Optimisation of the caco-2 permeability assay using experimental design methodology.

Purpose: This study used a Box-Behnken experimental design to optimise the experimental conditions in the Caco-2 assay for a series of p-hydroxybenzoate ester compounds (log P 1.96-5.69), as highly lipophilic compounds are not handled well in this system.

Methods: Caco-2 cells, passage 55-70, were cultured on Transwelltrade mark cell culture supports and permeability assays were performed on day 21. A three level three factorial experimental design was used to optimise the experimental conditions.

Results: Addition of BSA (4% w/v) in the medium increased the apparent permeability coefficients (Papp) of each of the parabens except the octyl ester. Increasing the stirring rate by 100 rpm increased Papp for all the parabens. Use of simulated intestinal fluid either increased (fasted state) or decreased (fed state) the Papp of methyl-butyl parabens.

Conclusions: The optimised conditions were; 1.55% w/v BSA, 215 rpm stirring rate and 3.02 mM sodium taurocholate in the simulated intestinal fluid; where octyl paraben (log P 5.69) had an Papp of 33.93 +/- 1.84 x 10(-6) cm/s, reflecting its rapid absorption in man. This study provides a systematic optimisation of the Caco-2 permeability assay to avoid the underestimation of the intestinal permeability of compounds with log P > 3.