DK172887B1 - Process for preparing high antithrombotic mucopolysaccharides - 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

in DK 172887 B1

The present invention relates to a process for producing high antithrombotic mucopolysaccharides.

It is known that mucopolysaccharide compounds of this type 5 can be obtained from heparin by alcoholic extraction or depolymerization by chemical or enzymatic route.

These processes yield mixtures or fractions consisting of mucopolysaccharides (MPS), wherein the chains contain at most approx. 25-30 repeating groups and possessing an anti-Xa activity, as measured by the Yin Wessler titer (YW), which is greater than the value for heparin, and a total anti-coagulation activity, expressed by the USP titer which is lower than the value for heparin, since this titer may even show a very low value, practically in the vicinity of 0.

These products are of interest as they exert a more specific activity than heparin at certain stages, which play a role in the coagulation process.

For a given treatment, the product will be selected as a function of the value of its YW titers and of the ratio of its YW to USP titers.

Methods have therefore been sought to make it possible to conveniently obtain compounds corresponding to a given profile in particular of their YW titers and their ratios of YW to USP titers. The studies carried out have led to the finding that if one works under predetermined reaction conditions, it is possible to isolate such products from other MPS compounds.

30 v DK 172887 B1 2

Thus, the present invention has for its object to provide a novel method for producing high antithrombotic mucopolysaccharides based on the treatment of another MPS product containing these, which allows convenient disposal of products of great utility.

The process of the invention is peculiar in that MPS mixtures consisting of a predominant amount of MPS chains having a molecular weight of about 10 are subjected. 1800 to approx. 8000 daltons, a higher YW titer and a greater YW / USP titre ratio than heparin, at least one fractionation step to selectively separate at least the majority of the molecular weight chains lower than about 15; 2000 and the chains having a higher molecular weight having a lower degree of sulfation than the average degree of sulfation of the chains in the mixture; for example, the fractions liberated from these chains recover which have a smaller YW / USP ratio than the starting materials but 20 larger than heparin and a higher USP titer than the starting mixture.

The fractionation step is preferably carried out by means of a mixture a) of water containing an inorganic salt, and b) an organic solvent selected from those wherein at least the majority of the products sought are selectively insoluble and precipitate.

The relative amount ratio of inorganic salt to solvent is adjusted depending on the pH of the medium to obtain the precipitation of the desired MPS chains.

In a preferred embodiment of the process of the invention, the organic solvent used is an alcoholic solvent, especially ethanol.

DK 172887 B1 3

In another preferred embodiment, the inorganic salt used is especially a salt miscible with the organic solvent used, such as sodium chloride or potassium chloride.

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

In a preferred embodiment of the invention, the MPS mixtures used for the fractionation exhibit a ratio of titers YW to USP of at least approx. 10 and a YW titer of approx. 200 to 250 units / mg.

The Yin Wessler titre is used as a measure of the anti-Xa activity of Yin E.T., Wessler S., Butler J.V. in J. Lab. Clin. with. 1973, 81, pages 298-310.

The MPS compounds used are advantageously obtained according to a process for partial depolymerization of heparin under the action of a chemical agent such as nitric acid. One is especially served by one

method described in FR

No. 2078646 of the inventor of the present invention.

Advantageously, a method of depolymerization of this type is employed; however, based on autoregulation of the depolymerization reaction, as described in FR Publication no.

2,503,714th

According to the most common aspect of this autoregulated depolymerization, heparin and nitric acid are reacted in aqueous medium at a pH of the order of 2-3, preferably 2.5, in such amounts that the heparin concentration is at least about And that the molar acidity of the nitric acid is from approx. 0.02 to 0.1 M, and after this depolymerization reaction is stopped by itself by consuming the nitric acid content, an MPS mixture of the type which can be precipitated is recovered by an organic solvent.

The nitric acid is formed in situ by the addition of an acid, which advantageously contains biologically acceptable anions, such as hydrochloric acid or acetic acid, to a: nitric acid derivative, especially a salt, an ether salt, and in particular an alkali metal salt or an alkaline earth metal salt. In particular, sodium nitrite is used with: a molarity of 0.03-0.05 M.

It is preferable to use NaNO4 at the concentration of 0.040-0.046 M in the implementation of the process of the invention, especially in the order of 0.043 M.

The MPS products, because of the manner in which they are obtained, possess at their reducing end an element of the 2,5-anhydromanno structure, preferably selected from 2,5-20 anhydromannose, 2,5-anhydromannitol and 2, The 5-anhydromannon acid groups.

