Covalently bound heparin - antithrombin-III complex, method for its preparation and its use for treating thromboembolism

Claim 2

A process for producing a covalently bound heparin - antithrombin-III complex which comprises the steps of: (a) contacting heparin containing components of varying degrees of activity with cyanogen bromide to obtain an activated heparin intermediate; and (b) contacting the activated heparin intermediate from step (a) with antithrombin-III.

Claim 3

A process according to claim 1 wherein the heparin used in step (a) is unfractionated heparin.

Claim 4

A process according to claim 1 or 2 including the additional step of: (c) processing the product obtained from step (b) to separate therefrom free heparin.

Claim 5

A process according to claim 3 wherein free heparin is separated from the said product by affinity chromatography techniques, preferably by using a lectin-containing, water-insoluble matrix as the column adsorbent.

Claim 6

A process according to any of claims 1 to 4 including the additional step of: (d) processing the product obtained from step (c) to separate therefrom free AT-III.

Claim 7

A process according to claim 5 wherein free antithrombin-III is separated by affinity chromatography techniques, preferably by using a heparin-containing, water-insoluble matrix as the column adsorbent.

Claim 8

A process according to claim 1 which comprises the steps of: (a) contacting unfractionated heparin containing components of varying degrees of activity with cyanogen bromide to obtain an activated heparin intermediate; (b) contacting the activated heparin intermediate from step (a) with antithrombin-III to obtain a mixture of a covalently bound complex of heparin and antithrombin-III (Hep - AT-III complex), free heparin and free antithrombin-III; (c) contacting the mixture of the covalently bound heparin - antithrombin-III complex, free heparin and free antithrombin-III from step (b) with a Concanavalin A (Con A)-Sepharose chromatographic column to obtain free heparin in the effluent and to retain the covalent Hep - AT-III complex and free AT-III bound on the Con A-Sepharose column; (d) eluting the Con A-Sepharose column having the covalent Hep - AT-III complex and free AT-III retained thereon from step (c) with a carbohydrate solution having sufficient carbohydrate concentration to effect separation of the covalent Hep - AT-III complex and free AT-III from the column. (e) contacting the covalent Hep - AT-III complex and free AT-III eluate from step (d) with a Heparin - Sepharose column to obtain in the eluate the free covalent Hep - AT-III complex and to retain the free AT-III bound on the Heparin - Sepharose column; and (f) recovering from the eluate of step (e) the covalently bound Hep - AT-III complex free of unbound, uncomplexed heparin and antithrombin.

Claim 9

A covalently bound heparin - antithrombin-III complex obtainable by a process of any of claims 1 to 7.

Claim 10

A pharmaceutical preparation comprising the complex of claim 8 and a pharmaceutically acceptable carrier.

Claim 11

Use of a complex according to claim 8 or a preparation according to claim 9 for treating thromboembolisn or for inhibiting coagulation of blood in a patient.