Vol. 280, Issue 2, 813-819, 1997

In Vivo and In Vitro Evidence for Nonrestricted Transport of 2,7-Bis(2-Carboxyethyl)-5(6)-Carboxyfluorescein Tetraacetoxymethyl Ester at the Blood-Brain Barrier

Tomoko Hirohashi, Tetsuya Terasaki, Minoru Shigetoshi and Yuichi Sugiyama

Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113, Japan

2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein tetraacetoxymethyl ester (BCECF-AM), a fluorescence reagent for the measurement of intracellular pH with a molecular weight of 809 Da, was used to test the hypothesis that the blood-brain barrier (BBB) does not restrict the influx of substrate with a molecular weight greater than 600 Da. Using cultured bovine brain capillary endothelial cells (BCEC), the influx rate of BCECF-AM was found to be 151 ± 2 µl/min/mg protein and was extrapolated to give 446 ± 7 µl/min/g brain as a BBB permeability surface area product (PS). No significant saturation was observed for the initial in vitro uptake of BCECF-AM into BCEC at concentrations 0.1, 1.0 and 5.0 µM. The apparent activation energy of the initial uptake of BCECF-AM was found to be 5.09 kcal/mol. These results suggest that BCECF-AM is transported into the BBB by passive diffusion. The in vivo BBB PS value was also found to be 295 ± 48 µl/min/g brain and 132 ± 24 µl/min/g brain by the in situ brain perfusion and the carotid artery injection methods, respectively. No significant efflux of BCECF-AM from the brain was observed over a 120 sec washout period, suggesting that BCECF-AM is immediately hydrolyzed to BCECF, a hydrophilic analogue, in the brain after crossing the BBB. The octanol/water partition coefficient of BCECF-AM was found to be 5.66 ± 0.27. The BBB PS value of BCECF-AM was predicted to be 105 µl/min/g brain, based on the relationship between the BBB PS value and the value of partition coefficient divided by the square root of the molecular weight. These results demonstrate that BCECF-AM transport across the BBB is not restricted despite its large molecular size.