A functional assay for quantitation of the apparent affinities of ligands of P-glycoprotein in Caco-2 cells

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 [H-3]-taxol, a known substrate of P-gp. The apparent affinities (K-I values) of the compounds were quantitatively determined based on the inhibitory effects of the compounds on the active transport of [H-3]-taxol. Intact Caco-2 cell monolayers were utilized for transport inhibition studies. Samples were analyzed by liquid scintillation counting. Results. [H-3]-Taxol (0.04 muM) showed polarized transport with the basolateral (BL) to apical (AP) flux sate being about 10-20 times faster than the flux rate in the AP-to-BL direction. This difference in [H-3]-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 [H-3]-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 [H-3]-taxol with resulting K-I 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 muM, respectively. With the exception of that of quinidine, these K-I 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.