Caco-2 Cell Monolayer Transport Research Articles

Evaluation of cytotoxicity of surfactants used in self-micro emulsifying drug delivery systems and their effects on paracellular transport in Caco-2 cell monolayer

The objective of this study was to examine the cellular effects of the members of two non-ionic amphiphilic tenside groups and their mixtures on human Caco-2 cell monolayers as dependent upon their chemical structures and physicochemical properties. The first group of polyethylene glycol esters is represented by Polysorbates and Labrasol alone and in blends, while the members of the second group. Capryol 90, Capryol PGMC, Lauroglycol 90 and Lauroglycol FCC were used as propylene glycol esters. They are increasingly used in SMEDDS as recent tensides or co-tensides to increase hydrophobic bioavailability of a drug. Critical micelle concentration was measured by determination of surface tension. CMC refers to the ability of solubilization of surfactants. Cytotoxicity tests were performed on Caco-2 cell monolayers by MTT and LDH methods. Paracellular permeability as a marker of the integrity of cell monolayers, was examined with Lucifer yellow assays combined with TransEpithelial Electrical Resistance (TEER) measurements. The effect of these surfactants on tight junctions as evidence for paracellular pathway was also characterized. The results of cytotoxicity assays were in agreement, and showed significant differences among the cytotoxic properties of surfactants in a concentration-dependent manner. Polysorbates 20, 60, 80 are the most toxic compounds. In the case of Labrasol, the degree of esterification and lack of sorbit component decreased cytotoxicity. If the hydrophyl head was changed from polyethylene glycol to propylene glycol the main determined factor of cytotoxicity was the monoester content and the length of carbon chain. In our CMC experiments, we found that only Labrasol showed expressed cytotoxicity above the CMC. It refers to good ability of micelle solubilization of Labrasol. In our paracellular transport experiments each of polyethylene glycol surfactants (Polysorbates and Labrasol) altered TEER values, but propylene glycol esters did not modify the monolayer integrity. Polyethylene glycol esters alone and in blends (0.05% Labrasol – 0.001% Polysorbates 20, 60, 80) were able to increase Lucifer yellow permeability significantly below the IC50 concentration. On the other hand Labrasol and Polysorbates 20 have expressed effect on tight junctions of Caco-2 monolayer. It could be concluded that polyethylene glycol ester-type tensides were able to enhance the paracellular permeability by the redistribution of junctional proteins. Our results might ensure useful data for selection of suitable tensides, co-tensides and tenside mixtures for SMEDDS formulations.

Transport in Caco-2 cell monolayers of flavone C-glycosides from a neotropical blueberry

Transport in Caco-2 cell monolayers of flavone C-glycosides from a neotropical blueberry

Transport in Caco-2 cell monolayers of cucurbitane triterpenoids from Momordica charantia

Transport in Caco-2 cell monolayers of cucurbitane triterpenoids from Momordica charantia

Effect of Structural Modification of α-Aminoxy Peptides on Their Intestinal Absorption and Transport Mechanism

A representative α-aminoxy peptide 1 has been demonstrated to have a potential for the treatment of human diseases associated with Cl(-) channel dysfunctions. However, its poor intestinal absorption was determined. The purpose of this study was to delineate the transport mechanism responsible for its poor absorption and also to prepare peptide analogues by structural modifications of 1 at its isobutyl side chains without changing the α-aminoxy core for retaining biological activity to improve the intestinal absorption. The poor intestinal absorption of 1 was proved to be due to the P-glycoprotein (P-gp) mediated efflux transport in Caco-2 cell monolayer, intestinal segments in Ussing chamber and rat single pass intestinal perfusion models. Four analogues with propionic acid (2), butanamine (3), methyl (4) and hydroxymethyl side chains (5) were synthesized and tested using the same models. Except for the permeability of 2, the absorbable permeability of the modified peptides in Caco-2 cell monolayer and their intestinal absorption in rats were significantly improved to 7-fold (3), 4-fold (4), 11-fold (5) and 36-fold (2), 42-fold (3), 55-fold (4), 102-fold (5), respectively, compared with 1 (P(app), 0.034 ± 0.003 × 10(-6) cm/s; P(blood), 1.61 ± 0.807 × 10(-6) cm/s). More interestingly, the structural modification remarkably altered transport mechanism of the peptides, leading to the conversion of the active transport via P-gp mediation (1, 2), to MRP mediation (3), MRP plus BCRP mediation (4) or a passive diffusion (5). Furthermore, P-gp mediated efflux transport of 1 and 2 was demonstrated to not alter the P-gp expression, while 1 but not 2 exhibited uncompetitive inhibitory effect on P-gp ATPase. The results demonstrated that intestinal absorption and transport mechanism of the α-aminoxy peptides varied significantly with different structures, and their absorption can be dramatically improved by structural modifications, which allow us to further design and prepare better α-aminoxy peptide candidates with appropriate pharmacokinetic fates, including intestinal absorption, for potential clinical use.

