Assessment of Transporter-Mediated and Passive Hepatic Uptake Clearance Using Rifamycin-SV as a Pan-Inhibitor of Active Uptake

Abstract

The use of in vitro data for the quantitative prediction of transporter-mediated clearance is critical. Central to this evaluation is the use of hepatocytes, since they contain the full complement of transporters and metabolic enzymes. In general, uptake clearance (CL_uptake) is evaluated by measuring the appearance of compound in the cell. Passive clearance (CL_pd) is often determined by conducting parallel studies at 4 °C or by attempting to saturate uptake pathways. Both approaches have their limitations. Recent studies have proposed the use of Rifamycin-SV (RFV) as a pan-inhibitor of hepatic uptake pathways. In our studies, we confirm that transport activity of all major hepatic uptake transporters is inhibited significantly by RFV at 1 mM (OATP1B1, 1B3, and 2B1 = NTCP (80%), OCT1 (65%), OAT2 (60%)). Under these incubation conditions, we found that the free intracellular concentration of RFV is ∼175 μM and that several major CYPs and UGTs can be reversibly inhibited. Using this approach, we also determined CL_uptake and CL_pd of nine known OATP substrates across three different lots of human hepatocytes. The scaling factors generated for these compounds at 37 °C with RFV and 4 °C were found to be similar. The CL_pd of passively permeable compounds like metoprolol and semagacestat were found to be higher at 37 °C compared to 4 °C, indicating a temperature effect on these compounds. In addition, our data also suggests that incorporation of medium concentrations into CL_uptake and CL_pd calculations may be critical for highly protein bound and highly lipophilic drugs. Overall, our data indicate that RFV, instead of 4 °C, can be reliably used to measure CL_uptake and CL_pd of drugs.