In Vitro Investigation On Human CYP2C8 Polymorphic Function And CYP2C8-genotype Dependent Drug-drug Interactions

Aim:The interin-dividual difference on the catalytic capacity of CYP2C8 is likely to be the result of CYP2C8 genetic polymorphisms. Systematic study on the enzymatic functional variations of important allelic CYP2C8 is significant to pharmacology and toxicology, and it can provide useful information and in vitro data to the personal therapy of clinical CYP2C8-metabolizing drugs. However, there has been no systematic analysis of the catalytic properties of the main CYP2C8 variants in the same expression system and under identical assay conditions. Moreover, the patients carrying CYP2C8 polymorphic genotype may be at increasing risk to CYP2C8-metabolic DDIs due to the interindividual difference of CYP2C8 catalytic properties. However, no report has been able to show whether and how CYP2C8 genetic variants affect the occurrence and the individual differences in the in vitro and in vivo DDIs with CYP2C8-cleared drugs. In this study, we systematically analysed these scientific problems on CYP2C8 genetic polymorphisms, their functional variations and CYP2C8 genotype-dependent DDIs, which have not been worked out.Method:Firstly, wild-type CYP2C8 (CYP2C8.1), three allelic CYP2C8s (CYP2C8.2, CYP2C8.3, CYP2C8.4) and two important CYP2C8 SNPs (R139K and K399R, carried by CYP2C8.3) were chosen, and then were expressed in the same expression system using in vitro recombinant and protein expression technology. We constructed the comprehensive CYP2C8 polymorphic gene library and protein library. Secondly, we investigated the difference on the catalytic properties of wild-type and variants CYP2C8s to the metabolism of anticancer drug (paclitaxel) and antimalaria drug (amodiaquine), using the constructed CYP2C8 polymorphic protein library, and systematically analysed the relationship between CYP2C8 genetic polymorphisms effects and the alteration on CYP2C8 enzymatic property. Thirdly, we evaluated in vitro inhibitory potency of 27 clinical drugs to the metabolism of paclitaxel by wild-type and 5 variant CYP2C8s. Fourthly, in vivo paclitaxel-detected drugs DDIs in different CYP2C8 genotype were predicted via in vitro inhibition data. Fifthly, we primarily developed a high-throughput fluorescent-based detection method for CYP2C8-mediated metabolic DDIs, which can detect the inhibitory potency of durgs or new chemical candidates to wild-type and variant CYP2C8 activities,Results:The investigation on function of wild-type and variant CYP2C8s indicated: 1. in vitro CYPs recombinant technology can be applied in characterization of CYP polymorphic function and in mechanistic study of in vitro drug metabolisms; 2. the fluorogenic substrate-based detection showed that all enzymes expressed in this system possess robust activity; 3. the investigation on in vitro metabolism of paclitaxel and amodiaquine by CYP2C8s showed that CYP2C8.1 has a normal Km value which is comparable to previous data and possesses normal catalytic property; 4. it reveales similar enzymatic activity in R139K and low activity in CYP2C8.2, CYP2C8.3, CYP2C8.4 and K399R compared with CYP2C8.1 and K399R shows the lowest enzymatic activity. The evaluation on DDIs indicated:1. the evaluation on the paclitaxel-detected drugs DDIs showed that IC50 values of the detected drugs to the metabolism of paclitaxel by CYP2C8 variants are significantly different from that of CYP2C8.1. CYP2C8-medicated metabolic DDIs may be affected by CYP2C8 genetic polymorphisms; 2. in vivo DDIs indices [I]/Ki which were calculated via IC50 values and their pharmacokinetic data showed CYP2C8 genotype may change the quality of in vivo DDIs, and we must pay attention to the CYP2C8 genotype effects on DDIs prediction; 3. in vitro recombinant CYPs and fluorogenic substrate-based high-throughput drug screening platform can be primarily applied in the in vitro DDIs study;Conclusions:This study systematically analysed the relationship between CYP2C8 genetic polymorphisms and CYP2C8 catalytic property from the pharmacogenomic and drug-drug interaction perspectives. Systematical analysis on the function of CYP2C8 variants can provide more homogeneous data for predicting CYP2C8 phenotypes and could be applied to personalised drug therapy, especially for paclitaxel and amodiaquine. The evaluation on DDIs between paclitaxel and 27 significantly clinical drugs can be used by pharmacologists in the design of in vivo studies to further assess and address the potential role of CYP2C8 genotype-dependent inhibition in clinical DDIs, and offers a new thinking to prediction on in vivo DDIs via in vitro inhibition data. We constructed a fluorogenic substrate-based CYP2C8 enzymatic-activity detecting and drug screening platform, which can provide a quick, sensitive and high-throughput method to detect CYP2C8 polymorphic activities and drug inhibition;...