Drug Metabolizing Enzymes In Humans And Animal Models

人类和动物模型中的药物代谢酶

• 批准号: 7967941
• 负责人: JOYCE GOLDSTEIN
• 金额: $ 162.72万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7967941
• 关键词: Accounting; Adenovirus Vector; Adverse effects; Affect; African American; Agonist; Alabama; Alleles; Animal Model; Anti-Inflammatory Agents; Antibodies; Anticoagulants; Antidiabetic Drugs; Antimalarials; Antineoplastic Agents; Arachidonic Acids; Arthritis; Asians; Bacteria; Binding; Binding Sites; Biochemical; Biological Assay; Biological Models; Brain; Brown Fat; CYP2C19 gene; CYP2C9 gene; Caco-2 Cells; Cardiovascular system; Caucasians; Caucasoid Race; Cell Culture Techniques; Cell Line; Cell model; Cells; Cessation of life; Chromatin; Chronic Kidney Failure; Circadian Rhythms; Clinical; Clinical Research; Coagulation Process; Code; Collaborations; Colon Carcinoma; Complex; Cyclophosphamide; Data; Development; Diastolic blood pressure; Disease; Distal; Dose; Drug Interactions; Drug Kinetics; Drug Tolerance; Electrophoretic Mobility Shift Assay; Elements; Enzymes; Epidemiologist; Epigenetic Process; Extrahepatic; Flurbiprofen; Gene Targeting; Genes; Genetic Polymorphism; Genetic Predisposition to Disease; Genetic Transcription; Genetic Variation; Genetic screening method; Genotype; Goals; Haplotypes; Heart Diseases; Hemorrhage; Hepatic; Hepatocyte; Hospitalization; Human; Hypotension; Hypoxia; In Vitro; Individual; Intestines; Ischemia; Kidney; Kidney Failure; Life; Ligands; Liver; Losartan; Luciferases; Lung; Malignant Epithelial Cell; Mammalian Cell; Measurement; Messenger RNA; Metabolism; Methylation; Methyltransferase; Minority Groups; Modification; Muscle; Mutate; Mutation; NCOA6 gene; Non-Insulin-Dependent Diabetes Mellitus; Non-Prescription Drugs; Normal tissue morphology; Nuclear; Nuclear Extract; Nuclear Receptors; Orphan; Outcome; Paclitaxel; Patients; Pharmaceutical Preparations; Physiological Processes; Platelet Factor 4; Promoter Regions; Proteins; Receptor Gene; Recombinants; Regulation; Retinoids; Rifampin; Risk; Role; Signal Transduction Pathway; Site; Small Interfering RNA; Stress; System; Testing; Therapeutic; Therapeutic Agents; Thymus Gland; Tissues; Tolbutamide; Toxic effect; Transcription Process; Translating; Translations; Treatment Efficacy; Universities; Up-Regulation; Urine; Variant; Warfarin; Warfarin Sodium; Western Blotting; Yeasts; blood pressure regulation; caucasian American; chromatin immunoprecipitation; chromatin modification; cofactor; constitutive active receptor; constitutive androstane receptor; cytochrome P-450 CYP2C subfamily; disorder risk; dosage; drug efficacy; drug metabolism; environmental change; environmental chemical; epidemiology study; exposed human population; high risk; histone methyltransferase; human tissue; immune function; in vitro Model; in vivo; interest; lipid metabolism; mRNA Expression; malignant breast neoplasm; man; overexpression; pregnane X receptor; prevent; promoter; prospective; prototype; receptor; receptor binding; repaired; response; urinary; vitamin K epoxide reductase; yeast two hybrid system

