(iv) Concentration equilibrium transport assay in MDCK cells.

Transport assays employing MDR1- or BCRP-expressing MDCK cells were performed on microporous polycarbonate membrane filters (3.0-μm pore size, 24-mm diameter; Transwell 3414; Costar, Corning, NY) as described previously (7, 8). The cells were seeded at a density of 1 × 10⁶ per insert and cultured for 4 days in standard cultivation medium, DMEM (Gibco) with 10% FBS, until reaching confluence, with daily replacement of cell culture medium. Thirty minutes before starting the experiment, cells were washed with prewarmed PBS and incubated with Opti-MEM with or without rilpivirine or control inhibitor. The transport assay was initiated by addition of [³H]abacavir (300 nM) with or without rilpivirine (or control inhibitor) to both apical and basal compartments, providing equal concentrations of the drug on both sides of the monolayer. For drug analysis, aliquots of 50 μl were collected from both compartments at 2, 4, and 6 h, and radioactivity was measured by liquid scintillation counting (Tri-Carb 2900 TR; PerkinElmer). The equilibrium assay concentration ratio (r_e) was calculated as described earlier (7, 8) by dividing the concentration of [³H]abacavir in the apical compartment by the concentration in the basal compartment at the end (6 h) of the experiment. The integrity of the monolayer was verified by analyzing the leakage of fluorescein isothiocyanate-dextran (accepting up to 1% per hour).