The MPS products used as starting material are dissolved in an amount of 5% w / v in water containing 10 g / liter NaCl.

After adjusting the pH to 3.8, the fractionation of these MPS products is carried out by means of an organic solvent which allows a selective precipitation from the mixture of the MPS compounds which possess the highest molecular weights and / or are the most sulfated.

DK 172887 B1 5

In particular, the organic solvent consists of an alcoholic solvent, in particular ethanol.

The elimination of the low molecular weight chains and the chains having a low sulfation degree results in 5 obtaining MPS fractions with higher USP titers while largely maintaining the YW titre.

The MPS mixtures recovered during the precipitation are generally characterized in that they are mainly formed by chains (1) having an average molecular weight of 4000-5000 10 daltons, in particular approx. 4500, (2) possesses a YW titer of approx.

180 to 200 units / mg and a ratio of titers YW / USP less than 10, especially about 6-3, especially in the order of 4.

These mixtures additionally consist of a predominantly 15 amount of chains terminated with units having the 2,5-anhydromanno structure when the compounds used as starting compounds are the result of a process for depolymerizing heparin with nitric acid.

The 20 compounds obtained by the process of the invention possess biological activities which, in particular, enable them to specifically regulate certain steps of blood coagulation.

Thus, they are particularly valuable in the manufacture of drugs useful especially for the prevention and treatment of thrombosis and the aging of the tissues.

These drugs are used in the forms of administration and with the posologies common to the type of indications in question.

The mucopolysaccharide compounds thus obtained are also useful in the preparation of biological reactants useful in the laboratory, especially as reference compounds for comparison use in the study of other products on which an anticoagulant activity is tested, especially with respect to the inhibition of factor Xa.

With regard to the products soluble in the mixture of water and organic solvent used in the fractionation, these are devoid of or practically devoid of antithrombotic activity.

These products can be recovered by gel filtration of the 10 soluble fraction. The use of a treatment step consisting of ion exchange chromatography makes it possible to separate various types of oligosaccharides from the product, especially hexasaccharidic chains, which possess a high inhibitory activity against neoangiogenesis.

The extraction of products from the fractions is advantageously carried out e.g. by alcoholic precipitation and lyophilization.

Other features and advantages of the present invention will become apparent from the following Examples 1-4. Examples 1 and 2 relate to the preparation of MPS compounds having a ratio of titers YW to USP on the order of 4, Examples 3 and 4 show the activity of products obtained in accordance with Examples 1 and 2, respectively, and Example 5 relates to obtaining products which are virtually devoid of antithrombotic activity.

Will DK 172887 B1 7 EXAMPLE 1

Process for obtaining MPS compounds having a ratio of the titers YW / USP of approx. 4

500 g of injection-quality heparin in the form of the sodium salt was dissolved in 4500 ml of demineralized water at a temperature of 18 ° C.

The YW / USP ratio of the heparin used was in the vicinity of 1, these titers representing a value in the range of 160-170.

The solution thus obtained was subjected to vigorous stirring and the pH thereof was lowered to 2.5 by the addition of concentrated hydrochloric acid. Then 15 g of sodium nitrite dissolved in 300 ml of water were added. The pH of the reaction mixture was adjusted to 2.5 by means of concentrated hydrochloric acid and the total volume of the solution was brought up to 5000 ml. The reaction was then allowed to continue for 45 minutes, securing the absence of residual nitrite in the reaction solution, e.g. using a 20 indicator paper impregnated with potassium iodide in conjunction with starch (developing a blue-violet color in the presence of Ν0Γ ions).

The reaction was then allowed to proceed to the complete disappearance of nitritions and the absence of reaction on 25 iodine starch paper by conducting control analysis every three or four minutes.

As these control analyzes had become negative, the reaction could be considered to have been completed. Then the pH of the solution was raised to 10 with concentrated sodium hydroxide and 5 g of sodium tetraborohydride was added.

DK 172887 B1 8

The solution was kept under stirring for 15 hours.

The unreacted sodium tetraborohydride was destroyed by lowering the pH to 3.0 by means of concentrated hydrochloric acid. The solution was stirred for 15 minutes, after which the pH was again adjusted to 7.0 by means of concentrated sodium hydroxide.

The reaction products were recovered by the addition of 10 liters of ethanol. After 48 hours of standing the product became! off-edge and the supernatant was removed.

