Validation of membrane vesicle-based breast cancer resistance protein and multidrug resistance protein 2 assays to assess drug transport and the potential for drug-drug interaction to support regulatory submissions

Xenobiotica. 2011 Sep;41(9):764-83. doi: 10.3109/00498254.2011.578761. Epub 2011 May 25.

Abstract

Breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) can play a role in the absorption, distribution, metabolism, and excretion of drugs, impacting on the potential for drug-drug interactions. This study has characterized insect cell- and mammalian cell-derived ABC-transporter-expressing membrane vesicle test systems and validated methodologies for evaluation of candidate drugs as substrates or inhibitors of BCRP or MRP2. Concentration-dependent uptake of BCRP ([³H]oestrone 3-sulfate, [³H]methotrexate, [³H]rosuvastatin) and MRP2 ([³H]oestradiol 17β-glucuronide, [³H]pravastatin, carboxydichlorofluorescein) substrates, and inhibitory potencies (IC₅₀) of BCRP (sulfasalazine, novobiocin, fumitremorgin C) and MRP2 (benzbromarone, MK-571, terfenadine) inhibitors were determined. The apparent K(m) for probes [³H]oestrone 3-sulfate and [³H]oestradiol 17β-glucuronide was determined in insect cell vesicles to be 7.4 ± 1.7 and 105 ± 8.3 µM, respectively. All other substrates exhibited significant uptake ratios. Positive control inhibitors sulfasalazine and benzbromarone gave IC₅₀ values of 0.74 ± 0.18 and 36 ± 6.1 µM, respectively. All other inhibitors exhibited concentration-dependent inhibition. There was no significant difference in parameters generated between test systems. On the basis of the validation results, acceptance criteria to identify substrates/inhibitors of BCRP and MRP2 were determined for insect cell vesicles. The approach builds on earlier validations to support drug registration and extends from those cell-based systems to encompass assay formats using membrane vesicles.

Publication types

  • Validation Study

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B / metabolism*
  • ATP Binding Cassette Transporter, Subfamily G, Member 2
  • ATP-Binding Cassette Transporters / metabolism*
  • Adenosine Triphosphate / metabolism
  • Animals
  • Biological Assay / methods*
  • Biological Transport
  • Buffers
  • Drug Approval
  • Drug Interactions*
  • Drug Stability
  • Drug and Narcotic Control*
  • Insecta
  • Kinetics
  • Membranes, Artificial*
  • Neoplasm Proteins / metabolism*
  • Quality Control
  • Reproducibility of Results
  • Time Factors

Substances

  • ABCG2 protein, human
  • ATP Binding Cassette Transporter, Subfamily B
  • ATP Binding Cassette Transporter, Subfamily G, Member 2
  • ATP-Binding Cassette Transporters
  • Buffers
  • Membranes, Artificial
  • Neoplasm Proteins
  • P-glycoprotein 2
  • Adenosine Triphosphate