NZ249165A

NZ249165A - Mixture of sulphated oligosaccharides and pharmaceutical compositions

Info

Publication number: NZ249165A
Application number: NZ249165A
Authority: NZ
Inventors: Andre Uzan
Original assignee: Rhone Poulenc Rorer Sa
Priority date: 1992-02-07
Filing date: 1993-02-04
Publication date: 1996-01-26
Also published as: NO942897D0; JP3218039B2; AU671817B2; WO1993016112A1; AU3505193A; ATE157991T1; JPH07503496A; HUT67878A; FR2687158B1; NO307466B1; MX9300584A; FI943642A; KR950700334A; FR2687158A1; CA2126241A1; HU214327B; NO942897L; CA2126241C; DE69313826D1; GR3024695T3

Abstract

A mixture of sulphated oligosaccharides which are components of heparin. At least 70 % has a molar mass of 5400-7800 daltons, and at least 5 % has a molar mass of over 6900 daltons.

Description

<div class="application article clearfix" id="description"> 249165 New Zealand No. 2 4 916 5 International No. PCT/FR93/00114 TO BE ENTERED AFTER ACCEPTANCE AND PUBUCAT10N Priority dates: 1- 2_* SZ. ;international fifing date: Lv"2-,c^ ;Classification: 008 Wl Ivca; Afelk.'&A-jg.g- ;Pubiicstion date: 2 6 JAN 1996 ;Journal No.: 1I4.O0 ;NEW ZEALAND PATENTS ACT 1953 ;COMPLETE SPECIFICATION ;Title of invention: ;Sulphated polysaccharides, preparation thereof, pharmaceutical composition and use thereof ;Name, address and nationality- of appiicant(s) as in international application form: ;RHONE-POULENC RORER SA, a French body corporate, of 20 Avenue Raymond Aron, F92160, Antony, France ;2491 ;WO 93/16112 1 PCT/FR93/00114 ;SULPHATED POLYSACCHARIDES, PREPARATION PROCESS, PHARMACEUTICAL COMPOSITION AND USE THEREOF ;The present invention relates to the field of low-molecular-weight polysaccharides. More 5 particularly, it relates to oligosaccharide compositions having excellent pharmacological and antithrombotic properties. ;Antithrombotic treatments generally make use of two large categories of agents, namely 10 anticoagulants and anti-platelet agents. ;Among the anticoagulants, anti-vitamin K compounds constitute a very important family. Since these compounds are active via the oral route, they are used in numerous indications. However, their use is 15 still limited by certain disadvantages, and in particular the risks of haemorrhaging which they cause and the difficulty of adapting the dosage to a long-term treatment. ;Heparins constitute the second category of 2 0 anticoagulants. They are biological substances of extraction from the family of glycosaminoglycans composed of oligosaccharides having variable chain lengths and degrees of sulphation. More precisely, the molecular weights of the chains constituting non-25 fractionated heparin are generally spread between 2,000 and 40,000 daltons. Heparins are used in different types of thrombosis, in particular in the treatment or prevention of venous thrombosis, possibly in ;249 1 ;2 ;combination with other therapies. ;The disadvantage of heparins resides in their high anticoagulant activity, which may bring about haemorrhaging, and in their sensitivity to certain 5 inhibitory factors of blood such as platelet factor 4, which necessitates the use of relatively large doses. ;Moreover, heparins are very heterogeneous products. It is thus difficult to evaluate their mechanism of action, to assess the contribution, of each 10 of the components in the overall activity of heparin and, consequently, to increase the antithrombotic activity without increasing the side effects. Developments in scientific knowledge in this field have revealed that the biological properties, that is to say 15 the effects on blood coagulation, as well as the pharmacological properties, that is to say the in vivo antithrombotic activities, of these oligosaccharides vary according to their molecular weight. ;A first solution to the abovementioned 2 0 disadvantages has been provided by low-molecular-weight heparins. These heparins are mixtures of sulphated oligosaccharides whose molecular weights are generally spread between 2,000 and 10,000 daltons. These heparins are obtained by fractionation of the oligosaccharide 2 5 chains of heparin (for example