Rats are extensively used as a preclinical model for assessing drug pharmacokinetics (PK) and tissue distribution; however, successful translation of the rat data requires information on the differences in drug metabolism and transport mechanisms between rats and humans. To partly fill this knowledge gap, we quantified clinically relevant drug-metabolizing enzymes and transporters (DMETs) in the liver and different intestinal segments of Sprague-Dawley rats. The levels of DMET proteins in rats were quantified using the global proteomics-based total protein approach (TPA) and targeted proteomics. The abundance of the major DMET proteins was largely comparable using quantitative global and targeted proteomics. However, global proteomics-based TPA was able to detect and quantify a comprehensive list of 66 DMET proteins in the liver and 37 DMET proteins in the intestinal segments of SD rats without the need for peptide standards. Cytochrome P450 (Cyp) and UDP-glycosyltransferase (Ugt) enzymes were mainly detected in the liver with the abundance ranging from 8 to 6502 and 74 to 2558 pmol/g tissue. P-gp abundance was higher in the intestine (124.1 pmol/g) as compared to that in the liver (26.6 pmol/g) using the targeted analysis. Breast cancer resistance protein (Bcrp) was most abundant in the intestinal segments, whereas organic anion transporting polypeptides (Oatp) 1a1, 1a4, 1b2, and 2a1 and multidrug resistance proteins (Mrp) 2 and 6 were predominantly detected in the liver. To demonstrate the utility of these data, we modeled digoxin PK by integrating protein abundance of P-gp and Cyp3a2 into a physiologically based PK (PBPK) model constructed using PK-Sim software. The model was able to reliably predict the systemic as well as tissue concentrations of digoxin in rats. These findings suggest that proteomics-informed PBPK models in preclinical species can allow mechanistic PK predictions in animal models including tissue drug concentrations.

ID 18183 Poster Board 577Kidney is one of the major drug-elimination organs. The total renal excretion of a compound is the net result of glomerular filtration, tubular secretion, and reabsorption. Tubular secretion is a transporter-mediated process, which is often mediated by organic cation transporters (OCT/Oct) that facilitate the active secretion of several cationic substrates including drugs such as metformin as well as endogenous cations 1 . Endogenous biomarkers of OCTs can help in the early assessment of drug-drug interaction without the administration of exogenous OCT probe substrates 2 . We hypothesize that administration of cimetidine, an OCT/Oct inhibitor, will lead to increased plasma levels and decreased renal clearance (CL R ) of endogenous OCT/Oct substrates. Such substrates can potentially act as OCT/Oct biomarkers. We carried out a rat pharmacokinetic (PK) study where metformin (5 mg/kg, IV) was administered as an exogenous substrate of OCT (positive control) to four Sprague-Dawley rats with and without cimetidine (100mg/kg, IP) in a cross-over study design with one week washing period. Blood samples were collected from the tail vein at different time points, i.e, pre-dose, 0.17, 0.5, 1, 2, 4, 6, and 8 h, and urine samples were collected at 0-4 and 4-8 h intervals. Rat blood and urine samples were analyzed for metformin and cimetidine levels by a validated method using liquid chromatography with tandem mass spectrometry (LC-MS/MS) (Waters Xevo-TQ-XS MS; Waters, Milford, MA). Metformin area under the blood concentration-time curve (AUC (0-8h) ) was significantly increased by 3.2 folds when co-administered with cimetidine (p-value, 0.003). Similarly, metformin CL R(0-8h) was significantly decreased in cimetidine arm by 3.7 folds (p-value, 0.029). Further, to investigate the effect of cimetidine on endogenous metabolites, we carried out untargeted metabolomics for rat blood and urine samples using Easy Spray 1200 series nanoLC coupled Q-Exactive HF MS (Thermo Fisher Scientific, Waltham, MA). The rat blood samples were analyzed in three groups, i.e., pooled (0.5, 1, and 2h), 0.5 h, and 1 h samples, while rat urine samples were analyzed at 0-4 h interval samples. The generated data were analyzed by open-access XCMS Online software (xcmsonline.scripps.edu). Greater than 18,000 features were detected in the blood which were shortlisted using optimized selection criteria, i.e., fold differences (with versus without cimetidine) of 1.9-10 fold, p-value <0.05, reproducible retention time, and quality of chromatogram peak. Out of the 85 shortlisted hits, 52 were detected by METLIN software, and 30 were common in blood and urine. Among several potential compounds predicted by METLIN for each mass-to-charge ratio (m/z) value, only compounds containing nitrogen atoms with mass error (ppm) less than 4 were selected. Two significant hits (m/z, 134.06, and 233.09 corresponding to putative oxindole and robustine, respectively) were consistently found in pooled, 0.5 h, and 1h samples (Figure 1). Other putative meta...

