IE58619B1 - A process for preparing mucopolysaccharidic compositions having a high antithrombotic activity - Google Patents

Abstract

1. Process for preparing mucopolysaccharides or MPS possessing high antithrombotic activity, characterized by the fact that compositions formed form a majority of MPS chains having a molecular weight of about 1800 to 8000 daltons, a YW titer and a ratio of the YW/USP titers higher than those of heparin to at least one fractionation step, in order to selectively separate from said compositions at least the major part of chains having MW lower than about 2000 and those having a higher MW but a sulfate content lower than the sulfate average contents of the mixture chain and that the fractions devoid from said chains are recovered, said fractions having a lower ratio of YW/USP titers than the starting compositions but higher than the one of heparin with USP titer higher than the one of the starting compositions.

Description

PROCESS FOR PREPARING MUCOPOLYSACCHARIDE COMPOSITIONS HAVING HIGH ANTITHROMBOTIC ACTIVITY.

BACKGROUND OF THE INVENTION The invention relates to a process for preparing mucopolysaccharide compositions endowed with high antithrombotic activity.

It is known that compositions cf this type have 10 been obtained from heparin by alcoholic extraction, or again, by depolymerisation by chemical or enzytr.atical route .

In general, these processes enable the production of compositions or fractions^ consisting of mucopolysaccha15 rides or MPS whose chains contain about 25 to 30 units at the most and possess an anti-Xa activity measured by the Yin Wessler titer (YW) higher thant that of heparin and an overall anti-coagulant activity, expressed by the USP titer, less than that of heparin, this titer can even have a value which is very low to practically nil.

These products have the interest of exerting a more specific activity than heparin on certain steps brought into play in the coagulation process.

In a given treatment, the product is chosen with regard to its YW titer and YW/USP ration. In that respect the inventors have studied means enabling the easy production of compositions corresponding to a given profil as regards, in particular, their YW titer and their ratio of YW to USP titers, Applicants have observed that by operat30 ing under particular conditions, it is possible to isolate such products from other MPS compositions.

It is therefore an object of the invention to provide a novel process for preparing compositions of MPS with high antithrombotic activity based on the treatment of another MPS composition containing them, whose simple employment enables products of great usefulness to be made easily available.

GENERAL DESCRIPTION OF THE INVENTION According to the invention there is a process provided characterized in that MPS compositions formed from a majority of MPS chains of molecular weight (MK) of about 1800 to 8000 daltons, having a YK titer and a ratio of YW/ USP titers higher than that of heparin are subjected to at least one fractionation step, in order to separate selectively from said compositions at least the major part of chains having MK lower than about 2000 and those having a higher MW but having a sulfate content lower than the sulfate average content of the mixture chains and that the fractions freed from said chains are recovered. These chains show a ratio of YK to USP titer smaller than the one of the starting compositions but higher than the one of heparin, with a USP titer higher than that of the starting compositions .

Preferably, the fractionation step is carried out by means of a mixture of water containing an inorganic salt and organic solvent, this solvent being selected from among those in which at least a majority of the products sought is selectively insoluble and precipitates.

The relative proportions of inorganic salt and of solvent are adjusted and this according to the pH of the medium to obtain the desired precipitation of the MPS chains.

According to a preferred feature of the invention the organic solvent is advantageously an alcoholic solvent, more especially ethanol.

According to another preferred feature, the organic salt used is constituted, particularly, by the sodium or potassium chloride or any other salt miscible in the organic solvent used.

According to another preferred feature, the pH of the reaction mixture is adjusted to a value corresponding to an acid pH, more especially to a pH less than 4.

According to a preferred embodiment of the invention, the MPS compositions employed for the fractionation contain a majority of chains of MW in the range of about 1800 to 8000, possessing a ratio of the YW to USP titers of at least about 10 and a YW titer of about 200 to 250 u/mg.

The Yin-Wessler titer is used for measuring the anti-Xa activity according to Yin et al in J. lab. Clin. Med. 1973, 81, p. 298.

These compositions are advantageously obtained by a partial depciymensa cion process of heparin under the action of chemical agents such as nitrous acid. Recourse is had particularly to the process described by Applicants in the second application for a certificate cf addition n* 80 06282 of 20.03.1980 to patent FR 78 31357 ox 6.11.1978. Advantageously, the depolymerisation process which is performed is based on self-regulation of the depolymerisation reaction such as described m patent application FR8107283 of 10.04.1981 in the name of Applicants.

