Caco 2 Cell

The aim of the present study was to improve the synthetic pathway of bioreversible dipeptide derivatives as well as evaluate the potential of using l-Glu-Sar as a pro-moiety for delivering three newly synthesised nucleoside and pyrimidine l-Glu-Sar derivatives. l-Glu(trans-2-thymine-1-yl-tetrahydrofuran-3-yl ester)-Sar (I), l-Glu(thymine-1-yl-methyl ester)-Sar (II) and l-Glu(acyclothymidine)-Sar (III) were synthesised and in vitro stability was studied in various aqueous and biological media. Affinity to and translocation via hPEPT1 was investigated in mature Caco-2 cell monolayers, grown on permeable supports. Affinity was estimated in a competition assay, using [14C] labelled Gly-Sar (glycylsarcosine). Translocation was measured as pHi-changes induced by the substrates using the fluorescent probe BCECF and an epifluorescence microscope setup. All dipeptide derivatives released the model drugs quantitatively by specific base-catalysed hydrolysis at pH > 6.0. II was labile in aqueous buffer solution, whereas I and III showed appropriate stability for oral administration. In 10% porcine intestinal homogenate, the half-lives of the dipeptide derivatives indicated limited enzyme catalyzed degradation. All compounds showed good affinity to hPEPT1, but the Compounds I and III showed not to be translocated by hPEPT1. The translocation of the l-Glu-Sar derivative of acyclovir, l-Glu(acyclovir)-Sar was also investigated and showed not to take place. Consequently, l-Glu-Sar seems to be a poor pro-moiety for hPEPT1-mediated transport.

Synchrony between mechanically separated biological systems is well known. We posed the question: can cells induce synchronous behavior in neighboring cells which are mechanically separated and which cannot communicate via chemical or electrical mechanisms. Caco-2 cell cultures were divided into three groups. "Inducer" cells were exposed to H 2 O 2 . "Detector" cells were placed in separate containers near the inducer cells but were not exposed to H 2 O 2 . Control cells were exposed to fresh media and were kept in a distant laboratory area. Samples were measured for total protein concentration, NFκB activation and structural changes, 10, 30 and 60 min after exposure respectively. Exposing inducer cells to H 2 O 2 resulted in a significant reduction in total protein content (−50%), an increase in nuclear NFκB activation (+38%), and structural damage (56%) compared to controls. There was a similar reduction in total protein content (−48%), increase in the nuclear fraction of NFκB (+ 35%) and structural damage (25%) in detector cells. These findings provide evidence in support of a non-chemical, non-electrical communication. This signaling system possibly plays a role in synchronous, stimulus-appropriate cell responses to noxious stimuli and may explain a number of cellular behaviors that are hard to explain based only on conventional cell signaling systems.

Cimetidine is known to enhance the survival of gastro-intestinal cancer patients, though the mechanisms involved are incompletely understood. Postulated modes of action include blocking the proliferative effect of tumors and inhibiting T suppressor cell activity, both of which are thought to be mediated by histamine type 2 receptors. Apoptotic cell death may offer an alternative explanation for reduced cell growth. We aimed to examine the effects of histamine, cimetidine, and ranitidine on in vitro proliferation and apoptosis in two human colorectal cancer cell lines, Caco-2 and LoVo. A cell proliferation assay was used as an index of cell growth. Histamine receptor status was determined by quantifying cyclic adenosine monophosphate and apoptosis via DNA fragmentation. Results show that histamine (10 −5 to 10 −9 M) had no effect on the growth of either cell line. The proliferation of Caco-2 was inhibited by ranitidine (10 −7 M) alone and in combination with histamine. Cimetidine (10 −5 M) only suppressed the growth of Caco-2 in the presence of histamine. The H 2 antagonists had no effect on LoVo irrespective of histamine. There was no accumulation of cyclic adenosine monophosphate in Caco-2 cells in response to histamine at a similar concentration. Apoptosis was induced in Caco-2 by both antisecretory drugs, and only ranitidine caused apoptotic cell death in LoVo cells. We conclude that cimetidine and ranitidine inhibit Caco-2 cancer cells in vitro, independently of the H 2 receptor. In addition, both drugs induce apoptosis in the same cell line. Growth inhibition and apoptosis are likely to contribute to the tumor regressive properties of cimetidine and ranitidine in vivo.

Human breast cancer resistance protein (BCRP/ABCG2) is an ABC-transporter that is present on the luminal membrane of intestinal epithelial cells and restricts absorption of anticancer drugs such as methotrexate, topotecan, mitoxantrone, and doxorubicin. The exact anatomic distribution of BCRP along the gastrointestinal (GI) tract, however, has not been determined before. The aim of this study was, therefore to investigate BCRP mRNA expression pattern along the GI tract in 14 healthy subjects. Furthermore, BCRP duodenal mRNA expression was compared with MDR1/ABCB1 mRNA. Additionally, BCRP mRNA expression was investigated in two human intestinal cell lines (Caco-2 and LS180). Since previous animal studies have suggested sex specific differences in BCRP expression, we analyzed intestinal BCRP expression with respect to sex. Biopsies were taken from different gut segments (duodenum, terminal ileum and ascending, transverse, descending and sigmoid colon). Gene expression was assessed by quantitative real-time PCR (Taqman). BCRP mRNA expression was maximal in the duodenum and decreased continuously down to the rectum (terminal ileum 93.7%, ascending colon 75.8%, transverse colon 66.6%, descending colon 62.8%, and sigmoid colon 50.1% compared to duodenum, respectively). BCRP expression in the duodenum was comparable to MDR1/ABCB1 gene expression. Caco-2 cells showed a comparable expression of BCRP as human duodenal tissue. Gender specific differences in BCRP expression were not observed. These findings represent the first systematic site-specific analysis of BCRP expression along the GI tract. This information might be helpful to develop target strategies for orally administered anticancer drugs. #

D-alpha-tocopheryl poly(ethylene glycol) 1000 succinate (TPGS 1000) is a widely used form of vitamin E. TPGS 1000 is comprised of a hydrophilic polar (water-soluble) head and a lipophilic (water-insoluble) alkyl tail. TPGS 1000 has been used as a solubilizer, an emulsifier and as a vehicle for lipid-based drug delivery formulations. Most recently, TPGS 1000 has been recognized as an effective oral absorption enhancer. An enhancing effect is consistent with a surfactant-induced inhibition of P-glycoprotein (P-gp), and perhaps other drug transporter proteins; however, the exact inhibition mechanism(s) remain unclear. Therefore, in an attempt to generate additional knowledge, we have synthesized and tested various TPGS analogs containing different PEG chain length (TPGS 200/238/400/600/1000/2000/3400/3500/4000/6000). These results demonstrate a relationship between TPGS PEG chain length and influence on rhodamine 123 (RHO) transport in Caco-2 monolayers, a relationship which may be illustrated using a Weibull distribution.

