CriticalSorb? Promotes Permeation of Flux Markers Across Isolated Rat Intestinal Mucosae and Caco-2 Monolayers

Purpose

CriticalSorb? is a novel absorption enhancer based on Solutol^? HS15, one that has been found to enhance the nasal transport. It is in clinical trials for nasal delivery of human growth hormone. The hypothesis was that permeating enhancement effects of the Solutol^?HS15 component would translate to the intestine.

Methods

Rat colonic mucosae were mounted in Ussing chambers and Papp values of [¹⁴C]-mannitol, [¹⁴C]-antipyrine, FITC-dextran 4000 (FD-4), and TEER values were calculated in the presence of CriticalSorb?. Tissues were fixed for H &; E staining. Caco-2 monolayers were grown on Transwells? for similar experiments.

Results

CriticalSorb?(0.01%?v/v) significantly increased the Papp of [¹⁴C]-mannitol, FD-4 [¹⁴C]-antipyrine across ileal and colonic mucosae, accompanied by a decrease in TEER. In Caco-2 monolayers, it also increased the Papp of [¹⁴C]-mannitol FD-4 and [¹⁴C]-antipyrine over 120?min. In both monolayers and tissues, it acted as a moderately effective P-glycoprotein inhibitor. There was no evidence of cytotoxicity in Caco-2 at concentrations of 0.01% for up to 24?h and histology of tissues showed intact epithelia at 120?min.

Conclusions

Solutol^? HS15 is the key component in CriticalSorb? that enables non-cytotoxic in vitro intestinal permeation and its mechanism of action is a combination of increased paracellular and transcellular flux.     

The mycotoxin patulin increases colonic epithelial permeability in vitro

The gastrointestinal lumen is directly exposed to dietary contaminants, including patulin, a mycotoxin produced by moulds. Patulin is known to increase permeability across intestinal Caco-2 monolayers. This study aimed to determine the effect of patulin on permeability, ion transport and morphology in isolated rat colonic mucosae. Mucosal sheets were mounted in Ussing chambers and voltage clamped. Apical addition of patulin (100–500 μM) rapidly reduced transepithelial electrical resistance (TEER) and increased permeability to [¹⁴C] mannitol (2.9-fold). Patulin also inhibited carbachol-induced electrogenic chloride secretion and histological evidence of mucosal damage was observed. To examine potential mechanisms of action of patulin on colonic epithelial cells, high-content analysis of Caco-2 cells was performed and this novel, quantitative fluorescence-based approach confirmed its cytotoxic effects. With regard to time course, the cytotoxicity determined by high content analysis took longer than the almost immediate reduction of electrical resistance in isolated mucosal sheets. These data indicate patulin is not only cytotoxic to enterocytes but also has the capacity to directly alter permeability and ion transport in intact intestinal mucosae. These data corroborate and extend findings in intestinal cell culture monolayers, and further suggest that safety limits on consumption of patulin may be warranted.     

The Effect of Monomers and of Micellar and Vesicular Forms of Non-ionic Surfactants (Solulan C24 and Solulan 16) on Caco-2 Cell Monolayers

Measurements of transepithelial electrical resistance (TEER), the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) test and monitoring of poly(ethylene glycol) (PEG) transport have been used to study the effects of the non-ionic surfactants Solulan C24 and Solulan 16, either free in solution or as an integral part of niosome bi-layers, on intestinal epithelial cells from man (Caco-2 cell monolayers). The effects on epithelial integrity and on the transport of the hydrophilic drug metformin depend on the concentration of the surfactants. At concentrations above 1% the effect on TEER of the surfactant in niosomal form and free in solution were equivalent whereas cell viability was preserved to a higher concentration of Solulans when the Solulans were present in the niosomal form. It was concluded that the toxic effect of niosomes arises from free surfactant present in the niosome suspension.     

Optimized conditions for prediction of intestinal drug permeability using Caco-2 cells.

