Permeability characteristics of tetragastrins across intestinal membranes using the Caco-2 monolayer system: Comparison between acylation and application of protease inhibitors

Purpose. Three types of acyl tetragastrin (TG), acetyl-TG (C-2-TG), butyryl-TG (C-4-TG) and caproyl-TG (C-6-TG) were synthesized and their in vitro intestinal permeability characteristics were examined using Caco-2 monolayers. Methods. The disappearance of acyl-TGs from the apical side of Caco-2 monolayers was estimated by analyzing degradation and permeation processes in terms of clearance. Results. The amount of native TG transported to the basolateral side was very low due to its large degradation clearance (CLd) on the apical side. Degradation of TG was reduced by chemical modification with fatty acids, which resulted in an increase in the transport of TG across Caco-2 monolayers. In addition, the permeation clearance (CLp) value of carboxyfluorescein (CF), a paracellular transport and undegradable marker, was increased in the presence of acyl-TGs. Furthermore, we investigated the effects of the protease inhibitors bacitracin and gabexate on the transport of TG across Caco-2 monolayers. In the presence of a low concentration (0.1 mM) of protease inhibitor, the CLd value of TG was reduced, but they did not affect its CLp value. However, a higher concentration (1.0 mM) of bacitracin significantly reduced TG degradation on the apical side, and further increased its CLp value. Conclusions. We demonstrated that acylation of TG made it resistant to intestinal proteases and caused it to enhance absorption of drugs, including itself, across Caco-2 monolayers. Further, bacitracin acted as both a protease inhibitor and an absorption enhancer.