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，CYP 2C 19和CYP 2C 8。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）。通过对临床已知的弱代谢者和种族多样性人群中的CYP 2C 9进行重新测序，我们共鉴定出8种CYP 2C 9等位基因，其中许多是缺陷型。在临床研究中，几个等位基因已被证明会影响人类的药物代谢和毒性（有时需要住院治疗）。通过对不同种族人群的DNA测序，在CYP 2C 9基因的启动子、外显子和内含子-外显子连接处发现了35个单核苷酸多态性（SNP），其中包括6个新的编码改变的等位基因。这些等位基因已在大肠杆菌中表达为重组酶。大肠杆菌表达系统，并检测了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）。CYP 2C 9 *11等位基因代谢甲苯磺丁脲的能力明显缺陷（90%）。CYP 2C 19代谢常见的抗溃疡药物奥美拉唑、抗惊厥药美芬妥英、抗焦虑药安定、巴比妥酸盐，激活某些抗疟药，并使农药甲拌磷亚砜化。我们已经报道了6个新的多态性。重组蛋白的表达表明，一个等位基因P227 L（2C 19 *10）在药物代谢中有缺陷（减少90%），而至少有两个其他等位基因表现出一定的损害。CYP 2C 9和CYP 2C 19的许多新的缺陷等位基因在非洲裔美国人或非洲人群中普遍存在，这表明这些人群在过去没有得到足够的研究。CYP 3A亚家族代谢约40%的所有已知药物和许多农药。通过重新测序发现了几个新的等位基因，并以重组形式表达。一个等位基因CYP 3A 5（F446 S）含有血红素结合区的变化，在代谢睾酮和高血压药物硝苯地平方面有95%的缺陷。 控制转录调控的因子对理解药物相互作用至关重要。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。我们正在研究CYP 2C亚家族，以阐明核受体如何通过结合基因调控区域内的特定元件来控制基因表达。我们的工作重点是人CYP 2C 9，CYP 2C 19和CYP 2C 8的启动子区域，以及小鼠CYP 2C 29和它们的调节核受体CAR（组成型雄烷受体），PXR（甾烷X受体），GR（糖皮质激素受体），RXR（类维生素A X受体），和转录因子C/EBP（CCAAT增强子结合蛋白）。 我们已经表明，用CAR转染HepG 2细胞上调CYP 2C 9，并且CYP 2C 9启动子中的两个CAR/PXR结合位点似乎负责这种上调。这些位点可以控制人肝脏中的组成型表达和对药物的诱导。CYP 2C 19在结构上与CYP 2C 9相关，但在人肝脏中，CYP 2C 19的表达水平远低于CYP 2C 19，且诱导性较低。我们在CYP 2C 19启动子中仅鉴定了一个CAR/PXR结合位点，而在CYP 2C 9启动子中鉴定了两个。我们正在使用删除结构，足迹，凝胶结合试验，以确定其他核受体结合位点，这可能会影响诱导。小鼠CYP 2C及其启动子是研究人CYP 2C功能和调控的可能模型。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。我们正在研究CAR对小鼠CYP 2C 29的调节，作为人类调节的可能模型。其他鼠CYP 2C启动子被用作进一步检查模型药物如四氯二苯并二恶英、PXR激动剂、CAR激动剂的作用的模型。CAR/PXR敲除小鼠的可用性使我们能够检查药物是否通过CAR或替代机制诱导CYP 2C。在体内用药物和激素治疗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.