Fukushi Hirayama

Antithrombotic effects of YM‐60828, a newly synthesized factor Xa inhibitor, in rat thrombosis models and its effects on bleeding time

The effects of YM‐60828, a newly synthesized factor Xa inhibitor, were investigated to analyse the relationship between its antithrombotic effects and its prolongation of template bleeding time in rats. YM‐60828 was compared with argatroban, heparin and dalteparin. All agents were intravenously administered as a bolus. In ex vivo studies, YM‐60828 and argatroban prolonged both prothrombin time and activated partial thromboplastin time in a dose‐dependent manner, while heparin and dalteparin prolonged only activated partial thromboplastin time. In a venous thrombosis model, all agents exerted antithrombotic effects in a dose‐dependent manner. The ID50 values of YM‐60828, argatroban, heparin and dalteparin were 0.0081 mg kg−1, 0.011 mg kg−1, 6.3 iu kg−1 and 4.7 iu kg−1, respectively. In an arterio‐venous shunt model, all agents exerted antithrombotic effects in a dose‐dependent manner. The ID50 values of YM‐60828, argatroban, heparin and dalteparin were 0.010 mg kg−1, 0.011 mg kg−1, 10 iu kg−1 and 4.2  iu  kg−1, respectively. In bleeding time studies, all agents prolonged template bleeding time in a dose‐dependent manner. ED2 values, the doses causing a 2 fold prolongation of bleeding time in the saline group, of YM‐60828, argatroban, heparin and dalteparin were 0.76 mg kg−1, 0.081 mg kg−1, 18 iu kg−1 and 25 iu kg−1, respectively. The ratio (ED2/ID50) of YM‐60828 was more than 30 fold greater than that of heparin and more than 10 fold greater than those of argatroban and dalteparin. These data show that YM‐60828 can exert its antithrombotic effects with little prolongation of bleeding time compared with the other currently used anticoagulant agents.

YM-60828, a novel factor Xa inhibitor: Separation of its antithrombotic effects from its prolongation of bleeding time

The antithrombotic effects of intravenous infusions of YM-60828 ([N-[4-[(1-acetimidoyl-4-piperidyl)oxy]phenyl]-N-[(7-amidino-2-nap hthyl)methyl]sulfamoyl]acetic acid dihydrochloride), a novel factor Xa inhibitor, argatroban, heparin and dalteparin in an arterio-venous shunt model were studied in comparison with their effects on template bleeding time. In an arterio-venous shunt model, all agents exerted antithrombotic effects in a dose-dependent manner. ID50 values of YM-60828, argatroban, heparin and dalteparin were 0.0087 mg/kg/h. 0.027 mg/kg/h, 22 IU/kg/h and 11 IU/kg/h, respectively. In bleeding time studies, all agents prolonged bleeding time in a dose-dependent manner. Doses (ED2) of YM-60828, argatroban, heparin and dalteparin, which caused 2-fold prolongation of bleeding time in the saline group, were 3.0 mg/kg/h, 0.25 mg/kg/h, 18 IU/kg/h and 26 IU/kg/h. respectively. The risk-benefit ratio (ED2/ID50) of YM-60828 was much greater than that of the other agents. These data suggest that the antithrombotic effect of YM-60828 is separate from its prolongation of bleeding time and that YM-60828 is much safer than conventional anticoagulant agents.

Relationship between the antithrombotic effect of YM-75466, a novel factor Xa inhibitor, and coagulation parameters in rats

The relationship between the antithrombotic effects of intravenous infusions of YM-75466 [N-[4-[(1-acetimidoyl-4-piperidyl)oxy]phenyl]-N-[(7-amidino-2-naph thyl)methyl] sulfamoyl]acetic acid monomethanesulfonate), a novel factor Xa (FXa) inhibitor, and various coagulation parameters (prothrombin time, activated partial thromboplastin time, thrombin-antithrombin III complex (TAT), anti-FXa activity and anti-thrombin activity) in rats was studied and compared with results for heparin. In the arterio-venous shunt model, both agents exerted antithrombotic effects in a dose-dependent manner. Coagulation parameters were studied simultaneously with antithrombotic effects. YM-75466 did not prolong coagulation time even at the dose which exerted significant antithrombotic effects, while it decreased TAT level in plasma in a dose-dependent manner. YM-75466 exerted anti-FXa activity but not anti-thrombin activity. In contrast, heparin prolonged activated partial thromboplastin time in a dose-dependent manner and decreased TAT level in plasma with increasing inhibition of thrombus formation. Heparin exerted both anti-FXa and anti-thrombin activity in a dose-dependent manner. These results suggest that TAT is a suitable parameter for monitoring the antithrombotic effect of YM-75466 in the arterio-venous shunt model in rats and that YM-75466, unlike heparin, exerts its antithrombotic effect through specific inhibition of FXa without any effect on thrombin.