Geum quellyon Sweet, a perennial herb of the Rosaceae family, has been used in the traditional medicine of the Mapuche Amerindians of Chile to treat tooth neuralgia, gastric inflammation, prostatitis and to regulate menstruation, and for its diuretic and aphrodisiac properties. Although many benefits have been claimed for this plant, few scientific studies are available in the literature. In this study, we investigated the antioxidant activity of a methanolic extract of Geum quellyon roots. We also examined the anticancer action of this plant on Caco-2 (colon adenocarcinoma cells), DU-145 (androgen-insensitive prostate cancer cells) and KB (oral squamous carcinoma cells) human tumor cell lines. Our data showed that Geum quellyon extract, containing tannins, exhibits interesting antioxidant properties, expressed by its capacity to scavenge 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH) and superoxide anion (O 2

The human intestinal Caco-2 cell line has been extensively used over the last twenty years as a model of the intestinal barrier. The parental cell line, originally obtained from a human colon adenocarcinoma, undergoes in culture a process of spontaneous di¡erentiation that leads to the formation of a monolayer of cells, expressing several morphological and functional characteristics of the mature enterocyte. Culture-related conditions were shown to in£uence the expression of these characteristics, in part due to the intrinsic heterogeneity of the parental cell line, leading to selection of sub-populations of cells becoming prominent in the culture. In addition, several clonal cell lines have been isolated from the parental line, exhibiting in general a more homogeneous expression of di¡erentiation traits, while not always expressing all characteristics of the parental line. Culturerelated conditions, as well as the di¡erent Caco-2 cell lines utilized in di¡erent laboratories, often make it extremely di¤cult to compare results in the literature. This review is aimed at summarizing recent, or previously unreviewed, data from the literature on the e¡ects of culture-related factors and the in£uence of line sub-types (parental vs. di¡erent clonal lines) on the expression of di¡erentiation traits important for the use of Caco-2 cells as a model of the absorptive and defensive properties of the intestinal mucosa. Since the use of Caco-2 cells has grown exponentially in recent years, it is particularly important to highlight these methodological aspects in order to promote the standardization and optimisation of this intestinal model.

Purpose. To investigate the underlying mechanism of low bioavailabilities of the water-soluble camptothecin derivatives, belotecan and topotecan. Methods. The bioavailability of belotecan and topotecan in rats was determined following oral administration of each drug at a dose of 5 mg/kg body weight. The vectorial transport of each drug was measured in Caco-2 and engineered MDCK II cells.

FXai, a direct inhibitor of the clotting factor Xa, provides high water solubility but poor membrane permeability due to multiple sites of ionization and a molecular weight exceeding 500 Da, making it a Class III drug according to the Biopharmaceutics Classification System. To overcome the ionization problem and increase the transcellular permeability, various ester and hydroxyamidine prodrugs exhibiting a reduced number of ionization sites were studied in the Caco-2 monolayer model for intestinal permeation. Alternatively, the potential transcellular permeation enhancement of Imwitor Ò 742 and the potential paracellular enhancement of three chitosan formulations were investigated in the same model. FXai has an apparent permeability (P app ) of about 1 nm/s, which is generally regarded as very low. The butylester-hydroxyamidine double-prodrug was found to provide a markedly increased permeability (40.4 nm/s) as did the co-application of chitosan (43.3 nm/s). Other prodrugs slightly increased permeability (1.3-9.2 nm/s) but were inferior to the previous attempts to enhance permeability while the Imwitor Ò admixture showed no effect (1.1 nm/s). Moreover, a bioactivating metabolism towards the hydroxyamidine mono-prodrug was detected in the Caco-2 cell permeation model. Although esterases were overexpressed and mainly located apically, an acceptable permeation was reached. In addition, the prodrugs triggered an efflux system that is not inhibited by verapamil but by quinidine, suggesting the involvement of an organic cation transporter.

The aim of this study was to investigate the adhesive phenotype of the human intestinal isolate Bifidobacterium bifidum MIMBb75 to human colon carcinoma cell lines. We have previously shown that the adhesion of this strain to Caco-2 cells is mediated by an abundant surface lipoprotein named BopA. In this study, we found that this strain adheres to Caco-2 and HT-29 cells, and that its adhesion strongly depends on the environmental conditions, including the presence of sugars and bile salts and the pH. Considerably more adhesion to a Caco-2 monolayer occurred in the presence of fucose and mannose and less when MIMBb75 grew in Oxgall bile salts compared to standard environmental conditions. In particular, growth in Oxgall bile salts reduced the adhesion ability of MIMBb75 and modified the SDS-PAGE profile of the cell wall associated proteins of the strain. The pH markedly affected both adhesion to Caco-2 and bacterial autoaggregation. Finally, experiments with sodium metaperiodate suggested that not only proteinaceous determinants are involved in the adhesion process of B. bifidum. In conclusion, it seems that the colonization strategy of this bacterium can be influenced by factors varying along the gastrointestinal tract, such as the presence of specific sugars and bile salts and the pH, possibly limiting the adhesion of B. bifidum to only restricted distal sites of the gut.

This study aimed i) to characterize the transepithelial transport of the mucolytic agent ambroxol hydrochloride across the intestinal barrier, ii) to classify the ambroxol according to Biopharmaceutics Classification System (BCS) and iii) to predict ambroxol absorption in humans. Transport of ambroxol (100, 300 and 1000 μmol/l) was studied in a human colon carcinoma cell line Caco-2 in apical to basolateral and basolateral to apical direction, under iso-pH 7.4 and pH-gradient (6 vs. 7.4) conditions. The relative contribution of the paracellular route was estimated using Ca 2+ -free transport medium. Ambroxol samples from receiver compartments were analysed by HPLC with UV detection (242 nm).

