Toshiyuki Funatsu

Effects of serotonin 5-HT3 receptor antagonists on stress-induced colonic hyperalgesia and diarrhoea in rats: a comparative study with opioid receptor agonists, a muscarinic receptor antagonist and a synthetic polymer.

Abstract  In this study, we examined the effects of serotonin (5‐HT)3 receptor antagonists (5‐HT3RAs) including ramosetron, alosetron, and cilansetron on colonic nociceptive threshold in rats. Furthermore, we established a restraint stress‐induced colonic hyperalgesia model in rats, and compared the inhibitory effects of 5‐HT3RAs on restraint stress‐induced colonic hyperalgesia and diarrhoea with those of loperamide, trimebutine, tiquizium and polycarbophil. The colonic nociceptive threshold was measured as the balloon pressure at the time the rat showed a nociceptive response during colonic distension by an intrarectally inserted balloon. Oral administration of ramosetron (3–30 μg kg−1), alosetron (30–300 μg kg−1), or cilansetron (30–300 μg kg−1) increased the colonic nociceptive threshold in a dose‐dependent manner in non‐stressed rats. Restraint stress for 1 h significantly decreased the colonic nociceptive threshold, but ramosetron (0.3–3 μg kg−1), alosetron (3–30 μg kg−1), cilansetron (3–30 μg kg−1) and trimebutine (100–1000 mg kg−1) significantly inhibited the decrease in the threshold. Loperamide (3–30 mg kg−1), tiquizium (100–1000 mg kg−1) and polycarbophil (1000 mg kg−1) did not affect the restraint stress‐induced decrease in the colonic nociceptive threshold. All drugs tested in this study showed dose‐dependent inhibition of restraint stress‐induced diarrhoea in rats. These results indicate that, unlike existing antidiarrhoeal and spasmolytic agents, 5‐HT3RAs have inhibitory effects on colonic nociception, and prevented restraint stress‐induced both diarrhoea and hyperalgesia at almost the same doses in rats. This suggests that the 5‐HT3RAs may be useful in ameliorating both colonic hyperalgesia and diarrhoea in patients with irritable bowel syndrome.

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.

Reduction in hepatic non-esterified fatty acid concentration after long-term treatment with atorvastatin lowers hepatic triglyceride synthesis and its secretion in sucrose-fed rats

The mechanism by which atorvastatin lowers plasma triglyceride (TG) levels is mainly through a decrease in hepatic TG secretion. However, it is not clear why atorvastatin, which does not inhibit TG synthesis in vitro, decreases hepatic TG secretion without a prospective increase in hepatic TG concentration. For the investigation of the mechanisms that underlie the hypotriglyceridemic effects of atorvastatin, we characterized the effect of either a single or an 11 day administration of atorvastatin in sucrose-induced hypertriglyceridemic rats. Atorvastatin (30 mg/kg p.o.) strongly decreased the rate of both very-low-density lipoprotein (VLDL)-TG and VLDL-apolipoprotein B secretion. The inhibitor also decreased hepatic TG concentration. Hepatic TG synthesis activity was also decreased by atorvastatin, and its activity was correlated with both hepatic and plasma TG concentration. There was also a strong correlation between the hepatic TG synthesis and hepatic non-esterified fatty acid (NEFA) concentration (r(2)=0.815). These effects required chronic administration of the inhibitor and were not observed by acute treatment. Repeated administration of atorvastatin also strongly reduced hepatic acyl-coenzyme A synthase mRNA levels. These results suggest that the reduced hepatic NEFA most likely lowers hepatic TG synthesis and TG secretion in sucrose-fed hypertriglyceridemic rats.

Pharmacological profile of ramosetron, a novel therapeutic agent for IBS.

Abstract.Ramosetron is a potent and selective serotonin (5-HT)3 receptor antagonist that has been shown to affect abnormal colonic function and abdominal pain in animals. Ramosetron (0.3 to 100 µg/kg, p.o.) has been found to significantly suppress abnormal defecation induced by conditioned-fear stress (CFS), restraint stress, corticotropin releasing factor (CRF) and 5-HT in rats and mice, and these effects were more potent than those of alosetron, cilansetron or loperamide. On the other hand, ramosetron (3,000 µg/kg, p. o., once daily for 7 days) did not inhibit normal defecation in dogs while tiquizium significantly inhibited it. Ramosetron (3 to 100 µg/kg, p. o.) also significantly prevented CFS-induced acceleration of colonic transit and CRF-induced abnormal water transport in rats, respectively. Moreover, ramosetron (0.3 to 3 µg/kg, p. o.) significantly suppressed restraint stress-induced decrease in colonic pain threshold, an effect not observed with loperamide. These results indicate that ramosetron produce beneficial clinical effects on IBS symptoms.

