Prothrombin derivatives

Details Prothrombin derivatives Prothrombin derivatives

1998-01-26

2000-07-11

Prouty, Rebecca E.

Drug, bio-affecting and body treating compositions

Enzyme or coenzyme containing

Hydrolases

435 696, 4352523, 4353201, 435440, 536 232, 530381, 530384, 424530, A61K 3848, C12P 2104, C12N 120, C12N 1500

Patent

active

060868716

DESCRIPTION:

BRIEF SUMMARY
The invention relates to new prothrombin mutants or derivatives thereof which may be utilized as antagonists of their natural functions.


BACKGROUND OF THE INVENTION

The mechanism of blood coagulation normally occurs in a cascade of two possible routes. One of the routes, the so-called extrinsic blood coagulation, starts with the liberation of thromboplastin and the activation of factor VII. Activated factor VII in turn activates factor X, followed by an activation of factor V and factor II (prothrombin). Factor IIa (thrombin) converts fibrinogen into fibrin at the end of the cascade.
The other route, the so-called intrinsic blood coagulation, occurs via an activation of factor XII by contact with and subsequent activation of factor XI, factor IX and factor X in the presence of calcium and factor VIII, followed by an activation of factor II to factor IIa which triggers the coagulation by cleaving fibrinogen to fibrin. Thus, factor IIa plays a central role in both routes of the blood coagulation cascade. Hitherto, there has been an intensive search for anticoagulants which may particularly be utilized in the treatment of septic shock, thromboses, embolisms, arteriosclerosis and cardiac infarctions, furthermore in case of blood transfusions or following surgery. One method of suppressing the coagulation of blood is the direct administration of substances which inhibit thrombin.
Hitherto, heparin or coumarin have been utilized as anticoagulants. They are, however, relatively systemic and increase the risk of inner hemorrhages. Hirudin, on the other hand, is extremely specific in its binding to thrombin and offers further advantages as compared to the other anticoagulants. It does not require endogenous cofactors, is pharmacodynamically inert, exhibits no effect on blood cells, plasma proteins (with the exception of thrombin) or enzymes, and is immunogenic on account of its small molecular size. Furthermore, hirudin is not stored in organs and is excreted unchanged in urine.
Hirudin is a single-chain polypeptide of 65 amino acids which is naturally formed by the medicinal leech (Hirudo medicinalis) in its secretory glands. Hirudin acts as extremely strongly binding and highly specific inhibitor for the protease thrombin and prevents blood coagulation. The mechanism of the effect of hirudin as thrombin inhibitor has been cleared up: The C-terminal part of hirudin binds to the anion binding sites of the thrombin and thus occupies the binding site of the fibrinogen chain on thrombin. In addition, the N-terminal part of hirudin blocks the active site of thrombin (Szyperski et al. 1992, J. Mol. Biol. 228: 1206-1211; Fenton et al. 1991, Blood Coagul. Fibrinol.2: 69-75; Rydel et al. 1990, Science 249: 277-280; Karshikov et al. 1992, Prot. Science 1: 727-735; Markwardt 1991, Thromb. Haemost. 66: 141-152). For this reason, there has already been an interest for quite some time in using hirudin as a specific anticoagulant.
Recently it has been possible to prepare large amounts of hirudin by a recombinant route, and to use them for pharmacological investigations (Rigel et al. 1993, Circl. Res. 72: 1091-1102; Loison et al. 1988, Biotechnol. 6: 72-77; Zawilska et al. 1993, Thromb. Res. 69: 315-320; Klocking et al. 1990, Blut 60: 129; Fareed and Walenga 1989, FASEB J. 3: 328; Markwardt et al. 1988, Pharmazie 43: 202-207). There result several clinical applications for hirudin: in hemodialysis, as an anticoagulant during the pulmonary transluminal coronary angioplasty (PTCA), for the prophylaxis of post-operative thrombosis, for the prevention of rethrombosis, for microvascular surgery, as anti-coagulant in hemodialysis and in case of extracorporeal circulation, as an admixture to thrombolytic agents, such as, e.g., plasminogen activators and streptokinase, as anticoagulant during surgery and for the clinical suppression of coagulation.
When administering anticoagulants, exact dosing, however, is difficult. For instance, the inhibition of thrombin in the circulation of blood caused by hirudin can lead to undesired complica

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