Probiotic bacteria play an important role in protecting the host from intestinal colonization of pathogenic bacteria. We have developed a new analytical approach based on a real-time PCR technique for quantifying Bifidobacterium adhesion to intestinal epithelial cells. Real-time PCR analysis showed that adhesion to enterocyte-like Caco-2 cells represented a variable phenotype in the genus Bifidobacterium, enabling classification of three adhesion behaviors: high adhesiveness (>40 bifidobacterial cells/Caco-2 cell); adhesiveness (5-40 bifidobacterial cells/Caco-2 cell); no adhesiveness (<5 bifidobacterial cells/Caco-2 cell). This molecular methodology was successfully used in competition studies in enteropathogens. All bifidobacterial strains examined evidenced displacement activity towards important enteropathogens (S. typhimurium, Y. enterocolitica and E. coli EPEC). Real-time PCR is a rapid, accurate and sensitive method for detecting and quantifying different bacterial genera and species simultaneously adhering to a epithelial cell monolayer. (P. Brigidi). colonization of enteric pathogens . From the host point of view, such a barrier effect can be considered as one of the main functions exerted by the gut microbiota. Three mechanisms of action are involved in the barrier effect played by the intestinal microflora: (i) prevention of enteropathogenic adhesion to the host enterocytes; (ii) favorable competition with exogenous pathogens for nutrient availability in the gastrointestinal ecological niches; (iii) inhibition of growth of pathogenic bacteria by bacteriocin production and lowering of the pH.

This study was aimed to synthesize polymeric excipients with improved mucoadhesive, cohesive and in situ-gelling properties to assure a prolonged retention time of dosage forms at a given target site, thereby achieving an increased uptake and improved oral bioavailability of certain challenging therapeutic agents such as peptides and proteins. Accordingly, poly(acrylic acid)-cysteine-2-mercaptonicotinic acid (PAAcys-2MNA) conjugates were synthesized by the oxidative SeS coupling of PAA-cys (100-, 250-and 450 kDa) with 2-mercaptonicotinic acid (2MNA). Unmodified PAAs, PAAs-cys (thiomers) and PAA-cys-2MNA (100-, 250-and 450 kDa) conjugates were compressed into tablets to perform disintegration tests, mucoadhesion studies and rheological measurements. Moreover, cytotoxicty of the polymers was determined using Caco-2 cells. The resulting PAA-cys-2MNA (100-, 250-and 450 kDa) conjugates displayed 113.5 AE 12.7, 122.7 AE 12.2 and 117.3 AE 4.6 mmol/g of 2-mercaptonicotinic acid, respectively. Due to the immobilization of 2MNA, the PAA-cys-2MNA (pre-activated thiomers) conjugates exhibit comparatively higher swelling properties and disintegration time to the corresponding unmodified and thiolated polymers. On the rotating cylinder, tablets based on PAA-cys-2MNA (100-, 250-and 450 kDa) conjugates displayed 5.0-, 5.4-and 960-fold improved mucoadhesion time in comparison to the corresponding unmodified PAAs. Results achieved from tensile studies were found in good agreement with the results obtained by rotating cylinder method. The apparent viscosity of PAA-cys-2MNA (100-, 250-and 450 kDa) conjugates was improved 1.6-, 2.5-and 206.2-fold, respectively, in comparison to the corresponding unmodified PAAs. Moreover, pre-activated thiomers/mucin mixtures showed a time dependent increase in viscosity up to 24 h, leading to 7.0-, 18.9-and 2678-fold increased viscosity in comparison to unmodified PAAs (100-, 250-and 450 kDa), respectively. All polymers were found non-toxic over Caco-2 cells. Thus, on the basis of achieved results the pre-activated thiomers seem to represent a promising generation of mucoadhesive polymers which are safe to use for prolonged residence time of drug delivery systems to target various mucosa. Biomaterials j o u r n a l h o m e p a g e : w w w . e l s e v i e r. co m/ lo ca t e / b i o m a t e ri a l s 0142-9612/$ e see front matter Ó

A theory of molecular absorption from the small intestine is outlined on the basis of macrotransport analysis. Certain features of the transport process that have not been previously considered in the literature are quantitatively described. These include complex interrelationships between lumen and membrane di!usion, convection, degradation and absorption mechanisms * and overall axial convection, dispersion, degradation and absorption rates. Therapeutic molecules are assumed to be introduced in the form of a bolus into the duodenum. They subsequently convect and di!use through the duodenum, jejunum, and ileum. Absorption into the systemic circulation across the epithelial barrier, as well as possible degradation or aggregation in the lumen or at the apical epithelial membrane, contribute to the disappearance of the therapeutic as the bolus travels through the lumen in an oral to caudal direction. Space-and time-varying lumen concentrations are predicted, as are time-varying systemic concentrations following introduction of the bolus. The inputs to the model are primarily anatomical or physicochemical characteristics that are either known or can be measured for a given therapeutic and animal model. A detailed parametric study is made, elucidating the individual roles of permeability and degradation rates. This leads to a simple paradigm for determining the two unknowns of the model (the membrane permeability and degradation rate constant) from systemic absorption data; it is shown that the membrane permeability constant can alternatively be estimated by independent in vitro measurements. Comparisons with published experimental systemic concentrations are made for molecules ranging from small lipophilic substances, such as ibuprofen, to polypeptides, such as calcitonin, and proteins, such as insulin. The deduced epithelial permeability values show reasonable agreement with values determined using alveolar epithelia and Caco-2 cell monolayers. By contrast, the membrane permeability values deduced from a simpli"ed model of absorption from the small intestine show relatively poor agreement with experimental values. The model may be useful as a numerical simulation tool for predicting (estimates of ) oral dose}response relationships in animals and humans given relatively limited in vivo data.

Sulfonamides and their derivatives inhibit the catalytic activity of carbonic anhydrases (CA, EC 4.2.1.1). Isozyme IX (CA IX) is a transmembrane isoform with the active site oriented toward the extracellular space. CA IX was recently shown to be a drug target, and it is highly overexpressed in hypoxic tumors with limited distribution in normal tissues. The present report deals with the drug design, synthesis, and biological investigation of a group of thioureido sulfonamides, which have been obtained by reaction of isothiocyanate-substituted aromatic sulfonamides with amines. These compounds have potent inhibitory properties against CA IX with K I values in the range of 10-37 nM and P app values > 0.34 · 10 À6 cm/s for the absorptive transepithelial transport in Caco-2 cells. In Caco-2 cells, one of these compounds (A6) was shown to be a substrate for efflux transporters such as P-glycoprotein (P-gp). P-gp activity is not likely to be rate-limiting for intestinal absorption, but might be useful when targeting hypoxic tumors expressing both P-gp and CA IX.

