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We compared the antithrombotic properties of a factor Xa inhibitor (DX-9065a) with those of a thrombin inhibitor (melagatran) in a rat disseminated intravascular coagulation model and a rat venous thrombosis model. Rat disseminated intravascular coagulation and venous thrombosis models were produced by injection of tissue factor and platinum wire placement, respectively. DX-9065a exerted antithrombotic effects dose dependently in both models. Melagatran was also effective in the venous thrombosis model, whereas it showed an aggravation in the disseminated intravascular coagulation model at low but not high doses. In the in vitro study, DX-9065a decreased the C max of the thrombin generation curve in plasma irrespective of whether protein C was present or not. However, melagatran increased the C max at low concentrations when protein C was present. This increase was not detected in protein C-deficient plasma. These results suggest that, unlike DX-9065a, melagatran in low doses aggravates disseminated intravascular coagulation by increasing thrombin generation, which may be partly due to suppression of negative feedback by activated protein C.

Antithrombin (AT) is a major physiological inhibitor of coagulation factors, primarily inhibiting thrombin and factor Xa (FXa). Binding of heparin and its related pentasaccharides, fondaparinux, to AT dramatically accelerates inhibition of thrombin and FXa. Entire AT-dependency of heparins may result in decreased anticoagulant effects in patients with inherited or acquired AT deficiencies. Objectives: We have developed an orally active direct (i.e. AT-independent) FXa inhibitor, DU-176b. The objectives of this study were to examine the anticoagulant and antithrombotic effects of DU-176b, fondaparinux, and heparin in heterozygous AT deficient (AT+/−) mice (Refs 1, 2), and to determine the impact of AT deficiency on the efficacies of these anticoagulants. Methods: [In vitro study] Plasma obtained from wild type (AT+/+, C57BL/6J) and AT+/− mice were subjected to measurement of levels of AT antigen and activity. The anticoagulant effects on prothrombin time (PT) and activated partial thromboplastin time (APTT) was measured and the drug concentrations were calculated required to double the clotting time (CT2). [In vivo study] Male AT+/+ and AT+/− mice were fasted over night. Thrombosis was induced in the inferior vena cava by applying filter paper (1 x 5 mm) presoaked in 15% FeCl3 for 10 min. Thrombus was removed 60 min after FeCl3 treatment and its protein content was assessed by Bradford method. DU-176b was orally administered 60 min before, fondaparinux was given s.c. 30 min before, and heparin was injected into the jugular vein 3 min before thrombus induction. Relative potencies of antithrombotic effects in AT+/− mice to those in AT+/+ mice were analyzed by parallel line assay. Results: [In vitro study] Plasma levels of AT antigen and activity in AT+/− mice were deceased to 40% compared with AT+/+ plasma. PT-CT2 of DU-176b was 0.72 μM in AT+/+ plasma and 0.74 μM in AT+/− plasma, respectively, indicating that anticoagulant activity of the direct FXa inhibitor was not affected by heterozygous AT deficiency. APTT-CT2 of fondaparinux and heparin in AT+/+ plasma was 3.8 μM and 14 mU/mL, respectively, whereas APTT-CT2 in AT+/− plasma was 9.2 μM and 20 mU/mL, respectively. Therefore, anticoagulant activities of such AT-dependent inhibitors were attenuated in AT+/− plasma. [In vivo study] All three anticoagulants inhibited venous thrombus formation of AT+/+ mice in dose-dependent manners. In AT+/− mice, the antithrombotic effects of fondaparinux and heparin were less potent than those in AT+/+ mice. In contrast, DU-176b prevented thrombus formation equipotently in both mice. Relative potencies of DU-176b, fondaparinux and heparin were 0.84, 0.40, and 0.70, respectively. Conclusion: DU-176b exerts a comparable antithrombotic effect even in individuals with low plasma AT antigens and activities. Thus, DU-176b may be prioritized over AT-dependent agents for use at the fixed dose in patients with lower plasma AT concentrations.

DU-176b is a novel potent, orally active and selective direct inhibitor of factor Xa (FXa). Direct FXa inhibitors have been reported to exert little effect on bleeding time at antithrombotic doses in animal studies. The aim of the present study was to compare the antithrombotic and hemorrhagic effects of DU-176b with unfractionated heparin (UFH), low molecular weight heparin (LMWH; dalteparin) and warfarin in rat models of thrombosis and hemorrhage. Rats were treated with DU-176b, UFH and LMWH by continuous intravenous infusion for 2 – 2.5 h, and with warfarin orally once daily for 4 days before thrombosis or hemorrhage. Thrombosis was induced by the insertion of a platinum wire into the inferior vena cava and left for 60 min. Tail template bleeding time was measured after an incision on the tail. DU-176b dose-dependently inhibited thrombus formation in the venous thrombosis model. The dose required for 50% inhibition (ED50) was 0.076 mg/kg/h. In contrast, the dose of DU-176b to double template bleeding time (BT2) was 0.75 mg/kg/h, indicating 10-fold dissociation of the doses of antithrombotic and hemorrhagic effects. UFH, LMWH and warfarin also prevented thrombus formation (ED50 = 56 U/kg/h, 66 U/kg/h and 0.16 mg/kg/day, respectively), but prolonged bleeding time at slightly higher doses (BT2 = 73 U/kg/h, 135 U/kg/h and 0.21 mg/kg/day, respectively) than the effective doses. The dissociation of the doses for these compounds was only 1.3, 2.0 and 1.3-fold, respectively. Moreover, the slope of dose-antithrombotic response curve of DU-176b was significantly slighter than those of UFH, LMWH and warfarin, indicating that the therapeutic dose range of DU-176b would be wider than those of the other anticoagulants. These results suggest that direct and selective inhibition of FXa by DU-176b is preferable for the treatment of thrombotic diseases in the aspect of lack of compromising primary hemostasis.

Factor Xa (FXa) is an attractive target for the treatment of thrombosis due to its crucial role in the blood coagulation cascade. Fondaparinux, a selective FXa inhibitor, has been approved for clinical use to prevent deep vein thrombosis after orthopedic surgery; however, it requires antithrombin (AT) to exert its antithrombotic effect. It is reported that AT dependent anticoagulants such as heparin are less effective to suppress platelet-rich arterial-type thrombus due to its inaccessibility to thrombus-bound FXa/thrombin. We have developed a potent direct (i.e. AT independent) FXa inhibitor, DU-176b. The objective of this study is to compare the antithrombotic properties of a direct selective FXa inhibitor, DU-176b, with an AT dependent selective FXa inhibitor, fondaparinux. We evaluated the antithrombotic effects of DU-176b and fondaparinux in rat models of arterial and venous thrombosis. The arterial and venous thrombosis was induced by topical application of ferric chloride to the carotid artery and by insertion of a platinum wire into the inferior vena cava, respectively. DU-176b (0.05 – 1.25 mg/kg/h) and fondaparinux (1 – 10 mg/kg/h for arterial thrombosis and 0.03 – 1 mg/kg/h for venous thrombosis) were intravenously administered as continuous infusions. DU-176b prevented both arterial and venous thrombosis in the same dose range. In contrast, the effective doses of fondaparinux markedly differed between these models. A higher dose of fondaparinux more than 100 times was required to inhibit arterial thrombosis compared with venous thrombosis. These results suggest that direct inhibition of FXa is a preferable strategy to AT dependent inhibition for the prevention of thrombus formation in the arteries.

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.