Correlation Between Oral Drug Absorption in Humans,and Apparent Drug Permeability in TC-7 Cells,A Human Epithelial Intestinal Cell Line: Comparison with the Parental Caco-2 Cell Line

Purpose. To determine and compare the relationship between in vivo oral absorption in humans and the apparent permeability coefficients (P _app ) obtained in vitro on two human intestinal epithelial cell lines, the parental Caco-2 and the TC-7 clone. Methods. Both cell lines were grown for 5–35 days on tissue culture-treated inserts. Cell monolayers were analysed for their morphology by transmission electron micrography, and for their integrity with respect to transepithelial electrical resistance, mannitol and PEG-4000 transport, and cyclosporin efflux. P _app were determined for 20 compounds exhibiting large differences in chemical structure, molecular weight, transport mechanisms, and percentage of absorption in humans. Results. The TC-7 clone exhibits morphological characteristics similar to those of the parental Caco-2 cell line, concerning apical brush border, microvilli, tight junctions and polarisation of the cell line. The TC-7 clone however appeared more homogenous in terms of cell size. Both cell lines achieved a similar monolayer integrity towards mannitol and PEG-4000. Monolayer integrity was achieved earlier for the TC-7 clone, mainly due to its shorter doubling time, i.e. 26 versus 30 hours for parental Caco-2 cells. When using cyclosporin A as a P-glycoprotein substrate, active efflux was lower in the TC-7 clone than in the parental Caco-2 cells. The P_app and mechanisms of transport (paracellular or transcellular routes, passive diffusion and active transport) were determined for 20 drugs. A relationship was established between the in vivo oral absorption in humans and P_app values, allowing to determine a threshold value for P_appof 2 10^–6 cm/sec, above for which a 100% oral absorption could be expected in humans. Both correlation curves obtained with the two cell types, were almost completely superimposable. These studies also confirmed that the dipeptide transporter is underexpressed in both cell lines. Conclusions. On the basis of morphological parameters, biochemical activity and drug transport characteristics, the TC-7 clone appeared to be a valuable alternative to the use of parental Caco-2 cells for drug absorption studies.     

Paracellular drug transport across intestinal epithelia: influence of charge and induced water flux.

The influence of drug charge and transepithelial water flux on passive paracellular drug transport was investigated in Caco-2 cell monolayers and rat ileal mucosa in vitro. Three small hydrophilic compounds with different net charges (creatinine, erythritol and foscarnet) were used as model drugs. A hypotonic glucose-rich solution was applied apically to induce epithelial absorption of water. In the Caco-2 monolayers, permeability to creatinine (positively charged) was 25-fold greater than to foscarnet (negatively charged), indicating a pronounced cation selective paracellular permeability. During apical exposure to the hypotonic glucose-rich solution, transport of all model drugs increased in both the absorptive and secretory directions. This enhanced transport coincided with a decrease in transepithelial resistance. Further, fluorescence and transmission electron microscopy indicated dilatations of the paracellular spaces but no damage to the cell membranes. These findings suggested that the enhancement in drug transport was attributable to increased paracellular tight junction permeability rather than to "solvent drag". In the ileal segments, mucosal exposure to the hypotonic glucose-rich solution had no effect on transepithelial resistance and only a marginal increase in drug transport was observed. Taken together, the modest absorption enhancement demonstrated in the in vitro models agrees with results obtained in vivo, supporting the conclusion that a more pronounced disruption of the tight junction barrier than that obtained through stimulation of epithelial absorption of water is required for efficient enhancement of paracellular intestinal drug absorption.     

Evidence for Diminished Functional Expression of Intestinal Transporters in Caco-2 Cell Monolayers at High Passages