The effect of the combined use of randomly methylated b-cyclodextrin (RAMEB), chitosan (CS), and bile components (dehydrocholic (DHCA) or ursodeoxycholic (UDCA) acids and their sodium salts) on solubility and permeability through Caco-2 cells of oxaprozin (a very poorly water-soluble non-steroidal antiinflammatory drug) has been investigated. Addition of CS, bile acids, and their sodium salts increased the RAMEB solubilizing power of 4, 2, and 5 times, respectively. Drug-RAMEB-CS co-ground systems showed very higher dissolution rate than corresponding drug-RAMEB systems. Addition of bile components further improved drug dissolution rate. The CS presence enabled a significant increase in drug permeability through Caco-2 cells with respect to drug-RAMEB systems. Moreover, CS and NaDHC showed a synergistic enhancer effect, enabling a 1.4-fold permeability increase in comparison with systems without bile salt. However, unexpectedly, no significant differences were found between physical mixtures and co-ground products, indicating that drug permeation improvement was due to the intrinsic enhancer effect of the carriers and not to drug-carrier interactions brought about by co-grinding, as instead found in dissolution rate studies. The combined use of RAMEB, CS, and NaDHC could be exploited to develop effective oral dosage forms of oxaprozin, with increased drug solubility and permeability, and then improved bioavailability.

Recombinant severe acute respiratory virus (SARS-CoV) variants lacking the group specific genes 6, 7a, 7b, 8a, 8b and 9b (rSARS-CoV-Δ[6-9b]), the structural gene E (rSARS-CoV-ΔE), and a combination of both sets of genes (rSARS-CoV-Δ[E,6-9b]) have been generated. All these viruses were rescued in monkey (Vero E6) cells and were also infectious for human (Huh-7, Huh7.5.1 and CaCo-2) cell lines and for transgenic (Tg) mice expressing the SARS-CoV receptor human angiotensin converting enzyme-2 (hACE-2), indicating that none of these proteins is essential for the viral cycle. Furthermore, in Vero E6 cells, all the viruses showed the formation of particles with the same morphology as the wt virus, indicating that these proteins do not have a high impact in the final morphology of the virions. Nevertheless, in the absence of E protein, release of virus particles efficacy was reduced. Viruses lacking E protein grew about 100-fold lower than the wt virus in lungs of Tg infected mice but did not grow in the brains of the same animals, in contrast to the rSARS-CoV-Δ[6-9b] virus, which grew almost as well as the wt in both tissues. Viruses lacking E protein were highly attenuated in the highly sensitive hACE-2 Tg mice, in contrast to the minimal rSARS-CoV-Δ[6-9b] and wt viruses. These data indicate that E gene might be a virulence factor influencing replication level, tissue tropism and pathogenicity of SARS-CoV, suggesting that ΔE attenuated viruses are promising vaccine candidates.

Introduction: The microemulsion concept was introduced in 1943 by Hoar and Schulman. Self-microemulsifying drug delivery systems [S(M)EDDS] are much more recent and can be described as isotropic solutions of oils and surfactants that form oil-in-water O/W microemulsions when they are poured into an aqueous medium. When they are presented as soft capsules for oral delivery, S(M)EDDS have the ability to considerably improve the intestinal absorption of agents that are incorporated into the S(M)EDDS. Forty per cent of newly discovered drug candidates have little or no water solubility and therefore have low and/or variable bioavailability profiles. Many of these drugs are good candidates for formulation into S(M)EDDS. Areas covered: This paper describes the preparation and assessment of these formulations and their current applications. The characterisation of this type of formulation has improved, and in vitro models (Caco-2 cell cultures, Ussing chambers, the everted sac technique, etc.) can be used for screening different formulations. It describes also marketed formulations [i.e., cyclosporine and saquinavir S(M)EDDS] and somes other formulations. Expert opinion: Actual applications of S(M)EDDS remain rare. The first drug marketed as a S(M)EDDS was cyclosporine, and it had significantly improved bioavailability compared with the conventional solution. In the last decade, several S(M)EDDS loaded with antiviral drugs (e.g., ritonavir, saquinavir) were tested for treatment of HIV infection, but the relative improvement in clinical benefit was not significant. The S(M)EDDS formulation of Norvir ® (soft capsules) has been withdrawn in some countries. The confusion is due to the fact that both self-microemulsifying systems (SMEDDS) and self-nanoemulsifying systems (SNEDDS) form dispersions with an emulsion particle size in the nanometre range [6]. In this review, these formulations are called "S(M)EDDS". Among lipid formulations, self-microemulsifying drug delivery systems [S(M)EDDS] are very specific. They are isotropic solutions of oils and surfactants that form O/W microemulsions when they are poured into an aqueous medium. Very gentle agitation allows dispersion of the isotropic solution into the aqueous medium. S(M)EDDS allow the drug to absorb without being digested, and they are classified Gibaud, S., & Attivi, D. (2012). Microemulsions for oral administration and their therapeutic applications. Expert Opinion on Drug Delivery, 9(8), 937-951. HS 15 [22] and Triton X-100 [23]. More lipophilic surfactants can also be used for O/W microemulsions. A particularly important class of surfactants consists of mono-and diglycerides such as glyceryl caprylatelcaprate (Capmul® MCM) or glyceryl monooleate (Maisine ® 35-1, Peceol ® ).

The essential oils of four Teucrium species were studied and 150 components, in all, were identified. All oils were rich in sesquiterpenes (50.1-55.8%). Spathulenol and d-cadinene were the main compounds of Teucrium brevifolium oil; caryophyllene and 4-vinyl guaiacol predominated in Teucrium flavum. Carvacrol and caryophyllene oxide predominated in Teucrium montbretii ssp. heliotropiifolium, while carvacrol and caryophyllene were the most abundant components in Teucrium polium ssp. capitatum. The oil which most effectively inhibited LPS-induced NO production in macrophage cell line RAW 264.7 was that from T. brevifolium (IC 50 = 7.1 lg/ml), followed by T. montbretii ssp. heliotropiifolium and T. polium ssp. capitatum (IC 50 = 16.5 and 29.4 lg/ml, respectively). The in vitro cytotoxic assay on three human cancer cell lines showed that the most antiproliferative oils were those from T. polium ssp. capitatum and T. montbretii ssp. heliotropiifolium on CACO-2 cell lines (IC 50 = 52.7 and 92.2 lg/ml, respectively). The T. brevifolium oil showed a selective cytotoxicity on COR-L23 while significant activity was exerted by T. polium oil on C32.

