depolymerisation products having a low mean molecular weight e.g. 5,300 to 4,500 daltons determined by ultracentrifuging) and an anticoagulating activity, (determined by the method U.S.P. No.XVII), of about 70 u.i./mg. These products of depolymerisation can be fractionated, by filtration over Sephadex gel, into fractions whose mean molecular weight, determined by ultracentrifuging, goes from 3200 to 5900 daltons and the anticoagulant activities from 45 to 95 u.i./mg (U.S.P. method). These depolymerisation products and the fractions which are obtained are active, taken orally as well as parenterally. It is also known (cf. LASKER already cited) 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 anticoagulant activity of 12 to 100 u.i./mg (U.S.P. method). Finally, it is known (cf. British Patent Application No. 2,002,406 A published on 21.2.1979) to prepare, by resulphatation _ 4 _ 197031 of depolymerisation products of heparin devoid of anticoagulant 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°, and an anticoagulant activity less than 50 u.i./ mg (U.S.P. method), which would be as active taken orally as parenterally for the prevention of thromboses. The present invention provides mixtures of sulphated polysaccharides of the formula: H R' G-O-U H NH-R- (I) wherein R is a hydrogen atom or a carboxylic group, in 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, R2 is a sulphonic group, in the free acid form or in the form of a salt, or an acetyl group, -0- is an oxygen bridge, the G linkages are the linkages of the glucosamine type appearing in the structure of heparin, the U linkages are the linkages of the uronic '.6- acid type^D-glucuronic acid, L-iduronic acid or sulphated - 5 - i o 7 n1 \ / i ^ w * P. L. B. & A. p«r >Z13 /Jtf L-iduronic acid, appearing in the structure of heparin and n is a whole number from 3 to 20, the acid groups of said polysaccharides being in free form or in the form of a salt and showing, in the state of the sodium salt, the following characteristics: percentage of sulphur by weight percentage of nitrogen by weight percentage of uronic acids by weight mean molecular weight 9% to 13.5% 1.8% to 2.5% 20% to 30% 2000 to 10,000 daltons specific rotatory power in aqueous solution at 20°C (A) 20 D : +25° to +55" The sulphur and nitrogen contents and the rotatory power indicated above have been determined by the methods of the U.S. pharmacopeia. XZjT, *^5. ' The content of uronic acids has been determined by the method of N. BLUMENKRANTZ et al. (Analytical Biochemistry, 54, 484, 1973). The mean molecular weight has been determined by gel permeation chromatography on gel of polyacrylamide-agarose by referring to the standard constituted by heparins of known mean molecular weight, according to the method of ANDERSSON et al. (Thrombosis Research, 9, 575, 1976). - o - I 97081 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 as previously defined, it has more particularly as its object those of these mixtures which, in the form of the sodium salt, show the additional characteristics indicated for the categories, I, II, III. in the Table A which follows: TABLE A P. L B. & A. par '4-/ 3 isi, Category I • Category II Category III Mean molecular weight (in daltons) 8000 to 10,000 3000 to 8000 2000 to 7000 Anticoagulant activity in vitro -Sodox KXSP 130 to 160 80 to 140 10 to 80 Activity in vitro anti-Xa 130 to 180 120 to 250 80 to 250 Activity in vitro A.P.T.T. 100 to 150 80 to 120 10 to 80 Ratio Activity in vitro anti-Xa 1 to 1.5 1.4 to 3 2 to 1C Activity in vitro A.P.T.T. |l 3 j, The anticoagulant activity Codex indicated in the Table above has been determined by the method of the -F yon eh '<f/3 m pharmacopeia, 8th odition^—hoparin monograph, Xvtl - 7 - 1 97 0 3 1 The anti-Xa and A.P.T.T. acitivities indicated in table A as well as those indicated in tables B and C further on have been determined by the method of TEIEN et al., already cited, according to the following procedures: a) Determination of the anti-Xa activity: This activity is determined on an ox plasma, free of platelets, by means of chromogenic substrate S 2222 [chromogenic peptide having the structure : (N-benzoyl)Ile-Glu-Gly-Arg-(p-nitro)anilide referring to the 3rd international standard. 100 ft-1 of citrated ox plasma, diluted from 2 to 5 with an aqueous buffer tris/EDTA pH 8,4, are added to 100^*1 of a solution of the product to be tested or of the standard in an aqueous buffer tris/ EDTA pH 8.4, said 100 ft 1 corresponding to 0,02 to 0.08 fj. g of product or standard. After 3 minutes of incubation at 37°C, 100 fJi± of an aqueous solution of Xa factor from ox, which correspond to 7 n Kat of factor Xa, are added. After a 30 seconds incubation period, 200 jA 1 of an aqueous solution of S 2222, which correspond to 0.2 ^mole of S 2222, are added. After a three minutes incubation period, 300 of acetic acid are added and the optical density of the solution is measured at 405 nm in 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 internation units per mg, is given by the formula: slope of the straight line relating to the product 173 slope of the straight line relating to the standard the number 173 corresponding to the value of the activity for the third international standard. - 8 - 197 031 b) Determination of the A.P.T.T. activity The product to be tested and the third international standard are dissolved in a 0.15 M aqueous solution of sodium chloride, then diluted with a citrated ox plasma, free