Permeability Of Caco-2 Cell Monolayers Research Articles

Nanofiber-boosted retrograded starch/pectin microparticles for targeted 5-Aminosalicylic acid delivery in inflammatory bowel disease: In vitro and in vivo non-toxicity evaluation

Incorporating 5-aminosalicylic acid (5-ASA) into a colon-specific carrier is crucial for treating inflammatory bowel diseases (IBD), as it enhances therapeutic efficacy, targets the affected regions directly, and minimizes side effects. This study evaluated the impact of incorporating cellulose nanofibers (CNF) on the in vitro and in vivo biological performance of retrograded starch/pectin (RS/P) microparticles (MPs) containing 5-ASA. Using Fourier Transform Infrared (FTIR) Spectroscopy, shifts in the spectra of retrograded samples containing CNF were observed with increasing CNF proportions, suggesting the establishment of new supramolecular interactions. Liquid absorption exhibited pH-dependent behaviors, with reduced absorption in simulated gastric fluid (∼269 %) and increased absorption in simulated colonic fluid (∼662 %). Increasing CNF concentrations enhanced mucoadhesion in porcine colonic sections, with a maximum force of 3.4 N at 50 % CNF. Caco-2 cell viability tests showed biocompatibility across all tested concentrations (0.0625–2.0000 mg/mL). Evaluation of intestinal permeability in Caco-2 cell monolayers demonstrated up to a tenfold increase in 5-ASA permeation, ranging from 29 % to 48 %. An in vivo study using Galleria mellonella larvae, with inflammation induced by LPS, showed reduction of inflammation. Given the scalability of spray-drying, these findings suggest the potential of CNF-incorporated RS/P microparticles for targeted 5-ASA delivery in IBD.

In vitro and in vivo preclinical pharmacokinetic characterization of aficamten, a small molecule cardiac myosin inhibitor

Aficamten, a small molecule selective inhibitor of cardiac myosin, was characterised in preclinical in vitro and in vivo studies. Protein binding in human plasma was 10.4% unbound and ranged from 1.6% to 24.9% unbound across species. Blood-to-plasma ratios ranged from 0.69 to 1.14 across species. Aficamten hepatic clearance in human was predicted to be low from observed high metabolic stability in vitro in human liver microsomes. Aficamten demonstrated high permeability in Caco-2 cell monolayers. Aficamten in vivo clearance was low across species at 8.8, 2.1, 3.3, and 11 mL/min/kg in mouse, rat, dog, and monkey, respectively. The volume of distribution was low-to-high ranging from 0.53 in rat to 11 L/kg in dog. Oral bioavailability ranged from 41% in monkey to 98% in mouse. Aficamten was metabolised in vitro to eight metabolites with hydroxylated metabolites M1a and M1b predominating. CYP phenotyping indicated multiple CYPs (2C8, 2C9, 2D6, and 3A4) contributing to the metabolism of aficamten. Human clearance (1.1 mL/min/kg) and volume of distribution (6.5 L/kg) were predicted using 4-species allometry employing ‘rule-of-exponents’. A predicted 69 hour half-life is consistent with observed half-life in human Phase-1. No CYP-based drug-drug interaction liability as a precipitant was predicted for aficamten.

Enhancing Oral Bioavailability of Domperidone Maleate: Formulation, In vitro Permeability Evaluation In-caco-2 Cell Monolayers and In situ Rat Intestinal Permeability Studies.

The domperidone maleate, a lipophilic agent classified as a Biopharmaceutical Classification System Class II substance with weak water solubility. Self- Emulsifying Drug Delivery System is a novel approach to improve water solubility and, ultimately bioavailability of drugs. This study aimed to develop and characterize new domperidone-loaded self-emulsifying drug delivery systems as an alternative formulation and to evaluate the permeability of domperidone-loaded self-emulsifying drug delivery systems by using Caco-2 cells and via single-pass intestinal perfusion method. Three self-emulsifying drug delivery systems were prepared and characterized in terms of pH, viscosity, droplet size, zeta potential, polydispersity index, conductivity, etc. Each formulation underwent 10, 100, 200, and 500 times dilution in intestinal buffer pH 6.8 and stomach buffer pH 1.2, respectively. Female Sprague Dawley rats were employed for in situ single-pass intestinal perfusion investigations. Results of the study revealed that the ideal self-emulsifying drug delivery systems formulation showed narrow droplet size, ideal zeta potential, and no conductivity. Additionally, as compared to the control groups, the optimum formulation had better apparent permeability (12.74 ± 0.02×10-4) from Caco-2 cell monolayer permeability experiments. The study also revealed greater Peff values (2.122 ± 0.892×10-4 cm/s) for the optimal formulation from in situ intestinal perfusion analyses in comparison to control groups (Domperidone; 0.802 ± 0.418×10-4 cm/s). To conclude, prepared formulations can be a promising way of oral administration of Biopharmaceutical Classification System Class II drugs.