Plant stanols and sterols of the 4-desmethyl family (e.g., sitostanol and sitosterol) effectively decrease LDL cholesterol concentrations, whereas 4,4-dimethylsterols ( ␣ -amyrin and lupeol) do not. Serum carotenoid concentrations, however, are decreased by both plant sterol families. The exact mechanisms underlying these effects are not known, although effects on micellar composition have been suggested. With a liver X receptor (LXR) coactivator peptide recruitment assay, we showed that plant sterols and stanols from the 4-desmethylsterol family activated both LXR ␣ and LXR ␤ , whereas 4,4-dimethyl plant sterols did not. In fully differentiated Caco-2 cells, the functionality of this effect was shown by the increased expression of ABCA1, one of the known LXR target genes expressed by Caco-2 cells in measurable amounts. The LXR-activating potential of the various plant sterols/stanols correlated positively with ABCA1 mRNA expression. Reductions in serum hydrocarbon carotenoids could be explained by the effects of the 4-desmethyl family and 4,4-dimethylsterols on micellar carotenoid incorporation. Our findings indicate that the decreased intestinal absorption of cholesterol and carotenoids by plant sterols and stanols is caused by two distinct mechanisms. -Plat, J., J. A. Nichols, and R. P. Mensink. Plant sterols and stanols: effects on mixed micellar composition and LXR (target gene) activation. J. Lipid Res. 2005. 46: 2468-2476.

The intestinal mucosa is not simply a barrier allowing entry of compounds such as nutrients or chemicals, and restricting that of others. Intestinal cells and activities perform selective absorption, biotransformations and efflux back to the lumen. Furthermore, food substances affect both bioavailability and intestinal function. Some are able to act as transcriptional regulators and enzyme modulators.

Proton-dependent oligopeptide transporter Peptide/histidine transporter 1 PHT1 Valacyclovir COS-7 Transient transfection a b s t r a c t Recently, the expression of the human peptide/histidine transporter (hPHT1, SLC15A4) mRNA was observed in the GI tract and in Caco-2 cells, suggesting that it may participate in the intestinal absorption of peptide-based agents. This study aims to elucidate the: (i) protein expression pattern of hPHT1 (SLC15A4) in human small intestine; (ii) cloning of the hPHT1 full-length sequence; (iii) functional characterization of hPHT1 in transiently transfected COS-7 cells. The expression of hPHT1 was measured using Western blot and immunohistochemical analysis. The hPHT1 full-sequence was amplified from BeWo cells, inserted into the pcDNA3.1-V5/His TOPO ® plasmid and transiently transfected into COS-7 cells to investigate the uptake kinetics of [ 3 H]histidine and [ 3 H]carnosine. Time, pH and sodiumdependent uptake studies were performed in mock (empty vector) and hPHT1-COS-7 cells.

Phytosterols have been shown to reduce cholesterol absorption in humans. Supplementing phytosterols in fat-free formulations, however, has yielded controversial results. In the present study, we investigated the effect of supplementing test meals with different fat-free phytosterol products on cholesterol incorporation into mixed micelles during simulated digestion and accumulation of micellar cholesterol by Caco-2 cells: control orange juice (OJ), orange juice supplemented with either multivitamin/multimineral tablets (MVT), multivitamin/multimineral tablets containing phytosterols (MVT+P), and phytosterol powder (PP). These combinations were added to Ensure-based test meals and spiked with cholesterol of natural isotopic composition or 13 C 2 -cholesterol to differentiate external from endogenous cholesterol. After simulated gastric/small intestinal digestion, micelle fractions were analyzed for cholesterol enzymatically (n ) 6-20/product) and by high-performance liquid chromatography-tandem mass spectrometry (n ) 12/product) and added to Caco-2 cells to determine the accumulation of 13 C 2 -cholesterol (n ) 10-24/product). As compared to OJ, PP and MVT+P significantly decreased cholesterol micellarization (determined enzymatically) by 70 ( 39 (mean ( SD) and 70 ( 39%, respectively (P < 0.001, Bonferroni). The stable isotope experiments revealed that both PP and MVT+P reduced cholesterol micellarization [by 25 ( 12 (P ) 0.055) and 21 ( 8% (P ) 0.020), respectively, Fisher's protected LSD test] and Caco-2 cell accumulation (by 28 ( 8 and 10 ( 8%, respectively; P < 0.010, Bonferroni). OJ+P did not inhibit micellarization or accumulation of cholesterol by Caco-2 cells. This study shows that fat-free phytosterol-containing products can significantly inhibit cholesterol micellarization and Caco-2 cell bioaccessibility, albeit to different extents depending on individual formulations. This is most likely explained by inhibition of cholesterol micellarization.

A cis-regulatory sequence also known as zone of polarizing activity (ZPA) regulatory sequence (ZRS) located in intron 5 of LMBR1 is essential for expression of sonic hedgehog (SHH) in the developing posterior limb bud mesenchyme. Even though many point mutations causing preaxial duplication defects have been reported in ZRS, the underlying regulatory mechanism is still unknown. In this study, we analyzed the effect on transcription factor binding of a novel ZRS point mutation (463T4G) in a Pakistani family with preaxial polydactyly and triphalangeal thumb. Electrophoretical mobility shift assay demonstrated a marked difference between wild-type and the mutant probe, which uniquely bound one or several transcription factors extracted from Caco-2 cells. This finding supports a model in which ectopic anterior SHH expression in the developing limb results from abnormal binding of one or more transcription factors to the mutant sequence.