Purpose. To investigate the effects of passaging on the intrinsic membrane transport parameters of compounds absorbed by means of passive and carrier-mediated processes in the Caco-2 cell line. Methods. Caco-2 cells at low (28–36) and high (93–108) passage numbers were used to evaluate the transport characteristics of model compounds for paracellular diffusion (mannitol), transcellular diffusion (progesterone) and carrier-mediated transport (cephalexin, cephradine, phenylalanine, proline, and taurocholic acid) using side-by-side diffusion chambers. Intrinsic intestinal transport parameters were determined by correcting the effective permeability for potential biases introduced by the microporous filter and aqueous boundary layer. Intrinsic maximal flux (J _max, Michaelis constant (K _m) and carrier permeability (P _c) were determined as a function of passage number. Results. Compared to the low passaged cells, the high passaged Caco-2 cells were characterized by less morphological heterogeneity, higher transepithelial electrical resistance, higher transcellular diffusion, lower paracellular diffusion, lower carrier-mediated transport and lower alkaline phosphatase activity. The use of effective transport parameters overestimated the K _m and underestimated P _c but had no effect on J _max. Conclusions. The current results provide experimental evidence that the passaging process significantly affects the biological characteristics and transport properties of Caco-2 cell monolayers. The effects are consistent with a reduction in the functional expression of a brush border enzyme and several transport proteins as passage number is increased. The underlying basis for this appears to be a selection of fast-growing subpopulations from the original heterogeneous Caco-2 cell line during passaging.     

Passive Transepithelial Absorption of Thyrotropin-Releasing Hormone (TRH) via a Paracellular Route in Cultured Intestinal and Renal Epithelial Cell Lines

Transport studies using intestinal brush-border membrane vesicles isolated from rats and rabbits have failed to demonstrate proton- or Na⁺-dependent carrier-mediated transport of thyrotropin-releasing hormone (TRH), despite a pharmacologically relevant oral bioavailability. To examine the hypothesis that reported levels of oral bioavailability reflect predominately a paracellular rather than transcellular route for transepithelial transport of TRH, we have studied TRH transport in cultured epithelial cell types of intestinal (Caco-2 and T₈₄) and renal (MDCK I, MDCK II, and LLC-PK₁ origin, whose paracellular pathways span the range of permeability values observed in natural epithelia. Transport of TRH across monolayers of intestinal Caco-2 cells was similar to the flux of mannitol (1–4% per 4 hr), and unlike other putative substrates for the di-/tripeptide carrier, apical-to-basolateral transport was not increased by the presence of an acidic pH in the apical chamber. TRH transport did not show saturation, being uneffected in the presence of 20 mM cold TRH. In each cell type studied TRH and mannitol transport were similar and positively correlated with the conductance of the cell layers, consistent with a passive mechanism of absorption. This evidence suggests that, providing that a peptide is resistant to luminal hydrolysis, small but pharmacologically significant amounts of peptide absorption may be achieved by passive absorption across a paracellular route.     

A Model of Human Small Intestinal Absorptive Cells. 1. Transport Barrier

The Caco-2 cell culture model of human small intestinal absorptive cells was used to investigate transepithelial transport. Transport of permeability markers such as mannitol demonstrated that Caco-2 monolayers became less permeable with increasing age in culture. Cells were routinely used for transport studies between day 18 and day 32. A transport index was determined for each compound by calculating the ratio of transport of the molecules under investigation to transport of an internal standard such as the permeability marker mannitol. Comparison of transport rates at 4 and 37°C was a simple approach for differentiating primary transport mechanisms (passive paracellular, passive transcellular, or transporter-mediated) but must be coupled with additional experimental manipulations for definitive determination of transport pathways. Compounds predicted to undergo predominantly paracellular transport (mannitol, FITC, PEG-900, and PEG-4000), transporter-mediated transcellular transport (glucose, biotin, spermidine, oralanine), or lipophilic transcellular transport (alprenolol, propranolol, clonidine, or diazepam) showed differential effects of temperature on rates of transport as well as the transport index.     

Novel oral absorption system containing polyamines and bile salts enhances drug transport via both transcellular and paracellular pathways across Caco-2 cell monolayers

The combinatorial use of spermine (SPM), a typical polyamine, and sodium taurocholate (STC), a typical bile salt, was found to be a promising safe preparation for improving the oral absorption of poorly water-soluble and/or poorly absorbable drug in our previous studies utilizing rats and dogs. To clarify the mechanisms behind the synergistic enhancement effect of the polyamine and bile salt, the transport of rebamipide, which is classified into Biopharmaceutics Classification System Class IV, was investigated in Caco-2 cell monolayers. The synergistic enhancement of rebamipide transport by SPM and STC was certainly observed in Caco-2 cells as well, while the separate use of either SPM or STC did not significantly improve the transport of rebamipide. The combinatorial use of SPM and STC significantly decreased the transepithelial electrical resistance (TEER) in Caco-2 cell monolayers, suggesting that the opening of paracellular pathway. On the other hand, it was also confirmed that the decrease in TEER was transient and reversible after removal of SPM and STC and that cell viability was maintained. Voltage-clamp study clearly showed that their combinatorial use improved rebamipide transport via both paracellular and transcellular pathways, and that the contribution of transcellular route could be larger than paracellular route.     