Protein-bound AGEs derived from methylglyoxal induce pro-inflammatory response and barrier integrity damage in epithelial cells by disrupting the retinol metabolism.

Advanced glycation end-products (AGEs) are complex and heterogeneous compounds widely present in processed foods. Previous studies evidenced the adverse effects of AGEs on gut homeostasis, but the precise pathological mechanisms and molecular pathways responsible for the disruption of intestinal barrier integrity by AGEs remain incompletely elucidated. In this study, protein-bound AGEs (BSA-MGO), the most common type of dietary AGE, were prepared by methylglyoxal-mediated glycation, and an in vitro human epithelial colorectal adenocarcinoma (Caco-2) cell model was employed to evaluate the impact of protein-bound AGEs on gut epithelial function. Results showed that exposure to BSA-MGO significantly increased the permeability of Caco-2 cell monolayers as evidenced by the decreased transepithelial electrical resistance value, increased paracellular transport of FITC-dextran, and down-regulated tight-junction proteins. In parallel, BSA-MGO induced pro-inflammatory responses and oxidative stress in the monolayers. Transcriptomic profiling further revealed that BSA-MGO disrupted the retinol metabolism, thereby contributing to the barrier integrity damage in epithelial cells. Overall, these results provide valuable insights into the disrupting effects of dietary AGEs on intestinal barrier function, and the perturbed pathways present potential targets for further exploration of the molecular mechanisms underlying the detrimental effect of processed foods on gut health.

Changes in wheat protein digestibility and allergenicity: Role of Pediococcus acidilactici XZ31 and yeast during dough fermentation

Wheat flour, as the most important source of food globally, is one of the most common causative agents of food allergy. This study aimed to investigate the effects of fermentation on wheat protein digestibility and allergenicity. Protein digestibility were evaluated using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and enzyme-linked immunosorbent assay. The effect of protein on intestinal permeability was investigated by Caco-2 cell monolayers. Co-culture fermentation with Pediococcus acidilactici XZ31 and yeast leads to improvement in digestibility of wheat protein compared to single strain fermentation. Fermentation leads to a decrease in albumin/globulin antigenicity and an increase in gluten R5 reactivity, with the most significant changes in the co-culture group. Digestion strengthen the decrease of protein antigenicity and counteracts the difference in antigenicity induced by fermentation between groups. However, pretreatment with P. acidilactici XZ31 reduces the amount of allergens across Caco-2 monolayer and attenuates the gluten-induced increase in permeability of Caco-2 cell monolayer by reducing actin polymerization and villous atrophy. Co-culture fermentation reduces gluten-induced cell monolayer damage to a greater extent than P. acidilactici XZ31 monoculture. These results gives valuable insight into the effects of P. acidilactici XZ31 fermentation on the allergenicity and toxicity of wheat proteins, which contribute to promoting the application of multi-strain leavening agent in hypoallergenic and gluten-free wheat products.

Wild blueberry (V. angustifolium) improves TNFα-induced cell barrier permeability through claudin-1 and oxidative stress modulation in Caco-2 cells.

