Evaluating the Utility and Constraints of the Competitive Counter Flow (CCF) Assay in OATP1B1 Substrate Profiling

The organic anion transporting polypeptide 1B1 (OATP1B1) plays a critical role in hepatic uptake of a wide range of high- and low-permeability, anionic molecules, particularly those in Extended Clearance Classification System (ECCS) Classes 1 and 3. However, conventional uptake assays using HEK293 cells overexpressing OATP1B1 can yield false-negative results, especially for highly permeable, lipophilic anionic or zwitterionic compounds, where passive diffusion and nonspecific binding may mask transporter-mediated uptake. In this study, we applied the competitive counterflow (CCF) assay to evaluate 58 compounds across different ECCS classes. The assay measures steady-state changes in intracellular [3H]-estradiol-17β-glucuronide retention following exposure to test compounds across multiple concentrations. Several known OATP1B1 substrates, exhibited more than 50% loss of intracellular [3H]-estradiol-17β-glucuronide at one or more concentrations compared to control. Using a three-tier classification based on tracer retention across multiple concentrations, the assay demonsrated high sensitivity for detecting compounds that are OATP1B1 substrates. Notably, the CCF assay enabled identification of four xenobiotics-bumetanide, candesartan, naringin and sulfasalazine-not previously recognized as OATP1B1 substrates using OATP1B1 uptake assay. Overall, this work provides a comprehensive evaluation of the CCF assay as a complementary, early‑stage screening and triage tool for OATP1B1 substrate assessment. The findings underscore the importance of an integrative strategy that combines CCF data with orthogonal in vitro and in vivo evidence to mitigate the risk of both false‑negative and false‑positive conclusions, particularly for ECCS Class 1B compounds where traditional overexpression systems show limited sensitivity.