Atorvastatin increases hepatic fatty acid beta-oxidation in sucrose-fed rats: comparison with an MTP inhibitor

We investigated the effects of atorvastatin, a widely used 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, and BMS-201038, a microsomal triglyceride transfer protein (MTP) inhibitor, in sucrose-fed hypertriglyceridemic rats to determine whether the activation of beta-oxidation by these compounds plays a role in their hypotriglyceridemic effect. The decrease in plasma triglyceride concentration and post-Triton very low-density lipoprotein (VLDL) triglyceride concentration, a measure of hepatic triglyceride secretion, by atorvastatin (30 mg/kg p.o.) for 2 weeks was to approximately the same degree as those by BMS-201038 (0.3 mg/kg). Atorvastatin (30 mg/kg) increased hepatic beta-oxidation activity by 54% (P < 0.01), while BMS-201038 (0.3 mg/kg) had no significant effect. Atorvastatin decreased hepatic triglyceride, fatty acid and cholesteryl ester concentrations by 21% to 39%, whereas BMS-201038 increased these variables by 28% to 307%. In the atorvastatin-treated groups, a significant relationship was seen not only between hepatic beta-oxidation activity and hepatic triglyceride concentration (R(2) = 0.535, P < 0.01), but also between hepatic and plasma triglyceride concentrations (R(2) = 0.586, P < 0.01). No effect of atorvastatin on hepatic fatty acid synthesis was observed. These results indicate that the activation of hepatic beta-oxidation by atorvastatin may contribute to the decrease in hepatic triglyceride concentration, leading to its hypotriglyceridemic effect.

Effects of serotonin 5-HT3 receptor antagonists on CRF-induced abnormal colonic water transport and defecation in rats

The effects of corticotropin releasing factor (CRF) and serotonin (5-HT)(3) receptor antagonists on intestinal water transport are not well understood. Hence, we established a CRF-induced abnormal water transport model in rat colon, and evaluated the effects of 5-HT(3) receptor antagonists including ramosetron, alosetron, and cilansetron, and the antidiarrheal agent loperamide, in this model. In addition, the effects of 5-HT(3) receptor antagonists and loperamide on abnormal defecation induced by CRF in rats were examined. Colonic water transport was measured in colonic loops in conscious rats. Centrally administered CRF (3-30 microg/kg) markedly decreased colonic fluid loss, whereas oral administration of ramosetron (3, 30 microg/kg), alosetron (300 microg/kg), cilansetron (300 microg/kg), or loperamide (3 mg/kg) significantly inhibited it. Ramosetron (1-10 microg/kg), alosetron (10-100 microg/kg), cilansetron (10-100 microg/kg), or loperamide (0.3-3 mg/kg) also showed dose-dependent inhibition of CRF-induced defecation in rats. These results suggest that 5-HT(3) receptors are involved in both abnormal colonic water transport and defecation induced by CRF, and that the inhibitory effects of 5-HT(3) receptor antagonists on CRF-induced abnormal defecation partly result from their ameliorating action on colonic water transport.

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.

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.

Bolus Injection of the Modified Tissue-Type Plasminogen Activator YM866 Versus Bolus Injection Plus Infusion of Alteplase: Comparison of Thrombolytic Activities

Abstract Objective To compare the thrombolytic activity of YM866, a modified tissue-type plasminogen activator (t-PA), with that of alteplase in dogs with induced coronary artery thrombi and rats with induced carotid artery thrombi. Background Although t-PA remains effective in many patients, exhibiting a coronary reperfusion rate of ⩾70%, its high dosing regimen increases the possibility of systemic bleeding and acute coronary artery reocclusion. In addition, infusion is more complicated and inconvenient than is bolus injection. Methods Adult male beagle dogs with copper coil-induced thrombosis and male rats with electrically induced thrombosis were used. Thrombolytic activity was determined, and plasma fibrinogen levels and bleeding times were measured. All drugs were administered in a volume of 0.5 mL/kg body weight. Time to reperfusion was expressed as the value observed for each animal. Results In dogs, either YM866 (0.1 mg/kg intravenous [IV] bolus injection) or alteplase (0.75 mg/kg IV bolus injection plus infusion), administered 3 hours after confirmation of coronary occlusion, produced reperfusion in all 5 animals in each group, with a median time to reperfusion of 5 and 10 minutes, respectively. In rats treated 10 minutes after carotid occlusion, YM866 (1 mg/kg IV bolus injection) produced reperfusion in 7 of 8 animals within a median time of 14.3 minutes, and alteplase (7.5 mg/kg IV bolus injection plus infusion) produced reperfusion in 8 of 9 animals within a median time of 13.2 minutes. In a separate experiment using conscious dogs, plasma fibrinogen levels decreased slightly after administration of YM866 (0.1 mg/kg IV bolus injection) or alteplase (0.75 mg/kg IV bolus injection plus infusion), but these recovered to baseline values within 24 hours after administration. YM866 and alteplase did not significantly affect bleeding time. Conclusions Although both YM866 and alteplase reperfused occluded coronary arteries in dogs with thrombi and occluded carotid arteries in rats with thrombi, only a single bolus injection of YM866 was necessary to achieve thrombolytic activities. YM866 slightly decreased plasma fibrinogen levels, but did not affect bleeding time. If future studies in humans support these data, YM866 shows promise as an improved clinical treatment for patients with acute myocardial infarction.