Drug Metabolizing Enzymes In Humans And Animal Models

我们已经确定了人类中β-内酰胺酶家族的新遗传多态性，这些多态性导致人类代谢特定药物和环境化学品的方式发生变化，从而改变人类对这些化学品的易感性。我们的实验室在确定细胞色素P450酶的多态性方面做出了重大贡献。目前研究进展主要集中在CYP 2C亚家族。这些包括CYP 2C 9和CYP 2C 19。CYP 2C 9代谢许多临床上重要的药物，包括苯妥英、甲苯磺丁脲、华法林、格列吡嗪和许多非甾体类抗炎药（NSAID）。通过对不同种族的人群进行重测序，我们发现了35个CYP 2C 9单核苷酸多态性（SNP），包括6个新的编码改变的CYP 2C 9等位基因，其中一些似乎是有缺陷的。这些等位基因已在大肠杆菌中表达为重组蛋白。大肠杆菌表达系统，并评估它们对甲苯磺丁脲的催化活性。新发现的CYP 2C 9等位基因包括氨基酸改变L19 I（2C 9 *7）、R150 H（2C 9 *8）、H251 R（2C 9 *9）、E272 G（2C 9 *10）、R335 W（2C 9 *11）和P489 S（2C 9 *12）。R335 W（CYP 2C 9 *11）等位基因在甲苯磺丁脲代谢方面非常缺陷（降低90%）。在亚洲人中发现的另外六个等位基因中，两个是无效的（不产生蛋白质），两个是催化缺陷的。这些新的CYP 2C 9缺陷等位基因中的大多数在非洲裔美国人或亚洲人中发现。CYP 3A亚家族代谢约40%的所有已知药物和许多农药。我们发现了一个新的等位基因CYP 3A 4 *17在代谢钙通道阻滞剂药物硝苯地平方面有99%的缺陷。该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特异性CYP 3A 5 *3连锁。已经针对新的等位基因开发了基因分型测试，并完成了甲泼尼龙弱代谢者的药物遗传学研究。 控制CYP 2C酶转录调节的因子对CYP 2C的调节至关重要，并产生药物-药物相互作用。文献中有证据表明，人CYP 2C可能因既往暴露于药物而上调，但机制尚不清楚。我们的工作重点是人类CYP 2C基因的启动子区域，以及它们的小鼠对应物，以及它们通过核受体CAR（组成型雄烷受体）、PXR（甾烷X受体）、GR（糖皮质激素受体）、RXR（维甲酸X受体）和其他受体的转录调控。我们已经表明，CYP 2C 9在HepG 2细胞中被hPXR配体药物上调。CYP 2C 9启动子中存在两个CAR/PXR位点。近端位点是必不可少的，而上游位点似乎有助于完全诱导。药物和草药，如利福平，圣约翰草和苯巴比妥似乎主要通过人类PXR（hPXR）上调。在CYP 2C 9中发现了肝核因子4 α位点，其似乎是CAR活化和利福平介导的PXR诱导所必需的。CYP 2C 8可由多种药物和草药诱导，如紫杉醇、利福平、苯巴比妥、苯妥英、贯叶金丝桃素和Citco（一种CAR激动剂）。这些化合物中的一些是PXR激动剂，而另一些是CAR激动剂，或与两种受体一起作用。CAR/PXR激活CYP 2C 8似乎通过上游约8.8 kb处的单个远端位点证实。鼠CYP 2C及其启动子是研究人CYP 2C功能和调节的可能模型，敲除小鼠表明cyp 2c 29是通过CAR受体诱导的。我们已经发现了新的小鼠CYP 2C，包括一个新的CYP 2C 44，立体特异性代谢花生四烯酸，被发现在肾脏和可能参与血管舒张在这个组织。定量PCR显示该P450比肝脏比肾脏更丰富（10倍）。定量PCR正在评估人体主动脉和心脏中的CYP 2C 9和CYP 2C 8，其中它们的代谢物也可能参与血管舒张。

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.