Bachmeier Corbin J, Trickler William J, Miller Donald W
Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Nebraska, USA.
Drug Metab Dispos. 2006 Jun;34(6):998-1003. doi: 10.1124/dmd.105.006999. Epub 2006 Mar 22.
The present study quantitatively compared the drug efflux transport kinetics of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and its fluorescent metabolite 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in various blood-brain barrier (BBB) models. BCECF-AM was exposed to freshly isolated bovine brain microvessels (BBM), primary cultured bovine brain microvessel endothelial cells (BBMEC), and MDCK-MDR1 cells for 30 min in the presence or absence of the P-glycoprotein (P-gp) inhibitor N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918). P-gp transport kinetics were determined indirectly by calculating the difference in BCECF accumulation when P-gp was functional and completely inhibited by GF120918 (3.2 microM). Multidrug resistance-associated protein (MRP) transport kinetics were determined by measuring the amount of BCECF transported out of the cell over time. For P-gp-related transport, Km values for BCECF-AM were approximately the same in all three models (around 2 microM), whereas the Vmax was 4-fold greater in the BBM than in the BBMEC or MDCKII-MDR1 cells. For MRP-related transport, Km values for BCECF varied widely among the three BBB models with a rank order of MDCKII-MDR1 < BBMEC < BBM. Like P-gp, the Vmax of BCECF for MRP-related transport was overwhelmingly higher in the BBM compared with the cultured cells. Because differences in the expression of P-gp, MRP5, and MRP6 were observed in the various BBB models using reverse transcription-polymerase chain reaction techniques, the disparity in transport kinetics between the BBB models may be linked to variations in the amount or type of drug efflux transporters expressed in each model. The present study introduces a method of quantitatively evaluating drug efflux transport kinetics in the BBB.
本研究定量比较了2',7'-双(2-羧乙基)-5(6)-羧基荧光素乙酰氧基甲酯（BCECF-AM）及其荧光代谢产物2',7'-双(2-羧乙基)-5(6)-羧基荧光素（BCECF）在各种血脑屏障（BBB）模型中的药物外排转运动力学。在存在或不存在P-糖蛋白（P-gp）抑制剂N-(4-[2-(1,2,3,4-四氢-6,7-二甲氧基-2-异喹啉基)乙基]-苯基)-9,10-二氢-5-甲氧基-9-氧代-4-吖啶甲酰胺（GF120918）的情况下，将BCECF-AM暴露于新鲜分离的牛脑微血管（BBM）、原代培养的牛脑微血管内皮细胞（BBMEC）和MDCK-MDR1细胞30分钟。通过计算P-gp发挥功能时和被GF120918（3.2 microM）完全抑制时BCECF积累的差异来间接测定P-gp转运动力学。通过测量随时间从细胞中转运出的BCECF量来测定多药耐药相关蛋白（MRP）转运动力学。对于与P-gp相关的转运，BCECF-AM的Km值在所有三种模型中大致相同（约2 microM），而BBM中的Vmax比BBMEC或MDCKII-MDR1细胞中的高4倍。对于与MRP相关的转运，BCECF的Km值在三种BBB模型中差异很大，顺序为MDCKII-MDR1 < BBMEC < BBM。与P-gp一样，与MRP相关转运的BCECF的Vmax在BBM中比在培养细胞中高得多。因为使用逆转录-聚合酶链反应技术在各种BBB模型中观察到了P-gp、MRP5和MRP6表达的差异，所以BBB模型之间转运动力学的差异可能与每个模型中表达的药物外排转运体的数量或类型的变化有关。本研究介绍了一种定量评估BBB中药物外排转运动力学的方法。
