DD239798A5 - METHOD FOR PRODUCING MUCOPOLYSACCHARIDE COMPOSITIONS WITH INCREASED ANTITHROMBOTIC ACTIVITY - Google Patents

Abstract

According to the method of the invention, mucopolysaccharide compositions having increased antithrombotic activity and a higher titre of YW / USP than that of heparin are subjected to at least one fractionation step to separate the chains containing no more than six structural arrangements.

Description

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Field of application of the invention

The invention relates to a process for the preparation of mucopolysaccharide compositions having increased antithrombotic activity. >

Characteristic of the known technical solutions

It is known that compositions of this type were obtained from heparin by alcoholic extraction or by depolymerization by chemical or enzymatic means.

By and large, these methods make it possible to obtain compositions or fractions of mucoplysaccharides, or MPS, whose chains comprise at most about 25 to 30 structural arrangements and which possess an anti-Xa activity, measured by the titer Yin Wessler (YW), higher than that of heparin, and have a general anti-coagulant activity, expressed by the USP titer and lower than that of heparin, this titer even having a very low value and can become virtually zero.

These products have the advantage of exerting a more specific effect than heparin on certain stages involved in coagulation processes.

For a treatment to be performed, the product is selected according to the value of its YW titer and the ratio of its titres YW and USP.

In this regard, the inventors have sought means to conveniently obtain compositions corresponding to a given profile and as regards this profile, particularly with respect to their YW-Tite; : And on the ratio of titers YW to USP. The work carried out has shown that, taking into account certain conditions, it is possible to isolate such products from other MPS compositions.

Object of the invention ₍

The aim of the present invention is to provide a novel process for the preparation of MPS compositions with increased antithrombotic activity.

Explanation of the essence of the invention

The invention has for its object to treat a suitable MPS composition so that in a simple manner advantageous products, especially those with increased antithrombic activity can be obtained.

The process of the invention is characterized in that MPS compositions formed from a plurality of MPS chains having a molecular weight (PM) of about 1800 to 8000 daltons and having a higher YW titer and a higher ratio of titers YW / USP as heparin, undergo at least one fractionation stage to selectively separate from these compositions at least most of the PM chains having less than about 2000 and the higher PM chain portion but having a lower degree of sulfation than the average grade of K These fractions have a lower ratio of titers YW to USP than the starting compositions, but higher than that of heparin, with a higher USP titer than the starting compositions

 Preferably, the stage of fractionation using a mixture of

a) water containing a mineral salt, and

b) an organic solvent

This solvent is selected such that at least the majority of the products sought are selectively insoluble and precipitate therein.

The relative proportions of mineral salt and solvent are adjusted as a function of the p-value of the medium in order to achieve the precipitation of the desired MPS chains.

According to a preferred embodiment of the invention, the organic solvent is advantageously an alcoholic solvent and in particular ethanol.

According to a further preferred embodiment, the mineral salt used consists in particular of a salt which is miscible in the organic solvent used, such as sodium or potassium chloride.

According to a further preferred embodiment, the pH of the reaction mixture is adjusted to a corresponding acid value, in particular to a pH below 4.

According to a preferred mode of implementation of the invention, the MPS compositions used for fractionation contain a plurality of molecular weight (PM) chains (1) of about 1800 to 8000, (2) have a ratio of YW to USP of at least about 10 and (3) a YW titer of about 200 to 250 μ / mg. It is recalled that the Yin-Wesseler titer is used as a measure of anti-Xa activity, according to Yin, E.T., Wesseler, S., Bülter, J.V. inJ.Lab.Clin.med. 1973, 81, p.298-310. '

The MPS compositions used are advantageously obtained by a partial depolymerization process of heparin under the action of a precipitated acid chemical agent. Reference was made in particular to a method described by the applicant in the Second Additional Application No. 8,006,282 of March 20, 1980 to FR-PS 7831357 of November 6, 1978.

In an advantageous manner, a depolymerization process of this type is carried out, but based on the self-regulation of the depolymerization reaction, as described in French Patent Application 81 07283 on April 10, 1981 in the name of the Applicant.

In a very general aspect of this autoregulated depolymerization, the reaction of heparin with nitrous acid in the aqueous medium is carried out at a pH of the order of 2 to 3, preferably 2.5, the concentration of heparin being at least about 8 μg After the depolymerization has come to a standstill by consumption of the nitrous acid itself, the MPS mixture of the type precipitated in an organic solvent is collected can.