Increasing evidence links the impairment of intestinal permeability (IP), a feature of the intestinal barrier, to numerous dysmetabolic and dysfunctional conditions. Several host and environmental factors, including dietary factors, can negatively and/or positively affect IP. In this regard, polyphenol-rich foods including berries have been proposed as potential IP modulators. However, the exact mechanisms involved are not yet fully elucidated. The aim of the present study was to evaluate the effect of a wild blueberry (WB; V. angustifolium) powder, naturally rich in polyphenols, to affect Caco-2 cell monolayer permeability and to identify the potential mechanisms in modulating the IP process. Caco-2 cells were incubated with TNF-α (10 ng mL-1), as a pro-inflammatory stimulus, and supplemented for 24 hours with different concentrations (1 and 5 mg mL-1) of WB powder. The integrity of the intestinal cell monolayer was evaluated by measuring the transepithelial electrical resistance (TEER) and the paracellular transport of FITC-dextran. In addition, the production of the tight junction proteins, such as claudin-1 and occludin, as well as protein carbonyl and 8-hydroxy 2 deoxyguanosine, as oxidative stress markers, were quantified in the supernatant by ELISA kits. Overall, the treatment with WB powder (5 mg mL-1) mitigated the loss of Caco-2 cell barrier integrity, as documented by an increase in TEER and a reduction in FITC values. This modulation was accompanied by an upregulation of claudin-1 and a reduction of 8-OHdG. Conversely, no effect was documented for the lower concentration (1 mg mL-1) and the other IP markers, as well as oxidative stress markers analysed. In conclusion, our findings suggest a potential role of WB in the modulation of cell barrier integrity. This modulation process could be attributed to an increase in claudin-1 expression and a reduction in 8-OHdG. Further studies should be performed to corroborate the results obtained. In addition, since the effects were observed at doses of WB achievable with the diet, these findings should be substantiated also through in vivo approaches.

Raptor knockdown concurrently increases the electrical resistance and paracellular permeability of Caco-2 cell monolayers

AimsAs a critical regulatory point of nutrient sensing, growth and metabolism, the mechanistic target of rapamycin complex 1 (mTORC1) is poised to influence intestinal homeostasis under basal conditions and in disease state. Intestinal barrier integrity ensures tissue homeostasis by closely regulating the permeability of the epithelium to lumenal contents. The role of mTORC1 in the regulation of intestinal barrier function and permeability remains to be fully elucidated. Materials and methodsIn this study, we employed lentivirus-mediated knockdown of mTORC1 signaling-associated proteins Raptor (regulatory-associated protein of mTOR) and TSC2 (tuberin) to ascertain the effects of constitutive activation or repression of mTORC1 activity on barrier function in Caco-2 cell monolayers. Key findingsResults showed that the loss of Raptor concomitantly raised the transepithelial electrical resistance (TEER) and para/transcellular permeability leading to a cell monolayer that is leaky for dextran yet electrically resistant to the movement of ions. Paracellular permeability was linked to the downregulation of tight junction protein expression and enhanced autophagy. Raptor-depleted cells had the highest abundance of myosin binding subunit MYPT1 concomitantly with the lowest abundance of p-MYPT1 (Thr696) and phosphorylated myosin light chain (p-MLC, Ser19) implying that MLC phosphatase activity was increased resulting in MLC relaxation. Although rapamycin suppressed mTORC1 activity and decreased the abundance of tight junction proteins in control cells, rapamycin caused a modest increase of TEER compared to Raptor knockdown. SignificanceThe study showed that epithelium paracellular permeability of small molecular weight dextran is dissociated from TEER.

Design, synthesis and evaluation of alpha lipoic acid derivatives to treat multiple sclerosis-associated central neuropathic pain

Multiple sclerosis-associated central neuropathic pain (MS-CNP) is difficult to alleviate with clinically used pain-killers and so there is a large unmet medical need for novel treatments for alleviating MS-CNP. Although (R)-alpha lipoic acid (ALA) evoked significant pain relief efficacy in a mouse model of multiple sclerosis-associated central neuropathic pain (MS-CNP), this dietary supplement has poor oral bioavailability due to low gastric stability. Eight ester prodrugs of the R enantiomer of ALA [(R)-ALA] were designed encompassing a range of biocompatible hydrophobic and hydrophilic features and synthesized in an effort to identify a prodrug candidate that was stable at gastric and upper gastrointestinal tract (GIT) pH, and that could be released (hydrolyzed by esterases) in the blood to (R)-ALA immediately after absorption into the portal vein (i.e., highly desirable features for pain relief development). These biocompatible hydrophobic and hydrophilic (R)-ALA pro-dugs underwent comprehensive preliminary screening to reveal PD-ALA4 HCl salt (10) as a promising candidate and PD-ALA 7 (8) could be a viable substitute, utilizing enzyme-free gastric and intestinal stability assessments, LogP evaluations, in vitro plasma stability and caco-2 cell monolayer permeability.

