The vesicular transport assay is an in vitro method used to evaluate whether a drug candidate is a substrate or inhibitor of ABC efflux transporters such as P-gp, BCRP, MRP2, or BSEP. It is one of the primary assays recommended by the FDA, EMA, and ICH M12 guidelines for drug-drug interaction (DDI) assessment during pharmaceutical development.<\/p>\n
Inside-out membrane vesicles are prepared from cells overexpressing a specific ABC transporter. Because the vesicles are inverted, the ATP-binding domain faces outward, allowing ATP to drive active transport of the test compound into the vesicle lumen. Transport is measured using radiolabeled substrates, LC-MS\/MS, or fluorescent probes, with and without ABC inhibitors as controls.<\/p>\n
Regulatory guidelines require vesicle assays for drugs that are candidates for P-gp, BCRP, BSEP, MRP2, or other transporter substrates. BSEP inhibition studies are particularly important when hepatotoxicity signals are observed in preclinical or clinical studies.<\/p>\n
The vesicle assay directly measures transport and is the gold standard for substrate identification. The ATPase assay measures ATP hydrolysis stimulated by the test compound and is faster and cheaper, but less definitive for regulatory purposes. Both are complementary tools in a DMPK screening cascade.<\/p>\n