The nitrous acid is generated in situ by adding an acid containing advantageously biologically, acceptable A "ions, such as hydrochloric acid or acetic acid to a derivative of the nitrous acid, particularly a salt, an Eth ·· ^-.!? 3a laundri particularly In particular, sodium nitrite is used in a molarity of 0.03 to 0.05 M. In order to carry out the method according to the present invention, it is preferable to use NaNO ₁ in a concentration of from 0.1 to 0.046M , in particular of the order of 0.043 M. Depending on the preparation method, the MPS compositions have at the reduction end an arrangement of the 2,5-anhydromanno structure, preferably selected from the groups 2,5-anhydromannose, 2,5-anhydromannitol or 2, 5-Anhydromannon sulphate a The starting MPS compositions used are solubilized in a ratio of 5% (w / v) in water containing 10 g / l NaC.

After adjusting the pH to 3.8, the fractionation of this MPS is carried out with the aid of an organic solvent which makes it possible to selectively precipitate the MPS having the highest molecular weight and / or the highest degree of sulfation.

In particular, the organic solvent consists of an alcoholic solvent, especially ethanol. Removal of the low molecular weight chains or having a small amount of salta ligand results in obtaining MPS fractions with further increased USP titer, with the YW titer remaining virtually unchanged. The MPS compositions collected in the precipitation are generally characterized as consisting essentially of (1) chains having an average molecular weight of from -4000 to 500 ° C daltons, especially about 4500, (2) a YW titer of about 180 to 200u / mg and have a ratio of titers YW / USP of lower 10, in particular from 6 to 3, especially in the order of 4.

These compositions are also formed from a plurality of chains bounded by 2,5-anhydromanno-structural arrangements when the starting compositions are derived from a nitrous acid depolymerization process of heparin.

The compositions obtained by the method according to the invention are seen to have biological effects which enable them to be used in particular for the specific control of certain stages of blood coagulation. They are therefore particularly valuable for the creation of drugs that can be used especially for the prevention and treatment of thrombosis and signs of aging of the tissue. These medicines are used in the usual forms and dosages for the intended type of indication.

i '

The mucopolysaccharide compositions obtained are also used for the preparation of biological reagents used in the laboratory, for example as reference titers for comparison with other products of which the anticoagulant activity is tested, in particular in the area of inhibition of factor Xa.

As for the products soluble in the water / organic solvent mixture used for the fractionation, they are without or practically without antithrombotic activity.

These products can be recovered by filtration of the soluble fraction over a gel. The use of one step of ion exchange chromatography allows the separation of these compounds, if desired, of various types of oligosaccharides, especially hexasaccharide chains, with an inhibitory effect on neoangiogenesis.

The recovery of the products from the fractions is carried out more advantageously by, for example, carrying out an alcoholic precipitation and lyophilization.

Embodiment ^v

The invention is explained in more detail below with reference to some examples. From the following examples, further features and advantages of the invention will become apparent. Examples 1 and 2 relate to the preparation of the MPS compositions with a ratio of the titres YW to USP of the order of 4, Examples 3 and 4 to the activity of the products obtained according to Examples 1 and 2 respectively, and Example 5 the recovery of the products having virtually no antithrombotic activity from the soluble fractions.

example 1

Process for the preparation of MPS compositions with a ratio of titres YW / USP of about 4.

500 g of injectable heparin in the form of the sodium salt are dissolved in 4500 ml of demineralized water at a temperature of 18 ° C in solution. The ratio YW / USP of the heparin used is approximately 1, these titres have a value in the order of 160-170.

The resulting solution is stirred vigorously and its pH is lowered to 2.5 by the addition of concentrated hydrochloric acid. Then add 15 g of sodium nitrite dissolved in 300 ml of water. The pH of the reaction is adjusted to 2.5 using concentrated hydrochloric acid and the total volume of the solution is increased to 5000 ml.

The reaction is allowed to proceed for 45 minutes and then the absence of residual nitrite ions in the reaction solution is checked, for example by means of indicator paper impregnated with potassium iodine starch (blue-violet coloration in the presence of NGv ions).

The reaction is allowed to continue until the disappearance of the nitrite ions and the absence of reaction on iodine starch.

Continue paper by performing the check every 3 or 4 minutes. _f

If these controls are negative, the reaction is considered complete. The pH of the solution is then raised to 10 with the aid of concentrated sodium hydroxide solution and 5 g of sodiumbordetrahydride are added.

Then the solution is stirred for 15 hours.

