De NovoIdentification and Validation of Human Endogenous Biomarkers of Renal Organic Anion Transporters for Predicting Interindividual Variability

Abstract ID 96852 Poster Board 433 Endogenous substrates of drug transporters can be used as phenotypic biomarkers to predict transporter-mediated drug-drug interactions (DDIs), drug toxicity, and interindividual variability in transport function.¹ Renal organic anion transporters (OAT1 and OAT3) and multidrug resistance-associated proteins (MRP2 and MRP4) are expressed on basolateral and apical membrane of kidney proximal tubule cells, respectively, facilitating the active secretion of several drugs and endogenous substrates. As with drugs, inhibition of OAT- and MRP-mediated transport can alter plasma and urine concentrations of endogenous transporter substrates. We identified and validated clinical biomarkers of OAT1/OAT3 and MRP2/MRP4 using a state-of-the-art metabolomics-based DMET biomarker discovery approach. To identify the biomarkers, a pharmacokinetic DDI study involving 16 healthy adult human participants (8 males and 8 females) was conducted using oral furosemide (5 mg) and probenecid (1000 mg) as the OAT/MRP substrate and inhibitor, respectively. Plasma (pooled samples collected at 1, 1.5, and 2 h) and urine (0-4 h) collections representing time points corresponding to maximum probenecid plasma concentrations were analyzed by LC-HRMS, followed by data analysis using XCMS Online software (xcmsonline.scripps.edu). Compared to the baseline (furosemide alone), probenecid increased furosemide plasma concentrations by >2.5-fold (p<0.05) and decreased renal clearance by >66%. Untargeted metabolomics analysis yielded >10,000 features (m/z values) that were either increased or decreased in pooled plasma and urine samples in the presence of probenecid. Features that were significantly increased in pooled plasma (1.5- to 10-fold) and were either decreased or non-significantly increased in urine samples were identified as putative OAT1/OAT3 biomarkers. Likewise, identification of putative MRP2/MRP4 biomarkers included features that were significantly decreased in urine (p<0.05) with a non-significant change in pooled plasma. The putative OAT1/OAT3 biomarkers (m/z range, 130-384) mainly consisted of fatty acids, glucuronide conjugates, peptides, steroids, and tryptophan metabolites. The putative MRP2/MRP4 biomarkers (m/z range, 110-510) consisted primarily of bile acids, fatty acids, glycosides, peptides, and steroids. These potential clinical biomarkers were then validated using high-throughput in vitro assays, wherein the uptake of endogenous substrates measured in human urine was compared between OAT1 over-expressing HEK293 cells and the mock cells. Of the 63 putative OAT1/OAT3 biomarkers identified in the clinical samples, 29 were confirmed as OAT1 substrates, i.e., uptake in OAT1 over-expressing cells was >1.4-fold higher (p<0.05) than that in mock cells (Fig. 1). Validation of putative OAT3, MRP2, and MRP4 biomarkers using cells overexpressing these transporters is ongoing. Once validated, structures of the compounds will be elucidated using MS/MS fragmentation approach and synthetic standards, followed by a detailed pharmacokinetic analysis (0-24 h) of these metabolites. The biomarkers identified and validated here using robust clinical and in vitro approaches can be used to assess the DDI potential and interindividual variability of renally secreted organic anionic drugs. Ref: Chu et al., 2018. CPT 104, 836-864. Funding: NIH/NICHD R01HD081299 and NIH/NCCIH U54AT008909.