Small-animal PET evaluation of [11C]MC113 as a PET tracer for P-glycoprotein

Background The radiolabelled inhibitor of the multidrug efflux transporter P-glycoprotein (P-gp) [C]elacridar was developed as a positron emission tomography (PET) tracer to assess expression levels of P-gp at the blood-brain barrier (BBB) [1]. [C]Elacridar was shown to interact specifically with P-gp at the rodent BBB, but its brain PET signal was very low, which was possibly caused by transport of [C]elacridar by P-gp [1]. In an attempt to gain a better understanding of the required properties of an effective P-gp PET tracer we evaluated C-labelled MC113, a structural analogue of elacridar, which was characterised as an unambiguous non-transported P-gp inhibitor and which possesses lower molecular weight, lower lipophilicity and higher potency for P-gp inhibition than elacridar (EC50 for inhibition of [ H]vinblastine transport in Caco-2 cell monolayers: 0.6 μM vs. 2.0 μM for elacridar) [2].

Comparison of bidirectional lamivudine and zidovudine transport using MDCK, MDCK–MDR1, and Caco-2 cell monolayers

Bidirectional transport studies were conducted using Caco-2, MDCK, and MDCK–MDR1 to determine P-gp influences in lamivudine and zidovudine permeability and evaluate if zidovudine permeability changes with the increase of zidovudine concentration and/or by association of lamivudine. Transport of lamivudine and zidovudine separated and coadministrated across monolayers based on these cells were quantified using LC–MS–MS. Drug efflux by P-gp was inhibited using GG918. Bidirectional transport of lamivudine and zidovudine was performed across MDCK–MDR1 and Caco-2 cells. Statistically significant transport decrease in B→A direction was observed using MDCK–MDR1 for zidovudine and MDCK–MDR1 and Caco-2 for lamivudine. Results show increased transport in B→A and A→B directions as concentration increases but data from Papp increase in both directions for both drugs in Caco-2, decrease in MDCK, and does not change significantly in MDCK–MDR1. Zidovudine transport in A→B direction increases when coadministrated with increasing lamivudine concentration but does not change significantly in B→A direction. Zidovudine and lamivudine are P-gp substrates, but results assume that P-gp does not affect significantly lamivudine and zidovudine. Their transport in monolayers based on Caco-2 cells increase proportionally to concentration (in both directions) and zidovudine transport in Caco-2 cell monolayer does not show significant changes with lamivudine increasing concentrations. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:4413–4419, 2009

The Effect of Hyperosmosis on Paracellular Permeability in Caco-2 Cell Monolayers

The intestinal epithelium is a significant barrier to oral absorption of hydrophilic compounds, and their passage through the intercellular space is restricted by the tight junctions. In this study we found that hyperosmosis is a significant factor altering paracellular transport in Caco-2 cell monolayers. Osmotic regulators, such as sodium chloride, mannitol, and raffinose, decreased transepithelial electrical resistance and enhanced lucifer yellow permeability. The effect of these osmotic regulators on Caco-2 cell monolayers was not likely to be caused by gross cytotoxicity. Although certain amino acids and oligosaccharides have been reported to have specific tight junction-modulating activity, we found that the increased paracellular permeability of Caco-2 monolayers induced by these compounds was at least partly due to the increased osmotic pressure of the test solutions. These findings provide a new potential precaution in the evaluation of paracellular permeability-modulating substances using the Caco-2 cell monolayer system.

Hepcidin inhibits apical iron uptake in intestinal cells

Hepcidin (Hepc) is considered a key mediator in iron trafficking. Although the mechanism of Hepc action in macrophages is fairly well established, much less is known about its action in intestinal cells, one of the main targets of Hepc. The current study investigated the effects of physiologically generated Hepc on iron transport in Caco-2 cell monolayers and rat duodenal segments compared with the effects on the J774 macrophage cell line. Addition of Hepc to Caco-2 cells or rat duodenal segments strongly inhibited apical (55)Fe uptake without apparent effects on the transfer of (55)Fe from the cells to the basolateral medium. Concurrently, the levels of divalent metal transporter 1 (DMT1) mRNA and protein in Caco-2 cells decreased while the mRNA and protein levels of the iron export transporter ferroportin did not change. Plasma membrane localization of ferroportin was studied by selective biotinylation of apical and basolateral membrane domains; Hepc induced rapid internalization of ferroportin in J774 cells but not in Caco-2 cells These results indicate that the effect of Hepc is cell dependent: in macrophages it inhibits iron export by inducing ferroportin degradation, whereas in enterocytes it inhibits apical iron uptake by inhibiting DMT1 transcription. Our results highlight the crucial role of Hepc in the control of intestinal iron absorption.

