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.

Identification of potent orally active factor Xa inhibitors based on conjugation strategy and application of predictable fragment recommender system.

Anticoagulant agents have emerged as a promising class of therapeutic drugs for the treatment and prevention of arterial and venous thrombosis. We investigated a series of novel orally active factor Xa inhibitors designed using our previously reported conjugation strategy to boost oral anticoagulant effect. Structural optimization of anthranilamide derivative 3 as a lead compound with installation of phenolic hydroxyl group and extensive exploration of the P1 binding element led to the identification of 5-chloro-N-(5-chloro-2-pyridyl)-3-hydroxy-2-{[4-(4-methyl-1,4-diazepan-1-yl)benzoyl]amino}benzamide (33, AS1468240) as a potent factor Xa inhibitor with significant oral anticoagulant activity. We also reported a newly developed Free-Wilson-like fragment recommender system based on the integration of R-group decomposition with collaborative filtering for the structural optimization process.

Novel strategy to boost oral anticoagulant activity of blood coagulation enzyme inhibitors based on biotransformation into hydrophilic conjugates.

The blood coagulation cascade represents an attractive target for antithrombotic drug development, and recent studies have attempted to identify oral anticoagulants with inhibitory activity for enzymes in this cascade, with particular attention focused on thrombin and factor Xa (fXa) as typical targets. We previously described the discovery of the orally active fXa inhibitor darexaban (1) and reported a unique profile that compound 1 rapidly transformed into glucuronide YM-222714 (2) after oral administration. Here, we propose a novel strategy towards the discovery of an orally active anticoagulant that is based on the bioconversion of a non-amidine inhibitor into the corresponding conjugate to boost ex vivo anticoagulant activity via an increase in hydrophilicity. Computational molecular modeling was utilized to select a template scaffold and design a substitution point to install a potential functional group for conjugation. This strategy led to the identification of the phenol-derived fXa inhibitor ASP8102 (14), which demonstrated highly potent anticoagulant activity after biotransformation into the corresponding glucuronide (16) via oral dosing.

Darexaban: anticoagulant effects in mice and human plasma in vitro, antithrombotic effects in thrombosis and bleeding models in mice and effects of anti-inhibitor coagulant complex and recombinant factor VIIa.

Here, we investigated the anticoagulant effects of darexaban in mice and human plasma in vitro, effects of darexaban in thrombosis and bleeding models in mice, and reversal effects of anti-inhibitor coagulant complex (ACC) and recombinant factor VIIa (rFVIIa) on anticoagulant effects of darexaban. In mice, darexaban inhibited FXa activity in plasma with an ED50 value of 24.8 mg/kg. Both darexaban and warfarin prolonged prothrombin time (PT) at 3 mg/kg and 0.3 mg/kg/day, respectively. PT and activated partial thromboplastin time (aPTT) prolonged by darexaban were dose-dependently reversed by intravenously-administered rFVIIa, significantly so at 1 mg/kg. In a pulmonary thromboembolism (PE) mouse model, both darexaban and warfarin dose-dependently reduced the mortality rate, significantly so at 10 mg/kg and 3 mg/kg/day, respectively. In a FeCl3-induced venous thrombosis (VT) mouse model, darexaban (0.3-10 mg/kg) dose-dependently decreased the thrombus protein content, significantly so at doses of 3 mg/kg or higher. In a tail-transection mouse model, darexaban had no significant effect on the amount of blood loss at doses up to 10 mg/kg, while warfarin showed a dose-dependent increase in blood loss, significantly so from 1 mg/kg/day. Darexaban and its metabolite darexaban glucuronide significantly prolonged PT and aPTT in human plasma in vitro, and while rFVIIa concentration-dependently reversed the prolonged PT in this plasma, ACC dose-dependently reversed both PT and aPTT changes prolonged by darexaban. Taken together, these results suggest that darexaban has a potential to be an oral anticoagulant with a better safety profile than warfarin, and that rFVIIa and ACC may be useful as antidotes to darexaban in cases of overdose.

Antithrombotic and anticoagulant effects of direct factor Xa inhibitor darexaban in rat and rabbit models of venous thrombosis.

The oral direct factor Xa inhibitor darexaban administered intraduodenally prevented venous thrombus formation in both rats and rabbits with no effect on bleeding. The indirect parenteral Factor Xa inhibitor fondaparinux exerted similar properties, only prolonging bleeding time at extremely high doses. In contrast, the thrombin inhibitor ximelagatran and low-molecular-weight heparin enoxaparin prolonged bleeding time at antithrombotic doses. Studies using human platelets showed darexaban glucuronide, a darexaban metabolite that predominantly determines darexaban antithrombotic effects in vivo, had no effect on platelet activation and aggregation, while heparin and enoxaparin activated platelets. Melagatran, heparin, and enoxaparin all inhibited thrombin-induced platelet aggregation at clinically relevant concentrations. Taken together, these results suggest that thrombin-inhibiting drugs may increase the risk of bleeding, while darexaban may have potential as an orally available antithrombotic agent with a wide therapeutic window.

Plasma factor Xa inhibition can predict antithrombotic effects of oral direct factor Xa inhibitors in rabbit atherothrombosis models.

