Identification of P-glycoprotein substrates using open tubular chromatography on an immobilized P-glycoprotein column: Comparison of chromatographic results with Caco-2 permeability

The Caco-2 cell monolayer model was used to classify 13 compounds as P-glycoprotein (Pgp) substrates or non-substrates. The apparent permeability coefficients (Papp) in the basal-to-apical direction (PappB–A) and in the apical-to-basal direction (PappA–B) were determined for each compound and a compound was designated as a Pgp substrate if PappB–A/PappA–B, the permeability ratio, exceeded 2.0. The same compounds were chromatographed on open tubular glass columns containing membranes from cell lines that either expressed Pgp (Pgp(+)-OT column) or did not express Pgp (Pgp(−)-OT column). The differential retentions in min, Δt values, of the compounds were determined using the following relationship Δt = t(Pgp(+)-OT) − t(Pgp(−)-OT). A statistically significant correlation was observed between the Δt values and the permeability ratios, r2 = 0.7749 (p = 0.0063), indicating that the differential chromatography approach could be used to quantitatively assess permeability ratios. The results also indicated that a Δt value ≥0.5 min was a reliable measure of a permeability ratios >2 and could be used as a rapid qualitative determination of whether a test compound was a Pgp substrate. The chromatographic study took 1 h to complete and a single pair of columns could be used to screen at least 150 compounds a week and 600 compounds during the 4-week lifetime of the columns.