Curcumin a poly-phenolic compound possesses diverse pharmacologic activities; however, its development as a drug has been severely impeded by extremely poor oral bioavailability. Poor aqueous solubility and extensive metabolism have been implicated for this but the role of membrane permeability has not been investigated. In the present study, permeability of curcumin was assessed using the Caco-2 cell line. Curcumin was poorly permeable with a P app (A ? B) value of 2.93 ± 0.94 Â 10 À6 cm/s. P app value in (B ? A) study was found out to be 2.55 ± 0.02 Â 10 À6 cm/s, thus ruling out the role of efflux pathways in poor oral bioavailability of curcumin. Studies using verapamil, a P-gp inhibitor, further confirmed this finding. Detailed mass balance studies showed loss of curcumin during transport. Further experiments using lysed cells revealed that 11.78% of curcumin was metabolized during transport. Studies using itraconazole, a CYP3A4 inhibitor, established its role in curcumin metabolism. Curcumin was also found to accumulate in cells as revealed by CLSM studies. Sorption and desorption kinetic studies further confirmed accumulation of curcumin inside the cells. Amount accumulated was quantitated by HPLC and found to be >20%. Thus, intestinal first-pass metabolism and intracellular accumulation played a role in poor permeability of curcumin. Based on its poor aqueous solubility and intestinal permeability, curcumin can be classified as a BCS Class IV molecule. This information can facilitate designing of drug delivery systems for enhancement of oral bioavailability of curcumin.

Azaspiracids cause severe damages in the epithelium of several organs. In this study we have investigated the effects of azaspiracid-1 (AZA-1) on two epithelial cell lines. Nanomolar concentrations of AZA-1 reduced MCF-7 cell proliferation and impaired cell-cell adhesion. AZA-1 altered the cellular pool of the adhesion molecule E-cadherin by inducing a dose-and timedependent accumulation of an E-cadherin fragment (E-cadherinrelated antigen [ECRA 100 ]), with a concentration inducing the half-maximal effect (EC 50 ) of 0.47nM. The immunological characterization of ECRA 100 revealed that it consists of an E-cadherin molecule lacking the intracellular domain, and these data showed that the effect induced by AZA-1 in MCF-7 cells is undistinguishable from that induced by yessotoxin (YTX) in the same experimental system. A comparison of toxin effects in MCF-7 and Caco 2 cells confirmed that the effects induced by AZA-1 and YTX are undistinguishable in these cells. Treatment of fibroblasts with AZA-1 did not affect the cellular pool of N-cadherin showing that the toxin effect is cadherin-specific. A comparison of the effects induced by AZA-1, YTX, and okadaic acid on F-actin and Ecadherin in MCF-7 and Caco 2 cells showed that 1nM AZA-1 did not cause significant changes in F-actin and that accumulation of ECRA 100 did not correlate with decreased levels of F-actin under our experimental conditions. Matching our results with those available in literature, we notice that, when molecular effects induced by AZA-1 and YTX have been studied in the same in vitro systems, experimental data show that they are undistinguishable in terms of sensitive cellular parameters, effective doses, and kinetics of responses in several cell lines. The possibility that azaspiracids and YTXs might share their molecular mechanism(s) of action in defined biological settings should be considered.

Twenty-nine Lactobacillus strains of dairy origin were examined in vitro for their probiotic potential. Only a few strains were able to survive at pH 1 or in the presence of pepsin, while all were unaffected by pH 3, pancreatin and bile salts. Strains exhibited variable bile salt hydrolase activity. None was haemolytic. The majority of strains were resistant to vancomycin

... 2.4. Characterization of phage resistant mutants. Phage resistance stability (Carminati, Zennaro, Neviani, &amp;amp;amp; Giraffa, 1993) and adsorption rates (Neviani, Carminati, &amp;amp;amp; Giraffa, 1992) were determined to characterize the phage resistance phenotype of the variants isolated. ...

Intestinal everted sac Intestinal uptake Lupinus albus a b s t r a c t A lupin seed glycoprotein, termed c-conglutin, has previously been found to display insulin-mimetic activity in myocyte models and reduce plasma glucose concentration when orally administered to both rats and humans. To envisage the possible metabolic fate of this bioactive protein, we used in vitro cell and ex vivo tissue models to monitor its transit through the intestinal barrier. Caco-2 cell monolayers and rat intestinal everted sacs were treated with purified c-conglutin and the protein was immunoassayed by chemi-luminescence-enhanced Western blotting. The in vitro approach showed that the intact protein can transit from the apical to the basolateral side of the cell monolayers. The unmodified lupin protein was also detected inside the intestinal everted sacs. Proper controls of cell monolayer and sac integrity ruled out the possibility of protein passive leakage.

Endomorphin 1 (Endo-1 = Tyr-Pro-Trp-Phe-NH 2 ), an endogenous opioid with high affinity and selectivity for μ-opioid receptors, mediates acute and neuropathic pain in rodents. To overcome metabolic instability and poor membrane permeability, the N-and C-termini of Endo-1 were modified by lipoamino acids (Laa) and/or sugars, and 2′,6′-dimethyltyrosine (Dmt) replacement of Tyr. Analogues were assessed for μ-opioid receptor affinity, inhibition of cAMP accumulation, enzymatic stability, and permeability across Caco-2 cell monolayers. C-terminus modification decreased receptor affinity, while N-terminus C8-Laa improved stability and permeability with slight change in receptor affinity. Dmt provided a promising lead compound: [C8Laa-Dmt 1 ]-Endo-1 is 9 times more stable (t ½ = 43.5 min), > 8-fold more permeable in Caco-2 cell monolayers, and exhibits 140-fold greater μ-opioid receptor affinity (K iμ = 0.08 nM).

This work aimed at showing the effect of pheromone plantaricin A (PlnA) by Lactobacillus plantarum DC400 towards other sourdough lactic acid bacteria and the potential of PlnA to protect the function of the human intestinal barrier. Growth and survival of sourdough lactic acid bacteria were differently affected by co-cultivation with L. plantarum DC400. Compared to mono-cultures, Lactobacillus sanfranciscensis DPPMA174 and Pediococcus pentosaceus 2XA3 showed growth inhibition and decreased viability when co-cultured with L. plantarum DC400. L. sanfranciscensis DPPMA174 induced the highest synthesis of PlnA. Survival of strain DPPMA174 only slightly varied by comparing the addition of PlnA to the culture medium and the co-cultivation with L. plantarum DC400. Compared to mono-culture, the proteome of L. sanfranciscensis DPPMA174 grown in co-culture with L. plantarum DC400 showed the variation of expression of 58 proteins (47 over expressed and 11 repressed). Thirty-four of them were also over expressed or repressed during growth of DPPMA174 with PlnA. Fiftyone of the above 58 proteins were identified. They had a central role in stress response, amino acid, energy and nucleotide metabolisms, membrane transport, regulation of transcription, and cell redox homeostasis. PlnA markedly increased the viability of human Caco-2/TC7 cells and the transepithelial electrical resistance.