Saturable transport of H2-antagonists ranitidine and famotidine across Caco-2 cell monolayers.

The purpose of this study was to investigate the mechanism by which the H2-antagonists ranitidine and famotidine interacted with the paracellular space during their transport across Caco-2 cell monolayers. Transport experiments with ranitidine and famotidine across Caco-2 cell monolayers were performed to determine the apical-to-basolateral flux at various concentrations. Kinetic analysis of the transport data showed that ranitidine and famotidine were transported by both saturable and nonsaturable processes. Na+, K+-ATPase inhibitor ouabain and metabolic inhibitors sodium azide + 2-deoxy-D-glucose did not affect ranitidine transport, suggesting that the active transport was not involved. Famotidine and some other guanidine-containing compounds, e.g., guanethidine, Arg-Gly, L-arginine methyl ester, and L-argininamide, inhibited the transport of ranitidine, whereas other guanidine-containing compounds with an additional negative charge, e.g., L-arginine, did not. 2,4, 6-Triaminopyrimidine (TAP), an inhibitor of paracelluar cationic conductance, also inhibited the transport of both ranitidine and famotidine. On the basis of these results, it is proposed that the saturable transport of ranitidine and famotidine across Caco-2 cell monolayers appears to be via a facilitated diffusion process mediated by the paracellular anionic sites. This mechanism is consistent with the observation that ranitidine and famotidine caused a concentration-dependent increase in transepithelial electrical resistance (TEER) across Caco-2 cell monolayers, presumably by blocking the paracellular anionic sites and thus inhibiting the flux of cations (e.g., Na+).     

Metabolism,Uptake, and Transepithelial Transport of the Diastereomers of Val-Val in the Human Intestinal Cell Line,Caco-2

Purpose. The purpose of this study was to determine whether the binding of the diastereomers of Val-Val to the apical oligopeptide transporter(s) could be correlated with their cellular uptake and transepithelial transport. Methods. The Caco-2 cell culture system was used for all experiments. The binding of the diastereomers of Val-Val was evaluated by determining their ability to inhibit [³H]cephalexin uptake. The stability of the diastereomers was determined in a homogenate of Caco-2 cells and in the apical bathing solution over Caco-2 cell monolayers. The cellular uptake and transepithelial transport properties of the individual diastereomers were studied using Caco-2 cell monolayers. Results. 10 mM concentrations of L-Val-L-Val, L-Val-D-Val, D-Val-L-Val and D-Val-D-Val inhibited cellular uptake of [³H]cephalexin (0.1 mM) by 92%, 37%, 70%, and 18%, respectively. When the cellular uptake of Val-Val diastereomers (1 mM) were evaluated, the intracellular concentrations of L-Val-D-Val and D-Val-L-Val were 15 and 50 times higher, respectively, than that of D-Val-D-Val. The cellular uptake of L-Val-D-Val and D-Val-L-Val was inhibited by Gly-Pro (10 mM) (>95%), whereas Gly-Pro had no effect on the cellular uptake of D-Val-D-Val. L-Val-L-Val was not detected in the Caco-2 cells, probably due to its metabolic lability. When the transepithelial transport of the Val-Val diastereomers (1 mM) was determined, L-Val-D-Val, D-Val-L-Val and D-Val-D-Val transport rates were similar. The transepithelial transport of L-Val-D-Val and D-Val-L-Val was inhibited by Gly-Pro (10 mM) 36% and 30%, respectively, while Gly-Pro inhibited carnosine (1 mM) transepithelial transport by 65%. Gly-Pro had no effect on the transepithelial transport of D-Val-D-Val. Conclusions. These results suggest that the major transepithelial transport route of L-Val-D-Val, D-Val-L-Val and D-Val-D-Val is passive diffusion via the paracellular route. The binding of Val-Val diastereomers to the oligopeptide transporter(s) is a good predictor of their cellular uptake, however, the binding is not a good predictor of their transepithelial transport. It appears that the stereochemical requirements for the transporter that mediates efflux of the peptide across the basolateral membrane may be different from the requirements for the apical transporter that mediates cellular uptake.     

