Yoshiyuki Iwatsuki

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.

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) 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.

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.

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.

Darexaban has high sensitivity in the prothrombin time clotting test

Most synthetic factor Xa (FXa) and thrombin inhibitors examined in pharmacokinetic studies and phase II/III clinical trials have had predictable pharmacokinetics and have been found to be effective and safe without the need for laboratory monitoring [1]. However, assessment of the degree of antithrombotic effect may be beneficial in certain patients, such as those with renal or liver impairment, or in emergency situations, although precisely how such assessments should be carried out is an open issue. Current pharmacodynamic markers for these agents include well-established clotting assays that measure prothrombin time (PT) and activated partial thromboplastin time (APTT), more sensitive methods that measure diluted PT (DPT), and specific enzyme assays for FXa inhibition and thrombin generation [1]. Changes in these markers correlate with the plasma concentration of the investigated inhibitor; however, their accuracy in predicting systemic exposure to inhibitors is unsatisfactory, owing to poor sensitivity or non-standardized procedures. Darexaban (YM150) is a novel, potent, oral direct FXa inhibitor that prevents the prothrombin activation induced by free and clot-associated FXa, prothrombinase, and whole blood clots [2]. After oral administration, darexaban is rapidly and almost entirely metabolized to darexaban glucuronide. Although darexaban and darexaban glucuronide are equipotent, darexaban glucuronide is the main active moiety in humans in vivo [3]. Consistent with this, a high correlation between darexaban glucuronide plasma concentration and PT prolongation has been observed, and relatively low doses of darexaban (20 mg twice-daily) result in a four-fold PT prolongation, indicating that PT is highly responsive to changes in darexaban glucuronide plasma concentrations [3]. Here, we compared the effects of darexaban on PT with those of other direct FXa inhibitors, using several thromboplastin reagents. We also examined the relationship between PT prolongation and FXa inhibition for each inhibitor. Human plasma was spiked with a 1/100 volume of vehicle, darexaban, darexaban glucuronide, rivaroxaban or apixaban to achieve different final concentrations, selected on the basis of reported clinical exposure concentrations during the treatment of venous thrombosis: approximately 0.3–3 lmol L (trough to peak concentrations) for darexaban glucuronide, and 0.03– 0.3 lmol L for rivaroxaban and apixaban [3–5]. The chromogenic assay for FXa activity in plasma was performed as described previously, with minor modifications [6]. PT was measured with three commercially available thromboplastin reagents, according to each manufacturer s protocol. The PT ratio (PTR) was calculated as the PT for the inhibitor-spiked plasma divided by the PT for the vehicle-spiked plasma. The local international sensitivity index values were determined to be 1.12, 0.91 and 0.84 for HemosIL PT-fibrinogen HS PLUS, PT-fibrinogen recombinant, andRecombiPlasTin, respectively. For theDPT test, thromboplastin reagents were diluted 16-fold as compared with conventional PT test quantities with 50 mmol L calcium chloride solution. All sample handling and FXa inhibitor-spiked plasma to vehicle-spiked plasma ratio calculations were otherwise similar to those applied in the conventional PT tests. Data are expressed as the mean ± standard error of the mean of four independent assays. ED50 values were calculated with a sigmoid-Emax model for the inhibition of FXa activity. Darexaban, darexaban glucuronide, rivaroxaban and apixaban inhibited FXa activity in human plasma in a concentration-dependent fashion, with typical Hill-shaped concentration–FXa curves, but different potencies (EC50 values and 95% confidence intervals: 3.4 [3.1–3.7], 1.1 [1.0–1.2], 0.12 [0.11–0.13], and 0.15 [0.14–0.15] lmol L, respectively). At clinical exposure concentrations, all tested FXa inhibitors prolonged the PT obtained when mixed with commercial thromboplastins (Fig. 1A). Furthermore, darexaban and its human metabolite darexaban glucuronide produced higher PTR values than rivaroxaban or apixaban, regardless of the thromboplastin reagent used. The relationship between PTR and FXa inhibition is shown in Fig. 1B. Interestingly, darexCorrespondence: Toshiyuki Funatsu, Applied Pharmacology Research Laboratories, Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba-shi, Ibaraki, Japan. Tel.: +81 3 5916 5329; fax: +81 3 5916 5606. E-mail: toshiyuki.funatsu@astellas.com

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.

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.