Characterization of Caco-2 and HT29-MTX cocultures in an in vitro digestion/cell culture model used to predict iron bioavailability

Cocultures of two human cell lines, Caco-2 and HT29-MTX mucus-producing cells, have been incorporated into an in vitro digestion/cell culture model used to predict iron bioavailability. A range of different foods were subjected to in vitro digestion, and iron bioavailability from digests was assessed with Caco-2, Caco-2 overlaid with porcine mucin, HT29-MTX or cocultures of Caco-2 and HT29-MTX at varying ratios. It was found that increasing the ratio of HT29-MTX cells decreased the amount of ferritin formed and resulted in an overall decline in the ability of the model to detect differences in iron bioavailability. At the physiologically relevant ratios of 90% Caco-2/10% HT29-MTX and 75% Caco-2/25% HT29-MTX, however, a mucus layer completely covered the cell monolayer and the in vitro digestion model was nearly as responsive to changes in sample iron bioavailability as pure Caco-2 cultures. The in vitro digestion/Caco-2 cell culture model correlates well with human iron bioavailability studies, but, as mucus appears to play a role in iron absorption, the addition of a physiologically realistic mucus layer and goblet-type cells to this model may give more accurate iron bioavailability predictions.     

Uptake of phenolic compounds from plant foods in human intestinal Caco-2 cells

In continuation of our studies on the bioaccessibility of phenolic compounds from food grains as influenced by domestic processing, we examined the uptake of phenolics from native/sprouted finger millet (Eleucine coracana) and green gram (Vigna radiata) and native/heat-processed onion (Allium cepa) in human Caco-2 cells. Absorption of pure phenolic compounds, as well as the uptake of phenolic compounds from finger millet, green gram, and onion, was investigated in Caco-2 monolayer model. Transport of individual phenolic compounds from apical compartment to the basolateral compartment across Caco-2 monolayer was also investigated. Sprouting enhanced the uptake of syringic acid from both these grains. Open-pan boiling reduced the uptake of quercetin from the onion. Among pure phenolic compounds, syringic acid was maximally absorbed, while the flavonoid isovitexin was least absorbed. Apparent permeability coefficient P_(app) of phenolic compounds from their standard solutions was 2.02 × 10^?6 cm/s to 8.94 × 10^?6 cm/s. Sprouting of grains enhanced the uptake of syringic acid by the Caco-2 cells. Open-pan boiling drastically reduced the uptake of quercetin from the onion. The permeability of phenolic acids across Caco-2 monolayer was higher than those of flavonoids.     

Correlation of in vitro and in vivo models for the oral absorption of peptide drugs

The aim of this study was to evaluate two in vitro models, Caco-2 monolayer and rat intestinal mucosa, regarding their linear correlation with in vivo bioavailability data of therapeutic peptide drugs after oral administration in rat and human. Furthermore the impact of molecular mass (Mm) of the according peptides on their permeability was evaluated. Transport experiments with commercially available water soluble peptide drugs were conducted using Caco-2 cell monolayer grown on transwell filter membranes and with freshly excised rat intestinal mucosa mounted in Using type chambers. Apparent permeability coefficients (P (app)) were calculated and compared with in vivo data derived from the literature. It was shown that, besides a few exceptions, the Mm of peptides linearly correlates with permeability across rat intestinal mucosa (R (2) = 0.86; y = -196.22x + 1354.24), with rat oral bioavailability (R (2) = 0.64; y = -401.90x + 1268.86) as well as with human oral bioavailability (R (2) = 0.91; y = -359.43x + 1103.83). Furthermore it was shown that P (app) values of investigated hydrophilic peptides across Caco-2 monolayer displayed lower permeability than across rat intestinal mucosa. A correlation between P (app) values across rat intestinal mucosa and in vivo oral bioavailability in human (R (2) = 0.98; y = 2.11x + 0.34) attests the rat in vitro model to be a very useful prediction model for human oral bioavailability of hydrophilic peptide drugs. Presented correlations encourage the use of the rat in vitro model for the prediction of human oral bioavailabilities of hydrophilic peptide drugs.     

