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.

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 ([(3)H] oestrone 3-sulfate, [(3)H] methotrexate, [(3)H] rosuvastatin) and MRP2 ([(3)H] oestradiol 17 beta-glucuronide, [(3)H] pravastatin, carboxydichlorofluorescein) substrates, and inhibitory potencies (IC(50)) of BCRP (sulfasalazine, novobiocin, fumitremorgin C) and MRP2 (benzbromarone, MK-571, terfenadine) inhibitors were determined. 3. The apparent K(m) for probes [(3)H] oestrone 3-sulfate and [(3)H] oestradiol 17 beta-glucuronide was determined in insect cell vesicles to be 7.4 +/- 1.7 and 105 +/- 8.3 mu M, respectively. All other substrates exhibited significant uptake ratios. Positive control inhibitors sulfasalazine and benzbromarone gave IC(50) values of 0.74 +/- 0.18 and 36 +/- 6.1 mu M, 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.