Transwell Assay Design

Transcellular Transport Across Efflux Transporter-Expressing Cells

MDR1 (P-gp) and BCRP are important efflux transporters involved in limiting the oral bioavailability of compounds, limiting the penetration of compounds through the BBB and in the excretion of compounds in the urine and bile. MDR1, and to a lesser extent BCRP, has been implicated in numerous drug-drug interactions. Interactions of compounds with MDR1 and BCRP is evaluated by measuring transcellular (bi-directional) transport of the compound or a probe substrate across MDR1 or BCRP transfected MDCKII and control cells. Transfected and control cells are cultured on a Transwell plate. Experiments can also be performed in Caco-2 cells.

Identification of a Substrate

The test compound is added to either the apical or basal side of the monolayer and the amount of compound permeating through the cell monolayers is measured by LC-MS/MS or liquid scintillation counting. The apparent permeability (Papp) of the compound is calculated in both the apical and basolateral (A to B) direction. The efflux ratio of permeability (B to A/A to B) is calculated. MDR1 of BCRP pump in the B to A direction. MDR1 or BCRP substrates exhibit a greater permeability in the B to A direction than the A to B direction and therefore have an efflux ratio greater than 2. Km is calculated from the relationship between the substrate concentration and the efflux ratio.

Identification of an Inhibitor

The ability of the test compound to inhibit the bi-directional transport of a probe substrate (e.g. digoxin or prasozin for P-gp or BCRP, respectively) is measured. The probe substrate is added to either the apical or basolateral side of the cell monolayer and test compound is added to both sides. The inhibitory effect of the drug candidate is evaluated by IC50 determination (concentration causing 50% inhibition of transporter-mediated transport).

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