Drug Metabolizing Enzymes In Humans And Animal Models

研究表明，将人肝细胞系和原代人肝细胞暴露于临床药物、草药和其他外源药物中，可以通过转录上调CYP2C亚家族（CYP2C8、2C9和2C19）的酶，从而增加许多临床和非处方药的代谢。这一数据与人体体内研究相一致，这些研究表明，接触某些药物和草药会减少许多药物的半衰期。这可能会产生对药物的耐受性或使患者治疗复杂化的药物-药物相互作用。在肝脏和肠道中，CYP2Cs可通过事先给药增加2-8倍。我们已经证明，人类CYP2C基因的启动子区域是由结合异种感应核受体CAR（组成型雄甾烷受体）、PXR（孕烷X受体）和肝脏富集受体（如HNF4 α）的元件调节的。药物如利福平、圣约翰草、苯巴比妥和其他外源药物与CAR和PXR相互作用，它们与启动子中的元件结合，增加转录。此外，启动子中的HNF4 α位点增强了CAR或PXR的诱导作用，并协同作用于肝细胞系和人肝细胞中增加CYP2C9。合作研究(1)表明，共激活子NCOA6负责将ASCOM复合体招募到启动子结合的FXR上，负责调控区域的表观遗传修饰，这被发现是激活/诱导胆汁盐输出泵（BSEP）的必要和先决条件。多药耐药相关蛋白2 （MRP2）和牛胆酸钠共转运多肽（NTCP）基因属于核受体调控的转运蛋白家族。相比之下，合作研究表明PXR通过改变染色质结构来抑制人硫转移酶SULT1E1(2)。染色质捕获实验表明，HNF4alpha结合到PXR响应元件上，并通过环化其靠近近端启动子的位置激活了增强子。由于肝脏富集受体HNF4 α在诱导药物代谢和各种生理疾病过程（如糖尿病和脂质代谢）中的重要性，我们使用酵母双杂交筛选来鉴定HNF4 α相互作用蛋白。去年（Rana et al, 2011），我们用酵母进行了两种杂交筛选和各种相互作用研究，结果表明介质复合体的成员Med 25是一种新的HNF4相互作用蛋白，可以将RNA聚合酶II带到起始位点，正如ChIP试验所显示的那样。我们利用原代人肝细胞过表达Med25，并利用shRNA沉默Med25。我们已经通过qPCR和微阵列分析表明，沉默Med25降低了HNF4 α对许多CYP mrna和蛋白质的上调。这包括上调各种CYP基因，包括药物代谢酶，如CYP2C9， CYP3A4, CYP2B6，以及参与脂肪酸代谢的酶，但不包括参与葡萄糖调节的酶。因此，med25选择性地启动一些但不是全部由HNF4 α控制的通路的转录。今年Zhang et al . 2012(3)研究发现CYP2C蛋白（特别是CYP2C8>CYP2C19>CYP2C9）受microRNAs 103和107的转录后调控。CYP2C8代谢重要的化合物，如抗癌药物紫杉醇、几种II型糖尿病药物和某些他汀类药物。MicroRNAS （miRNAs）是一类新的小非编码RNA基因（22个核苷酸），通过结合各种基因（通常是靶RNA的3-非编码区或编码区）的互补序列来调节许多基因。目前已鉴定出1000多种mirna。它们通常通过结合靶rna的3-非编码区或编码区来影响多个基因靶标的翻译。我们使用miRBase Targets数据库和Target Base进行在线搜索，在CYP2C8和其他CYP2C mrna的3-region中发现了潜在的microRNA应答元件（MREs）。其中，miR103和miR107在转录后调控CYP2C8。miR103/107前体异位表达可降低人原代肝细胞中CYP2C8 >CYP2C19>CYP2C9的表达。另一方面，锁定核酸修饰的反义核苷酸（AsOs）被发现沉默人原代肝细胞中的miR103和miR107，增加CYP2C8蛋白，并在较小程度上增加CYP2C19蛋白。这些mirna对mRNA水平没有影响，这与它们对这些蛋白质的翻译控制的影响一致。当将3个推测的CYP2C8 MREs拷贝插入荧光素酶表达报告基因的下游并转染到原代人肝细胞时，miR103和miR107的前体降低了荧光素酶活性，而ASOs提高了这些构建体的荧光素酶活性。在肝细胞制备的多余肝组织中，CYP2C8蛋白水平与CYP2C8 mRNA没有很好的相关性。然而，（蛋白/mRNA）的翻译效率与miR103和miR107呈负相关。已知miR103和miR107是从控制乙酰辅酶a合成的PANK基因的内含子转录而来，并对胰岛素敏感性、葡萄糖耐量、脂质代谢、细胞代谢、应激和各种疾病有反应。这提示了调节CYP2C酶的新途径。

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.