Glucose but Not Fructose Alters the Intestinal Paracellular Permeability in Association With Gut Inflammation and Dysbiosis in Mice.

A causal correlation between the metabolic disorders associated with sugar intake and disruption of the gastrointestinal (GI) homeostasis has been suggested, but the underlying mechanisms remain unclear. To unravel these mechanisms, we investigated the effect of physiological amounts of fructose and glucose on barrier functions and inflammatory status in various regions of the GI tract and on the cecal microbiota composition. C57BL/6 mice were fed chow diet and given 15% glucose or 15% fructose in drinking water for 9 weeks. We monitored caloric intake, body weight, glucose intolerance, and adiposity. The intestinal paracellular permeability, cytokine, and tight junction protein expression were assessed in the jejunum, cecum, and colon. In the cecum, the microbiota composition was determined. Glucose-fed mice developed a marked increase in total adiposity, glucose intolerance, and paracellular permeability in the jejunum and cecum while fructose absorption did not affect any of these parameters. Fructose-fed mice displayed increased circulation levels of IL6. In the cecum, both glucose and fructose intake were associated with an increase in Il13, Ifnγ, and Tnfα mRNA and MLCK protein levels. To clarify the relationships between monosaccharides and barrier function, we measured the permeability of Caco-2 cell monolayers in response to IFNγ+TNFα in the presence of glucose or fructose. In vitro, IFNγ+TNFα-induced intestinal permeability increase was less pronounced in response to fructose than glucose. Mice treated with glucose showed an enrichment of Lachnospiracae and Desulfovibrionaceae while the fructose increased relative abundance of Lactobacillaceae. Correlations between pro-inflammatory cytokine gene expression and bacterial abundance highlighted the potential role of members of Desulfovibrio and Lachnospiraceae NK4A136 group genera in the inflammation observed in response to glucose intake. The increase in intestinal inflammation and circulating levels of IL6 in response to fructose was observed in the absence of intestinal permeability modification, suggesting that the intestinal permeability alteration does not precede the onset of metabolic outcome (low-grade inflammation, hyperglycemia) associated with chronic fructose consumption. The data also highlight the deleterious effects of glucose on gut barrier function along the GI tract and suggest that Desulfovibrionaceae and Lachnospiraceae play a key role in the onset of GI inflammation in response to glucose.

Gualou Xiebai Decoction ameliorates increased Caco-2 monolayer permeability induced by bile acids via tight junction regulation, oxidative stress suppression and apoptosis reduction.

Gualou Xiebai Decoction (GXD), a classic prescription, is widely used to dealing with inflammatory diseases in China for thousands of years. Abnormal metabolic state of bile acids (BAs) is confirmed to cause intestinal epithelial barrier dysfunction. In preliminary work, we observed that GXD could decrease intestinal permeability in hyperlipidemia mice. The present study aimed to explore the protective effect of GXD on intestinal mucosa in vitro. Caco-2 cell monolayer permeability among different groups was determined by measuring the concentrations of FITC-dextran in the lower compartments and transepithelial electrical resistance (TEER). Meanwhile, mRNA and protein expressions of tight junctions (TJs) were investigated. Generation of intracellular reactive oxygen species (ROS) and the ratio of cell apoptosis induced by BAs were assessed by fluorescence probe and flow cytometry. GXD was shown to keep the cell monolayer in low permeable status, increase TEER and mRNA and protein expressions of occludin (Ocln) and zonula occluden 2 (ZO2) remarkably in cells challenged with cholic acid (CA), deoxycholic acid (DCA) and glycocholic acid (GCA). However, no significant effects were uncovered against the pathological effects of taurocholic acid (TCA). Meanwhile, generation of ROS and increased levels of apoptotic cells caused by CA, DCA and GCA were dramatically decreased by GXD, which were not observed on TCA. GXD could significantly attenuate intestinal barrier dysfunction induced by BAs via TJs regulation, oxidative stress suppression and cell apoptosis decrease, but such effects and behind mechanisms differed among different kinds of BAs.

