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A Functional Assay for Quantitation of the Apparent Affinities of Ligands of P-Glycoprotein in Caco-2 Cells

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Abstract

Purpose. To develop a facile functional assay for quantitative determination of the apparent affinities of compounds that interact with the taxol binding site of P-glcoprotein (P-gp) in Caco-2 cell monolayers.

Methods. A transport inhibition approach was taken to determine the inhibitory effects of compounds on the active transport of [3H]-taxol, a known substrate of P-gp. The apparent affinities (KI values) of the compounds were quantitatively determined based on the inhibitory effects of the compounds on the active transport of [3H]-taxol. Intact Caco-2 cell monolayers were utilized for transport inhibition studies. Samples were analyzed by liquid scintillation counting.

Results. [3H]-Taxol (0.04 μM) showed polarized transport with the basolateral (BL) to apical (AP) flux rate being about 10-20 times faster than the flux rate in the AP-to-BL direction. This difference in [3H]-taxol flux could be totally abolished by inclusion of (±)-verapamil (0.2 mM), a known inhibitor of P-gp, in the incubation medium. However, inclusion of probenecid (1.0 mM), a known inhibitor for the multidrug resistance associated protein (MRP), did not significantly affect the transport of [3H]-taxol under the same conditions. These results suggest that P-gp, not MRP, was involved in taxol transport. Quinidine, daunorubicin, verapamil, taxol, doxorubicin, vinblastine, etoposide, and celiprolol were examined as inhibitors of the BL-to-AP transport of [3H]-taxol with resulting KI values of 1.5 ± 0.8, 2.5 ± 1.0, 3.0 ± 0.3, 7.3 ± 0.7, 8.5 ± 2.8, 36.5 ± 1.5, 276 ± 69, and 313 ± 112 μM, respectively. With the exception of that of quinidine, these KI values were comparable with literature values.

Conclusions. This assay allows a facile quantitation of the apparent affinities of compounds to the taxol-binding site in P-gp; however, this assay does not permit the differentiation of substrates and inhibitors. The potential of drug-drug interactions involving the taxol binding site of P-gp can be conveniently estimated using the protocol described in this paper.

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Gao, J., Murase, O., Schowen, R.L. et al. A Functional Assay for Quantitation of the Apparent Affinities of Ligands of P-Glycoprotein in Caco-2 Cells. Pharm Res 18, 171–176 (2001). https://doi.org/10.1023/A:1011076217118

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