by gel permeation according to Barrowcliffe et al., Thrombosis research 12 (1977) 27-36), or by fragmentation (depolymerization) of the oligosaccharide chains of ;249 1 ;3 ;heparin using chemical or enzymatic agents. In particular, the depolymerization has been described by treatment of a heparin ester in the presence of a strong base (EP 40144). It may also be performed by 5 treatment of heparin in the presence of nitrous acid, or by the action of a heparinase (EP 64452). These various processes lead to mixtures of oligosaccharides having the general structure of the polysaccharides constituting heparin, but having a lower weight-average 10 molecular weight. These modifications are generally reflected in a better bioavailability and a minimal effect on bleeding relative to non-fractionated heparin. ;More particularly, research efforts have 15 essentially been oriented towards mixtures derived from heparin having very short oligosaccharide chains. Thus, Patent EP 27089 indicates that mixtures of oligosaccharides derived from heparin not containing more than 8 saccharide units have a specific 2 0 antithrombotic activity which is superior to heparin. ;In the same way, hexasaccharides have been prepared and their antithrombotic properties studied (EP 64452). There may also be mentioned the more recent Patents EP 84 999 and EP 301 618 on polysaccharides such as 25 hexa-, penta- and tetrasaccharides derived from heparin. ;However, the products hitherto described have not made it possible to resolve the problems ;24 9 1 6 5 ;4 ;encountered with heparins in a totally satisfactory manner. In particular, most of the low-molecular-weight heparins have an inhibitory action towards thrombin (anti-IIa activity) which is more than 6 times lower 5 than that of heparin. Experimentally on a standard thrombosis model such as that of Wessler in rabbits, the antithrombotic doses are very much greater than those of heparin. ;The Applicant has now shown that it is 10 possible to obtain, from native or depolymerized heparins, mixtures of oligosaccharides which have excellent biological and pharmacological properties and, consequently, better therapeutic potential. ;Indeed, the Applicant has unexpectedly found 15 that when a mixture of oligosaccharides is prepared for which the molecular distribution is characterized by a proportion equal to or greater than 70 % of sulphated oligosaccharides of molecular weight between 5,400 and 7,800 daltons and at least 5 % of sulphated 2 0 oligosaccharides of molecular weight greater than ;6, 900 daltons, having an anti-IIa activity (determined in the manner herein after described) greater than approximately 60 IU/mg and preferably equal to or greater than approximately 70 IU/mg, this preparation retains the advantages of good bioavailability and of ;25 negligible effects on bleeding of the low-molecular- ;weight heparins, but has the additional advantage of an increased inhibitory power towards thrombin, and ^ ;superior antithrombotic activity with an at le^as ;2491 ;5 ;duration of action. ;One subject of the invention thus resides in a mixture of sulphated oligosaccharides having the general structure of the oligosaccharides constituting 5 heparin, characterized in that it comprises at least 70 % of oligosaccharides having a molecular weight between 5,400 and 7,800 daltons, at least 5 % of oligosaccharides having a molecular weight greater than 6,900 daltons, and in that it has an anti-IIa activity 10 greater than approximately 6 0 IU/mg and preferably equal to or greater than approximately 7 0 IU/mg. ;Such a mixture unites for the first time the advantages of non-fractionated heparin and those of low-molecular-weight heparins. ;15 Compared with native heparin, the mixtures of the invention additionally have a less pronounced activity to the inhibitory factors of blood such as platelet factor 4, which increases their therapeutic potential. ;2 0 Other advantages of the mixtures of the invention reside in particular in the reduction of certain undesirable side effects, such as the thrombocytopenic effect. One of the disadvantages