Permeability and modulation of the intestinal epithelial barrier in vitro

The bioavailability of all ingested compounds is to a great extend determined by the ability of these compounds to pass the intestinal epithelium. A high bioavailability is guaranteed for most nutrients and electrolytes since they are actively absorbed by the epithelium. The same epithelium, however, renders the entrance of non-nutrient (potentially harmful) hydrophilic (macro-) molecules, viruses and bacteria into the systemic circulation very low by presenting an almost impermeable barrier to these agent. The functional and structural properties of the single cell layer forming the intestinal epithelium determine the transepithelial permeability, which is an important factor in determining the bioavailability of all ingested compounds. Thus, insight in this transepithelial permeability is of considerable interest. However, the complexity of the gastrointestinal tract, and of the pre-and post-absorptive kinetics hampered the collection of accurate data. Therefore, a simple, accurate, reproducible, and cheap in vitro model was needed to screen the vast amount of newly developed, or discovered compounds for their intestinal transepithelial permeation.One very popular model consists of the human colonic carcinoma cell line Caco-2 grown in a two-compartment system. Transport experiments with this Caco-2 system yielded in vitro permeation rates which allowed prediction of the oral bioavailability in humans for many different compounds. However, it is generally assumed that the Caco-2 cells restrict the paracellular permeation (extracellular passage along the cells) of hydrophilic compounds such as intact peptides to a greater extent than the small intestinal epithelium in vivo . Thus especially for the modeling of hydrophilic macromolecular permeation, an additional model would be of great value. We posed the question whether the small intestinal epithelium derived cell line IEC-18 could be that model. We found that whereas the Caco-2 cells presented a good model for a general screen on intestinal permeation for a wide variety of compounds, the IEC-18 cells did allow for a better discrimination between the hydrophilic compounds on the basis of their molecular weight. Consequently, experiments with the filter grown IEC-18 cells yielded discriminative permeation rates for the paracellular permeation of hydrophilic compounds with differing bioavailabilities. Thus, data presented in this thesis suggest that the filter grown IEC-18 cells could be valuable as model for paracellular permeability in the small intestine.However, the cell morphology and brush border enzyme activities of IEC-18 cells cultured under standard conditions showed that the cells obtained only a low status of differentiation, while addition of dexamethasone to the culture medium triggered the cells to acquire a more enterocyte-like phenotype. Concomitant with the morphological changes, a tightening of the paracellular barrier was found which suggests that IEC-18 cells might present a model to study the effects of intestinal epithelial cell differentiation on paracellular permeability development along the intestinal crypt-villus axis.Additionally, since it has been recognized that the intestinal epithelial barrier can be compromised by compounds such as drug absorption enhancers or ingested toxins, we studied the effects of a proposed drug absorption enhancer (palmitoyl carnitine) and the bio-active heavy metal cadmium, on structural and functional features of the two intestinal cell lines. We concluded that for studies to modulation of intestinal epithelial barrier function, the Caco-2 is a more convenient model than the IEC-18 cells due to the high basal barrier function for hydrophilic molecules of the Caco-2 cells relative to the low barrier function of IEC-18 cells. The occurrence of increased hydrophilic macromolecular permeability concomitant with membrane damage but without concurrent alterations in the expression of the tight junctional protein ZO-1, as was found after palmitoyl carnitine exposure, strongly suggests the enhancement of transcellular permeability. On the other hand, increased hydrophilic macromolecular permeability and a decreased transepithelial electrical resistance, concomitant with alterations in junctional ZO-1 expression, but without concurrent membrane damage, as was found after cadmium exposure, strongly suggests the enhancement of the paracellular pathway.In conclusion, the two intestinal cell lines Caco-2 and IEC-18, together, offer the possibility to obtain in vitro permeation rates, enabling bioavailability modeling for virtually all classes of compounds. Furthermore, the in vitro systems and research strategies presented in this thesis might help to elucidate modes of action of newly developed compounds on the barrier function of the intestinal mucosa and might thus help to assess the effectivity and safety of absorption enhancers, and to indicate mucosal toxicity of orally ingested compounds such as drugs or food related products.