Comparative Pharmacokinetic Study Of Clopidogrel And Analogs

Purpose:Clopidogrel was an oral antiplatelet agent used to prevent blood clots in coronary artery disease,peripheral vascular disease,and cerebrovascular disease.But there were still many drawbacks in clinical application of Clopidogrel.(1)low bioavailability: Clopidogrel was primarily metabolized to inactive carboxylic acid derivative,generated by hydrolysis.(2)affected by genetic polymorphisms: clopidogrel was metabolized to the the pharmacologically active metabolite(AM)via an inactive 2-oxoclopidogrel by a two-step cytochrome P450 oxidation process.The enzymes involved in the oxidation reaction were “weak metabolite enzymes.” In view of these drawbacks of clopidogrel in clinical application,we modify the structure of clopidogrel: deuterium modification and esterification modification.In order to compare the difference betweeen the active metabolite of clopidogrel and analogs,a quantitative method for the active metabolite was established based on LC-MS/MS.Method:Clopidogrel and analogs were synthesized.These compounds were administered to rats and beagle dogs by gavaged and divided into three groups: clopidogrel and D-clopidogrel?Vicagrel and D-vicagrel?clopidogrel and D-vicagrel.Plasma samples were immediately centrifuged at low temperature after collected.The supernatant immediately added alkylating reagent 2-bromo-3?-methoxyacetophenone(MPB)to stabilize the thiol group of clopidogrel active metabolite.The LC separation was performed by using water containing 0.1% formic acid and acetonitrile as mobile phases with gradient elution at a flow rate of 1 ml/min.Quantitative analysis was achieved after the chromatographic separation on an Ascentis C18 column(5 cm × 4.6 mm,5?m).The electrospray ionization(ESI)source in positive mode and multiple reaction monitoring(MRM)were used for the detection of clopidogrel AM derivative(MP-AM).The ion transition of m/z 504.1? m/z 354.1 was selected for the analyst,and the ion transition of m/z 436.0? m/z386.9 was selected for bipheny diester which was used as internal standard(IS).The linear rang of MP-AM was 1~1000 ng/ml.The specificity,linearity,accuracy,precision,the lower limit of quantification,extraction recovery,matrix effect,residual effect and stability were evaluated for method validation according to CFDA.Rseult:(1)Method established: LC-MS/MS method was established and validated in different matrix.The method was accurate,sensitive and simple,the peaks were narrow and symmetrical.The method has no matrix interference whatever in rat or beagle dog plasma.The intra-assay/inter-assay accuracy and precision were all acceptable,and this method meets the needs of clopidogrel pharmacokinetic studies.(2)Comparative pharmacokinetic study of clopidogrel and analogs: By calculating the pharmacokinetic parameters,the results were shown: in the group of clopidogrel and D-clopidogrel or Vicarel and D-vicagrel,the AUC of MP-DAM was 20% higher than MP-AM whatever in rat or beagle dog plasma;in the group of clopidogrel and D-vicagrel,the AUC of MP-DAM was four times higher than MP-AM whatever in rat or beagle dog plasma.It is shown that the artificially designed by strong(Deuterization)or weak(Esterification)can reduce the effect of metabolic enzymes and improve the bioavailability of clopidogrel.