Caco-2 permeability assay

For determination of NOV202 permeability, Caco-2 cell monolayers were grown to confluence on collagen-coated, microporous, polycarbonate membranes in 12-well transwell plates (Corning Inc., New York, USA). The permeability assay buffer was Hanks Balanced Salt Solution containing 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 15 mM glucose at pH 7.4. The buffer in the receiver chamber also contained 1% bovine serum albumin. The dosing solution concentration was 5 µM test compound in the assay buffer. Cell monolayers were dosed on the apical side (A-to-B) or basolateral side (B-to-A) and incubated at 37°C with 5% CO₂ in a humidified incubator. Samples were taken from the donor and receiver chambers at 120 min. Each determination was performed in duplicate. The coapplied Lucifer yellow flux was also measured for each monolayer to out rule any cell monolayer damage during the flux period. All samples were assayed by liquid chromatography-mass spectrometry and tandem mass spectrometry using electro-spray ionization.

The apparent permeability, P_app, and percent recovery were calculated as follows:

where dCr/dt is the slope of the cumulative concentration in the receiver compartment versus time in µM/s; V_r is the volume of the receiver compartment in cm³; A is the area of the insert (1.13 cm² for 12-well plate); C_A is the average of the nominal dosing concentration and the measured 120 min donor concentration in µM.

Interpretations of the results were done based on the following thresholds: Permeability classification: (P_app A-to-B) <1.0×10⁻⁶ cm/s: low; (P A-to-B) ≥1.0×10⁻⁶ cm/s: app high. Significant efflux: efflux ratio >3.0 and (P_app B-to-A) ≥1.0×10⁻⁶ cm/s.