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, Yuichi Sugiyama

Research output: Contribution to journalArticlepeer-review

18 Citations (Scopus)

Abstract

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 μVmin/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.

Original languageEnglish
Pages (from-to)813-819
Number of pages7
JournalJournal of Pharmacology and Experimental Therapeutics
Volume280
Issue number2
Publication statusPublished - 1997 May 3
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

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