Toshiro Shibano

Comparison of antithrombotic and haemorrhagic effects of edoxaban, an oral direct factor Xa inhibitor, with warfarin and enoxaparin in rats

INTRODUCTIONnFactor Xa (FXa) is a key serine protease in the coagulation cascade and a promising target for a new antithrombotic agent. Edoxaban is an oral, selective and direct FXa inhibitor. The objective of this study was to compare the antithrombotic and haemorrhagic effects of edoxaban with clinically available anticoagulants, warfarin and enoxaparin, in rat models of thrombosis and haemorrhage.nnnMETHODSnRats were treated with single oral administration of edoxaban, repeated oral dosing of warfarin for 4 days and single subcutaneous administration of enoxaparin before thrombosis or haemorrhage induction. Thrombosis was induced by the insertion of a platinum wire into the inferior vena cava for 60 min. Tail template bleeding time was measured after making an incision on the tail.nnnRESULTSnEdoxaban at 0.3, 1 and 3mg/kg exerted dose-dependent and significant inhibition of venous thrombus formation. The 50% thrombus inhibition dose (ED(50)) was 1.9 mg/kg. At supra-therapeutic doses (10 and 20mg/kg), edoxaban significantly but moderately (less than 2-fold) prolonged bleeding time. Warfarin and enoxaparin also dose-dependently inhibited venous thrombosis and prolonged bleeding time. The ED(50) values of warfarin and enoxaparin were 0.12 mg/kg and 500 IU/kg, and the 2-fold bleeding time prolongation doses (BT2) were 0.16 mg/kg and 1700 IU/kg, respectively. The safety margin (ratio of BT2 to ED(50)) of edoxaban (>10.5) was greater than those of warfarin (1.3) and enoxaparin (3.4).nnnCONCLUSIONSnEdoxaban inhibited venous thrombosis comparably to warfarin and enoxaparin, and the attendant bleeding risk of edoxaban was lower than that of warfarin and enoxaparin in rats.

Synthesis of azido derivatives of semotiadil, a novel 1,4-benzothiazine calcium antagonist, for photoaffinity probes of calcium channels

Abstract Aliphatic and aromatic azido derivatives of semotiadil ( 1 ), a novel calcium antagonist with a 1,4-benzothiazine skeleton, were synthesized for developing photoaffinity probes of L type calcium channels. The azidophenoxy derivative 12 proved to be a potent calcium antagonist and its [ 3 H]-labeled compound 16 would be a useful tool to clarify the binding sites of 1 to the calcium channels.

Platelet glycoprotein Ib alpha polymorphisms affect the interaction with von Willebrand factor under flow conditions

Interaction of platelet glycoprotein (GP) Ibα with von Willebrand factor (VWF) is essential for thrombus formation, particularly under high shear conditions. Previous case–control studies indicated that two GPIbα polymorphisms, 145Thr/Met and/or variable number (1–4) tandem repeats of 13 amino‐acid sequences, are associated with arterial thrombosis. The 145Met‐allele and the 3R‐ or 4R‐allele is associated with increased risk. However, there is little clear experimental data to support this association. To elucidate the functional effects of these polymorphisms, we prepared recombinant GPIbα fragments and tested them in vitro. The dissociation constants of ristocetin‐induced 125I‐labelled VWF binding to two forms of soluble recombinant GPIbα [1His–302Ala, either 145Thr (145T) or 145Met (145M)] were not different. Four types of Chinese hamster ovary cells expressing full‐length GPIbαβ/IX, 145T with one repeat (T1R), 145M with one repeat (M1R), 145T with four repeats (T4R), and 145M with four repeats (M4R), were prepared, and cell interactions with immobilized‐VWF were examined under various shear conditions. The cell rolling velocity of M4R under a shear condition of 114/s was significantly slower than that of T1R. Intermediate values were obtained with M1R and T4R. The results suggest that M4R interacts more strongly with VWF under flow conditions.

