WO2008006925A2

WO2008006925A2 - Process for depolymerisation of heparin using glycidol molecules

Info

Publication number: WO2008006925A2
Application number: PCT/ES2007/000423
Authority: WO
Inventors: Jean Sarkis Mardiguian; Ochoa Maria Teresa Gomez; Gregorio Roncero Andres
Original assignee: Laboratorios Farmaceuticos Rovi S. A.
Priority date: 2006-07-14
Filing date: 2007-07-13
Publication date: 2008-01-17
Also published as: WO2008006925A3; ES2292351B1; ES2292351A1

Abstract

The present invention relates to a simple and economic procedure for depolymerising heparin using glycidol molecules that offers an alternative to known heparin depolymerisation methods.

Description

NEW PROCEDURE FOR DEPOLIMERIZATION OF HEPARINE

FIELD OF THE INVENTION:

The present invention falls within the field of biochemistry. The present invention relates to a new method of depolymerization of heparin. The present invention thus provides a new simple and economical process that poses an alternative to the known methods of depolymerization of heparins.

STATE OF THE TECHNIQUE

Heparin is a mucopolysaccharide that is composed of polymers of alternating derivatives of D-glucosamine (N-sulfated or N-acetylated) and uric acids (L-iduronic or D-glucuronic acids), linked by glycosidic bonds, in varying proportions. Heparin has, among other properties, the prolongation of blood clotting time, mainly through the formation of an intermediate compound with plasma antithrombin. This enhances thrombin inactivation and inhibits other proteases that act in the coagulation process such as Activated Factor X. The limiting step in the cascade reaction is the activation of Factor X, which is involved in the extrinsic and intrinsic pathways. Small amounts of Heparin in combination with Antithrombin III inhibit thrombosis by deactivating Factor X and inhibiting the conversion of prothrombin to thrombin. This bases its use in prophylaxis with Heparin in small doses. Once the active thrombosis has developed, larger amounts of Heparin can inhibit new coagulations by inactivating the thrombin by preventing the conversion of fibrinogen to fibrin. In combination with Antithrombin III, Heparin inactivates Factors IXa, Xa, XIa, XIIa and thrombin, inhibiting the transformation of fibrinogen to fibrin. Heparin also prevents the formation of a stable fibrin clot by inhibiting the activation of Factor XIII (the fibrin stabilizing factor). Heparin surprisingly inhibits the reactions that lead to coagulation, but does not significantly alter the levels of coagulation factors in the blood. Heparin has no fibrinolytic activity; It does not cause lysis of existing clots, but it can prevent the spread of existing clots. Heparin also increases the release of lipoprotein lipases, which eliminates circulating lipids from the plasma, increases the amount of circulating free fatty acids and reduces lipoprotein levels. According to its molecular structure, heparin is a sulfated mucopolysaccharide formed by units of uronic acid (U) (D-glucuronic or L-iduronic) alternated with units (Gl) of D-glucosamine, as shown by the compound of formula general I:

U - Gl - U - Gl - U

Compound of general formula I

The uronic acid (U) is normally 2-sulfated and the glucosamine (Gl) can be N-sulfated or 6-sulfated, but also N-acetylated or 3-sulfated.

Heparin is used in the form of sodium salt (sodium heparin), but it can also be in the form of calcium salt (calcium heparin) or magnesium.

The depolymerization of heparin can be carried out enzymatically by heparinases [FR20020011724; WO1996US15593; US19950004622P] or chemical route [RU19970117697; JP19830159102; JP19810046288; JP19800051602; JP19760159896; JP19800051601] either for structure studies or to prepare low molecular weight heparins that are used in antithrombotic therapy. The chemical depolymerization processes in aqueous medium use sodium nitrite in acidic medium or peroxides, or the alkaline treatment of the benzyl ester of heparin. In a non-aqueous medium, a benzyl ester can be treated with a base or directly a quaternary ammonium salt of heparin with a strong base.

During the course of recent years, different chemical methods have been used to depolymerize heparin, such as:

- treatment with sodium nitrite in acid 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, etc. These methods allow, with variable yields, to obtain mixtures of heparin fragments in which the average molecular weight and the anticoagulant activity varies according to the procedure and the operating conditions [E99500184; P9901671; P8700020].

In the state of the art there is also a selective and specific method of depolymerization of heparin, protected by the holder of the present invention, which is carried out in a non-aqueous medium with a strong base. The process begins by subjecting the unfractionated heparin to the quaternary ammonium treatment. Thus, a heparin salt is formed that particularly protects the key groups of the HNF molecule. The chain is then depolymerized and fractionated by unprotected sites, obtaining up to 75% of homogeneous fragments, of molecular weight between 2,000 and 6,000 daltons. After the controlled depolymerization process proceeds to purification and freeze drying with which different LMWH Weight including Bemiparina LMWH 2 ^to generation 3,600 daltons medium PM [P9901671] are obtained.

DESCRIPTION OF THE INVENTION

Normally all these procedures have a number of disadvantages and involve several stages, among which an additional stage of delicate final purification stands out. In addition, these procedures need strict control of working conditions.

The present invention provides a practical solution and an alternative to the heparin depolymerization methods employed so far. By means of a novel solution that includes the novel use of glycidol molecules for the first time in heparin depolymerization processes, a simple and substantially economical process is obtained, since being more selective entails fewer reaction steps.

