Eyer Klaus, Herger Michael, Krämer Stefanie D, Dittrich Petra S
ETH Zurich, Department of Chemistry and Applied Biosciences, Bioanalytics, Laboratory of Organic Chemistry, Wolfgang-Pauli-Str. 10, 8093, Zurich, Switzerland.
Pharm Res. 2014 Dec;31(12):3415-25. doi: 10.1007/s11095-014-1431-2. Epub 2014 Jun 14.
The membrane protein P-glycoprotein (P-gp) plays key roles in the oral bioavailability of drugs, their blood brain barrier passage as well as in multidrug resistance. For new drug candidates it is mandatory to study their interaction with P-gp, according to FDA and EMA regulations. The vast majority of these tests are performed using confluent cell layers of P-gp overexpressing cell lines that render these tests laborious. In this study, we introduce a cell-free microfluidic assay for the rapid testing of drug- P-gp interactions. Cell-derived vesicles are prepared from MDCKII-MDR1 overexpressing cells and immobilized on the surface of a planar microfluidic device. The drug is delivered continuously to the vesicles and calcein accumulation is monitored by means of a fluorescence assay and total internal reflection fluorescence (TIRF) microscopy. Only small amounts of compounds (~10 μl) are required in concentrations of 5, 25 and 50 μM for a test that provides within 5 min information on the apparent dissociation constant of the drug and P-gp. We tested 10 drugs on-chip, 9 of which are inhibitors or substrates of P-glycoprotein and one negative control. We benchmarked the measured apparent dissociation constants against an alternative assay on a plate reader and reference data from FDA. These comparisons revealed good correlations between the logarithmic apparent dissociation constants (R(2) = 0.95 with ATPase assay, R(2) = 0.93 with FDA data) and show the reliability of the rapid on-chip test. The herein presented assay has an excellent screening window factor (Z'-factor) of 0.8, and is suitable for high-throughput testing.
膜蛋白P-糖蛋白（P-gp）在药物的口服生物利用度、血脑屏障通透性以及多药耐药性方面发挥着关键作用。根据美国食品药品监督管理局（FDA）和欧洲药品管理局（EMA）的规定，对于新的候选药物，研究它们与P-糖蛋白的相互作用是必不可少的。这些测试绝大多数是使用过表达P-糖蛋白的细胞系的汇合细胞层进行的，这使得这些测试既费力又耗时。在本研究中，我们引入了一种无细胞微流控分析方法，用于快速检测药物与P-糖蛋白的相互作用。从过表达MDCKII-MDR1的细胞中制备细胞衍生囊泡，并将其固定在平面微流控装置的表面。药物持续输送到囊泡中，并通过荧光分析和全内反射荧光（TIRF）显微镜监测钙黄绿素的积累。对于一项能在5分钟内提供药物与P-糖蛋白表观解离常数信息的测试，仅需要少量化合物（约10微升），浓度分别为5、25和50微摩尔。我们在芯片上测试了10种药物，其中9种是P-糖蛋白的抑制剂或底物，一种为阴性对照。我们将测量的表观解离常数与酶标仪上的另一种分析方法以及FDA的参考数据进行了比较。这些比较显示，对数表观解离常数之间具有良好的相关性（与ATP酶分析的R(2) = 0.95，与FDA数据的R(2) = 0.93），并表明了芯片上快速测试的可靠性。本文介绍的分析方法具有出色的筛选窗口因子（Z'因子），为0.8，适用于高通量测试。
