@article {Chien356, author = {Huan-Chieh Chien and Arik A. Zur and Tristan S. Maurer and Sook Wah Yee and John Tolsma and Paul Jasper and Dennis O. Scott and Kathleen M. Giacomini}, title = {Rapid Method To Determine Intracellular Drug Concentrations in Cellular Uptake Assays: Application to Metformin in Organic Cation Transporter 1{\textendash}Transfected Human Embryonic Kidney 293 Cells}, volume = {44}, number = {3}, pages = {356--364}, year = {2016}, doi = {10.1124/dmd.115.066647}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {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 {\textpm} 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{\textpm} 0.07 and 1.25{\textpm}0.06, respectively, were used to determine the intracellular metformin concentrations. After incubation of the cells with 5 {\textmu}M metformin, the intracellular concentrations were 26.4 {\textpm} 7.8 μM and 268 {\textpm} 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 {\textpm} 3.8 μM and 198.1 {\textpm} 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.}, issn = {0090-9556}, URL = {https://dmd.aspetjournals.org/content/44/3/356}, eprint = {https://dmd.aspetjournals.org/content/44/3/356.full.pdf}, journal = {Drug Metabolism and Disposition} }