Drug Metabolizing Enzymes In Humans And Animal Models

我们已经在人类的CYP家族中发现了新的基因多态，这些基因多态负责在人类代谢特定药物和环境化学品的方式上引入变异性，从而改变人类对这些化学品的敏感性。我们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。在细胞色素P450 2 C亚家族中取得了最大的进展。这些药物包括细胞色素P450 C9、细胞色素C19和细胞色素C8。CYP2C9代谢多种临床重要药物，包括苯妥英钠、甲苯丁胺、华法林、格列吡嗪和许多非类固醇抗炎药。通过对临床上已知的代谢不良者和种族多样性人群中的CYP2C9进行重新测序，我们总共鉴定了8个CYP2C9等位基因，其中许多是缺陷的。在临床研究中，一些等位基因已经被证明影响药物代谢和人类的毒性(有时需要住院治疗)。来自不同种族的DNA测序发现了35个单核苷酸多态(SNPs)，其中包括6个新的编码变化的等位基因。这些等位基因已经在大肠杆菌表达系统中以重组酶的形式表达，并检测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L19I(2C9*7)、R150H(2C9*8)、H251R(2C9*9)、E272G(2C9*10)、R335W(2C9*11)和P489S(2C9*12)。CYP2C9*11等位基因代谢甲苯丁胺的能力明显缺陷(90%)。临床研究将在未来解决这些等位基因的问题。在亚洲人身上发现了新的等位基因。在非裔美国人或非洲人群体中发现了许多新的有缺陷的等位基因，这表明过去对这些群体的研究还不够。CYP3A亚家族代谢了大约40%的已知药物和许多杀虫剂。一个新的等位基因CYP3A4*17在代谢钙通道阻滞剂硝苯地平方面存在99%的缺陷。在东欧、土耳其和美国的某些高加索少数民族中，该等位基因似乎与CYP3A5*3连锁。已经对新的等位基因进行了基因分型测试。 控制细胞色素P450酶转录调控的因子对细胞色素P2磷酸酶的调控和药物与药物的相互作用起着关键作用。文献中有证据表明，预先接触药物可能会上调人类细胞色素P450 2 C的表达，但其机制尚不清楚。我们正在研究CYP2C亚家族，以阐明核受体如何通过结合基因调节区内的特定元件来控制基因表达。我们的工作主要集中在人的细胞色素P450_2C9、细胞色素P_2_2_C19和细胞色素P_2C_8的启动子区域，以及小鼠的细胞色素Pyp2c29及其核受体CAR(构成雄烷受体)、PXR(孕烷X受体)、GR(糖皮质激素受体)、RXR(维甲酸X受体)和转录因子C/EBP(CCAAT增强子结合蛋白)。 我们已经证明，在转染hPXR的细胞中，药物上调了CYP2C9在HepG2细胞中的表达。在CYP2C9启动子中有两个位点，在CYP2C19启动子中有一个。药物和草药如利福平、圣约翰？S麦汁和苯巴比妥似乎是通过人类PXR而不是人类汽车上调的。在HepG2细胞中，CYP2C9表达上调~2-4倍，而CYP2C19表达仅上调2倍。这与人体活体数据是一致的。肝脏核因子4在CYP2C9中已被发现。CYP2C19在结构上与CYP2C9相关，但在人体肝脏中的表达水平比CYP2C19低得多，而且诱导性较差。我们在CYP2C19启动子中只发现了一个CAR/PXR结合位点，而在CYP2C9启动子中发现了两个结合位点。CYP2C8似乎含有至少一个远上游位点和另一个近端位点。HPXR和HCAR参与这种酶的诱导正在被研究，相比之下，在小鼠中，CAR基因敲除研究和启动子构建表明，CYP2C29主要由小鼠CAR上调。CAR/PXR基因敲除小鼠的出现使我们能够研究药物是通过CAR还是通过替代机制诱导细胞色素P450 2 C的表达。小鼠细胞色素P450 2 C及其启动子是研究人类细胞色素P2 2蛋白功能和调控的可能模型。我们在肾脏中发现了新的小鼠细胞色素P450，其中包括一种立体特异性代谢花生四烯酸的新细胞色素P450，它可能参与了肾脏组织的血管扩张。定量聚合酶链式反应显示该P450比肝脏丰富(10倍)。模型系统。总共发现了15个小鼠基因和假基因，它们对花生四烯酸具有不同的底物特异性。其中一种是代谢维生素A，在ah受体基因敲除模型中缺乏，导致肝脏维生素A毒性。在合作研究中，已经培育出了一种CYP2C29基因敲除小鼠，并将解决其生理后果

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, CYP2C19, and CYP2C8. CYP2C9 metabolizes numerous clinically important drugs including phenytoin, tolbutamide, warfarin, glipizide and numerous nonsteroidal antiinflammatory drugs (NSAIDs). By resequencing CYP2C9 in clinically known poor metabolizers and racially diverse populations, we have identified a total of 8 alleles of CYP2C9 many of which are defective. Several alleles have been shown to affect drug metabolism and toxicity (sometimes requiring hospitalization) in humans in clinical studies. Sequencing DNA from diverse racial populations have identified 35 single nucleotide polymorphisms (SNPs) in the promoter, exons, and intron-exon juctions of CYP2C9 including 6 alleles with new coding changes. These alleles have been expressed as recombinant enzymes in an E. coli expression system, and their catalytic activity toward tolbutamide examined. 