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

Abstract

1. 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.

2. 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.

3. 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.

4. 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.

Keywords::

• Transporters
• in vitro
• DDI
• drug development
• breast cancer resistance protein
• multidrug resistance protein 2
• substrate
• inhibitor

Acknowledgements

The authors would like to thank Johan (Karlsson) Palm (AstraZeneca R&D Mölndal) and Glynis Nicholls (AstraZeneca R&D Alderley Park) for scientific discussions regarding the design of the validation studies.

Declaration of interest

Lisa Fox and David Stresser are employees of Becton Dickinson which is the commercial supplier of the BD Gentest BCRP, MRP2 and control vesicle test systems utilized in this study.