The effects of various experimental conditions on in vitro drug permeability to Caco-2 monolayers were investigated to determine the optimized conditions for the prediction of intestinal drug absorption. Concerning the pH of the transport medium in the Caco-2 study, two different pH values, 6.0 and 7.4, were tested for the apical medium with the pH of the basolateral medium fixed to 7.4. The change in the apical pH showed pronounced effects on the permeability of both passively and actively transported drugs. It was found that the transport study under the condition of an apical pH value of 6.0 showed a better prediction of in vivo drug absorption in human. The appropriate conditions for determining the permeability of poorly soluble drugs were also examined. First, the effects of bile acids, surfactant and some agents used for solubilizing drugs on the permeability and transepithelial electrical resistance (TEER) of Caco-2 monolayers were investigated. Taurocholic and cholic acid showed no effects on the permeability of 3H-Dexamethasone (DEX) and TEER at 10 mM concentration, suggesting the possibility of use in the Caco-2 study. Polyethyleneglycol-400 and dimethylsulfoxide reduced the permeability of DEX concentration dependently, whereas ethanol induced no significant changes in the permeability. Furthermore, it was demonstrated that the addition of plasma protein (bovine serum albumin) to the basolateral medium apparently facilitated the transport of poorly soluble drugs with high lipophilicity across Caco-2 monolayers. These findings clearly suggest the importance of considering the physiological conditions of in vivo drug absorption in optimizing the in vitro experimental conditions for transport study using Caco-2 cells, in order to obtain a satisfactory in vitro-in vivo correlation.     

Enhanced transport of P-glycoprotein substrate saquinavir in presence of thiolated chitosan

It was the aim of this study to investigate the effect of chitosan-4-thiobutylamidine (Ch-TBA) and reduced glutathione (GSH) on the absorption of P-glycoprotein (P-gp) and multidrug resistance protein (MRP) substrate saquinavir in vitro and in vivo. Bidirectional transport studies were performed with Caco-2 cell monolayers and additionally with freshly excised rat small intestinal mucosa mounted in Ussing type chambers. Furthermore, a delivery system based on Ch-TBA and GSH was evaluated in vivo in rats. The functional activity of the efflux pumps in Caco-2 cells and rat intestinal mucosa during the experiment was proven by the efflux ratio of saquinavir, which was 6.4 for Caco-2 cells and 2.1 for rat intestinal mucosa, respectively. Ch-TBA and particularly the combination of Ch-TBA with GSH enhanced apical (AP) absorption and decreased the secretory transport of saquinavir. In presence of 0.5% Ch-TBA and 0.5% GSH, the uptake of saquinavir was 1.6-fold improved in Caco-2 monolayer and 2.1-fold improved in rat intestinal mucosa. In vivo, the area under the plasma concentration time curve (AUC) of saquinavir was 1.4-fold and Cmax 1.6-fold increased, in comparison with control. Results of this study showed that Ch-TBA in combination with GSH can be an interesting tool for increasing the oral bioavailability of actively secreted compounds.     

Comparison of impact of the different hydrophilic carriers on the properties of piperazine-containing drug.

The objective of this study was to determine the impact of a series of nonionic surfactants on the solubility of piperazine-containing drug (meclizine, MZ) in comparison to that of natural cyclodextrins (alpha-CD and beta-CD) and dimethyl-beta-cyclodextrin (DM-beta-CD). The solubility of the drug was studied in either CDs solutions or nonionic surfactant solutions. Three classes of nonionic surfactants were used namely; polyoxyethylene (POE) sorbitan fatty acid esters (polysorbates), POE fatty acid esters (Myrjs) and polyethylene oxide (PEO) fatty alcohol ethers (Brijs and Eumulgins). The solubility of MZ was increased linearly with the increasing surfactant concentration, indicating that micellar solubilization follows the partition model. It was found that the longer the hydrocarbon chain in a homologous series, the more efficient is the solubilizing power of surfactant. For example, polysorbate 80 (Tween-80) is a more efficient solubilizer than polysorbate 20 (Tween-20), indicating that the drug was incorporated in the core of micelle more than the capsular region of the micelle. On the other hand, in case of POE fatty acid esters, the solubilizing power increased with decreasing polyoxyethylene chain as Myrj 53 was more efficient than Myrj 59. In class of PEO fatty alcohol ethers, the shorter the hydrophilic chain and longer lipophilic chain, the more efficient was the solubilizing capacity. Thus, Brij 58 was more efficient solubilizer than Brij 35 and Eumulgin C1000 was more active than Eumulgin C1500. Comparatively, Eumulgin C1000 had the highest solubilizing power for MZ among the studied PEO fatty alcohol ethers and other groups of surfactants. The solubility action of surfactants toward MZ was increased by raising the temperature of the surfactant solutions from 30 to 45 degrees C. Hydrophilic macromolecules (PEG 1000 and PEG 6000) or cosolvents (glycerol and propylene glycol) have a very slight effect on the solubility of MZ and confirm the predominance of hydrophobic interaction between the drug and nonionic surfactants. A(L)-type phase solubility diagrams were obtained for the drug with alpha-, beta- and DM-beta-CDs showing that the solubility of MZ was enhanced through inclusion complexation. Comparatively, DM-beta-CD had the highest solubilizing efficiency for the drug among the investigated CDs, which could be attributed to its larger hydrophobic cavity size.     

