OATP2B1 is an intestinal and hepatic drug uptake transporter. Intestinal OATP2B1 has been elucidated as the mechanism of unexpected clinical drug−drug interactions (DDIs), where drug exposure was unexpectedly decreased with unchanged half-life. Hepatic OATP2B1 may be an understudied clinical DDI mechanism. The aim of the present work was to understand the prevalence of clinically relevant intestinal and hepatic OATP2B1 inhibitors in marketed drug space. HEK293 cells stably overexpressing human OATP2B1 or vector control were generated and cultured for 72 h in a 96-well format. OATP2B1-mediated uptake of dibromofluorescein (DBF) was found to be optimal at 10 μM concentration and 30 min incubation time. A total of 294 drugs (top 300 marketed drugs, excluding biologics and restricted drugs, supplemented with ∼100 small-molecule drugs) were screened for OATP2B1 inhibition at 10 μM. Drugs demonstrating ≥50% inhibition in this screen were advanced for IC 50 determination, which was extrapolated to clinical intestinal and hepatic OATP2B1 inhibition as per 2017 FDA DDI guidance. Of the 294 drugs screened, 67 elicited ≥50% inhibition of OATP2B1-mediated DBF uptake at 10 μM screening concentration. For the 67 drugs flagged in the singleconcentration inhibition screen, upon evaluation of a full concentration range, IC 50 values could be determined for 58 drugs. OATP2B1 IC 50 values established for these 58 drugs were extrapolated as potentially clinically relevant at the intestinal level for 38 orally administered drugs (I gut /IC 50 ≥ 10), and 17 were flagged as potential clinical inhibitors of hepatic OATP2B1 uptake (1 + I in,max,u /IC 50 ≥ 1.1). This analysis of 294 drugs demonstrated prevalence of clinically relevant intestinal and hepatic OATP2B1 inhibitors to be 13 and 6%, respectively. As OATP2B1-inhibitor drugs are not exceedingly rare, these results suggest that clinical OATP2B1 DDIs have been rarely observed because OATP2B1 is uncommonly the predominant determinant of drug disposition.

OATP2B1, a member of the solute carrier (SLC) transporter family, is an important mechanism of substrate drug uptake in the intestine and liver and therefore a determinant of clinical pharmacokinetics and site of drug–drug interactions. Other SLC transporters have emerged as pharmacology targets. Studies of SLC transporter uptake to-date relied on radioisotope- or fluorescence-labeled reagents or low-throughput quantification of unlabeled compounds in cell lysate. In this study, we developed a cell-based MALDI MS workflow for investigation of OATP2B1 cellular uptake by optimizing the substrate, matrix, matrix–analyte ratio, and matrix application and normalization method. This workflow was automated and applied to characterize substrate transport kinetics and to test 294 top-marketed drugs for OATP2B1 inhibition and quantify inhibitory potencies necessary for extrapolation of clinical drug–drug interaction potential. Intra-assay reproducibility of this MALDI MS method was high (CV < 10%), and results agreed well (83% overlap) with previously published radioisotope assay data. Our results indicate that fast and robust MALDI MS cellular assays could emerge as a high-throughput label-free alternative for direct assessment of drug transporter function in DDIs and toxicities as well as enable drug discovery for transporters as pharmacology targets.