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.
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 microl/min/mg protein and was extrapolated to give 446 +/- 7 microl/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 microM. 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 microl/min/g brain and 132 +/- 24 microl/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 microl/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.
Hirohashi
T
T
Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Japan.
Terasaki
T
T
Shigetoshi
M
M
Sugiyama
Y
Y
D020001:1-Butanol; D019325:3-O-Methylglucose / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D001812:Blood-Brain Barrier; D000440:Butanols / Q000493:pharmacokinetics; D002151:Calorimetry; D002196:Capillaries; D002339:Carotid Arteries; D002417:Cattle; D002478:Cells, Cultured; D002560:Cerebrovascular Circulation; D004058:Diffusion; D004730:Endothelium, Vascular / Q000378:metabolism*; D005452:Fluoresceins / Q000008:administration & dosage / Q000493:pharmacokinetics*; D007269:Injections, Intra-Arterial; D008297:Male; D008353:Mannitol / Q000493:pharmacokinetics; D051381:Rats; D017208:Rats, Wistar
D000440:Butanols; D005452:Fluoresceins; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; D019325:3-O-Methylglucose; D008353:Mannitol; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D020001:1-Butanol