Yumiko Moritani

Biological properties of a specific Gαq/11 inhibitor, YM-254890, on platelet functions and thrombus formation under high-shear stress

1 The effects of YM‐254890, a specific Gαq/11 inhibitor, on platelet functions, thrombus formation under high‐shear rate condition and femoral artery thrombosis in cynomolgus monkeys were investigated. 2 YM‐254890 concentration dependently inhibited ADP‐induced intracellular Ca2+ elevation, with an IC50 value of 0.92±0.28 μM. 3 P‐selectin expression induced by ADP or thrombin receptor agonist peptide (TRAP) was strongly inhibited by YM‐254890, with IC50 values of 0.51±0.02 and 0.16±0.08 μM, respectively. 4 YM‐254890 had no effect on the binding of fibrinogen to purified GPIIb/IIIa, but strongly inhibited binding to TRAP‐stimulated washed platelets. 5 YM‐254890 completely inhibited platelet shape change induced by ADP, but not that induced by collagen, TRAP, arachidonic acid, U46619 or A23187. 6 YM‐254890 attenuated ADP‐, collagen‐, TRAP‐, arachidonic acid‐ and U46619‐induced platelet aggregation with IC50 values of <1 μM, whereas it had no effect on phorbol 12‐myristate 13‐acetate‐, ristocetin‐, thapsigargin‐ or A23187‐induced platelet aggregation. 7 High‐shear stress‐induced platelet aggregation and platelet‐rich thrombus formation on a collagen surface under high‐shear flow conditions were concentration dependently inhibited by YM‐254890. 8 The antithrombotic effect of YM‐254890 was evaluated in a model of cyclic flow reductions in the femoral artery of cynomolgus monkeys. The intravenous bolus injection of YM‐254890 dose dependently inhibited recurrent thrombosis without affecting systemic blood pressure or prolonging template bleeding time. 9 YM‐254890 is a useful tool for investigating Gαq/11‐coupled receptor signaling and the physiological roles of Gαq/11.

Biochemical and pharmacological profile of darexaban, an oral direct factor Xa inhibitor.

Darexaban (YM150) is an oral factor Xa inhibitor developed for the prophylaxis of venous and arterial thromboembolic disease. This study was conducted to investigate the biochemical and pharmacological profiles of darexaban and its active metabolite darexaban glucuronide (YM-222714), which predominantly determines the antithrombotic effect after oral administration of darexaban. In vitro activity was evaluated by enzyme and coagulation assays, and a prothrombin activation assay using reconstituted prothrombinase or whole blood clot. In vivo effects were examined in venous thrombosis, arterio-venous (A-V) shunt thrombosis, and bleeding models in rats. Both darexaban and darexaban glucuronide competitively and selectively inhibited human factor Xa with Ki values of 0.031 and 0.020 μM, respectively. They showed anticoagulant activity in human plasma, with doubling concentrations of darexaban and darexaban glucuronide for prothrombin time of 1.2 and 0.95 μM, respectively. Anticoagulant activity was independent of antithrombin. Darexaban and darexaban glucuronide inhibited the prothrombin activation induced by prothrombinase complex or whole blood clot with similar potency to free factor Xa. In contrast, prothrombinase- and clot-induced prothrombin activation were resistant to inhibition by enoxaparin. In venous and A-V shunt thrombosis models in rats, darexaban strongly suppressed thrombus formation without affecting bleeding time, with ID₅₀ values of 0.97 and 16.7 mg/kg, respectively. Warfarin also suppressed thrombus formation in these models, but caused a marked prolongation of bleeding time at antithrombotic dose. In conclusion, darexaban is a selective and direct factor Xa inhibitor and a promising oral anticoagulant for the prophylaxis and treatment of thromboembolic diseases.

