Species Difference Of Asarinin Metabolism In Vitro And Its Effect On The Activity Of Cytochrome P450 Enzymes

ObjectiveThe study was to investigate the metabolic behaviors of asarinin in different species,to find animal models with similar metabolic pathways to human beings,and to identify the metabolic enzymes involved in asarinin metabolism.Meanwhile,the interactions between asarinin and cytochrome P450 s were studied to reveal the potential risks of asarinin when being used together with other drugs,so as to provide correct guidance for clinical use of asarinin and evaluate the safety of asarinin.MethodsAsarinin was incubated with seven different liver microsomes(humans,rabbits,mini pigs,dogs,monkeys,rats and mice)in phase I and phase II metabolic reaction systems,the metabolites of asarinin were detected by HPLC,and the changes of product production were observed.The enzymatic kinetics of liver microsomes from different species were determined to study the interspecies differences of asarinin metabolism in vitro.Meanwhile,asarinin was incubated with 13 CYP enzyme subtypes(CYP1A1、CYP1A2、CYP2A6、CYP2B6、CYP2C8、CYP3A4、 CYP2C19、 CYP3A5、 CYP1B1、 CYP2C9、CYP2D6 、 CYP2E1 、 CYP4F2).The changes of asarinin metabolites were determined by HPLC,and the metabolic enzymes involved in asarinin were identified.In addition,the effects of asarinin(0-100 μM)on enzymes activity were determined by incubating six CYP enzymes in human liver microsomes,the interaction between asarinin and cytochrome P450 enzymes was studied,the inhibition type(non-competitive inhibition or competitive inhibition)was determined by drawing Dixon and Lineweaver-Burk curve.The Ki value was calculated by the slope of Lineweaver-Burk curve and drug concentration.ResultsThe metabolic profiles exhibited that there were important differences in the metabolism of asarinin among the seven different species.Rats,pigs,and monkeys liver microsomes exhibited stronger metabolic capacities in phase I reaction system.Monkeys showed high metabolic capacities in phase I and phase II reaction system,respectively.In vitro inhibition experiments showed that asarinin could inhibit the activity of CY2C9,CYP3A4 and CYP2E1.CYP3A5、 CYP2C8、 CYP1A1 and CYP4F2 had been identified to be the main cytochrome P450 isoforms that catalyzed asarinin metabolism.Asarinin inhibited the activity of CYP2C9 and CYP2E1 competitively,and inhibited the activity of CYP3A4 noncompetitively.ConclusionsThe study found that dogs had similar metabolic pathways to humans,and could be used as animal models to investigate the metabolism of asarinin.Liver was the main place of asarinin metabolism.CYP3A5、 CYP2C8、 CYP1A2 and CYP4F2 were proved to be the major CYP450 enzymes involved in asarinin metabolism.Asarinin could inhibit the activity of CYP2C9,CYP2E1 and CYP3A4,which indicated that there were potential drug-drug interactions between asarinin and some drugs metabolised by CYP2C9,CYP2E1,and CYP3A4.