of platelets, so as to obtain concentrations of product to be tested (or of standard) from 0 to 1+jig/ml, 100 i+l of the reagent "automated APTT Precibio" (reagent based on phospholipids of rabbit brain and micronized silica) are added to 100 of the solution so obtained. After a five minutes incubation period at 37°C, 100 i*,i of a 0.025 M aqueous solution of calcium chloride are added. The clotting time is measured by means of a Bio-Merieux fibrometer. By 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: slope of the straight line relating to the product 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 the Tables A, B and C are expressed in international units per mg (u.i./mg), referring to , the 3rd international standard. The mixtures according to the invention are prepared by i process in which a heparin ester resulting from the partial or total esterification of the carboxylic acid groups of heparin is reacted with a base, in an inert organic solvent of the said ester, the product of depolymerisation thus formed - 9 - 197081 in which the carboxylic acid groups are still esterified, being isolated as an alkali metal salt either directly, if fi i'd q a ' ' " A- the original ester is in the form of an alkali metal salt, per &/S> ***** L* 77T or by adding an alkali acetate, if the original ester is 15 J&f T in the form of an alkaline earth metal salt or a quaternary ammonium salt, and hydrolysis being effected by at least 0.1N aqueous solution of sodium hydroxide at a temperature between 0° and +5°. In this ester, the groups of the 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 the sodium salt, an alkaline earth salt such as the calcium salt, a magnesium salt or quaternary ammonium salt with a long chain such as the benzethonium salt. In this case where the heparin ester is soluble in water (for example where the non-esterified acid groups are in the form of the sodium salt), the 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 the bases soluble in water and in particular sodium hydroxide, potassium hydroxide, alkali metal carbonates, triethylamine, triethylenediamine, quinuclidine, 1,5-diazabicyclo [4.3.0] 5-nonene and 1,5-diaza-bicyclo [5.4.0] 5-undecene. Once the reaction finishes, the product of depolymerisation formed is isolated, for example by precipitation by addition of sodium chloride then methanol. has . in _ 1 o*y no-? ± ■o t b os. The ethylenic double bond at one of the extremities of each chain is formed by a reaction of - elimination: + R-OH H R' P. L. B. & A p#r: 2^/3 W under the action of a nucleophileagent or strong base, there fee*u\r\cj qroup is elimination of the group leaving R-0 with formation of an ethylenic double bond and saponification of the ester function. The reaction between the ester and the base may also be effected, particularly when the non-esterified acid groups of the ester are in the form of quaternary ammonium salt with a long chain, in an inert organic solvent for the said ester, such as for example dichloromethane, dimethylformamide, formamide or tetrahudrofuran, at a temperature preferably of 20°C to 80°C. - 11 - KZ. il iH6\ ii yQ4 2' 197031 Bases which may be used are the bases soluble in the solvent used and in particular 1, 5-diaza-bicyclo |4>3«o| 5-nonene, quinuclidine and 1,5-diaza-bicyclo |j5.4»oj 5-undecene. Once the reaction has ended, the product of depolymerisation formed in which the carboxylic acid groups are still esterified is isolated in the form of an alkaline salt, and is hydrolysed P L B & A ]i\' ' ' by an aqueous solution of alkali metal hydroxide, especially °f" o o soc^um hydroxide, at least IN, at i-ew temperature 0 C 10 +5 C . "The final product is separated, for example by precipitation by addition of sodium chloride then methanol. Heparin esters which may be used as starting products for preparing the mixtures of polysaccharides according to the invention may be non-selective esters or selective esters. By "non-selective esters' is meant 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' is meant heparin esters wherein are esterified, partially or wholly, either 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. Heparin esters which may be used as starting products in P. L. B. & A. processes according to the invention are in particular the iJJlUf heparin esters described in the Frcnok Patent No. 15Q> 724 ^-^^Sg|and in the British Patent No. 1,501,095 as well as the methyl, artCSP ethyl, ethoxycarbonylmethyl, cyanaoraethyl, benzyl and 2 - 12 - 197031 I • L. B. & A. substituted benzyl (especially 4-chloro-benzyl, 4-nitro-benzyl) esters of heparin. Preferably, the benzyl or substituted benzyl esters of heparin are used as a starting product. The heparin esters used as starting substances in the processes according to the invention may come from heparin of any origin (e.g. ox lung heparin, heparin from pigs mucous membrane, heparin from cattle intestines) . The non-selective methyl, ethyl, ethoxycarbonylmethyl, cyanomethyl, benzyl and substituted benzyl esters of heparin may be obtained, for example, by the action of a neutral r^,n quaternary ammonium or amino salt of heparin with a halogenated pGr..0r.