Regulation of drug metabolizing enzymes by miRNAs

描述（由申请人提供）：可变的药物处置是药物有效性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的个体差异的机制，并最终利用这些信息来个性化药物治疗。虽然一些可变性可归因于药物相互作用和遗传变异，但其大部分原因尚不清楚。在目前的资助期内，我们已经确定了人类miRNAs肝脏表达的巨大发育变化（从胎儿到儿童到成人）。其中许多被预测以药物处置基因为目标。其中一些mirna在肝脏表达中具有显著的个体间差异。利福平（一种已知能改变药物代谢的药物）也改变了肝细胞中的miRNA表达。我们还表明，miRNA靶向调节药物代谢和处置的途径，miRNA靶基因的遗传变异似乎影响药物代谢。在这些发现的基础上，我们还在与等位基因特异性表达相关的肝脏调节基因的预测miRNA结合位点的种子序列中发现了额外的snp。我们的中心假设是，肝脏mirna调节发育变化，促进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miRNA表达改变对肝细胞药物代谢的影响。原代人肝细胞将转染miRNA模拟物，显示肝脏表达的发育变化和显著的个体间差异。肝细胞反应将通过测量特定临床重要细胞色素P450酶的代谢探针底物和全局mRNA表达来确定。我们的第二个目标是测试对药物处置很重要的基因的预测miRNA结合位点的遗传变异。我们开发了一种新的高通量生物测定方法来检测miRNA结合位点上的大量3' UTR snp，以确定那些影响miRNA靶向和基因表达的snp。我们的最后一个目标是确定血浆mirna通过五种临床重要的细胞色素P450酶预测肝脏代谢的能力。这将使用来自2个已完成的前瞻性临床试验的血浆样本。这些试验旨在测量5种探针药物的药代动力学，这些药物可测定CYP3A4/5、CYP2B6、CYP2C19、CYP2C9和CYP1A2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靶点的功能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mirna在发育中的肝脏调节机制中的作用。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.