Enhanced transport of P-glycoprotein substrate saquinavir in presence of thiolated chitosan

It was the aim of this study to investigate the effect of chitosan-4-thiobutylamidine (Ch-TBA) and reduced glutathione (GSH) on the absorption of P-glycoprotein (P-gp) and multidrug resistance protein (MRP) substrate saquinavir in vitro and in vivo. Bidirectional transport studies were performed with Caco-2 cell monolayers and additionally with freshly excised rat small intestinal mucosa mounted in Ussing type chambers. Furthermore, a delivery system based on Ch-TBA and GSH was evaluated in vivo in rats. The functional activity of the efflux pumps in Caco-2 cells and rat intestinal mucosa during the experiment was proven by the efflux ratio of saquinavir, which was 6.4 for Caco-2 cells and 2.1 for rat intestinal mucosa, respectively. Ch-TBA and particularly the combination of Ch-TBA with GSH enhanced apical (AP) absorption and decreased the secretory transport of saquinavir. In presence of 0.5% Ch-TBA and 0.5% GSH, the uptake of saquinavir was 1.6-fold improved in Caco-2 monolayer and 2.1-fold improved in rat intestinal mucosa. In vivo, the area under the plasma concentration time curve (AUC) of saquinavir was 1.4-fold and C_max 1.6-fold increased, in comparison with control.

Results of this study showed that Ch-TBA in combination with GSH can be an interesting tool for increasing the oral bioavailability of actively secreted compounds.     

Comparison of ceftibuten transport across Caco-2 cells and rat jejunum mounted on modified Ussing chambers

Ceftibuten uptake into Caco-2 cells and intestinal brush border membrane vesicles is mediated by the dipeptide transport system (PEPT1). The apical to basolateral transport characteristics of ceftibuten across Caco-2 cells and rat jejunum mounted on a modified Ussing chamber was examined. Mannitol was used as a paracellular marker along with trans-epithelial electrical resistance (TEER) for monitoring tight junction permeability. Transport across Caco-2 cells and rat jejunum mounted on a modified Ussing chamber was linear across the concentration range 0.25-10 mM. The net flux of mannitol and ceftibuten was higher across rat jejunum compared with Caco-2 cells. At a donor concentration of 0.25 mM, ceftibuten transport across Caco-2 cells was found to be pH dependent. Glycyl proline, a dipeptide, and 2,4- dinitrophenol, an energy poison, caused a reduction in the permeability of 0.25 mM ceftibuten across Caco-2 cells. Benzoic acid and adipic acid also inhibited transcellular transport of ceftibuten. At a donor concentration of 0.25 mM, passive paracellular transport accounts for about 60% and the active carrier mediated mechanism accounts for about 40% of ceftibuten transport across Caco-2 cells. None of the inhibitors however, had a significant effect on ceftibuten transport across rat jejunum mounted on a modified Ussing chamber at a donor concentration of 0.25 mM. In the concentration range 0.25-10 mM, ceftibuten is predominantly transported by paracellular mechanisms across rat jejunum and a mixture of active and passive transport across Caco-2 cells.     

Transport of anti-allergic drugs across the passage cultured human nasal epithelial cell monolayer.

