Drug Metabolizing Enzymes In Humans And Animal Models

我们已经在人类CYP家族中发现了新的遗传多态性，这些多态性负责在人类代谢特定药物和环境化学物质的方式中引入变异，从而改变人类对这些化学物质的易感性。我们的实验室在鉴定细胞色素P450酶的多态性方面做出了重大贡献。在CYP2C亚家族中取得了大部分进展。其中包括CYP2C9和CYP2C19。CYP2C9代谢许多临床重要药物，包括苯妥英、甲苯丁胺、华法林、格列吡嗪和许多非甾体抗炎药（NSAIDs）。通过对不同种族的人群进行重测序，我们发现了35个CYP2C9单核苷酸多态性（snp），其中包括6个具有新编码变化的CYP2C9等位基因，其中一些等位基因似乎存在缺陷。这些等位基因已在大肠杆菌表达系统中以重组蛋白的形式表达，并评估了它们对甲苯丁酰胺的催化活性。新发现的CYP2C9等位基因包括氨基酸变化的L19I （2C9*7）、R150H （2C9*8）、H251R （2C9*9）、E272G （2C9*10）、R335W （2C9*11）和P489S （2C9*12）。R335W （CYP2C9*11）等位基因对甲苯丁胺的代谢有很大缺陷（降低90%）。在亚洲人身上发现的另外6个等位基因中，2个是无效的（不产生蛋白质），2个是催化缺陷的。这些新的CYP2C9缺陷等位基因大多在非洲裔美国人或亚洲人身上发现。CYP3A亚家族代谢大约40%的已知药物和许多杀虫剂。我们发现一个新的等位基因CYP3A4*17在钙通道阻滞剂硝苯地平的代谢中有99%的缺陷。这种等位基因与来自东欧、土耳其和美国的某些高加索少数民族的CYP3A5*3基因缺陷有关。已经开发了新的等位基因的基因分型测试，并完成了甲基强的松龙不良代谢物的药物遗传学研究。

We have identified new genetic polymorphisms in the CYP family in humans which are responsible for introducing variability in the way humans metabolize specific drugs and environmental chemicals and thus alter susceptibility of humans to these chemicals. Our laboratory has made substantial contributions in identifying polymorphisms in cytochrome P450 enzymes. Most progress has been made in the CYP2C subfamily. These include CYP2C9 and CYP2C19. CYP2C9 metabolizes numerous clinically important drugs including phenytoin, tolbutamide, warfarin, glipizide and numerous nonsteroidal antiinflammatory drugs (NSAIDs). By resequencing racially diverse populations, we have identified 35 single nucleotide polymorphisms (SNPs) in CYP2C9 including 6 alleles with new coding changes alleles of CYP2C9 several of which appear to be defective. These alleles have been expressed as recombinant proteins in an E. coli expression system, and their catalytic activity toward tolbutamide assessed. The newly discovered CYP2C9 alleles include the amino acid changes L19I (2C9*7), R150H (2C9*8), H251R (2C9*9), E272G (2C9*10), R335W (2C9*11) and P489S (2C9*12). The R335W (CYP2C9*11) allele is very defective in its metabolism of tolbutamide (90% decrease). Of six additional alleles discovered in Asians, Two are null (producing no protein) and two are catalytically defective. Most of these new defective alleles of CYP2C9 were found in African-Americans or Asian. The CYP3A subfamily metabolizes approximately 40% of all known drugs and many pesticides. We discovered a new allele CYP3A4*17 is 99% deficient in metabolizing the calcium channel blocker drug nifedipine. This allele is linked to the deective CYP3A5*3 in certain Caucasian ethnic minorities from Eastern Europe, Turkey and the US. Genotyping tests have been developed for the new alleles and pharmacogenetic studies of a poor metabolizer of methlprednisolone completed. Factors controling transcriptional regulation of the CYP enzymes are critical to regulation of the CYP2Cs and produce drug-drug interactions. There is evidence in the literature that the human CYP2Cs may be upregulated by prior exposure to drugs, but the mechanism has been unknown. Our work focuses on the promoter regions of the human CYP2C genes as well as their murine counterparts and their transcriptional regulation by the nuclear receptors CAR (constitutive androstane receptor), PXR (pregnane X receptor), GR (glucocorticoid receptor), RXR (retinoid X receptor) and other receptors. We have shown that CYP2C9 is upregulated in HepG2 cells by drugs which are hPXR ligands. Two CAR/PXR sites are present in the CYP2C9 promoter. The proximal site is essential while the upstream site appears to contribute to full induction. Drugs and herbal remedies such as rifampicin, St John's Wort and phenobarbital appear to upregulate primarily via human PXR (hPXR). Hepatic nuclear factor 4 alpha sites have been discovered in CYP2C9 which appear to be necessary for CAR activation and for PXR mediated induction via rifampicin. CYP2C8 is inducible by a variety of drugs and herbal remedies such as paclitaxel, rifampicin, phenobarbital, phenytoin, hyperforin and Citco (a CAR agonist). Some of these compounds are PXR agonists while others are CAR agonists, or act with both receptors. CYP2C8 activation by CAR/PXR appears to be confirmed by a single distal site at ~8.8 kb upstream. The murine CYP2Cs including their promoters are possible models for studying function and regulation of the human CYP2Cs and knockout mice indicate that cyp2c29 is induced via CAR receptors. We have discovered new murine CYP2Cs, including a new CYP2C44 which stereospecifically metabolize arachidonic acid, is found in kidney and may be involved in vasodilation in this tissue. Quantitative PCR shows this P450 is more abundant than liver than kidney (10X). Quantitative PCR is assessing CYP2C9 and CYP2C8 in human aorta and heart where their metabolites may also be involved in vasodilation.