Seiji Kaku

Flavocetin-A and -B, two high molecular mass glycoprotein Ib binding proteins with high affinity purified from Trimeresurus flavoviridis venom, inhibit platelet aggregation at high shear stress

Two high molecular mass proteins, flavocetin-A and flavocetin-B, were purified from Trimeresurus flavoviridis venom. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the apparent molecular mass of flavocetin-A and -B were 149 and 139 kDa, respectively, under nonreducing conditions. On reduction, flavocetin-A showed two distinct subunits (17 and 14 kDa), and flavocetin-B three distinct subunits (17, 15 and 14 kDa). At 1 microgram/ml, flavocetin-A and -B (flavocetins) inhibited the von Willebrand factor (vWF)-dependent aggregation of fixed human platelets. However, flavocetins (10 micrograms/ml) had no effect on ADP- and collagen-induced platelet aggregation in PRP. Flavocetins (3 micrograms/ml) also inhibited shear-induced platelet aggregation at high shear stress. Furthermore, flavocetin-A completely inhibited the aggregation of and ATP release from washed platelets stimulated with a low concentration of thrombin. Flavocetin-A specifically bound to platelet with high affinity (Kd = 0.35 +/- 0.13 nM) at 21,500 +/- 1760 binding sites per platelet. The N-terminal amino acid sequences of the subunits of flavocetin-A show a high degree of homology with those of echicetin, botrocetin, alboaggregin-B and factor IX/factor X-binding protein. These results suggest that flavocetins may be a useful tool for further investigation of the GPIb-vWF interaction.

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.

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.

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.

Effect of mirabegron, a novel β3-adrenoceptor agonist, on bladder function during storage phase in rats

Mirabegron, a selective β3-adrenoceptor agonist, facilitates urine storage function by exerting a relaxing effect on bladder smooth muscle. Here, we investigated the effect of mirabegron on bladder function during the storage phase. We assessed the effect of mirabegron on the resting intravesical pressure in anesthetized rats and also tested antimuscarinics (oxybutynin and tolterodine) under the same experimental conditions. Mirabegron dose-dependently decreased the resting intravesical pressure, while oxybutynin and tolterodine showed no statistically significant effects on resting intravesical pressure. We also investigated the effect of mirabegron on bladder function using cystometry technique in conscious rats with bladder outlet obstruction. While mirabegron dose-dependently decreased the frequency of nonvoiding contractions, considered an index of abnormal response in bladder storage, no significant effects were noted on the amplitude of nonvoiding contractions, micturition pressure, threshold pressure, voided volume, residual volume, or bladder capacity. Neither oxybutynin nor tolterodine affected the frequency of nonvoiding contractions; however, oxybutynin increased residual volume and tended to decrease voided volume in a dose-dependent manner, and tolterodine dose-dependently decreased voided volume. Taken together, these results shed light on the suggestion of mirabegron as a therapeutic agent, compared with antimuscarinics, with its most prominent effect being the facilitation of bladder storage.

Interleukin-11 acts synergistically with bone morphogenetic protein-2 to accelerate bone formation in a rat ectopic model

We previously demonstrated that recombinant human interleukin-11 (rHuIL-11) induced osteoblast differentiation of C3H10T1/2 progenitor cells and also acted synergistically with recombinant human bone morphogenetic protein-2 (rHuBMP-2) in performing the same function. In this study, we investigated the effect of rHuIL-11 and rHuBMP-2 on bone formation in a rat ectopic model. When placed in rats, implants consisting of polymer-coated gelatin sponges containing various concentrations of rHuBMP-2 showed a dose-dependent increase in calcium content. This was confirmed by radiographic analysis of the implants. Although implants containing rHuIL-11 alone did not accumulate calcium, implants containing a combination of rHuBMP-2 and rHuIL-11 had significantly higher calcium levels than those containing rHuBMP-2 alone. This increase was rHuIL-11 dose dependent. The synergistic effect of 20 micrograms rHuIL-11 and 6 micrograms rHuBMP-2 on bone formation was estimated to be 1 week in advance of that of 6 micrograms rHuBMP-2 alone. Histologic examination revealed that the combination of rHuIL-11 and rHuBMP-2 caused spindle cells to accumulate around implants and induced cell infiltration into implants. Bone formation occurred faster in implants with the combination of rHuIL-11 and rHuBMP-2 compared with rHuBMP-2 alone. These results suggest that rHuIL-11 acts synergistically with rHuBMP-2 to more rapidly stimulate bone formation compared with rHuBMP-2 alone. This novel combined therapy may be of great clinical benefit in bone healing.

