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OtherDRUG METABOLISM AND DISPOSITION

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
Journal of Pharmacology and Experimental Therapeutics February 1997, 280 (2) 813-819;
Tomoko Hirohashi
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Tetsuya Terasaki
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Minoru Shigetoshi
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Yuichi Sugiyama
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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 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.

Footnotes

  • Send reprint requests to: Dr. Yuichi Sugiyama, Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan.

  • Abbreviations:
    BBB
    blood-brain barrier
    BCEC
    brain capillary endothelial cells
    BCECF
    2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
    BCECF-AM
    2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein tetraacetoxymethyl ester
    DMSO
    dimethyl sulfoxide
    EDTA
    ethylenediaminetetraacetic acid
    HEPES
    4-(2-hydroxyethyl)-piperazineethanesulfonic acid
    MDR
    multidrug-resistant
    MEM
    minimum essential medium
    3OMG
    3-O-methyl-d-glucose
    PBS
    phosphate buffered saline
    PC
    partition coefficient
    PS
    permeability surface area product (μl/min/g brain)
    SDS
    sodium dodecyl sulfate
    TCA
    trichloroacetic acid
    • Received October 23, 1995.
    • Accepted October 31, 1996.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics
Vol. 280, Issue 2
1 Feb 1997
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OtherDRUG METABOLISM AND DISPOSITION

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
Journal of Pharmacology and Experimental Therapeutics February 1, 1997, 280 (2) 813-819;

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OtherDRUG METABOLISM AND DISPOSITION

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
Journal of Pharmacology and Experimental Therapeutics February 1, 1997, 280 (2) 813-819;
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