| Objective1.To develop a LC-MS/MS method for simultaneous determination of scutellarin,clopidogrel and its carboxylic acid metabolite SR26334 in rat plasma,and to investigate the combined effects of clopidogrel and dengzhan shengmai capsules on pharmacokinetics in rats.2.To evaluate the combined effects of clopidogrel and dengzhan shengmai capsules by studying two drugs interaction on the platelet aggregation in rats.3.To investigate the safety on the combined effects of clopidogrel and dengzhan shengmai capsules by studying two drugs interaction on the bleeding and clotting time in mice.Methods1.A accurate and sensitive LC-MS/MS method was developed for simultaneous determination of scutellarin,clopidogrel and its carboxylic acid metabolite(SR26334)in rat plasma.SD rats were randomly divided into three groups,clopidogrel group,dengzhan shengmai capsule group,or dengzhan shengmai capsule with clopidogrel group.All the rats were administered respectively to three groups by gavage.2.56 male SD rats were randomly divided into eight groups,control group(equal volume of distilled water),clopidogrel group,low-dose dengzhan shengmai capsule group,middle-dose dengzhan shengmai capsule group,high-dose dengzhan shengmai capsule group,low-dose dengzhan shengmai capsule with clopidogrel group,middle-dose dengzhan shengmai capsule with clopidogrel group and high-dose dengzhan shengmai capsule with clopidogrel group.Each group had 7 rats.All the rats were administered for a week by gavage and drawn blood from abdominal aorta for determination of platelet aggregation rate at 1h after the last administration.3.80 half male and half female BALB/c mice were randomly divided into eight groups,control group(equal volume of distilled water),clopidogrel group,low-dose dengzhan shengmai capsule group,middle-dose dengzhan shengmai capsule group,high-dose dengzhan shengmai capsule group,low-dose dengzhan shengmai capsule with clopidogrel group,middle-dose dengzhan shengmai capsule with clopidogrel group and high-dose dengzhan shengmai capsule with clopidogrel group.Each group had 10 mice.All the mice were administered for 6 days by gavage and determined bleeding and clotting time at 1h after the last administration.Results1.The calibration curve of clopidogrel was linear in the concentration range of 0.14?72.00ng·mL-1 and the correlation coefficient was better than 0.999.The lowest limit of quantification was 0.14ng·mL-1.The calibration curve of SR26334 was linear in the concentration range of 8.80?4500.OOng·mL-1 and the correlation coefficient was better than 0.999.The lowest limit of quantification was 8.80ng · mL-1.The calibration curve of scutellarin was linear in the concentration range of 1.31?670.OOng·mL-1 and the correlation coefficient was better than 0.999.The lowest limit of quantification was 1.31 ng · mL-1.The matrix effect of clopidogrel,SR26334 and scutellarin were within the range of 86?101%.The recovery,intra-day and inter-day variances met the requirement of biological sample analysis.2.The plasma concentration of clopidogrel increased rapidly after oral administration of clopidogrel or clopidogrel combining with dengzhan shengmai capsules,and then reached the maximum blood concentration at 0.5h.Comparing with clopidogrel group,AUC0-t decreased,while MRT0-?,and t1/2 increased significantly.There was no statistically significant between clopidogrel group and dengzhan shengmai capsule group(P>0.05).Meanwhile,Cmax decreased and MRT0-t increased obviously,there were statistically significant(P<0.01 or P<0.05).The result suggested that the maximum blood concentration of clopidogrel decreased and its mean retention time prolonged after oral administration of clopidogrel combining with dengzhan shengmai capsules.3.The plasma concentration of SR26334 increased rapidly after oral administration of clopidogrel or clopidogrel combining with Dengzhan shengmai capsules,and then reached the maximum blood concentration at 0.5h.Comparing with clopidogrel group,MRT0-t and AUC0-? of SR26334 had no obvious change,AUC0-t and Cmax decreased,while MRT0-??Tmax and t1/2 increased obviously.There was no statistical significance between clopidogrel group and dengzhan shengmai capsule group(P>0.05).The