(T1130-02-08) In Vitro Selectivity of Several Bile Acid Glucuronides as OATP1B Endogenous Biomarkers

Purpose: Organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 are major hepatic uptake transporters which are the determinant for hepatic elimination of many important drugs, such as statins. Inhibition of OATP1B may cause clinically significant drug-drug interactions (DDIs). Therefore, evaluation of the risk for OATP1B-mediated DDIs has been required by the regulatory agencies. In recent years, rapid progress has been made to identify, characterize, and use the endogenous substrates of OATP1B as biomarkers to assess the risk for DDI in early drug development. Several bile acid glucuronides, including glycochenodeoxycholate 3-O-glucuronide (GCDCA-3G) and glycodeoxycholate 3-O-glucuronide (GDCA-3G) have been identified as potential biomarkers to assess OATP1B-mediated DDIs (1,2). To quantitatively assess their selectivity to OATP1B1 and OATP1B3, we applied the relative activity factor (RAF) method (3) to determine the relative contribution of OATP1B1 and OATP1B3 on the hepatic uptake of several bile acid glucuronides including GCDCA-3G, GDCA-3G, and chenodeoxycholate 24-glucuronide (CDCA-24G).

Methods: In vitro uptake studies of GCDCA-3G, GDCA-3G, and CDCA-24G were conducted in OATP1B1 or OATP1B3 stably transfected human embryonic kidney (HEK) 293 cells. For uptake time course studies, uptake was initiated by the addition of bile acid glucuronides (0.1 µM GCDCA-3G, 0.1 µM GDCA-3G, and 0.1 µM CDCA-24G). For uptake kinetic studies, uptake was initiated by the addition of various concentrations of the bile acid glucuronides at indicated time point based on their linear uptake in the respective transporter transfected cell lines. The uptake of the test compounds was quantified by LC-MS/MS. The RAF value, which was defined as the ratio of active and transporter specific uptake clearance of respective reference compound in hepatocytes to that in transporter transfected cells (3), was used to determine the relative contribution of OATP1B1 versus OATP1B3. In this study, the RAF values obtained by Chan et al (3) were re-calibrated with uptake clearance of reference compounds, pitavastatin in OATP1B1- and cholecystokinin (CCK-8) in OATP1B3-transfected cells, respectively. Intrinsic uptake clearance (CL_{int, uptake}, V_max/K_m) of the reference compounds and biomarkers in transfected cells mediated by the transporter of interest was determined via measuring initial uptake rate and uptake kinetics. The total active hepatic uptake clearance of biomarkers was calculated by the sum of intrinsic uptake clearance of biomarker mediated by individual transporter calibrated according to corresponding RAF. The % contribution by specific transporter was the percentage of intrinsic uptake clearance of biomarker mediated by individual transporter to total active hepatic intrinsic clearance of biomarkers.

Results: Uptake of GCDCA-3G, GDCA-3G, and CDCA-24G in transporter transfected cells was time-dependent and 4- to 61-fold higher than that in control cells, indicating that these biomarkers are substrates of OATP1B1 and OATP1B3. Transporter-mediated CL_{int, uptake} (V_max/K_m) in OATP1B1 transfected cells was 0.013, 0.023, and 0.011 µL/µg/min for GCDCA-3G, GDCA-3G, and CDCA-24G, respectively, while CL_{int, uptake} in OATP1B3 transfected cells was 0.006, 0.004, and 0.218 µL/µg/min for GCDCA-3G, GDCA-3G, and CDCA-24G. Using the RAF method, our studies suggested that OATP1B1 demonstrated higher contribution than OATP1B3 to active hepatic uptake of both GCDCA-3G (86.5%–88.2% in lot HCE and MMN) and GDCA-3G (87.0%–88.6% in lot HCE and MMN). These in vitro observations are in line with clinical pharmacogenomic studies (1). In contrast, OATP1B3 was a primary transporter for active hepatic uptake of CDCA-24G (84.5%–86.4% in lot HCE and MMN).

Conclusion: Our studies suggested that GCDCA-3G and GDCA-3G are more selective biomarker for OATP1B1, while CDCA-24G is more selective to OATP1B3. The present data, combined with other preclinical and clinical pharmacogenomic data provide important information on the selection of appropriate OATP1B biomarkers and quantitative translation of biomarker data for DDI prediction.

References: (1) Neuvonen, Mikko, et al. "Identification of glycochenodeoxycholate 3‐O‐glucuronide and glycodeoxycholate 3‐O‐glucuronide as highly sensitive and specific OATP1B1 biomarkers." Clinical Pharmacology & Therapeutics 109.3 (2021): 646-657.
(2) Hämäläinen, Kreetta, et al. "Non‐targeted metabolomics for the identification of plasma metabolites associated with organic anion transporting polypeptide 1B1 function." Clinical and Translational Science 17.3 (2024): e13773.
(3) Chan, Grace Hoyee, et al. "Evaluation of the selectivity of several organic anion transporting polypeptide 1B biomarkers using relative activity factor method." Drug Metabolism and Disposition 51.9 (2023): 1089-1104.