Regulation of drug metabolizing enzymes by miRNAs

描述（由申请人提供）：可变药物分布是药物疗效和安全性的关键决定因素。我们的长期目标是更好地了解药物处置和效果个体间差异的机制，并最终利用这些信息来个性化药物治疗。虽然一些变异性可归因于药物相互作用和遗传变异，但其中大部分的原因仍然未知。在目前的资助期间，我们已经确定了人类miRNAs肝脏表达的大的发育（胎儿到儿科到成人）变化。其中许多被预测为靶向药物处置基因。这些miRNAs中的一些在肝脏表达中具有显著的个体间变异性。肝细胞中的miRNA表达也被利福平改变，利福平是一种已知可以改变药物代谢的药物。我们还发现，miRNA靶向调节药物代谢和处置的途径，并且miRNA靶基因中的遗传变异似乎影响药物代谢。根据这些发现，我们还在与等位基因特异性表达相关的肝脏调控基因的预测miRNA结合位点的种子序列中鉴定了额外的SNP。我们的中心假设是，肝脏miRNAs调节发育变化，并有助于关键药物代谢酶表达的个体间差异，从而改变药物暴露。我们的第一个目标是确定改变的miRNA表达对肝细胞药物代谢的影响。原代人肝细胞将用显示发育变化和肝脏表达的显著个体间变异性的miRNA模拟物转染。将通过测量特定临床重要细胞色素P450酶和总体mRNA表达的代谢探针底物来确定肝细胞反应。我们的第二个目标是测试预测的miRNA结合位点的基因变异，这些位点对药物处置很重要。我们已经开发了一种新的高通量生物测定法来测试miRNA结合位点中的大量3' UTR SNP，以鉴定影响miRNA靶向和基因表达的那些SNP。我们的最后一个目标是确定血浆miRNA通过五种临床上重要的细胞色素P450酶预测肝脏代谢的能力。这将使用来自2项已完成的前瞻性临床试验的血浆样本进行。这些试验旨在测量5种探针药物的药代动力学，这些药物可确定CYP 3A 4/5、CYP 2B 6、CYP 2C 19、CYP 2C 9和CYP 1A 2酶的活性。通过完成这些研究，我们期望1）了解肝脏miRNA表达的发育变化对药物代谢的功能影响，2）鉴定改变药物代谢的miRNA靶位点中的功能SNP，3）发现预测肝脏药物代谢的血浆miRNA模式。这将有助于更好地理解miRNAs在发育中肝脏调控机制中的作用。最终，我们期望它将改善整个发育连续体中药物代谢变异性的预测。

DESCRIPTION (provided by applicant): Variable drug disposition is key determinant of drug efficacy and safety. Our long-term goal is to better understand the mechanisms responsible for the interindividual variability in drug disposition and effect, and eventually use this informationto personalize drug therapies. Although some of the variability is attributable to drug interactions and genetic variants, the cause of much of it remains unknown. During the current funding period, we have identified large developmental (fetal to pediatric to adult) changes in hepatic expression of human miRNAs. Many of these are predicted to target drug disposition genes. Some of those miRNAs have substantial interindividual variability in hepatic expression. miRNA expression in hepatocytes was also changed by rifampin, a drug known to alter drug metabolism. We have also shown that miRNAs target pathways that regulate drug metabolism and disposition and that genetic variants in miRNA target genes appear to influence drug metabolism. Following up on these findings, we have also identified additional SNPs in the seed sequences of predicted miRNA binding sites of hepatic regulatory genes that are associated with allele-specific expression. Our central hypothesis is that hepatic miRNAs regulate developmental changes and contribute to the interindividual variability in the expression of key drug metabolizing enzymes, and thereby, alter drug exposure. Our first aim will be to determine the effect of altered miRNA expression on hepatocyte drug metabolism. Primary human hepatocytes will be transfected with miRNA mimics that show developmental changes and substantial interindividual variability in hepatic expression. Hepatocyte responses will be determined by measuring the metabolism probe substrates for specific clinically important cytochrome P450 enzymes and global mRNA expression. Our second aim will test genetic variants in predicted miRNA binding sites of genes that are important for drug disposition. We have developed a novel high throughput bioassay to test large numbers of 3' UTR SNPs in miRNA binding sites to identify those which affect miRNA targeting and gene expression. Our last aim will determine the ability of plasma miRNAs to predict hepatic metabolism by five clinically important cytochrome P450 enzymes. This will be done using plasma samples from 2 completed prospective clinical trials. Those trials were designed to measure the pharmacokinetics of 5 probe drugs that determine the activity of CYP3A4/5, CYP2B6, CYP2C19, CYP2C9, and CYP1A2 enzymes. By completing these studies, we expect to 1) understand the functional impact of the developmental changes in hepatic miRNA expression on drug metabolism, 2) identify functional SNPs in miRNA target sites that alter drug metabolism, and 3) discover plasma miRNA patterns that predict hepatic drug metabolism. This should lead to a better understanding of the role of miRNAs in regulatory mechanisms of the developing liver. Ultimately, we expect that it will improve the prediction of variability in drug metabolism across the developmental continuum.