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Factor Xa (FXa) is a key serine protease in the coagulation cascade and is a promising target enzyme for developing a new antithrombotic agent. Our first clinical candidate for a small molecular direct FXa inhibitor DX-9065a potently inhibits FXa (Ki = 41 nM) and exerts antithrombotic effects in animal models. However, due to its poor bioavailability (10% in monkeys) the compound is used only as an injectable formulation in clinical studies. Here we report in vitro characteristics of serine proteases inhibition, anticoagulant effects and in vivo antithrombotic efficacy of DU-176b, a novel, potent and orally active direct FXa inhibitor. DU-176b competitively inhibited human FXa with a Ki value of 0.561 nM, indicating 70-fold increase in FXa inhibitory activity compared with DX-9065a. DU-176b demonstrated 10,000-fold selectivity relative to inhibition of thrombin (Ki = 6.00 μM), and had no effects on the enzymatic activities of factor VIIa, t-PA, plasmin, trypsin and chymotrypsin. In human plasma, DU-176b prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT) in a concentration-dependent manner. Its concentrations for doubling these clotting times were 0.256 and 0.508 μM, respectively. After oral administration of DU-176b to rats, significant anti-Xa activity was observed in plasma over 4 h. The oral bioavailability of DU-176b (approximately 50%) was significantly higher than that of DX-9065a (10%) in monkeys. The antithrombotic efficacy of DU-176b was examined by oral administration to rats 30 minutes prior to thrombogenic stimuli. In a venous stasis thrombosis model, DU-176b (0.5 – 12.5 mg/kg, p.o.) dose-dependently inhibited thrombus formation, prolonged PT, and revealed plasma anti-Xa activity. DU-176b also exerted significant anticoagulant effect in a rat model of tissue factor-induced disseminated intravascular coagulation at doses of 0.1 – 2.5 mg/kg, p.o. These results demonstrate that DU-176b is a potent and selective factor Xa inhibitor that possesses antithrombotic effect after oral administration. DU-176b has the potential to be clinically useful for prophylaxis and treatment of several thrombotic diseases.

Rebound like recurrent thrombotic events are concerns about anticoagulant therapies. Withdrawal of heparins and a direct thrombin inhibitor is reported to be associated with evidence of rebound coagulation phenomenon in patients with coronary artery diseases (Ref 1). Previously we have shown that low-dose administration of a direct thrombin inhibitor, melagatran, enhances coagulation induced by tissue factor (TF) in rats (Ref 2). Objectives: To determine whether anticoagulants enhance thrombin generation in human plasma, and whether the negative-feedback system [thrombin-thrombomodulin (TM)-protein C] contributes to the enhancement. Methods: Thrombin generation in pooled human plasma was assayed by means of the calibrated automated thrombography (CAT) with the thrombinoscope software in vitro. Thrombin generation was induced by 2.5 pM tissue factor (TF) and 4 μM phospholipids. The effects of following anticoagulants were assessed: antithrombin (AT)-independent thrombin inhibitors [melagatran, recombinant hirudin (lepirudin), and active site blocked thrombin (IIai)], AT-dependent anticoagulants (heparin, dalteparin, and fondaparinux), and AT-independent FXa inhibitors (DU-176b and DX-9065a). Results: Melagatran, lepirudin, and IIai increased peak levels of thrombin generation in the presence of 8 nM recombinant human soluble TM. The effects reached maximal at 200 nM of melagatran (2.3-fold), 8.95 nM of lepirudin (1.6-fold), and 405 nM of IIai (2.2-fold). At higher concentrations, melagatran and lepirudin turned to suppress thrombin generation. Heparin, dalteparin, fondaparinux, DU-176b, and DX-9065a did not enhance thrombin generation, just exerted inhibitory effects. In the absence of TM, the enhancement by melagatran of peak thrombin generation was only 1.2-fold, suggesting the significant role of the negative-feedback system in this aggravation of thrombin generation. Since thrombin acts both the pro- and anti-coagulant, the inhibition of the negative-feedback system by these thrombin inhibitors may cause enhancement of thrombin generation. To test this hypothesis, we examined thrombin generation in protein C-deficient plasma. AT-independent thrombin inhibitors failed to enhance thrombin generation in protein C-deficient plasma. Conclusions: These results indicate that AT-independent thrombin inhibitors at low concentrations enhance thrombin generation probably due to suppression of the negative feedback system by inhibiting protein C activation. This in vitro aggravation of thrombin generation may be a possible explanation of hypercoagulation by melagatran in a rat model of TF-induced intravascular coagulation. Furthermore this phenomenon would contribute to clinical rebound like recurrent thrombotic events associated with anticoagulant therapies with these inhibitors. In contrast, AT-independent FXa inhibitors like DU-176b are less prone to induce the rebound because of lack of increase in thrombin generation.

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