Quantitative Contribution of Six Major Transporters to the Hepatic Uptake of Drugs: "SLC-Phenotyping" Using Primary Human Hepatocytes

Hepatic uptake transporters [solute carriers (SLCs)], including organic anion transporting polypeptide (OATP) 161, OATP1B3, OATP2B1, sodium-dependent taurocholate cotransporting polypeptide (NTCP), and organic anion (OAT2) and organic cation (OCT1) transporters, play a key role in determining the systemic and liver exposure of chemically diverse drugs. Here, we established a phenotyping approach to quantify the contribution of the six SLCs, and passive diffusion, to the overall uptake using plated human hepatocytes (PHHs). First, selective inhibitor conditions were identified by screening about 20 inhibitors across the six SLCs using sing le-transfected human embryonic kidney 293 cells. Data implied rifamycin SV (20 mu M) inhibits three OATPs, while rifampicin (5 mu M) inhibits OATP1B1/163 only. Further, hepatitis B virus nnyristoylated-preS1 peptide (0.1 mu M), quinidine (100 mu M), and ketoprofen (100-300 mu M) are relatively selective against NTCP, OCT1, and OAT2, respectively. Second, using these inhibitory conditions, the fraction transported (f(t)) by the individual SLCs was characterized for 20 substrates with PHH. Generally, extended clearance classification system class 1A/3A (e.g., warfarin) and 16/3B compounds (e.g., statins) showed predominant OAT2 and OATP1B1/163 contribution, respectively. OCT1-mediated uptake was prominent for class 2/4 compounds (e.g., metformin). Third, in vitro f(t) values were corrected using quantitative proteomics data to obtain "scaled f(t)." Fourth, in vitro-in vivo extrapolation of the scaled OATP1B1/163 f(t) was assessed, leveraging statin clinical drug-drug interaction data with rifampicin as the perpetrator. Finally, we outlined a novel stepwise strategy to implement phenotypic characterization of SLC-mediated hepatic uptake for new molecular entities and drugs in a drug discovery and development setting.