We evaluated the relationship between antithrombotic effects and pharmacodynamic (PD) marker changes produced by the novel factor (F)Xa inhibitors darexaban (YM150) and rivaroxaban in a rabbit model of plaque disruption-induced arterial thrombosis. Animals were subjected to catheter-induced endothelial denudation via the femoral artery followed by a two-week high-cholesterol diet. Plaque disruption was induced by balloon angioplasty, and then stasis was achieved by ligation at the distal side of the injured segment. Darexaban and rivaroxaban were administered orally 1 hour (h) before and 9 h after plaque disruption, and their antithrombotic effects were evaluated 24 h after the initiation of ligation. Prothrombin time (PT), activated partial thromboplastin time (APTT), and plasma FXa activity were measured using blood samples collected before and 1h after administration. Darexaban and rivaroxaban significantly reduced thrombus formation. The thrombus weight obtained in the 30 mg/kg darexaban group was comparable to that in the 1 mg/kg rivaroxaban group (2.17 ± 0.63 and 3.23 ± 1.64 mg, respectively, vs. 8.01 ± 1.08 mg in the control group). Plasma FXa activity correlated with the antithrombotic effects of darexaban and rivaroxaban, while PT only correlated with those of darexaban. Our findings suggest that the degree of plasma FXa inhibition may be useful for predicting antithrombotic effects of darexaban and rivaroxaban in arterial thrombosis. PT may also be useful in evaluating antithrombotic effects of darexaban in particular.

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.

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.

Renoprotective properties of pirfenidone in subtotally nephrectomized rats.

Renal fibrosis is the final common pathway of chronic kidney disease, and its progression predicts the degree of renal dysfunction. We investigated the renoprotective properties of pirfenidone in a remnant kidney model of chronic renal failure to determine its pharmacological potency compared to enalapril. Five-sixths nephrectomized rats were fed diet containing pirfenidone (approximately 700mg/kg/day) for 8weeks. Pirfenidone steadily inhibited the progression of proteinuria, but not to a significant degree. Pirfenidone prevented the elevation of plasma creatinine and blood urea nitrogen. At the end of the experiment, pirfenidone had reduced systolic blood pressure by means of its renoprotective effect. In a histological study, pirfenidone improved interstitial fibrosis in the renal cortex. These effects were supported by the suppression of the expression of TGF-beta and fibronectin in the mRNA of the kidney. In contrast, pirfenidone had little effect on the expression of alpha-smooth muscle actin, which is one of the proteins responsible for epithelial-mesenchymal transition. This property was confirmed by the TGF-beta-induced transdifferentiation observed in cultured normal rat kidney tubular epithelial NRK52E cells. These results suggest that pirfenidone improves the progression of chronic renal failure via its antifibrotic action, although pirfenidone has less effective TGF-beta-induced epithelial to mesenchymal transdifferentiation.

AKR-501 (YM477) a novel orally-active thrombopoietin receptor agonist.

Thrombopoietin (TPO) is the principal physiologic regulator of platelet production. We have searched for small molecule compounds that mimic the action of TPO by using human TPO receptor-expressed in Ba/F3 cells, resulting in the discovery of AKR-501 (YM477). AKR-501 specifically targeted the TPO receptor and stimulated megakaryocytopoiesis throughout the development and maturation of megakaryocytes just as rhTPO did. AKR-501, however, was shown to be effective only in humans and chimpanzees with high species specificity. Therefore, we examined the in vivo platelet-increasing effect of AKR-501 in human platelet producing non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice transplanted with human fetal liver CD34(+) cells. Daily oral administration of AKR-501 dose-dependently increased the number of human platelets in these mice, with significance achieved at doses of 1 mg/kg and above. The peak unbound plasma concentrations of AKR-501 after administration at 1 mg/kg in NOD/SCID mice were similar to those observed following administration of an active oral dose in human subjects. These results suggest that AKR-501 is an orally-active TPO receptor agonist that may be useful in the treatment of patients with thrombocytopenia.

AKR-501 (YM477) in combination with thrombopoietin enhances human megakaryocytopoiesis.

OBJECTIVE: AKR-501 (YM477) is an orally active thrombopoietin (TPO) receptor agonist that mimics the biological effect of TPO in vitro and in vivo. Here, we report that AKR-501 in combination with TPO has additive effect on megakaryocytopoiesis. MATERIALS AND METHODS: Granulocyte colony-stimulating factor-mobilized human peripheral blood CD34+ cells were cultured with AKR-501, TPO, or a combination of the two in serum-free liquid culture system. The numbers of hematopoietic progenitor cells, megakaryocytic progenitor cells, and megakaryocytes were measured using flow cytometry. Further, the effect of AKR-501 on TPO binding to TPO receptor was examined. RESULTS: Both AKR-501 and TPO alone increased the number of megakaryocytes, and the maximum activities of AKR-501 and TPO were similar. Interestingly, in the presence of TPO concentrations producing maximal stimulation, the addition of AKR-501 increased the number of megakaryocytes to about 200% of that generated with TPO only. In the time course experiment, the combination of AKR-501 and TPO augmented the numbers of hematopoietic progenitor cells and colony-forming unit in culture in the early stages. Thus, the combination of AKR-501 and TPO enhanced not only the differentiation into megakaryocytes, but also the expansion of human hematopoietic progenitor cells. Further, AKR-501 did not inhibit TPO binding to the TPO receptor. This result indicated the possibility that AKR-501 and TPO may act simultaneously on the TPO receptor, and this could be responsible for their additive effect of on megakaryocytopoiesis. CONCLUSIONS: This study suggests that AKR-501 would be useful for the treatment of thrombocytopenia even at high plasma levels of endogenous TPO following chemotherapy.