^_ I jderivative of formula Hal - CH2 - in which Hal represents a chlorine, bromine or iodine atom and R represents a hydrogen atom or a methyl, ethoxycarbonyl, cyano, phenyl or substituted phenyl group. This reaction is effected in solution or in suspension in an inert solvent such as dimethylformamide, methylene chloride, dimethylsulphoxide, tetrahydrofuran or acetone, at a temperature between -20°C and +60°C. The methyl, ethyl, ethoxycarbonylmethyl, cyanomethyl, benzyl and substituted benzyl esters of heparin wherein are esterified, partially or wholly, either 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 are obtained by reacting a hiogen derivative of the above formula (II) with an acid F L B & A .\r' quaternary ammonium salt of heparin in which are salified, OLfltdll-Koo-Vo ' I y'jjj ^bboidoo the sulfate groups, either only the carboxyl groups of 7,Tv:'- the D-glucuronic acid linkages, or only the carboxyl groups of the D-gluquronic acid and unsulphated L-iduronic acid linkages, t^ie ot^er carboxyl groups being in the free acid form. This - 13 - 197031 reaction is carried out in the same conditions as the reaction of the halogen derivative of the formula (II) with a neutral quaternary ammonium salt of heparin. P. L. B. & A. cj a The acid quaternary ammonium salts of heparin, in which f IS /£& are salified,^l»eoidco the sulfate groups, only the carboxyl groups of the D-glucuronic acid linkages, are prepared by reacting a quaternary ammonium salt with heparin, in an aqueous medium the pH of which is between 3 and 4* p. L. B. & r^g acj_cj quaternary ammonium salts of heparin, in which £V$ adcil'Hcio *+r> ^r"T — are salified, booidoa the sulfate groups, only the carboxyl i ft m groups of the D-glucuronic acid and unsulphated L-iduronic acid linkages, are 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 sulfate groups are salified (In practice the pH is from 2 to 2.5), then selectively neutralising the carboxyl groups of the D-glucuronic acid and unsulphated L-iduronic acid linkages of the product so obtained by 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 said product having a known weight. The methyl, ethyl, ethoxycarbonylmethyl, cyanomethyl, benzyl and substituted benzyl esters of heparin in which are esterified, partially or wholly, either 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 prepared by reacting an alcohol of the - \ formula HO - CH2 - R(III), in which R is a hydrogen atom or a methyl, ethoxycarbonyl, cyano, phenyl or substituted phenyl group, - 14 - 197031 lP. L. B. S A. with heparin, in an aqueous medium, in presence of a water-soluble condensation agent of the carbodiimide 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 in the range 2-3 in the second case. As alcohol of formula (III) which can be used, may be particularly mentioned methanol and ethanol, in which case one obtains respectively a selective methyl ester of heparin and a selective ethyl ester of heparin. The processes according to the invention using the action of a mineral or organic base on a heparin ester, which are processes by p - elimination, produce a partial and controlled "depolymerisation of the heparin without altering its general structure. The mixtures of sulphated polysaccharides according to the invention possess anticoagulant and antithrombotic activities and a hypolipemiant activity. For mixtures having a sufficiently low mean molecular weight (in practice less than or equal to 7000 daltons), the antithrombotic activity is greater than the anticoagulant- activity. The mixtures according to the invention have little toxicity. For example, the ?o r.G.m. q , I j^ product of example g is not toxic at a dose 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 pharmaceutically acceptable salts, especially in the form of the sodium, calcium or magnesium salt," can be used, as anticoagulant and antitfrombotic agents, for the preventior and treatment of thrombosis. i 97081 They are also utilisable for the treatment of hyperlipemia. Advantageously they can be substituted for heparin for such 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 fewer secondary effects (Hemorragic effects) then heparin. They can be administered, in admixture with a pharmaceutically acceptable vehicle, intravenously, subcutanously, 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 (antithrombotic or hypolipemiant effect). The following examples illustrate the invention without it being restricted thereto. t ? The neutral benzethonium salt of heparin or benzethonium -i r; ,-,^-,,heparinate used as the starting-substance in the examples . * Ok u 1* to 3, 10, 12 to 15 comes from a pig's intestine heparin having .> ,ft£ C&v:*;o ' ~~—the following characteristics: - Weight mean Molecular weight : 16,000 daltons - Specific rotatory power in aqueous solution at 20°C : p B.&A. £<*] 20 : +41° Per-^.iA- Anticoagulant acitivty : 157 u.i./mg The neutral benzethonium salt of heparin or benzethonium heparinate used as starting product in the Examples 4 "to 9 and 11 comes from a heparin from cattle intestines having the following characteristics: - mean molecular weight by weight : 11,400 daltons W 2S : +37° - 16 - 'W 3 /X(^ 1970S - 17 - P. L. B. & A. ,45lP fr^j^ - anticoagulant activity GogTox : 128 u.i./mg The neutral benzethonium salt of heparin or benzethonium heparinate used as the starting product in the example 16 comes p , „ from a pig's mucosa heparin having a weight mean molecular r. L. o. & A. weight 16,000 daltons, a specific rotatory power in aqueous 'W5 solut ion at 20°C of +44° and a GodoK anticoagulant activity 180 u.i./mg. The sodium salt of heparin used as starting product in examples 17 to 19 corresponds to the above pig's mucosa heparin. Example 1 30 g of (4-chloro)benzyl chloride are added to a solution of 30 g of benzethonium heparinate in 600 ml of dimethylformamide. After solution, the reactantsare left in contact for 60 hours at the ambiant temperature (about 20°C), then 600 ml 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. 