Copper treatment alters the barrier functions of human intestinal Caco-2 cells: involving tight junctions and P-glycoprotein

This study investigated the effects of copper on paracellular permeability and P-glycoprotein (P-gp) in Caco-2 cells. Apical treatment with 100-300 microM CuSO4 in Hanks' balanced salt solution (HBSS, up to 3 hours) induced a time- and concentration-dependent increase in permeability of Caco-2 cell monolayers monitored by transepithelial electrical resistance (TEER). Copper treatment also induced a concentration-dependent reduction of F-actin stain, but not of tight junctional protein ZO-1. In addition, without any adverse effects on TEER, apical treatment with 300 microM CuSO4 in complete medium (for 24 hours) could reduce basolateral-to-apical transport, and increase apical-to-basolateral transport of rhodamine-123 (Rho-123) and accumulation of Rho-123 in Caco-2 cells. Treatment with 10-100 microM CuSO4 in HBSS (up to 3 hours) also induced a time- and concentration-dependent increase in accumulation of Rho-123 in Caco-2 cells. The results indicated that copper treatment increased the paracellular permeability probably by perturbing F-actin skeleton, and inhibited P-gp, thus altering the barrier functions of Caco-2 cells.     

Enhancement of paracellular transport of heparin disaccharide across Caco-2 cell monolayers

The enhancement of paracellular transport of heparin disaccharide using several absorption enhancers across Caco-2 cell monolayers was tested. The cytotoxicity of these enhancers was also examined. The enhancing effects by Quillaja saponin, dipotassium glycyrrhizinate, 18beta-glycyrrhetinic acid, sodium caprate and taurine were determined by changes in transepithelial electrical resistance (TEER) and the amount of heparin disaccharide transported across Caco-2 cell monolayers. Among the absorption enhancers, 18beta-glycyrrhetinic acid and taurine decreased TEER and increased the permeability of heparin disaccharide in a dose-dependent and time-dependent manner with little or negligible cytotoxicity. Our results indicate that these absorption enhancers can widen the tight junction, which is a dominant paracellular absorption route of hydrophilic compounds. It is highly possible that these absorption enhancers can be applied as pharmaceutical excipients to improve the transport of macromolecules and hydrophilic drugs having difficulty in permeability across the intestinal epithelium.     

Influence of t-butylhydroquinone and beta-naphthoflavone on formation and transport of 4-methylumbelliferone glucuronide in Caco-2/TC-7 cell monolayers

Human Caco-2 cells have been established as a model system for intestinal biotransformation and permeability. When grown on Transwell polycarbonate filters they develop morphologic and biochemical characteristics of enterocytes with well separated apical and basolateral surfaces. In addition, Caco-2/TC-7 cells have proven to be useful to study regulation of human UDP-glucuronosyltransferases (UGTs) by Ah receptor agonists and antioxidant-type inducers such as beta-naphthoflavone (BNF) and t-butylhydroquinone (TBHQ). In the present investigation, formation and transport of 4-methylumbelliferone glucuronide was studied in intact Caco-2 cell monolayers. The following results were obtained: when loaded with 50-200 microM MUF either apically or basolaterally, MUF-GA was the major metabolite which was mostly released (80%) at the basolateral surface, probably via the multidrug resistance protein isoform MRP3; MUF sulfate formation was low (5 +/-2%). Pretreatment of cells with 80 microM TBHQ or 50 microM BNF for 72 hr before addition of 100 microM MUF enhanced basolateral secretion of MUF-GA 1.4- and 1.7-fold, respectively. However, at >200 microM MUF, MUF-GA secretion and induction was smaller, probably due to inhibition of intracellular UGT activity. MRP3 protein was localized to the basolateral surface of Caco-2 cells but was not induced by TBHQ or BNF. The results suggest that MUF-GA is mostly secreted basolaterally in Caco-2 cell monolayers. Treatment with TBHQ or BNF significantly enhanced MUF-GA formation in the intact cell.     