The extract of the fern Polypodium leucotomos (PL, Fernblock Ò ) is an oral photoprotectant with strong antioxidative properties. Recent studies to determine its chemical composition have shown 4-hydroxycinnamic acid (p-coumaric), 3 methoxy-4-hydroxycinnamic acid (ferulic), 3,4-dihydroxycinnamic acid (caffeic), 3-methoxy-4-hydroxybenzoic acid (vanillic) and 3-caffeoilquinic acid (chlorogenic) to be among its major phenolic components. No conclusive data are available, however, on the H 2 O 2 -scavenging capacity of these compounds, or on their absorption and metabolism following their oral intake. In the present work, their antioxidative capacity was assessed by the luminol/H 2 O 2 assay, their absorption studied using Caco-2 cells to resemble the intestinal barrier, and their metabolism investigated using cultured primary rat hepatocytes. The antioxidant capacity of PL components increased in a concentration-dependent manner, with ferulic and caffeic acids the most powerful antioxidants. The apparent permeability results correspond to a human post-oral administration absorption of 70-100% for all tested substances. Coumaric, ferulic and vanillic acids were metabolized by CYP450-dependent mono-oxygenases and partially conjugated to glucuronic acid and sulfate. These phenolic compounds may contribute to the health benefits afforded by this oral photoprotectant.

Malachite green (MG) is still illegally used as a fungicide in aquaculture. In Wsh it is absorbed and metabolised to its major reduced metabolite, leucomalachite green (LMG). This latter represents the main residue found in Wsh tissues and may persist for several months. Since MG, suspected to act as a tumour promoter in vitro and in vivo, might be also present as a residue in Wsh, the present study was undertaken to ascertain the in vitro toxicity of both compounds in two human tumour cell lines (Caco-2 and HEp-2).

We previously developed an in vitro model to estimate the relative bioavailability of carotenoids from a meal prepared using commercial baby foods. The general applicability of this model was tested using a stir-fried meal consisting of fresh spinach, fresh carrots, tomato paste, and vegetable oil. After in vitro digestion of the cooked meal, the aqueous fraction was separated from residual oil droplet and solids by centrifugation to quantify micellarized carotenoids. The percentages of lutein, lycopene, ␣-carotene, and ␤-carotene transferred from the meal to the micellar fraction were 29.0 Ϯ 0.6, 3.2 Ϯ 0.1, 14.7 Ϯ 0.3, and 16.0 Ϯ 0.4, respectively. Carotenoid transfer from the meal to the aqueous fraction was inhibited when bile extract was omitted from the intestinal phase of digestion. The bioavailability of the micellarized carotenoids was validated using differentiated cultures of Caco-2 human intestinal cells. All four carotenoids were accumulated in a linear manner throughout a 6-hr incubation period. Metabolic integrity was not compromised by exposure of cultures to the diluted aqueous fraction from the digested meal. The addition of 500 mol/L ␣-tocopherol to test medium significantly improved the stability of the micellar carotenoids within the tissue culture environment. These results support the utility of the in vitro digestion procedure for estimating the bioavailability of carotenoids from foods and meals. (J. Nutr.

Micronutrient malnutrition, especially selenium (Se), iron (Fe), and zinc (Zn) deficiency, is a major global health problem. Previous attempts to prevent micronutrient malnutrition through food fortification, supplementation, and enrichment of staple crops has had limited success. Canadian grown lentils are rich in micronutrients Fe (73–90 mg kg−1), Zn (44–54 mg kg−1), Se (425–673 μg kg−1), and have very low concentrations of phytic acid (2.5–4.4 mg g−1). Our preliminary studies using a Caco-2 cell model show that the uptake of Fe from lentils is relatively greater than that of most other staple food crops. Moreover, preliminary results from our human nutrition study in Sri Lanka show an increased trend in blood Se concentration after lentil consumption. This article briefly overviews our previously published results as well as data from international lentil field trials, and describes the potential for biofortified lentil to provide a whole food solution to combat global human micronutrient malnutrition.

Thunbergia laurifolia Lindl. or Rang Chuet (RC) is widely described in Thai traditional medicine for protection against dietary and environmental toxicants with little substantiation. To better access the potential of RC as a medicinal plant, extracts were prepared by infusion with water, ethanol and acetone.

Caco-2 cells, an intestinal cell line derived from a human colorectal carcinoma that spontaneously differentiates under standard culture conditions, lends it- self to the in vitro study of human gut in view of its efficient intestinal transport processes. Among its multi- ple biological functions are those related to the absorp- tion, transport, and metabolism of lipids and lipoproteins. Despite their

Polydimethylsiloxane (PDMS) silicone elastomer is extensively used in soft lithography processes to fabricate microscale or nano scale systems for microfluidic or cell culture applications. Though PDMS is biocompatible, it is not an ideal material for cell culture due to its poor cell adhesion properties. In this study, PDMS surfaces were modified to promote intestinal cell adhesion, in the interest of testing feasibility of using microfabricated PDMS systems for high throughput drug screening. Modification techniques included changing chemical composition of PDMS (i.e., varying curing to mixing agent ratio, and oxidization of PDMS surface by oxygen plasma), surface treatment of PDMS by coating with charged molecules (i.e., poly-D-lysine, L-a-phosphatidylcholine, and a layer by layer coating), and deposition of extracellular matrix (ECM) proteins (i.e., laminin, fibronectin, and collagen). The influence of these modifications on PDMS properties, including elastic modulus and surface properties (wettability, chemical composition, topography, and protein adsorption) were characterized. Modification techniques were all found to change PDMS properties and influence the attachment and proliferation of Caco-2 cells over three days of culture to varying degrees. Generally, Caco-2 cells preferred to attach on collagen-coated, fibronectin-coated, and fibronectin-coated oxygen-plasma treated PDMS. The results highlight the importance of considering multiple physical and chemical factors that may be influenced by biomaterial modification and result in altered cell attachment to microfabricated systems, including surface hydrophobicity, chemical composition, stiffness, and topography. This study provides a foundation for further miniaturization, utilizing soft lithography techniques, of Caco-2 cell-based system for high-throughput screening of drug intestinal absorption during lead optimization in drug discovery. The understanding of different surface modifications on adjusting cell adhesion on PDMS allows systemic design of Biomicroelectromechanical Systems (BioMEMS) with tunable cell adhesion properties.

