1. Field of the Invention
This invention relates to thrombin inhibitors and substrates.
2. Description of the Related Art
Thrombin, the last enzyme in the coagulation system, cleaves soluble fibrinogen to fibrin, which is then crosslinked and forms an insoluble gel forming the matrix for a thrombus. When a vessel is damaged, the above process is necessary to stop bleeding. Under normal circumstances there is no measurable amount of thrombin present in plasma. Increase of the thrombin concentration can result in formation of clots, which can lead to thromboembolic disease, one of the most common serious medical problems of our time.
Thrombin contributes to haemostatic control by means of several biological reactions. In addition to its primary function, the conversion of fibrinogen to fibrin, thrombin activates Factor XIII, which is responsible for the crosslinking of fibrin. Thrombin also acts by means of a positive feedback mechanism involving the activation of Factors V and VIII, which both are necessary for its own formation from prothrombin. Thrombin has another essential role: its binding to platelets initiates platelet release and aggregation which is responsible for primary haemostasis.
Fibrinolysis is the process which causes an enzymatic dissolution of fibrinogen and fibrin clots. Plasma contains a protein, plasminogen, which under the influence of various activators is converted to plasmin, a proteolytic enzyme, the activity of which resembles that of fibrin. Plasmin breaks down fibrin to fibrin degradation products.
Under normal conditions, the fibrinolysis system is in balance with the coagulation system. Small thrombi formed in the blood stream can be dissolved enzymatically and the circulation through the vessels can be restored by the activation of the fibrinolytic system in the body. If the fibrinolytic activity is too high, it may cause or prolong bleeding and if it is too low compared to the activity of the coagulation system, there is a risk of thrombosis.
The reactions of thrombin are further controlled by natural inhibitors in plasma. The most important of these are antithrombin III and heparin. These two compounds have been isolated and are therapeutically and prophylactically used in conditions where there is an imbalance in the haemostatic mechanisms with risk for prothrombin activation.
Mainly two types of therapeutic agents are used for the prevention of thrombosis. The heparins act by accelerating the inhibition of thrombin by antithrombin III. Coumarin derivatives, the oral anticoagulants, e.g. Warfarin, prevent the generation of thrombin by blocking the post-translational vitamin K-dependent .gamma.-carboxylation in the synthesis of prothrombin. Neither Heparin nor Warfarin are ideal. Heparin must be given parenterally and as it functions as a cofactor to antithrombin III it has no effect without this inhibitor. The effect of Warfarin develops very slowly and individual doses must be adjusted by frequent tests. None of these anticoagulants is specific for thrombin, they also inhibit other serine proteases and both of them may cause bleeding if the doses are not correctly balanced.
Thus, direct acting, specific thrombin inhibitors, having oral activity would be useful alternatives to the above anticoagulants. Much research in this area has resulted in the synthesis of different kinds of inhibitors of thrombin.
By imitating amino acid sequences of fibrinogen, the important natural substrate of thrombin, several good short peptide substrates for thrombin have been synthesized. The very first developed sequence with affinity for the active site of thrombin was Phe-Val-Arg 1! which mimics the fibrinogensequence preceding the bond split by thrombin. This sequence has later been improved to give D-Phe-Pro-Arg and D-Phe-Pip-Arg which have been used in chromogenic substrates, e.g. D-Phe-Pro-Arg-pNA and D-Phe-Pip-Arg-pNA 1! and in inhibitors of thrombin, e.g. the peptide aldehyde D-Phe-Pro-Arg-H 2!, the irreversible inhibitor D-Phe-Pro-Arg-CH.sub.2 Cl 3!, inhibitors with a ketomethylene bond e.g. D-Phe-Pro-Arg-k-Gly-piperidide 4! and in the recently synthesized peptide boronic acid inhibitors e.g. Z-D-Phe-Pro-boroArg 5! and the nitrile: Boc-D-Phe-Pro-ArgCN 6!.
Thus, D-Phe-Pro-Arg has been considered the best sequence for about 15 years, and it has been shown to have very good affinity for the active site of thrombin, in substrates (Km around 10.sup.-6 M) as well as in inhibitors (Ki 10.sup.-7 M to 10.sup.-9 M).