MXPA99009805A - Compositions comprising very low molecular weight heparin - Google Patents

Abstract

Compositions of heparins of very low molecular weight, with the formula I, in which, n may vary between 1 and 12;R1=H or SO3Na;R2=SO3Na or COCH3. Such compositions of heparin are composed of mixtures of oligosaccharides or fragments of heparin and are characterised byhaving anti-Xa activity and anti-factor IIa activity and because they can be used as anti-thrombic medicaments.

Description

COMPOSITIONS OF HEPARINAS OF VERY LOW WEIGHT MOLECULAR FIELD OF THE INVENTION This invention relates to new compositions of low molecular weight heparins (LMWH) constituted by a limited number of heparin fragments that possess a 4-enopyranosyl uronate group at its non-reducing end: Heparin is a sulfated mucopolysaccharide of animal origin, intestine or mammalian lung extract (cow, lamb, pig) and used for a long time in human therapy for the prevention and treatment of thromboembolic diseases. It is well known that the use of heparin is accompanied by very troublesome hemorrhagic effects and that the daily administration, three subcutaneous or intravenous injections, constitutes a considerable inconvenience. DESCRIPTION OF THE PREVIOUS TECHNIQUE During the last years, various chemical methods have been used to depolymerize heparin, such as: - treatment with sodium nitrite in acidic medium - alkaline treatment of esters - use of free radicals generated in the presence of hydrogen peroxide - treatment of a quaternary ammonium salt of heparin in a non-aqueous medium with a strong base according to a beta-elimination mechanism. These methods make it possible to obtain, with variable yields, mixtures of heparin fragments in which the average molecular weight and the anticoagulant activity vary according to the procedure and the conditions of the operation. The low molecular weight heparins (LMWH) described in the state of the art or marketed are obtained according to various depolymerization processes. Its average molecular weight (Mw) is between 4,000 and 6,000 daltons. It is now recognized that the antithrombotic activity of LMWH is mainly due to its ability to activate antithrombin III, plasma protein, potent inhibitor of activated factor X and thrombin. Thus, it is possible to measure the antithrombotic activity of heparin by specific tests of the inhibition of these factors. The investigations carried out by different authors in recent years, show that fragments or oligosaccharides of heparin constituted by short chains of average molecular weight < 4,800 daltons have a selective action on activated factor X and have little effect on global coagulation measured with pharmacopoeia methods. It has been found that if it is desired to obtain fragments of very low molecular weight and having a strong anti Xa activity it is preferable to use a method of selective depolymerization in a non-aqueous medium, such as that described in patent USP 4,981,955 which does not have the risk of attacking the antithrombin III binding site. DETAILED DESCRIPTION OF THE INVENTION In view of the background and state of the art set forth above, the present application has developed, using a non-aqueous medium, the controlled depolymerization of heparin that allows obtaining a new family of HBMP rich in oligosaccharides. of low molecular mass that exhibit a high anti-Xa activity and a low anti-Ha activity, and that respond to the general formula: Where: n can vary between 1 and 12 R2 = SO3Na or COCH3 Said heparin of very low molecular weight, is obtained by selective depolymerization of heparin in non-aqueous medium according to a beta elimination procedure. The very low molecular weight heparin compositions according to this invention are characterized by an average molecular weight between 2,000 and 4,000 daltons, an anti-factor Xa activity at least equal to 100 U. IJmg and contain a high proportion, up to 75%, of oligosaccharides of low degree of polymerization that extends from hexasaccharide (n = 1) to dodecasaccharide (n = 4). These compositions are useful in the prophylaxis and treatment of venous and arterial thrombosis. They can be used as antithrombotic drugs. Known and commercially exploited LMWHs contain small proportions of oligosaccharides of low molecular mass, notably the oligosaccharides whose degree of polymerization goes from hexasaccharide to dodecasaccharide. The main characteristic of the heparin compositions of the present invention is that they contain a high proportion, up to 75%, of such oligosaccharides. In addition, these oligosaccharides have a high anti Xa activity, (>; 100 U.IJmg) which gives them a high long-term antithrombotic activity. Said heparin compositions have an anti Xa activity comprised between 100 and 150 U. IJmg and whose anti-factor Ha activity is less than or equal to 10 U.IJmg.

