Drug Metabolizing Enzymes In Humans And Animal Models

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Studies exposure of human liver cell lines and primary human hepatocytes to clinical drugs, herbal remedies and other xenobiotics have been shown to transcriptionally upregulate the CYP2C subfamily (CYP2C8, 2C9, and 2C19) of enzymes increasing metabolism of many clinical and over-the counter drugs. This data is consistent with in vivo studies in humans which have shown that exposure to certain drugs and herbal remedies decrease the half-life to many drugs. This could produce tolerance to drugs or drug-drug interactions complicating patient therapy. In liver and intestine the CYP2Cs can be increased >2-8 fold by prior administration of drugs. We have shown that the promoter regions of the human CYP2C genes are regulated by elements which bind xenosensing nuclear receptors CAR (constitutive androstane receptor), PXR (pregnane X receptor), and and liver-enriched receptors such as HNF4 alpha. Drugs such as rifampicin, St Johns Wort, phenobarbital and other xenobiotics interact with CAR and PXR which bind to elements in the promoter increasing transcription. Moreover HNF4 alpha sites in the promoter enhance inducibility by CAR or PXR, and act synergistically to increase CYP2C9 in liver cell lines and in human hepatocytes. Collaborative studies (1) have shown that coactivator NCOA6 is responsible for the recruitment of the ASCOM complex to the promoter bound FXR, responsible for epigenetic modification of the regulatory region which was found to be essential and prerequisite for the activation/induction of the bile salt export pump (BSEP), multi drug resistance associated protein 2 (MRP2) and sodium taurcholate cotransporting polypeptide (NTCP) genes which belong to the transporter family of genes regulated by nuclear receptors. In contrast, collaborative studies have shown that PXR represses human sulfotransfease SULT1E1 by altering chromatin structure (2). Chromatin capture experiments showed that HNF4alpha bound to the PXR responsive element and activated the enhancer by looping its position close to the proximal promoter. Because of the importance of the liver enriched receptor HNF4alpha in induction of drug metabolism and various physiological disease processes such as diabetes and lipid metabolism, we have used yeast two-hybrid screens to identify HNF4 alpha interacting proteins. Last year (Rana et al, 2011) we showed with yeast two hybrid screens and various interaction studies that Med 25, a member of the Mediator Complex, is a new HNF4 interacting protein which brings RNA polymerase II to the start site as shown with ChIP assays. Using primary human hepatocytes we over expressed of Med 25 and silenced Med25 using shRNA. We have shown with qPCR and microarray analysis that silencing the Med25 decreases up regulation of a number of CYP mRNAs and proteins by HNF4 alpha. These include up regulation of various CYP genes including drug metabolizing enzymes such as CYP2C9, CYP3A4, CYP2B6 and also enzymes involved in fatty acid metabolism but not those involved in glucose regulation. Thus Med 25 selectively initiates transcription of some but not all pathways controlled by HNF4 alpha. This year Zhang et al, 2012 (3) showed that the CYP2C proteins (particularly CYP2C8>CYP2C19>CYP2C9) are regulated postranscriptionally by microRNAs 103 and 107. CYP2C8 metabolizes important compounds such as the anticancer drug taxol as well as several type II diabetes drugs and certain statins. MicroRNAS (miRNAs) are a new class of small noncoding RNA genes (22 nucleotides) that regulate many genes by binding to complementary sequences in various genes (often the 3-noncoding region or the coding region of target RNAs. More than 1000 miRNAs have been identified. They most often affect the translation of multiple gene targets by binding to the 3-noncoding region or coding region of target RNAs. We used online searches with miRBase Targets database and Target Base and found potential microRNA response elements (MREs) in the 3-region of CYP2C8 and other CYP2C mRNAs. Of these, miR103 and miR107 were found to regulate CYP2C8 postranscriptionally. Ectopic expression of precursors for miR103/107 were found to decrease expression of CYP2C8 >CYP2C19>CYP2C9 in primary human hepatocytes. On the other hand locked nucleic acid modified antisense nucleotides (AsOs) were found to silence miR103 and miR107 in primary human hepatocytes and increase CYP2C8 protein and to a lesser degree CYP2C19 protein. These miRNAs had no effect on mRNA levels consistent with their effect on translational control of these proteins. When 3 copies of the putative CYP2C8 MREs were inserted downstream of a luciferase expression reporter and transfected into primary human hepatocytes, precursors for miR103 and miR107 decreased luciferase activity while ASOs increased luciferase activity of these constructs. CYP2C8 protein levels did not correlate well with CYP2C8 mRNA in excess liver tissue from hepatocyte preparations. However, the translational efficiency of (protein/mRNA) was inversely correlated with miR103 and miR107. miR103 and miR107 are known to be transcribed from the introns of the PANK genes which control acetyl CoA synthesis and are responsive to insulin sensitivity, glucose tolerance, lipid metabolism, cellular metabolism, stress and various diseases. This suggests new ways in which the CYP2C enzymes may be regulated. Collaborative studies with Dr Prough (4) have shown that murine aldehydehydrogenase is upregulated by homodimers of c-Jun through AP-1 sites. Aldehyde dehydrogenase 1a1 (ALDH1A1) is upregulated by acroline as well as butylated hydroxyanisole via these sites. AP-1 sites are now being studied in human CYP2C9 in our laboratory. CYP3A4 and CYP3A5 are two of the most abundant P450s in human liver. Activity depends on ratios of reductase and b5 and we documented the optimum conditions for studying these enzymes (5). Collaborative studies with Dr Masons group (6) showed that the purinergic receptor P2X7-NADPH oxidase is the primary mediator of oxidative stress-induced exacerbation of inflammatory liver injury in obese mice via NADPH dependent enzymes. Quantitative PCR studies show increase in p47 phox mRNA in CCl4-treted mouse liver and Kupfer cells. Finally collaborative clinical studies examined the role of polymorphisms of P-glycoprotein, CYP2B6 and CYP3A4/5 in various clinical situations. The first study found a polymorphism in P-glycoprotein was associated with long term dose requirements of sirulimus in patients receiving renal transplants (7). A second clinical study (8) found pharmacokinetics of cyclohosphamide and 4-OH cyclophosphamide in patients with lupus nephritis and small vessel vasculitis were similar. A CYP2B6*9 and ABCB1 polymorphism both alter the pharmacokinetics of cyclophosphamide and 4-OH-cyclophosphamide.