Impact of nonspecific binding to membrane vesicles on unbound IC50 estimation for P-glycoprotein and breast cancer resistance protein inhibitors and implications for drug-drug interaction risk assessment.

P-glycoprotein and breast cancer resistance protein are key efflux transporters relevant to drug-drug interactions (DDIs) for orally administered drugs. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use M12 guidance recommends using unbound in vitro inhibition potency (IC50,u) for clinical risk assessment, yet the influence of nonspecific binding to membrane vesicles remains uncharacterized. Herein, vesicle binding for a panel of efflux transporter inhibitors was systematically quantified using equilibrium dialysis and compared with conventional microsomal binding. Nonspecific binding to vesicles reduced free inhibitor concentrations, resulting in right-shifted IC50 values, particularly for high-binding compounds (itraconazole and dronedarone) compared with low-binding compounds (telmisartan) in breast cancer resistance protein assays with Sf9 control vesicles. Similar right shifts were observed for P-glycoprotein inhibition assays with human embryonic kidney 293 control vesicles, although less pronounced. Correction of IC50 values across different vesicle concentrations generally resulted in aligned IC50,u values. Microsomal binding correlated strongly (Pearson correlation coefficient = 0.965) with vesicle binding, supporting its use as a surrogate for binding correction. Incorporating binding-corrected IC50 values into static DDI risk calculations increased false positives but did not affect false negatives. These findings demonstrate that accounting for fraction unbound in vesicle-based assays can substantially influence the estimation of in vitro inhibitory potency and subsequent static DDI risk assessment. SIGNIFICANT STATEMENT: This study systematically characterizes nonspecific binding to membrane vesicles in P-glycoprotein and breast cancer resistance protein inhibition assays and demonstrates its substantial impact on IC50 determination. The strong correlation between vesicles and microsomal binding provides a practical surrogate for implementing International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use M12 unbound potency recommendations for vesicle-based inhibition assays.