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%)。CYP2C19代谢常见的抗溃疡药物奥美拉唑、抗癫痫药美苯妥英、抗焦虑安定、巴比妥酸盐，激活某些抗疟疾药物，并亚磺化杀虫剂甲拌磷。我们报道了6个新的多态。重组蛋白的表达表明，1个等位基因P227L(2C19*10)存在药物代谢缺陷(下降90%)，另有至少2个等位基因存在一定程度的药物代谢缺陷。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。通过对种群和的重新测序，发现了几个新的等位基因，并以重组形式表达。一个含有血红素结合区改变的等位基因CYP3A5(F446S)在代谢睾酮和高血压药物硝苯地平方面存在95%的缺陷。 控制CYP酶转录调控的因素对于理解药物之间的相互作用至关重要。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，用CAR基因转导HepG2细胞后，细胞色素P450受体9表达上调，并且在细胞色素P2受体9启动子中的两个CAR/PXR结合位点似乎是导致这种上调的原因。这些位点可能控制人类肝脏的结构性表达和对药物的反应诱导。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。我们正在使用缺失构建、足迹和凝胶结合分析来确定可能影响诱导性的其他核受体结合位点。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。我们发现了新的小鼠细胞色素P450，包括一种新的能立体定向代谢花生四烯酸的细胞色素P450。我们正在研究汽车对小鼠体内小鼠细胞色素P450 2C29的调节，作为一种可能的人类调节模型。其他小鼠CYP2C启动子正被用作进一步检验模型药物如四氯二苯二恶英、PXR激动剂、CAR激动剂的效果的模型。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。在体内用药物和激素治疗小鼠模型的能力也提供了作为模型系统的优势。

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% ). CYP2C19 metabolizes the common antiulcer drug omeprazole, the anticonvulsant mephenytoin, the anxiolytic valium, barbiturates, activates certain antimalarials, and sulfoxidizes the pesticide phorate . We have reported 6 new polymorphisms. Expression of the recombinant proteins revealed that one allele P227L (2C19*10) was defective ( 90% decrease) in metabolizing drugs while at least two other alleles showed some impairment. 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. Several new alleles were discovered by resequencing populations and and expressed in recombinant form . One allele CYP3A5(F446S) containing a change in the heme binding region was 95% defective in metabolizing testosterone and the hypertensive drug nifedepine. Factors controling transcriptional regulation of the CYP enzymes are critical to understanding 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 transfection of HepG2 cells with CAR upregulates CYP2C9, and that two CAR/PXR binding sites in the CYP2C9 promoter appear to be responsible for this upregulation. These sites may control both constitutive expression in human liver and induction in response to drugs. 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 promote. We are using deletion constructs, footprinting, and gel binding assays to identify other nuclear receptor binding sites, which may influence inducibility. The 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. We are investigating the regulation of murine CYP2C29 in mouse by CAR as a possible model for human regulation . Other murine CYP2C promoters are being used as a model to further examine the effects of model drugs such as tetrachlorodibenzodioxin, PXR agonists, CAR agonists. The availability of CAR/PXR knockout mice enables us to examine whether drugs induce the CYP2Cs via CAR or alternate mechanisms. The ability to treat a murine model with drugs and hormones in vivo also offers advantages as a model system.