(M1230-05-31) Impact of Pre-incubation on BCRP and P-gp Transporter Inhibition in the Membrane Vesicle Assay

Purpose: The effects of pre-incubation on IC50s in the HEK-293 vesicle system has not been well established. For SLC transporters, pre-incubation of inhibitors has been shown to change IC50 values, however this has not been extensively evaluated in ABC transporters using the membrane vesicle assay. This study aims to evaluate the impact of inhibitor pre-incubation on BCRP and PGP IC50s in HEK-293 vesicles.

Methods: To determine the effect of pre-incubation on BCRP and PGP IC50 values, 7 known inhibitors were preincubated for 0 or 30 minutes with BCRP or PGP-HEK-293 vesicles, followed by co-incubation with probe substrates, rosuvastatin or N-methylquinidine, respectively. Alkaline phosphatase (ALP) activity in HEK-293 vesicles was also assessed for the phosphate prodrug, fostamatinib. Transporter IC50s of fostamatinib were compared to IC50s generated in the presence of the ALP inhibitor, sodium orthovanadate. Active drug formation was determined in the absence and presence of sodium orthovanadate when pro-drug was incubated with mock HEK-293 vesicles.

Results: The IC50s were determined with and without pre-incubation for 7 compounds for each transporter, and no major differences were observed with the exception of the pro-drug, fostamatinib. All compounds had IC50s within 2 fold except for elacridar and fostamatinib (4.2 fold, 20.6 -23.5 fold ). When fostamatinib was pre-incubated with vesicles, the IC50 was approximately equal to the IC50 of the active drug R406. When fostamatinib was pre-incubated with sodium orthovanadate the IC50s shifted approximately 5-6 fold. Rate of formation of R406 when incubated with mock vesicles decreased by 13.3 fold in the presence of sodium orthovanadate.

Conclusion: Based on the compound set tested, a 30 minute preincubation of inhibitors with ABC transporters P-gp and BCRP does not affect transporter activity. However, HEK29-based vesicles may retain alkaline phosphatase activity and should be considered if preincubating phosphate prodrugs in this assay system.