title: 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.
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 [(3)H]-inulin distribution volume (extracellular space, ECS) from the [(14)C]-urea distribution volume (total water space, TWS). Values obtained for intracellular space (mean ± S.E.M.; μl/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 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.
authors.affiliation: Department of Bioengineering and Therapeutic Sciences, Schools of Pharmacy and Medicine, University of California San Francisco, San Francisco, California (H.C.C., A.A.Z., S.W.Y., K.M.G.); Systems Modeling and Simulation (T.S.M.) and Cardiovascular and Metabolic Disease Research Unit (D.O.S.), Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research and Development, Cambridge, Massachusetts; RES Group, Inc. (J.T., P.J.) Needham, Massachusetts.
authors.forename: Huan-Chieh
authors.initials: HC
authors.lastname: Chien
authors.affiliation: Department of Bioengineering and Therapeutic Sciences, Schools of Pharmacy and Medicine, University of California San Francisco, San Francisco, California (H.C.C., A.A.Z., S.W.Y., K.M.G.); Systems Modeling and Simulation (T.S.M.) and Cardiovascular and Metabolic Disease Research Unit (D.O.S.), Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research and Development, Cambridge, Massachusetts; RES Group, Inc. (J.T., P.J.) Needham, Massachusetts.
authors.forename: Arik A
authors.initials: AA
authors.lastname: Zur
authors.affiliation: Department of Bioengineering and Therapeutic Sciences, Schools of Pharmacy and Medicine, University of California San Francisco, San Francisco, California (H.C.C., A.A.Z., S.W.Y., K.M.G.); Systems Modeling and Simulation (T.S.M.) and Cardiovascular and Metabolic Disease Research Unit (D.O.S.), Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research and Development, Cambridge, Massachusetts; RES Group, Inc. (J.T., P.J.) Needham, Massachusetts.
authors.forename: Tristan S
authors.initials: TS
authors.lastname: Maurer
authors.affiliation: Department of Bioengineering and Therapeutic Sciences, Schools of Pharmacy and Medicine, University of California San Francisco, San Francisco, California (H.C.C., A.A.Z., S.W.Y., K.M.G.); Systems Modeling and Simulation (T.S.M.) and Cardiovascular and Metabolic Disease Research Unit (D.O.S.), Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research and Development, Cambridge, Massachusetts; RES Group, Inc. (J.T., P.J.) Needham, Massachusetts.
authors.forename: Sook Wah
authors.initials: SW
authors.lastname: Yee
authors.affiliation: Department of Bioengineering and Therapeutic Sciences, Schools of Pharmacy and Medicine, University of California San Francisco, San Francisco, California (H.C.C., A.A.Z., S.W.Y., K.M.G.); Systems Modeling and Simulation (T.S.M.) and Cardiovascular and Metabolic Disease Research Unit (D.O.S.), Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research and Development, Cambridge, Massachusetts; RES Group, Inc. (J.T., P.J.) Needham, Massachusetts.
authors.forename: John
authors.initials: J
authors.lastname: Tolsma
authors.affiliation: Department of Bioengineering and Therapeutic Sciences, Schools of Pharmacy and Medicine, University of California San Francisco, San Francisco, California (H.C.C., A.A.Z., S.W.Y., K.M.G.); Systems Modeling and Simulation (T.S.M.) and Cardiovascular and Metabolic Disease Research Unit (D.O.S.), Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research and Development, Cambridge, Massachusetts; RES Group, Inc. (J.T., P.J.) Needham, Massachusetts.
authors.forename: Paul
authors.initials: P
authors.lastname: Jasper
authors.affiliation: Department of Bioengineering and Therapeutic Sciences, Schools of Pharmacy and Medicine, University of California San Francisco, San Francisco, California (H.C.C., A.A.Z., S.W.Y., K.M.G.); Systems Modeling and Simulation (T.S.M.) and Cardiovascular and Metabolic Disease Research Unit (D.O.S.), Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research and Development, Cambridge, Massachusetts; RES Group, Inc. (J.T., P.J.) Needham, Massachusetts.
authors.forename: Dennis O
authors.initials: DO
authors.lastname: Scott
authors.affiliation: Department of Bioengineering and Therapeutic Sciences, Schools of Pharmacy and Medicine, University of California San Francisco, San Francisco, California (H.C.C., A.A.Z., S.W.Y., K.M.G.); Systems Modeling and Simulation (T.S.M.) and Cardiovascular and Metabolic Disease Research Unit (D.O.S.), Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research and Development, Cambridge, Massachusetts; RES Group, Inc. (J.T., P.J.) Needham, Massachusetts kathy.giacomini@ucsf.edu.
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Giacomini
mesh_terms: D001692:Biological Transport / Q000502:physiology; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008687:Metformin / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D027702:Organic Cation Transporter 1 / Q000378:metabolism*; D014162:Transfection / Q000379:methods
chemical_list: D027702:Organic Cation Transporter 1; D008687:Metformin