The essential oils of four Teucrium species were studied and 150 components, in all, were identified. All oils were rich in sesquiterpenes (50.1-55.8%). Spathulenol and d-cadinene were the main compounds of Teucrium brevifolium oil; caryophyllene and 4-vinyl guaiacol predominated in Teucrium flavum. Carvacrol and caryophyllene oxide predominated in Teucrium montbretii ssp. heliotropiifolium, while carvacrol and caryophyllene were the most abundant components in Teucrium polium ssp. capitatum. The oil which most effectively inhibited LPS-induced NO production in macrophage cell line RAW 264.7 was that from T. brevifolium (IC 50 = 7.1 lg/ml), followed by T. montbretii ssp. heliotropiifolium and T. polium ssp. capitatum (IC 50 = 16.5 and 29.4 lg/ml, respectively). The in vitro cytotoxic assay on three human cancer cell lines showed that the most antiproliferative oils were those from T. polium ssp. capitatum and T. montbretii ssp. heliotropiifolium on CACO-2 cell lines (IC 50 = 52.7 and 92.2 lg/ml, respectively). The T. brevifolium oil showed a selective cytotoxicity on COR-L23 while significant activity was exerted by T. polium oil on C32.

In this contribution, from a coumarin library consisting of 29 compounds including natural and synthetic derivatives, an active acetylcholinesterase (AChE) inhibitor (coumarin 106) was found. This circumstance leaded us to continue with the pharmacological characterization of coumarin 106. The first study with the coumarin library was performed using a 96-microtiter well plate assay based on Ellman's reaction. Coumarins were assayed at 5 and 30 M, and coumarin 106 was found the most active inhibitor at both concentrations. The follow-up analysis using kinetic studies demonstrated that coumarin 106 displays mixed-type AChE inhibition with a pIC 50 = 4.97 ± 0.09 and K i = 2.36 ± 0.17 M. The ability of this molecule to interact with AChE was further confirmed through computational studies, in which a primary binding was proved to occur at the active gorge site, while a secondary binding was demonstrated at the peripheral anionic site. Also, coumarin 106 was shown to inhibit butyrylcholinesterase (BChE) with slightly lower potency (pIC 50 = 4.56 ± 0.06), and found to be non-toxic in Caco-2 cells. The combination of these findings makes coumarin 106 an attractive molecule for further investigation. This is the first report where AChE inhibitory activity has been associated with coumarin 106, and proof has been given of its convenience as a lead molecule.

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Objectives Flavonoids are phenolic compounds found in most edible fruits and vegetables. Previous studies have demonstrated their biological and beneficial effects on human health. However, their bioavailability and, in particular, their intestinal absorption mechanism have not yet been clearly identified. The aim of our work was to quantify and to characterize in vitro the nature of the transport of two flavonoids distinguished by their physicochemical and pharmacological properties: quercetin, a flavan-3-ol, and naringenin, a flavanone. Methods Differentiated and polarized Caco-2 human intestinal epithelial cell lines were used for this purpose. Key findings In our experimental conditions, quercetin and naringenin were poorly absorbed by Caco-2 cells. Quercetin was absorbed by passive diffusion and a pHdependent mechanism mediated by the organic anion transporting protein B (OATP-B). It was not a multidrug resistance associated protein (MRP)1 substrate, but was substrate of the MRP2 efflux transporter and not P-glycoprotein (P-gp). Intestinal permeability from the apical to the basolateral side was higher for naringenin than for quercetin, which was partly explained by naringenin's physicochemical characteristics. Naringenin, partially absorbed by passive diffusion, was also an ATP-dependent transport substrate mediated by MRP1, but was not an OATP-B substrate. However, naringenin was secreted via active P-gp and MRP2 efflux transporters. Conclusions The contribution of ATP-dependent efflux transporters (MRP2 and P-gp) to the permeability of these compounds in the apical side could explain their low bioavailability. In conclusion, knowledge of the absorption mechanism of these two flavonoids was used to determine the intake level that has a beneficial effect on human health and their putative role in food-drug interactions.

Vitamin D is enzymatically modified to more than 35 metabolites. While many of these are thought to represent degradation products, some have been shown to exhibit biological activity. We tested whether 3-epi-1α,25-dihydroxyvitamin D3 (3-epi-1α,25(OH)2D3), 1α,25-dihydroxy-24-oxo-vitamin D3 (1α,25(OH)2-24-oxo-D3), and 1α,25(OH)2D3-26,23-lactone can stimulate transcription of vitamin D responsive genes. MC3T3-E1 cells transfected with a 25-hydroxyvitamin D 24-hydroxylase (CYP24) promoter construct displayed a

This study aimed to investigate dose effects of dimethyl sulfoxide (DMSO) (0.05-1%) on the intestinal inflammatory response in confluent-and differentiated-Caco-2 cells stimulated with interleukin (IL)-1␤ or a pro-inflammatory cocktail for 24 h. Cyclooxygenase-2 (COX-2) activity was assayed by incubating inflamed cells with arachidonic acid and then measuring prostaglandin-E 2 (PGE 2 ) produced. Soluble mediators (IL-8, IL-6, macrophage chemoattractant protein-1 (MCP-1), and COX-2-derived PGE 2 ) were quantified by enzyme immunoassays and mRNA expression of 33 proteins by high throughput Taq-Man Low Density Array. Data showed that DMSO decreased induced IL-6 and MCP-1 secretions in a dose-dependent manner (P < 0.05), but not IL-8; these effects were cell development-and stimulusindependent. Moreover, in IL-1␤-stimulated confluent-cells, DMSO dose-dependently reduced COX-2derived PGE 2 (P < 0.05). DMSO at 0.5% decreased significantly mRNA levels of 14 proteins involved in the inflammatory response (including IL-6, IL-1␣, IL-1␤, and COX-2). Thus, DMSO at low concentrations (0.1-0.5%) exhibits anti-inflammatory properties in the in vitro intestinal Caco-2 cell model. This point is important to be taken into account when assessing anti-inflammatory properties of bioactive compounds requiring DMSO as vehicle, such as phenolic compounds, in order to avoid miss-interpretation of the results.