Background and objectives Testosterone metabolism by the highly polymorphic UDP‐glucuronosyltransferase 2B17 (UGT2B17) is widely recognized, but the metabolic fate of testosterone in the carriers of UGT2B17 gene deletion is not well understood. Similarly, testosterone glucuronide (TG) is prone to secretion into intestinal lumen (1); however, the role of microbiome in activation of testosterone is not studied. Therefore, the aims of this study were: i) to elucidate the alteration in the liver proteome and its effect on steroid metabolism pathways (i.e., metabolic switching) in the UGT2B17 gene deletion, and ii) to assess the TG deconjugation by gut β‐glucuronidases. Methods Human liver S9 fraction with gene deletion and high expression (n=3) were used. Untargeted proteomics data was acquired using Easy nano‐LC 1200 coupled with the high‐resolution QE‐HF Orbitrap. The proteomic data were analyzed, processed and pathway mapping was performed using MaxQuant, Perseus and STRING, respectively. In vitro studies in liver S9 fraction of UGT2B17 gene deletion were carried out to investigate testosterone metabolism by aldo‐keto reductases (AKRs). The glucuronidation pathway of 5β‐dihydrotestosterone (5β‐DHT) and 5β‐tetrahydrotestosterone (5β‐THT) were identified using recombinant UGT2B isoforms. These data were verified using testosterone incubations in human hepatocyte and serum levels of 5β‐THTG after oral testosterone dosing (800 mg) in human. Activation mechanism TG to testosterone in the human gut was investigated by incubating TG with recombinant human gut microbial β‐glucuronidases and human fecal extracts. Results A total of 1002 liver proteins were quantified in the S9 fraction, of which 14 and 39 proteins were exclusively from the deletion and high‐expressor groups, respectively, whereas 949 proteins were common. The following pathways were upregulated in UGT2B17 deletion group: oxidation‐reduction, drug metabolic, steroid metabolic, and fatty acid metabolic process. Notable proteins that were upregulated in the gene deletion groups were: AKR1D1, AKR1C4, AKR7DA3, AKR1A1, DHCR7, ADH6, ADH1C, ALDH1A1, ALDH9A1, and ALDH5A1 A deeper molecular pathway analysis revealed that the AKR1D1 and AKR1C4 that metabolize testosterone to inactive 5β‐DHT and 5β‐THT, respectively (2) (Figure 1) were significantly upregulated in UGT2B17 deletion group. UGT2B7 is primarily responsible for glucuronidation of 5β‐THT (Figure 1). Finally, incubation with purified bacterial β‐glucuronidases and human fecal extracts confirmed facile but variable deconjugation of TG to testosterone in human gut. Conclusion Novel mechanisms of testosterone metabolic switching in UGT2B17 gene deletion and testosterone activation by gut microbiome have been identified. These findings have a potential to provide deeper insights into the mechanisms of testosterone associated pathophysiological conditions, such obesity, insulin sensitivity, and cancer. References Li, C. Y. et al. J Steroid Biochem Mol Biol (2019). Rižner et al. Steroids (2014).

No abstract

No abstract