The present invention relates to a new method of depolymerization of heparin. It is a simple and economical procedure, which consists of treating an aqueous solution of heparin with a certain amount of glycidol of at least 0.4 ml per gram of heparin, at a temperature of 40 ° C to 80 ° C, for a period of 4 to 24 hours.

CH ₂ - CH - CH ₂ OH

Glycidol

In this way it is possible to isolate the products formed, by precipitation or by any other procedure of the state of the art.

The procedure described in the present invention comes from beta-elimination, with formation of an enopyranosyluronic moiety.

R = H or SO ₃

Enopyranosyluronic rest

The reaction may eventually be accompanied by the formation of residues of a compound of general formula A in small amounts. However, by optimizing working conditions, this secondary reaction can be minimized, and even avoided.

- O— CH ₂ - CH-CH ₂ OH

OH

Remains of general formula A It is also possible to limit the attack of glycidol to the antithrombin binding site of heparin, which is responsible for its anticoagulant activity, working with a concentrated solution of heparin, at 40-50 ° C.

More generally, other molecules defined by the general formula B can be used, but it seems that glycidol is the most reactive.

CH ₂ - CH - CH ₂ - R

\ /

Remains of general formula B

In accordance with a first aspect, the present invention relates to a heparin depolymerization process in which an aqueous solution of heparin of a concentration range between 10% and 40% is treated with glycidol in a proportion of at least 0, 4 ml per gram of heparin, at a temperature range of 40 ° C to 80 ° C, for a period of 4 to 24 hours.

According to another important aspect, the present invention relates to a heparin depolymerization process in which an aqueous solution of heparin of a concentration range between 20% and 40% is treated with an amount of glycidol in a proportion between 0 , 4 ml to 2 ml per gram of heparin, at a temperature range of 40 ° C to 50 ° C, for a period of 6 to 24 hours.

According to a second important aspect, the present invention relates to the use of glycidol molecules to depolymerize heparin, in which glycidol is used in a proportion of at least 0.4 ml per gram of heparin. EXAMPLES OF REALIZATION:

The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations to the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application. The products described have been obtained from heparin of porcine origin (examples 3 and 4) or bovine (examples 1 and 2) of pharmaceutical quality. They have been isolated by precipitation in alcohol, by adding methanol to the aqueous solution containing 10% sodium chloride, and then filtration of the precipitate and drying.

The products obtained have a characteristic absorption spectrum in the ultraviolet, with a maximum of approximately 232 nm. Its average molecular weight was determined by the method of the European Pharmacopoeia (Ph. Eur. 5 Edition).

Its anti-factor Xa activity has been measured according to a chromogenic method (Ph. Eur. 5 ^a

Edition) and has been expressed in international units per mg.

Example 1

20 g of heparin are dissolved in 80 ml of water, then 24 ml of glycidol are added, the solution is brought to 50 ° C for 24 hours and then cooled and allowed to return to room temperature, and by precipitation in Usual alcohol is isolated 13.2 g of depolymerized heparin.

Example 2

5 g of heparin are dissolved in 20 ml of water, 6 ml of glycidol are added and the solution is brought to 40 ° C for 24 hours. After cooling and returning to room temperature, by precipitation in alcohol, 4 g of depolymerized heparin are obtained. Example 3

20 g of heparin are dissolved in 80 ml of water and 12 ml of glycidol are added. The solution is brought to 50 ° C for 24 hours. After cooling, 19.5 g of depolymerized heparin are isolated as in the previous examples.

Example 4

10 g of heparin are dissolved in 40 ml of water and 12 ml of glycidol are added. The solution is brought to 50 ° C for 4 hours and then cooled to room temperature. 8.9 g of depolymerized heparin are obtained.

Characteristics of the products described (see figure 1):

PM (1) Activity (2) UV spectrum (3)

Example 1 2757 60 max. at 232 nm

Example 2 3594 65 max. at 232 nm

Example 3 4427 73 max. at 232 nm

Example 4 5467 83 max. at 232 nm

(1) average molecular mass (daltons)

(2) anti-Xa activity (Ul / mg)

(3) UV spectrum in 0.01 N HCI

Claims

1. Heparin depolymerization process characterized in that an aqueous solution of heparin of a concentration range between 10% and

40% with glycidol at a rate of at least 0.4 ml per gram of heparin, at a temperature range of 40 ° C to 80 ° C, for a period of 4 to 24 hours.

2. Heparin depolymerization process according to claim 1, characterized in that an aqueous solution of heparin of a concentration range between 20% and 40% is treated with an amount of glycidol in a proportion less than or equal to 2 ml per gram of heparin at a temperature range of 40 ° C to 50 ° C for a period of ₁ 6 to 24 hours.

3. Heparin depolymerization process according to claim 2, characterized in that the heparin treated is sodium heparin.

4. Method according to claim 1, wherein the heparin is in the form of calcium or magnesium salt.

5. Heparin depolymerization process according to any of claims 2 and 3 characterized in that the structure of the oligosaccharides obtained has an enopyranosyluronic moiety and a residual anti-factor Xa activity greater than 40 Ul / mg.

6. Use of glycidol molecules to depolymerize heparin.

7. Use of glycidol molecules to depolymerize heparin according to claim 6, in a proportion of at least 0.4 ml per gram of heparin.