Derivatives of K5 polysaccharide having high anticoagulant activ

Details Derivatives of K5 polysaccharide having high anticoagulant activ Derivatives of K5 polysaccharide having high anticoagulant activ

1998-11-06

2000-12-19

Wilson, James O.

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

514 56, 536 21, 536 53, 536 54, 536 55, 536 551, 536 552, A61K 31715

Patent

active

061627976

DESCRIPTION:

BRIEF SUMMARY
PRIOR ART

It is known that the product mainly used in anticoagulant therapy is the heparin obtained by extraction from animal organs. However the production of heparin from animal organs employs great amounts of solvents and chemical agents involving disposal and therefore potential environmental pollution problems. Moreover the final product may contain residues of biological substances normally or exceptionally present in the animal tissues as viruses or prions. O-sulfation processes carried out on derivatives of the K5 polysaccharide (B. Casu et al., Carbohydrate Letters, 1, 107-114 (1994)) are also known.
The publications by B. Casu et al. refer to O-sulfations carried out on K5 which result in products showing an anticoagulant strength lower then the commercial heparin.
This is also due to the fact that the entirety of uronic acids is represented by glucuronic acids. The glucuronic acid gives to the polysaccharidic chain a lower flexibility towards the target proteins such as for example the antithrombin III and then a lower anticoagulant activity (B. Casu, M. Petitou, M. Provasoli and P. Sinay (1988). Conformational flexibility: a new concept for explaining binding and biological properties of iduronic acid containing glycosaminoglycans. Trends Biochem. Sci. 13, 221-225).


SUMMARY

Now a process for the preparation of new derivatives of the K5 polysaccharide which allow to overcome the drawbacks of the prior art has been found.
Said process includes the following steps: at least 50% of iduronic acid with respect to the total of uronic acids, and it is characterized in that the product obtained in the step c) is further treated according to the following steps: through a column containing a cation exchange resin; product is redissolved in an organic solvent and treated with a sulfating agent to obtain the O-sulfation; distilled water and dialyzed against distilled water; a sulfating agent in order to obtain the N-resulfation of the in case N-desulfated groups.
Optionally the product obtained after the step h) is depolymerized by controlled nitrous acid degradation according to known technology (i.e. U.S. Pat. No. 5,019,649).
The products according to the present invention have, beside new characteristics, a high anticoagulant activity, greater than the anticoagulant activity of the heparin obtained by extraction from animal tissues.
FIG. 1 represents the data relative to the affinity of heparin for antithrombin III.
FIG. 2 represents the data relative to the affinity of the derivative of Example 1 for antithrombin III.
FIG. 3 represents the data relative to the affinity of the derivative of Example 1 for antithrombin III.
FIG. 4 represents the data relative to the affinity of the derivative of Example 3 for antithrombin III.


DETAILED DESCRIPTION OF THE INVENTION

The characteristics and the advantages of the derivatives of the K5 polysaccharide and the relative preparation process according to the present invention will be mostly pointed out during the following detailed description. The starting material for the achievement of said derivatives is the K5 polysaccharide obtained from E. Coli as described by M. Manzoni, S. Bergomi and V. Cavazzoni (Journal of Bioactive and Compatible Polymers. Vol. VIII, July 1993, 251-257). The K5 polysaccharide is first of all treated as in the following steps: N-sulfated K5 from 25 to 100%; L-iduronyl C5 epimerase extracted from bovine liver in order to obtain a product having a L-iduronic acid content from 50 to 90% with respect to the total of the uronic acids.
The N-deacetilation of the step a) is carried out by treatment with a hydrazine and hydrazine sulfate mixture or in an alkaline environment with sodium hydroxide or potassium hydroxide. Subsequently the N-sulfation of the step b) is carried out by treatment with triethylamine-sulfur trioxide or with trimethylamine-sulfur trioxide. The N-deacetilation and N-sulfation reactions are carried out according to the known techniques, for example according to the Patent WO 92/17507.
The N-sulfated product is then

REFERENCES:
patent: 5019649 (1991-05-01), Lormeau et al.
patent: 5550116 (1996-08-01), Lormeau et al.
Carbohydrate Letters, vol. 1, pp. 107-114, 1994.
TIBS 13, Jun. 1998, pp. 221-225.

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