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.

The discovery of YM-60828: a potent, selective and orally-bioavailable factor Xa inhibitor.

Since Factor Xa (FXa) is well known to play a central role in thrombosis and hemostasis, inhibition of FXa is an attractive target for antithrombotic strategies. As a part of our investigation of a non-peptide, orally available FXa inhibitor, we found that a series of N-[(7-amidino-2-naphthyl)methyl]aniline derivatives possessed potent and selective inhibitory activities. Structure--activity relationship (SAR) of the substituent (R(1)) on the central aniline moiety suggested that increasing lipophilicity caused a detrimental effect on anticoagulant activity (prothrombin time assay) in plasma. Several compounds bearing a hydrophilic substituent in R(1) showed not only potent FXa inhibitory activities but also high anticoagulant activities. The best compound in this series was sulfamoylacetic acid derivative (YM-60828) which was a potent, selective and orally bioavailable FXa inhibitor and was chosen for clinical development.

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.

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 effect of YM-75466 is separated from its effect on bleeding time and coagulation time

The antithrombotic effects of YM-75466 ([N-[4-[(1-acetimidoyl-4-piperidyl)oxy]phenyl]-N-[(7-amidino-2-nap hthyl)methyl]sulfamoyl]acetic acid monomethane sulfonate), a novel orally-active factor Xa inhibitor, and its effects on bleeding time and coagulation time were studied in rats and compared with those of warfarin. Both agents were orally administered. In the venous thrombosis model, YM-75466 and warfarin inhibited thrombus formation dose-dependently, with ID50 values of 3.3 and 0.56 mg/kg, respectively. Ex vivo study showed that both YM-75466 and warfarin prolonged prothrombin time dose-dependently, with doses, causing a two-fold prolongation of prothrombin time in the control group, of 89 and 0.38 mg/kg, respectively. In bleeding time studies, YM-75466 and warfarin prolonged bleeding time dose-dependently, with doses, causing a two-fold prolongation of bleeding time in the control group, of > 100 and 0.43 mg/kg, respectively. These results show that the antithrombotic effects of YM-75466 are markedly separate from its effects on bleeding time and coagulation time compared with warfarin.

Antithrombotic effects of YM-60828 in three thrombosis models in guinea pigs.

The antithrombotic effects of a novel factor Xa inhibitor, YM-60828 ([N-[4-[(1-acetimidoyl-4-piperidyl)oxy]phenyl]-N-[(7-amidino-2-nap hthyl)methyl]sulfamoyl]acetic acid dihydrochloride), in three thrombosis models in guinea pigs were studied in comparison with its effect on bleeding time. The antithrombotic effects of YM-60828 were most pronounced in the venous thrombosis and the arterio-venous shunt models but YM-60828 showed 10-fold weaker effects in the carotid thrombosis model. However, YM-60828 prolonged bleeding time at a much higher dose than that required in all thrombosis models. In conclusion, YM-60828 exerted its antithrombotic effects without prolonging bleeding time in all thrombosis models and may be of clinical value not only in venous thrombosis but also in arterial thrombosis.

Design, synthesis and biological activity of YM-60828 derivatives: potent and orally-bioavailable factor Xa inhibitors based on naphthoanilide and naphthalensulfonanilide templates.

Factor Xa (FXa) is a serine protease which plays a pivotal role in the coagulation cascade. The inhibition of FXa has received great interest as a potential target for the development of new antithrombotic drug. Herein we describe a series of novel 7-amidino-2-naphthoanilide and 7-amidino-2-naphthalensulfonanilide derivatives which are potent FXa inhibitors. These scaffolds are rigid and are allowed to adopt an L-shape conformation which was estimated as the active conformation based on a docking study of YM-60828 with FXa. Optimization of the side chain at the central aniline nitrogen of 7-amidino-2-naphthoanilide has led to several potent and orally active FXa inhibitors. 5h (YM-169964), the best compound of these series, showed potent FXa inhibitory activity (IC(50)=3.9nM) and effectively prolonged prothrombin time by 9.6-fold ex vivo at an oral dose of 3mg/kg in squirrel monkeys.