Experimental model of lower limb ischemia in rats and the effect of YM466, an oral direct factor Xa inhibitor.

A simple, quantitative, and reproducible model of lower limb ischemia was developed. Vascular injury was induced by ferric chloride (FeCl(3)) solution to the rat iliac artery, after which blood flow in all of the lower limbs were continuously monitored using a scanning laser Doppler blood flowmeter. After FeCl(3) injury, a distinct decrease in blood flow in the ischemic lower limb was observed and blood flow did not recover during the 30 min after vascular injury. YM466, an oral direct factor Xa inhibitor, dose-dependently inhibited the reduction of peripheral blood flow. The area under the blood flow-time curve during 30 min after vascular injury improved dose-dependently, with significance at doses of 3 and 10 mg/kg. These results suggest that factor Xa inhibitors are effective in patients with peripheral arterial disease, and that this vascular injury model is a useful tool for the screening and evaluation of the efficacy of new antithrombotic agents.

Prodrug-based design, synthesis, and biological evaluation of N-benzenesulfonylpiperidine derivatives as novel, orally active factor Xa inhibitors.

We describe here our investigation of a new series of orally active fXa inhibitors based on a prodrug strategy. Solid-phase parallel synthesis identified a unique series of fXa inhibitors with a substituted benzenesulfonyl group as a novel S4 binding element. This series resulted in compound 39, which exhibited potent inhibitory activity against fXa (IC50 = 13 nM) and excellent selectivity over thrombin (>7000-fold). The masking of its highly hydrophilic groups led to the creation of related prodrug 28, which demonstrated an anticoagulant effect after oral dosing.

Synthesis and biological activity of novel 1,2-disubstituted benzene derivatives as factor Xa inhibitors.

Factor Xa (fXa) is a serine protease that plays a pivotal role in the coagulation cascade. High-throughput screening of the Yamanouchi compound library yielded lead compound 1 with the ability to inhibit fXa at micromolar concentrations. To improve its fXa inhibitory activity and its oral anticoagulant activity, the linker between benzamidine and the central benzene ring was modified and a carboxyl group was introduced at the central benzene ring. The resulting compounds 40b (YM-203552), 41a (YM-202054), and 41c (YM-203558) exhibited potent fXa inhibitory activity and oral anticoagulant activity. In particular, YM-203558 exhibited the most potent oral anticoagulant activity, prolonging PT more than 3-fold at 0.5 and 2.0 h. Additionally, these compounds showed a high degree of selectivity for other serine proteases.

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.

Orally active factor Xa inhibitor: synthesis and biological activity of masked amidines as prodrugs of novel 1,4-diazepane derivatives.

Factor Xa (fXa) is a serine protease, which plays a pivotal role in the coagulation cascade. To improve the oral anticoagulant activity of fXa inhibitors containing a 1,4-diazepane moiety as the P4 part, a prodrug strategy was examined. Among the compounds evaluated in this study, amidoxime prodrugs bearing an ester moiety, such as compounds 21 and 30, showed effective oral anticoagulant activity in mice.

Rhythmical contractions in pulmonary arteries of monocrotaline-induced pulmonary hypertensive rats.

Rhythmical contractions accompanied by an increase in cytosolic Ca2+ concentrations were produced in ring preparations of endothelium-denuded pulmonary arteries from monocrotaline-treated rats, but not in those from vehicle-treated rats, 2-3 h after a resting tension of 15 mN (150-180% of the initial wall length of the artery) was applied. The rhythmical contractions were abolished by nicardipine and ryanodine. Cyclopiazonic acid reduced the relaxation phase of the rhythmical contractions, finally leading to a sustained contraction. Similarly, apamin caused a sustained contraction, whereas charybdotoxin increased the amplitude of the rhythmical contractions. Glibenclamide had no apparent effects on them. Indomethacin and the prostaglandin H2/thromboxane A2 receptor antagonist SQ29548 abolished the rhythmical contractions and reduced the tension, but the thromboxane synthase inhibitor ozagrel had no effect. These results suggest that optimal stretch induces rhythmical contractions in the pulmonary arteries of monocrotaline-induced pulmonary hypertensive rats, to which both Ca2+ influx through voltage-operated Ca2+ channels and Ca2+ release from the endoplasmic reticulum seem to contribute. It is also suggested that small-conductance Ca(2+)-activated K+ channels participate in the relaxation phase of rhythmical contractions. Furthermore, prostaglandin H2 released from nonendothelial cells is likely to play a pivotal role in the induction of rhythmical contractions.