10.75 g of the 4-chloro-benzyl 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 aqueous 0.4N solution of sodium hydroxide at 25°C. At the ,;7C;•• pCE end of 2 hours, neutralisation is effected by addition of a 0.4N Jaclueous solution of hydrochloric acid and precipitation is ««——r* """'effected by 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 30 g of benzyl chloride art. tcioed to a solution of 30 g of. benezethonium heparinate in 600 ml of dimethylformamide. After solution, the reactants are left in contact for 60 hours at the ambiant temperature, and precipitation is effected by 1970S addition of 600 ml of a 10$ 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. 3 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°C - 25°C. Then the solution is neutralised by addition of a 0.4N aqueous solution of hydrochloric acid and precipitation is effected by adding 2 volumes of methanol. 2.23 g of depolymerised heparin are thus isolated, in the form of the sodium salt. Example 3 10 g of benzyl chloride are added to a solution of 10 g of benzethonium heparinate in 250 ml of dichloromethane. After solution, the mixture is left 24 hours at ambiant temperature, then the solvent is evaporated in vacuo. The residue is dissolved in 150 ml of dimethylformamide and precipitation is effected by addition of 150 ml of a 10$ 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 0.1N aqueous solution of sodium hydroxide at 60°C. After cooling, the solution is neutralised by addition of a 0.IN aqueous solution of hydrochloric acid, then precipitation is effected by addition of 2 volumes of methanol. 1.54 g of depolymerised heparin are thus obtained, in the form of the sodium salt. I* - j>cr - 1 9?0s 1 EXAMPLE 4 5 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 ambiant temperature. The solvent is evaporated under vacuum, the residue is taken up with 75 ml of dimethylformamde and precipitation is effected by 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 g of the above ester are treated with 43 ml of a 0.1 N aqueous solution of sodium hydroxide at 60°C, with stirring, for two hours. After cooling, the solution is neutralised by addition of a O.IN solution of hydrochloric acid and precipitation is effected by addition of two volumes of methanol. 1.33 g of depolymerised heparin are isolated by filtration, in the-form of the sodium salt. ft - ^ - 1 9700 1 Example 5 10 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 ambiant temperature, then the solvent is evaporated in vacuo. The residue is taken up with 150 ml of dimethlformamide and precipitation is effected by addition of 150 ml of a lOfo solution of sodium acetate in methanol. After filtration, washing the precipitate with methanol and drying in-vacuo, 3«$4 , of the (4-chloro)-benzyl ester of heparin are isolated in the form of the sodium salt. 2 g of the above ester are treated with 50 ml of a 0.1N aqueous solution of sodium hydroxide at 60°C, with stirring, for two hours. After cooling and neutralisation with a 0.1N aqueous solution of hydrochloric acid, precipitation is effected by addition of two volumes of methanol. The precipitate is isolated by filtration, washed with methanol and dried in vacuo. I.38 g of depolymerised heparin are thus obtained, in the form of the sodium salt. Example 6 5 g of (4-nitro)benzyl chloride are added to a solution of 5 g of benzethoni-um heparinate in 125 ml of dichloromethane and dissolved by stirring. The solution is then left for 3 days at ambiant temperature, then the solvent is evaporated in vacuo and the residue is dissolved in 75 ml of dimethylformamide. The ester formed is precipitated by addition of 75 ml of a 10% solution of sodium acetate in methanol. The precipitate is collected by filtration, washed in methanol, and dried in vacuo. 1 97 08 1 1.89 g of the (4-nitro)-benzyl ester of heparin are thus obtained, in the form of the sodium salt. 1.8$ g of the above ester are treated with 46 ml of a O.IN aqueous solution of sodium hydroxide at 60°C for two hours, with stirring. After cooling, neutralisation is effected by addition of a O.IN aqueous solution of hydrochloric acid, and precipitation is effected by addition of two volumes of methanol. The precipitate is isolated by filtration, washed with methanol and dried in vacuo. 1.13 g of depolymerised heparin are thus obtained, in the form of the sodium salt. Example 7 30 g of benzyl chloride are added to a solution of 30 g of benzethonium heparinate in 500 ml of dichloromethane and dissolved by stirring. The solution is then left at ambiant 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. 30 g of the benzyl ester of heparin are thus obtained, in the form of benzethonium salt. This ester is dissolved in 200 ml of ciichl or erne thane containg 8 ml of 1,5-diaza-bicyclo 4.3*0 5-nonene. The solution is refluxed for 3 h 30, then the 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 IN aqueous solution of sodium hydroxide. After neutralisation, precipitation is effected by addition of two volumes of methanol. The precipitate is isolated by filtration, washed with methanol and dried in vacuo. 