Nami Hisamichi

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.

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.

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.

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.

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.

Cholesterol-lowering effect of YM-16638 in cynomolgus monkeys

YM‐16638 was found in preclinical studies to be an orally active leukotriene antagonist. Because LY‐171883, another leukotriene receptor antagonist with a similar structure to YM‐16638, showed a triglyceride‐lowering effect with a peroxisomal proliferative effect in monkeys fed a normal diet, we investigated whether YM‐16638 also showed a serum triglyceride‐lowering effect by examining serum and hepatic lipid levels in cynomolgus monkeys fed a normal diet supplemented with YM‐16638 for 4 weeks at a daily dose of 3.75 mg (8.5 μmole), 30 mg (67.7 μmole) or 60 mg (135.4 μmole)/kg body weight. Monkeys given YM‐16638 showed a dose‐dependent decrease in serum total cholesterol. At 2 weeks of treatment, serum LDL‐ and HDL‐cholesterol in the YM‐16638 group showed marked decreases of 35% and 32%, respectively. However, serum triglyceride levels did not change. By contrast, hepatic cholesterol and cholesterol ester levels in this group were only slightly increased, without any effect on hepatic triglyceride level. In vitro investigation of the effect of YM‐16638 on LDL‐receptor activity and mRNA expression in the human hepatoma cell line HepG2 cells showed that YM‐16638 increased LDL‐receptor activity in a dose‐dependent manner at 44 h of treatment. mRNA level in these cells was also increased 1.7‐fold at 8 h of treatment. These results suggest that the decrease in serum cholesterol level in monkeys treated with YM‐16638 may be due to an increase in hepatic LDL‐receptor activity. Furthermore, they suggest that YM‐16638 may represent a potent hypocholesterolemic drug without serious side effects.

Species specificity in the blood cholesterol-lowering effect of YM-16638

1 . The compound YM‐16638, [[5‐[[3‐(4‐acetyl‐3‐hydroxy‐2‐propylphenoxy)propyl]thio]‐1,3,4‐thiadiazol‐ 2‐yl]thio] acetic acid was developed in a series of in vitro and in vivo studies as a leukotriene D4 receptor antagonist. 2 . In a clinical trial as a leukotriene antagonist drug, this compound was found to have a potent serum cholesterol lowering effect in normolipidaemic healthy male volunteers. 3 . In the present study, we investigated the serum cholesterol lower effect of this compound in various species of experimental animals. 4 . Administration of YM‐16638 did not cause a significant decrease in serum total cholesterol (TC) in mice (up to 200 mg kg−1, body weight per day for 28 days), rats (200 mg kg−1 for 15 days) or rabbits (90 mg kg−1 for 18 days). In hamsters, administration of YM‐16638 orally or by peritoneal injection at 50 mg kg−1 or more daily for 7 days caused a significant decrease in serum TC and the rate of body weight gain. In monkeys, serum TC did not change in YM‐16638‐administered squirrel monkeys (50 mg kg−1 daily for 3 weeks), but a significant decrease in serum TC was observed in cynomolgus monkeys (33% decrease at 30 mg kg−1 for 4 weeks) and rhesus monkeys (27% decrease at 30 mg kg−1 for 3 weeks) without any serious decrease in body weight. These results were consistent with those in a phase I study in human subjects. In contrast, serum alanine aminotransferase (ALT) level decreased in all animals after YM‐16638 treatment. 5 . From these results, we conclude that YM‐16638 has a potent hypocholesterolaemic effect, but that this effect if species‐specific and is only recognized clearly in human subjects and old‐world monkeys.

Pharmacological properties of YM-57029, a novel platelet glycoprotein IIb/IIIa antagonist

The pharmacological properties of YM-57029 [4-[4-(4-carbamimidoylphenyl)-3-oxopiperazin-1-yl]piperidino]acetic acid monohydrochloride trihydrate), a novel glycoprotein IIb/IIIa antagonist were examined in this study. YM-57029 inhibited fibrinogen binding to purified glycoprotein IIb/IIIa, 1000-fold more potently than the tetrapeptide arginine-glycine-aspartic acid-serine (RGDS). YM-57029 concentration-dependently inhibited ADP-, collagen- and high shear stress-induced platelet aggregation, strongly inhibited ATP release from platelets activated by ADP, and enhanced deaggregation of ADP-induced platelet aggregates. In a pro-aggregatory activity study, RGDS or SC-54701A ((S)-3-[3-[(4-amidinophenyl)carbamoyl]propionamido]-4-pentynoic acid monohydrochloride) caused prominent small aggregate formation. At a higher concentration, RGDS induced medium and large size aggregates, and SC-54701A induced medium aggregates. In contrast, YM-57029 produced only a few small and no larger size aggregates. Ex vivo ADP-induced platelet aggregation and platelet retention to collagen-coated plastic beads were dose-dependently inhibited by YM-57029 after intravenous bolus injection in guinea pigs. YM-57029 produced dose-dependent antithrombotic effects in carotid artery thrombosis and arterio-venous shunt thrombosis models in guinea pigs at 10 and 30 microg/kg, respectively. At these doses, YM-57029 prolonged template bleeding time. These results suggest that YM-57029 is a potent glycoprotein IIb/IIIa antagonist which has less pro-aggregatory effect. It may be a promising antiplatelet agent for thromboembolic diseases, and a good prototype for developing an orally active compound.

Synergistic effect of aurintricarboxylic acid and triflavin in a photochemically induced thrombosis model in rats.

We report here the synergistic antithrombotic effect of aurintricarboxylic acid in combination with a snake venom-derived disintegrin, triflavin, in a photochemically induced thrombosis model in rats. The time to initiation of thrombus was prolonged by i.v. bolus injection of aurintricarboxylic acid at 10 mg/kg. In contrast, time to occlusion was dose-dependently prolonged by both agents, this prolongation being significant with aurintricarboxylic acid at 10 mg/kg i.v. and with triflavin at more than 3 mg/kg i.v. Interestingly, the combination of aurintricarboxylic acid at 3 mg/kg i.v. and triflavin at 1 mg/kg i.v. prolonged not only the initiation of thrombus, but also the time to occlusion.