Concurrent determination of topotecan and model permeability markers (atenolol, antipyrine, propranolol and furosemide) by reversed phase liquid chromatography: utility in Caco-2 intestinal absorption studies

A simple, sensitive, specific and high-resolution reversed-phase liquid chromatographic method utilizing ultraviolet detection has been developed and validated for simultaneous determination of topotecan and four intestinal permeability markers (atenolol, antipyrine, propranolol and furosemide) as suggested by US-FDA. Chromatography was carried out on C-18 column with mobile phase comprising water (pH 3.0) and acetonitrile gradient pumped at a flow rate of 1 ml min(-1). The validation parameters included specificity, accuracy, precision, sensitivity and stability studies. Topotecan, an anti-cancer drug widely used in metastatic carcinoma, is a P-glycoprotein substrate having oral bioavailability of 30% with large inter-patient variability. The present method was successfully applied for demonstrating P-gp mediated transport of topotecan and its inhibition using verapamil in Caco-2 cell monolayer. The method can be used in identification of novel P-gp inhibitors for topotecan and estimating the contribution of P-gp in affecting oral bioavailability of topotecan. The other applications of method include its use in validation of Caco-2 monolayer assay for getting biowaiver based on Biopharmaceutic Classification System and its extrapolation to in situ and/or in vivo studies.     

Proposing a Caco-2/HepG2 cell model for in vitro iron absorption studies

The Caco-2 cell line is well established as an in vitro model for iron absorption. However, the model does not reflect the regulation of iron absorption by hepcidin produced in the liver. We aimed to develop the Caco-2 model by introducing human liver cells (HepG2) to Caco-2 cells. The Caco-2 and HepG2 epithelia were separated by a liquid compartment, which allowed for epithelial interaction. Ferritin levels in cocultured Caco-2 controls were 21.7±10.3 ng/mg protein compared to 7.7±5.8 ng/mg protein in monocultured Caco-2 cells. The iron transport across Caco-2 layers was increased when liver cells were present (8.1%±1.5% compared to 3.5%±2.5% at 120 μM Fe). Caco-2 cells were exposed to 0, 80 and 120 μM Fe and responded with increased hepcidin production at 120 μM Fe (3.6±0.3 ng/ml compared to 2.7±0.3 ng/ml). The expression of iron exporter ferroportin in Caco-2 cells was decreased at the hepcidin concentration of 3.6 ng/ml and undetectable at external addition of hepcidin (10 ng/ml). The apical transporter DMT1 was also undetectable at 10 ng/ml but was unchanged at the lower concentrations. In addition, we observed that sourdough bread, in comparison to heat-treated bread, increased the bioavailability of iron despite similar iron content (53% increase in ferritin formation, 97% increase in hepcidin release). This effect was not observed in monocultured Caco-2 cells. The Caco-2/HepG2 model provides an alternative approach to in vitro iron absorption studies in which the hepatic regulation of iron transport must be considered.     

Low-molecular-weight hyaluronan permeates through human intestinal Caco-2 cell monolayers via the paracellular pathway

The intestinal permeability of low-molecular-weight hyaluronan (LMW-HA) was investigated by using cultured monolayers of Caco-2 cells. The amount of LMW-HA that permeated the Caco-2 monolayers was measured by a carbazole assay. The permeability of LMW-HA increased inversely with the molecular size and was dose-dependent. The transport was observed to be energy-independent, and was correlated with the tight junction (TJ) permeability. These results suggest that LMW-HA permeated the Caco-2 cell monolayers via the paracellular pathway.     

An exploratory study of two Caco-2 cell models for oral absorption: a report on their within-laboratory and between-laboratory variability, and their predictive capacity

In 2005, the European Centre for the Validation of Alternative Methods (ECVAM) sponsored a study aimed at evaluating the reproducibility (between-laboratory and within-laboratory variability) and the predictive capacity of two in vitro cellular systems--the Caco-2/ATCC parental cell line and the Caco- 2/TC7 clone--for estimating the oral fraction absorbed (Fa) in humans. Two laboratories, both of which had experience with Caco-2 cultures, participated in the study. Ten test chemicals with documented in vivo oral absorption data were selected. Atenolol, cimetidine and propranolol were included as reference compounds for low, medium and high intestinal absorption, respectively. Transport experiments were independently carried out in the two laboratories, according to an agreed protocol. The apparent permeability coefficient (Papp) was calculated in either the apical to basolateral (absorption) or the basolateral to apical (efflux) direction. To investigate the involvement of possible active transport processes, experiments were also performed in the presence of sodium azide plus 2-deoxy-D-glucose in the donor compartment. Before performing the permeability experiments, the highest concentration that did not impair barrier integrity was identified for each test chemical in both cell models, by applying the chemicals together with a marker of the paracellular pathway. In addition, barrier integrity was assessed by measuring the trans-epithelial electrical resistance. All the permeability data obtained were independently analysed. Reproducibility was assessed for the seven substances for which sufficient data were available. Within-laboratory variability was based on coefficient of variation (CV) values. Median CV values of 10.4% and 14.7% were found for the two laboratories. Concerning between-laboratory reproducibility, comparable response levels were obtained for the three reference compounds and for paracetamol, while, for the other chemicals, the results were less reproducible--in particular, for compounds known to be actively transported. The Papp values obtained for both cell lines were comparable for identical experimental conditions. Despite the limited number of substances tested, the predictive capacity was investigated by using two mathematical models available in the literature. Good estimations of the human Fa were obtained for five well-absorbed compounds, while moderately and poorly absorbed compounds were overestimated. It is proposed that a confirmatory study addressing the main results, including power considerations, would now be useful.     