The purpose of this study was to investigate the nasal absorption characteristics of a series of anti-allergic drugs across the human nasal epithelial cell monolayer, which was passage cultured by the liquid-covered culture (LCC) method on Transwell. Characterization of this cell culture model was achieved by bioelectric measurements and morphological studies. The passages 2--4 of cell monolayers exhibited the TEER value of 1731+/-635 Omega cm(2) after 2 days of seeding and maintained high TEER value for 4--6 days. Morphological study by TEM and SEM showed the existence of the tight junctions, and the cuboidal shaped epithelial cells monolayer. A series of anti-allergic drugs, albuterol hemisulfate, albuterol, fexofenadine HCl, dexamethasone, triamcinolon acetonide, and budesonide were selected as model compounds for transport studies. All the drugs were assayed using reversed-phase HPLC under isocratic conditions. Results indicated that within the logP (apparent 1-octanol/water partition coefficient) range from --1.58 (albuterol) to 3.21 (budesonide), there existed 100-fold difference in the apparent permeability coefficients (P(app)). A log-linear relationship was shown between the drug logP and the P(app) across passaged human nasal epithelial monolayers. The amount of fexofenadine HCl and dexamethasone across passaged human nasal cell monolayers was concentration-dependent in the direction of apical to basolateral. The direction dependent transport studies were investigated among all these drugs and no significant difference in the two directions was observed. In conclusion, this LCC passaged human nasal epithelial culture model may be a useful in vitro model for studying the passive transport processes in nasal drug delivery.     

Effects of capsaicin on cellular damage and monolayer permeability in human intestinal Caco-2 cells

Recent studies suggest that capsaicin (Cap), a major constituent of hot pepper, may affect the function and permeability of the intestinal mucosa in vitro. However, the relationships between the dose of Cap and the barrier and/or transporter functions on intestinal epithelial cells are unknown. The aim of this study was to investigate whether Cap initiates cellular injury and alter epithelial permeability in Caco-2 cells. Cellular toxicity, as measured using a lactate dehydrogenase release assay, was not observed at high concentrations of Cap (up to 300 microM). When cell viability was measured by a WST-1 assay (tetrazolium salt-based assay), damage to Caco-2 monolayers was observed at doses of 200 and 300 microM of Cap. The barrier function of tight junctions was assessed by measuring transepithelial electrical resistance (TEER) in Caco-2 cells. Treatment of Caco-2 cells with Cap at doses above 100 microM significantly decreased the TEER compared to treatment with buffer alone for 2 h (p<0.05). We next examined the effects of Cap on the activity of P-glycoprotein (P-gp) found on transcellular transporters. At doses of 100 and 200 microM, Cap inhibited the transport of rhodamine 123 by P-gp-mediated efflux in Caco-2 cells. Cap thus exhibited inhibitory effects on P-gp. The results of this study indicate that Cap, a dietary phytochemical, causes functional and structural changes in Caco-2 cell monolayers at noncytotoxic doses (less than 100 microM of Cap). The concomitant administration of Cap with drugs that are substrates of P-gp might increase the plasma concentrations of such drugs.     

Transport of octreotide and evaluation of mechanism of opening the paracellular tight junctions using superporous hydrogel polymers in Caco-2 cell monolayers

The purpose of this study was to investigate the mechanism of opening of tight junctions in Caco-2 cell monolayers using superporous hydrogel (SPH) and SPH composite (SPHC) polymers as permeation enhancers for peptide drug delivery. Moreover, the transport of octreotide across Caco-2 cell monolayers was assessed by application of SPH and SPHC polymers on Caco-2 cell monolayers. In these experiments, N,N,N-trimethyl chitosan chloride with 60% quaternization (TMC60) was used as a positive control for opening of tight junctions. Transepithelial electrical resistance (TEER) studies showed that all three polymers (TMC60, SPH, and SPHC) were able to decrease TEER values to approximately 30% of the initial values, indicating the ability of these polymers to open the tight junctions. Recovery TEER studies showed that the effects of the polymers on Caco-2 cell monolayers were reversible, indicating viability of the cells after incubation with polymers. Both SPH and SPHC (compared with TMC60) were able to increase the paracellular transport of octreotide by their mechanical pressures on tight junctions. The mechanistic studies showed that junctional proteins, including actin, occludin, and claudin-1, were influenced by application of SPH and SPHC polymers to the Caco-2 cell monolayers. SPH and SPHC induced clear changes in the staining pattern of all three proteins compared with the control, indicating that the expression of these proteins in the tight junctions was increased, most likely due to the mechanical pressure of the polymers on the junctional proteins.     