Transepithelial Transport Properties of Peptidomimetic Thrombin Inhibitors in Monolayers of a Human Intestinal Cell Line (Caco-2) and Their Correlation to in Vivo Data

Peptidomimetic thrombin inhibitors (TI), derived from L-Asp-D-Phe were examined in confluent monolayers of a human colon carcinoma cell line (Caco-2) to elucidate their transepithelial transport properties. Effect availabilities, based on activated partial thromboplastin time (aPTT) measurements in rats, after peroral administration of five TI correlated reasonably well with permeability coefficients obtained from in vitro transport studies in Caco-2 monolayers, whereas physicochemical properties, such as molecular mass, solubilities, pK_a and octanol-buffer partition coefficients failed to yield meaningful relationships. Substitution of the -carboxylic group of L-Asp leads to analogues which are mainly transported by passive diffusion, while an unsubstituted carboxylic group favours carrier-mediated active transport. The effects of concentration, temperature, competitive inhibitors and direction dependence on in vitro transport were investigated. The results obtained are compatible with a saturable carrier-mediated transport, operating parallel to a passive paracellular route. The Michaelis-Menten parameters for the active transport component (K_m = 1.67 mM, V_max = 26.5 pmol min^–1 mg protein^–1) indicate an involvement of the intestinal di/-tripeptide transport system for one of the TI. The Caco-2 transport model may be helpful for the design of perorally active peptidomimetics.     

Metabolism,Uptake, and Transepithelial Transport of the Stereoisomers of Val-Val-Val in the Human Intestinal Cell Line,Caco-2

Purpose. The purpose of this study was to determine the stereospecificity of the apical oligopeptide transporter(s) for the stereoisomers of Val-Val-Val and to determine whether the interaction of these molecules with this transporter(s) could be correlated with their cellular uptake and/or transepithelial transport. Methods. The interactions of these stereoisomers with this transporter(s) were evaluated by determining their ability to inhibit [³H]cephalexin uptake into Caco-2 cells. The metabolism of these stereoisomers was determined in a homogenate of Caco-2 cells and in the apical bathing solution over Caco-2 cell monolayers. The cellular uptake and transepithelial transport properties of these stereoisomers were studied using the Caco-2 cell monolayers. Results. The L-L-L tripeptide was totally degraded within 1 h in the Caco-2 cell homogenate and within 2 h when applied to the apical side of a Caco-2 cell monolayer. In contrast, 36.7 ± 1.3% and 69.7 ± 0.9% of L-Val-L-Val-D-Val remained after 2 h in the cell homogenate and in the apical bathing solution, respectively. The other six stereoisomers of Val-Val-Val were completely stable in the Caco-2 cell homogenate. Five of the stereoisomers (L-L-L, L-L-D, L-D-L, D-L-L, D-D-L) significantly inhibited the cellular uptake of [³H]cephalexin (91%, 62%, 14%, 45%, 16%, respectively). The other stereoisomers had no effect on the [³H]cephalexin uptake. When the cellular uptake of the stereoisomers was determined, the D-L-L and L-D-L tripeptides showed the highest intracellular concentrations (1.32 ± 0.25 and 0.62 ± 0.20 nmol/mg protein after a 2-h incubation, respectively). In contrast, the intracellular concentrations of the other stereoisomers were less than 0.1 nmol/mg protein. Moreover, the cellular uptake of the D-L-L and L-D-L tripeptides was inhibited by Gly-Pro by 82% and 68%, respectively, whereas Gly-Pro showed moderate to no inhibitory effect on the cellular uptake of the other stereoisomers. The permeability coefficients of the stereoisomers across the Caco-2 cell monolayers were very low (1.8 to 3.1 × 10^–7 cm/sec) and almost identical. Gly-Pro had no effect on their transepithelial transport. Conclusions. These results suggest that the interaction of the Val-Val-Val stereoisomers with the apical oligopeptide transporter(s) could be a good predictor of their cellular uptake. However, since the major transepithelial transport mechanism of Val-Val-Val stereoisomers is passive diffusion via the paracellular route, the binding of these molecules to the oligopeptide transporter(s) is not a good predictor of their transepithelial transport. It appears that the stereochemical requirements for the transporter that mediates permeation of the peptide across the basolateral membrane may be different from the requirements for the apical transporter that mediates cellular uptake.     

Transepithelial Transport and Metabolism of Thyrotropin-Releasing Hormone (TRH) in Monolayers of a Human Intestinal Cell Line (Caco-2): Evidence for an Active Transport Component?

