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Vol. 288, Issue 1, 348-357, January 1999
School of Pharmacy, Martin-Luther-University Halle-Wittenberg,
Halle (Saale), Germany (S.D., C.H., H.S.-L.);
Astra Hässle AB,
Mölndal, Sweden (S.D., P.L., C.G.R., T.B.A.); and
School of
Pharmacy, Johannes Gutenberg University, Mainz, Germany (P.L.)
Interaction with the exsorptive transporter P-glycoprotein
(P-gp) is a possible source of peculiarities in drug pharmacokinetics, including dose-dependent absorption, drug-drug interactions,
intestinal secretion, and limited permeability of the blood-brain
barrier. Among the established in vitro methods of the analysis of drug interactions with P-gp, none directly quantifies the affinity of
ligands with P-gp. Instead, they measure the result of a membrane permeation and a receptor-binding process; this may lead to
difficulties in the interpretation of results. An assay for
quantification of drug affinity to the transporter is presented on the
basis of the radioligand-binding assay principle. This has the
advantage of directly quantifying the interaction between drugs and
P-gp. Because of the reversible and competitive interaction of
numerous substrates with P-gp, a radioligand-binding assay was
developed by taking [3H]verapamil and
[3H]vinblastine as radioligands and the human intestinal
Caco-2 cells, overexpressed with P-gp by culturing in the presence of vinblastine or transfecting with multidrug resistance gene MDR-1 as receptor preparation. The assay was performed in 96-well
plates and has the potential to be used as a high-throughput method. A
clear induction of the expression of P-gp was demonstrated in the
Caco-2 cells grown in the presence of vinblastine, as well as in the
transfected cells, although to a lesser extent. Both radioligands were
shown to bind to P-gp. Verapamil was the radioligand of choice for
further investigations due to its lower nonspecific binding to the
transporter preparation. Kinetics as well as specificity of the binding
of verapamil to the P-gp preparation were demonstrated. A two-affinity
model was found to adequately describe the data derived from saturation
as well as from competition experiments, in accordance with previous
findings on two exsorption sites for P-gp. The binding properties of
[3H]verapamil and [3H]vinblastine to a P-gp
preparation derived from induced Caco-2 cells are described. The
concentration-dependent displacement of the radioligand by nonlabeled
substrates for P-gp should be a suitable principle for the
determination of drug affinity to the respective binding sites at the
human intestinal multidrug transporter P-gp.
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