According to the most general aspect of this autoregulated depolymerisation, heparin is reacted with nitrous acid in an aqueous medium at a pH cf about 2 to 3, advantageously of 2.5, m amounts such that heparin concentration is of at least about 8% m weight and nitrous acid molarity of about 0.02M to 0.1M.

When the depolymerisation reaction stops by itself, after having consumed all the nitrous acid, the MPS mixture of the kind of those which can be precipitated by an organic solvent is recovered.

Nitrous acid is generated m situ by adding an acid having advantageously biologically acceptable anions, such as HCI or CH^COOH. to a nitrous acid derivative, particularly a salt, an ether-salt and more especially an alkaline or an alkaline-earth salt. NaNO^ is more particularly used at a molarity of 0.03 to 0.05M.

The process according to the present invention is preferably performed with a NaNO^molarity of 0.040M to 0.046M more particularly of about 0.043M.

Preferably, the MPS compositions employed possess at the reducing end, a unit of 2,5 anhydromanno structure, preferably selected from among 2,5-anhydromannonic acid groups.

The starting MPS compositions corresponding to the character ist ics of YW and USP titers given above are placed in solution in a proportion of 5% w/v in water containing 10g/l of NaCl.

After adjustment of the pH to 3.8, the fractionation of these MPS is carried out by means of an organic solvent enabling the selective precipitation of the MPS chains possessing the highest molecular weights and/or the most sulfated of the mixture chains.

Preferably the organic solvent is an alcoholic solvent, more especially ethanol.

The elimination of low MW chains and of the low sulfate content chains results in fractions of a higher USP titer, the YW titer being practically maintained.

The MPS compositions recovered on precipitation are characterised in that they are essentially formed of -chains (1) of an average molecular weight of 4000 to 5000 daltons, particularly about 4500 (2) possessing a YW titer of about 180 to 200 u/mg and a ratio of the YW/USP titers, less than 10, particularly of about 6 to 3 more especially of about 4. Said compositions are formed of a majority of chains which are terminated by units of 2.5-anhydromanno structure when the starting compositions are obtained by a nitrous heparin depolymerisation process.

The compositions obtained according to the process of the invention are endowed with biological activities enabling them in particular to control specifically certain steps of blood clotting. These compositions are hence particularly valuable for developing medicaments which can be used, particularly, for the prevention of thrombosis and aging of the tissues.

Said medicaments are used under the usual administration forms and doses for a given indication.

These titers are expressed in Yin-Wessler units.

MPS compositions obtained according to the invention are useful for the constitution of biological reagents useful in the laboratory, particularly as a comparison reference for the study of other products of which the anticoagulant activity is tested, particularly at the level of inhibition of the Xa factor.

Regarding the products soluble in the water organic solvent mixture used in the fractionation step, they are devoid or practically devoid of antithrombotic activity.

Said products can be recovered by gel filtrating the soluble fraction.

By using an exchange ion chromatography. different kinds of oligosaccharides can be separated, particularly hexosaccharidic chains which have a high activity on inhibition of neoangiogenesis. The recovery of the oligosaccharides from said fractions are advantageously performed by alcoholic precipitation for example and lyophalisation.

Other characteristics and advantages of the invention will appear in the examples which follow. Examples 1 and 2 relate to the preparation of MPS compositions possessing a ratio of the YW to USP titers of the order cf 4. examples 3 and 4 to the activity of the products respectively obtained according to examples 1 and 2 and example 5 to the obtention from the soluble fractions of products practically devoid of antithrombotic activity.

EXAMPLE 1: Process for obtaining MPS compositions with YW/ USP ration of about 4. 500 g of injectable heparin, in the form of sodium salt, are placed in solution in 4500 ml of demineralized water, at a temperature of 18°C.

The YW/USP ratio of the heparin used is in the vicinity of I, these titers having a value of the order cf 160-170.

The solution obtained is subjected to vigorous stirring, and its pH is lowered to 2.5 by the addition of concentrated hydrochloric acid. Then 15 g of sodium nitrite dissolved in 300 ml of water are added. The pH of the reaction is adjusted to 2.5 by concentrated hydrochloric acid, and the total value of the solution is brought to 5000 ml. The reaction is left to take place for 45 minutes and then the absence of residual nitrous ions in the reaction solution is checked, by means of indicator paper impregnated with starch potassium iodide (development of a blue-viclet colour in the presence of N02~ ions).

The reaction is allowed to continue up to the total disappearance of nitrous ions and the absence of reaction with iodo-starch paper, by carrying out checks every 3 or 4 minutes.