Comparison of the antiplatelet effect of YM337 and abciximab in rhesus monkeys

We directly compared the effects of YM337, the Fab fragment of the humanized monoclonal antibody C4G1, on platelet aggregation and template bleeding time with those of abciximab, the Fab fragment of the human/murine chimeric monoclonal antibody 7E3, in rhesus monkeys. The duration of inhibition of platelet aggregation by abciximab after i.v. bolus injection was much longer than that by YM337. Although YM337 significantly prolonged template bleeding time at 5 min after i.v. bolus injection, this action recovered within 1 h after injection. In contrast, although abciximab also prolonged template bleeding time, the duration of this effect was sustained. In a dose-escalating continuous infusion study, we evaluated the relationship between inhibition of platelet aggregation and prolongation of template bleeding time. Platelet aggregation was inhibited by over 80% by both agents at 3 microg/kg per min, and template bleeding time was prolonged to about 30 min at 30 microg/kg per min for YM337 and 10 microg/kg per min for abciximab. Interestingly, plasma concentrations between inhibition of platelet aggregation and prolongation of template bleeding time did not overlap with YM337, but did overlap with abciximab. These results suggest that YM337 allows easier control of antiplatelet activity with less effect on bleeding time than abciximab, and has a wider therapeutic window than abciximab.

Design, synthesis and biological activity of YM-60828 derivatives. Part 2: potent and orally-bioavailable factor Xa inhibitors based on benzothiadiazine-4-one template

Compound YM-60828 was previously characterized in our laboratory as a potent, selective and orally-bioavailable Factor Xa (FXa) inhibitor. The L-shape conformation of this compound in the active site of FXa was recognized as an important factor in displaying its FXa inhibitory activity. This led to the exploration of conformationally restricted cyclic scaffolds bearing a similar active conformation. The current study investigated a novel series of benzothiadiazine-4-one based compounds as FXa inhibitors. Structure-activity relationship (SAR) investigations revealed some potent FXa inhibitors that were selected for further in vitro and ex vivo anticoagulant studies. Among them, compound 6j (YM-169920) was proved to be most effective anticoagulant in this series. The synthesis and SAR in addition to docking studies of this class of inhibitors are described.

Thrombolytic activity of YM866, a novel modified tissue-type plasminogen activator, in a photochemically induced platelet -rich thrombosis model

We compared the thrombolytic activity of a novel modified tissue-type plasminogen activator (t-PA; del 92-173, 275Arg-->Glu), YM866, with that of t-PA in a platelet-rich thrombosis model. Thrombus was induced in guinea pig mesenteric artery by irradiation with filtered light in combination with intravenous (i.v.) administration of fluorescent dye. When occlusion by the thrombus extended to 99% of the luminal area of the vessel, test drug (YM866, t-PA, or saline) was administered by i.v. bolus injection under heparinization. Both YM866 and t-PA exhibited dose-dependent thrombolytic activity; however, the improvement in occlusion rate and the incidence of successful thrombolysis induced by YM866 were three times higher than those induced by t-PA. With YM866 1 mg/kg, alpha 2-plasmin inhibitor levels decreased significantly to 58% of saline group values, but no change was noted in fibrinogen levels. YM866 antigen levels at this dose were seven times higher than those of t-PA. These results suggest that YM866 in single bolus injection is a thrombolytic agent superior to t-PA in platelet-rich thrombi without systemic fibrinolytic activation and that this efficacy is due to the prolonged half-life (t1/2) of the drug.

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