Effects of fondaparinux and a direct factor Xa inhibitor TAK-442 on platelet-associated prothrombinase in the balloon-injured artery of rats.

Endothelial damage triggers platelet adhesion and platelet-associated prothrombinase formation at the point of injury, resulting in the progression of thrombus formation. The present study compared the inhibitory effects of fondaparinux, an indirect factor Xa (FXa) inhibitor, and TAK-442, a direct FXa inhibitor, on platelet-associated prothrombinase activity in the balloon-injured rat artery. TAK-442 and fondaparinux inhibited endogenous FXa activity in platelet-poor human [half-maximal inhibitory concentration (IC(50)): 53 nM, TAK-442; 11 nM, fondaparinux] and rat (IC(50): 32 nM, TAK-442; 19 nM, fondaparinux) plasma. TAK-442 inhibited in vitro reconstituted human prothrombinase (system included FXa, calcium, and washed platelets) with an IC(50) value of 51 nM, whereas fondaparinux exhibited only weak inhibition (IC(50): 1700 nM). In a rat model of balloon injury, thrombin activity on the surface of injured vessels increased to 3.2-, 22-, and 5.8-fold the activity on the surface of the intact aorta at 5 minutes, 1 hour, and 24 hours after the injury, respectively. At approximately 1 hour after the injury, TAK-442 blocked platelet-associated thrombin generation on the surface of injured aortas with an IC(50) value of 19 nM, whereas fondaparinux showed no significant inhibition at the highest concentration tested (IC(50): >300 nM). These results suggest a possible limitation of fondaparinux in inhibiting platelet-associated prothrombinase activity and resultant thrombus formation as compared with TAK-442.

Differential effects of TAK-442, a novel orally active direct factor Xa inhibitor, and ximelagatran, a thrombin inhibitor, on factor V-mediated feedback on coagulation cascade and bleeding.

Thrombin amplifies the blood coagulation via factor V (FV)-mediated positive feedback loop. We hypothesised that factor Xa (FXa) inhibitors would interfere more gradually with this feedback activation loop than thrombin inhibitors, thereby achieving a better balance between haemostasis and prevention of thrombosis. In this study, we compared the effects of TAK-442, a novel FXa inhibitor, versus ximelagatran, a thrombin inhibitor, on FV-mediated positive feedback, venous thrombosis and bleeding. In normal plasma, TAK-442 delayed the onset of tissue factor-induced thrombin generation and prolonged prothrombin time (PT) with more gradual concentration-response curve than melagatran, the active form of ximelagatran. The effect of melagatran on the onset of thrombin generation decreased in an FVa-concentration-dependent manner in FV-deficient plasma supplemented with FVa. Furthermore, in FV-deficient plasma, the PT-prolonging potency of melagatran was markedly increased with a change in its concentration-response curve from steep to gradual. In the rat venous thrombosis model, TAK-442 (10 mg/kg, p.o.) prevented thrombus formation by 55% with 1.2 times prolongation of PT; a similar effect was observed in ximelagatran-treated (3 mg/kg, p.o.) rats. TAK-442 at 100 mg/kg prolonged PT by only 2.1 times with no change in bleeding time (BT), whereas ximelagatran at 10 mg/kg prolonged PT by 3.9 times and significantly increased BT. These results suggest that the differential effects of the two agents on FV-mediated amplification of thrombin generation may underlie the observation of a wider therapeutic window for TAK-442 than for ximelagatran.

Synergistic effect of a factor Xa inhibitor, TAK-442, and antiplatelet agents on whole blood coagulation and arterial thrombosis in rats.

INTRODUCTION: Activated platelets facilitate blood coagulation by providing factor V and a procoagulant surface for prothrombinase. Here, we investigated the potential synergy of a potent factor Xa/prothrombinase inhibitor, TAK-442, plus aspirin or clopidogrel in preventing arterial thrombosis and whole blood coagulation. METHODS: Thrombus formation was initiated by FeCl(3)-induced rat carotid injury. Bleeding time was evaluated with the rat tail transection model. Whole blood coagulation was assessed by thromboelastographic examination (TEG) for which blood obtained from control, aspirin-, or clopidogrel-treated rats was transferred to a TEG analyzer containing, collagen or adenosine diphosphate (ADP), and TAK-442 or vehicle. RESULTS: TAK-442 (3mg/kg, po), aspirin (100mg/kg, po) or clopidogrel (3mg/kg, po) alone had no significant effect on thrombus formation, whereas the combination of TAK-442 with aspirin and clopidogrel remarkably prolonged the time to thrombus formation without additional significant prolongation of bleeding time. TEG demonstrated that the onset of collagen-induced blood coagulation were slightly longer in aspirin-treated rats than control; however, when the blood from aspirin-treated rats was subsequently treated in vitro with 100 nM TAK-442, the onset of clotting was significantly prolonged. In contrast, only marginal prolongation was observed with TAK-442 treatment of blood from control animals. The onset time of ADP-induced blood coagulation was slightly longer in clopidogrel-treated rats compared with control, and it was further extended by TAK-442 treatment. CONCLUSION: These results demonstrate that blood coagulation can be markedly delayed by the addition of TAK-442 to antiplatelets treatment which could contribute to synergistic antithrombotic efficacy in these settings.

Antithrombotic and anticoagulant profiles of TAK-442, a novel factor Xa inhibitor, in a rabbit model of venous thrombosis.