The weight-average molecular mass of the heparin compositions of the present application is between 2,000 and 4,000 daltons and because: they contain from 25 to 60% of oligosaccharides of molecular mass less than 2,000 daltons; - contain from 40 to 75% of oligosaccharides of molecular mass between 2,000 and 6,000 daltons; and - contain less than 15% oligosaccharides of molecular mass greater than 6,000 daltons.

The heparin compositions of the present invention are composed of mixtures of oligosaccharides or heparin fragments. The percentage of the fragments that form part of the present are the following: - it contains less than 10% fragments in which n is between 10 and 12; - contains from 80 to 90% fragments in which n is between 1 and 6; and - contains less than 15% fragments in which n is between 7 and 9. The present invention is illustrated by the following examples, without these examples limiting the scope thereof.

The molecular mass (Mw), the molecular distribution, as well as the activities anti factor Xa and anti factor lia have been determined according to the techniques described in the monograph n ° 828"Low molecular weight heparin" of the 3rd Edition of the European Pharmacopoeia .

EXAMPLE 1: After dissolving 1 kg of unfractionated sodium heparin in 7 liters of purified water, 4.4 liters of benzalkonium chloride solution in 50% w / v water are added to the heparin solution and with stirring. It is completed with water up to an approximate volume of 30 liters and it is left to decant. Then the supernatant is removed, water is added up to 30 liters and it is left to decant. Once decanted, the supernatant is removed and the precipitate is lyophilized. Approximately 2.7 kg of benzalkonium salt of heparin are obtained (Product A). After dissolving 100 g of product A in 300 ml of dichloromethane, Triton B is added in three stages: - 25 ml of Triton B are added and left at 25 ° C. for 8 h - 25 ml of Triton B are added and 16 at 25 ° C - add 25 ml of Triton B and leave 8 h at 25 ° C. The above solution is precipitated on 600 ml of sodium acetate solution in 10% w / v methanol and the precipitate is collected by centrifugation by washing with methanol . The product obtained is dissolved in 500 ml of water, neutralized with 0.1 N HCl, sodium chloride is added to a concentration of 10% w / v and precipitated by the addition of 1.25 liters of methanol. The precipitate is then collected by filtration, washing with methanol and drying under vacuum at 35 ° to obtain 33 g of Product B, which are dissolved in water at 10% w / v. It is tempered at 25 ° C and sodium chloride is added to a concentration of 10% w / v. It is precipitated by the addition of 2.5 volumes of methanol. The precipitate is then collected by filtration by washing with methanol and dried under vacuum at 35 ° C to obtain 26 g of purified product which are dissolved in water at 5% w / v. The pH is adjusted to 6.6 with 0.1 N HCl and sodium chloride is added to a concentration of 5% w / v. It is precipitated by the addition of 0.8 volumes of methanol. After collecting the precipitate by filtration by washing with methanol, it is dried under vacuum at 35 ° C. The supernatant is precipitated with 1.6 volumes of methanol. Collect said precipitate by filtration by washing with methanol and drying under vacuum at 35 ° C. Finally, 22 g of Product are obtained.