of the known mixtures derived from heparin originates from 25 the fall which they may cause in the number of platelets. This undesirable effect is strongly decreased when the mixtures of the invention are employed. ;2491 ;6 ;The abovementioned properties make possible a particularly effective pharmacological use, especially in the prophylaxis and treatment of venous or arterial thrombosis. In addition, they should make it possible 5 to use larger doses in vivo without increasing the risks of haemorrhaging. ;The mixtures of the invention more preferably comprise at least 10 % of oligosaccharides having a molecular weight greater than 5,900 daltons. 10 Moreover, preferred mixtures of the invention comprise 70 % of oligosaccharides having a molecular weight between 5,70 0 and 7,500 daltons. ;Even more preferably, the mixtures of the invention comprise at least 60 % of oligosaccharides 15 having a molecular weight between 5,7 00 and 6,900 daltons. ;Moreover, the anti-IIa activity of the mixtures of the invention is preferably lower than their anti-Xa activity. ;2 0 In a preferred mode, the mixtures of the invention are more particularly fractions of depolymerized heparin. As indicated above, ;depolymerized heparin may be obtained by any chemical, enzymatic or other technique, known to a person' skilled 25 in the art, which makes it possible to fragment the oligosaccharide chains of heparin. In particular, the processes described in Patents EP 40144, EP 64452, ■EP 37319 or EP 337327 are suitable for the invention. ;249 1 6 5 ;7 ;Even more preferably, the mixtures of the invention consist of oligosaccharides having a ;2-O-sulpho-4-enopyranosuronic acid moiety at one of their ends. ;5 ;A particularly advantageous mixture consists of a heparin fraction depolymerized by the action of a base on a heparin ester. ;process for the preparation of a mixture as defined 10 above, characterized in that a heparin or a depolymerized heparin is fractionated by gel filtration. ;parameters, the control of which makes it possible to 15 calibrate the molecular weight and molecular distribution of the final mixture. In particular, these parameters are the ionic strength of the eluent and the nature of the support used. ;20 characterized in that the steps consisting of (i) dissolving the starting heparin or depolymerized heparin in the eluent, (ii) passing the solution thus obtained through a column containing at least the solid support for the gel filtration, which has been 25 equilibrated beforehand with the same eluent, and (iii) recovering the fractions of desired molecular weight. ;Another subject of the invention relates to a ;The process of the invention involves several ;More preferably, the fractionation is are successively performed. ;In a particular embodiment of the inv ;2491 ;8 ;a depolymerized heparin is used as starting heparin. Such a heparin may be obtained according to the processes described in Patents EP 40144, EP 64452, EP 37319 or EP 337327. ;5 Even more preferably, a heparin is used which has been depolymerized by the action of a base on a heparin ester, such as for example according to the technique described in Patent EP 40 144. In particular, the depolymerization may be performed in an aqueous 10 medium or in an inert organic solvent," under the action of an organic or inorganic base such as for example sodium hydroxide or potassium hydroxide, an alkali metal carbonate or a tertiary amine (triethylamine, triethylenediamine, etc.). The action of the base on 15 the ester makes it possible to perform a partial and controlled depolymerization of heparin without altering its general structure. ;As eluent which may be used in the process of the invention, there may be mentioned various types of 20 saline solutions, such as sodium chloride solutions. However, the Applicant has shown that, in order to obtain fractions having the best properties, it is particularly advantageous to perform the fractionation using an eluent chosen from phosphate buffers, in 25 particular such as potassium phosphate, sodium phosphate or NH4H2P04. It is also