Effect of YM-254890, a specific Galphaq/11 inhibitor, on experimental peripheral arterial disease in rats

The protective effect of YM-254890, a specific Galphaq/11 inhibitor, on laurate-induced peripheral arterial disease in rats was compared with those of prostaglandin E1 (PGE1), beraprost, and clopidogrel. YM-254890 inhibited ADP-induced ex vivo rat platelet aggregation at a dose of 3 microg/kg. Furthermore, YM-254890 strongly inhibited phenylephrine-, serotonin- and endothelin-1-induced contractions in the rat aorta, and improved dermal blood flow after the laurate injection. The intra-arterial single bolus administration of YM-254890 15 min after the laurate injection dose-dependently inhibited the progression of the lesion, with significance, at 3 microg/kg without affecting systemic blood pressure. PGE1 and beraprost, when administered before the laurate injection, were effective, but their potencies were less than that of YM-254890. Clopidogrel significantly suppressed lesion progression when administered at 30 mg/kg twice a day for 3 days, which completely inhibited platelet aggregation. These results suggest that the local administration of YM-254890 may be useful for treating peripheral arterial disease.     

YM-254890, a novel platelet aggregation inhibitor produced by Chromobacterium sp. QS3666

A novel platelet aggregation inhibitor, YM-254890, was isolated from the culture broth of strain QS3666. This strain was isolated from a soil sample collected at Okutama, Tokyo, Japan, and was identified as Chromobacterium sp. by morphological and physiological criteria. YM-254890 was purified from the culture supernatant by solvent extraction, ODS and silica gel flash chromatography, followed by preparative HPLC. YM-254890 inhibited ADP-induced platelet aggregation in human platelet-rich plasma with an IC50 value below 0.6 microM by blocking the P2Y1 receptor-signal transduction pathway.     

Examination of signalling pathways involved in muscarinic responses in bovine ciliary muscle using YM-254890, an inhibitor of the Gq/11 protein

BACKGROUND AND PURPOSE: In the ciliary muscle, the tonic component of the contraction produced by cholinergic agonists is highly dependent on Ca2+ provided by influx through non-selective cation channels (NSCCs) opened by stimulation of M3 muscarinic receptors. We examined effects of YM-254890 (YM), a Gq/11-specific inhibitor, on contraction, NSCC currents and [Ca2+]i elevation induced by carbachol (CCh). EXPERIMENTAL APPROACH: Isometric tension was recorded from ciliary muscle bundles excised from bovine eyes. In ciliary myocytes dispersed with collagenase and cultured for 1-5 days, whole-cell currents were recorded by voltage clamp and the intracellular free Ca2+ concentration [Ca2+]i was monitored using the Fluo-4 fluorophore. Existence and localization of M3 receptors and the alpha subunit of Gq/11 (Galpha(q/11)) were examined by immunofluorescence microscopy using AlexaFluor-conjugated antibodies. KEY RESULTS: Both phasic and tonic components of contractions evoked by 2 microM CCh were inhibited by YM (3-10 microM) in a dose-dependent manner. In the cultured cells, CCh (0.05-10 microM) evoked an NSCC current as well as an elevation of the [Ca2+]i. Both initial and sustained phases of these CCh-evoked responses were abolished by YM (3-10 microM). Immunostaining of the cytoplasmic side of the plasma membrane of ciliary myocytes revealed a dense distribution of M3 receptors and Galpha(q/11). CONCLUSIONS AND IMPLICATIONS: The tonic as well as phasic component of the ciliary muscle contraction appears to be under control of signals conveyed by a G(q/11)-coupled pathway. YM is a useful tool to assess whether Gq/11 is involved in a signal transduction system.     

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.     