6.6 g of depolymerised heparin are obtained, in the form of the sodium salt. _ 22-- ^ - 1 9700 1 EXAMPLE 8 10 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 ambiant 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-chlor)-benzyl ester of heparin are thus obtained, in the form of the benzethonium salt. 5 g of this product are dissolved in 100 ml of dichloromethane containing 1.5 ml of 1,5-diaza-bicycl [4.3.0] 5-nonene and refluxed for 4 hours. Then the solvent is evaporated in vacuo, the residue is taken up with 30 ml of dimethylformamide and 100 ml of a 10$ 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 addition of a IN solution of hydrochloric acid and precipitation is effected by 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. Example 9 30 g of benzyl chloride are added to a solution of 30 g of benzethonium heparinate in 600 ml of dimethylformamide. After solution, the reactants are left in contact for 60 hours at the ambiant temperature, then the ester formed is precipitated by addition of 1200 ml of a 10$ solution of sodium acetate in methanol. The precipitate is isolated by filtration, washed with methanol and d ed in vacuo. 11.4 g of the benzyl ester t 97 0 8 1 of heparin are thus obtained, in the form of the sodium salt. 10 g of the above ester are treated with 250 ml of a O.IN aqueous solution of sodium hydroxide at 60°C for two hours, with stirring. After cooling, the solution is neutralised by a O.IN aqueous solution of hydrochloric acid and precipitation is effected by addition of two volumes of methanol. The precipitate is filtered, washed with methanol and dried in vacuo. 6.65 g of depolymerised heparin are thus obtained, in the form of the sodium salt. EXAMPLE 10 120 g of (4-chloro)benzyl chloride are added to a solution of 120 g benzethonium heparinate 2.4 1 of dimethyl-formamide 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 forms is separated by filtration, washed with methanol and dried in vacuo. 46 g of the (4-chloro)-benzyl ester of heparin are thu: obtained, in the form of the sodium salt. 20 g of the above ester are treated with 500 ml of a O.IN aqueous solution of sodium hydroxide at 60°C, for two hours, with stirring. After cooling and neutralisation, precipitatior is effected by 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 30 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 48 hours at the ambiant temperature, then the solvent is evaporated in vacuo. The residue is taken up with 450 ml of dimethylformami' and precipitation is effected by 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 o sodium salt. 3 1 2 g of the above ester are treated with $0 ml of a O.IN aqueous solution of sodium hydroxide at 60°C, with stirring, for two hours. After cooling, the pH of the solution is brought to about 4«5 by stirring with a carboxylic ion exchange resin in the H+ form. The resin is then separated by filtration and washed with water. The collected aqueous phases are neutralised by addition of a dilute aqueous solution of sodium hydroxide, and are then lyophilised. 2 g of depolymerised heparin are thus obtained, in the form of the sodium salt. EXAMPLE 12 3 g of the (4-chloro)-benzyl ester of heparin obtained in Example 10 are treated with 120 ml of a 10$ aqueous solution of disodium carbonate at 60°C, for two hours, with stirring. After cooling, the solution is neutralised by a 0.4N aqueous solution of hydrochloric acid and precipitation is effected by addition of two volumes of methanol. 1.57 g of depolymerised heparin are isolated by filtration, in the form of the sodium salt EXAMPLE 13 30 g of ethyl chloracetate are added to a solution of 30 g of benzethonium heparinate in 600 ml of dimethylforaiamide. After solution, the substances are left in contact for 60 hours at ambiant temperature, then 600 ml of a 10$ solution of sodium acetate in methanol is added. The precipitate formed is separated by filtration, washed with methanol and dried in vacuo. 10.78 g of the carbethoxymethyl ester of heparin are thus obtained, in the form of the sodium salt. 2S~ - 1 97 1 3 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 by addition of an aqueous solution of hydrochloric acid, then precipitation is effected by addition of 2 volumes of methanol. 2.5 g of depolymerised heparin are isolated by filtration, in the form of the sodium salt. EXAMPLE 14 5 g of chloracetonitrile are added to a solution of 5 g of benzethonium heparinate in 125 ml of dichloromethane and dissolved by stirring. The solution is left for 4& hours at ambiant temperature, then the solvent is evaporated in vacuo. The residue is dissolved in 75 ml of dimethylformamide and precipitation is effected by 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 O.IN aqueous solution of sodium hydroxide at 60°C, with stirring, for two hours. After cooling, the solution is neutralised by addition of a O.IN aqueous solution of hydrochloric acid, then precipitation is effected by 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 3 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 disodium carbonate. After two hours f 9708 1 - j^- stirring at a temperature from 20°C to 25°C, the pH of the solution is brought to 6 by 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 is isolated by filtration and 2.1 g of the (4-chloro)-benzyl ester of heparin are thus obtained. 