Drug Metabolizing Enzymes In Humans And Animal Models

点击翻译按钮获取中文摘要

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.