TY - JOUR T1 - Simultaneous Assessment in vitro of Transporter and Metabolic Processes in Hepatic Drug Clearance: Use of a media loss approach JF - Drug Metabolism and Disposition JO - Drug Metab Dispos DO - 10.1124/dmd.117.079590 SP - dmd.117.079590 AU - James Harrison AU - Tom De Bruyn AU - Adam S Darwich AU - J Brian Houston Y1 - 2018/01/01 UR - http://dmd.aspetjournals.org/content/early/2018/02/07/dmd.117.079590.abstract N2 - Hepatocyte drug depletion-time assays are well established for the determination of metabolic clearance in vitro. The present study focuses on the refinement and evaluation of a "media loss" assay, an adaptation of the conventional depletion assay involving centrifugation of hepatocytes prior to sampling, allowing estimation of uptake in addition to metabolism. Using experimental procedures consistent with a high throughput, a selection of 12 compounds with a range of uptake and metabolism characteristics (atorvastatin, cerivastatin, clarithromycin, erythromycin, indinavir, pitavastatin, repaglinide, rosuvastatin, saquinavir and valsartan, together with two control compounds - midazolam and tolbutamide) were investigated in the presence and absence of the cytochrome P450 inhibitor 1-aminobenzotriazole and organic anion transporter protein inhibitor rifamycin SV. Data generated simultaneously for a given drug provided, through the use of a mechanistic cell model, clearance terms characterising metabolism, active and passive uptake, together with intracellular binding and partitioning parameters. Results were largely consistent with the particular drug characteristics, with active uptake, passive diffusion and metabolic clearances ranging between 0.4 - 777, 3 - 383 and 2 - 236 μL/min/mg protein, respectively. The same experiments provided total and unbound drug cellular partition coefficients ranging between 3.8 - 254 and 2.3 - 8.3, respectively and intracellular unbound fractions between 0.014 - 0.263. Following in vitro to in vivo extrapolation, the lowest prediction bias was noted using uptake clearance, compared to metabolic clearance or apparent clearance from the media loss assay alone. This approach allows rapid and comprehensive characterisation of hepatocyte drug disposition valuable for prediction of hepatic processes in vivo. ER -