possible to use solutions of NaCl04 or NH4N03, which allow mixtures having excellent characteristics to be obtained. ;249 1 ;9 ;The concentration of the eluent, and thus its ionic strength, are adapted to the desired final mixture. In particular, the concentration of the eluent is advantageously less than 1 M and even more 5 preferably between 0.1 and 0.5 M. ;When a phosphate buffer is used, it is particularly advantageous to work at concentrations close to 0.2 M. ;In the second step of the process of the 10 invention, the support used is generally chosen according to the average molecular weight of the starting product (native, depolymerized etc. heparin), the final product desired, and the behaviour of the starting mixture in the eluent used. A gel of 15 polyacrylamide-agarose type is advantageously used as support. By way of example, there may be mentioned the gels AcA 54, AcA 202, sephadex G25 or G50, or alternatively Biogel P30, which give good results. ;In a first particularly advantageous 2 0 embodiment of the process of the invention, during the second step of the fractionation, the solid support is distributed in several columns arranged in series. This variant of the invention makes it possible to employ large final amounts of support for the gel filtration, 25 without the disadvantages of the prior art, namely, essentially, the packing phenomena. In this way, the separation is much cleaner, including the high molecular weight range, in a single fractionation ;249165 ;10 ;operation, and the supports may more readily be regenerated. ;The number of columns used is adapted by a person skilled in the art, depending on the volume and 5 nature of the gel employed, so as to obtain the best balance between the efficiency of the separation and the harmful effect due to the packing of the gel. ;For practical implementation reasons, it is generally preferred to use a number of columns less 10 than 2 0 in the second step of the process. ;As a guide, 40 litres of AcA 202 gel may be distributed between 10 columns of 4 litres, or 130 litres between 10 columns of 13 litres. ;In another particularly advantageous 15 embodiment of the process of the invention, at least 2 types of support are used successively during the second step of the fractionation, which supports have . different separation characteristics. This variant of the invention allows a better quality of final 20 fractionation to be obtained. ;By way of example, the fractionation may be performed on the following sequence of gels: ;AcA 202 - AcA 54 - AcA 202. ;For a better implementation of the invention, 25 it is important to employ large amounts of gel, in order to achieve a cleaner separation and to obtain greater homogeneity. Taking into account, however, the ■ fairly slow flow rates used for this type of gel ;249165 ;11 ;filtration, the volume of gel should be adapted to the amount of product to be separated, in order to obtain the best balance between the separation and the longitudinal diffusion effect. ;In the process of the invention, the starting heparin (g) /volume of gel (1) ratio is advantageously-less than 2 and even more preferably between 0.5 and 1.5. ;Another aim of the invention relates to a pharmaceutical composition having a mixture as defined above as active principle. Such a composition may be used particularly advantageously in the prophylaxis or the treatment or the prevention of thrombotic accidents. More precisely, it may be used: ;- in the prevention of venous thrombosis in risk situations, ;- in the prevention of arterial thrombotic accidents, in particular in the case of myocardial infarction, ;- in post-operative care, in the prevention of venous thrombosis in surgical patients, or alternatively ;- in the prevention of clotting of blood in surgical equipment. ;The present invention will be described more fully with the aid of the examples which follow, ;should be considered as a guide and non-limiting these examples, the physicochemical, biological ;249165 ;12 ;pharmacological characteristics of the mixtures were determined according to the following