We have examined the effects of genetic polymorphisms of CYP2C9 (an important drug metabolizing enzyme (CYP2C9) and polymorphisms in the vitamin K epoxide reductase complex 1 on the dose of warfarin required to control cardiovascular (thromboembolytic) disease and risk for life-threatening side effects in a large prospective epidemiology study of 302 Caucasian Americans and 273 African-American patients treated with warfarin in over a 2 year period, in a collaboration with Limdi et al (University of Alabama)(1-5). We developed new rapid pyrosequencing genotyping tests for known or suspected defective alleles of human CYP2C9 alleles (*2, *3, *5, *6, 10, and 11 variants) which occur in Caucasians and African Americans. Individuals with aberrant CYP2C9 alleles required lower doses of warfarin. CYP2C9 and VKORC1 accounted for 30% and 18% of the variability of required warfarin dose in American Caucasians and 10% in African-Americans (1). Among VKORC haplotypes, VKORC1 was the most important. The risk of overcoagulation was higher among Caucasian Americans with variant VKORC1 allele and marginally higher among those with variant CYP2C9 genotypes. We then examined the effects of polymorphisms of CYP2C9 on serious and life threatening side effects (serious hemorrhage and death). The presence of variant alleles of CYP2C9 conferred a 5-fold increase in risk for major hemorrhage before stabilization of dose, and a 2-fold risk even after stabilization of the dose (3). The variant VKORC1 genotype did not confer a significant increase in risk for major hemorrhages. Chronic kidney disease (CKD) was found to significantly lower required warfarin dosages and these individuals were at high risk for overcoagulation (5). We also identified a new coding allele of CYP2C9 (Asp360Glu) in an African-American hospitalized for life-threatening hemorrhage (6). CYP2C9 metabolizes losartan which is used for anti-proteinuric and blood pressure control in chronic kidney disease (CDK). In another collaborative clinical study (7) we found patients with CDK who had defective CYP2C9 genotypes had less favorable lowering of urinary protein and less favorable effects on diastolic blood pressure. In studies of the regulation of the CYP2C enzymes, we studied the ability of retinoid-related orphan receptors (ROR) on transcriptional up regulation of the human CYP2Cs in liver (8). Three ROR genes, alpha, beta, and gamma, are differentially expressed in liver, kidney, lung, muscle, brown fat, thymus, and brain where they are involved in the regulation of many physiological processes such as immune function, brain development, circadian rhythm, and lipid metabolism. ROR alpha1, 2, and gamma1 are expressed in liver. We examined whether RORs activate the upstream promoter regions of the human CYP2C drug metabolizing enzymes in HepG2 cell (liver cell line) and a human colon carcinoma cell line (Caco 2 cells) and whether they up regulate the mRNAs for the CYP2Cs. Overexpression of ROR and ROR in HepG2 cells increased luciferase activity of a 3kb CYP2C8 promoter construct, but not that of CYP2C9 or CYP2C19 promoter constructs. CYP2C8 