Radioligand-binding assay employing P-glycoprotein-overexpressing cells: testing drug affinities to the secretory intestinal multidrug transporter

Pharm Res. 1998 Jul;15(7):1001-6. doi: 10.1023/a:1011965707998.


Purpose: To develop a rapid and reliable system for affinity determination of conventional as well as newly synthesized compounds to P-gp.

Methods: The principles of radioligand-binding assay were adapted to the human intestinal P-gp. Acceptor protein was obtained from the human carcinoma cell line Caco-2, where overexpression of P-gp was induced by growing cells in the presence of the cytostatic drug vinblastine. 3H-Verapamil was chosen as radioligand.

Results: The saturability and specificity of 3H-verapamil as the radioligand for the binding to P-gp was demonstrated. From concentration dependence of displacement of the radioligand by various non-labeled ligands for P-gp, affinity constants to P-gp binding sites were calculated. The binding results obtained were in agreement with those published earlier where influx and efflux experiments with cell monolayers had been conducted in order to functionally characterize the P-gp -drug interaction.

Conclusions: A radioligand-binding assay on the basis of P-gp overexpressing Caco-2 cells has been developed. The method might be suitable for high-throughput screening of drug interaction with human P-gp. It will allow modeling of the interaction of drugs with the human multidrug transporter and has also the potential to serve as a high-throughput screening tool to detect compounds prone to P-gp mediated intestinal secretion and potential P-gp related drug/drug interactions in drug discovery and early development.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / biosynthesis
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism*
  • Adrenergic beta-Antagonists / metabolism
  • Adrenergic beta-Antagonists / pharmacokinetics
  • Antineoplastic Agents, Phytogenic / metabolism
  • Antineoplastic Agents, Phytogenic / pharmacokinetics
  • Binding, Competitive
  • Caco-2 Cells / metabolism
  • Calcium Channel Blockers / metabolism*
  • Calcium Channel Blockers / pharmacokinetics
  • Fluorescent Dyes / metabolism
  • Fluorescent Dyes / pharmacokinetics
  • Humans
  • Kinetics
  • Propanolamines / metabolism
  • Propanolamines / pharmacokinetics
  • Radioligand Assay / methods*
  • Reproducibility of Results
  • Rhodamine 123
  • Rhodamines / metabolism
  • Rhodamines / pharmacokinetics
  • Tritium
  • Verapamil / metabolism*
  • Verapamil / pharmacokinetics
  • Vinblastine / metabolism
  • Vinblastine / pharmacokinetics


  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Adrenergic beta-Antagonists
  • Antineoplastic Agents, Phytogenic
  • Calcium Channel Blockers
  • Fluorescent Dyes
  • Propanolamines
  • Rhodamines
  • Tritium
  • Rhodamine 123
  • talinolol
  • Vinblastine
  • Verapamil