| BackgroundIschemic heart disease,also known as coronal atherosclerosis heart disease,is the deadliest disease of the world today,which severely impairs humanity health.Once coronary atherosclerosis ruptured,it could lead to the activation of platelet,subsequently lead to the formation of the thrombus and a range of serious symptoms,even to death.Platelet P2Y12 receptor plays an crucial role in platelet activation and ADP is the nature ligand.At present,clopidogrel,an antiplatelet medicine which target P2Y12 receptor,is widely used in the clinic forits precise curative effect and good tolerance.However,clopidogrel is a prodrug with no activity until it is been metabolized by cytochrome P450 enzyme oxidization.For this reason,clopidogrel has some defects as fellows:slow onset(6h after administration),long elimination(5-7d),clopidogrel resistance and so on.A series of 2-hydroxyetrahydrothienopyridine derivatives were designed and synthesized as prodrugs of clopidogrel thiolactone.Compound 3(vicagrel)was found to inhibit platelet aggregation during preliminary screening.The antiplatelet activities of vicagrel were evaluated in several animal models,and the preliminary antiplatelet activity of 20 metabolites of vicagrel were studied in this paper.MethodsPart one:antiplatelet drug screeningCompounds were prepared into suspension by 1%CMC-Na,intragastrically administrated for wistar rats(3mg/kg).After two hours,platelet rich plasma(PRP)and platelet poor plasma(PPP)was extracted with sodium citrate(1:9)from abdominal aortic of rats anesthetized with urethane.Platelet aggregation were observed with inducing agent ADP(final concentration of 1.2μM),and the inhibition of platelet aggregation was calculated.Part two:pharmacodynamic investigation of vicagrel1.Time-effect relationship:Using clopidogrel(10mg/kg,30mg/kg),prasugrel(lmg/kg,3mg/kg)as positive control,1%CMC-Na as negative control,vicagrel(lmg/kg,3mg/kg)as experimental groups,intragastrically administrated in wistar rats.Blood plasma were collected from abdominal aortic at 0.5h,1h,2h,3h,4h,12h with 3.8%sodium citrate(1:9)after administration,PRP and PPP were prepared by centrifugation.Platelet aggregation were observed with inducing agents ADP(final concentration of 1.2 μM),and the inhibition of platelet aggregation at various time points was calculated.2.Dose-response relationship:Using clopidogrel(lmg/kg,3mg/kg,10mg/kg,30mg/kg),prasugrel(O.lmg/kg,0.3mg/kg,lmg/kg,3mg/kg)as positive control,1%CMC-Na as negative control,vicagrel(O.lmg/kg,0.3mg/kg,lmg/kg,3mg/kg)as experimental groups,intragastrically administrated in wistar rats.Blood plasma were collected from abdominal aortic after 3h with 3.8%sodium citrate(1:9),PRP and PPP were prepared by centrifugation.Platelet aggregation were observed with inducing agents ADP(final concentration of 1.2μM),and the inhibition of platelet aggregation was calculated.3.The antithrombotic effects of vicagrel on giving electric stimulus to rats’common carotid artery:Using clopidogrel(10mg/kg),prasugrel(3mg/kg)as positive control,1%CMC-Na as negative control,vicagrel(O.lmg/kg,0.3mg/kg,lmg/kg,3mg/kg)as experimental groups,intragastrically administrated in rats for three days.Giving 1.5mA electric stimulus to rats’ common carotid artery to bring on thrombosis after three hours of the last administration.Occlusion time(OT)of different groups were measured.4.The antithrombotic effects of vicagrel on arteriovenous shunt thrombosis model:Using clopidogrel(10mg/kg),prasugrel(3mg/kg)as positive control,1%CMC-Na as negative control,vicagrel(0.1mg/kg,0.3mg/kg,1mg/kg,3mg/kg)as experimental groups,intragastrically administrated in wistar rats for three days.After three hours of the last administration,the left carotid arteries and the right external jugular vein were isolated,the arteriovenous shunt was established.The weight of thrombus in bypass was measured.5.The effects of vicagrel on bleeding time in mouse model:Using clopidogrel(16mg/kg),prasugrel(1.6mg/kg)as positive control,1%CMC-Na as negative