! The precipitate was redissolved in 10 liters of demineralized 1 water. 100 g of sodium chloride was added and the pH of the solution was lowered to 3.8 using concentrated hydrochloric acid.

The volume was adjusted to exactly 10 liters by means of demineralized water and added under vigorous. Stirring 10 liters of ethanol. Allow to stand for 48 hours.

Then, with a swollen supernatant, it was aspirated, which was discarded. The precipitate was recovered, washed with ethanol, ground and dried under vacuum.

230 g of a product having the following characteristics were obtained: USP titers = 52 international units / mg

Yin / Wessler titer = 225 international units / mg

Average molecular weight = 4000-5000 daltons.

EXAMPLE 2 Process for obtaining high YW titers and USP titers MPS fractions at a mutual ratio of approx. 4

5000 g of injection-quality heparin was dissolved in 45 liters of demineralized water at 18 ° C.

is DK 172887 B1 9

By analogy to Example 1, the quantities of reactants were multiplied by 10, i.e.: - adding 150 g of sodium nitrite in the solution in 3 liters of water, 5 - adjusting the volume of the reaction mixture to 50 liters, - precipitating by using 100 liters of ethanol, - redissolving in 90 liters of water, - adding 1000 g of sodium chloride, 10 - adjusting the final volume to 100 liters, - finally adding 100 liters of ethanol.

In this way, 2230 g of a product having the following characteristics were obtained: USP titers * 54 international units / mg 15 Yin / Wessler titers = 232 international units / mg

Average molecular weight = 4000-5000 daltons.

The reaction products were recovered by adding 10 liters of ethanol, standing for 48 hours, decanting and removing the supernatant.

The precipitate was redissolved in 9 liters of demineralized water.

100 g of sodium chloride was added and the pH of the solution was lowered to 3.8 with concentrated hydrochloric acid.

The volume was adjusted to exactly 10 liters by means of demineralized water and 10 liters of ethanol were added with vigorous stirring. Allow to stand for 48 hours. The supernatant was swallowed with swab and discarded.

DK 172887 B1 10

The precipitate was recovered, washed with ethanol, ground and dried under vacuum.

230 g of a reaction product were obtained which exhibited the following characteristics: 5 USP titers = 52 international units / mg in Yin / Wessler titers = 225 international units / mg * Average molecular weight = 4000-5000 daltons.

In the following Example 3, pharmacological test results which have been carried out with the reaction product of Example 1 are reported.

In Example 3

In vitro study of the activity of the reaction products of Example 1 In the accompanying Figures 1-4 are shown the curves obtained by examining the in vitro variations in the coagulation time induced in human blood plasma by increasing doses of a commercially available heparin product and the reaction product of Example I.

In the experiments corresponding to the results shown in FIG. 3 20 and 4, plasma released from platelets and with reduced and very low content of factor XI has been used.

From these figures, the variations of thrombin times expressed in seconds (Fig. 1), of cephalin-kaolin times (Fig. 2), of coagulation in the presence of concentrated thromboplastin (Fig. 3), and of diluted thromboplastin (Figs. 4) which were induced by the products tested, viz. of the reaction product of Example 1 (curves a1, a2, a3 and a4) and of heparin (curves b1, b2, b3 and b4) plotted as a function of the respective dose used.

Γ 1 «11 DK 172887 B1

The thrombin times and the cephalin-kaolin times both constitute two types of measurements, reflecting above all the effect of the respective investigated preparations on the inhibition of activated factor II and of global coagulation. The study of the curves of FIG. 1 and 2 show that the MPS of the invention exerts a significantly more reduced effect than the effect of heparin on the inhibition of the activation of prothrombin and with respect to global coagulation. FIG. 3 and 4, which are representative of 10 phenomena more directly related to the sequence of the enzymatic reactions characteristic of the external coagulation dependent (especially the relative absence of factor X1a), exhibit the beneficial effect of MPS produced by the method of invention 15 relative to heparin.

Example 4 described below relates to in vivo experimental results on rabbits with MPS from Example 2.

EXAMPLE 4

Examination of the antithrombotic activity of the product of Example 2 on rabbits

Administration of 500 units of YW to the rabbit induces significant anti-Xa activity, while overall anticoagulant activity remains relatively weak.

Two hours after administration, the YW activity was in the order of 0.88 units / ml, while the cephalin-kaolin time was 0.09 international units / ml.

Thus, in vitro and in vivo experiments show the clearly more selective effect of MPS produced by the method of the invention, especially with respect to inhibition of 30 factor Xa, in comparison with heparin.

