PT - JOURNAL ARTICLE 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 TI - 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 AID - 10.1124/dmd.115.066647 DP - 2016 Mar 01 TA - Drug Metabolism and Disposition PG - 356--364 VI - 44 IP - 3 4099 - http://dmd.aspetjournals.org/content/44/3/356.short 4100 - http://dmd.aspetjournals.org/content/44/3/356.full SO - Drug Metab Dispos2016 Mar 01; 44 AB - 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.