Abstract
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 μM). 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 μM), 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.
Footnotes
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These studies were supported by funds from National Institutes of Health Grant R01-CA93558. C.J.B. was supported through the McDonald/Bukey and Blanche Widaman graduate studies fellowships.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.105.006999.
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ABBREVIATIONS: BBB, blood-brain barrier; P-gp, P-glycoprotein; MRP, multidrug resistance-associated protein; BCRP, breast cancer resistant protein; BBM, bovine brain microvessel(s); BBMEC, bovine brain microvessel endothelial cell(s); MDCKII-MDR1, Madin-Darby canine kidney epithelial cell(s) transfected with the human MDR1 gene; BCECF-AM, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; GF120918, 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; BCECF, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; RT-PCR, reverse transcription-polymerase chain reaction; EBG, estradiol-17β-d-glucuronide.
- Received August 23, 2005.
- Accepted March 15, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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