Caco-2 Cell Permeability of Flavonoids and Saponins from Gynostemma pentaphyllum: the Immortal Herb.

Gynostemma pentaphyllum (the immortal herb) has been an important component of Chinese Traditional Medicine for millennia. Recent clinical studies have revealed that the plant exhibits numerous beneficial biological activities, making it of interest to the pharmaceutical industry. An extract of the herb contains over 200 individual secondary metabolites including flavonol glycosides and dammarane saponins. To focus attention on the compounds most likely to be responsible for the biological activities, this study predicts the potential oral bioavailability of nine dammarane saponins and five flavonol glycosides from G. pentaphyllum using the Caco-2 cell monolayer permeability model. Two flavonoids, 8 and 9, and four saponins, 10, 11, 12, and 14, exhibited high permeability across the monolayers. The results indicated that a higher degree of glycosylation-facilitated permeability, suggestive of active transport. This study demonstrates the utility of the Caco-2 permeability assay as a method of identifying possible bioavailable compounds from medicinal herbal extracts.

Anisakis simplex products impair intestinal epithelial barrier function and occludin and zonula occludens-1 localisation in differentiated Caco-2 cells.

BackgroundAnisakis spp. are nematode parasites found in a wide range of marine organisms. Human beings may accidentally become infected, showing the symptoms of anisakiasis and allergic responses. There has been evidence of increased intestinal permeability in A. simplex–sensitized subjects and that specific IgE titres increase in some allergic patients when fishery products are re-introduced into their diet. The aims of this work were to study the effect of A. simplex crude extract on the intestinal integrity and permeability by using Caco-2 cell monolayer. To analyse the capacity of Ani s 4 allergen to cross the epithelial barrier.Methodology/Principal findingsCellular bioenergetics, transepithelial electrical resistance, viability, permeability, reactive oxygen species generation and immunofluorescent staining of tight junction proteins were analysed. A. simplex crude extract compromises the Caco-2 cell monolayer integrity in a dose-dependent manner. This effect is detected at 1 hour of culture and integrity is recovered after 24 hours of culture. The epithelial barrier disruption is accompanied by an increase in paracellular permeability and reactive oxygen species production and by a delocalization of occludin and zonula occludens-1. Finally, Ani s 4, a thermostable and resistant to digestion allergen with cystatin activity, is able to cross the epithelial barrier in Caco-2 monolayer and reach a cumulative mean percentage of 22.7% of total concentration in the basolateral side after 24 hours of culture.Conclusions/SignificanceOur results demonstrate that A. simplex induces an early and reversible alteration of integrity and permeability of Caco-2 cell monolayer and that an underlying mechanism of this effect would involve the oxidative stress and disruption of epithelial tight junctions. Additionally, it has been shown that Ani s 4 allergen is able to cross the epithelial barrier. These findings could explain the increased intestinal permeability observed in Anisakis-sensitized patients, the changes over time in IgE sensitization to A. simplex allergens, and the specific IgE persistence in Anisakis allergy.

Structure–bioavailability relationship study of genistein derivatives with antiproliferative activity on human cancer cell