双苯氟嗪对血小板聚集和血栓形成的影响(英文)

在体内外实验中观察我国首创合成的新钙拮抗剂双苯氟嗪(Dip)对血小板聚集和实验性血栓形成的作用。Dip iv 1—2mg·kg~(-1)和1—100μmol·L~(-1)体外温育可剂量或浓度依赖性地抑制ADP和AA诱发的家兔血小板聚集。Dip iv 2.5—10 mg·kg~(-1)和ip 50—100 mg·kg~(-1)可抑制大鼠体内血栓形成;Dip iv 10mg·kg~(-1)和200μmol·L~(-1)体外给药可抑制体外血栓形成。提示,减轻紊乱的血小板与血管壁反应是其抗血栓作用的主要因素,Dip的作用强于Cin。     

甲基莲心碱对大鼠血压、血小板聚集和血栓形成的影响

采用比浊法研究甲基莲心碱对大鼠血压及血小板聚集、血栓形成的影响，结果显示甲基莲心碱在降低血压的同时，能剂量依赖性地抑制ＡＤＰ和胶原诱导的正常大鼠、肾性高血压大鼠和高脂喂养大鼠血小板聚集；甲基莲心碱亦能明显延长电刺激大鼠颈动脉闭塞性血栓形成时间。此结果表明甲基莲心碱具有降压与抗血小板聚集和血栓形成的双重作用。     

Understanding the mechanism of platelet thrombus formation under blood flow conditions and the effect of new antiplatelet agents

Platelet aggregation induced by stirring of a platelet suspension after activation by the exogenous addition of stimulants, such as ADP, epinephrine and thrombin, has long been used as a platelet function test, and for the screening of antiplatelet agents. Since platelet aggregation has been demonstrated to be mediated exclusively by the binding of plasma fibrinogen to the activated GP IIb/IIIa, anti-GP IIb/IIIa agents, which can completely inhibit platelet aggregation, were expected to become among the most potent of antithrombotic agents. The strong preventive effects of anti-GP IIb/IIIa agents against thrombotic complications after coronary intervention, and the weaker preventive effects of the same agents against the onset of coronary thrombosis in unstable angina pectoris patients point to both the efficacy and the limitations of anti-GP IIb/IIIa antithrombotic agents. Recently, many investigators have reported that platelets play a major role in thrombus formation at sites exposed to blood flow. Several platelet-function assay systems have been developed to elucidate the mechanism of thrombus formation under blood flow conditions. Numerous studies have demonstrated that von Willebrand factor (VWF) and its interaction with its receptors on platelets, including GP Ibalpha and GP IIb/IIIa, play essential roles not only in platelet activation, but also in platelet adhesion and possibly platelet cohesion. These findings prompted us to explore whether the VWF-mediated process of thrombus formation could be exploited as a target for potential antiplatelet agents. Moreover, recent studies have demonstrated that multiple synergistic signals, including those mediated by catecholamine receptors, purine nucleotide receptors, as well as some types of collagen receptors are involved in the process of VWF-mediated platelet thrombus formation. We now have new antiplatelet agents on the horizon, targeted against thrombus formation under blood flow conditions.     

Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases

Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide++ + dihydrochloride] is widely used as a specific inhibitor of the Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) family of protein kinases. This study examined the inhibition mechanism and profile of actions of Y-27632 and a related compound, Y-30141 [(+)-(R)-trans- 4-(1-aminoethyl)-N-(1H-pyrrolo[2, 3-b]pyridin-4-yl)cyclohexan-ecarboxamide dihydrochloride]. Y-27632 and Y-30141 inhibited the kinase activity of both ROCK-I and ROCK-II in vitro, and this inhibition was reversed by ATP in a competitive manner. This suggests that these compounds inhibit the kinases by binding to the catalytic site. Their affinities for ROCK kinases as determined by K(i) values were at least 20 to 30 times higher than those for two other Rho effector kinases, citron kinase and protein kinase PKN. [(3)H]Y-30141 was taken up by cells in a temperature- and time-dependent and saturable manner, and this uptake was competed with unlabeled Y-27632. No concentrated accumulation was found, suggesting that the uptake is a carrier-mediated facilitated diffusion. Y-27632 abolished stress fibers in Swiss 3T3 cells at 10 microM, but the G(1)-S phase transition of the cell cycle and cytokinesis were little affected at this concentration. Y-30141 was 10 times more potent than Y-27632 in inhibiting the kinase activity and stress fiber formation, and it caused significant delay in the G(1)-S transition and inhibition of cytokinesis at 10 microM.     