Pharmacological properties of YM-254890, a specific G α q/11 inhibitor, on thrombosis and neointima formation in mice

The pharmacological properties of YM-254890, a specific G(alpha)q/11 inhibitor, on acute thrombosis and chronic neointima formation after vascular injury have been investigated. FeCl3 was used to induce vascular injury in the carotid artery of mice. For the thrombosis studies, the test drug was either intravenously or orally administered before vascular injury. For the neointima studies, the test drug was orally administered 1 h before and twice daily for 1 week after vascular injury. Histological analysis was then performed 3 weeks later. YM-254890 significantly inhibited ex vivo platelet aggregation 5 min after intravenous bolus injection at 0.03 mg/kg or more, and 1 h after oral administration at 1 mg/kg. YM-254890 significantly inhibited thrombus formation after intravenous bolus injection at 0.03 mg/kg as well as after oral administration at 1 mg/kg, but tail transection bleeding time was significantly prolonged at 0.1 mg/kg for intravenous injection and 3 mg/kg for oral administration. Furthermore, oral administration of YM-254890 dose-dependently inhibited neointima formation 3 weeks after vascular injury with significant effects at 1 mg/kg twice daily for 1 week. Clopidogrel also significantly inhibited neointima formation at its antithrombotic dose, but its inhibitory potency was less than that of YM-254890. However, YM-254890 significantly reduced systemic blood pressure at doses 3 times higher than those that produced significant inhibitory effects on thrombosis and neointima formation. Though the systemic use of YM-254890 may be limited, owing to its narrow therapeutic window, this unique compound is a useful research tool for investigating the physiological roles of G(alpha)q/11 .

Discovery of N-[2-Hydroxy-6-(4-methoxybenzamido)phenyl]-4- (4-methyl-1,4-diazepan-1-yl)benzamide (Darexaban, YM150) as a Potent and Orally Available Factor Xa Inhibitor

Inhibitors of factor Xa (FXa), a crucial serine protease in the coagulation cascade, have attracted a great deal of attention as a target for developing antithrombotic agents. We previously reported findings from our optimization study of a high-throughput screening (HTS) derived lead compound 1a that resulted in the discovery of potent amidine-containing FXa inhibitors represented by compound 2. We also conducted an alternative optimization study of 1a without incorporating a strong basic amidine group, which generally has an adverse effect on the pharmacokinetic profile after oral administration. Replacement of 4-methoxybenzene with a 1,4-benzodiazepine structure and introduction of a hydroxy group at the central benzene led to the discovery of the potent and orally effective factor Xa inhibitor 14i (darexaban, YM150). Subsequent extensive study revealed a unique aspect to the pharmacokinetic profile of this compound, wherein the hydroxy moiety of 14i is rapidly transformed into its glucuronide conjugate 16 (YM-222714) as an active metabolite after oral administration and it plays a major role in expression of potent anticoagulant activity in plasma. The distinctive, potent activity of inhibitor 14i after oral dosing was explained by this unique pharmacokinetic profile and its favorable membrane permeability. Compound 14i is currently undergoing clinical development for prevention and treatment of thromboembolic diseases.

Antithrombotic and thrombolytic efficacy of YM-254890, a Gq/11 inhibitor, in a rat model of arterial thrombosis

We examined the antithrombotic and thrombolytic effects of the G(q/11) inhibitor YM-254890 in an electrically-induced carotid artery thrombosis model in rats. YM-254890 dose-dependently inhibited ex vivo ADP-induced platelet aggregation after i.v. bolus injection. In the thrombosis study, YM-254890 dosedependently prolonged time to occlusion at doses of 3 and 10 g/kg i.v. and decreased occlusion rate at 10 g/kg i.v. In the thrombolysis study, YM-254890 at 30 micro g/kg i.v. shortened the time to reperfusion and prevented reocclusion after thrombolysis with a modified tissue-type plasminogen activator. YM-254890, at 10 micro g/kg and more, significantly improved carotid patency status after thrombolysis. However, at 30 micro g/kg and more, YM-254890 decreased systemic blood pressure. These results suggest that YM-254890 may be effective for treating G(q)-mediated diseases, and that YM-254890 is a useful tool for investigating the biological roles of G(q/11).

