HU197931B - Process for producing mucopolysaccharide compositions with high 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

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

Compositions of this type are known to be prepared from heparin by alcoholic extraction or by chemical or enzymatic depolymerization.

In general, these known methods allow the preparation of compositions or fractions having a chain of up to about 25-30 units, which have a higher anti-Xa activity measured with the so-called Yin-Wessler titer (YW) than heparin (heparin YW and USP-). titer (about 160 units / mg) and USP titer (as defined by U.S. Pharmacopoeia, 16th January, 1985, edition 16) have a lower total anticoagulant activity than heparin, and this titer may be very low , even zero.

For example, U.S. Patent No. 2,944,792. The German Patent Publication discloses fractions of mucopolysaccharide having a YW titer of about 160 units / mg and a USP titer of about 45 units / mg, resulting in an activity ratio of about 3.55, i.e., higher than the starting heparin (1). However, the yield of the alcoholic extraction process for their preparation is very low, about 1%. For products obtained by the process according to PCT Publication No. WO 82 03 627, the YW / USP ratio is always at least 10, in this particular example 13, i.e. much higher than the starting heparin. Thus, it is an object of the invention to produce high stability mucopolysaccharides in high yields.

The products of the present invention are valuable for being more specific with heparin during each step of the coagulation process.

In a given treatment, the product is selected based on the YW titer and YW / USP titer ratio. In this regard, experiments have therefore been conducted on the ease of preparation of compositions of a given profile, that is, of a given YW titer and YW / USP titer. It has been observed that it is possible to isolate such products from other mucopolysaccharide compositions, subject to certain operational parameters.

It is therefore an object of the present invention to provide a novel process which makes it possible to prepare mucopolysaccharide compositions having a high antibiotic activity by treating other mucopolysaccharide compositions and to produce very useful products easily and readily available.

It has been found that this object can be achieved by subjecting the compositions of mucopolysaccharide chains of predominantly YW titer and YW / USP with a molecular weight of about 1800 to about 8,000 tons to at least one fractionation step and thereby selectively removing At least the greater part of chains with a molar mass of less than 2000, and at least a greater part of those chains having a sulphate content lower than the sulfate content of the chain mixture, and finally from these chains. the liberated fractions were separated. These said fractions have a lower YW / USP titer ratio than the parent compositions, but higher than the heparin, while having lower USP titers than the parent compositions.

Preferably, the fractionation step is carried out with a mixture of water and an organic solvent containing an inorganic salt, the organic solvent being one in which at least most of the desired products are selectively insoluble and precipitates.

The relative ratio of the inorganic salt to the organic solvent is adjusted and the desired precipitation of the mucopolysaccharide chains is promoted by providing an appropriate pH of the reaction medium.

According to a preferred embodiment of the process of the invention, the organic solvent is preferably an alcoholic solvent, ethanol.

In another preferred embodiment of the process of the invention, the inorganic salt is sodium or potassium chloride or any other salt miscible with the organic solvent used.

In yet another preferred embodiment of the process of the invention, the pH of the reaction mixture is adjusted to an acid value, particularly preferably less than 4.

In the process of the present invention, the starting material for fractionation is mucopolysaccharide compositions wherein the majority of the chains have a molecular weight of about 1800-8000, a YW / USP titer of at least 10, and a YW titer of about 200-250 units / mg. The YW titer, i.e. the Yin-Wessler liter, is used to measure anti-Xa activity by Yin et al., J. Clin. Med., 81, 298 (1973).

These mucopolysaccharide compositions are preferably prepared by partial depolymerization of heparin with a chemical agent such as nitric acid. Reference is made here in particular to our French Patent Application No. 80 06 282 to French Patent 78 31357, as literature describing such a process. Preferably, the depolymerization process carried out is based on self-regulation of the depolymerization reaction as described in French Patent No. 81 07 283.

In the broadest aspect of this self-regulated depolymerization, the heparite is reacted with nitric acid for about 2 to 3, preferably

In an aqueous medium of pH 2.5, the concentration of heparin is adjusted to at least about 8% by weight and the molar ratio of nitric acid to about θ.Ο2 to 0.1.

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After the depolymerization reaction has ceased spontaneously, after complete depletion of nitric acid, it is possible to isolate a type of mucopolysaccharide mixture which can be precipitated with an organic solvent.