is important in the metabolism of clinical drugs such as paclitaxel (breast cancer), certain antidiabetic drugs for type II diabetes, and antimalarial drugs as well as metabolizing arachidonic acid to active metabolites which are important in cardiovascular regulation. We identified the regulatory sites for ROR to one essential site in the CYP2C8 promoter using mutational assays, showed binding of RORs in gel-shift assays. Overexpression of RORs using adenoviral constructs increased CYP2C8 mRNA in human primary hepatocytes as well as HepG2 cells. siRNA to ROR alpha and gamma decreased CYP2C8 mRNA expression in HepG2 cells. These data show a role for RORs in upregulation of CYP2C8 expression in liver and possibly extrahepatic tissues in response to stress, hypoxia and diurnal rhythm. In other studies exposure of patients or human liver cells to clinical drugs transcriptionally has been shown to upregulate CYP2C8, 2C9, and 2C19 enzymes increasing metabolism of drugs metabolized by these enzymes in vivo in man. 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, producing higher metabolism in exposed individuals leading to tolerance. We have previously shown that the promoter regions of the human CYP2C genes are regulated by elements which bind the nuclear receptors CAR (constitutive androstane receptor), PXR (pregnane X receptor), and liver-enriched receptors such as HNF4 alpha. New data shows that HNF4 sites in the promoter enhance inducibility by CAR or PXR, and HNF4 and CAR act synergistically to increase CYP2C9 in liver cell lines. We theorize that various coactivators may be involved in forming a bridge between the distal CAR site and the proximal HNF4 site in the CYP2C9 promoter. Results of yeast two hybrid screens identified NCOA6 as a new HNF4 interacting protein as well known coactivators such as PGC-1(10). We overexpressed CAR and HNF4 in adenoviral vectors and performed pull downs with GST-HNF4 and GST-CAR. CAR was identified as part of a complex with GST-HNF4 complex in nuclear extracts of HepG2 cells by Western blotting and by mass spectrometric analysis. Nuclear cofactors such as NCOA6 and PGC-1 were also identified in the complex. When NCOA6 and PGC-1 were translated in vitro, they could also interact with HNF4-GST and were identified by Western blotting showing direct interaction. Using promoter assays and mRNA measurement, the synergy between HNF4 and CAR in the presence of their respective drug ligands could be blocked by adenoviral constructs to siRNAs for NCOA6, the new HNF4 interacting protein. In chromatin immunoprecipitation (ChIP) assays antibodies to CAR pulled down both the CAR binding site and the HNF4 binding site of the CYP2C9 promoter showing these factors bind to the promoter in vivo. NCOA6 was pulled down at the HNF4 site and the CAR site. Adenoviral constructs containing siRNA to NCOA6 did not affect HNF4 binding to its site but blocked NCOA6 binding and binding of other coregulators such as PGC-1, and PMT (an O-methyltransferase which repairs ASN and ASP residues). Therefore NCOA6 appears to be an essential cofactor that helps form bridge