EXAMPLE 5

Process for obtaining MPS fractions practically liberated from antithrombotic activity

The soluble fraction of Example 1 is used.

5 This fraction contains chains with 2-14 repeat groups.

A further alcoholic fractionation step on this fraction results in a precipitate which is separated and a soluble fraction from which the mixture γ is recovered. This mixture consists mainly of chains containing 6-8 repeat groups and exhibits a YW titer lower than I 70 and a USP titer lower than 5.

By subjecting the mixture y to a gel filtration step in accordance with classical methods, hexasaccharide mixtures z are recovered. These compounds are almost completely devoid of global anticoagulant activity and YW activity.

EXAMPLE 6 Activity of mixtures y and z on neoangiogenesis

In vitro experiments were conducted on anti-angiogenesis activity on the chorio-allantoin membrane of 6-day-old chicken embryos by growing in petri dishes.

Pills were formed by admixing 5 µg of 25 hydrocortisone and 10 µc of methyl cellulose, and then adding mixtures γ and z at doses increasing from 5 µg to 100 µg. The pellets thus formed were dried and then applied to chorio-allantoin membranes to which they adhered. In each experiment 4 eggs were used.

DK 172887 B1 13

After 48 hours of incubation, the presence of avascular zones was investigated. A significant decrease in the number of vascular zones was observed. These results have been achieved remarkably with as little as 5 and 6 µg of the tested compounds.

EXAMPLE 7

Effect of the hexasaccharidic mixture z on a B 16 mouse melanoma

The experiments were performed on mice C57 BL / 6, which were carriers 10 of melanomas B 16.

The treatment was carried out over a period of 15 days.

To each mouse, twice daily subcutaneous injection, 0.15 mg of the hexasaccharide mixture z and of varying doses of cortisone acetate, which decreased during the 15 treatment period, was administered from 250 mg / kg / day to 40-50 mg / kg / day. At the end of the 15 day period, the growth and development of the tumors was halted.

A favorable regression of these 20 tumors was also observed.

Claims (9)

A process for the preparation of high antithrombotic action mucopolysaccharides (MPS), known g-; net by subjecting mucosaccharide mixtures 5 consisting of a predominant amount of MPS chains of 1800-8000 daltons molecular weight, a higher YW titer and a greater YW / USP titre ratio than heparin, at least one fractionation step for selective separation of it! at least the majority of the molecular weight chains lower than 2000 and the higher molecular weight chains having a lower sulphation rate than the average sulphation rate of the chains in the mixture, and recovering the fractions liberated for these chains which have a smaller YW / USP ratio than the starting mixture, but in 15 greater than heparin, and a higher USP titer than the starting mixture. Process according to claim 1, characterized in that the fractionation is carried out by means of a mixture of water containing an inorganic salt and of -organic solvent, wherein at least the majority of the desired mucopolysaccharides are selectively insoluble. Process according to claim 2, characterized in that the fractionation is carried out by means of an organic solvent enabling the selective precipitation of MPS with chains of medium molecular weight 4000-5000 daltons, especially in the order of 4500 and having a YW / USP conditions less than approx. 10, preferably approx. 6 to approx. 3, especially near 4, and a YW titer of approx. 180-1. 30 200 units per unit. mg. Process according to claim 2 or 3, characterized in that the organic solvent used is an alcoholic solvent, especially ethanol. as m DK 172887 B1 Process according to claim 2, characterized in that the inorganic salt used is a salt miscible with the organic solvent used, such as sodium chloride or potassium chloride. Process according to any one of claims 2-5, characterized in that the pH of the reaction mixture is adjusted to a value corresponding to an acidic pH, in particular to a pH lower than 4. Process according to any one of claims 1-6, characterized in that the MPS mixtures used for the fractionation exhibit a YW / USP ratio of at least approx. 10 and a YW titer of approx. 200-250 units / mg. Process according to claim 1, characterized in that 5% (w / v) MPS mixtures are dissolved in water containing 20 g / liter NaCl, adjusted to pH 3.8, carried out the fractionation by adding a volume of ethanol. and recover the precipitate thus formed. Process according to any one of claims 1-8, ke n-20, characterized in that the MPS mixtures used as starting compounds at the reducing end have a link with 2,5-anhydromanno structure, preferably selected from 2,5-anhydromannose. -, 2,5-anhydromannitol and 2,5-anhydromannonic acid groups.