The nitric acid is liberated in situ by adding an acid, preferably a biologically acceptable anion, such as hydrochloric acid or acetic acid, to a nitric acid derivative, more particularly a salt, an ether salt, and in particular an alkali metal or alkaline earth metal salt. The most preferred nitrite salt is sodium nitrite in the range of 0.03 to 0.05 molar.

It is particularly advantageous to work in a system containing 0.040 to 0.046 molar, especially about 0.043 molar sodium nitrite.

The mucopolysaccharide compositions used as starting materials in the process according to the invention preferably have at their reducing end a moiety derived from 2,5-anhydromannitol, particularly preferably a moiety having 2,5-anhydromannic acid. Detailed description of each end group and their conversion to each other is given in Hungarian Patent No. 188,657.

The YW and USP titrated starting mucopolysaccharide compositions described above are dissolved in 10 g / l of water containing sodium chloride to form a 5% w / v solution.

After adjusting the pH to 3.8, the fractionation of these mucopolysaccharides is carried out with an organic solvent which allows the selective precipitation of the mucopolysaccharide chains with the highest molecular weight and / or the most sulfated chains.

It is preferred to use an alcoholic solvent of the organic type, especially ethanol.

Removal of low molecular weight and sulfate-containing mucopolysaccharide chains results in higher USP titer and essentially the same YW titer fractions.

The mucopolysaccharide compositions which can be isolated by precipitation are characterized by (i) having an average molecular weight of 4000 to 5000, in particular about 4500;

(fi) 180-23.0 units / mg YW titers; and (iii) a YW / USP titre ratio of less than 10, more particularly about 3 to 6, especially about 4.

Compositions having the above properties are predominantly chains which contain units of structure derived from 2,5-anhydromannitol as the end group when the initial compositions are prepared by depolymerization of heparin with nitric acid.

The mucopolysaccharide compositions of the present invention are capable of specifically affecting each step of blood coagulation due to their biological activity. Thus, these compositions are extremely valuable in the development of pharmaceutical compositions which are primarily used to prevent thrombosis and tissue aging.

These pharmaceutical compositions are used in conventional dosage forms at concentrations depending on the particular disease.

Titers are given in Yin-Wessler units.

The mucopolysaccharide compositions of the present invention may also be used in the preparation of laboratory biological reagents, in particular as comparators used to determine the level of anticoagulant activity of other substances, in particular factor Xa inhibition.

As for the products soluble in the mixture of organic solvents and water used for fractionation, they are free of virtually no antithrombotic activity. Otherwise, these products may be isolated by gel filtration of the soluble fraction.

By using ion exchange chromatography, various oligosaccharides can be isolated, in particular hexosaccharide chains, which are highly active in inhibiting neoangiogenesis. Separation of these oligosaccharides from said fractions can be accomplished, for example, by alcohol precipitation and lyophilization.

Other features and advantages of the process of the invention will be apparent from the embodiments. Examples 1 and 2 relate to the preparation of mucopolysaccharide compositions having a YW / USP titer ratio of about 4, Examples 4 and 5 describe the activity of the products obtained in Examples 1 and 2, while Example 6 refers to the separation of soluble fractions practically devoid of antithrombotic activity. Example 3 relates to the determination of the properties of the reaction mixture obtained after the decomposition of nitrite. The process of the invention is based on the above reaction mixture.

Example 1

A process for preparing mucopolysaccharide compositions having a YW / USP titer ratio of about 4

At 18 ° C, 500 g of injectable quality heparin (containing molecular weight chains of 2000 to 5000 dalions) are dissolved in 4500 ml of deionized water as the sodium salt.

The YW / USP titer ratio of heparin used is about 1, since both titers are 160-170.

The resulting solution was first stirred vigorously and then the pH was reduced to 2.5 by the addition of concentrated hydrochloric acid. A solution of 15 g of sodium nitrite in 300 ml of water is then added. The pH of the reaction mixture was again adjusted to 2.5 with concentrated hydrochloric acid and the total volume was increased to 5000 ml. Then re

The action mixture was left to stand for 45 minutes and the absence of nitration was checked using indicator paper impregnated with a mixture of starch and potassium iodide (blue-violet color develops in the presence of NO3 ions).

The reaction mixture is allowed to stand until the nitration has completely disappeared, checking every three or four minutes with said indicator paper.

Whenever a negative result is observed during these checks, the instant of reaction is considered to be complete.