2 g of the above ester are treated, with stirring, for two hours with 50 ml of a O.IN aqueous solution of sodium hydroxide at 60°C. After cooling, the solution is neutralised by addition of a O.IN aqueous solution of hydrochloric acid, then precipitation is effected by 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 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 16 10 g of (4-chloro)benzyl chloride are dissolved with stirring in a solution of 10 g of benzethonium heparinate in 250 ml of dichloromethane. The solution is left for 24 hours at the ambiant temperature, then a 10$ solution of sodium acetate in methanol is added. The precipitate formed is filtered, washed with methanol. 3*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 of formamide is treated at 60°C, for 5 hours, with 0.5 ml of 1,5-diaza-bicyclo [4.3. °] 5-nonene. After cooling, 70 ml of acetone are added. O.364 g of a precipitate are collected by VI t 97 08 1 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 addition of a N aqueous solution of hydroxide. The aqueous phase is neutralised by addition of a N aqueous solution of hydrochloric acid and the concentration of NaCl in the medium is adjusted to 10$ by addition of sodium chloride. Precipitation is effected by adding two volumes of. methanol. 0.263 g of depolymerised heparin are thus obtained, in the fora 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 by centrifugation, washed with water and dried. 19.67 g of benzethonium acid heparinate are obtained. 11 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 ambiant 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. 4 g 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 centrifugation, washed with water and dried under vacuum. The (4-chloro)-benzyl ester of heparin is thus obtained, in the form of the benzethonium salt. o* 1 07 1 ' - j£-- " " V;- U J 1 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 £4,3,03 5-nonene at 60°C for five hours. After cooling, 50 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«$ ml of a N aqueous solution of sodium hydroxide. The aqueous phase is neutralised by addition of a N aqueous solution of hydrochloric acid and the concentrdion of NaCl in the medium is adjusted to 10$ by addition of sodium chloride. Precipitation is effected by adding two volumes of methanol. The precipitate is filtered, washed with methanol. 0.253 g of depolymerised heparin are thus obtained in the form of the sodium salt. EXAMPLE. 18 2.5 ml 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 centrifugation, washed with water and dried under vaccum. 20.5 g of benzethonium acid heparinate are thus obtained. 2.95 g of the above product are dissolved in 60 ml of dinethylformamide, then 5*9 ml of 0.1N 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 ambiant temperature. 74 ml of a 10$ solution of sodium acetate in methanol are added. 1.32 g of (4-chloro)-benzyl ester of heparin are isolated by filtration, in the form of the sodium salt. - - f 97 0 8 1 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 centrifugation, washed with water and dried under vacuum. The (4-chloro)-benzyl ester of heparin is thus obtained, in the form of the benzethonium salt. 1 g of the above ester (benzethonium salt) is dissolved in 20 ml of dichloromethane and 1 ml of 1,5-diaza-bicyclo [WJ 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 15 ml of dimethylformamdie and 20 ml of a 10$ solution of sodium acetate in methanol are added. The precipitate formed is separated by filtration and washed with methanol. 0.405 g are thus obtained of a product which 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 addition of a N. aqueous solution of hydrochloric acid and the conentration of NaCl in the medium is adjusted to 10$ by addition of sodium chloride. The precipitation is effected by 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 7 ml of water and the pH of the solution is adjusted to 3«5 by addition of a N aqueous solution of hydrochloric acid. 0.300 g of l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide are added and, after dissolution, the solution is left for one hour at the ambiant temperature. 2.5 ml of an aqueous solution of sodium chloride containing 280 g per litre, then 15 ml of methanol are added. " I 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 50$, are thus obtained in the form of the sodium salt 0.300 g of the above ester are dissolved in 7.5 ml of a O.IN aqueous solution of sodium hydroxide and the solution is heated at 60°C for 2 hours. After cooling, the solution is neutralised by addition of a 0.1 N aqueous solution of hydrochloric acid, the concentration of NaCl in the medium is adjusted to 10$ by adding sodium chloride and the precipitation is effected by 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 the 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 indicated in these Tables have been determined by the methods previously described. In the column dispersion of molecular weights are given the approximative extreme values of the molecular weights of the polysaccharides constituting the mixtures, such as determined by gel permeation chromatography on gel of polyacrylamide-agarose. In the viscosity column, the viscosities at 25°C of the 10$ aqueous solutions of the products are shown. In the UV absorption column are shown the absorptions by a 1 cm thickness of the 1$ solutions of the products in 0.01 N HC1, said absorptions being measured at the wave-length of the absorption maximum appearing in the range 220-232 nm. i 97081 TABLE C " -• B. P< A. P 'V 5 / Vlf Example Mean Molecular Weight (in daltons) Absorption UV Anticoagulant Activity Anti-Xa