techniques: Molecular weight and molecular distribution The molecular weights and the molecular distributions 5 of the products are determined by high pressure liquid chromatography with two columns in series, for example those marketed under the name TSK G3 0 00 XL and TSK G2000 XL coupled with a refractometric detector. ;The eluent used is 0.5 M lithium nitrate and 10 the flow rate is 0.6 ml/minute. The system is calibrated with monodisperse standards obtained by fractionation of enoxaparine (Pharmuka) by exclusion chromatography on agarose-polyacrylamide (IBF) ;according to the technique described by Barrowcliffe et 15 al., Thromb. Res., 12, 27-36 (1977-78) or D.A..Lane et al., Thromb. Res. 12, 257-271 (1977-78). The results are calculated using the GPC6 program (Perkin Elmer). Anti-Xa activity ;The technique used is that described by Teien A.N. and 20 Lie M. in Thrombosis research ljO (1977) 399-410, with as standard the first international standard for low-molecular-weight heparins (WHO LMWH1). ;Anti-IIa activity ;The technique used is that described by Anderson L.O. 25 et al. , in Thrombosis research JL5 (1979) 531-541, with as standard the first international standard for low-molecular-weight heparins (WHO LMWH1). ;249 1 ;13 ;APTT ;The technique used is the "Actimat" technique (Bio Merieux) as described by Bell W.N. ana Alton H.G. in Nature 174 (1954) 880-881. ;5 Venous thrombosis in rabbits ;The technique used is that described by Wessler S. in Journal of Clinical investigation 3_4 (1955) 647-651. Bleeding time in rats ;The technique used is that described by Palm M. et al., 10 in Thrombosis and haemostasis 64(1) (1990) 127-132. Key to the figures: ;Figure 1: in vitro APTT activity on human plasma of the mixtures of the invention compared with other heparins. Figure 2 (a and b): kinetics of the anti-Xa activity of 15 the mixtures of the invention in rabbits: comparison with another low-molecular-weight heparin. ;Figure 3 (a and b): kinetics of the anti-IIa activity of the mixtures of the invention in rabbits: comparison with another low-molecular-weight heparin. ;20 Example 1:.Preparation of a depolymerized heparin. ;To a solution of 10 g of sodium heparinate in 100 ml of water is added a solution of 25 g of benzethonium chloride in 125 ml of water. The product obtained at room temperature is filtered, washed with water and 25 then dried. 15 g of benzethonium heparinate thus obtained are dissolved in 75 ml of methylene chloride, to which are added 15 ml of benzyl chloride. The solution is heated at a temperature between 25 and 35°C ;249 ;14 ;for 25 hours. 90 ml of 10 % sodium acetate solution in methanol are then added, followed by filtration, ;washing with methanol and drying. 10 g of heparin benzyl ester obtained under the conditions described 5 above, in the sodium salt form, are dissolved in 250 ml of water. To this solution, heated to approximately 6 0°C, is added 0.9 g of sodium hydroxide. The temperature is maintained for 1 h 30 at approximately 60°C, and the reaction mixture is subsequently cooled 10 to about 20°C and neutralized by addition of dilute hydrochloric acid. The concentration of the medium is then adjusted to a 10 % content of sodium chloride, and the product is precipitated in 750 ml of methanol, filtered off and dried. ;15 Example 2: Preparation of the mixture Ml ;10 glass columns 100 mm in diameter and 50 cm in height, each containing 4 litres of AcA 202 gel (gel in the form of polyacrylamide-agarose beads, of diameter between 60 and 140 m), are used in series for 20 the fractionation: ;A solution containing 30 g of heparin which has been depolymerized under the conditions described in Example 1 is placed at the head of the first column and eluted with a mobile phase consisting of 0.2 M 25 KH2P04 solution, at a flow rate of 300 ml/hour. The fractions are collected at the end of the tenth column. ;This treatment allows the oligosaccharide chains to be separated efficiently according to their ;2491 ;15 ;molecular weight, and to combine them subsequently in order to obtain mixtures having the desired characteristics. The mixture Ml was thus obtained, for which the in vitro biological and physicochemical 5 characteristics are given in Table 1. ;Example 3: Preparation of the mixture M2. ;The mixture M2 was obtained by following the same fractionation procedure as that described in Example 2, using 50 g of depolymerized heparin. 