The impact of protein on Caco-2 permeability of low mass balance compounds for absorption projection and efflux substrate identification

This study was to evaluate the mechanistic effect of protein to help better interpret the permeability results for compounds with low mass balance in Caco-2 permeability assay. The absorptive or bi-directional permeability of lipophilic compounds with mass balance were measured across Caco-2 cell monolayers as well as the empty transport devices with or without protein (4% bovine serum albumin, BSA) added to the receiver side. The results from empty transport device study indicated that the filter membrane is a permeability barrier for the low mass balance compounds and protein increases permeability by improving the compound diffusivity through the filter membrane. Caco-2 permeability measured with protein provided better absorption projection. Assuming the amount of compound associated with cells as transported did not correlate with absorption. For efflux substrate identification using Caco-2 bi-directional permeability assay, protein at the receiver side had no significant effect on the conclusion regarding the tested compounds as efflux substrate but increased the permeability measurement from both transport directions. In conclusions, Caco-2 permeability results measured using protein-containing buffer at the receiver side for low mass balance compound seems to provide better correlation with in vivo absorption. The fact that protein at receiver side has minimal effect on efflux substrate identification provides scientific basis for further specific transporter characterization (such as P-gp or BCRP) using specific inhibitors, in which same concentration of inhibitor is used in both sides of the Caco-2 cell system and protein for optimal permeability assessment has to be avoided.