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Research ArticleArticle

Characterization of Binding Properties to Human P-Glycoprotein: Development of a [3H]Verapamil Radioligand-Binding Assay

Stephan Döppenschmitt, Peter Langguth, Carl Gunnar Regårdh, Tommy B. Andersson, Constanze Hilgendorf and Hildegard Spahn-Langguth
Journal of Pharmacology and Experimental Therapeutics January 1999, 288 (1) 348-357;
Stephan Döppenschmitt
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Peter Langguth
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Carl Gunnar Regårdh
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Tommy B. Andersson
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Constanze Hilgendorf
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Hildegard Spahn-Langguth
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Abstract

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.

Footnotes

  • Send reprint requests to: Prof. Dr. Peter Langguth, School of Pharmacy, Biopharmaceutics and Pharm. Tech., Staudingerweg 5, D-55099 Mainz, Germany.

  • 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
    2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein:d-TUB, 2′-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
    • Received February 20, 1998.
    • Accepted July 20, 1998.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 288 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 288, Issue 1
1 Jan 1999
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Research ArticleArticle

Characterization of Binding Properties to Human P-Glycoprotein: Development of a [3H]Verapamil Radioligand-Binding Assay

Stephan Döppenschmitt, Peter Langguth, Carl Gunnar Regårdh, Tommy B. Andersson, Constanze Hilgendorf and Hildegard Spahn-Langguth
Journal of Pharmacology and Experimental Therapeutics January 1, 1999, 288 (1) 348-357;

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Research ArticleArticle

Characterization of Binding Properties to Human P-Glycoprotein: Development of a [3H]Verapamil Radioligand-Binding Assay

Stephan Döppenschmitt, Peter Langguth, Carl Gunnar Regårdh, Tommy B. Andersson, Constanze Hilgendorf and Hildegard Spahn-Langguth
Journal of Pharmacology and Experimental Therapeutics January 1, 1999, 288 (1) 348-357;
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