A novel paracellular transport mechanism of hydrophilic cations across intestinal epithelium

This dissertation work has elucidated contribution of a paracellular mechanism in absorptive transport of orally available hydrophilic cations across Caco-2 cell monolayers. Previous studies demonstrated saturable absorptive transport of model compounds for such hydrophilic cations, ranitidine and famotidine, across Caco-2 cell monolayers. In this dissertation, distinct approaches have been developed to evaluate paracellular contribution to saturable absorptive transport of model hydrophilic cations across Caco-2 cell monolayers. Firstly, a fluorescent analogue of famotidine (FAF-1) was synthesized to visualize absorptive transport pathway of this compound using confocal laser scanning microscopy. Optical images of cross-sections of Caco-2 cells revealed that fluorescence of FAF-1 was restricted primarily to the paracellular region, providing direct evidence that FAF-1 exhibits saturable absorptive transport mechanism via the paracellular route across Caco-2 cell monolayers. Based on inhibition of FAF-1 transport by famotidine and ranitidine, these results also support the hypothesis that saturable transport of famotidine and ranitidine can be mediated by paracellular mechanism. Secondly, U73122 was utilized to specifically breach paracellular barrier to overall transport of compounds across Caco-2 cell monolayers. Enhancement factor (Ef) was developed to evaluate the transcellular vs. paracellular contribution. Ef successfully differentiated compounds exhibiting three distinct transport mechanisms. An inverse relationship was observed between Ef and Jmax(app)/Km(app) (i.e., transporter efficiency) for intestinal transporter substrates evaluated in this work. Interestingly, data for hydrophilic cations such as ranitidine, famotidine and metformin did not fit into inverse relationship established with transporter substrates. Instead, Ef values for those tested hydrophilic cations were comparable to those of paracellularly transported compounds. These results indicate that the saturable absorptive transport of tested hydrophilic cations may be mediated primarily by paracellular mechanism. Lastly, this dissertation work tested a hypothesis that hydrophilic compounds sharing the saturable absorptive transport mechanism of ranitidine would be well absorbed in humans. Extent of inhibition of the saturable absorptive transport of ranitidine by selected hydrophilic compounds showed a significant correlation (r2 = 0.78) to their fraction of oral dose absorbed in humans, suggesting that inhibition of ranitidine absorptive permeability across Caco-2 cell monolayers may be utilized to evaluate in vivo absorption potential for hydrophilic compounds.