10 The in vitro physicochemical and biological characteristics of the mixture M2 are given in Table 1. Example 4: Preparation of the mixture M5 and of the mixture M6 ;The mixture M5 was obtained by following the 15 same fractionation procedure as that described in Example 2, from 3 0 g of depolymerized heparin, but using 10 columns 9 cm in diameter and 50 cm in height. ;The mixture M6 was obtained by a first fractionation performed according to the procedure of 2 0 Example 2, from 15 0 g of depolymerized heparin on ;10 columns 18 cm in diameter and 50 cm in height, with a flow rate of the mobile phase of 1.1 1/hour. The fractions of interest were pooled and the oligosaccharides precipitated by addition of methanol. 2 5 The recovered solid was subsequently dissolved and then refractionated on the same chromatographic system. The fractions were recovered at the end of the tenth ■column. ;?4Q165 ;16 ;The in vitro biological and physicochemical characteristics of the mixtures M5 and M6 are given in Table 1. ;Example 5: Comparative Example: Preparation of the 5 mixture M3 and of the mixture M4. ;The mixture M3 was obtained by following the same fractionation procedure as that described in Example 2. For the mixture M4, after fractionation according to the procedure of Example 2, it was 10 obtained by mixing a fraction coming off at ;5,530 daltons (63 %) and a fraction coming off at 7,770 daltons (37 %). ;The in vitro biological and physicochemical characteristics of the mixtures M3 and M4 are given in 15 Table 1. ;Example 6: in vitro study of the APTT activity on human plasma of the mixtures of the invention. ;The results obtained are presented in Figure 1. They show clearly that the mixtures of the 20 invention (mixture Ml) cause the APTT, on human plasma, to vary much more markedly in vitro than with non-fractionated heparin. ;10 ;249165 ;17 Table 1 ;Product ;Ml ;M2 ;Ex. 1 ;M3 ;M4 ;M5 ;M6 ;Average molecular weight ;6410 ;6340 ;4280 ;5550 ;6360 ;6420 ;6780 ;Molecular distribution (% MW) ;- > 7,500 ;1 ;5.5 ;9 ;0 ;22 ;3 . 5 ;3 . 5 ;- 7,200+300 ;12 . 5 ;15 ;3 . 5 ;0 ;10 . 5 ;16.75 ;35.25 ;- 6,600+300 ;47 ;30.5 ;5 ;9 ;10 ;37 .5 ;50.75 ;- 6,000+300 ;35 ;32.5 ;6.5 ;30 ;19.5 ;31.75 ;9 . 5 ;- 5, 400 + 300 ;4 . 5 ;13 .5 ;7 . 5 ;40.5 ;25 ;9.5 ;1 ;- < 5,100 ;0 ;3 ;68.5 ;20.5 ;13 ;1 ;0 . 1 ;Xn vitro activitv ;- anti-Xa ;109 ;127 ;94 . 25 ;116 . 6 ;130 ;126 ;106 ;- anti-IIa ;75 ;80 ;27 .75 ;41. 8 ;78 ;68 ;70 ;Example 7: Kinetic study of the anti-Xa and anti-IIa 20 activities in rabbits ;This study was performed according to the following procedure: ;- rabbits: New Zealand (3.5 kg) ;- volume injected: subcutaneous route: 0.5 ml/kg ;2 5 (abdominal region) ;- blood samples: 3 ml of blood are withdrawn on 3.1 % sodium citrate solution (1/10) from the median artery of the ear at times 0 min, 45 min, 90 min, 3 h, 4 h, ;6 h, 8 h. ;3 0 After subcutaneously injecting the rabbit ;2491 ;18 ;with the mixtures Ml, M5 and M6 according to the invention, or with depolymerized heparin according to Example 1, at equal anti-Xa doses, anti-Xa and anti-IIa activities are obtained in the plasma which are: 5 - parallel and comparable for anti-Xa (Figure 2) ;- much superior with the mixture of the invention for the anti-IIa activity (Figure 3). ;The same comparison, made with respect to the mixture M4 described in Example 5, shows that the anti-10 ■ Xa activities of the plasma remain parallel but that there is a very large difference for the anti-IIa activity (Figures 2 and 3) . ;Therefore, these results surprisingly show that the mixtures of the invention have persistent, 15 sustained and high in vivo anti-Xa and anti-IIa activities. These mixtures are accordingly particularly advantageous with respect to non-fractionated heparin, whose in vivo anti-Xa and anti-IIa activities are very short-lived, but also with respect to the low-20 molecular-weight .heparins described in the prior art, whose in vivo anti-IIa activity is very short-lived. Example 8: Study of the in vivo activity of the mixtures of the invention on venous thrombosis in rabbits after intravenous injection. 