Role of hepatocyte nuclear factor 1alpha and 1beta in the transcriptional regulation of human dipeptidyl peptidase IV during differentiation of Caco-2 cells

Caco-2 cells undergo differentiation to an enterocytic-like cell when maintained in a post-confluent state for 1-2 weeks. During this period Caco-2 cells begin to express high levels brush border membrane associated enzymes such as dipeptidyl peptidase IV. Using the dipeptidyl peptidase IV gene promoter in electrophoretic mobility shift assays, we have shown for the first time that levels of hepatocyte nuclear factor 1alpha increase three- to fourfold during Caco-2 cell differentiation. Transient cotransfection experiments with 3T3 cells using dipeptidyl peptidase IV promoter constructs and expression vectors containing hepatocyte nuclear factor 1alpha and beta show that the ratio of alpha and beta modulates reporter gene expression. These results suggest that the increase in levels of hepatocyte nuclear factor 1alpha that occur during intestinal cell differentiation, are important for expression of dipeptidyl peptidase IV and other intestinal proteins.     

Development,Characterization and Permeability Assessment Based on Caco-2 Monolayers of Self-Microemulsifying Floating Tablets of Tetrahydrocurcumin

Novel self-microemulsifying floating tablets were developed to enhance the dissolution and oral absorption of the poorly water-soluble tetrahydrocurcumin (THC). Their physicochemical properties and THC permeability across Caco-2 cell monolayers were assessed. The self-microemulsifying liquid containing THC was adsorbed onto colloidal silicon dioxide, mixed with HPMC, gas-generating agents (sodium bicarbonate and tartaric acid), lactose and silicified-microcrystalline cellulose and transformed into tablets by direct compression. The use of different types/concentrations of HPMC and sodium bicarbonate in tablet formulations had different effects on the floating characteristics and in vitro THC release. The optimum tablet formulation (F2) provided a short floating lag time (∼23 s) together with a prolonged buoyancy (>12 h). About 72% of THC was released in 12 h with an emulsion droplet size in aqueous media of 33.9 ± 1.0 nm while that of a self-microemulsifying liquid was 29.9 ± 0.3 nm. The tablet formulation was stable under intermediate and accelerated storage conditions for up to 6 months. The THC released from the self-microemulsifying liquid and tablet formulations provided an approximately three- to fivefold greater permeability across the Caco-2 cell monolayers than the unformulated THC and indicated an enhanced absorption of THC by the formulations. The self-microemulsifying floating tablet could provide a dosage form with the potential to improve the oral bioavailability of THC and other hydrophobic compounds.KEY WORDS: Caco-2 cells, controlled release, permeability, self-microemulsifying floating tablets, tetrahydrocurcumin     

Caco-2 cell permeability and stability of two d-glucopyranuronamide conjugates of thyrotropin-releasing hormone

Caco-2 cell permeability and stability assays were used as an in vitro model to study the intestinal epithelial transport and stability of two analogues of thyrotropin-releasing hormone (TRH; Pyr-His-Pro-NH2). Peptide 1 (Pyr-His-Pro-D-glucopyranuronamide) was more permeable across the Caco-2 cell monolayer compared with the permeability of the parent TRH peptide (Papp=5.10+/-1.89x10(-6) cm/s c.f. Papp=0.147+/-0.0474x10(-6) cm/s respectively). The permeability of peptide 1 was improved threefold by attaching a 2-aminooctanoic acid moiety to the N-terminus to form peptide 2 (2-aminooctanoic acid-Gln-His-Pro-D-glucopyranuronamide) (Papp=16.3+/-2.47x10(-6) cm/s). The half-life for both peptide 1 and peptide 2 was approximately 20 min in a homogenate of Caco-2 cells compared with the half-life of TRH which is approximately 3 min. It was concluded that the permeability of peptides 1 and 2 was enhanced because of their increased stability, while the higher permeability of peptide 2 compared with peptide 1 may be attributed to its increased lipophilicity which results in enhanced passive diffusion.     

