Study On The Cytochrome P450Isoforms Involved In α-hydroxylation And O-demethylation Metabolism Of Metoprolol

Background：Metoprolol is a selective β₁adrenoceptor antagonist, is used in clinical practicefor the treatment of myocardial infarction,heart failure,and arterial hypertension.Study show that metoprolol are mainly metabolized by hepatic cytochrome P450viaseveral metabolic pathway in liver,65%via the O-demethylation pathway andmetabolic product is O-demethylmetoprolol,10%via the α-hydroxylation pathwayand metabolic product is α-hydroxymetoprolol.As CYP2D6is involved in themetabolism of metoprolol,but foreign scholars have found that, maybe exceptCYP2D, CYP3A is still involved in the metabolism of metoprolol in rat.the researchwill study on if any other cytochrome P450isoforms involved in the metabolism ofmetoprolol except CYP2D6, and indentify what kinds of the cytochrome P450isoforms is involved in α-hydroxylation and O-demethylation pathway in themetabolism of metoprolol.Objectives：We observe the metabolic of MET in human liver microsomal incubationsystem, Then we confirme the metabolic pathway of metoprolol using recombinantenzymes. To study the metabolism produce differences and its molecular mechanismof metoprolol in wild-type and different points mutant CYP3A4recombinant enzyme;analysis the experimental data of metoprolol in vitro studies, To provide a theoreticalbasis for in vivo experiments and the individual drugs in clinical.Methods：1.To establish the incubation method of metoprolol in human liver microsomesand CYP3A4recombinant enzyme and the sample processing methods,set up theHPLC-Flu analyse method of metoprolol,active metabolites α-hydroxymetoprolol andO-demethylmetoprolol, a full verification methodology with reference to biologicalsample analysis.2.Analysis enzymatic kinetic of metoprolol in human liver microsomes in vitro study. The experiment was divided into control group and test group, total volume ofincubation is200μL in human liver microsomes,test group add NADP reactionsystem60μL（MgCl₂5mM,G-6-P10mM,G-6-PD2U,NADP⁺1mM）intoincubation system, control group add PBS instesd, and final concentration of humanliver microsomes in the incubation system is1mg/ml, each group were added a seriesof concentrations of metoprolol10μL, final concentration of the metoprolol are5、10、20、40、80、100、160μM. The reaction time is60min after add100μL ice acetonitrileto terminate the reaction, samples were treated with HPLC-Flu method for thedetermination of the amount of metoprolol and their active metabolitesα-hydroxymetoprolol and O-demethylmetoprolol in the reaction system, and comparemetoprolol and the metabolism and pharmacokinetics in human liver microsomes,using the Michaelis-Mentent equation model and cartography of Lineweaver-Burk tocompare the enzyme kinetics parameters V_max, K_mand CL_intof active metabolitesα-hydroxymetoprolol and O-demethylmetoprolol in human liver microsomes.3.Analysis the effect of enzyme inhibitor of CYP450on the metabolies ofmetoprolol,the active metabolites O-demethylmetoprolol in human livermicrosomes.and The experiment was divided into control group and test group, add aseries of enzyme inhibitor of CYP450in test group,and final concentration of themetoprolol is40μM. add a series of enzyme inhibitor of CYP450such as CYP1A2inhibitor α-Naphthoflavone （α-Nap）, CYP2E1inhibitor Disulfiram （Dis）, CYP2C9inhibitor sulfaphenazole （Sul）, CYP2C19inhibitor S-mephenytoin （S-mep）,CYP2D6inhibitor Quinidine （Qui）, CYP3A4inhibitor Troleandomycin （Tro）,CYP3A4&CYP2D6inhibitor Ketoconazole（KTZ）10μL, final concentration in each inhibitor intest group is α-Nap5μM、Dis10μM、Sul30μM、S-mep50μM、Qui5μM、Tro50μM、KTZ3μM, control group add PBS instesd, and each test group were used toinvestigate the inhibit effects on the metabolism of metoprolol, the active metabolitesα-hydroxymetoprolol and O-demethylmetoprolol in human liver microsomes.4.Analysis enzymatic kinetics of metoprolol in the wild-type and different pointmutant CYP3A4recombinant enzyme in vitro study. The experiment was dividedinto control group, CYP3A4^*1CYP3A4^*3, CYP3A4^*5, CYP3A4^*16, andCYP3A4^*18recombinase group, total volume of incubation is200μL,test group add NADP reaction system60μL（MgCl₂5mM,G-6-P10mM,G-6-PD2U,NADP⁺1mM）into incubation system, control group add PBS instesd, and final concentrationof CYP3A4recombinant enzyme in the incubation system is1mg/ml, each groupwere added a series of concentrations of metoprolol10μL, final concentration of themetoprolol are5、10、20、40、80、100、160μM. The reaction time is60min after add100μL ice acetonitrile to terminate the reaction, samples were treated with HPLC-Flumethod for the determination of the amount of metoprolol and their active metabolitesα-hydroxymetoprolol and O-demethylmetoprolol in the reaction system,and comparemetoprolol