Melittin as a Permeability Enhancer II: <Emphasis Type="BoldItalic">In Vitro</Emphasis> Investigations in Human Mucus Secreting Intestinal Monolayers and Rat Colonic Mucosae

Purpose Melittin has shown potential as a non-cytotoxic absorption enhancer in Caco-2 monolayers. Our objectives were to assess in vitro efficacy and cytotoxicity of melittin in two intestinal permeability models and investigate the potential mechanism by which melittin might enhance gastrointestinal absorption. Materials and methods The effects of melittin were examined in the mucus-secreting intestinal cell monolayers, HT29-MTX-E12 (E12), using transepithelial electrical resistance (TER), transmission electron microscopy (TEM) and the MTT viability assay. The effects of melittin on TER, permeability and short circuit current (Isc) were also investigated in rat colon mucosae mounted in Ussing chambers. Ion transporting capacity of tissue was measured in response to secretagogues as surrogate markers of cytotoxicity. Melittin stability was examined by a means of a hemolytic assay. The mechanism by which melittin decreases TER across the rat mucosa was examined with a range of enzymatic inhibitors. Results Apical addition of melittin resulted in a reversible non-cytotoxic concentration-dependent decrease in TER across E12 monolayers, which was independent of the presence of mucus. Apical addition of melittin reduced TER and increased the permeability of [¹⁴C]-mannitol across rat colonic mucosae. The melittin-induced drop in TER in rat colon was significantly attenuated by W7 suggesting partial mediation by calmodulin. Conclusions The rapid and reversible nature of melittin’s permeation enhancing properties and its limited cytotoxicity in polarized intestinal epithelia, suggests a potential drug delivery role for the peptide in oral formulations of poorly absorbed drugs.     

Novel potential drug against T. cruzi and its interaction with surfactant micelles

The interaction of 5-nitro-2-furfurilylidene benzhydrazide (5NFB), potential anti-trypanosomal compound, with micellar solutions was studied. The results indicated that 50 mug of 5NFB completely kills 20 million T. cruzi epimastigote cells within 3 days, whereas the same amount of benznidazole kills 30% of the cells after 4 days. 5NFB solubility in surfactants solutions (SDS, DTAB, C12EO8) increased linearly with surfactant concentration. According to small angle X-ray scattering (SAXS), 5NFB does not affect micellar structural features. A comparison between C12EO8 effects on T. cruzi epimastigote cells and on erythrocytes showed that surfactant lytic effect is stronger in parasite cells, enlightening the potential of 5NFB micellar formulations.     

Effect of self-microemulsifying drug delivery systems containing Labrasol on tight junctions in Caco-2 cells

The aim of this study was to investigate the effect of two novel self-microemulsifying drug delivery systems (SMEDDS) containing Labrasol with different dilutions on tight junctions. Changes in barrier properties of Caco-2 cell monolayers, including transepithelial electrical resistance (TEER) and permeability to the paracellular marker, i.e., mannitol, were assessed in response to dilutions and surfactants contents within formulations. The cytotoxicity of SMEDDS and the effect of surfactants on Caco-2 cells were evaluated by the MTT. Changes in subcellular localization of the tight junction proteins, ZO-1 and F-actin, were examined by confocal laser scanning microscopy. Results demonstrated that negatively charged SMEDDS with different dilutions had no effect on the TEER, but significantly increased the permeability of mannitol. In contrast, the positively charged formulation showed a dilution-dependent reduction in TEER. A corresponding increase in mannitol permeability of up to 29.4-fold to 64.7-fold greater than the control was also observed across the monolayer. Labrasol with the concentration of 0.1 and 1% was shown to increase the permeability of mannitol by 4.6-fold and 33.8-fold, respectively. The mechanism of opening of tight junctions was found to involve F-actin-related changes and redistribution of ZO-1.     