Effects of benzo(e)pyrene and benzo(a)pyrene on P-glycoprotein-mediated transport in Caco-2 cell monolayer: A comparative approach

The previous studies from our laboratory reported that benzo(a)pyrene (Bap) influenced efflux transport of rhodamine 123 (Rho-123) by induction of P-glycoprotein (P-gp) in Caco-2 cells. The present study investigated whether induction of P-gp and the enhanced efflux transport of Rho-123 were caused by benzo(e)pyrene (Bep), which has a structure similar to Bap, but is not a carcinogenic compound. In Caco-2 monolayer exposed to 50 μM Bep for 72 h, the ratio of the apparent permeability coefficient ( P app) of Rho-123 efflux increased significantly compared to that of the control monolayer. Similarly, a significant increase in expression of MDR1 mRNA and of P-gp at the protein level were detected by RT-PCR and by Western blot analysis, respectively, in Caco-2 cells exposed to Bep, compared to that of the control. Caco-2 cells exposed to Bep showed oxidative stress that was detected by fluorescence microscopy using aminophenyl fluorescein. However, the oxidative stress was weaker compared with that of Bap. The cellular GSH content was decreased to 80% or 59% of control cells, respectively, in Caco-2 cells exposed to either Bep or Bap. Our results further show that Bep or Bap-induced P-gp in Caco-2 cells might have been the result of oxidative stress rather than DNA damage.

Improved intestinal delivery of salmon calcitonin by Lys 18-amine specific PEGylation: Stability, permeability, pharmacokinetic behavior and in vivo hypocalcemic efficacy

Peptides like salmon calcitonin (sCT) are subjected to aggressive proteolytic attack by various intestinal enzymes, and fractions that enter the systemic circulation via the intestinal route are rapidly inactivated by tissue accumulation and glomerular filtration. Here, we describe the beneficial effects of the Lys 18-amine specific PEGylation of sCT on the intestinal delivery of sCT. Two key properties were enhanced by the PEGylation process: (i) the resistance of sCT to intestinal enzymes and (ii) the systemic clearance of sCT that had entered the circulation. Initially, we evaluated the cAMP-secreting activities of PEG 2K-sCT isomers substituted at Cys 1-, Lys 11- or Lys 18-amine position in T47D cells, and found that sCT PEGylated at Lys 18-amine (Lys 18-PEG 2K-sCT) had the highest bioactivity. We then investigated the stability of Lys 18-PEG 2K-sCT in the presence of intestinal enzymes, its abilities to traverse the intestinal membrane, its pharmacokinetic behavior and in vivo hypocalcemic efficacy. Results show that Lys 18-PEG 2K-sCT has significantly increased resistance to pancreatic peptidases and brush-border peptidases. Despite the molecular size increase caused by PEGylation, Lys 18-PEG 2K-sCT was found to have an intestinal permeability similar to that of unmodified sCT ( p > 0.59) over an apical concentration range 12.5–100 μM in a Caco-2 cell monolayer transport system. In particular, tissue distribution results showed that 125I-labeled Lys 18-PEG 2K-sCT markedly resists liver accumulation and glomerular filtration; levels were reduced by 75% and 50% vs. sCT. Finally, the hypocalcemic efficacy of intestinally administered Lys 18-PEG 2K-sCT, measured as total serum calcium in a rat model, was 5.8 and 3.0 times that of sCT at 100 and 200 IU/kg ( p < 0.025). Our findings suggest that this site-specific conjugation of peptides with PEG of proper size enhances pharmacokinetic properties by increasing their abilities to resist both proteolysis and systemic clearance without significantly reducing their membrane permeabilities or bioactivities. We believe that this concept, namely, dual effects by PEGylation, has great potential value because it presents a practical means of enhancing the efficacies of the peroral/intestinal pharmacologic route.