in CYP3A4wild type and the metabolism and pharmacokinetics betweenthe wild-type and mutant CYP3A4, using the Michaelis-Menten equation model andcartography of Lineweaver-Burk to compare the enzyme kinetics parameters V_max,K_m, CL_int,of the active metabolites α-hydroxymetoprolol and O-demethylmetoprololin the wild-type and different point mutant CYP3A4recombinant enzyme.5.The statistical analysis of data. All data are deviate by mean±standard （mean±SD）, using SPSS12.0software for data processing, the difference between the groupswith the design of the t-test, statistical results P<0.05was considered the difference issignificant.Results：1.Established successfully incubation method of metoprolol in human livermicrosomes and CYP3A4recombinant enzyme and sample processing methods,Established the HPLC-Flu analyse method of metoprolol and the active metabolitesα-hydroxymetoprolol and O-demethylmetoprolol with high sensitivity and specificity,biological samples analysis methodology conform to its requirements.2.Enzymatic kinetics of metoprolol in human liver microsomes under theincubation condition of37℃,120rpm/min, between30-180minutes, metabolic ofmetoprolol present linear eliminate,the best incubation time is60min,and finalconcentration of human liver microsomes in the incubation system is1mg/ml, theconcentration of metoprolol between5-40μM, metabolic of α-hydroxymetoprolol andO-demethylmetoprolol present linear generate, while in100μM, active metabolitehave no change, metabolism of metoprolol in human liver microsomes comply withthe typical enzyme kinetics characteristics.using the Michaelis-Mentent equation model and cartography of Lineweaver-Burk to compare the enzyme kineticsparameters V_max,K_mand CL_intof α-hydroxymetoprolol and O-demethylmetoprolol inhuman liver microsomes. Results of research enzyme kinetics parameters, values ofV_maxare1.74±1.32pmol/min/mg,111±4.10pmol/min/mg; values of K_mare22.5±1.33μM,35.44±2.23μM, values of CL_intare0.97ml/min/μg,3.13ml/min/μg.3.Test group with CYP2D6inhibitor Quinidine （Qui）, CYP3A4inhibitorTroleandomycin （Tro）,CYP3A4&CYP2D6inhibitor Ketoconazole （KTZ） effect onthe metabolies of metoprolol, compared with control group, metabolic rate ofmetoprolol have significant differences（P <0.05）, furthmore, with CYP2D6inhibitorQuinidine （Qui）, CYP3A4inhibitor Troleandomycin （Tro）,CYP3A4&CYP2D6inhibitor Ketoconazole（KTZ） effect on the formation of the active metabolites, Quiand KTZ inhibit formation of α-hydroxymetoprolol,and Qui,KTZ and Tro inhibitformation of O-demethylmetoprolol,compared with control group, formation rate ofα-hydroxymetoprolol and O-demethylmetoprolol have significant differences（P<0.05）.4.Enzymatic kinetics of metoprolol in the wild-type and different point mutantCYP3A4recombinant enzyme under the incubation condition of37℃,120rpm/min,between0-60minutes, metabolic of metoprolol present linear eliminate,the bestincubation time is30min,and final concentration of CYP3A4recombinant enzyme inthe incubation system is1mg/ml, using the Michaelis-Mentent equation model andcartography of Lineweaver-Burk to compare the enzyme kinetics parameters V_max,K_m, CL_intof the active metabolites O-demethylmetoprolol in the wild-type anddifferent point mutant CYP3A4recombinant enzyme. Results of research areCYP3A4^*1enzyme kinetics parameters of the K_m, V_max, CL_intvalues are35.25±5.36μM,103.45±7.28pmol/min/mg,2.93ml/min/μg; Enzymatic kinetic parametersK_m,V_maxand CL_intvalues of CYP3A4^*3are28.13±4.27μM,111.11±4.37pmol/min/mg,3.95ml/min/μg, its activity is significantly greater than the wild type; enzymekinetics parameters K_m,V_max,CL_intvalues of CYP3A4^*5are39.61±3.13μM,116.32±9.14pmol/min/mg,2.94ml/min/μg, its activity slightly larger than the wildtype;CYP3A4^*16enzymatic kinetic parameters of the K_m,V_maxand CL_intvaluesare43.60±5.67μM,129.43±6.32pmol/min/mg,2.97ml/min/μg,its activity slightly larger than the wild type; CYP3A4^*18enzyme kinetics parameters of the K_m,V_maxand CL_intvalues are60.33±6.27μM,91.38±5.15pmol/min/mg,1.51ml/min/μg, its activity lowerthan the wild type.Conclusions：In this subject,analysis metabolism of metoprolol in human liver microsomes invitro study, find which CYP450enzyme related with vitro metabolism of metoprolol,we infer that CYP2D6involved in α-hydroxylation pathway and CYP2D6andCYP3A4involved in O-demethylation pathway in the metabolism of metoprolol inhuman.we infer except for CYP2D6, metabolism of metoprolol also related withCYP3A4; as well as wild type and different sites of CYP3A4mutant in vitrometabolism study, to elucidate the effect of CYP3A4gene polymorphism on themetabolism of metoprolol,compared with CYP3A4^*1, the activity of CYP3A4^*3andare higher, the activity of CYP3A4^*5and CYP3A4^*16are on behalf similar,theactivity of CYP3A4^*18is lower than the wild-type of CYP3A4.