Characterization of binding properties to human P-glycoprotein: Development of a [H-3]verapamil radioligand-binding assay

Interaction with the exsorptive transporter P-glycoprotein (P-gp) is a poss ible source of peculiarities in drug pharmacokinetics, including dose-depen dent absorption, drug-drug interactions, intestinal secretion, and limited permeability of the blood-brain barrier. Among the established in vitro met hods of the analysis of drug interactions with P-gp, none directly quantifi es 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 diffi culties in the interpretation of results. An assay for quantification of dr ug affinity to the transporter is presented on the basis of the radioligand -binding assay principle. This has the advantage of directly quantifying th e interaction between drugs and P-gp. Because of the reversible and competi tive interaction of numerous substrates with P-gp, a radioligand-binding as say was developed by taking [H-3]verapamil and [H-3]vinblastine as radiolig ands and the human intestinal Caco-2 cells, overexpressed with P-gp by cult uring in the presence of vinblastine or transfecting with multidrug resista nce gene MDR-I as receptor preparation. The assay was performed in 96-well plates and has the potential to be used as a high-throughput method. A clea r 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 i ts lower nonspecific binding to the transporter preparation. Kinetics as we ll as specificity of the binding of verapamil to the P-gp preparation were demonstrated. A two-affinity model was found to adequately describe the dat a derived from saturation as well as from competition experiments, in accor dance with previous findings on two exsorption sites for P-gp. The binding properties of [H-3]verapamil and [H-3]vinblastine to a P-gp preparation der ived from induced Caco-2 cells are described. The concentration-dependent d isplacement of the radioligand by nonlabeled substrates for P-gp should be a suitable principle for the determination of drug affinity to the respecti ve binding sites at the human intestinal multidrug transporter P-gp.