Inhibition of OATP1B1-mediated uptake of [³H]E₁S (A), [³H]E₂G (B), atorvastatin (C), pitavastatin (D), and rosuvastatin (E) after preincubation with CsA. The cells were washed with transport buffer prewarmed at 37°C and were then pretreated for 5 minutes with the transport buffer without substrates or inhibitors. The medium was then replaced with the transport buffer containing inhibitors at the various concentrations of CsA, and cells were further preincubated for 0 or 30 minutes at 37°C. After the preincubation, the transport buffer containing inhibitors was removed, and the reaction was then started by applying prewarmed fresh transport buffer containing [³H]E₁S, [³H]E₂G, atorvastatin (0.3 μM), pitavastatin (0.5 μM), or rosuvastatin (3 μM) and the inhibitors to the cells. At 0.5 minutes ([³H]E₁S and [³H]E₂G) or 1 minute (atorvastatin, pitavastatin, and rosuvastatin), the reaction solution was removed by aspiration, and cells were washed with ice-cold transport buffer. The OATP1B1-mediated uptake was obtained by subtracting the uptake in HEK293/mock cells from that in HEK293/OATP1B1 cells. Data are shown as percentage mean uptake volume values ± S.E. of three to six wells in one to two independent experiments compared with those of the control.

Inactivation of OATP1B1-mediated uptake of [³H]E₁S (A), [³H]E₂G (B), atorvastatin (C), pitavastatin (D), and rosuvastatin (E) after preincubation with CsA. HEK293/OATP1B1 cells were preincubated with CsA at 0 (〇, black), 0.02 (△, blue), 0.05 (□, green), 0.1 (●, orange), 0.2 (▲, red), and 0.5 μM (■, purple) for [³H]E₁S and [³H]E₂G and 0 (〇, black), 0.005 (△, blue), 0.01(□, green), 0.02 (●, orange), 0.05 (▲, red), and 0.1 μM (■, purple) for atorvastatin, pitavastatin, and rosuvastatin for the designated periods. After preincubation, the cells were washed once with prewarmed transport buffer and incubated with each substrate. At 0.5 ([³H]E₁S and [³H]E₂G) or 1 minute (atorvastatin, pitavastatin, and rosuvastatin), the reaction solution was aspirated, and the cells were washed twice with ice-cold transport buffer, followed by determination of their uptake. Uptake values are represented as the percentage of the activity obtained in the vehicle control samples and are plotted vs. the preincubation period. Each symbol represents the mean ± S.E. of three wells in a single experiment.

Observed k_obs,app values of OATP1B1-mediated uptake of [³H]E₁S (A), [³H]E₂G (B), atorvastatin (C), pitavastatin (D), and rosuvastatin (E) vs. CsA concentration. The k_obs,app values were determined as the negative slopes of the natural logarithm (0–20 seconds for [³H]E₁S, [³H]E₂G, pitavastatin, and rosuvastatin; 0–3 minutes for atorvastatin) shown in Fig. 2. Solid lines represent the fitting curves of the observed k_obs,app vs. CsA concentration by nonlinear regression analysis based on eq. 2. Each symbol represents the mean ± S.E. of three wells in a single experiment.

Recovery of OATP1B1 activity after preincubation with CsA. HEK293/OATP1B1 cells were preincubated with culture medium containing CsA (0.3 μM) for 30 minutes. The medium was replaced with fresh culture medium and further incubated in the absence of CsA for the designated periods. The cells were then washed with prewarmed transport buffer once, and the transport buffer, including [³H]E₁S (〇), [³H]E₂G (△), atorvastatin (●), pitavastatin (▲), and rosuvastatin (■), was added, followed by determination of their uptake. Data are normalized with the control value obtained without the inhibitor and are shown as means ± S.E. of three wells in a single experiment.