Transporter Inhibition Assay Protocol

OATP1B1, OATP1B3, NTCP and vector control cell lines were cultured and subsequent transporter assays conducted as described above using the same transporter‐specific probe substrates and incubation times. For OATP1B1, the probe substrate was [³H]‐estradiol 17β‐D‐glucuronide (0.02 μM) incubated for 2 min, with rifamycin SV (100 μM) as positive control inhibitor. For IC₅₀ determinations, the only difference to the method performed above was that the 15‐min pre‐incubation step contained a range of six concentrations of protease inhibitor drug, and subsequent incubations were conducted with the same six concentration levels of drug rather than with a single concentration. All four protease inhibitor drugs were studied against OATP1B1 using pre‐incubation/incubation concentrations of either 0.1, 0.3, 1, 3, 10 and 30 μM for atazanavir and darunavir, or 0.03, 0.1, 0.3, 1, 3 and 10 μM for lopinavir and ritonavir. Based upon the results determined from the inhibition screen, only atazanavir and lopinavir were studied against OATP1B3 or NTCP using concentrations of 0.1, 0.3, 1, 3, 10 and 30 μM and 0.03, 0.1, 0.3, 1, 3 and 10 μM, or 0.3, 1, 3, 10, 30 and 50 μM and 0.1, 0.3, 1, 3, 10 and 20 μM, respectively.
For each drug, determined percentage (vehicle) control transport activity was plotted against nominal inhibitor concentration and fitted using SigmaPlot 12.5 (Systat Software Inc., Chicago, IL; four parameter logistic equation) to determine the concentration that produces half‐maximal inhibition of probe substrate transport (IC₅₀; equivalent to K_i assuming competitive inhibition as probe substrate concentration in the assay is at least 10‐times lower than its K_m).