The anticoagulant and antithrombotic profiles of TAK-442, a direct factor Xa (FXa) inhibitor, were investigated. TAK-442 showed potent inhibition of human FXa (Ki = 1.8 nM) and high specificity, with a 440-fold greater selectivity than thrombin and negligible effects on trypsin, plasmin, and tissue plasminogen activator (K(i) > 30 microM). [corrected] In human plasma, TAK-442 doubled FXa-induced clotting time, prothrombin time (PT), and activated partial thromboplastin time at 0.19, 0.55, and 0.59 microM, respectively. The relative PT-prolonging potencies of TAK-442, rivaroxaban, and apixaban were 1, 2.0-2.6, and 0.46-1.3, respectively, in 4 different PT reagents. In a rabbit model of venous thrombosis, 50- and 100-micrograms/kg [corrected] TAK-442 (intravenous bolus followed by 1-hour infusion) reduced thrombus formation by 50% and 81%, with plasma anti-FXa activity of 23%-26% and 34%-38%, respectively, and only marginal prolongation of PT and activated partial thromboplastin time. Melagatran, a thrombin inhibitor, showed similar antithrombotic activity to TAK-442. However, 500-micrograms/kg [corrected TAK-442 did not affect bleeding time (BT), whereas the same dose of melagatran significantly prolonged BT by 3.6-fold compared with vehicle control. These findings suggest that TAK-442 has similar antithrombotic effects as melagatran but does not cause BT prolongation, and plasma anti-FXa activity may reliably predict its potency.

Discovery of a tetrahydropyrimidin-2(1H)-one derivative (TAK-442) as a potent, selective, and orally active factor Xa inhibitor.

Coagulation enzyme factor Xa (FXa) is a particularly promising target for the development of new anticoagulant agents. We previously reported the imidazo[1,5-c]imidazol-3-one derivative 1 as a potent and orally active FXa inhibitor. However, it was found that 1 predominantly undergoes hydrolysis upon incubation with human liver microsomes, and the human specific metabolic pathway made it difficult to predict the human pharmacokinetics. To address this issue, our synthetic efforts were focused on modification of the imidazo[1,5-c]imidazol-3-one moiety of the active metabolite 3a, derived from 1, which resulted in the discovery of the tetrahydropyrimidin-2(1H)-one derivative 5k as a highly potent and selective FXa inhibitor. Compound 5k showed no detectable amide bond cleavage in human liver microsomes, exhibited a good pharmacokinetic profile in monkeys, and had a potent antithrombotic efficacy in a rabbit model without prolongation of bleeding time. Compound 5k is currently under clinical development with the code name TAK-442.

Discovery of imidazo[1,5-c]imidazol-3-ones: weakly basic, orally active factor Xa inhibitors.

The coagulation enzyme factor Xa (FXa) has been recognized as a promising target for the development of new antithrombotic agents. We previously found compound 1 to be an orally bioavailable FXa inhibitor in fasted monkeys; however, 1 showed poor bioavailability in rats and fed monkeys. To work out the pharmacokinetic problems, we focused our synthetic efforts on the chemical conversion of the 4-(imidazo[1,2- a]pyridin-5-yl)piperazine moiety of 1 to imidazolylpiperidine derivatives (fused and nonfused), which resulted in the discovery of the weakly basic imidazo[1,5- c]imidazol-3-one 3q as a potent and selective FXa inhibitor. Compound 3q showed favorable oral bioavailability in rats and monkeys under both fasted and fed conditions and antithrombotic efficacy in a rat model of venous thrombosis after oral administration, without a significant increase in bleeding time (unlike warfarin). On the basis of these promising properties, compound 3q was selected for further evaluation.

Discovery of piperazinylimidazo[1,2-a]pyridines as novel S4 binding elements for orally active factor Xa inhibitors.

We have recently reported the discovery of orally active sulfonylalkylamide Factor Xa (FXa) inhibitors, as typified by compound 1 (FXa IC(50)=0.061 microM). Since the pyridylpiperidine moiety was not investigated in our previous study, we conducted detailed structure-activity relationship studies on this S4 binding element. This investigation led to the discovery of piperazinylimidazo[1,2-a]pyridine 2b as a novel and potent FXa inhibitor (FXa IC(50)=0.021 microM). Further modification resulted in the discovery of 2-hydroxymethylimidazo[1,2-a]pyridine 2e (FXa IC(50)=0.0090 microM), which was found to be a selective and orally bioavailable FXa inhibitor with reduced CYP3A4 inhibition.

Discovery of sulfonylalkylamides: A new class of orally active factor Xa inhibitors.

Factor Xa (FXa) is a trypsin-like serine protease involved in the coagulation cascade and has received great interest as a potential target for the development of new antithrombotic agents. Most of amidine-type FXa inhibitors reported have been found to show extremely poor oral bioavailability. Compound 1 is one of the first reported non-amidine type FXa inhibitors. To discover novel and orally active FXa inhibitors, we investigated flexible linear linkers between the 6-chloronaphthalene ring and the 1-(pyridin-4-yl)piperidine moiety of 1 and found the orally active sulfonylalkylamide 2f with an FXa IC(50) of 0.05 microM, comparable with that of 1. Further modification to reduce the CYP3A4 inhibitory activity of 2f resulted in the potent, selective, and orally active 2-methylpyridine analogue 2s (FXa IC(50) of 0.061 microM), for which the liability of CYP3A4 inhibition was significantly weakened compared to 2f. Compound 2s also showed long lasting anticoagulant activity in cynomolgus monkeys.