When these checks become negative, the reaction is considered as having been completed.

The pH of the solution is then raised to 10 by means of concentrated soda, and 5 g of sodium-tetrahydrideborate is added.

The solution is kept under stirring for 15 hours.

The unreacted sodium tetrahydrideborate is destroyed by lowering the pH to 3 by means of concentrated hydrochloric acid. The solution is subjected to stirring for 15 minutes, then the pH is readjusted to 7.0 by means of concentrated soda.

The reaction products are recovered by the addition of 10 1 of ethanol. After 48 hours standing, the product is decanted and the supernatant liquor removed.

The precipitate is redissolved in 9 litres of demineralized water. 100 g of sodium chloride are added, and the pH of the solution is lowered to 3.8 by means of concentrated hydrochloric acid. The volume is adjusted exactly to 10 litres by means of demineralized water, and with vigorous stirring IO litres of ethanol are added.

This is left to stand 48 hours. The supernatant liquor is siphoned off and put aside. The precipitate is recovered, washed with ethanol, ground, dried under vacuum. 230 grams of product having the following characteristics are obtained : USP titer = 52 ul/mg Yin and Kessler titer = 225 ul/mg Average molecular weight = 4000 to 5000 daltons EXAMPLE 2: 5000 g of injectable heparin are dissolved in 45 litres of demineralized water, at 18°C.

In similar manner to example 1 the procedure is followed multiplying the amounts of reagents by 10, that is to say: - by adding 150 g of sodium nitrite in solution in three litres of water. - by adjusting the volume of the reaction to 50 litres - by adding 50 g of tetrahydrideborate. - by precipitating by means of 1O0 litres of ethanol. - by redissolving 90 1 of water. - by adding 1000 g of sodium chloride. - by adjusting the final volume to 100 litres. - by finally adding lOO litres of ethanol.

In this way 2230 g of product are obtained having the following characteristics : USP titer = 54 ul/mg Yin and Wessler titer = 232 ul/mg Average molecular weight = 4000 to 5000 daltons The products of the reaction are recovered by addition of 10 1 of ethanol, left to stand 48 hours, decanted and the supernatant liquor removed.

The precipitate is redissolved in 9 litres of dimineralized water. 1O0 g of sodium chloride are added, and the pH of the solution is lowered to 3.8 by means of concentrated hydrochloric acid. The volume is adjusted exactly to 10 litres by means of dimineralized water, and with vigorous stirring 10 litres of ethanol are added. It is left to stand 48 hours. The supernatant liquor is syphoned of_and put aside : the precipitate is recovered, washed with ethanol, ground, dried under vacuum. 230 grams of product having the following characteristics are obtained : USP titer = 52 ul/mg Yin and Wessler titer = 225 ul/mg Average molecular weight = 4000 to 5000 daltons In Example 3 given below, the results of pharmacological tests carried out with the product of example 1, are reported.

EXAMPLE 3: In vitro activity of the composition of example 1: On figures 1 to 4 are reported the curves obtained by studying, in vitro, the variation of the clotting times induced in human blood plasmas by increasing doses of commercial heparin and the product of example 1.

In the tests corresponding to the results given in figures 3 and 4, plasmas free of platelets were used, and impoverished in factor XI.

These figures show the variation in seconds of thrombin time (figure 1), of cephalin-kaolin (figure 2), of coagulation in presence of concentrated thromboplastin (figure 3) and of dilute thromboplastin (figure 4), induced by the tested products, namely, the product of example 1 (curves al, a2, a3 and a4) and of heparin (curves bl, b2, b3, b4) according to the doses respectively used.

The thrombin times and the cephalin-kaolin times both constitute types of measurement reflecting rather the action of the preparation studied respectively on the inhibition of the activated factor II and the overall coagulation. Study of the curves of figures 1 and 2 shows that the MPS according to the invention exert a distinctly smaller effect than that of heparin on the inhibition of activation of prothrombin and at the level of the overall coagulation. Figures 3 and 4 which are representative of phenomena more directly connected with the sequence of enzymatic reactions, characteristics of extrinsic coagulation (particularly in the relative absence of factor XIa) , show the advantageous effect of the MPS of the invention with respect to heparin.

The example 4 below concerns the results of tests in vivo in the rabbit with the MPS of example 2.

EXAMPLE 4: Study of the antithrombotic activity of the product of example 2 in the rabbit.

The administration of 500 u YW t-o the rabbit causes a considerable anti-Xa activity whilst the overall anticoagulant activity remains relatively low.

