Characterization of Binding Properties to Human P-Glycoprotein: Development of a [H]Verapamil Radioligand-Binding Assay

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 [H]verapamil and [H]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 [H]verapamil and [H]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. More than 20 years ago, the modulating effect of P-glycoprotein (P-gp) on drug permeability across cellular membranes was discovered (Juliano and Ling, 1976) and its role in the development of multidrug resistance in cancer chemotherapy identified. In spite of the known amino acid and gene sequence of P-gp (Chen et al., 1986), its functional properties are not yet fully understood, neither with respect to the ATP-driven mechanism of transport across the plasma membrane (Hsing et al., 1992) nor to the fact that a vast number of structurally unrelated compounds seem to be recognized by the transporter. In addition to its expression in cancer cells (Chen et al., 1986), P-gp has been shown to be present under physiological conditions in different tissues, where it is supposed to contribute to the “compartmentation” in the body, e.g., at the gut wall or the blood-brain barrier (Schinkel et al., 1994; van Asperen et al., 1997). Two major fields of research have originated from these pathophysiological and physiological findings. The first field focuses on strategies of overcoming multidrug resistance in cancer treatment (Ayesh et al., 1996) by the use of drugs modulating the function of P-gp. The other more recent field is dedicated to investigating the influence of P-gp expression on the absorption in the gastrointestinal tract, distribution, and excretion of drugs (Schinkel et al., 1996; Hunter and Hirst, 1997; Spahn-Langguth et al., 1998). Received for publication February 20, 1998. 1 This study was supported by the Fonds der Chemischen Industrie, Frankfurt and Dr. Robert Pfleger Stiftung, Bamberg (to H.S.L.). ABBREVIATIONS: P-gp, P-glycoprotein; RBA, radioligand-binding assay; FACS, fluorescence-activated cell sorting; R123, rhodamine 123; BCECF, 29,79-bis(2-carboxyethyl)-5(6)-carboxyfluorescein: d-TUB, 29-deoxytubercidine; MES, 2-(N-morpholino)ethanesulfonic acid: PBS, phosphate-buffered saline; HBSS, Hanks’ balanced salt solution; HPLC, high-performance liquid chromatography; PBS-BSA-G, bovine serum albumin; OCT, organic cation transporter. 0022-3565/99/2881-0348$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 288, No. 1 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 288:348–357, 1999 348 at A PE T Jornals on A ril 0, 2017 jpet.asjournals.org D ow nladed from Because of the growing interest in the determination of drug affinity to P-gp, various assay systems have been developed based on the influence of potential substrates on the ATPase activity of P-gp (Al-Shawi and Senior, 1993), the accumulation or efflux of fluorescent dyes transported by P-gp (Broxterman et al., 1997), or the modulation of toxicity of cytotoxic P-gp substrates (Scala et al., 1997). None of these assays allow interpretation of the results with respect to drug affinity to P-gp, although direct binding of substrates to P-gp has been shown with the use of photoaffinity labeling analogs of vinblastine and colchicine. This labeling was competitively inhibited by P-gp substrates (Beck and Qian, 1992; Bruggemann et al., 1992). Consequently, the observed binding characteristics seem to correspond well to the theory of competitive receptor–ligand interaction and may therefore be used for the development of a radioligand-binding assay (RBA) (Crevat-Pisano, 1986; Spahn et al., 1989). In this investigation, the human colon carcinoma cell line Caco-2, which has previously been characterized with respect to its differentiation and expression of P-gp (Pinto et al., 1983; Anderle et al., 1998), was used. To increase its P-gp content, this cell line was adapted to grow in the presence of vinblastine. The vinblastine-induced cell line was characterized functionally by transport studies with polarized monolayers, saturation experiments, and fluorescence-activated cell sorting (FACS) analysis. Furthermore, Caco-2 cells transfected with MDR-1 were characterized with respect to the amplification of the expression of P-gp by transport and saturation experiments, as well as by Northern blotting. Important variables for the binding experiments such as cell permeabilization procedures have been optimized. Furthermore, binding characteristics of radioligands, as well as association and dissociation kinetics, were determined. Specificity of the radioligand binding was tested by competition experiments with ligands for other potential binding sites; moreover, the presence of cytochrome P-450 3A was investigated by using Western blotting. Materials and Methods Cell Culture Materials Dulbecco’s modified Eagle’s medium, fetal calf serum, L-glutamine (200 mM), penicillin/streptomycin (10,000 U/ml, 10,000 mg/ml), trypsin/EDTA, minimum essential medium, nonessential amino acids, Hanks’ balanced salt solution (HBSS), and phosphate-buffered saline (PBS) were obtained from Life Technologies (Paisley, UK). Vinblastine sulfate and trypan blue solution (0.4%) were purchased from Sigma (Malmö, Sweden). Transwell cell culture inserts used for transport experiments (24 mm, 0.4-mm pore size, polycarbonate membrane) and all other cell culture materials were obtained from Costar (Cambridge, MA). The Caco-2 cells used were obtained from the American Type Culture Collection (Rockville, MD).