Relationship between structure and permeability of tryptophan derivatives across human intestinal epithelial (Caco-2) cells

L-Trp and its derivatives were used as model compounds to clarify structural factors which influence the intestinal epithelial permeation and metabolism of amino-acid derivatives. Permeability of model compounds through Caco-2 cells was used as an in vitro absorption model for human intestinal epithelial cells. The influence of compound concentration, the effects of various transporter substrates on permeability coefficients, and pH dependency of permeability coefficients were investigated. The transcellular permeability of Trp and Trp-NH2 in the direction from the apical side to the basolateral side, in which nutrients and drugs were ordinarily absorbed, declined with increasing concentration and saturated at more than 1 and 0.4 mM, respectively. The permeability coefficients for N-terminal protected Trp derivatives and Ac-Trp-NH2 showed similar and constant values in both from the apical-to-basolateral and basolateral-to-apical directions. In addition, significant inhibition of the apical-to-basolateral permeation of Trp by Leu and Phe was observed. The permeability coefficient ratio at pH 6.3 to that at pH 7.3 was explained by the ratio of the ionic form to the neutral form of the compounds. Based upon these results and the partition coefficients in the 1-octanol/water system, possible absorption mechanism of Trp and its derivatives across Caco-2 cells was proposed.     

Permeation of Insulin,Calcitonin and Exenatide across Caco-2 Monolayers: Measurement Using a Rapid, 3-Day System

Objectives

Caco-2 monolayers are one of the most widely used in vitro models for prediction of intestinal permeability of therapeutic molecules. However, the conventional Caco-2 monolayer model has several drawbacks including labor-intensive culture process, unphysiological growth conditions, lack of reproducibility and limited throughput. Here, we report on the use of 3-day Caco-2 monolayers for assessing permeability of polypeptide drugs.

Methods

The 3-day monolayers were grown in a commercially available transwell set-up, which facilitates rapid development of the Caco-2 monolayers in an intestinal epithelial differentiation mimicking environment. This set-up included use of serum-free medium of defined composition with supplements such as butyric acid, hormones, growth factors, and other metabolites, reported to regulate the differentiation of intestinal epithelial cells in vivo. We measured permeability of 3 different therapeutic polypeptides; insulin, calcitonin, and exenatide across the monolayer.

Results

Preliminary validation of the monolayer was carried out by confirming dose-dependent permeation of FITC-insulin and sulforhodamine-B. Transport of insulin, calcitonin, and exenatide measured at different loading concentrations suggests that the permeability values obtained with 3-day cultures resemble more closely the values obtained with ex vivo models compared to permeability values obtained with conventional 21-day cultures.

Conclusions

Short-term 3-day Caco-2 monolayers provide new opportunities for developing reproducible and high-throughput models for screening of therapeutic macromolecules for oral absorption.     

Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers

Permeability coefficients across monolayers of the human colon carcinoma cell line Caco-2, cultured on permeable supports, are commonly used to predict the absorption of orally administered drugs and other xenobiotics. This protocol describes our method for the cultivation, characterization and determination of permeability coefficients of xenobiotics (which are, typically, drug-like compounds) in the Caco-2 model. A few modifications that have been introduced over the years are incorporated in the protocol. The method can be used to trace the permeability of a test compound in two directions, from the apical to the basolateral side or vice versa, and both passive and active transport processes can be studied. The permeability assay can be completed within one working day, provided that the Caco-2 monolayers have been cultured and differentiated on the permeable supports 3 weeks in advance.     

Different responses of Fe transporters in Caco-2/HT29-MTX cocultures than in independent Caco-2 cell cultures

The human intestinal epithelium is composed of several cell types, mainly enterocytes and goblet (mucin-secreting) cells. This study compares the cellular response of Fe transporters in Caco-2, HT29-MTX, and Caco-2/HT29-MTX co-culture models for Fe bioavailability. Caco-2 cells in vitro differentiate into enterocyte-like cells and HT29-MTX cell lineage into a mucin-secreting cellular population. Cell cultures were exposed to digests of Fe⁺³, Fe⁺³/ascorbic acid, cooked fish (high-available Fe) or white beans (low-available Fe). Cell responses as shown by mRNA expression of the main Fe transporters, DMT1 and DcytB, and cell ferritin formation were monitored. In Caco-2/HT29-MTX co-cultures, the mucin layer lowered the pool of free Fe to diffuse towards the cell brush border membrane of enterocytes, which was accompanied of an upregulation of DMT1 mRNA expression. In contrast, cultures exposed to digests of fish or white beans showed no significant differences in the regulation of Fe transporters.     

Molecular characterization and intracellular regulation of the human serotonin transporter in Caco-2 cells.