Effect of benzo[a]pyrene on P-glycoprotein-mediated transport in Caco-2 cell monolayer

The main exposure pathway of benzo[a]pyrene (Bap) for humans is considered to be via the daily diet. The purpose of this study was to investigate the effect of BaP on the intestinal transport of chemicals mediated by P-glycoprotein (P-gp). The intestinal epithelial membrane transport of rhodamine-123 (Rho-123), a substrate of P-gp, was examined using a monolayer of the human Caco-2 cell line grown in transwells. In the monolayer exposed to Bap for 72 h before transport experiments, the ratio of the apparent permeability coefficients ( P app) of Rho-123 efflux increased compared to that of the control. The permeability of rhodamine-B (Rho-B), not a substrate of P-gp, showed no difference between the monolayers. Treatment with quinidine or cyclosporine A, which are P-gp inhibitors, decreased the P app of Rho-123 to the same degree in both monolayers. The transport of Rho-123 was not influenced by the presence of Bap. Thus, Bap seemed not to act directly on the efflux activity of P-gp and be a binding site competitor of Rho-123. In the Caco-2 cells that enhanced the efflux of Rho-123 by the treatment with Bap, an increase in mRNA expression of MDR 1 (P-gp) was confirmed compared to that of control by RT-PCR. Furthermore, Western blot analysis using a monoclonal antibody, C219, demonstrated the increase of P-gp in Caco-2 cells exposed to Bap, compared with controls. It was inferred that Bap exposure induced the expression of P-gp, which led to the observed increase in efflux transport of Rho-123. The possibility was suggested that Bap might affect the disposition of medicines by increasing P-gp expression.

Campylobacter jejuni inhibits the absorptive transport functions of Caco-2 cells and disrupts cellular tight junctions

Caco-2 cells are models of absorptive enterocytes. The net transport of fluid from apical to basolateral surfaces results in 'domes' forming in differentiated monolayers. Here, the effect of Campylobacter jejuni on this process has been examined. C. jejuni caused no changes in short-circuit current upon infection of Caco-2 cell monolayers in Ussing chambers. Thus, no active secretory events could be demonstrated using this model. It was therefore hypothesized that C. jejuni could inhibit the absorptive function of enterocytes and that this may contribute to diarrhoeal disease. C. jejuni infection of fluid-transporting ('doming') Caco-2 cells resulted in a significant reduction in dome number, which correlated with a decrease in tight junction integrity in infected monolayers, when measured as transepithelial electrical resistance. Defined mutants of C. jejuni also reduced dome numbers in infected monolayers. C. jejuni also altered the distribution of the tight junction protein occludin within cell monolayers. The addition to monolayers of extracellular gentamicin prevented these changes, indicating the contribution of extracellular bacteria to this process. Thus, tight junction integrity is required for fluid transport in Caco-2 cell monolayers as leaky tight junctions cannot maintain support of transported fluid at the basolateral surface of infected cell monolayers. Inhibition of absorptive cell function, changes in epithelial resistance and rearrangement of tight junctional proteins such as occludin represent a potential diarrhoeal mechanism of C. jejuni.

Transepithelial Taurine Transport in Caco-2 Cell Monolayers

Here we characterized transepithelial taurine transport in monolayers of cultured human intestinal Caco-2 cells by analyzing kinetic apical and basolateral uptake and efflux parameters. Basolateral uptake was Na(+)- and Cl(-)- dependent and was inhibited by beta-amino acids. Uptake by this membrane showed properties similar to those of the apical TauT system. In both membranes, taurine uptake fitted a model consisting of a non-saturable plus a saturable component, with a higher half-saturation constant and transport capacity at the apical membrane (K(m), 17.1 micromol/L; V(max), 28.4 pmol.cm(-2).5 min(-1)) than in the basolateral domain (K(m), 9.46 micromol/L; V(max), 5.59 pmol.cm(-2).5 min(-1)). The non-saturable influx component, estimated in the absence of Na(+) and Cl(-), showed no significant differences between apical and basolateral membranes (K(D), 89.2 and 114.7 nL.cm(-2) . 5 min(-1), respectively). Taurine efflux from the cells is a diffusive process, as shown in experiments using preloaded cells and in trans-stimulation studies (apical K(D),72.7 and basolateral K(D), 50.1 nL.cm(-2).5 min(-1)). Basolateral efflux rates were significantly lower than passive influx rates. We conclude that basolateral taurine uptake in Caco-2 cells is mediated by a transport mechanism that shares some properties with the apical system TauT. Moreover, calculation of unidirectional and transepithelial taurine fluxes reveals that apical influx of this amino acid is higher than basolateral efflux rates, thereby enabling epithelial cells to accumulate taurine against a concentration gradient.

Structural effects of phenolic acids on the transepithelial transport of fluorescein in caco-2 cell monolayers.