Two hours after the administration, the YK activity is of the order of 0.88 u/ml whilst the cephalin kaolin time is 0.09 ui.ml.

The in vitro and in vivo tests show therefore the distinctly more selective action of the MPS of the invention, particularly at the level of inhibition of the Xa factor, than that of heparin.

EXAMPLE 5: Process for obtaining MPS fractions practically devoid of antithrombotic activity.

The soluble fraction of example 1 is recovered.

Said fraction contains chains with 2 to 14 units.

Additional alcoholic fractionation step of said fraction results in a precipitate, which is separated, and a soluble fraction from which is recovered composition y^. Said composition comprises chains with 6 to 8 units at most and has a YW titer lower than 70 and a USP titer lower than 5.

By gel filtrating composition v, according to the usual techniques, hexasaccharidic compositions £ devoid of anticoagulant activity are recovered. Said compositions inhibit neoangogenisis .

Example 6: Activity on the neoangiogenisis of compositions y and z .

The trials in vitro concerning anti angiogenisis were carried out on a chicken embryo chorioallantoic membrane 6 days old in culture in petri dishes.

Pellets are formed by mixing 5 pg of hydrocortisone with 10 pi of methylcellulose, compositions y and z are added in amounts varying from 5ug to 100 pg. The pellets obtained are dried and placed on the chorioallantoic membrane to which they adhere. For each test 4 eggs are used.

After 48 hours of incubation, avascular zones are found and an important drop in the vascular zones can be seen. These results are reached in a remarkable way using only 6 pg of the tested products.

Example 7: Action of a hexasaccharidic z composition on a B16 mouse melanoma.

Tests carried out on C5 7 BL/6 mice carrying B16 melanomas .

The treatment carried out during a period of 15 days, administered by sub-cutaneous injection twice a day to each mouseof 0.15 mg of the hexasaccharide composition of acetate of cortisone in varying doses reduced during the treatment from 250 mg/kg/j to 40-50 mg/kg/j. At the end of the 15 day period the growth of the tumours has ceased.

A regression of the tumours is advanteously observed.

Claims (12)

1. Process for preparing mucopolysaccharides or MPS possessing high antithrombotic activity, said process comprising subjecting compositions formed from a majority of MPS chains (MW) having a molecular weight of about 1800 to 8000 daltons, and a ratio of the YW/USP titers higher than that of heparin to at least one fractionation step, in order to selectively separate frcm said compositions at least the major part of chains having MA lcwar than about 2000 and those having a higher MA but having a sulfate content lower than the sulfate average content of the mixture chains and that the fractions devoid of said chains are recovered, said fractions having a lower ratio of YW/USP titers than the starting compositions with a higher USP titer.

2. Process according to claim 1, wherein the fractionation is carried out by means of a mixture of water containing an inorganic salt and organic solvent, this solvent being selected from among those in which at least the majority of •the desired products is selectively insoluble.

3. Process according to claim 2, wherein the organic solvent is an alcoholic solvent, more especially ethanol.

4. Process according to claim 2, wherein the inorganic salt is constituted particularly of sodium or potassium chloride or any other salt miscible in the organic solvent used.

5. Process according to claim 2, wherein the pH of the reaction mixture is adjusted to an acid value.

6. Process according to claim 5, wherein the pH is adjusted to a value below 4.

7. Process according to claim 1, wherein the MPS compositions employed for the fractionation possess a ratio of the YP to USP titers of at least about 10 and a YW titer of about 200 to 250 u/mg.

8. Process according to claim 5, wherein the fractionation is carried out by means of an organic solvent permitting the selective precipitation of the MPS possessing a ratio of the YW to USP titers less than 10, preferably than about 6 to 3, and more especially in the vicinity of 4.

9. Process according to claim 2, comprising placing in solution in water 5% w/v of MPS containing 10 g/1 of ethanol and after adjustmentof the pH to 3.8, effecting the fractionation by the addition of one volume of ehtanol and recovering the precipitate formed.

10. Process according to claim 1, wherein the MPS compositions employed possess at the reducing end a unit of 2,5-anhydromanno structure selected preferably from among the groups 2,5-anhydromannose, 2,5-anhydromannitol or 2,5-anhydro-

11. A process according to Claim 1 for preparing a mucopolysaccharide or MPS possessing high antithrombotic activity, substantially as hereinbefore described with particular reference to Examples 1 and 2 of the accompanying Examples.

12. A mucopolysaccharide or MPS possessing high antithrombotic activity, whenever prepared by a process claimed in a preceding claim.