in Vitro A.P.T.T. in Vitro Oe do.it in Vitro 16 4200 6 140 63 60 17 5500 7.96 165 80 70 18 4400 6.3 110 60 40 19 5000 10.9 150 45 50 TABLE C (continued) Example % Uronic Acids % S % N Dispersion of molecular weights Viscosit 16 25.1 11.3 2.2 + 41° 1000 - 10,000 1.75 17 24.6 11.5 2.1 + 44° 2000 - 12,000 1.90 18 26.0 11.2 2.2 + 43° 2000 - 9,000 1.60 19 25.4 11.1 2.3 4- -P-o 2000 - 12,000 1.90 TABLE B p ~r t\ ri 5, Example % % S %N Mean Uronic Molecular Acids Weight (in daltons) 1 23.0 11.5 2.1 + 44° 7300 2 26.0 11.6 2.0 + 44° 6500 3 25.4 12.0 2.2 + 44° 6400 4 23.9 10.9 2.1 + 35° 5500 5 22.8 12.2 2.2 + 39° 5000 ' 6 24.5 10.8 2.3 + 33° 4200 7 25.2 11.3 2.0 + 33° 4100 ' 8 23.7 11.1 2.2 + 35° 4500 9 26.0 11.8 2.2 + 38° 4000 10 25.2 10.9 2.1 + 41° 3800 11 23.8 11.0 2.3 + 27° 2800 12 23.9 11.5 2.0 + 40° 4000 13 24.3 11.8 2.2 + 45° 9000 14 25.6 12.0 2.1 + 44° 8500 15 25.3 11.8 2.3 + 45° 4000 Dispersion of Molecular Weights 1600 1800 3000 2200 2000 1000 1700 1000 2000 1000 1000 2000 3000 3000 2000 12,00 13,00 11,00 13,00 13,00 10,00 10,00 10,00 10,00 9,00 8,00 9,00 20,00 20,00 20,00 - 31 - TABLE B (continued) 1970S! P. L. B. & A. P«ri?i/& '47 J Jtlf Example Viscosity (in centi-poises) Absorption UV Anticoagulant Anti-Xa Activity Activity MSP Oodojc in Vitro in vitro (u.i./mg) (u.i./mg) A.P .T.T. Activity in Vitro (u.i./mg 1 2.37 7.5 127 180 105 2 2.25 7.S 116 ' 148 100 3 2.OS 7.9 112 163 89 4 1.93 8.2 33 130 52 5 1.91 9.7 75 118 43 6 1.74 12.5 60 140 33 7 1.64 15.0 50 130 30 8 1.61 16.4 54 110 33 9 1.62 14.0 80 159 45 10 1.57 15.4 62 159 40 11 1.50 22.8 40 90 20 12 1.60 14.4 75 140 35 13 2.60 5.9 135 .140 130 14 2.50 6.1 138 130 130 15 1.65 10.64 110 170 86 EXAMPLE 20 The product of Example 1 on the one hand and a commercial heparin on the other hand have been administered, at different times, by subcutaneous way to five healthy volunteers, at a dose of 5000 u.i. Codex. On the blood samples taken 1 hour, 3 hours, 5 hours and 7 hours after the administration, there -j__ was measured the plasmatic anticoagulant activity by means of ; the anti-Xa and A.P.T.T. tests previously defined. The results — obtained have been expressed in international units per ml of - plasma, by referring to a staadard curve traced from tests effected on a control plasma to which has been added known quantities of reference heparin (3rd international standard) . M TSMAR K34 - 32 - </div>

Claims

<div class="application article clearfix printTableText" id="claims"> 1 ^ 7 3 8 1 The average results obtained are collected in the following Table D_: TABLE D Time elapsing between the administration and the sampling Anti-Xa activity A.P.T.T. activity Product of Example 1 Commercial Heparin Product of Example 1 Commercia Heparin 1 hour 0.20 0.04 0.04 0.05 3 hours 0.27 0.08 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 exerts an anti-Xa effect much more intense than that of the commercial heparin. - - i 97OS 1 - 34 - WHAT WE CLAIM IS:

1. Mixtures of sulphated polysaccharides of the formula H R' CH2-R, •0—^ih ty H.OH H NH-R2 (I) P. L. B. & A. p«rE^ / z a w r-f <n 6 !0\ V-X \ ,'f X*aV fa . X. ■\ •• h. X;- - \ v! ■ \ s \ wherein R is a hydrogen atom or a carboxylic group, in 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, R2 is a sulphonic group, in the free acid form or in the form of a salt, or an acetyl group, -0- is an oxygen bridge, the G linkages are the linkages of the glucosamine type appearing in the structure of heparin, the U linkages are the linkages of the urc*\ic acid type,^-glucuronic acid, L-iduronic acid or sulphated L-iduronic acid, appearing in the structure of heparin and n is a whole number from 3 to 20, the acid groups of said polysaccharides being in free form or in the form of a salt and showing, in the state of the sodium salt, the following characteristics: percentage of sulphur by weight percentage of nitrogen by weight X percentage of uronic acids by weight \V. V'-jmean molecular weight specific rotatory power in aqueous solution at 20°C 9% to 13.5% 1.8% to 2.5% 20% to 30% 2000 to 10,000 daltons (<£ ) 20 D +25° to +55' - 35 - i 97081

2. Mixturer: according to Claim 1 which, in the form of the sodium salt, shows the following characteristics ^ - mean molecular weight : 8000 to 10,000 daltons - anticoagulant activity in vitro •Sodox: 150 to 160 u.i./mg - activity in vitro anti-Xa : 150 to 180 u.i./mg - activity in vitro A.P.T.T. : 100 to 150 u.i./mg activity in vitro anti-Xa c. i +•« i q - ratio activity in vitro A.P.T.T. ' P- L B. & A. /Ltl

3 It if n 9 • ^ • <*■ f\. 9 F Mixture- according to Claim 1 which show£ in the /ifl J ify, ' ' form of the sodium salt, the following characteristics: p. l. b. & a. _ mean molecular weight : 5000 to 8000 daltons perFV^' /j / 7-T"""""" - anticoagulant activity in vitro Qodex: 80 to 140 u.i./mg ' HI 5 I'Qf - activity in vitro anti-Xa : 120 to 250 u.i/mg - activity in vitro A.P.T.T. : 80 to 120 u.i./mg activity in vitro anti-Xa : 1.4 to 5 -'ratio activity in vitro A.P.T.T. '" p. L. 3. 2= a. ^

