OATP1B1-Mediated Transport Kinetics

Transport studies in HEK293 stable cell lines and in human SCH were conducted similarly to those described previously.^{41 (link),42 (link)} HEK293-OATP1B1, -FLAG-OATP1B1, and -Mock cells were seeded at a density of 0.8–1.1 × 10⁵ cells/well in 24-well culture plates precoated with poly-l-lysine and were cultured for 48–72 h. Cells from stable cell lines or human SCH were either pretreated with culture medium containing CQ, monensin, bafilomycin A1, or vehicle control for the designated times or not pretreated. 0.1% DMSO was used as vehicle control for monensin and bafilomycin A1 treatment. As CQ stock (50 mM) was resolved in water, CQ-free fresh medium was used as vehicle control for CQ pretreatment. To determine the effects of CQ on OATP1B1-mediated transport after prolonged treatment, HEK293-OATP1B1 cells were preincubated with CQ-free (CTL) or 25 μM CQ-containing medium for 2 h. At the end of pre-incubation, the culture medium was aspirated and CTL cells were cultured in CQ-free medium and cells pretreated with 25 μM CQ were cultured in medium containing 1.5 μM CQ, for the indicated time, up to 24 h.
At the time of uptake experiments, after rinsing with prewarmed (37 °C) HBSS buffer (pH 7.4) three times, cells were incubated with HBSS containing [³H]E₂17G (1 μM, 2 min) or [³H]pitavastatin (1 μM, 0.5 min) in the absence or presence of testing drugs (CQ or rifampicin). At the end of incubation, the buffer was aspirated rapidly, and the cells were rinsed with ice-cold HBSS three times and then lysed with Triton X-100 (0.5% v/v) in DPBS. An aliquot of the lysate was subjected to liquid scintillation counting (LS6500 scintillation counter, Beckman Coulter, Brea, CA). Substrate accumulation was normalized to protein concentration determined by BCA assay (Pierce Chemical, Rockford, IL) and corrected for nonspecific binding of the substrate by including a non-overlaid poly-l-lysine coated blank plate for uptake studies in stable cell lines and a Matrigel overlaid blank plate for human SCH, respectively.
To determine [³H]E₂17G transport kinetic parameters, the maximal transport velocity (V_max) and the affinity constant (K_m), [³H]E₂17G accumulation (0.1–40 μM, 2 min) was determined in HEK293-OATP1B1 and -Mock cells following pretreatment with CQ(25 μM) or CTL. Values of [³H]E₂17G accumulation in Mock cells were subtracted from those in HEK293-OATP1B1 cells. The V_max and K_m values of E₂17G transport were estimated by fitting the Michaelis–Menten equation (eq 2) to the data using Phoenix WinNonlin, v6.3 (Certara, St. Louis, MO), where ν is E₂17G transport velocity and S is E₂17G concentration.