羟基红花黄色素A对大鼠血栓形成和血小板聚集功能的影响

一类新药羟基红花黄色素A为从传统中草药红花中提取的有效成分,为探讨其抗缺血性脑损伤机制是否与抗血栓形成和抑制血小板聚集有关,我们参照文献[1,2]测定其对大鼠血小板聚集功能和体内动静脉旁路血栓形成的影响.     

Effect of physical states of binders on high-shear wet granulation and granule properties: a mechanistic approach toward understanding high-shear wet granulation process, part 3: effect of binder rheological properties

Ternary blends consisting of efavirenz (a model drug compound), lactose monohydrate, and a polymeric binder were investigated to verify the "physical state theory" in which granulation occurs only when binders undergo transition from glassy state to rubbery solution state. Furthermore, it was found that the rheological properties of the binders can significantly affect the granulation process. The blends with binders of viscous flow [polyvinylpyrrolidone (PVP) K17, PVP K25, and PVP K29/32 after exposure to 96% RH] showed an increase in particle size with both binder concentration and mixing time. On the contrary, binders with viscoelastic properties, such as hydroxypropylcellulose (HPC) EXF and PVP K90, did not flow well and thereby, the blends with HPC and PVP K90 did not show much effect of binder concentration and mixing time on their granule size. Moreover, the friability of granules made with HPC EXF and PVP K90 is low, indicating that the strength of the granules largely depends on the viscosity of the binders, as the binders of higher viscosity tend to produce stronger granules. Finally, the viscoelastic state of the polymeric binders upon absorbing water was analyzed using the glass-rubber transition model, which shows five regions with different viscoelastic properties.     

Effect of YM-53601, a novel squalene synthase inhibitor,on the clearance rate of plasma LDL and VLDL in hamsters

1. To better understand how it decreases plasma cholesterol and triglyceride, we evaluated the effect of YM-53601 ((E-2-[2-fluoro-2-(quinuclidin-3-ylidene) ethoxy]-9H-carbozole monohydrochloride) on the clearance rate of low density lipoprotein (LDL) and very low density lipoprotein (VLDL) in hamsters. 2. Treatment with YM-53601 at 50 mg kg(-1) for 5 days in hamsters fed a normal diet enhanced the disappearance of 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-VLDL and DiI-LDL. This effect on DiI-LDL was lost in the early phase after DiI-methyl(met)-LDL, chemically modified to block LDL receptor binding, was injected in hamsters, but was retained in the late phase. Pre-treatment with protamine sulphate, which inhibits the activity of LPL, also failed to enhance DiI-VLDL clearance rate by YM-53601. 3. Even on single oral administration at 30 mg kg(-1), YM-53601 enhanced the disappearance of the high concentration of plasma triglyceride after injection of intrafat, an emulsion of fat. Plasma triglyceride was significantly decreased as soon as 1 h after single administration of YM-53601 in hamsters fed a normal diet. 4. These results indicate that the decrease in plasma total cholesterol and triglyceride after the treatment with YM-53601 is due to its enhancement of the clearance rate of LDL and VLDL, respectively. Moreover, YM-53601 may be effective in decreasing plasma triglyceride levels early in the course of treatment of hypertriglyceridaemia in humans.     