The unreacted sodiumbordetrahydride is then decomposed by lowering the pH to 3.0 with concentrated hydrochloric acid. The solution is then stirred for 15 minutes and then brought back to a pH of 7.0 by means of concentrated sodium hydroxide solution.

The reaction products are obtained by adding 10 liters of ethanol. After standing for 48 hours, the product is decanted and the supernatant is removed.

The precipitate is redissolved in 9 liters of demineralised water. 100 g of sodium chloride are added and the pH of the solution is lowered to 3.8 using concentrated hydrochloric acid. The volume is then made up to 10 liters with demineralized water and 10 liters of ethanol are added with vigorous stirring. Then let stand for 48 hours. The supernatant liquid is drawn off with a siphon and removed. The precipitate is collected, washed with ethanol, crushed and dried under vacuum. 230g of product are obtained with the following characteristics:

Titre USP = 52ul / mg

Titre Yin and Wesseler = 225ul / mg

average molecular weight = 4000 to 5000 daltons

Example 2

Process for the preparation of MPS fractions with a ratio of titres YW / USP of about 4.

5000g of injectable heparin are dissolved in 45 liters of demineralized water at a temperature of 18 ° C.

The procedure is repeated in the same way as described in Example 1 by multiplying the amounts of the reactants by 10, that is to say:

With the addition of 150 g of sodium nitrite in solution of 3 liters of water,

With replenishment of the reaction volume to 50 liters,

With the addition of 50 g of sodiumbordetrahydride,

With precipitation with the aid of 100 liters of ethanol,

- with redissolution in 90 liters of water,

With the addition of 100 g of sodium chloride,

- filled to a final volume of 100 liters,

- with final addition of 100 liters of ethanol.

This gives 2230 g of product with the following characteristics: titre USP = 54ul / mg

Titre Yin and Wesseler = 232u! / Mg

average molecular weight = 4000 to 5000 daltons

The reaction products are recovered by adding 10 liters of ethanol, leaving to stand for 48 hours, decanting and removing the supernatant.

The precipitate is redissolved in 9 liters of demineralised water. 100 g of sodium chloride are added and the pH of the solution is lowered to 3.8 using concentrated hydrochloric acid. The volume is then made up to 10 liters with demineralized water and 10 liters of ethanol are added with vigorous stirring. Then let stand for 48 hours. The supernatant liquid is drawn off with a siphon and removed. The precipitate is collected, washed with ethanol, crushed and dried under vacuum.

230g of product are obtained with the following characteristics:

Titre USP = 52ul / mg

Titer Yin and Wesseler = 225ul / mg

average molecular weight = 4000 to 5000 daltons

In the following Example 3, reference is made to the results of the pharmacological investigations carried out with the product of Example 1.

Example 3

Examination of the activity of the products of Example 1 in vitro.

Figures 1 to 4 show the curves obtained in the in vitro experiment, namely the change in times of coagulation induced in human blood plasma by increasing dosages of commercial heparin and the product of Example 1.

In the experiments corresponding to the results given in FIGS. 3 and 4, plasma was used by platelets and diluted by the factor XI.

These figures show the changes in seconds of thrombin (Figure 1), cephalin kaolin (Figure 2), coagulation in the presence of concentrated thromboplastin (Figure 3) and dilute thromboplastin (Figure 4) induced by the products tested, viz the product of Example 1 (curves a 1, a 2, a3 and a4) and heparin (curves b1, b2, b3 and b4) depending on the doses used.

The times of thrombin and that of cephalin kaolin represent types of measurements that reflect rather the effect of each of the preparations tested on the inhibition of the active factor II and the total coagulation. The experiment of curves Figures 1 and 2 shows that the MPS according to the invention exert a significantly lower effect on the inhibition of activation of prothrombin in the region of total coagulation than is the case with heparin. Figures 3 and 4, which are characteristic of phenomena more directly related to the sequence of enzymatic reactions as a feature of external coagulation (especially in the relative absence of Factor XIa), demonstrate the beneficial effect of the MPS of the present invention on heparin

 Example 4 below relates the results of rabbit in vivo experiments with the MPS of Example 2.

Example 4

Investigation of the antithrombotic activity of the product of Example 2 in rabbits.

Administration of 50 Ou YW to the rabbit provokes significant anti-Xa activity, while overall anticancer activity remains relatively low.

Two hours after the administration, the YW activity is on the order of 0.88 μ / ml, while the time of cephalin kaolin is 0.09 μL / ml.

