Development Of Human Cytochrome 2B6 Polymorphic Enzymes Yeast Expression System And Characterization Of The Polymorphism Effecting On Metabolic Analysis And Drug Screen In Vitro

Human Cytochrome P450 2B6(CYP2B6) belongs to the superfamily of Cytochrome P450 enzymes which catalyze a vast variety of biotransformations,mainly oxidations,of numerous endogenous substrates and xenobiotics.Although cytochrome P450 2B6(CYP2B6) accounts for 5%on average of total hepatic P450 content.The enzyme metabolizes important pharmaceuticals which almost account for 7%of all drugs.Drugs predominantly metabolized by this enzyme include the anticancer prodrug cyclophosphamide,antineoplastic tamoxifen, the narcotic propofol,the antidepressant bupropion,imipramine,the reverse transcriptase inhibitor efavirenz.,and et al.The factor of race,gender,age and disease may affect hepatic expression or activity of the CYP2B6 protein,but the genetic polymorphisms is the fundamental reason to interindividual variability of CYP2B6 drugs metablism.In our study,we aimed to evaluate the activities of five CYP2B6 recombined variants (CYP2B6.1,CYP2B6.4,CYP2B6.6,CYP2B6.9 and CYP2B6.13) in vitro and test 22 drugs to validate the influence of alleles on drug metabolism by using high-throughput screening assays.We cloned the cDNA of CYP2B6^*1(prototype) and four alleles polymorphism including CYP2B6^*4,CYP2B6^*6,CYP2B6^*9 and CYP2B6^*13.The prototype cDNA(1.5kb) of CYP2B6 was obtained by PCR from commercial plasmid then was cloned into pYES2/CT vector for galactose-inducible expression in yeast.The cDNA of prototype was subsequently used as a template to introduce SNP by site-directed mutagenesis.CYP2B6 prototype and variants were co-expressed with human cytochrome P450 reductase in Saccharomyces cerevisiae.Protein expression was confirmed by western blot.Fluorogenic substrate(Vivid^? BOMCC) was employed to determine the activities of CYP2B6 enzymes, Km,Vmax and Vmax/Km values were calculated,and the result was 18.67±3.4 μM,4.06±0.76 nmol/min/mg and 0.22±0.06 mL/min/mg respectively.The inhibition assay was developed by incubation a mixture containing enzymes,BOMCC at the concentration equal to its apparent Kin,and test drugs which were 2-fold serial diluted from 128 to 0.0625μM.The half-maximal(50%) inhibitory concentration(IC50) of each drug was calculated.22 drugs were screened with high throughput fluorometry assay.Finally,we established stable human CYPs heterogenous expression system and drug screen system successfully.As shown by Western Blot,each enzyme was expressed well and the microsomes were obtained successfully.The Km,Vmax,and Vmax/Km of BOMCC to CYP2B6.1 were 18.67±3.4μM,4.06±0.76 nmol/min/mg and 0.22±0.06 mL/min/mg,respectively.The values of parameters were comparable to reported data.The parameters of the four CYP2B6 variants were compared and the results are,CYP2B6.6＜.1＜.9＜.13＜.4 for Km,CYP2B6.4＜.6＜.13＜.9＜.1 for Vmax,and CYP2B6.4＜.6＜.13＜.9＜.1 for Vmax/Km.The kinetic parameters of.4,.6 and.13 were significantly different from.1(prototype enzyme)(p＜0.01). The inhibition assays demonstrated that the antidepressants(sertraline,tranylcypromine, fluvoxamine),antineoplastics(thiotepa,raloxifen),and ketoconazole,ticlopidine inhibited CYP2B6.1 strongly.The IC50 values of the tested drugs to CYP2B6.1 are comparable to reported data.While there was different inhibitory potentials of these drugs to CYP2B6.1 and other alleles but not very obviously.The most interesting was that inhibitory potentials of some drugs to CYP2B6.1 and Alleles were obviously different,for example,quinidine, Fluoxetine,clomipramine,amitriptyline and tamoxifen had stronger inhibition on allele3 but did not inhibit CYP2B6.1 and other alleles.Drug-drug interactions resulting from inhibition or induction of CYP2B6 can have serious consequences in the case of substrate drugs with a narrow therapeutic index,such as cyclophosphamide.And the conclusion is that the recombinant CYP2B6 polymorphic enzymes could be used as tools for the metabolism-based drug-drug interactions screening. CYP2B6 allelic variants could lead to different inhibitory potential of drugs.These findings may provide useful information for predicting the potential drug-drug interactions with consideration of CYP2B6 polymorphic enzymes.