Sch 38519, a novel platelet aggregation inhibitor produced by a Thermomonospora sp. Taxonomy, fermentation, isolation, physico-chemical properties, structure and biological properties

The complex containing a new platelet aggregation inhibitor, Sch 38519, was recovered from the fermentation filtrate of Thermomonospora sp. SCC 1793. A chemically defined medium was developed which favored the production of Sch 38519. The antibiotic was isolated from the fermentation filtrate by absorption on macroreticular resin and further purified by ion exchange chromatography and reverse phase HPLC. Sch 38519 is an isochromanequinone structurally related to medermycin, lactoquinomycin and granaticin. It inhibits thrombin-induced aggregation of human platelets with an IC50 of 68 micrograms/ml. Sch 38519 is also active against Gram-positive and Gram-negative bacteria.     

Amelioration of collagen-induced arthritis in mice by a novel phosphodiesterase 7 and 4 dual inhibitor, YM-393059

YM-393059 is a novel phosphodiesterase (PDE) 7 and PDE4 dual inhibitor that inhibits PDE7A with high potency (IC50=14 nM) and PDE4 with moderate potency (IC50=630 nM). It inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha production in mice with an ED50 value of 2.1 mg/kg [Yamamoto, S., Sugahara, S., Naito, R., Ichikawa, A., Ikeda, K., Yamada, T., Shimizu, Y., 2006. The effects of a novel phosphodiesterase 7A and -4 dual inhibitor, YM-393059, on T-cell-related cytokine production in vitro and in vivo. Eur. J. Pharmacol. 541, 106-114.]. In this study, we investigated the therapeutic potential of YM-393059 for the treatment of rheumatoid arthritis in several animal models. YM-393059 was found to inhibit LPS-induced interleukin (IL)-1beta production in mice with an ED50 value of 16.6 mg/kg, but it had only a slight effect on IL-6 production. YM-393059 and cyclosporine significantly suppressed arthritis development at doses of 30-100 mg/kg and 20 mg/kg, respectively, in the mice collagen-induced arthritis model. YM-393059 (100 mg/kg) significantly inhibited increases in the serum immunoglobulin G level that occurred in response to autoantigenic collagen in arthritic mice, whereas cyclosporine (20 mg/kg) did not. In contrast, cyclosporine completely suppressed the acute rejection of cardiac allografts in rats, whereas YM-393059 did not, even at a dose of 100 mg/kg. YM-393059 potently inhibited proinflammatory cytokine production and selectively suppressed the response to the autoantigen without affecting the response to alloantigens. These results suggest that YM-393059 is an attractive compound for the treatment of autoimmune disorders such as rheumatoid arthritis.     

Enhancement of platelet aggregation and thrombus formation by arsenic in drinking water: a contributing factor to cardiovascular disease

Arsenic in drinking water is a worldwide health problem that is associated with cardiovascular disease, but the cause is currently unknown. Arsenic effects on platelets, which are important in development of cardiovascular disease, were examined in vitro and in a drinking water study using a rat animal model. Trivalent inorganic arsenic (arsenite) induced in vitro aggregation when platelets were exposed to subthreshold challenge by thrombin and several other agonists in a concentration-dependent manner, with arsenite being the most potent form tested. Arsenite also induced significant increases in serotonin secretion, thromboxane A(2) formation, and adhesion protein expression in platelets. Consistent with the in vitro studies, 4-week ingestion of arsenite-contaminated drinking water resulted in enhanced arterial thrombosis. Human platelets showed similar responses, suggesting that the effects seen in animal experiments are applicable to humans. These results will provide new insights into the mechanism of arsenic-induced cardiovascular disease. They will also allow regulatory agencies to estimate risk from arsenic-induced cardiovascular disease and to determine if drinking water regulatory levels based on human cancer studies will protect against noncancer effects associated with cardiovascular disease.     

Pharmacokinetic studies on ditazole, a novel inhibitor of platelet aggregation.