25 The results obtained are collated in Table 2. ;This table shows that the mixture M3 (Example 4), whose molecular distribution is different from that claimed, ■and depolymerized heparin (Example 1) are less active ;2 ;19 ;than the mixture Ml of the invention. In particular, the latter gives an anti-IIa activity which is clearly superior. ;^vamnle 9: Study of the in vivo activity of the 5 mixtures of the invention on venous thrombosis in rabbits after subcutaneous injection. ;The results obtained are collated in Tables 3 and 4. These tables show that the mixtures Ml, M5 and M6 are more active than a depolymerized heparin of the 10 prior art (Example 1), and that they have an at least equivalent duration of action. ;Example 10: Study of the in vivo activity on the bleeding time in rats. ;The results presented in the following 15 Table 5 show clearly that the mixtures of the invention (illustrated by the mixtures Ml, M5 and M6) cause less bleeding than the other low-molecular-weight heparins. The results given correspond with the increase in the bleeding time in minutes with respect to control 2 0 values. By way of comparison, at 2 mg/kg, heparin induces an increase in the bleeding time of 13 [lacuna] . ;249165 ;20 Table 5 ;Dose\Product ;Ml ;M5 ;M6 ;Ex. 1 ;M4 ;4 mg/kg ;1.07 + 0 . 4 ;4 + 1.1 ;8 . 6 + 3 . 6 ;8 mg/kg ;3.0±0.7 ;4.0+0.5 ;9.6±1.8 ;7.9 + 2. 1 ;33.7+8 ;EXAMPLE 1 ;Anti-Xa = 94.25 IU/mg Anti-IIa = 27.75 IU/mg ;MIXTURE Ml ;109 IU/mg 75 IU/mg ;MIXTURE M3 ;Anti-Xa = 116.6 u/mg Anti-IIa = 41.8 u/mg ;Control ;Mg/kg ;Doses anti-Xa ;/*/kg anti-IIa M/kg 318.00 30.00 8 . 80 265.00 25 . 00 7.30 212.00 20.00 5.90 183.50 20 . 00 13 . 80 257 .3 30 10 .75 214.4 25 8.9 6 171.5 20 7 . 1 107 12 . 5 4.5 0 0 0 n 5 5 8 8 5 8 8 5 8 Weight of the thrombus 2.24± 2 . 24 12 . 4±8.4 53.2±25 6.44± 3.44 27 ,74± 17 . 96 27 . 5± 12 . 66 50.5±15 7 5 . 3 ± 2 5 120. 7± 34.1 Number of rabbits without thrombus 4/5 2/5 3/8 5/8 2/5 2/8 1/8 0/5 0/8 Anti-Xa plasma /i /ml 0.370± 0.026 0.356± 0.039 0.193± 0.015 0.268± 0.02 0 .363± 0 . 023 0.296± 0 . 008 0 .241± 0 . 022 0.125± 0 . 011 (n = 4 ) Anti-IIa plasma ;x/ml 0.100± 0.004 0.069± 0.011 0 . 062± 0.004 0.196+ 0 . 009 0.100± 0 . 008 0 .130± 0 . 006 0.093± 0.008 0.054+ 0.005 (n = 4) to no K«B& a> CJT f • • # Table 3 Control EXAMPLE 1 Anti-Xa = 94.25 IU/mg Anti-IIa = 27.75 IU/mg MIXTURE Ml 109 IU/mg 7 5 IU/mg Time 90 minutes 240 minutes 360 minutes 90 minutes 240 minuteB 360 minutes anti-Xa IU/kg Doses anti-IIa IU/kg mg/kg 300.00 88.33 3 .18 300.00 88.33 3 .18 300.00 88.33 3 .18 200.00 137 .98 1. 83 200.00 137 .98 1. 83 200.00 137.98 1.83 n 8 5 5 5 5 5 5 Weight of the thrombus 73 .10 62 .70 41.00 68.00 114.00 100 . 60 339.70 166 . 90 0 . 00 13 . 60 4 .10 0 . 00 10 .40 0.00 47 . 20 0 .00 22 . 00 3 . 60 91.00 19 . 50 2 . 90 0.00 15 . 20 0 . 00 44 . 80 18 .50 3 .20 0 . 00 0 . 00 19 . 00 0 . 00 23 .40 0 . 00 111.50 12 .20 60.40 7 . 10 0 . 00 Mean £ SEM 120±34 5 . 62±2.75 14 . 56±9.12 25 . 72±16.72 13 . 30±8.60 8.48±5.24 38 . 24±21.20 Student test p <0.05 p <0.05 NS p <0.05 p <0.05 NS Number of rabbits without thrombus 0/8 2/5 2/5 1/5 2/5 3/5 1/5 Anti-Xa plasma /i/ml 0.8 9±0.10 0.89±0 .125 0 . 47±0.005 0.61±0.06 0 . 61±0.06 0 . 40±0 . 04 (N = 4) Anti-IIa plaema /i/ml 0.16±0.02 0 .16±0 . 023 0.12±0.013 0 . 55±0.04 0.55±0 . 04 0.32±0.02 (n = 4 ) # Table 4 CONTROL MIXTURE M5 MIXTURE M6 Time 90 minuteQ 90 minutes 90 minutes 90 minutes Anti-Xa doses IU/kg 200.00 300.00 200 .00 300.00 n 6 8 5 8 2 126 . 9 28 . 4 31.1 0 77.2 77 . 3 0 135 0 110.2 25 . 9 44 . 5 24 . 6 Weight of the thrombus 150 . 7 0 0 0 180 . 8 0 0 0 223 .0 0 0 102 83 . 9 0 0 23 . 2 Mean ± SEH 144 . 8±21 26.93±14.38 8 . 9±8.9 3 9.4 8±18.06 0 Number of rabbits 0/6 4/8 4/5 3/8 0/2 without thrombus TNO DIIIBI^I CD CJl 249165 WO 93/16112 24 PCT/FR93/00114 </div>

