Permeability Screen Using Caco-2 or MDCK Cell Monolayers

Purpose

Assessment of the intestinal permeability of compounds intended for oral administration plays an important role in selecting candidates for drug development. Ranking a series of lead compounds in order of absorption potential facilitates compound selection and optimization. A currently accepted method for investigation of the absorption potential of compounds within a series is by comparison of the apparent permeabilities through Caco-2 or MDCK monolayer cultures (Artusson and Borchardt, Pharm. Res. 1997, 14, 1655-1657). These absorption models are also useful for understanding any absorption issues associated with compounds further advanced in development, including those involved with active transport mechanisms.

Assay Protocol

Monolayer cultures, suitable for investigation of compound permeability, are grown on either 24- or 96-well polycarbonate membrane inserts. The monolayers are maintained at 37°C in a 5% CO₂ atmosphere at 95% relative humidity until confluent. The maturity and membrane integrity of the monolayers are confirmed by measurement of the trans-epithelial electrical resistance (TEER) or the apparent permeability of the fluorescent marker compound lucifer yellow.

Apparent permeabilities of a series of test articles and of selected marker compounds are determined in duplicate at a single concentration of 10 mM in the apical to basolateral direction. The transport investigations are initiated by the addition of the test article to the apical compartment and the plates are maintained under culture conditions during the course of the experiment. The basolateral compartments following 30 and 60 minutes of exposure and the final apical compartments are collected and analyzed for test article content by LC-MS/MS. The recovery and apparent permeability of each test article are calculated from these data.

Confirmation Data

The apparent permeabilities of a selection of compounds with various absorption properties were determined through Caco-2 and MDCK monolayers. The results obtained agree well with those quoted in the literature, including Liang et. al. J. Pharm. Sci. 2000, 89, 342, and confirm that Ricerca cell lines are comparable to the industry standard.

The apparent permeabilities of the radiolabeled versions of four of the compounds were also determined, using liquid scintillation counting to determine concentrations instead of LC-MS/MS. Both analytical techniques yield similar results confirming the acceptability of the LC-MS/MS method of analysis.

Mean Apparent Permeabilities of Various Compounds
Through MDCK and Caco-2 Monolayers

Comparison of LSC Quantification to LC-MS/MS Quantification
for Determination of Apparent Permeabilities of Compounds
Through Caco-2 Monolayers