Transepithelial Permeation of Tolbutamide across the Human Intestinal Cell Line, Caco-2

Abstract

Sulfonylurea hypoglycemic agents have interindividual variability in the gastrointestinal absorption rate. However, the absorption mechanism at the intestinal epithelium has not yet been clarified. To elucidate contribution of the specific mechanism for transepithelial transport of sulfonylureas, the apical-to-basolateral and basolateral-to-apical transport studies of tolbutamide were carried out using Caco-2 cell monolayers cultured on the polycarbonate membrane. The transported amounts of the substrate were measured by HPLC to estimate the apparent permeability coefficients (P(app)). In the apical-to-basolateral flux, the transport activity of tolbutamide was facilitated when the pH of the apical medium was more acidic than the basolateral one. ATP-depletion decreased the P(app) of tolbutamide. The kinetic analysis of the permeation rate indicated that the saturable process largely contributed to the tolbutamide flux. The P(app) of tolbutamide was lowered by an ionophore and monocarboxylic acids, while dicarboxylic acids and the inhibitor for the anion exchanger had no effect. In addition, mutual inhibition with benzoic acid was observed in transepithelial transport of tolbutamide. On the other hand, the permeation rate of tolbutamide from the basolateral to apical side was concentration-independent and neither affected by metabolic inhibitors, probenecid nor inhibitors for P-glycoprotein. In conclusion, these results suggest that apical-to-basolateral transport of tolbutamide across the Caco-2 cell monolayers is mediated by the pH-dependent specific system, presumably shared with other organic anions such as benzoic acid.

Determination of the active transport of fucoidan derived from Okinawa Mozuku across the human intestinal Caco-2 cells as assessed by size-exclusion chromatography

“…2). The respiration inhibitor ofazide (at 10 mM) is found to inhibit thisfucoidantransport; i.e., this inhibition of transport is found to be significant(p < 0.05; Mann-Whitney's U test; two-tailed test).Similar observation of 77% inhibition (p < 0.001; n = 3~4) by azide (10 mM) onto the transport of a small molecule of tolbutamide (a sulphonylurea drug for type II diabetes; Mr 270.4) in Caco-2 cells has been reported by Nishimura et al [9].…”

“…5. FCCP inhibits the fucoidan transport by 33% (significance p< 0.05; Mann-Whitney's U test; one-tailed test), but ClCCPdoes not (about 13% inhibition, but not significant).Nishimura et al alsohave reported the observation of 12% inhibition (p < 0.05; n = 3~4) by FCCP (0.05mM) onto the transport oftolbutamide in Caco-2 cells [9].The inhibition on the fucoidan transport byClCCPhas not reached to the significant level on the contrary to theprevious observation in the bacterial artificial membrane vesicles [10]. Thisdiscrepancy may be due to the chemical differences in the membranes between bacterial cells and human colon cancer cells.Since this fucoidan transport demonstrated byCaco-2 cells monolayeris inhibited by both the respiration inhibitor of azide and theuncoupler of FCCP, thetype of fucoidan transport by Caco-2 cells is considered to be a typical energy-requiringactive-transport.…”

“…Uncouplers of FCCP [9] and ClCCP [10]are known to uncouple between the energy (NADH for the case of bacteria [10]) obtainedfrom the degradationof glucose and theactive transportsystem. Then, uncouplers are tested, and the result of the inhibitiontest by FCCP is shown in Fig.…”

Effects of Pomegranate Juice on Human Cytochrome P450 2C9 and Tolbutamide Pharmacokinetics in Rats

“…Because pomegranate juice is an inhibitor of CYP3A (Hidaka et al, 2005), the juice may be also an inhibitor of P-glycoprotein in the intestine and enhance the absorption of drugs. However, Nishimura et al (2004) reported that basolateralto-apical transport of tolbutamide across the Caco-2 cell monolayers was not operated by the carrier-mediated transport system including P-gp efflux. Therefore, the inhibition of P-glycoprotein by pomegranate juice likely would not be associated with the increased bioavailability of tolbutamide.…”

A Novel and Facile Approach to Imaging Nanoparticles Transport across Transwell Filter Grown Cell Monolayer in Real-Time and in Situ under Confocal Laser Scanning Microscopy

“…However, the visualization of the cellular transport process occurring on Transwell system in real-time and in situ has not been realized yet. Up to now, the main method used to verify the transported substance using Transwell system is to sample the basolateral medium at predetermined time points and quantify the transported substance by HPLC 16) or other quantitative instruments. 17,18) In the studies of interaction with cells, fluorescent reagents are often used include Coumarin 6 (C6) 19) etc.…”