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.

Abstract

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 (IC_50,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 IC_50,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.