The serotonin transporter (SERT) has shown itself to be an effective pharmacological target in the treatment of mood disorders and some kinds of gastrointestinal syndromes. Most of the molecular studies of SERT in humans have been carried out using heterologous models. In this work, we have investigated the human enterocyte-like Caco-2 cell line as a potential "in vitro" model to study the human SERT. The results show that these cells express a SERT mRNA identical to the human brain SERT, and a 70 kDa protein immunodetected using a specific antibody. The SERT activity levels in Caco-2 cells increased in correlation with the onset and maintenance of the morphological and functional differentiation of the cells. Caco-2 SERT was also shown to be a high affinity (Kt=0.216 microM) saturable, Na(+) -dependent transporter that was inhibited by fluoxetine (IC(50)=17.6 nM). In addition, SERT activity was inhibited by the intracellular modulators protein kinase C and cAMP, either after short or long-term treatment. In short, the expression and molecular characteristics of the human SERT in Caco-2 cells indicate that this cell line may be an ideal tool to study in vitro the physiology and pharmacology of human SERT.     

Alpha,Beta-cyclic-beta-benzamido hydroxamic acids: Novel oxaspiro[4.4]nonane templates for the discovery of potent, selective, orally bioavailable inhibitors of tumor necrosis factor-alpha converting enzyme (TACE)

Two novel oxaspiro[4.4]nonane beta-benzamido hydroxamic scaffolds have been synthesized in enantio- and diasteriomerically pure form. These templates proved to be exceptional platforms that have led to the discovery of potent inhibitors of TACE that are active in a cellular assay measuring suppression of LPS-induced TNF-alpha. Furthermore, these inhibitors are selective against related MMPs, demonstrate permeability in a Caco-2 assay, and display good oral bioavailability.     

Transport mechanism and metabolism of olive oil hydroxytyrosol in Caco-2 cells

3,4-dihydroxyphenylethanol (hydroxytyrosol; DPE) is the major phenolic antioxidant present in extra virgin olive oil, either in a free or esterified form. Despite its relevant biological effects, no data are available on its bioavailability and metabolism. The aim of the present study is to examine the molecular mechanism of DPE intestinal transport, using differentiated Caco-2 cell monolayers as the model system. The kinetic data demonstrate that [(14)C]DPE transport occurs via a passive diffusion mechanism and is bidirectional; the calculated apparent permeability coefficient indicates that the molecule is quantitatively absorbed at the intestinal level. The only labelled DPE metabolite detectable in the culture medium by HPLC (10% conversion) is 3-hydroxy-4-methoxyphenylethanol, the product of catechol-O-methyltransferase; when DPE is assayed in vitro with the purified enzyme a K(m) value of 40 microM has been calculated.     

Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells.

Monolayers of a well differentiated human intestinal epithelial cell line, Caco-2, were used as a model to study passive drug absorption across the intestinal epithelium. Absorption rate constants (expressed as apparent permeability coefficients) were determined for 20 drugs and peptides with different structural properties. The permeability coefficients ranged from approximately 5 x 10(-8) to 5 x 10(-5) cm/s. A good correlation was obtained between data on oral absorption in humans and the results in the Caco-2 model. Drugs that are completely absorbed in humans had permeability coefficients greater than 1 x 10(-6) cm/s. Drugs that are absorbed to greater than 1% but less than 100% had permeability coefficients of 0.1-1.0 x 10(-6) cm/s while drugs and peptides that are absorbed to less than 1% had permeability coefficients of less than or equal to 1 x 10(-7) cm/s. The results indicate that Caco-2 monolayers can be used as a model for studies on intestinal drug absorption.     

Relationship between structure and permeability of dipeptide derivatives containing tryptophan and related compounds across human intestinal epithelial (Caco-2) cells

The permeability of dipeptide derivatives containing tryptophans and indole derivatives through Caco-2 cells was used as an in vitro intestinal absorption model in order to clarify structural factors which influence their intestinal epithelial permeation and metabolism. Most peptide derivatives were hydrolysed not only by the cytosolic enzymes in Caco-2 cells during permeation but also by enzymes released to the apical solution before cell permeation. The N-terminal blocked dipeptides were more resistant to hydrolases expressed in the Caco-2 cells and indole derivatives were not entirely degraded. Based on compound concentration dependency and comparison of permeability coefficients in apical-to-basolateral and basolateral-to-apical directions, the main absorption mechanism of compounds were determined. Compounds were then classified into three groups; (1) passively transported compounds, (2) actively transported compounds and (3) compounds excreted by P-glycoprotein.