The pH of the reaction mixture (in which the depolymerized fraction has an average molecular weight of 250 Daltons) is then raised to 10 by the addition of concentrated sodium carbonate solution and 5 g of sodium borohydride are added.

The reaction mixture was stirred for 15 hours, and then the unreacted sodium borohydride was quenched by adjusting the pH of the reaction mixture to 3 with concentrated hydrochloric acid. The reaction mixture was stirred for 15 minutes and then adjusted to pH 7.0 with concentrated sodium carbonate solution. Thus, the 2,5-anhydromannnose end groups are converted to 2,5-anhydromannnite end groups.

The reaction products were isolated by adding 10 liters of ethanol. After 48 hours, the product was isolated by decantation and the supernatant liquid removed. The product obtained had a USP titer of 32 IU / mg, Yin and Wessler a titer of 164 IU / mg, giving a ratio of 5.1. (IU stands for International Unity.)

The precipitate was redissolved in deionized water (9 L), then sodium chloride (100 g) was added and the pH was reduced to 3.8 with concentrated hydrochloric acid. The volume is then adjusted to exactly 10 liters with deionized water and then 10 liters of ethanol are added with vigorous stirring. The resulting mixture was allowed to stand for 48 hours. The supernatant fluid is aspirated and discarded. After separation, the precipitate is washed with ethanol, ground and dried in vacuo.

230 g of product having the following parameters are obtained.

USP titer = 52 IU / mg

Yin-Wessler titer = 225 IU / mg

Ratio of YW, USP titers = 4.3

Average molecular weight = 4000-5000 dalion

The removal of lower than average sulfate chains is demonstrated by the following experiments.

let's compare

1) Glucose amino glucan chains in the product of Example 1 (hereinafter referred to as CY 216 D) and

2) the degree of sulfation of the chains remaining in the supernatant fluid obtained by fractionation with a mixture of water and ethanol containing sodium chloride.

The chains of (1) and (2) are separated by gel filtration and the degree of sulfation is determined by conductometry.

Experimental method

The supernatant obtained in the fractionation of Example 1 was adjusted to pH 7.0 with 5N aqueous sodium carbonate solution, followed by the addition of 19 liters of ethanol. The resulting mixture was allowed to stand for 48 hours, then washed with ethanol, pulverized and dried in vacuo. 120 g of an oligosaccharide mixture are obtained.

60 g of the resulting mixture are dissolved in 500 ml of 0.5 M aqueous sodium chloride and applied to the top of an Ultragel AcA 202 column (manufactured by IBF, Villeneuve La Garenne, France). The absorbance of ultraviolet light at 214 nm is directly recorded. The different oligosaccharide fractions were separated and precipitated with twice the volume of ethanol.

The degree of polymerization of the obtained fractions was determined by high performance liquid chromatography. It is determined on a column containing Waters-60 (manufactured by Waters Millipore, St. Quentin en Yvelines, France) using oligosaccharides of known molecular weight for calibration.

The sulfate / carboxyl ratio of each fraction was determined by Casu, B. and Gennaro, V. in Carbohydr. Gap

39. 168-176 (1975).

The procedure described above also applies to CY 216 D.

Results

The characteristics of the oligosaccharides isolated from CY 216 D and the supernatant fluid are given in the following table.

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F o r k i n g 1 '1 } ss 5 6 7 l'ol imer i zác i o degrees disaccharide tetra s 2 a c h a - r id hexa * s z a c n a - r i tí octactactiar id decaszachar i d dodeca- saccharide dodeca- sucrose Ridna On the long Sabbat Weight (g) 1, 2 5.6 12 9.8 S 6, 2 4, 3 Swim weight i 2.54 11.0 2 5, 4 20.0 16.9 13.1 9.1 'Ozolol t / w -Ί Γ ti 1.1 X 11 ratio 0.9 0.97 1.15 1.53 1.45 1, Λ8 1, 55 weight (g) 0 0.42 1.8 4, 6 6.6 6.9 20 Total weight train included \ 0 0,8é 5, 7 9.5 13.7 14.3 58 Parental / carboxyl gold 1 /. 1, 2 T, 34 1.45 1, 54 1, 61 1, 82

findings

The table data shows that

1) the supernatant liquid phase contains essentially tetra-, hexa-, octa- and decasaccharides, whereas CY 216 D contains predominantly longer than decacaccharides, i.e. ethanol fractionation also selects for the length of the molecules, and

2) for the same degree of polymerization the oligosaccharides formed from CY 216 D have a higher sulphate / carboxyl ratio than the oligosaccharides isolated from the supernatant, i.e., for each oligosaccharide having a homogeneous degree of polymerization, the degree of sulphation is performed by ethanol fractionation.

