Comparative Study On The Effect Of 4 Different CYP2D6 Enzymes On Drug Metabolism And Drug-drug Interactions

Objective:CYP450?cytochrome P450?is a major enzyme in the liver,which involves in the metabolism of varieties of endogenous and exogenous substances,and is responsible for the activation and clearance of drugs in vivo.CYP2D6 is a one of the most important subfamily in CYP450,which exhibits extensive genetic polymorphism.Generally,the change of the enzymatic activity in CYP2D6 mutants,affecting the metabolism and interaction of drugs,is the main genetic factor causing adverse drug reactions and side effects.Therefore,the research referring to the effect of CYP2D6 mutant enzymes on drug metabolism and drug interactions is of great significance for dose adjustment,drug combination and individualized threapy.Method:In our study,four most representative CYP2D6 allelic variants in Asian population were selected as follows:CYP2D6*1?wild-type?,CYP2D6*2,CYP2D6*10 and CYP2D6*39.And six drugs,comprising of quinidine,propafenone,amitriptyline,risperidone,fluvoxamine and metoprolol,were chosen as inhibititors.Then,using metoprolol as substrate to establish the kinetic study and dextromethorphan as substrate for inhibition study,we investigate the difference of CYP2D6 mutant enzymes in participation of drug metabolism and drug interactions.We select the metoprolol as probe,and the oncentration of?-hydroxymetoprolol as a measurement for the kinetic study.Firstly,three incubation parameters including substate concentration,incubation time and enzyme concentration was optimized to establish CYP2D6 enzymes-metoprolol incubation systems in vitro.Then,all incubation samples were processed,LC-MS/MS method was adopted for the determination of HM in samples.Lastly,using software to calculate the values of K_m,V_max,and the difference in the interaction between CYP2D6 mutant enzymes and metoprolol was characterized by these values.To explore the difference of CYP2D6mutant enzymes in drug interaction,six inhibitors,including quinidine,propafenone,amitriptyline,risperidone,fluvoxamine and metoprolol were tested.We select dextromethorphan as probe,and oncentration of dextrorphan as a measurement for the inhibition study.Firstly,three incubation parameters including substate concentration,incubation time and enzyme concentration was optimized to establish CYP2D6 enzymes-dextromethorphan incubation systems in vitro.Then,all incubation samples were processed,LC-MS/MS method was adopted for the determination of dextrorphan in samples.Finally,using software to calculate the values of half maximal inhibitory concentration(IC₅₀)of various drugs towards CYP2D6 enzymes.IC₅₀0 were used as indication for the comoarison of CYP2D6mutant enzymes in drug interaction.Computer simulation was used for detecting the molecular docking of four CYP2D6 enzymes and sustrates,inhibitors to analyze the effect of different mutants on structure of CYP2D6.Results:1.The valuses of K_m,V_max and CL_int in CYP2D6*1?wild type?incubation system are 11.31±1.21?M,85.06±4.12 nM/min,7.52 nM/min/?M,its relative clearance(CL_int*1)was defined as 100.00%.Three parameters in CYP2D6*2?variants?incubation system are 28.13±4.27?M,78.23±5.49 nM/min,5.63nM/min/?M,CYP2D6*2 demonstrated the second high catalytic activity(CL_int*2/*1=74.87%).Three parameters in CYP2D6*10?variants?incubation system are 31.46±6.20?M,21.08±2.55 nM/min?0.67 nM/min/?M,CYP2D6*10 showed minimal catalytic activity(CL_int*10/*1=8.91%).Three parameters in CYP2D6*39?variants?incubation system are 18.56±1.83?M,41.38±2.08 nM/min,2.23nM/min/?M,CYP2D6*39 also showed minimal catalytic activity(CL_int*39/*1=29.65%).2.Compared with IC₅₀0 values towards CYP2D6*1 for each drug,IC₅₀ values towards CYP2D6*10 were 2.5-6.7 fold higher.Whereas that towards CYP2D6*1,CYP2D6*2 and CYP2D6*39 exhibited minor difference.3.The value of IC₅₀0 of each drug towards DXM was slightly affected by two mutants of R296C and S486T,while the value of IC₅₀0 were greatly influned by the mutant of P34S.Conclusion:1.Kinetics study revealed that the activity of CYP2D6*2 and CYP2D6*39 was not significantly different from that of the wild type,while the catalytic activity of CYP2D6*10 was obvious lower than that of the wild type.This comprehensive in vitro data suggested the prominent influence of CYP2D6 polymorphisms on the metabolism of MET.The results could predict the drug metabolism and adverse effects of CYP2D6 variants,which offer valuable guidance for personalized administration of MET in clinic.2.Inhibition study showed that there was no significant difference in the inhibitory effect of each drug on CYP2D6*1,CYP2D6*2 and CYP2D6*39.However,the weakest inhibitory action of all inhibitors towards CYP2D6*10 metabolizing DXM was discovered.These data could predict DDIs related to CYP2D6 enzyme and also supply reliable theory and reference for drug combination.3.The influence of R296C and S486T were not significant on the active site in CYP2D6 enzyme.However,the active stite in CYP2D6 enzyme was affected by P34S.