Guanosine 5′-O-(3-thiotriphosphate) causes endothelium-dependent, pertussis toxin-sensitive relaxations in porcine coronary arteries

To determine whether direct stimulation of endothelial G-proteins causes relaxations of the underlying vascular smooth muscle, the effects of guanosine 5-O-(3-thiotriphosphate) (GTP gamma S) and sodium fluoride were studied in porcine coronary arteries and endothelial cells. Isometric tension was measured in coronary rings contracted with prostaglandin F2 alpha. GTP gamma S (in the presence of saponin) and sodium fluoride (in the presence of AlCl3) relaxed rings with, but not those without endothelium. The responses were inhibited by nitro-L-arginine and pertussis toxin. In membrane fractions of coronary endothelial cells, GTP gamma S and sodium fluoride inhibited the ADP-ribosylation of G-proteins catalyzed with [32P]-NAD and pertussis toxin. These data suggest that direct stimulation of G-proteins in endothelial cells by GTP gamma S and sodium fluoride causes a pertussis toxin-sensitive relaxation which may be attributed to the release of nitric oxide.

Evidence that 5-HT2A receptors are not involved in 5-HT-mediated thermoregulation in mice

To determine the role of 5-hydroxytryptamine2A (5-HT2A) receptors in 5-HT-mediated thermoregulation in mice, we studied the effects of a 5-HT2A receptor agonist and 5-HT2A receptor antagonists on the body temperature, and the effects of selective 5-HT2A receptor and nonselective 5-HT receptor antagonists on hypothermia induced by 5-hydroxytryptophan (5-HTP). (+/-)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane (DOI), a 5-HT2A receptor agonist, did not change body temperature in mice at doses of 1 and 5 mg/kg, intraperitoneally (IP), which induced head twitch response. Three 5-HT2A receptor antagonists, ketanserin (1 mg/kg, orally), ritanserin (1 and 10 mg/kg, orally), and DV-7028 (10 mg/kg, orally), also failed to alter body temperature, although these three 5-HT2A receptor antagonists at > or = 1 mg/kg, orally, inhibited head twitch response induced by 5-HTP (200 mg/kg, IP), a precursor of 5-HT. Ketanserin (1 mg/kg, orally), ritanserin (1 and 10 mg/kg, orally), and DV-7028 (10 mg/kg, orally) did not inhibit hypothermia induced by 5-HTP (200 mg/kg, IP). A nonselective 5-HT receptor antagonist, methysergide (1 mg/kg, subcutaneously), attenuated hypothermic response to 5-HTP. These results suggest that in mice, 5-HT2A receptors are unlikely to be involved in 5-HT-mediated thermoregulation.

Impact of antithrombin deficiency on efficacy of edoxaban and antithrombin-dependent anticoagulants, fondaparinux, enoxaparin, and heparin

INTRODUCTIONnOral factor Xa (FXa) inhibitors are a novel class of anticoagulants that, unlike heparins, are expected to demonstrate antithrombotic effects independent of plasma antithrombin (AT) concentrations. We utilized heterozygous AT-deficient (AT+/-) mice to determine the impact of AT deficiency on anticoagulant and antithrombotic effects of edoxaban, a direct FXa inhibitor, and compared with heparins (fondaparinux, enoxaparin, and unfractionated heparin [UHF]).nnnMATERIALS AND METHODSnThe effects of edoxaban and heparins on in vitro prothrombin time and activated partial thromboplastin time were measured in plasma obtained from wild type (AT+/+) and AT+/- male mice. To assess the antithrombotic effects of these anticoagulants in vivo, venous thrombosis was induced in the inferior vena cava by FeCl3 treatment. Potency ratios of antithrombotic effects in AT+/- compared with AT+/+ mice were analyzed by a parallel line assay.nnnRESULTSnIn vitro studies demonstrated that the clotting-time prolongation effects of edoxaban were not affected by heterozygous AT deficiency whereas those of AT-dependent anticoagulants were attenuated. In AT+/- mice, the antithrombotic effects of AT-dependent anticoagulants were less potent than those in AT+/+ mice. In contrast, edoxaban was equipotent in preventing thrombus formation in both wild-type and AT-deficient mice. The attenuated antithrombotic effects of fondaparinux, enoxaparin, and UFH in AT-deficient mice were restored by AT supplementation. Edoxaban exerts a comparable antithrombotic effect even in mice with low plasma AT antigen and activity to that in wild-type mice.nnnCONCLUSIONnEdoxaban may potentially be prioritized over AT-dependent anticoagulants in patients with lower plasma AT concentration.

