In vitro permeability assay using an epithelial model of Caco-2/HT29-MTX/Raji-B cells

Abstract

The oral route is one of the main routes for administration of drugs, however, the gastrointestinal tract is a hostile environment due to pH variation, presence of several digestive enzymes and the intestinal barrier that undermines the permeation of drugs. The aim of this study was to evaluate the ability of papain complexed with β-cyclodextrin to enhancer the permeation of furosemide, as a model drug, in a triple co-culture of Caco-2, HT29-MTX and Raji cells. Papain and the papain-β-cyclodextrin complex were evaluated at 0.3, 0.7 and 1.0 μM and the biophysical integrity of the cell layer was evaluated by RET (Transepitelial Electrical Resistance) at 0, 4, 24, 48 and 72 hours. The epithelium was also stained using DAPI and Alexa Fluor™ 488 Phalloidin. HPLC was employed to quantification of furosemide. The RET results at initial time for all the samples and control were in a range of 267.63 to 318.28 Ω*cm2 and after 72 h this values were raised to a range of 365.14 to 492.64 Ω*cm2. There was a decrease of RET after samples’ application, nevertheless, the results showed that the epithelium presents a recovery, proportional to the time of cell replication, and that this recovery occurs in all samples tested with no significant statistical difference. The RET recovery implies that papain, complexed or not, was not able to kill the cells, corroborating the hypothesis that the action mechanism is the disruption of the tight junction. In addition, the triple co-culture presents a higher resistance to papain action, in comparison with the Caco-2 monolayer assays, emphasizing the importance of testing new drugs, potential candidates for oral formulations, in epitheliums that faithfully mimics what actually happens in in vivo systems. The fluorescent microscopy observation of the cells stained with DAPI and the junctions stained with Alexa Fluor™ 488 Phalloidin, showed that co-culture exhibits microvilli inherent to the intestinal tissue. The results obtained in the triple co-culture model bi-directional transport experiments confirmed the significant increase in furosemide transport indicating the importance of the paracellular route. In conclusion, the triple co-culture model was successfully standardized and papain complexed with β-cyclodextrin acts probably over the tight junctions enhancing the permeation of furosemide.