The promise of pharmacogenomics - identification of the right dose of the right drug the first time for everyone - has yet to be fully realized in part because we lack a complete understanding of the mechanisms underlying drug response variability. Recent studies suggest that microRNAs (miRs) - small noncoding RNAs that regulate gene expression post-transcriptionally - could be an important determinant of interindividual variability in drug metabolism and disposition. Here we propose to use a variety of novel approaches to identify miRs that are responsible for drug metabolizing enzyme variability. The focus of the application will be the UDP- glucuronosyltransferase (UGT) 1A enzymes that are critical to the elimination of many clinically important drugs with a narrow therapeutic index. Importantly, al 9 UGT1A enzymes (out of 18 total human UGTs) are encoded by spliced mRNAs that share an identical 3'-untranslated region (UTR). Although we have evidence that these enzymes are co-regulated by a post-transcriptional mechanism, virtually nothing is known regarding the role of miRs in UGT1A expression. Our overarching hypothesis is that specific miRs regulate UGT1A gene expression through the conserved UGT1A-3'-UTR mRNA region and/or indirectly by modulating regulatory transcription factor levels. To test this hypothesis, th following specific aims are proposed.
In Aim 1 candidate miRs associated with UGT1A glucuronidation will be identified by multiple independent approaches, including bioinformatics, functional genomics screen, and whole genome (miR-transcriptome) association studies. The novel functional genomics screen we propose enables rapid identification of miRs that suppress a fluorescent- tagged miR 3'-UTR reporter from a pooled lentivirus library of all known human miRs. This method should have broad application outside of our field enabling a cost-effective rapid approach to describing of the """"""""miR interactome"""""""". The reference UGT1A-3'-UTR allele will be evaluated as well as the major variant (*1b) associated with decreased bilirubin glucuronidation in vivo. miRs that are overexpressed in human liver bank samples with a low UGT1A activity and translational efficiency phenotype will also be identified by miR transcriptomics (arrays and Next gen small RNA-seq).
In Aim 2 we wil confirm the functional impact of candidate miRs on UGT1A glucuronidation through overexpression (lenti-miRs) and knockdown (lenti- antagomiRs) of candidate miRs in human model cell lines and primary hepatocytes.
In Aim 3 we will determine the mechanism of UGT1A regulation by functional miRs through use of UGT1A-3'UTR luciferase-reporter constructs and through studying effects of miR overexpression and knockdown in model cell lines and primary human hepatocytes on specific candidate transcription factor proteins (PXR and HNF1) as well as the entire proteome (iTRAQ LC-MS/MS method). This work will ultimately lead to development of validated predictive biomarkers (miR-associated polymorphisms and/or miR levels in blood) that would be incorporated into the pharmacogenetic algorithms currently being developed to guide dosing of low therapeutic index drugs.

Public Health Relevance

A variety of drugs used to treat cancer (e.g. irinotecan), HIV infection (e.g. raltegravir), and prevent organ rejection (e.g. mycophenolate) have a low margin of safety and show highly variable effects between patients. These studies will identify microRNAs (small noncoding RNAs) that regulate the levels of the enzymes that metabolize and clear these drugs (i.e. the UGT1A enzymes). The ultimate goal is to use blood levels of these microRNAs and/or microRNA associated genetic variants to predict the safest and most effective drug dose for a particular patient.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
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Okita, Richard T
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Tufts University
Schools of Medicine
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Perez Jimenez, Tania E; Mealey, Katrina L; Grubb, Tamara L et al. (2016) Tramadol metabolism to O-desmethyl tramadol (M1) and N-desmethyl tramadol (M2) by dog liver microsomes: Species comparison and identification of responsible canine cytochrome P-450s (CYPs). Drug Metab Dispos :
Gammal, R S; Court, M H; Haidar, C E et al. (2016) Clinical Pharmacogenetics Implementation Consortium (CPIC) Guideline for UGT1A1 and Atazanavir Prescribing. Clin Pharmacol Ther 99:363-9
Slovak, J E; Mealey, K; Court, M H (2016) Comparative metabolism of mycophenolic acid by glucuronic acid and glucose conjugation in human, dog, and cat liver microsomes. J Vet Pharmacol Ther :
Redmon, Joanna M; Shrestha, Binu; Cerundolo, Rosario et al. (2016) Soy isoflavone metabolism in cats compared with other species: urinary metabolite concentrations and glucuronidation by liver microsomes. Xenobiotica 46:406-15
Papageorgiou, Ioannis; Freytsis, Marina; Court, Michael H (2016) Transcriptome association analysis identifies miR-375 as a major determinant of variable acetaminophen glucuronidation by human liver. Biochem Pharmacol 117:78-87
Chen, S; Laverdiere, I; Tourancheau, A et al. (2015) A novel UGT1 marker associated with better tolerance against irinotecan-induced severe neutropenia in metastatic colorectal cancer patients. Pharmacogenomics J 15:513-20
Court, Michael H; Almutairi, Fawziah E; Greenblatt, David J et al. (2014) Isoniazid mediates the CYP2B6*6 genotype-dependent interaction between efavirenz and antituberculosis drug therapy through mechanism-based inactivation of CYP2A6. Antimicrob Agents Chemother 58:4145-52
Court, Michael H; Peter, Inga; Hazarika, Suwagmani et al. (2014) Candidate gene polymorphisms in patients with acetaminophen-induced acute liver failure. Drug Metab Dispos 42:28-32
Kwara, Awewura; Cao, Lei; Yang, Hongmei et al. (2014) Factors associated with variability in rifampin plasma pharmacokinetics and the relationship between rifampin concentrations and induction of efavirenz clearance. Pharmacotherapy 34:265-71
Haas, David W; Kwara, Awewura; Richardson, Danielle M et al. (2014) Secondary metabolism pathway polymorphisms and plasma efavirenz concentrations in HIV-infected adults with CYP2B6 slow metabolizer genotypes. J Antimicrob Chemother 69:2175-82

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