Potential Excipient Effect on Tight Junctions

The intercellular tight junction is one of the major barriers to the paracellu-lar transport of macromolecules and polar compounds (Denker and Nigam, 1998). They have two physiological functions: first, they constitute the principal barrier to passive movement of fluid, electrolytes, macromolecules, and cells through the paracellular pathway (the "gate" function), and secondly, they contribute to transepithelial transport of compounds promoting epithelial cell polarity (Madara, 1987; Citi, 1992; Citi and Denisenko, 1995). Tight junction's structure and permeability can be regulated by many potential physiological factors, including the concentration of cyclic AMP (cAMP) (Duffey et al., 1981), intercellular calcium concentration (Palant et al., 1983), and transient mucosal loads (Madara et al., 1986). Several studies have shown that one of the possible mechanisms of penetration enhancers is to loosen the tight junctions of epithelial membranes thereby increasing the paracellular transport of poorly absorbable drugs (Gonzales-Mariscal and Nava, 2005). Hence the issue of tight junction's regulation by absorption enhancers appears to be crucial for macromolecular drug absorption.

In a study by Thanou (2000), the tight junction's membrane protein occludin was visualized by immunocytochemistry staining in the presence and absence of Trimethyl Chitosan 60 (TMC60) (degree of quaternization 60%) using con-focal laser scanning microscopy (CLSM). Additionally, the effects of TMC60 on cytoskeletal F-actin were determined by visualization using CLSM. The transmembrane protein occludin displayed a disrupted pattern after incubation with 1.0% (w/v) TMC60, suggesting that the interaction of TMC60 with the tight junction's protein is the major mechanism for opening of the tight junctions and subsequent increased paracellular permeability. These observations were quite similar to images of Caco-2 cells with 0.1% (w/v) chitosan, but the effect appeared to be stronger than for the reported 0.1% (w/v) chitosan. Chitosan treated cells showed a thickened pattern of occludin at the cell periphery and not a disrupted one, which might be due to the ten-fold difference in concentration or to an effect exclusively related to the quaternized derivative of chitosan, TMC60. Additionally it was observed that TMC60 provoked a redistribution of the cytoskeletal F-actin, a phenomenon that appeared to correlate well with the opening of epithelial tight junctions.

Calcium depletion by chelating agent (e.g., EDTA, EGTA) has been reported to increase paracellular permeability. These agents induce general changes in the cell physiology such as disruption of actin filaments and adherence junctions, diminished cell adhesion, and activation of protein kinases (Citi, 1992). It was proposed that EGTA provokes alterations on the tight junctions, being a consequence of its effects on Ca2+ dependent adhesion molecules (which are concentrated in adherence junctions), through a contraction of the junction-associated microfilament cytoskeleton (Citi and Denisenko, 1995). It has been demonstrated that serosal rather than apical Ca2+ levels play a more important role in this process (Collares et al., 1994). Basolateral Ca2+ levels vary and a particular chelation enhancer cannot accomplish full depletion of the calcium ions from the adherence junction to provoke the paracellular widening. Therefore, the approach of using chelating agent as permeation enhancers leads to variable results, even in controllable in vitro conditions like the Caco-2 cell system (LeCluise and Sutton, 1997).

Functional polymers such as polyacrylic acid derivatives and chitosan (derivatives) appear to be a valuable alternative solution to increase exclusively the paracellular permeation and absorption of hydrophilic drugs. Being high molecular weight and hydrophilic polymers it is assumed that their intrinsic absorption and related to this their toxicity is minimal and they are not expected to show systemic adverse side effects (Junginger and Verhoef, 1998; Thanou et al., 2001c). These functional polymers will be discussed in the next sections of this chapter.

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