The experiments in vivo and in vitro thus show a significantly more selective action of the MPS according to the invention, in particular at the level of inhibition of the factor Xa, than is the case with heparin.

Example 5

Process for the preparation of MPS fractions with virtually no antithrombotic activity.

Collect the soluble fraction of Example 1. This fraction contains chains of 2 to 4 structural arrangements.

A stage of additional alcoholic fractionation of this fraction leads to a precipitate which is separated into a soluble fraction from which the composition y is collected. This composition is formed essentially of chains of at most 6 to 8 structural arrangements and has a YW titer of less than 70 μl. ·) A USP titer of less than 5.

By subjecting composition y to a gel filtration step according to classical techniques, hexasaccharide compositions are obtained. These compositions are virtually completely devoid of total anticoagulation activity.

and without YW activity. ',

Examples

Activity of compositions y and ζ on neoangiogonese. The anti-angiogenesis-related experiments in vi; :

were performed on the chorioallantoic membrane of 6 day old chicken embryo in Petri dish culture.

Make lozenges by mixing 5μg of hydrocortisone to 10 / ml of methylcellulose, then add the mix. ir <i; e ::

y and ζ according to dosages of 5 / ng to 100 / ^ g added. The finished lozenges are dried and then placed on the chorio-allantoin membrane, to which they stick.

For each experiment you use four eggs. After 48 hours of incubation, the presence of avascular zoner is checked; A significant reduction of the vascular zones is observed. These results will be remarkably satisfied with only 6μg of the products tested.

Example 7. '

Effect of hexasacchand compositions ζ on a type B16 melanoma in the mouse. These experiments are carried out on mice C57 BI / 6 carrying melanomas B16.

The treatment is carried out for 15 days. Each mouse is administered by subcutaneous injection twice daily with 0.15 mg hexasaccharide compositions and cortisone acetate at dosages which decrease in the course of the treatment from \ 250 mg / kg / day to 40-50 mg / kg / day.

At the end of the 15 days, a developmental arrest of the tumor is detected.

Even a regression of this tower is observed in favorable cases.

Claims (9)

Entitlement to the invention: A process for the preparation of mucopolysaccharides or MPS having increased antithrombotic activity, characterized in that compositions formed from a plurality of MPS chains having a PM of about 1800 to 8000 daltons and having a higher YW titer and έτη having a higher ratio of titers YW / USP than heparin undergoes at least one fractionation stage to selectively separate from these compositions at least most of the PM chains having less than about 2000 and the higher PM chain content but having a lower degree of sulfation; than the average degree of the chains of the mixture and that one collects the fractions freed from these chains, these fractions having a lower ratio of the titres YW to USP than the starting compositions, but higher than that of heparin, with a higher USP titer than that of the starting compositions. * 2. The method according to item!, Characterized in that the fractionation is carried out with the aid of a water mixture comprising a mineral salt and an organic solvent in which at least the majority of the products sought is selectively insoluble. 3. The method according to item 2, characterized in that one carries out the fractionation with the aid of an organic solvent, which allows the selective precipitation of the formed MPS consisting of PM chains on average from 4000 to 5000 daltons, in particular of the order of 4500 daltons having a ratio of titres YW to USP of less than about 10, preferably about 6 to 3, and especially close to 4, with a YW titer of about 180 to 200 u / mg. . 4. The method according to item 2 or 3, characterized in that the organic solvent is an alcoholic solvent, in particular ethanol. _# 5. The method according to item 2, characterized in that the mineral salt used in particular consists of a miscible salt in the solvent used, such as sodium or potassium chloride. 6. The method according to any one of the items 2 to 5, characterized in that adjusting the pH of the reaction mixture to a corresponding acidic pH, in particular to a pH below 4. A method according to any one of items 1 to 6, characterized in that the MPS compositions used for the fractionation have a ratio of titres YW to USP of at least about 10 and a YW titer of about 200 to 250 μ / mg. 8. The method according to item 1, characterized in that 5% (w / v) MPS compositions containing 20g / l NaCl bring in an aqueous suspension and after adjusting the pH to 3.8 the Fractionation is carried out by adding a volume of ethanol, after which collects the precipitate formed. A process according to any one of the preceding claims, characterized in that the starting MPS compositions at the reduction end have an arrangement of the 2,5-anhydromanno structure, preferably selected from the groups 2,5-anhydromannose, 2,5-anhydro mannitol or 2,5-anhydromannonic acid.