GSK-3β negatively regulates megakaryocyte differentiation and platelet production from primary human bone marrow cells in vitro

Glycogen synthase kinase (GSK)-3, a constitutively active serine-threonine kinase, acts as a key regulator of major signaling pathways, including the Wnt, Hedgehog, and Notch pathways. Although a number of studies have demonstrated that GSK-3 plays a critical role in several cellular processes, such as differentiation, growth, and apoptosis, the effects of GSK-3 on platelet production have not been explored. There are two GSK-3 isoforms, GSK-3α and GSK-3β. GSK-3β is more highly expressed in platelets. In the present study, primary human bone marrow cells were cultured for 12 days in megakaryocyte lineage induction (MKLI) media to induce their differentiation into megakaryocyte (MK) lineage cells, in the presence or absence (+/−) of TWS119, a GSK-3β inhibitor, during MK differentiation from stem cells and subsequent platelet production. MK maturation, MK production, and subsequent platelet production were markedly enhanced in cells cultured in TWS119 (+) compared with cells cultured in TWS119 (−). These effects on MK lineage cells were thrombopoietin (TPO)-dependent. We next performed the experiment focusing on the inhibitory effect of GSK-3β on platelet production. Bone marrow cell-derived CD41 (+)/CD42b (+)/propidium iodide (−) cells in the large (MK)-sized cell population (day 8), as living mature MKs, were further cultured in the MKLI media in TWS119 (+/−) for 6 days. Platelet production from mature MKs in TWS119 (+) was approximately two-fold higher than that in TWS119 (−). The mature MKs were cultured in MKLI media in TWS119, in TPO (+/−), and platelet production was markedly decreased in TPO (−). This indicated that the GSK-3β inhibition affects thrombopoiesis under these conditions with TPO. To identify the target(s) of GSK-3β inhibition during differentiation into MK lineage cells, we performed a differential gene expression study and subsequent pathway analysis of the large (MK)-sized CD41 (+)/propidium iodide (−) cells cultured in TWS119 (+/−) for 3 days. The results of the analysis indicated that GSK-3β inhibition during differentiation into MK lineage cells affected factors related to transcription, apoptosis, cell division, cell cycle, blood coagulation, lipid transport, keratin filament, metabolic processes, and the Wnt signaling and transforming growth factor-β signaling pathways. These observations suggest that GSK-3β inhibition and TPO treatment affect both megakaryopoiesis and thrombopoiesis in an in vitro differentiation system for primary human bone marrow cells.

The effects of warfarin and edoxaban, an oral direct factor Xa inhibitor, on gammacarboxylated (Gla-osteocalcin) and undercarboxylated osteocalcin (uc-osteocalcin) in rats

INTRODUCTIONnOsteocalcin plays a role in bone homeostasis. The vitamin K cycle is essential for the gamma-carboxylation of glutamic acid residues in osteocalcin. Some evidence suggests that long-term warfarin therapy, which inhibits the vitamin K cycle and prevents gamma-carboxylation, is associated with increased bone-fracture risk. The aim of this study was to determine the effects of warfarin and edoxaban, a direct factor Xa inhibitor, on the serum concentration of total, gamma-carboxylated (Gla-osteocalcin) and undercarboxylated osteocalcin (uc-osteocalcin) in rats.nnnMATERIALS AND METHODSnRats received orally administered warfarin or edoxaban, and 24h later serum and plasma were prepared. Osteocalcin level in serum was measured with ELISA. A Gla-osteocalcin was precipitated by the addition of hydroxyapatite, and the resulting supernatant was used for measuring uc-osteocalcin. Prothrombin time (PT) of plasma was also measured.nnnRESULTSnWarfarin at 1mg/kg (a dose which prolonged PT 2.62-fold) markedly increased the serum level of uc-osteocalcin and slightly increased the total osteocalcin level compared with control in rats. Serum Gla-osteocalcin significantly decreased by warfarin. Edoxaban at 1mg/kg (an antithrombotic dose) and 54mg/kg (a dose which prolonged PT 2.25-fold) had no effects on total, uc-, and Gla-osteocalcin levels.nnnCONCLUSIONSnThis study demonstrates that warfarin impaired the carboxylation of osteocalcin in rats. In contrast, edoxaban at or higher doses than needed for an antithrombotic effect sustained the circulating Gla-osteocalcin level. These findings suggest that edoxaban has no effects on the production of Gla-osteocalcin and thus, may have a lower risk of adverse effects on bone health.

