Comparative Study Of Drug Metabolisms And Drug-drug Interactions Of Human Cytochrome P450 3A And Its Alleles In Vitro

Objective: The cytochrome P450 (P450) enzymes form a superfamily of heme-thiolate proteins in human liver, and are the major catalysts involved in the metabolism of drugs.They also are called mixed-function oxygenases. CYP3A are regarded as predominant functional forms of human P450 in many organs, nearly 30% total P450s in the liver. They are involved in oxidation, peroxidation and reduction of approximately 45-60% of commonly used drugs. CYP3A4 and 3A5 are regarded as predominant functional forms of human CYP3A in liver and intestine. The substrates metabolized by CYP3A4 and 3A5 are generally overlapped, but the kinetic parameters are usually different. In addition, the content ratios of CYP3A4 to CYP3A5 in different individuals are variable, thus may result in different drug responses. The expression of CYP3A4 varies 40-fold in individual human livers and metabolism of CYP3A4 substrates varies at least 10-fold in vivo may because of the factors like: gene polymorphism, age, disease et al.Between them the most important factor is the gene polymorphism.Today, several CYP3A4 alleles have been found and may be a source of inter-individual drug metabolism difference. To study the differences between the 3A4 wild type ,3A5 wild type and 3A4 four alleles (CYP3A4^*3, CYP3A4^*4, CYP3A4^*17, CYP3A4^*18) in drug metabolisms and drug drug interactions in vitro and to assess the application of the study in the clinic and new drug discovery are very important.Methods: The cDNAs of the CYP3A4 wildtype and CYP3A5 wildtype were applied in our lab. The mutants were introduced into the wildtype 3A4 gene by PCR mutagenesis method. After that, we used two enzymes Kpnâ… and Xhoâ… to digest PCR products and the yeast expression vector pYES2/CT. And then we jointed the insert and the vector for cloning. We used the ampicilin resistance to choose the recon.Then we used the PCR method for premie screening.Following sequencing, recombinants were transport to the yeast strain with POR gene.The P450 enzyme expressed in yeast by galactose induction. Recombinant 3A enzymes' microsome fractions containing were isolated by differential centrifugation.The enzyme were used to test the enzymatic activities in real-time kinetic assays in two reactions, dibenzylfluorescein (DBF) -O-dealkylation and CYP3A4-specific substrate nifedipine oxidization. The kinetic parametres were determined by nonlinear regression analysis for determination of K_m and V_max values. Furthermore, 19 chemical drugs were two-fold serial diluted and added to the two reactions for IC₅0 determination.Results: From the results we could concluded budding yeast S.cerevisiae was a suitable host to express human CYP3A enzymes. Except the F189S, the other allelic enzymes could metabolize DBF and nifedipine to the relevant metabolites. Both substrates affinities for CYP3A5 were lower than that for CYP3A4 (with 2- to 3-folds higher K_m values than CYP3A4). The CL_int were 2- and 3-folds lower than CYP3A4 (p<0.05, n≥3). Furthermore, in drugs IC₅₀ determination experiments, Ketoconazole strongly inhibited the catalylic ability of CYP3A4.IC₅₀ values to DBF-O-dealkylation and nifedipine oxidization are 0.012 and 0.036μM,respectively. However, CYP3A5 was less inhibited by ketoconazole in those two reactions, which IC₅₀ values are 0.040 and 0.049μM.To the other 18 drugs,IC₅₀ values of some drugs were unavailable in the concentration range we test, except those ,IC₅₀ of rest drugs to CYP3A5 were on average, 4-fold higher than that to CYP3A4 no matter what probe substrate was tested.In the comparative study of 3A4 and its alleles, DBF affinities for the alleles were similar to that for WT. The intrinsic clearance (CL_int) of ^*3 and ^*18 were 3.1 and 3.8 times (p<0.05) higher than WT respectively. The CL_int of ^*4 was 1.32 times (p=0.1010) higher than WT. Nifedipine affinities for there alleles(^*3,^*4,^*18)were lower to that for WT (with 1.6,2.0 and 1.3-folds higher K_m values than WT). The CL_int of ^*18 was 2.6 times (p<0.05) higher than that of WT. The CL_int of ^*4 was 0.7 times (p<0.05) lower than WT. The CL_int of ^*3 was similar to wildtype (p=0.7676). The drugs display the following rank order of in vitro potency against WT and allelic enzymes as measured by their inhibition DBF-O-dealkylation: ketoconazole>diltiazem> troglitazone> Sertraline> verapamil> Fluoxetine>diflucan> Omeprarole> erythromycin> lescol> avandia> pioglitazone> pravachol= lipitor= warfarin= celebrex= zocol=vioxx= climetidine.The IC₅₀ values of these enzymes by 19 chemical compounds were arranged as ^*3>^*18>WT>^*4.In the nifedipine oxidization inhibitions, the IC₅₀ values of drugs are arrange as ketoconazole>diltiazem> verapamil>diflucan> celebrex> vioxx, the inhibition of these enzymes by chemical compounds showed remarkable enzyme-selective inhibitory effects. The IC50 values of the allelic enzymes were arranged as WT> ^*18> ^*3 > ^*4.There were no correlation between the two substates in IC₅₀ values.Conculsions: The kinetic properties of these enzymes exhibited significantly different compared with each other. And the enzymes of each allele showed drug-specific altered susceptibility to inhibition. This study showed budding yeast S.cerevisiae was a suitable host to express human CYP3A enzymes. The recombinant enzyme' characters were stable and well.Meanwhile this study established two drug screening platforms to study the CYP3A4 genetic polymorphism. They were fluorescent and HPLC method. In new drug R & D process, the two platforms can be carried out to screening of drug candidates to reduce the failure possibility in the post-drug clinical trials and clinical application stage. Furthermore,the two platforms also establish a foundation method to study CYP450 research.The two methods can be used not only as methods in preclinical high-throughput screening,but also can help promote the clinical development of individualized medicine.