The structural specificity of the monocarboxylic acid transporter (MCT) for the transport of phenolic acids was investigated by measuring the inhibitory effect on the fluorescein transport in Caco-2 cell monolayers. Although most of the monohydroxylated derivatives had an inhibitory effect, the di- and tri-hydroxylated ones did not. The methoxylated derivatives were more inhibitory than the hydroxylated ones in all the meta-substituted derivatives, suggesting that meta-hydroxylation of the substrate would decrease the affinity for MCT.

Carbapenem antibiotics inhibit valproic acid transport in Caco-2 cell monolayers

The concomitant use of carbapenem antibiotics with valproic acid has been prohibited because carbapenems induced a decrease in plasma concentration of valproic acid in epileptic patients during valproic acid therapy. Our previous in vivo study in rats proposed that inhibition by carbapenem of the intestinal absorption of valproic acid might be a possible mechanism for the drug–drug interaction. To demonstrate the hypothesis, we examined the effects of imipenem and panipenem on intestinal transepithelial transport of valproic acid using Caco-2 cell monolayers. Imipenem and panipenem inhibited the transport of [ 14C]–valproic acid across the Caco-2 cell monolayers from apical-to-basolateral side in a concentration-dependent manner, although they had no effect on the uptake of [ 14C]–valproic acid by Caco-2 cells. The inhibition by the carbapenems of the valproic acid transport was found even when they were added to only the basolateral side. From these results, the carbapenems may inhibit the absorption of valproic acid at the basolateral membrane of intestinal epithelial cells, which contributes to the decrease in plasma concentration of valproic acid after oral administration.

Inhibition of the CYP3A4-mediated metabolism and P-glycoprotein-mediated transport of the HIV-1 protease inhibitor saquinavir by grapefruit juice components.

Cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp) are both expressed in the intestinal mucosa and present a barrier to oral drug delivery. CYP3A4 and P-gp share both overlapping tissue distribution and substrate specificity. Grapefruit juice interactions with CYP3A4 substrates are well documented and occur as a consequence of down regulation of intestinal CYP3A4. The aim of the present study was to screen grapefruit juice components against the CYP3A4-mediated metabolism and P-gp mediated transport of the HIV-1 protease inhibitor saquinavir. Five grapefruit juice components: quercetin, naringin, naringenin, 6', 7'-dihydroxybergamottin and bergamottin were screened as potential inhibitors of the metabolism of saquinavir by human liver microsomes. The known CYP3A4 inhibitor ketoconazole was also screened for inhibitory potential. These compounds were also screened as modulators of P-gp activity by assessing the directional transport of saquinavir across Caco-2 cell monolayers which express P-gp. The effect of verapamil, a known modulator of P-gp function, was also determined in these cell lines. On preincubation, 6', 7'-dihydroxybergamottin and bergamottin inhibited the metabolism of saquinavir, with IC50 values of 0.33+/-0.23 muM and 0.74+/-0.13 muM, respectively (n=3). Ketoconazole achieved an IC50 of 0. 55+/-0.12 muM (n=4). The other compounds studied failed to reach IC50 at concentrations of up to 100 muM. The transport of saquinavir in the basolateral-->apical (BL-->AP) direction exceeded that in the apical -->basolateral direction (AP-->BL), with apparent permeability coefficients of 199.2+/-15.8x10-7 cm s-1 and 8.00+/-1. 13x10-7 cm s-1, respectively (n=3) which is indicative of a polarized efflux mechanism. The ratio of BL-->AP/AP-->BL for saquinavir was 25, but in the presence of verapamil and ketoconazole this ratio was reduced to 3.6 and 4.0, respectively (n=3), indicating extensive inhibition of P-gp mediated saquinavir efflux. Of the grapefruit juice components studied only naringin and 6', 7'-dihydroxybergamottin had any appreciable effect, reducing the ratio to 7.6 and 7.1, respectively (n=3); but this was due solely to increased AP-->BL transport. Grapefruit juice components inhibit CYP3A4-mediated saquinavir metabolism and also modulate, to a limited extent, P-gp mediated saquinavir transport in Caco-2 cell monolayers. The in vivo effects of grapefruit juice coadministration are most likely the result of effects on CYP3A4 (inhibition and down regulation) and only to a minor extent on modulation of P-gp function.

Rapid modulation of electrolyte transport in Caco-2 cell monolayers by enteropathogenic Escherichia coli (EPEC) infection

Background and aims—The pathophysiology of enteropathogenic Escherichia coli (EPEC) diarrhoea remains uncertain. EPEC adhere to enterocytes and transduce signals which produce a characteristic “attaching and effacing” (A/E) lesion in the...

Cellular and paracellular Ca ++ transport in CACO-2 CELL monolayers: effect of vitamin D

Cellular and paracellular Ca ++ transport in CACO-2 CELL monolayers: effect of vitamin D