EXAMPLE 2 The production of Product B from Example 1 is repeated. 20 g of product B are dissolved in 150 ml of water and 100 ml of benzalkonium chloride solution in 50% w / v water are added thereto. It is then completed with water to an approximate volume of 500 ml and left to decant. Once decanted, the supernatant is removed, water is added to 500 ml and it is left to decant. After removing the supernatant, the precipitate is lyophilized to obtain 50 g of benzalkonium salt. Dissolve 20 g of the salt obtained in 60 ml of dichloromethane. Add 5 ml of Triton B and leave for 8 hours at 35 ° C. The above solution is then precipitated over 120 ml of sodium acetate solution in 10% w / v methanol and the precipitate is collected by centrifugation by washing with methanol. The product obtained is dissolved in 100 ml of water, it is neutralized with 0.1 N HCl, sodium chloride is added to a concentration of 10% w / v and precipitated by the addition of 250 ml of methanol. The precipitate is collected by filtration by washing with methanol and dried under vacuum at 35 ° C. Finally, 6.3 g of Product are obtained. EXAMPLE 3: Initially, 5 g of the product obtained in Example 2 are dissolved in 5% w / v water. The pH is adjusted to 6.6 with 0.1 N HCl and sodium chloride is added to a concentration of 5% w / v. It is precipitated by the addition of 1.5 volumes of methanol. The precipitate is then collected by filtration by washing with methanol and drying under vacuum at 35 ° C. Finally, 3 g of Product are obtained. EXAMPLE 4 The production of product B of the example is repeated. 20 g of product B are dissolved in 150 ml of water and 100 ml of benzalkonium chloride solution in 50% w / v water are added thereto. Then it is completed with water up to an approximate volume of 500 ml. After removing the supernatant, water is added to 500 ml and it is left to decant. The supernatant is removed, water is added to 500 ml and it is left to decant. The supernatant is removed again and the precipitate is lyophilized to obtain 50 g of benzalkonium salt. After dissolving 20 g of the salt obtained above in 60 ml of dichloromethane, Triton B is added in two stages: - 5 ml of Triton B are added and left for 8 hours at 35 ° C. - add 5 ml of Triton B and leave 16h at 35 ° C. The above solution is precipitated over 120 ml of sodium acetate solution in 10% w / v methanol and the precipitate is collected by centrifugation by washing with methanol. The product obtained is dissolved in 100 ml of water, neutralized with 0.1 N HCl, sodium chloride is added to a concentration of 10% w / v and precipitated by the addition of 250 ml of methanol. The precipitate is then collected by filtration by washing with methanol and drying under vacuum at 35 ° C. Finally 8.7g of product are obtained. EXAMPLE 5 Initially 5 g of the product obtained in Example 4 are dissolved in 5% w / v water. The pH is adjusted to 6.6 with 0.1 N HCl and sodium chloride is added to a concentration of 5% w / v. It is then precipitated by the addition of 0.93 volumes of methanol.

After collecting the precipitate by filtration, washing with methanol, drying under vacuum at 35 ° C. The supernatant is precipitated with 2 volumes of methanol. The precipitate is collected by filtration by washing with methanol and dried under vacuum at 35 ° C. Finally, 4 g of product are obtained.

ANALYSIS OF THE PRODUCTS: 5 *% of oligosaccharides of molecular mass less than 2,000 daltons **% of oligosaccharides of molecular mass between 2,000 and 6,000 daltons ***% of oligosaccharides of molecular mass greater than 6,000 daltons NOVELTY OF THE INVENTION Having been described the present invention is considered as a novelty, and therefore, claiming as property is contained in the following claims:

Claims (6)

1. REVINDICATIONS: 1.- Compositions of heparin characterized by being of very low molecular weight, with the following general formula: in which: n can vary between 1 and 12 R, = H or SO3Na R2 = SO3Na or COCH3
2. 2. - Heparin compositions according to claim 1, characterized by being composed of mixtures of oligosaccharides or fragments of heparin
3. 3. - Heparin compositions according to claims 1 and 2, characterized by containing less than 10% fragments in which n is between 10 and 12; from 80 to 90% fragments in which n is between 1 and 6; less than 15% fragments in which n is between 7 and 9.
4. 4. - Compositions of heparin according to claims 1 and 2, characterized in that the weight average molecular mass (Mw) is between 2000 and 4000 daltons and because they contain from 25 to 60% of oligosaccharides of molecular mass less than 2000 daltons, from 40 to 75% of oligosaccharides of molecular mass between 2000 and 6000 daltons and less than 15% of oligosaccharides of molecular mass greater than 6000 daltons.
5. 5. - Compositions of heparin according to claims 1 and 2, characterized in that the anti Xa activity is comprised between 100 and 150 U.IJmg and whose anti-factor Ha activity is less than or equal to 10 U.IJmg.
6. 6. Compositions of heparin according to claims 1 to 5, characterized in that they are usable as antithrombotic drugs