between CAR and HNF4 (the bridge may contain other coregulators as mentioned). We recently reviewed all studies available on regulation of the CYP2C enzymes (11). Recently we have shown in cultured primary human hepatocytes have shown that binding sites for liver-specific HNF4 sites are necessary for the upregulation of the CYP2C8 and CYP2C9 promoter of the CYP2Cs genes. Mutation of these sites prevents induction by the PXR agonist rifampicin. Mutation of either the PXR sites or HNF4 sites abolishes induction. siRNA to HNF4 almost abolishes induction of CYP2C8, 2C9 or 2C19 mRNA in primary human hepatocytes which are the best in vitro model we have for human liver.

在一项大型前瞻性流行病学研究中，我们研究了CYP2C9基因多态性（一种重要的药物代谢酶（CYP2C9））和维生素K酰氯还原酶1基因多态性对控制心血管（血栓溶栓）疾病所需的华法林剂量和危及生命的副作用风险的影响，该研究对302名白种美国人和273名非裔美国人进行了为期2年的华法林治疗。与Limdi等人（阿拉巴马大学）合作（1-5）。我们开发了一种新的快速焦磷酸测序基因分型检测方法，用于检测白种人和非裔美国人CYP2C9等位基因（*2、*3、*5、*6、10和11变体）中已知或疑似缺陷的等位基因。具有异常CYP2C9等位基因的个体需要较低剂量的华法林。CYP2C9和VKORC1分别占美国白种人华法林所需剂量变异的30%和18%，非裔美国人占10%(1)。在VKORC单倍型中，VKORC1是最重要的。VKORC1等位基因变异的美国白种人发生过凝血的风险较高，CYP2C9基因型变异的美国白种人发生过凝血的风险略高。然后，我们检查了CYP2C9多态性对严重和危及生命的副作用（严重出血和死亡）的影响。CYP2C9变异等位基因的存在使得在剂量稳定之前发生大出血的风险增加了5倍，甚至在剂量稳定之后风险也增加了2倍(3)。变异的VKORC1基因型并没有显著增加大出血的风险。慢性肾脏疾病（CKD）可显著降低华法林所需剂量，这些患者有高凝血风险(5)。我们还在一位因危及生命的出血住院的非裔美国人身上发现了CYP2C9 （Asp360Glu）的一个新的编码等位基因(6)。CYP2C9代谢氯沙坦，用于慢性肾脏疾病（CDK）的抗蛋白尿和血压控制。在另一项合作临床研究(7)中，我们发现CYP2C9基因型缺陷的CDK患者尿蛋白降低效果较差，舒张压降低效果较差。在CYP2C酶调控的研究中，我们研究了类维生素a相关孤儿受体（ROR）在肝脏中上调人CYP2C转录的能力(8)。三个ROR基因，α、β和γ，在肝脏、肾脏、肺、肌肉、棕色脂肪、胸腺和大脑中有差异表达，参与许多生理过程的调节，如免疫功能、大脑发育、昼夜节律和脂质代谢。ROR α 1、α 2和γ 1在肝脏中表达。我们在HepG2细胞（肝细胞系）和结肠癌细胞系（Caco 2细胞）中检测了RORs是否激活了人类CYP2C药物代谢酶的上游启动子区域，以及它们是否上调了CYP2C的mrna。在HepG2细胞中，过表达ROR和ROR会增加3kb CYP2C8启动子结构体的荧光素酶活性，而CYP2C9或CYP2C19启动子结构体的荧光素酶活性则不会增加。CYP2C8在紫杉醇（乳腺癌）、某些II型糖尿病降糖药、抗疟药等临床药物的代谢以及花生四烯酸代谢为活性代谢物中具有重要的心血管调节作用。我们通过突变分析确定了CYP2C8启动子中ROR的一个重要位点的调控位点，并在凝胶转移分析中显示了ROR的结合。使用腺病毒构建的RORs过表达人原代肝细胞和HepG2细胞中的CYP2C8 mRNA增加。siRNA转ROR α和γ可降低HepG2细胞中CYP2C8 mRNA的表达。这些数据表明，RORs在应激、缺氧和昼夜节律反应中上调肝脏和肝外组织中CYP2C8的表达。在其他研究中，患者或人肝细胞暴露于临床药物转录已被证明上调CYP2C8、2C9和2C19酶，增加这些酶在体内代谢的药物在人体内的代谢。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可通过先前给药增加2-8倍，使暴露个体代谢增加，从而产生耐受性。我们之前已经表明，人类CYP2C基因的启动子区域是由结合核受体CAR（组成型雄烷受体）、PXR（孕烷X受体）和肝脏富集受体（如HNF4 α）的元件调节的。新的数据显示，启动子中的HNF4位点增强了CAR或PXR的诱导性，并且HNF4和CAR协同作用增加肝细胞系中CYP2C9的表达。我们推测多种共激活因子可能参与了在CYP2C9启动子的远端CAR位点和近端HNF4位点之间形成桥梁的过程。酵母两种杂交筛选的结果表明，NCOA6是一种新的HNF4相互作用蛋白，与PGC-1等已知的共激活因子相互作用（10）。我们在腺病毒载体上过表达CAR和HNF4，并用GST-HNF4和GST-CAR进行拉下。通过Western blotting和质谱分析，在HepG2细胞的核提取物中鉴定出CAR是与GST-HNF4复合物的一部分。核辅因子如NCOA6和PGC-1也在该复合体中被鉴定。当NCOA6和PGC-1在体外翻译时，它们也可以与HNF4-GST相互作用，并通过Western blotting鉴定为直接相互作用。通过启动子分析和mRNA测量，在各自药物配体存在的情况下，HNF4和CAR之间的协同作用可以被腺病毒构建的NCOA6 sirna阻断，NCOA6是新的HNF4相互作用蛋白。在染色质免疫沉淀（ChIP）实验中，CAR抗体拉低了CYP2C9启动子的CAR结合位点和HNF4结合位点，表明这些因子在体内与启动子结合。NCOA6在HNF4位点和CAR位点被拉低。含有NCOA6 siRNA的腺病毒构建体不影响HNF4与NCOA6位点的结合，但阻断了NCOA6与其他共调节因子如PGC-1和PMT（一种修复ASN和ASP残基的o -甲基转移酶）的结合。因此，NCOA6似乎是一个重要的辅助因子，有助于在CAR和HNF4之间形成桥梁（桥梁可能包含其他协同调节因子，如前所述）。我们最近回顾了所有关于CYP2C酶调节的研究（11）。最近，我们在培养的原代人肝细胞中表明，肝脏特异性HNF4位点的结合位点对于cyp2c基因的CYP2C8和CYP2C9启动子的上调是必需的。这些位点的突变阻止了PXR激动剂利福平的诱导。PXR位点或HNF4位点的突变均可消除诱导作用。siRNA to HNF4几乎消除了原代人肝细胞中CYP2C8、2C9或2C19 mRNA的诱导作用，而原代人肝细胞是目前最好的体外人肝模型。