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
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
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DOI：
10.1124/dmd.115.066647
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发表时间：
2016-03-01
影响因子：
3.9
通讯作者：
Giacomini, Kathleen M.
中科院分区：
文献类型：
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作者：
Chien, Huan-Chieh;Zur, Arik A.;Giacomini, Kathleen M.
由于细胞内未结合药物浓度在预测体内浓度中的重要性，体内浓度是药物功效和毒性的决定因素，因此已经开发了许多测定法来评估药物的体外未结合浓度。在这里，我们提出了一种快速的方法来确定细胞内未结合的药物浓度在培养的细胞中，我们应用该方法沿着与一个机械模型来预测二甲双胍浓度的稳定转染的人胚肾293（HEK 293）细胞的亚细胞隔室。通过从[C-14]-尿素分布体积（总水空间，TWS）中减去[H-3]-菊粉分布体积（细胞外空间，ECS）来计算细胞内空间（ICS）。获得的细胞内空间值（平均值+/- S.E.M.;使用HEK细胞（HEK-空载体[EV]）和过表达人有机阳离子转运蛋白1（HEK-OCT 1）的单层细胞（分别为1.21 +/- 0.07和1.25 +/- 0.06）测定细胞内二甲双胍浓度。用5 μ M二甲双胍孵育细胞后，HEK-EV和HEK-OCT 1中的细胞内浓度分别为26.4 +/- 7.8 μ M和268 +/- 11.0 μ M。此外，在HEK-OCT 1细胞中，与正常K+缓冲液（5.4 mM KCl）相比，高K+缓冲液（140 mM KCl）中的细胞内二甲双胍浓度较低（分别为54.8 +/- 3.8 μ M和198.1 +/- 11.2 μ M; P < 0.05）。我们的机制模型表明，根据假设的生理值的可信范围，带正电荷的二甲双胍在内质网和/或线粒体中积累到特别高的水平。该方法与计算模型一起可用于确定细胞内未结合浓度，并预测药物在其他复杂系统（如原代细胞）中的亚细胞蓄积。
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.