Glycosaminoglycans having high antithrombotic activity

Details Glycosaminoglycans having high antithrombotic activity Glycosaminoglycans having high antithrombotic activity

1999-09-20

2001-03-06

Lee, Howard C. (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C514S056000, C514S822000, C536S124000

Reexamination Certificate

active

06197943

ABSTRACT:

FIELD OF THE INVENTION
Object of the present invention are glycosaminoglycans having high antithrombotic activity in vitro, obtained by various kinds of glycosaminoglycans supersulfated by the preparation of the salt of an organic base of the starting supersulfated glycosaminoglycan, by partial solvolytic desulfation of said salt and N-resulfation of said partially desulfated product.
PRIOR ART
Heparin is a polysaccharide extracted by animal organs, employed for more than lo fifty years as an anticoagulant and antithrombotic agent. Together with heparan sulfate, it belongs to the family of the glycosaminoglycans, consisting of alternate sequences of an uronic acid (iduronic or glucuronic) and glucosamine, variously sulfated depending on the tissue and the animal species from which they have been obtained and, to a certain extent, on the isolation processes too. The structure of heparin may be represented in statistical terms by formula
where the U units represent the iduronic (IdoA) or glucuronic (GIcA) acid and the. A units represent the N-sulfated (GIcNAc) glucosamine; R
2
, R
3
and R
6
represent sulfate groups or hydrogen and R′ represents SO
3
or Ac. The sequences mostly represented in the heparins used in clinic are those of the trisulfated disaccharide (IdoA2SO
3
-GlcNSO
3
6SO
3
). On the contrary, a minor pentasaccharidic sequence contained only in about one third of the chains forming the common heparins and characterized by a glucosaminic unit sulfated in position 3 (GIcNSO
3
3SO
3
) and forming the active site of heparin and heparan sulfate for the antithrombin III is essential for the expression of significant anticoagulant and antithrombotic activities. The 3-O-sulfated glucosamine unit is considered the marker of said active site and the antithrombotic activity, generally expressed by the inhibition capability of the X activated (Xa) and thrombin (IIa) coagulation factors, is correlable with the content percent of said unit in the heparins.
The anticoagulant and antithrombotic properties of heparin and heparan sulfate are also modulated by the length of the polysaccharidic chains composing them. For example the heparins having low molecular weight (LMWH) have lower anticoagulant activity but antithrombotic activity similar to the traditional heparins one, and they are inclined to substitute the latter ones in several therapeutic applications, especially in order to reduce the haemorrhagic risks and other side effects of heparin, such as the thrombocytopenia. In addition, the LMWH are characterized by a bioavailability better than the traditional heparins one when they are administered by subcutaneous route, as it is common in the prevention of the venous thrombosis. (B. Casu Heparin structure, Hemostasis 20/1, 66-73 (1990)); (D. A. Lane, J. Björk, U. Lindall (Ed. s). Heparin and Related Polysaccharides, Plenum Press, New York, 1992).
Since, as above remembered, the pentasaccharidic sequences of heparin mostly responsible of the antithrombotic activity are contained only in about one third of the natural heparin chains, it is of practical interest to strengthen said activity concentrating the chains containing said sequences, or generating new active sites either in the chains which already contain one, or in those ones lacking active sites. The first goal, attainable by affinity chromatography on antithrombin III or (less effectively) by treatment of heparin with cationic resins, is still considered expensive, also because it does not use a substantial (about two thirds) part of the common heparins.
The goal to generate further active sites for antithrombin is not on the other hand attainable by sulfation of heparin with classical methods, which result in structures wherein the active site for antithrombin is masked by an excess of sulfate groups. Even if fortuitously they show an anticoagulant activity greater than the starting heparin one (for the most part by action mechanisms different from the ones mediated by antithrombin), said products are generally less active than heparin as antithrombotic agents, and they may generate undesirable side effects due to the aspecific interactions with other plasmatic proteins. [B. Casu, Structure and biological activity of heparin, Advances Carbohydr. Chem. Biochem. 43, 51-134 (1985)].
On the other hand, attempts to “reconstruct” the active sequences of heparin by resulfation of partially or totally desulfated heparins result in products with a reduced, rather than strengthened, antithrombotic activity. This is due, in particular, to the tendency of the iduronic units to sulfate in position 3 rather than in position 2, and/or to insufficient sulfation in position 3 of the glucosaminic units. [R. N. Rej, K. G. Ludwig-Baxter, A. S. Perlin, Carbohydr. Res. 210, 299-310 (1991)].
SUMMARY OF THE INVENTION
Now we have found a process which makes possible to obtain glycosaminoglycans having high antithrombotic activity in vitro, containing at least 20% of iduronic units sulfated in position 2 and not sulfated in position 3 and at least 30% of sulfaminoglucosaminic units sulfated in position 3 and in position 6, and having a sulfates/carboxyls molar ratio ranging from 2.0 to 3.5. Said process employs as starting materials various kinds of supersulfated glycosaminoglycans and it is characterized by the following steps:
a) preparation of the salt of an organic base of the starting supersulfated glycosaminoglycan;
b) partial solvolytic desulfation of the salt of the organic base of the step a);
c) N-resulfation of the partially desulfated product of the step b);
d) possible 6-O-resulfation of the product of the step c).
The obtained product may be used as an active substance in the preparation of pharmaceutical compositions suitable to the antithrombosis treatment.
DETAILED DESCRIPTION OF THE INVENTION
The characteristics and the advantages of the glycosaminoglycans having high antithrombotic activity according to the present invention and the related preparation process will be mostly illustrated during the following detailed description.
In the process according to the present invention as starting materials may be used several kinds of supersulfated glycosaminoglycans and in particular may be employed: low molecular weight supersulfated heparins (ssLMWH), from 1,500 to 8,000 daltons, high molecular weight supersulfated heparins, from 8,000 to 20,000 daltons, low molecular weight supersulfated heparan sulfates, from 1,500 to 8,000 daltons, high molecular weight supersulfated heparan sulfates, from 8,000 to 25,000 daltons, “biotechnological” supersulfated heparan sulfates and heparins, epimerized and not epimerized, obtained from the N-sulfated K5 polysaccharide. As it is known, the supersulfated glycosaminoglycans are glycosaminoglycans in which all the hydroxylic hydrogens (or the most part of them) have been substituted with SO
3
— groups, and they are prepared according to procedures described by various authors (M. L. Wolfrom et al, J. Am. Chem. Soc. 75, 1519 (1953); Nagasawa et al., Carbohydr. Res. 158, 183-190 (1986); EP 214,879 (1986); U.S. Pat. No. 4,727,063; Naggi et al., Biochem. Pharmacol. 36, 1895-1900 (1987); Ogamo et al., Carbohydr. Res. 193, 165-172 (1989)).
The process is carried out according to the following steps:
a) Preparation of the salt of an organic base of the starting supersulfated glycosaminoglycan, said organic base being selected among pyridine, tetramethyl ammonium and tetrabutyl ammonium salts.
The starting supersulfated glycosaminoglycan in form of a sodium salt is dissolved in distilled water and passed through a cationic exchange column.
To the obtained solution the organic base is added at room temperature and in an amount such that pH 9 is reached obtaining the salt which is then lyophilized.
b) Partial solvolytic desulfation of the step a). The organic base salt obtained in the step a) is treated with a solution of an aprotic polar solvent containing methanol or H
2
O in a percentage ranging from 5 to 10%, preferably DMSO containing 10% of methanol, with a ratio by w

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