Cell culture models for gastrointestinal transport and metabolism are important mechanistic tools. Our studies of Caco-2 monolayers demonstrate heterogeneity in transport characteristics depending on passage number and origin of the cells. In accordance with data obtained in animals and humans, TRH shows a carrier-mediated, saturable transport component, which operates parallel to a passive pathway in Caco-2 cells at passage number 89-99. At low TRH concentrations (<3 mM) active transport becomes prominent, as demonstrated by the temperature dependence of TRH transport and inhibition experiments. The Michaelis-Menten parameters of the active, saturable transport component are: K_m = 1.59 mM and V_max = 1.84 µM/min. The pH optimum was determined to be at pH 6.0. On the other hand an exclusively paracellular passive route was found with Caco-2 cells at passage number 30-34. These results are also in agreement with observations made by others in cell culture experiments. The aspect of rigorously characterizing the specific Caco-2 clone under investigation is emphasized, especially when active transport mechanisms are suspected.     

Utilization of a Human Intestinal Epithelial Cell Culture System (Caco-2) for Evaluating Cytoprotective Agents

Human intestinal epithelial cells (Caco-2) were cultured as confluent monolayers on polycarbonate membranes in Transwells for investigating their applicability in evaluating the cytoprotective activity of sucralfate. The control experiments established a reproducible chemical method (using 0.5 mM indomethacin in Hanks' balanced salt solution) for inducing damage to the Caco-2 cell monolayers. Damage was determined by measuring changes in transepithelial electrical resistance (TEER). Twenty-day-old Caco-2 cell monolayers were significantly and reproducibly damaged (compared to buffer alone) (P < 0.001) by application of 0.5 mM indomethacin to the apical side for 1 hr. While sucralfate, at a 0.5, 2, or 5 mg/mL concentration in the buffer, was shown not to reverse (treat) the damage caused by indomethacin in this cellular model, it was able to protect (prevent) the cells from indomethacin-induced damage (P < 0.001). We observed that indomethacin-induced damage to the Caco-2 cell monolayers greatly affected the paracellular pathway since the percentage transport of [³H]methoxyinulin was significantly elevated. In contrast, protection of the Caco-2 cells with 5 mg/mL sucralfate in the presence of the damaging agent resulted in transport of the paracellular marker similar to that in the control (HBSS-treated) cell monolayers. This direct cytoprotective effect was thus independent of vascular factors at neutral pH and was observed to be dose dependent (0.5 to 5 mg/mL) when sucralfate was applied to the cells in the presence of the damaging agent. These findings, which are consistent with those observed for sucralfate in vivo (Okabe et aL, Digest. Dis. Sci. 28:1034-1042, 1983), demonstrate the feasibility of using Caco-2 cell monolayers as an in vitro cell culture system which may serve to identify and rapidly screen the cytoprotective activity of potential drugs and their pharmaceutical formulations.     

Parallel artificial membrane permeability assay (PAMPA) combined with a 10-day multiscreen Caco-2 cell culture as a tool for assessing new drug candidates

The parallel artificial membrane permeability assay (PAMPA) is extensively used for the evaluation of early drug candidates. It is high throughput, low cost and is amenable to automation. This method has been shown useful in assessing transmembrane, non-energy dependent, diffusion of drugs such that reasonable predictability with in vivo (passive) absorption is possible. Cell cultures mimicking the gastrointestinal tract such as the CACO-2 cultures have the advantage of taking into account other transport mechanism including paracellular and carrier-mediated uptake but are lower throughput and labor-intensive. In this study, the applicability of two high throughput permeability assays namely PAMPA (PSR4p, pION Inc.) and 96-well Caco-2 cell assay (MultiScreen, Millipore) were used to rank drug permeability as well as to predict passive and active drug absorption/secretion for a series of marketed drugs as well as a collection of structurally diverse drug candidates. CACO-2 cells were cultured using MultiScreen hardware over a period of 10 days with the integrity of the cells assessed using transepithelial electrical resistance (TEER) and by the ability of the monolayer to the transport a paracellular marker, sodium fluorescence. Effective permeability (Peff) data were calculated using spectrophotometric data and were binned based on a pre-defined cut-off values as either highly and poorly permeable. A comparison of a well characterized drug training set indicate at least 85% concordance between the data generated from PAMPA and Caco-2 MultiScreen. The values obtained using the MultiScreen approach were also similar to data obtained from the literature using the conventional 21-day Caco-2 cell assay. Differences between PAMPA and CACO-2 ranking were useful indicators of either drug efflux (PAMPA (Peff) > CACO-2 (Peff)) or absorptive transport (CACO-2 (Peff) > PAMPA (Peff)). These results indicate that PAMPA combined with the MultiScreen Caco-2 cell culture may be a useful high throughput screening for predicting passive diffusion and active transport of new drugs.     