Melagatran, a direct thrombin inhibitor, but not edoxaban, a direct factor Xa inhibitor, nor heparin aggravates tissue factor-induced hypercoagulation in rats

There are concerns that some anticoagulants can paradoxically increase thrombogenesis under certain circumstances. We have shown that low-dose administration of a direct thrombin inhibitor, melagatran, significantly worsens the coagulation status induced by tissue factor injection in rats. We compared the effect of inhibition of thrombin and factor Xa for their potential to aggravate tissue factor-induced coagulation in rats. Hypercoagulation was induced by the injection of 2.8 U/kg tissue factor after administration of melagatran, heparin and edoxaban in rats. Blood samples were collected 10min after tissue factor injection. Platelet numbers, thrombin-antithrombin complex concentrations and plasma compound concentrations were measured. Though a high dose of melagatran (1mg/kg, i.v.) suppressed platelet consumption and thrombin-antithrombin complex generation induced by tissue factor, lower doses of melagatran (0.01, 0.03 and 0.1mg/kg, i.v.) significantly enhanced platelet consumption and thrombin-antithrombin complex generation. In addition, although melagatran (3mg/kg, i.v.) improved coagulation status when tissue factor was given 5min after the drug administration, and 2, 4 and 8h after melagatran dosing, it deteriorated coagulation status. These results were well explained by the plasma melagatran concentration. Low concentrations (15-234ng/ml) of melagatran aggravated coagulation status whereas it was mended by high concentrations (1190ng/ml or more) of the compound. In contrast, edoxaban and heparin did not show any exacerbation under these examination conditions. These results show that subtherapeutic concentrations of melagatran are associated with coagulation pathway activation, whereas factor Xa inhibition with edoxaban has a low risk of paradoxical hypercoagulation.

Comparison of antithrombotic and hemorrhagic effects of edoxaban, a novel factor Xa inhibitor, with unfractionated heparin, dalteparin, lepirudin and warfarin in rats

BACKGROUNDnEdoxaban is a novel, potent and orally active direct Factor Xa (FXa) inhibitor under development for prophylaxis and treatment of thromboembolic diseases. Properties of dose response and margin of safety of anticoagulants are the key factors for a positive risk/benefit of novel oral anticoagulants.nnnOBJECTIVESnTo compare the dose response of antithrombotic effect and margin of safety between antithrombotic and hemorrhagic effects of edoxaban with conventional anticoagulants, unfractionated heparin (UFH), dalteparin (low molecular weight heparin), lepirudin, and warfarin in rat models of thrombosis and hemorrhage.nnnMETHODSnRats were treated with edoxaban, UFH, dalteparin, and lepirudin by continuous intravenous (iv) infusion, or with oral warfarin for 4 days before inducing thrombosis or bleeding. Thrombosis was induced by inserting a platinum wire into the inferior vena cava for 60 minutes. Tail template bleeding time was measured after making an incision on the tail.nnnRESULTSnIn rats, iv infusion of edoxaban inhibited venous thrombosis in a dose-dependent manner. The other anticoagulants also exerted dose-dependent antithrombotic effects. The slopes of the dose-response curves of edoxaban were significantly shallower than the slopes of UFH, dalteparin, and warfarin. At supratherapeutic doses, edoxaban prolonged bleeding time in a rat tail bleeding model. To determine bleeding risk, the margins between antithrombotic and bleeding-time prolongation were compared. The margins of safety of edoxaban were wider than those of UFH, dalteparin, lepirudin, and warfarin.nnnCONCLUSIONSnThese results suggest that edoxaban may be more easily controlled and has the potential for a more positive risk/benefit ratio compared to conventional anticoagulants.