Example 2

5000 g of injectable heparin are dissolved in 45 liters of deionized water at 18 ° C. In a similar manner to that described in Example 1, the reagents were increased tenfold, i.e., a solution of 150 g of sodium nitrite in 3 liters of water was added, - the volume of the reaction mixture was adjusted to 50 liters, boron tetrahydride is used, - precipitation is carried out with 100 liters of ethanol, - redissolved in 90 liters of water, - 1000 g of sodium chloride is added, - the final volume is adjusted to 100 liters, and - finally 100 liters of ethanol are added.

The product obtained after treatment with sodium borohydride tetrahydride or precipitation with ethanol had a USP titre of 38 IU / mg and a Yin-Wessler titre of 178 IU / mg, resulting in a ratio of 4.68.

This gives 2230 g of product having the following parameters:

USP titer = 54 IU / mg

Yin-Wessler titer = 232 IU / mg

Ratio of YW / USP titers = 4.3 average molecular weight = 4000-5000 daltons

Example 3

a. but after the nitrite decomposition, the product was isolated and 43 g of the product were dissolved in 1200 ml of desiccated water at room temperature. To the resulting solution was added potassium borohydride (7 g) with vigorous stirring. The resulting reaction mixture was stirred at room temperature for 2 hours and then added with acetic acid at a rate to adjust the pH to 4.0 to decompose excess potassium borohydride.

After stirring for 30 minutes, the pH was adjusted to 7.5 with 5N aqueous sodium hydroxide solution and 5 volumes of ethanol were added. The precipitate was collected, the mother liquor was suctioned off, the precipitate was washed with pure ethanol and

Dry in vacuo at 60 ° C.

46.5 g of product containing 2,5-anhydromannyl end groups are obtained ^{65 with} the following characteristics:

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USP titer = 17 IU / mg

YW titer = 250 IU / mg

b) Dissolve 40 g of the starting material isolated in part a) in 400 ml of distilled water at room temperature. The resulting solution was adjusted to pH 8-5 with 5N aqueous sodium hydroxide solution and then a solution of 2 g of potassium permanganate in 40 ml of water was added.

The resulting reaction mixture was stirred vigorously for 15 hours. During stirring, the pH was maintained at 8.5 by the addition of 5N aqueous sodium hydroxide solution as needed.

After 1 hour, 90 mL of ethanol was added to reduce unreacted potassium permanganate.

After standing for 5 hours, the manganese dioxide precipitate was removed by centrifugation. The product is then precipitated with ethanol, washed and dried.

35 g of product containing 2,5-anhydro-D-mannanoic acid end groups are obtained with the following characteristics:

USP titer = 12 IU / mg

YW titer = 750 IU / mg

Example 4 reports the results of pharmacological tests with the product of Example 1.

Example 4

In vitro activity of the composition of Example 1

1-4. FIGS. 4A-5B show the curves obtained from in vitro studies of the change in clotting time in human plasma using increasing doses of commercially available heparin and the product of Example 1.

In the experiments corresponding to the results shown in Figures 3 and 4, they are platelet-free and shown in Figure XI. Factor enriched plasma was used.

These figures show the changes in thrombin (Fig. 1), cephalin-kaolin (Fig. 2), concentrated thromboplastin (Fig. 3) and dilute thromboplastin (Fig. 4) expressed in securitis, which were used at appropriate doses in the particular cases. experimental products, i.e. the product of Example 1 (curves al, a2, a3 and a4) and heparin (b1, b2, b3 and b4) were induced.

Both thrombin times and cephalin-kaolin times were representative of types of measurements that primarily involved activated II. and inhibition of total coagulation. Examination of the curves of Figures 1 and 2 shows that the mucopolysaccharides produced by the process of the invention have a markedly less potency than heparin in inhibiting the activity of prothrombin and inhibiting the level of total coagulation. Figures 3 and 4 (which are representative of phenomena more directly related to the sequence of enzymatic reactions characteristic of extrinsic coagulation, in particular relative lack of factor Xla) show the beneficial effects of the mucopolysaccharides of the present invention over heparin.