control,vicagrel(0.16mg/kg,0.48mg/kg,1.6mg/kg)as experimental groups,intragastrically administrated for three days.Bleeding time(BT)was determined by cutting-tail method in mice after two hours of the last administration.6.The effects of vicagrel on pulmonary embolism caused by arachidonic acid KM mice were randomly divided into 8 groups:control group(1%CMC-Na,0.1M Na2CO3),model group(1%CMC-Na,AA),vicagrel group(2.5mg/kg,5mg/kg,10mg/kg,20mg/kg),prasugrel group(10mg/kg)and clopidogrel group(100mg/kg).Continuously administrate drugs by drenching for three days.Two hours later after the last administrated,control group were injected Na2CO3(0.1 M)by caudal vein,other groups were injected arachidonic acid.Mortality in three minutes was observed.Part three:antiplatelet activities screening of vicagrel’s metabolitesCompounds were prepared into suspension by 1%CMC-Na,intragastrically administrated for wistar rats(3mg/kg).After two hours,PRP and PPP was extracted with sodium citrate(1:9)by abdominal aortic method from rats anesthetized with urethane.Platelet aggregation were observed with inducing agents ADP(final concentration of 1.2 μM),and the inhibition of platelet aggregation was calculated.ResultsPart one:antiplatelet drug screeningFourteen compounds showed significant antiplatelet activity in rats.Compound 3(vicagrel)shows the highest activity.Part two:Pharmacodynamic evaluation of vicagrel on antiplatelet activities1.Time-effect relationship:vicagrel showed marked inhibition of platelet aggregation and time-effect relationships.The effect of each drug enhanced with the dose increased.The inhibition of platelet aggregation started at one hour after administration and strengthened with time.It kept strongest antiplatelet effect between 1 and 4 hours and decreased at 12 hours later.2.Dose-response relationship:vicagrel,clopidogrel and prasugrel showed marked inhibition of platelet aggregation and dose-effect relationship.IC50:vicagrel 2.03mg/kg;prasugrel 1.91mg/kg;clopidogrel 15.03mg/kg.3.The antithrombotic effects of vicagrel on giving electric stimulus to rats’common carotid artery:vicagrel(0.3mg/kg,1mg/kg,3mg/kg)prolonged occlusion time obviously,indicating that vicagrel had the inhibition of thrombosis in this model.4.The antithrombotic effects of vicagrel on arteriovenous shunt thrombosis.model:vicagrel(0.3mg/kg,1mg/kg,3mg/kg)reduced the weight of thrombosis in arteriovenous shunt,indicating that vicagrel inhibited thrombosis in this model.The IC50 of vicagrel is 0.56mg/kg.5.The effects of vicagrel on bleeding time in mice model:vicagrel(0.48mg/kg,1.6mg/kg)prolonged bleeding time of mice significantly,indicating that vicagrel inhibited the hemostatic function of platelet.6.The effects of vicagrel on fatality rate of pulmonary embolism caused by arachidonic acid:vicagrel group(2.5mg/kg,5mg/kg,10mg/kg,20mg/kg),prasugrel group(10mg/kg)and clopidogrel group(100mg/kg)are failed to reduce death rate,indicating that these drugs had no significant effect on fatality rate of pulmonary embolism caused by arachidonic acid.Part three:antiplatelet activity screening of vicagrel’s metabolitesSeven metabolites inhibited platelet aggregation in rat model,indicating that some metabolites of vicagrel had orally antiplatelet activities.ConclusionVicagrel showed strong activities of anti-platelet aggregation and the effects appeared at one hours after administration.The IC50 of vicagrel on anti-platelet aggregation in rats was 2.03mg/kg,which was much better than clopidogrel(15.03mg/kg)and similar with prasugrel(1.91mg/kg).Vicagrel inhibited the formation of experimental thrombus in animal models and the efficacy was closed to prasugrel.Vicagrel showed no significant effect on fatality rate of pulmonary embolism caused by arachidonic acid.The possible reason may be that vicagrel targets P2Y12 ADP receptor and rarely affects TXA2 receptor.Seven metabolites showed significant antiplatelet activity among twenty metabolites in rat model. |
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