| Objectives:Inhibitive potencial of drugs on CYP450 enzymes was the most important factor causing drug-drug interactions, which manifested that one drug slow down the metabolism of the combination-drug by decreasing the activity of hepatic microsomal enzyme, so that enhanced efficacy or produced toxicity of the combination-drug, leading to adverse reactions. Study of the inhibitive potential on CYP450 of innovative drugs should be conducted early in the development to predict the in vivo drug-drug interactions and to develop more safe and more effective drugs, and to guide clinical medication. Fluorescence combined with the recombinase was the traditional method which can not be used to study the drugs with the interference of fluorescence, and the acquired results can not deduce to the in vivo directly for that the high activity of the recombinase. The aim of this study was to develop an LC-MS/MS method to analysis the corresponding metabolites of CYP450, and taking human microsomes as the study model to establish a rational, accurate and reliable in vitro evaluation system by optimizing a set of related issues. The system was applied to assess the inhibitive potencial on 7 CYP450 enzymes of innovative drugs and to predict the in vivo drug-drug interactions, provide information for further clinical study. Methods:Establish an LC-MS/MS method to quantify the corresponding metabolites of CYP450 enzymes specific substrates, and conduct a fully validation including: selectivity/specificity, residue of sample, interfering of the internal standard and the analytes, the linearity and lower limit of quantification, accuracy and precision, matrix effects, extraction recovery, stability and dilution effects, etc. Establish an in vitro inhibition evaluation system of CYP450 enzymes using human liver microsomes by investigating the inhibitive potencial of 7 specific inhibitors to verify the reliability of the system. The system was applied to study the inhibitive potential on the 7 CYP450 enzymes of human liver microsomes by three innovative drugs, and to predict the in vivo drug-drug interactions. Results:The LC-MS/MS method showed good specificity, each metabolite showed good linearity(r>0.99) in the matrix of human liver microsomes. The lowest limit of quantitation were 50/10/10/50/10/50/20 nM for(CYP1A2/CYP2C19/CYP2D6/ CYP3A4/CYP2B6/CYP2C8/CYP2C9), the accuracy(RE) of the 7 metabolites was ranged from-3.20 to 8.22%, and the intra- and inter-day precision(RSD) were 1.84~10.2% and 0.900~14.9% at three levels. The 7 specific inhibitors showed significant inhibition on the corresponding CYP450 enzymes in the evaluation system, the measured IC50 values were approximate with the reported values in the literatures. The system was applied to study the inhibitive potential on the 7 CYP450 enzymes of human liver microsome by three innovative drugs, and the results showed that the IC50 of candidate B on CYP2D6 was 21.7 ?M, the rest of the values were >30 ?M. Conclusions:The established LC-MS/MS method was sensitive, rapid, specific and reliable to quantify the corresponding metabolites of CYP450 enzymes in the incubation system simultaneously. The 7 specific inhibitors showed significant inhibition on the corresponding CYP450 enzymes in the evaluation system, which showed that the system was reliable to evaluate the inhibition. The system was applied to study the inhibitive potential on the 7 CYP450 enzymes of human liver microsome by three innovative drugs(A, B, C), the results showed that candidate drug B showed inhibition to some extent(IC50 21.7 ?M), the rest of the values were >30 ?M, which showed low possibility to inhibit CYP450 enzymes. The established evaluation system can be successfully applied in the study the inhibitive potential on the 7 CYP450 enzymes of human liver microsomes by innovative drugs. |
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