result showed that the peak time,the mean retention time and half-life of SR26334 would be prolonged after oral administration of clopidogrel combining with dengzhan shengmai capsules.4.The average plasma concentration-time curve of scutellarin presented multiple-peak phenomenon.Comparing with dengzhan shengmai capsule group,t1/2?Tmax and Cmax of scutellarin had no obvious change,AUC0-t and MRT0-t increased,AUC0-? and MRT0-? increased obviously.There was a great individual difference in laboratory animals that causes AUC0-? and MRT0-? had no statistical significance.The result indicated that there was a great individual difference on pharmacokinetic behavior of scutellarin.5.The rats platelet aggregation result showed that the administration group could significantly reduce the rate of platelet aggregation in rats.There were statistically significant(P<0.05).Comparing with clopidogrel group,middle-dose and high-dose dengzhan shengmai capsule with clopidogrel group reduced platelet aggregation significantly in rats.There were statistically significant(P<0.05).The result showed that the combined using of clopidogrel and dengzhan shengmai capsules could enhance the inhibition of ADP-induced platelet aggregation of clopidogrel.While the dosages of dengzhan shengmai capsules were increasing,the inhibition of ADP-induced platelet aggregation was enhancing.6.Comparing with control group,the administration group affected the bleeding time in mice.There were statistically significant(P<0.01).Comparing with clopidogrel group,high-dose dengzhan shengmai capsule group and all dosages of dengzhan shengmai capsule with clopidogrel group prolonged the bleeding time of mice.There were statistically significant(P<0.05 or P<0.01).The mice bleeding time experiment result showed that the combined using of clopidogrel and dengzhan shengmai capsules would prolong the bleeding time and enhanced the anti-coagulation effect.Comparing with control group,the administration group affected the clotting time in mice.There were statistically significant(P<0.01).Comparing with clopidogrel group,middle-dose and high-dose dengzhan shengmai capsule with clopidogrel group prolonged the clotting time of mice.There were statistically significant(P<0.05 or P<0.01).The mice clotting time experiment result implied that the combined using of clopidogrel and low-dose dengzhan shengmai capsules would reduce or avoid adverse reaction of bleeding.The combined using of clopidogrel and high dosages of dengzhan shengmai capsules would prolong the bleeding time and increase the risk of hemorrhage.ConclusionA LC-MS/MS method was developed for simultaneous determination of scutellarin,clopidogrel and its carboxylic acid metabolite SR26334 in rat plasma.It was investigated that combined effects of clopidogrel and dengzhan shengmai capsules on pharmacokinetics in rats.Pharmacokinetics study showed that combined using of clopidogrel and dengzhan shengmai capsules had certain effect on pharmacokinetic behavior in rats.Cmax of clopidogrel decreased and MRT0-t increased obviously,there were statistically significant.It was indicated that combined using of those two drugs would decrease the maximum blood concentration and prolong the mean retention time of clopidogrel.Combined using of dengzhan shengmai capsules and clopidogrel could enhance the inhibition of ADP-induced platelet aggregation.While the dosages of dengzhan shengmai capsules were increasing,the inhibition of ADP-induced platelet aggregation was enhancing.Combined using of those two drugs could enhance the anticoagulant effect,too.With the dosages of dengzhan shengmai capsules were increasing,the risk of bleeding was increasing.The results showed that the combined using of clopidogrel and dengzhan shengmai capsules induced drug-drug interaction.It was implied that doctors should perform individual medication scheme for patients and pay close attention to the adverse reaction when combined using of clopidogrel and dengzhan shengmai capsules for ischemic stroke therapy.Doctors should reduce the dosages of dengzhan shengmai capsules as lower as possible for decreasing or avoiding the adverse reaction of bleeding. |
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