| Assay Detail |
Prior to the start of the assay, the cell monolayer was washed and pre-incubated at 37 ºC and 5% CO2 for 30 min with HBSS-HEPES buffer containing 1% DMSO. TEER values were measured before starting and after completion of the assay. For the apical to basolateral (A to B) assay, 0.5 mL buffer solution containing one of the above combinations was added to the apical compartment and 1.5 mL buffer containing 1% DMSO was added to the basolateral compartment. The assay plate was incubated at 37 ºC and 5% CO2. 100 μL samples from apical side at 0 and 120 min and 150 μL samples from the basolateral compartment at 30, 60, 90 and 120 min were withdrawn into acetonitrile containing 96-well plate. Similarly, for the basolateral to apical (B to A) assay, 0.5 mL buffer containing 1% DMSO and 1.5 mL buffer solution containing one of the above combinations were added to the apical compartment and basolateral compartment respectively. The assay plate was incubated at 37 ºC and 5% CO2. 150 μL samples from basolateral side at 0 and 120 min and 100 μL samples from the apical compartment at 30, 60, 90 and 120 min were withdrawn into a 96-well plate containing acetonitrile. At the end of the assay, the cell monolayer was washed with 500 μL buffer containing 1% DMSO and the washings were collected. Finally, the monolayer was washed with 500 μL acetonitrile to lyse the cells and the lysate was collected in tubes.
A fit-for-purpose LC-MS/MS method was used for the estimation of peptide(s) as well as the positive controls. Cumulative amount of peptides and Digoxin (Q) transported at teach time point was plotted as a function of time. The slope was used to calculate the rate of appearance of peptides and Digoxin in the basolateral compartment. Apparent permeability (Papp) was calculated using the following formula: Papp = (dQ/dt)/(A*C0) where, dQ/dt is the rate of appearance of the peptides and digoxin in the receiver compartment. A is the surface area of the membrane (1.12 cm2). C0 is the initial concentration of the test items and Digoxin. The efflux ratios were compared with and without Elacridar to assess whether the peptides are substrates of efflux transporter.
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