New and better protocols for a short-term Caco-2 cell culture system

The aim of the present study was to develop new and better protocols for a short-term Caco-2 cell culture system for use in rapid screening of intestinal drug absorption. Caco-2 cells were cultured according to several protocols for short-term cell culture to obtain monolayers. The effects of serum (fetal bovine serum, FBS) in the culture medium and of the period of cell culture on the barrier function and transporter activities of the monolayers were examined. The barrier function was estimated both from the transepithelial electrical resistance (TEER) and the permeability of [(14)C]mannitol. Transporter activities were monitored by measuring the permeability of [(14)C]glycylsarcosine for oligopeptide transporter (PepT1) and of rhodamine 123 for P-glycoprotein (P-gp). Caco-2 monolayers obtained by 3-day culture in the BIOCOAT HTS Caco-2 Assay System, developed by Becton Dickinson Bioscience, showed much higher permeability to hydrophilic compounds, such as mannitol, compared with those obtained by the standard 21-day culture system, due to the leaky structure of cell junctions. The newly developed 3-day protocol, which includes 10% FBS in the culture medium during the first day of culture, markedly enhanced TEER and lowered mannitol permeability of the monolayers. This protocol allowed us to better determine the rank order of permeability of compounds, giving results equivalent to those in the 21-day culture system. The longer culture period gave tighter monolayers, and the maximum value of TEER was obtained with 5 days in culture. However, after 5 days in culture, the integrity of monolayers decreased gradually. The highest activities of transporters, PepT1 and P-gp, in monolayers were obtained at days 5 or 6 of culture by the new protocol with FBS-containing medium. These results indicate that by a simple modification of the short-term culture protocol, it is possible to obtain Caco-2 monolayers with better barrier properties and higher activity of transporters that are equivalent to those found in the 21-day Caco-2 culture system.     

Lack of Effect of Ammonium Glycyrrhizinate on the Morphology of Ovine Nasal Mucosa in Vitro

Glycyrrhetinic acid derivatives are reported to be nasal absorption promoters (1). Effects of ammonium glycyrrhizinate (AMGZ) on the in vitro morphology of ovine nasal mucosa were therefore examined by light and electron microscopy. Nasal mucosa was stripped from the submucosa and mounted in Ussing chambers. Exposure of the apical surface to 2% ammonium glycyrrhizinate (24 mM) for 90 min caused no histopathological changes to the nasal epithelium. Epithelial integrity remained intact as evidenced by the continued presence of morphologically intact junctional complexes. No sloughing of the epithelial layer from the basement membrane was observed, and cilia and microvilli were not affected by treatment with AMGZ. The results indicate that short-term exposure in vitro to ammonium glycyrrhizinate caused no overt morphological damage to ovine nasal mucosa.     

Transepithelial electrical resistance is not a reliable measurement of the Caco-2 monolayer integrity in Transwell

The significance of monitoring transepithelial electrical resistance (TEER) value during the study on drug absorption through Caco-2 monolayers in Transwells was re-evaluated. TEER value was monitored before, during, and after the absorption of Streptokinase (45 KD). Four enhancers--disodium ethylenediaminetetracetate (disodium EDTA), sodium cholate (NaC), sodium taurocholate (NaTC), and sodium caprate along with alpha-hemolysin (a cell membrane pore-forming toxin)--were used to signify the outcome of this study. Modified trypan blue exclusion technique was used to examine the Caco-2 cell viability throughout the absorption studies. The enhancers at the used concentration exhibited toxic effect on the Caco-2 cells as evident from the trypan blue exclusion studies. This toxic effect was not reflected by the TEER profile because TEER value dropped after the addition of the absorption enhancers. But it came back to its initial value after the cell culture media was replaced by enhancer-free media. This toxic effect was confirmed by the antiproliferation studies on the four enhancers and alpha-hemolysin against Caco-2 cells. Therefore, we concluded that the measurement of TEER is not a reliable method to determine the absorption enhancers toxicity or integrity of the Caco-2 monolayers in the Transwells.     

Anti-inflammatory effects of budesonide in intestinal epithelial cells.

