TY - JOUR T1 - Rapid Method To Determine Intracellular Drug Concentrations in Cellular Uptake Assays: Application to Metformin in Organic Cation Transporter 1–Transfected Human Embryonic Kidney 293 Cells JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 356 LP - 364 DO - 10.1124/dmd.115.066647 VL - 44 IS - 3 AU - Huan-Chieh Chien AU - Arik A. Zur AU - Tristan S. Maurer AU - Sook Wah Yee AU - John Tolsma AU - Paul Jasper AU - Dennis O. Scott AU - Kathleen M. Giacomini Y1 - 2016/03/01 UR - http://dmd.aspetjournals.org/content/44/3/356.abstract N2 - Because of the importance of intracellular unbound drug concentrations in the prediction of in vivo concentrations that are determinants of drug efficacy and toxicity, a number of assays have been developed to assess in vitro unbound concentrations of drugs. Here we present a rapid method to determine the intracellular unbound drug concentrations in cultured cells, and we apply the method along with a mechanistic model to predict concentrations of metformin in subcellular compartments of stably transfected human embryonic kidney 293 (HEK293) cells. Intracellular space (ICS) was calculated by subtracting the [3H]-inulin distribution volume (extracellular space, ECS) from the [14C]-urea distribution volume (total water space, TWS). Values obtained for intracellular space (mean ± S.E.M.; μl/106 cells) of monolayers of HEK cells (HEK-empty vector [EV]) and cells overexpressing human organic cation transporter 1 (HEK-OCT1), 1.21± 0.07 and 1.25±0.06, respectively, were used to determine the intracellular metformin concentrations. After incubation of the cells with 5 µM metformin, the intracellular concentrations were 26.4 ± 7.8 μM and 268 ± 11.0 μM, respectively, in HEK-EV and HEK-OCT1. In addition, intracellular metformin concentrations were lower in high K+ buffer (140 mM KCl) compared with normal K+ buffer (5.4 mM KCl) in HEK-OCT1 cells (54.8 ± 3.8 μM and 198.1 ± 11.2 μM, respectively; P < 0.05). Our mechanistic model suggests that, depending on the credible range of assumed physiologic values, the positively charged metformin accumulates to particularly high levels in endoplasmic reticulum and/or mitochondria. This method together with the computational model can be used to determine intracellular unbound concentrations and to predict subcellular accumulation of drugs in other complex systems such as primary cells. ER -