Example 5 describes the effect of the mucopolysaccharides prepared in Example 2 on in vivo experiments in rabbits.

Example 5

Study of the antithrombotic activity of the product prepared in Example 2 in rabbits

The administration of 500 units of YW to rabbits results in significant anti-Xa activity, while the total anti-coagulant activity remains relatively low.

At 2 hours after administration, YW activity was of the order of 0.88 U / ml and cephalin-kaolin time was 0.09 IU / ml.

Thus, the in vitro and in vivo assays clearly show a much more selective effect of the mucopolysaccharides produced by the process of the invention than heparin, particularly at the level of factor Xa inhibition.

Example 6

Preparation of mucopolysaccharide fractions practically devoid of antibiotic activity

The soluble fraction of Example 1 was isolated. This fraction contains chains of 2 to 14 units.

If this fraction is subjected to additional alcoholic fractionation, a precipitate (this is separated) and a soluble fraction are obtained and the composition y is separated from the latter. This composition consists of chains of up to 6-8 units. It has a YW titer of less than 70 and a USP titer of less than 5.

If the y composition is subjected to gel filtration in the usual manner, a z-hexosaccharide composition free of anticoagulant activity is obtained. These compositions inhibit neonangiogenesis.

Example 7

Activity of y and z compositions for neoangiogenesis

In vitro experiments related to anti-angiogenesis are performed on a 6-day culture with choroidalanthoid membrane of germ embryo.

Pellets are prepared by mixing 5 µg of hydrocortisone with 10 µl of methyl cellulose and then adding 5 µg to 100 µg of azy and the compositions in varying amounts. The thus obtained pellets are dried and placed on a chorioallantoic membrane to which they are adhered. Four eggs were used for each experiment.

after an hour of incubation, vascular zones are observed and vascular zones are significantly reduced. These results are already remarkable

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Example 8

Effect of τ hexosaccharide composition on B16 murine melanoma

The experiments were performed on C5 7 BL / 6 mice with B16 melanoma.

The treatment is continued for 15 days, with each mouse being injected subcutaneously with 0.15 mg hexosaccharide composition and cortisone acetate twice daily, decreasing from 250 mg / kg / day to 40-50 mg / kg / day. administered. At the end of the 15-day period, the growth of tumors ceases and, more preferably, a decrease in tumors is observed.

Claims (2)

A process for preparing mucopolysaccharide compositions comprising chains of average molecular weight of 4000-5000, preferably 4500 Daltons, having a YW titer of 180-230 units / mg and a YW / USP titer ratio of less than 10, preferably 3 to 6, especially 4, and their end groups have the structure 2,5-anhydromannnitol, 2,5-anhydromannnose or 2,5-anhydromannanic acid, characterized in that they have a predominantly YW / USP titer ratio of 1800 to 8000 or at least 150 units Compositions consisting of YW titer mucopolysaccharide chains are subjected to at least one fractionation step with a mixture of an inorganic salt water and an alcohol type organic solvent to selectively remove at least most of the lower molecular weight chains and those with a lower sulfate content. at the average sul72 tree content of the chain mixture, maid eventually from these fractions with a lower YW / USP titer ratio and higher USP titer than the starting compositions were separated. 2. A process according to claim 1 wherein the organic solvent is a C 1-4 alkanol. 3. The process according to claim 1, wherein the inorganic salt is sodium or potassium chloride or another salt miscible with the organic solvent used. The process of claim 1, wherein the pH of the reaction mixture is acidic Set to 15. 5. The process of claim 4, wherein the pH of the reaction mixture is adjusted to less than 4. 6. The process of claim 1, wherein the starting material is mucopolysaccharide compositions having a titer ratio of at least 10 YW / USP and a titre of 200-250 units / mg YW. 2g 7. The process according to claim 2, wherein the alcoholic solvent for the fractionation is ethanol. 8. A process according to claim 1, wherein 5% w / v solution of mucopolysaccharide 30 is prepared with water containing 10 g / l ethanol and the pH of this solution is adjusted to 3.8, one volume of ethanol was added and the precipitate was collected. 3® 9. Method according to claim, characterized in that the starting material contains preferably 2.5 mu-anhydro-mannose, 2,5-anhydro-mannitol or 2,5-anhydro-mannonic acid structure units ⁴⁰ reducing végegységként used kopoliszacharid compositions. 2 sheets drawing, figure 4