Combined effects of a factor Xa inhibitor YM466 and a GPIIb/IIIa antagonist YM128 on thrombosis and neointima formation in mice

Thrombosis and neointima formation limit the efficacy of coronary angioplasty. Factor Xa inhibitors and GPIIb/IIIa antagonists have shown to be effective on acute thrombosis and late neointima formation, however, their combined effects remain to be elucidated. Vascular injury was induced by FeCl(3) in the carotid artery in mice. For thrombosis studies, the test drug was orally administered 1 hour before vascular injury. For neointima studies, the test drug was orally administered 1 hour before and twice daily for 1 week after vascular injury, and then histological analysis was performed 3 weeks after vascular injury. YM466 inhibited thrombotic occlusion at 30 mg/kg with prolongation of prothrombin time (PT), and tail transection bleeding time (BT) was affected at 100 mg/kg. YM466 also inhibited neointima formation at 10 mg/kg. YM128 inhibited thrombotic occlusion and neointima formation at 10 and 30 mg/kg, respectively, with inhibition of platelet aggregation and prolongation of BT. In contrast, the combination of 10 mg/kg YM466 and 3 mg/kg YM128 inhibited thrombotic occlusion and neointima formation without affecting PT, platelet aggregation and BT. Concomitant inhibition of factor Xa and GPIIb/IIIa may provide a safer and more effective therapeutic regimen for treatment of coronary angioplasty.

Pharmacological properties of YM-57029, a novel platelet glycoprotein IIb/IIIa antagonist

The pharmacological properties of YM-57029 [4-[4-(4-carbamimidoylphenyl)-3-oxopiperazin-1-yl]piperidino]acetic acid monohydrochloride trihydrate), a novel glycoprotein IIb/IIIa antagonist were examined in this study. YM-57029 inhibited fibrinogen binding to purified glycoprotein IIb/IIIa, 1000-fold more potently than the tetrapeptide arginine-glycine-aspartic acid-serine (RGDS). YM-57029 concentration-dependently inhibited ADP-, collagen- and high shear stress-induced platelet aggregation, strongly inhibited ATP release from platelets activated by ADP, and enhanced deaggregation of ADP-induced platelet aggregates. In a pro-aggregatory activity study, RGDS or SC-54701A ((S)-3-[3-[(4-amidinophenyl)carbamoyl]propionamido]-4-pentynoic acid monohydrochloride) caused prominent small aggregate formation. At a higher concentration, RGDS induced medium and large size aggregates, and SC-54701A induced medium aggregates. In contrast, YM-57029 produced only a few small and no larger size aggregates. Ex vivo ADP-induced platelet aggregation and platelet retention to collagen-coated plastic beads were dose-dependently inhibited by YM-57029 after intravenous bolus injection in guinea pigs. YM-57029 produced dose-dependent antithrombotic effects in carotid artery thrombosis and arterio-venous shunt thrombosis models in guinea pigs at 10 and 30 microg/kg, respectively. At these doses, YM-57029 prolonged template bleeding time. These results suggest that YM-57029 is a potent glycoprotein IIb/IIIa antagonist which has less pro-aggregatory effect. It may be a promising antiplatelet agent for thromboembolic diseases, and a good prototype for developing an orally active compound.

Inhibitory effects of ASP6537, a selective cyclooxygenase-1 inhibitor, on thrombosis and neointima formation in rats

INTRODUCTION Percutaneous coronary interventions (PCIs), such as balloon angioplasty and stent placement, are effective in the treatment of coronary artery disease. PCI has drawbacks, however, including acute thrombosis after the procedure and restenosis of the vascular lumen due to abnormal neointimal hyperplasia. ASP6537 is a selective COX-1 inhibitor that has been investigated as a possible candidate for clinical development as an antiplatelet agent. In the present study, we evaluated the in vivo antithrombotic effect of ASP6537 and its effect on neointima formation after balloon angioplasty. MATERIAL AND METHODS The antithrombotic effect of ASP6537 was examined using an arteriovenous shunt thrombosis model in rats while the effect of ASP6537 on neointima formation was evaluated in a rat carotid arterial balloon angioplasty model. RESULTS In the thrombosis study, ASP6537 dose-dependently decreased the protein content of the thrombus, while no prolongation of template bleeding time was observed. In the neointima study, ASP6537 reduced neointima formation. CONCLUSIONS ASP6537 may be a promising agent for the prevention of acute thrombosis and restenosis after PCI in place of aspirin.