Regulation of drug metabolizing enzymes by miRNAs

描述(申请人提供)：可变药物处置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导致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。尽管部分变异可归因于药物相互作用和遗传变异，但其中大部分原因仍不清楚。在目前的资助期间，我们已经确定了人类miRNAs在肝脏的表达发生了巨大的发育性变化(从胎儿到儿童到成人)。其中许多基因被预测为针对药物处置基因。其中一些miRNAs在肝脏中的表达具有很大的个体间差异。肝细胞中miRNA的表达也被利福平改变，利福平是一种已知的改变药物代谢的药物。我们还表明，miRNAs的目标是调节药物新陈代谢和处置的途径，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，我们还在预测的肝脏调节基因的miRNA结合位点的种子序列中发现了额外的SNPs，这些位点与等位基因特异性表达相关。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。我们的第一个目标将是确定miRNA表达改变对肝细胞药物代谢的影响。原代人类肝细胞将被导入miRNA模拟物，这些模拟物显示出发育变化和肝脏表达的显著个体差异。肝细胞的反应将通过测量特定的临床重要细胞色素P450酶的代谢探针底物和全球mRNA表达来确定。我们的第二个目标将测试对药物处置重要的基因的预测miRNA结合位点中的遗传变异。我们建立了一种新的高通量生物检测方法来检测miRNA结合位点上的大量3‘UTRSNPs，以确定那些影响miRNA靶向和基因表达的SNPs。我们的最后一个目标将通过五种临床上重要的细胞色素P450酶来确定血浆miRNAs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来完成。这些试验旨在测量5种探针药物的药代动力学，这些药物确定了细胞色素P3A4/5、细胞色素P450 2 B6、细胞色素P2 C19、细胞色素P2 C9和细胞色素P1A2酶的活性。通过完成这些研究，我们希望1)了解肝脏miRNA表达的发育变化对药物代谢的功能影响，2)确定miRNA靶点上改变药物代谢的功能SNPs，以及3)发现预测肝脏药物代谢的血浆miRNA模式。这应该会导致更好地理解miRNAs在发育中的肝脏调节机制中的作用。最终，我们预计它将改善对整个发育过程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.