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 CYP2C9*11 allele is markedly defective in its ability to metabolize tolbutamide (90% ). Clinical studies will address these alleles in the future. New alleles have been discovered in Asians. Many new defective alleles of CYP2C9 and CYP2C19 were prevalent in African-American or African populations, suggesting that these populations have not received sufficient study in the past. The CYP3A subfamily metabolizes approximately 40% of all known drugs and many pesticides. One new allele CYP3A4*17 is 99% deficient in metabolizing nifedipine a calcium channel blocker drug. This allele appears to be linked to CYP3A5*3 in certain Caucasian ethnic minorities from Eastern Europe, Turkey and the US. Genotyping tests have been developed for the new alleles. 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. We are investigating the CYP2C subfamily to elucidate how nuclear receptors control gene expression by binding specific elements within gene regulatory regions. Our work focuses on the promoter regions of the human CYP2C9, CYP2C19, and CYP2C8, as well as murine cyp2c29 and their regulation by the nuclear receptors CAR (constitutive androstane receptor), PXR (pregnane X receptor), GR (glucocorticoid receptor), RXR (retinoid X receptor), and the transcription factor C/EBP (CCAAT enhancer binding protein). We have shown that CYP2C9 is upregulated in HepG2 cells by drugs in cells transfected with hPXR. Two sites are present in the CYP2C9 promoter and one in the CYP2C19 promoter. Drugs and herbal remedies such as rifampicin, St John?s Wort and phenobarbital appear to upregulate via human PXR (HPXR) rather than human CAR. CYP2C9 is upregulated ~2-4 fold in HepG2 cells while CYP2C19 is upregulated only two fold. This is consistent with in vivo data in man. Hepatic nuclear factor 4 sites have been discovered in CYP2C9. CYP2C19 is structurally related to CYP2C9, but CYP2C19 is expressed at much lower levels than CYP2C19 in human liver, and appears less inducible. We identified only one CAR/PXR binding site in the CYP2C19 promoter versus two in the CYP2C9 promoter. CYP2C8 appears to contain at least one far upstream site and one more proximal site. The involvement of hPXR and hCAR in induction of this enzyme is being examined In contrast, in the mouse, CAR knockout studies and promoter constructs show that CYP2C29 is upregulated primarily by mouse CAR. The availability of CAR/PXR knockout mice enables us to examine whether drugs induce the CYP2Cs via CAR or alternate mechanisms. Thee murine CYP2Cs including their promoters are possible models for studying function and regulation of the human CYP2Cs. 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). model system. A total of 15 murine genes and pseudogenes have been discovered with different substrate specificity toward arachidonic acid. One metabolizes vitamin A and is deficient in the Ah receptor knockout model, causing hepatic vitamin A toxicity. A knockout mouse for CYP2C29 has been produced in collaborative studies and the physiological consequences will be addressed