The glucocorticosteroid budesonide is one of the mainstay treatments in inflammatory bowel disease. The aim of this study was to investigate its effects on Caco-2 cells upon coculture with LPS stimulated macrophages, or with conditioned medium (CM) from the same. The mRNA expression of the chemokines IL-8 and ENA-78 were upregulated in Caco-2 cells, which was always more pronounced in the coculture, as compared to the CM situation, with the ENA-78 induction being higher than of IL-8. Addition of budesonide to the apical side of Caco-2 cells, decreased both IL-8 and ENA-78 mRNA levels in a dose dependent manner. A reduction in trans epithelial electrical resistance (TEER) and an increase in the apical-basolateral transport of fluoresceinated sulfonic acid (FS) were observed in the Caco-2 cells both in cocultures and CM experiments. The reduction in TEER was counteracted by budesonide, whereas it had no effect on the FS transport. We conclude that budesonide exert an anti-inflammatory effect directly on intestinal epithelial cells, which may contribute to the overall action of budesonide in the treatment of inflammatory bowel diseases.     

Effect of Rhamnolipids on Permeability Across Caco-2 Cell Monolayers

Purpose

This report describes the effect of rhamnolipids (RLs), an amphiphilic biosurfactant produced by the bacterium Pseudomonas aeruginosa, on the integrity and permeability across Caco-2 cell monolayers.

Methods

We measured the trans-epithelial electrical resistance (TEER) and permeability of [¹⁴C]mannitol across Caco-2 cell monolayers upon incubation with 0.01–5.0% v/v RLs as a function of incubation time (30, 60, 90, and 120 min). We also studied the recovery of RL-treated Caco-2 cell monolayers upon incubation with Kaempferol, which is a natural flavonoid that promotes the assembly of the tight junctions.

Results

TEER of Caco-2 cell monolayers incubated with 0.01–5.0% v/v RLs solution dropped to 80–28% of that of untreated cells. Decline in TEER was associated with an increase in [¹⁴C]mannitol permeability as a function of RLs concentration and incubation time with Caco-2 cells. Incubation of RLs-treated Caco-2 cell monolayers with normal culture medium for 48 h did not restore barrier integrity. Whereas, incubation of a RLs-treated Caco-2 cells with culture medium containing Kaempferol for 24 h restored barrier function indicated by the higher TEER and lower [¹⁴C]mannitol permeability values.

Conclusions

These results show the ability of RLs to modulate the integrity and permeability of Caco-2 cell monolayers in a concentration- and time-dependent fashion, which suggest their potential to function as a non-toxic permeation enhancer.     

In vitro and in vivo evaluation of effects of sodium caprate on enteral peptide absorption and on mucosal morphology

Sodium salts of medium-chain fatty acids, sodium caprate (C10) in particular, have been used as absorption-enhancing agents to promote transmucosal drug absorption. In this study, we conducted both in vitro and in vivo experiments to investigate the effects of C10 on intestinal permeabilities and mucosal morphology. Mucosal addition of C10 (13-25 mM) reduced the transepithelial electric resistance (TEER) of cultured monolayers of the human intestinal cell line Caco-2 by 40-65% and, upon removal of C10, a marked tendency of TEER recovery was recorded. C10 added mucosally at 13-50 mM increased the transports of mannitol and polyethylene glycol (PEG) 900 across Caco-2 in a dose-dependent manner. In contrast, the transport of a model D-decapeptide was maximally enhanced with 20-25 mM C10. No noticeable morphological alteration of the Caco-2 monolayers was observed after a 1-h mucosal pretreatment with C10. Co-delivery with C10 (0.05-0.5 mmol/kg) into the rat terminal ileum increased the D-decapeptide bioavailability (BA) dose-dependently. With 0.5 mmol/kg C10 co-administered, D-decapeptide percent BA was elevated from 2 to 11%. Following a 1-h incubation with 0.5 mmol/kg C10 (in liquid or powder form) non-invasively delivered into the rectal lumen, no signs of histological change in the rectal mucosa were detected. These results demonstrate that C10 can promote intestinal absorption of a small peptide without causing detrimental alterations of the intestinal mucosa. C10 thus seems to be a good candidate as an enhancing agent for improving the oral BA of small therapeutic peptides.