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Efflux Transporters

• Differentiated MDCKII cell monolayers expressing selected drug transporter for substrate and inhibition assays
• Vesicular transport assays for efflux transporter inhibition

A family of ATP binding cassette (ABC) transporters is an important group of membrane transporters involved in drug absorption, distribution and elimination. Regulatory authorities (EMA, FDA) recommend investigating the potential roles of ABC superfamily members ABCB1 (MDR1, P-glycoprotein), and ABCG2 (BCRP) for transporter-mediated drug-drug interactions (DDI), by evaluating whether the new drug candidate is a substrate (potential DDI victim) or an inhibitor (potential DDI perpetrator) towards drug transporters. Additionally, evaluation of inhibition towards bile salt export pump (BSEP, ABCB11) is recommended due to safety concerns associated with inhibition of this transporter.

In substrate assays the permeability of the test compound is measured through human efflux transporter expressing MDCKII cell monolayers to both directions in the presence and absence of chemical transporter inhibitor. Active transport is observed as higher apparent permeability in basolateral to apical than in apical to basolateral direction and confirmed by reduction of efflux ratio by a known chemical inhibitor and/or by a smaller efflux ratio with control cells lacking the transfected human transporter. Similarly, in the inhibition assay the impact of the test compound on active transport of a probe substrate is evaluated. Assays are conducted with ready-to-use transfected MDCKII monolayers and LC/MS/MS is used as an analytical tool.

Inhibition of transporters P-gp, BCRP and BSEP can also be studied with inside-out membrane vesicles prepared from mammalian cells expressing a single human efflux transporter. The inhibition is evaluated by measuring ATP dependent vesicular uptake of a probe substrate into membrane vesicles in the presence and absence of test compound using LC/MS/MS for analysis.