4. Fixture according to Claim 1 .which show$y in the "/ 3 fttf form of the sodium salt, the following characteristics: jo 7 n01 - 36 - " p. l!~B. & A. p«r.EjJk _ mean molecular weight : 2000 to 7000 daltons ^3 _ anticoagulant activity in vitro ^ocfox: 10 to 80 u.i./mg - activity in vitro anti-Xa: 80 to 250 u.i./mg - activity in vitro A.P.T.T. : 10 to 80 u.i./mg p. l. b. & a. activity in vitro anti-Xa . p -f-n to - ratio activity in vitro A.P.T.T. ■ ; $. Mixture according to Claim 5, which shows, in the form w^/3 lrip of the sodium salt, the following characteristics: - mean molecular weight : 4-200 daltons P. L r p \ ~ rotatory power (<X ) 20 = + 33° k & a d /cfl5 !(« - anticoagulant activity in vitro Codex : 60 u.i./mg - activity in vitro anti-Xa : 14-0 u.i./mg - activity in vitro A.P.T.T. : 33 u.i./mg F. I. B. & A. | f? . £ t If. j ^ V« Mixture according to Claim y which shows, in the form of the sodium salt, the following characteristics 7 - mean molecular weight : 4100 daltons - rotatory power ((X ) ^ = + 33° ' ® ?■?/,*> a a. d x . ^ v P'srCV^ ^ _ anticoagulant activity in vitro ffodox : 50 u.i./nT ^ 3 !il r activity in vitro anti-Xa : 130 u.i./mg - activity in vitro A.P.T.T. : 30 u.i./mg P. L. B. & A. d 1■ * f &. Mixture according to Claim which shows, in the J HI 3 /tfy * form of the sodium salt, the following characteristics: - mean molecular weight : 4500 daltons - rotatory t>ower (*0^° = + 35° P. L. B. a a. D - anticoagulant activity in vitro Oodox : 5^ u.i./mg 'h/ 3 fry - activity in vitro anti-Xa : 110 u.i./mg - activity in vitro A.P.T.T. : 33 u.i./mg 97031 P.1R. & A. klj. - 37 % Mixture according to Claim 5*, which shows^ in the form of the sodium salt, the following characteristics: - mean molecular weight : 4000 daltons D - rotatory power (oC ) = + 38° 1 • L. 8. &. A. D p«r£.■££...._ _ anticoagulant activity in vitro •Toctox: 80 u.i./mg l^3 Ulo - activity in vitro anti-Xa : 159 u.i./mg P. L. B. & A. p- L. B. & A. ' t-tf 3 }<if, m - activity in vitro A.P.T.T. : 45 u.i./mg rV6 ^ $ %). Mixture according to Claim >, ; i^/ 3 tfif Mixture according to Claim >, which shows, in'the form of the sodium salt, the following characteristics: - mean molecular weight : 3800 daltons P- L B. & a. - rotatory power (<£ ) =+41° — - anticoagulant activity in vitro : 62 u.i./me - activity in vitro anti-Xa : 159 u.i./mg - activity in vitro A.P.T.T. • 40 u.i./mg — T<i. Mixture according to Claim Jfc, which shows, in the form of the sodium salt, the following characteristics: - mean molecular weight : 2800 daltons P. Lb 3 a ~ ^atory power (oi) = +27 ■ ■ A. D ^ USP 1 3 / fij. - anticoagulant activity in vitro Gotiox: 40 u.i./rng - activity in vitro anti-Xa : 90 u.i./mg - activity in vitro A.P.T.T. : 20 u.i./mg ■F'b. a a. » i* Mixture according to Claim 5s, which shows, in the '4V 3 /fij- form of the sodium salt, the foil owing characteristics : - mean molecular weight : 4000 daltons , N on o NZ.PA7i-:;rfC;,T!CE-- rotatory power (CL ) _ = +40 * D i 1 6 MAR i?04 i i 97031 - 38 - p. la. - anticoagulant activity in vitro &odeK : 75 u.i./mg ^ 3 _ activity in vitro anti-Xa : 140 u.i./mg - activity in vitro A.P.T.T. : 35 u.i./mg 12,. Mixture according to Claim 3, which shows, in the form of the sodium salt, the following characteristics: L- B. & A. psr mean molecular weight : 4000 daltons 20 £rotatory power (<£ ) ^ = + 45° anticoagulant activity in vitro QuJlm: : 110 u.i./mg activity in vitro anti-Xa : 170 u.i./mg activity in vitro A.P.T.T. : 86 u.i./mg

13. A process for the preparation of the mixtures according to Claim 1, in which a water-soluble heparin ester resulting from the partial or total esterification of the carboxylic acid groups of heparin is reacted, in water, at a temperature from 20°C to 80°C, with a water-soluble mineral or organic base, and the product of depolymerisation thus formed is isolated.

14. A process according to Claim 13, in which the molar concentration of the base in the medium is 0.1 to 0.6.

15. A process for the preparation of the mixtures according to Claim 1, in which a heparin ester resulting from the partial or total esterification of the carboxylic acid groups of heparin is hydrolysed with a base, in an inert organic solvent of the said ester, the product of depolymerisation thus—formed in s&z /l r f-' * * i <r\ n o -? i. o i vO^. - 39 - which the carboxylic acid groups are still esterified, being P. L B & a ^so^-ate(^ as an alkali metal salt either directly, if the Dor original ester is in the form of an alkali metal salt, or by *«h»l ^ /3 adding an alkalL acetate, if the original P.L.B.&A. * form of an alkaline earth metal salt or a quaternary ammonium w w ester is in the A. * o*\, 42J\ j j salt, and hydrolysis being effected by^at least O.IN aqueous solution of sodium hydroxide at a temperature between 0° and + 5' c

16. A process according to Claim 15, ii) which the reaction of the heparin ester with the base is effected at a temperature from 20°C to 80°C.

17. A medicament for the prevention and treatment of thromboses and for the treatment of hyperlipemia, which contains as active ingredient a mixture of sulphated polysaccharides as defined in any one of Claims 1 to 12, the acid groups of the said polysaccharides being in the form of pharmaceutically acceptable salts.

18. A medicament according to Claim 17 in which the acid groups of the sulphated polysaccharides are in the form of sodium salts, calcium salts or magnesium salts.

19. Mixtures of sulphated polysaccharides, as claimed in Claim 1 and substantially as hereinbefore described with reference to any one of Examples 1-19. o "7 n o 1 ✓ / u u I - 40 -

20. A process for the preparation of the mixtures of sulphated polysaccharides as claimed in Claim 1, substantially as hereinbefore described with reference to any one of Examples 1-19. PHARMINDUSTRIE by their authorised agents P.L. BERRY & ASSOCIATES per rL% J^PATENTOFFfCg ? 2'MAR 1984 i 197OS 1 291 CHrRj G-O-U H,OH H NH-R. </div>