Fibrinolytic and anti-thrombotic cleavable dimers

Details Fibrinolytic and anti-thrombotic cleavable dimers Fibrinolytic and anti-thrombotic cleavable dimers

1992-06-03

1995-07-18

Walsh, Stephen G.

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

530350, 530402, 435 697, 424 9464, C12N 970, C07K 1300

Patent

active

054340730

DESCRIPTION:

BRIEF SUMMARY
This application is the US national stage under 37 CFR 371 of PCT/GB90/01911, filed 7 Dec. 1990.
This invention relates to proteinaceous compounds which can be cleaved to release fibrinolytic and/or anti-thrombotic activity. It also relates to nucleic acid (DNA and RNA) coding for all or part of such compounds. In preferred embodiments, the invention relates to fusion proteins produced by linking together fibrinolytic and/or anti-thrombotic proteins with a cleavable linker, their preparation, pharmaceutical compositions containing them and their use in the treatment of thrombotic disease.
The fibrinolytic system is the natural counterpart to the clotting system in the blood. In the process of blood coagulation, a cascade of enzyme activities are involved in generating a fibrin network which forms the framework of a clot, or thrombus. Degradation of the fibrin network (fibrinolysis) is accomplished by the action of the enzyme plasmin. Plasminogen is the inactive precursor of plasmin and conversion of plasminogen to plasmin is accomplished by cleavage of the peptide bond between arginine 561 and valine 562 of plasminogen. Under physiological conditions this cleavage is catalysed by tissue-type plasminogen activator (tPA) or by urokinase-type plasminogen activator (uPA).
If the balance between the clotting and fibrinolytic systems becomes locally disturbed, intravascular clots may form at inappropriate locations leading to conditions such as coronary thrombosis and myocardial infarction, deep vein thrombosis, stroke, peripheral arterial occlusion and embolism. In such cases, the administration of fibrinolytic and anti-thrombotic agents has been shown to be a beneficial therapy for the promotion of clot dissolution.
Fibrinolytic therapy has become relatively widespread with the availability of a number of plasminogen activators such as tPA, uPA, streptokinase and the anisoylated plasminogen streptokinase activator complex, APSAC. Each of these agents has been shown to promote clot lysis, but all have deficiencies in their activity profile which makes them less than ideal as therapeutic agents for the treatment of thrombosis (reviewed by Marder and Sherry, New England Journal of Medicine 1989, 318: 1513-1520).
A major problem shared by all of these agents is that at clinically useful doses, they are not thrombus specific as they activate plasminogen in the general circulation. The principal consequence of this is that proteins such as fibrinogen involved in blood clotting are destroyed and dangerous bleeding can occur. This also occurs with tPA despite the fact that, at physiological concentrations, it binds to fibrin and shows fibrin selective plasminogen activation.
Another important shortcoming in the performance of existing plasminogen activators is that re-occlusion of the reperfused blood vessel commonly occurs after cessation of administration of the thrombolytic agent. This is thought to be due to the persistence of thrombogenic material at the site of thrombus dissolution. Anti-thrombotic proteins may be used in the treatment or prophylaxis of thrombosis either alone or as an adjunct to fibrinolytic agents. Suitable anti-thrombotic proteins include hirudin, activated protein C and anti-thrombin III.
An alternative approach to enhancing fibrinolysis and inhibition of blood clotting has now been devised which is based on the use of fusion proteins cleavable to achieve release of fibrinolytic and/or anti-thrombotic activity at the site of blood clotting. To achieve this, proteins involved in fibrinolysis or inhibition of coagulation are joined by a linker region which is cleavable by an enzyme involved in blood clotting. Examples of proteins which may be incorporated into such a cleavable protein include tPA, uPA, streptokinase, plasminogen, activated protein C, hirudin and anti-thrombin III. Fusion of such proteins to a protein with a favourable property not directly related to dissolution of blood clots, for example albumin which has a long plasma half-life, may also be beneficial. An advantage of this

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