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

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 [H-3]-inulin distribution volume (extracellular space, ECS) from the [C-14]-urea distribution volume (total water space, TWS). Values obtained for intracellular space (mean +/- S.E.M.; mu l/10(6) 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 mu M metformin, the intracellular concentrations were 26.4 +/- 7.8 mu M and 268 +/- 11.0 mu M, respectively, in HEK-EV and HEK-OCT1. In addition, intracellular metformin concentrations were lower in high K+ buffer (140 mMKCl) compared with normal K+ buffer (5.4 mM KCI) in HEK-OCT1 cells (54.8 +/- 3.8 mu M and 198.1 +/- 11.2 mu M, respectively; P < 0.05). Our mechanisticmodel 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.