Usefulness of a novel Caco-2 cell perfusion system. I. In vitro prediction of the absorption potential of passively diffused compounds

A simple, reliable, and user friendly system was established to cultivate Caco-2 cell monolayer for epithelial transport studies. After an initial growth period of 1 week in a CO(2) incubator, Caco-2 cells were cultivated in an automated continuous perfusion system (Minucells and Minutissue, Germany). Medium was constantly renewed at the apical and basal side of the monolayers, which resulted in a continuous supply of nutrients as well as in a continuous removal of metabolite wastes. The monolayers obtained with the new perfusion culture system were evaluated to estimate the passive transport properties of a series of model compounds. The results produced were compared to those of monolayers obtained with the standard 21-day system. The integrity of cell monolayers was checked by measuring transepithelial electrical resistance (TEER) and by the transport of the paracellular leakage marker sodium fluorescein. The results of confocal microscopy as well as TEER measurements indicated the formation of a monolayer on various support filters. The growth and differentiation of Caco-2 cells were highly dependent upon the individual support filters and extracellular matrix proteins used for Caco-2 attachment. The permeability coefficients of several model compounds across Caco-2 cells obtained with the perfusion system were approximately two-fold higher than those obtained using the traditional 21-day Snapwell-based cultures. A good correlation was found between the transport of passively diffused drugs across Caco-2 monolayers differentiated in the perfusion system and the transport according to the standard method. The rank ordering of high permeable model compounds tested through Caco-2 monolayers, differentiated in a perfusion system, was similar to the standard 21-day culture method.     

Intestinal Absorption of Miltefosine: Contribution of Passive Paracellular Transport

Purpose This study aimed to characterize the transepithelial transport of miltefosine (HePC), the first orally effective drug against visceral leishmaniasis, across the intestinal barrier to further understand its oral absorption mechanism. Materials and Methods Caco-2 cell monolayers were used as an in vitro model of the human intestinal barrier. The roles of active and passive mechanisms in HePC intestinal transport were investigated and the relative contributions of the transcellular and paracellular routes were estimated. Results HePC transport was observed to be pH-independent, partially temperature-dependent, linear as a function of time and non-saturable as a function of concentration. The magnitude of HePC transport was quite similar to that of the paracellular marker mannitol, and EDTA treatment led to an increase in HePC transport. Furthermore, HePC transport was found to be similar in the apical-to-basolateral and basolateral-to-apical directions, strongly suggesting that HePC exhibits non-polarized transport and that no MDR-mediated efflux was involved. Conclusions These results demonstrate that HePC crosses the intestinal epithelium by a non-specific passive pathway and provide evidence supporting a concentration-dependent paracellular transport mechanism, although some transcellular diffusion cannot be ruled out. Considering that HePC opens epithelial tight junctions, this study shows that HePC may promote its own permeation across the intestinal barrier.     

Transport of Proteolytic Enzymes Across Caco-2 Cell Monolayers

Purpose. To investigate the mechanisms by which proteolytic enzymes, such as trypsin, chymotrypsin, papain, and bromelain, are able to cross the intestinal mucosal barrier after oral administration to man. Methods. Filter-grown Caco-2 cell monolayers were incubated with proteolytic enzymes and then the transepithelial electrical resistance (TEER) and the transport of the paracellular marker fluorescein were monitored. The effects of the enzymes on the cells were investigated by light microscopy and by biochemical assays. Transport of intact proteases across the cells was verified by monitoring the proteolytic activity and MALDI-TOF mass spectroscopic identification of undegraded trypsin. Results. Depending on time, concentration, and side of exposure to Caco-2 cell monolayers, all proteases decreased the TEER and increased the transport of fluorescein. Some morphological and metabolic changes were observed. The effects were reversible, but until 24 hours after removal of the proteases. Under the conditions of this in-vitro model, approximately 10% of the apically applied dose reached the basolateral compartment as biologically active, non-degraded molecules. Conclusions. Proteolytic enzymes were found to exert considerable effects on the barrier function of Caco-2 monolayers, facilitating the transport of normally non-absorbable compounds. This suggests the also reported, but so far unexplained, systemic absorption of proteolytic enzymes after oral administration in vivo may occur by self-enhanced paracellular transport.