Several cultured epithelial cell lines of human or animal origin can model the passive absorption of compounds across intestinal tissue in vivo. The Caco-2 human colonic cell line is perhaps the most widely used cell line for this purpose. Caco-2 cells adopt a morphology and express many intestinal transport proteins and other enzymes characteristic of epithelial cells of the small intestine, when cultured under proper conditions. They also form tight junctions with each other. These limit the paracellular permeability or the “leakiness” of cell monolayers grown to confluence on polycarbonate membrane filters. These properties make Caco-2 monolayers a good test system for characterizing the passive absorption and active transport of test compounds. In a typical permeability assay, Caco-2 cells are grown to confluence on a polycarbonate membrane separating two chambers filled with culture medium. The cells adopt a polarized epithelial cell morphology under these culture conditions with distinct apical and basolateral sides. This process typically takes about three weeks from the time the cells are initially seeded onto the membranes until they adopt the proper morphology and form intercellular tight junctions. Protocols for more rapid production of cell monolayers have been published, but our experience has been that these protocols lead to an unacceptably high fraction of monolayers that have poor barrier properties and, thus, poor discrimination between transcellular and paracellular permeation. Prior to a permeability assay, the culture media is replaced with a physiologically balanced buffer supplemented with an energy source, such as glucose. Then the electrical resistance across each cell monolayer is measured to confirm proper barrier function. Monolayers that pass are arrayed into permeability assay plates that can hold between 12 and 96 individual monolayers depending on their configuration. The apical and basolateral wells of the assay plate are filled with buffer. Then the test compound is added to the apical well. Samples are taken from the basolateral well at various times over a two-hour period. At the end of the experiment the apical well is sampled in order to measure recovery of the test compound. The parameter that is usually calculated from this data is the apparent permeability (P_app). This is the slope of the basolateral concentration versus time curve divided by the concentration of compound in the apical dosing chamber and the total area of the Caco-2 cell monolayer. The exact value of P_app is influenced by the conditions under which the assay is conducted. Therefore, a validation set of compounds with established human fractional absorption must be run through the assay under conditions similar to those used for test compounds in order to establish a relative ranking score for P_app values. A plot of human fractional absorption versus P_app is then constructed. This plot is sigmoidal with a sharp break between the P_app values for compounds with fractional absorption of greater than 90% and those with values between 50% and 90%. For example, under our assay conditions the difference between the P_app values for atenolol, which has a fractional absorption of about 50%, and propranolol, which has a fractional absorption greater than 90%, is about thirty-fold. Thus, the Caco-2 assay system and similar cell-based systems are tools for classifying absorption potential into high, medium and low categories but human fractional absorption is not linearly related to P_app values obtained from these assays. In addition to these cultured cell models, Absorption Systems offers:

• In vitro assays for absorption across human or animal intestinal strips
• Oral bioavailability studies in rodents, dogs, pigs and non-human primates
• Intestinal loop perfusion studies in rats