The distribution of ditazole in blood and tissues of rats was determined by a simple GLC technique. Ditazole, after intravenous injection in rats (20 mg/kg), entered preferentially into the brain, the liver, and the heart in decreasing order. In the epididymal adipose tissue, the drug was present only in small amounts. Ditazole disappeared from the rat organs 4 hr after the treatment. The apparent ditazole half-life in rat blood was 41 min, the volume of distribution was 2.068 liters/kg, and the body clearance was 0.0345 liter/kg/min.     

Eptifibatide: a potent inhibitor of the platelet receptor integrin,glycoprotein IIb/IIIa

Platelet aggregation is intimately involved in the pathophysiology of acute coronary syndromes. Blockade of the platelet glycoprotein IIb/IIIa receptor, the mediator of platelet aggregation induced by all physiologic agonists, prevents arterial thrombosis in animal models far more effectively than aspirin. Eptifibatide (Integrilin ?) is a rapidly reversible competitive inhibitor of glycoprotein IIb/IIIa studied in a broad range of ischaemic coronary conditions, including percutaneous coronary intervention, ST-segment and non-ST-segment acute myocardial infarction and unstable angina. In each case, therapy with eptifibatide has reduced acute ischaemic complications without any increased risk of life-threatening bleeding or adverse events. Based on data from the Integrelin to Minimize Platelet Aggregation and Coronary Thrombosis (IMPACT) II study, a salutary benefit in the range of a 20 - 25% reduction in adverse clinical events can be expected in patients undergoing coronary intervention. Few significant pharmacological effects other than inhibition of platelet aggregation and the effect on bleeding time have been reported. Future research will focus on alternative doses, infusion regimens and combinations with other therapies to improve further cardiovascular outcomes.     

Eptifibatide: a potent inhibitor of the platelet receptor integrin, glycoprotein IIb/IIIa

Platelet aggregation is intimately involved in the pathophysiology of acute coronary syndromes. Blockade of the platelet glycoprotein IIb/IIIa receptor, the mediator of platelet aggregation induced by all physiologic agonists, prevents arterial thrombosis in animal models far more effectively than aspirin. Eptifibatide (Integrilintrade mark) is a rapidly reversible competitive inhibitor of glycoprotein IIb/IIIa studied in a broad range of ischaemic coronary conditions, including percutaneous coronary intervention, ST-segment and non-ST-segment acute myocardial infarction and unstable angina. In each case, therapy with eptifibatide has reduced acute ischaemic complications without any increased risk of life-threatening bleeding or adverse events. Based on data from the Integrelin to Minimize Platelet Aggregation and Coronary Thrombosis (IMPACT) II study, a salutary benefit in the range of a 20 - 25% reduction in adverse clinical events can be expected in patients undergoing coronary intervention. Few significant pharmacological effects other than inhibition of platelet aggregation and the effect on bleeding time have been reported. Future research will focus on alternative doses, infusion regimens and combinations with other therapies to improve further cardiovascular outcomes.     

The effects of siguazodan, a selective phosphodiesterase inhibitor, on human platelet function

1. The effects of siguazodan (SK&F 94836) a selective phosphodiesterase (PDE) inhibitor with inotropic and vasodilator activity, were studied on human platelets. 2. Siguazodan selectively inhibited the major cyclic AMP-hydrolysing PDE in human platelet supernatants. The inhibited enzyme has been variously termed cyclic GMP-inhibited PDE or PDE-III. 3. In platelet-rich plasma (PRP), siguazodan inhibited U46619-induced aggregation more potently than that induced by ADP and collagen. Treatment of the PRP with aspirin had no effect on the potency of siguazodan. 4. In washed platelets, siguazodan increased cyclic AMP levels and reduced cytoplasmic free calcium [( Ca2+]i). ADP decreased the ability of siguazodan to raise cyclic AMP and this may explain its lower potency in inhibiting responses to ADP. 5. Siguazodan has anti-platelet actions over the same concentration range that it is an inotrope and vasodilator.