Claims

<div class="application article clearfix printTableText" id="claims"> CLAIMS

1. Mixture of sulphated oligosaccharides having the general structure of the constituent oligosaccharides of heparin, characterized in that it 5 comprises at least 70 % of oligosaccharides having a molecular weight between 5,400 and 7,800 daltons, and at least 5 % of oligosaccharides having a molecular weight greater than 6,900 daltons, and in that it has an anti-

IIa activity, determined in the manner herein before described, greater than 60 IU/mg. 10 2. Mixture according to claim 1, characterized in that it comprises at least 7 0 % of oligosaccharides having a molecular weight between 5,700 and 7,5 00 daltons.

3. Mixture according to claim 2, 15 characterized in that it comprises at least 60 % of oligosaccharides having a molecular weight between 5,700 and 6,900 daltons.

4. Mixture according to any one of claims 1 to 3, characterized in that it comprises at least 10 % of 2 0 oligosaccharides having a molecular weight greater than 6,900 daltons.

5. Mixture according to any one of claims 1 to 4, characterized in that it has an anti-IIa activity equal to or greater than 70 IU/mg. 25

6. Mixture according to any one of claims 1 to 5, characterized in that it is a fraction of heparin.

7. Mixture according to any one of clait 5, characterized in that it is a fraction of 24 9 1 6 5 25 depolymerized heparin.

8. Mixture according to claim 7, characterized in that it consists of oligosaccharides having a 2-O-sulpho-4-enopyranosuronic acid moiety at one of their ends.

9. Mixture according to claim 8, characterized in that it is a heparin fraction which has been depolymerized by the action of a base on a heparin ester.

10. Process for the preparation of a mixture according to any one of claims 1 to 9, characterized in that heparin or depolymerized heparin is fractionated by gel filtration.

11. Process according to claim 10, characterized in that the fractionation comprises successively the steps consisting of (i) dissolving the starting heparin or depolymerized heparin in the eluent, (ii) passing the solution thus obtained through a column containing at least the solid support for the gel filtration which has been equilibrated beforehand with the same eluent, and (iii) recovering the fractions of desired molecular weight.

12. Process according to claim 11, characterized in that depolymerized heparin is used.

13. Process according to claim 12, characterized in that heparin is used which has been depolymerized by the action of a base on a hepari ester. 24 916 5 26

14. Process according to claim 11, characterized in that, during the second step of the fractionation, the support is distributed in several columns arranged in 5 series.

15. Process according to claim 11, characterized in that the second step is performed by successively using at least 2 types of support, having different separation characteristics. 10

16. Pharmaceutical composition having as active principle a mixture of oligosaccharides according to any one of claims 1 to 9.

17. Pharmaceutical composition according to claim 16, which is intended for the treatment and for the 15 prevention of venous and arterial thrombosis.

18. Pharmaceutical composition according to claim 16, which is intended for the prevention of arterial thrombotic accidents.

19. Pharmaceutical composition according to claim 2 0 18 which is intended for the prevention of arterial thrombotic accidents in the case of myocardial infarction.

20. Pharmaceutical composition according to claim 16, which is intended for use in post-operative the prevention of venous thrombosis in surgical 25

21. Use of a mixture of oligosaccharides to any one of claims 1 to 9 in the prevention of of blood in surgical equipment. DATED THISs-Effi^Y OF «V) 19°pT / A. J. PARK & SON Pffi / . /\j QaJ ' A0ENTS FOR THE APPLICANTS </div>