Caco drug absorption experiments

Cell monolayers grown on permeable culture inserts form confluent mono-layers with barrier properties and can be used for drug absorption experiments. The most well-known cell line for the in vitro determination of intestinal drug permeability is the human colon adenocarcinoma Caco-2 [20, 21], The utility of the Caco-2 cell line is due to its spontaneous differentiation to enterocytes under conventional cell culture conditions upon reaching confluency on a porous membrane to resemble the intestinal epithelium. This cell model displays small intestinal carriers, brush borders, villous cell model, tight junctions, and high resistance [22], Caco-2 cells express active transport systems, brush border enzymes, and phase I and II enzymes [22-24], Permeability models... 

Compared to in vivo studies, the Caco-2 model substantially increases the speed at which absorption potential can be estimated and reduces the amount of drug substance needed. However, manually performed assays are still too slow and labor intensive compared to biological high-throughput screening assays. Caco-2 cells take about 3 weeks to form monolayers of fully differentiated cells. At this point, Caco-2 monolayers are used to evaluate absorption potential under a variety of permeability protocols. In order to further expedite the process of absorption potential assessment, efforts have been made to increase the throughput of Caco-2 transport experiments. 

With the difficulties associated with accurate estimation of permeability based only on physicochemical properties, a variety of methods of measuring permeability have been developed and used, among which are (l)cul-tured monolayer cell systems, such as Caco-2 or MDCK ( 2 diffusion cell systems that use small sections of intestinal mucosa between two chambers (3) in situ intestinal perfusion experiments performed in anesthetized animals such as rats and (4)intestinal perfusion studies performed in humans (40,54-62). All of these methods offer opportunities to study transport of drug across biological membranes under well-controlledconditions. Caco-2 mono-layer systems in particular have become increasingly commonly used in recent years and human intestinal perfusion methods are also becoming more commonly available. Correlations between Caco-2 permeability and absorption in humans have been developed in several laboratories (63-72). As shown in Fig. 

The lanthaninn teehnique was used to study the epithelial permeability in the rat small intestine (Madara and Trier 1982). Dense lanthanum precipitates in TJ and paracellular spaces were restricted to a subpopulation of villous goblet cells and were not found between villous absorptive cells. These TJ were also permeable to barium, but not to macromolecular tracers such as microperoxidase, eytochrome c and horseradish peroxidase. It was also shown that palmitoylcamitine (PCC) opens TJ in a monolayer of Caco-2 colon carcinoma cells this phenomenon appears to be responsible for the significant enhancement of the absorption of hydrophilic drugs across intestinal mucosa caused by PCC and other long-chain acylcamitines (Hochman, Fix et al. 1994).In an experiment on rats, it was demonstrated that immobilisation stress induced a significant (but reversible) increase in epithelial permeability to the lanthanum tracer (Mazzon, Stumiolo et al. 2002). 

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