The present study assesses the in vitro and in vivo bioavailability of genistein derivatives, hydroxyalkyl- and glycosyl alkyl ethers (glycoconjugates). Studies were carried out using compounds that exhibit higher in vitro antiproliferative activity in comparison with the parent isoflavone.Based on in vitro experiments using the Parallel Artificial Membrane Permeability Assay (PAMPA) and the Caco-2 cell monolayer permeability model, we found that modification of the isoflavone structure by O-alkylation improved bioavailability in comparison to genistein. Additionally, the structure of the substituent and its position on genistein influenced the type of mechanism involved in the transport of compounds through biological membranes. The PAMPA assay showed that the structure of glycoconjugates had a significant influence on the passive transport of the genistein synthetic derivatives through a biological membrane. Preferentially the glycoconjugates containing O-glycosidic bond were transported and the transport rate decreased as the carbon linker increased. For glycoconjugates, determination of their transport and metabolism through the Caco-2 membrane was not possible due to interaction with the membrane surface, probably by the change of compound structure caused by contact with the cells or degradation in medium.The intestinal absorption and metabolism of genistein and three derivatives, Ram-3, Ram′-3 and Ram-C-4α (Fig. 1), were tested in vivo in rats. We found that in comparison to genistein, glycoconjugates were metabolized more slowly and to a lesser extent. As part of the in vivo research, we performed analysis of compound levels in plasma samples after enzymatic hydrolysis, but in the collected samples, analytes were not observed. We hypothesize that glycoconjugates compounds bind plasma proteins and were removed from the sample.In conclusion, we show that O-functionalization of the natural, biologically active isoflavone genistein can affect biological activity, bioavailability, and the rate of compound metabolism. The position of the substituent, the length of the linker and the structure of sugar moieties provides a tool for the optimization of the derivative's biological properties.

Flavonol glycosides of Rosa multiflora regulates intestinal barrier function through inhibiting claudin expression in differentiated Caco-2 cells

Eijitsu, the fruits of Rosa multiflora Thunberg, is a traditional Japanese natural medicine and used as purgatives. The active constituents were identified as flavonol glycosides, multiflorin A (MF), and multinoside A (MSA), but mechanism of the purgative action is still unknown. We hypothesized that the flavonol glycosides 1 and 2 may exhibit the purgative actions through modulating intestinal epithelial barrier function. Then, this study aimed to investigate their effects on intestinal epithelial barrier function and possible molecular mechanisms in human intestinal Caco-2 cells. MF and MSA decreased transepithelial electrical resistance and increased paracellular permeability of Caco-2 cell monolayers. Expression of claudins (CLDNs) involved in paracellular permeability of ions and low-molecular substances was significantly decreased by the treatment with MF or MSA. The compounds increased the ratio of N-cadherin/E-cadherin, expression of transforming growth factor-β and Slug, and phosphorylation level of Smad3, suggesting epithelial-mesenchymal transition activation, and epithelial-mesenchymal transition inhibition by transforming growth factor-β receptor kinase inhibitors completely recovered the decreased CLDNs expression caused by MF and MSA. Moreover, the increased paracellular permeability and the decreased CLDNs expression by the treatment with MF or MSA for 24 hours recovered to the same extent as the untreated group with the compounds by continuous culture in the growth medium alone for 48 hours. These results suggest that Eijitsu may be effective in preventing or relieving constipation symptoms, unless used chronically.

Multicomponent self nano emulsifying delivery systems of resveratrol with enhanced pharmacokinetics profile

Resveratrol is a drug with high potential for clinical application based on experimental models. Though, resveratrol translation to clinical use has not been successful yet due to its poor pharmacokinetics, related to poor solubility and fast metabolism. The use of drug delivery systems, namely self-emulsifying drug delivery systems (SEDDS), may be a viable strategy to overcome the poor in vivo performance of resveratrol. In this work, a rational development of two different ternary SEDDS was conducted. Experimental data showed that quantitative variations on SEDDS composition impacted dispersion and robustness to dilution of SEDDS, as well as loading capacity and droplet size. Formulations composed of Lauroglycol® 90/Labrasol®/Capryol® PGMC (12.5/75.0/12.5) (Lau/Lab/Cap) and Tween® 80/Transcutol®/Imwitor® 742 (33.3/33.3/33.3) (T80/Trans/Imw) featured improved performance and were selected for further studies. T80/Trans/Imw formulation yield faster emulsification and originated smaller droplet size, with lower cumulative percentile of 90% of particles (D90) (below 200 nm), as compared to the than Lau/Lab/Cap formulation. Higher resveratrol permeation rate was observed in Caco-2 cell monolayer permeability studies for both formulations as compared to the free drug. Reduction of the metabolization and/or efflux of resveratrol was also noticed in the case of SEDDs, as suggested by the increased recovery of total drug. Plasmatic drug concentrations in rats observed after oral gavage indicate that both formulations provided faster resveratrol absorption than free drug, resulting in shorter Tmax values (30 min vs. 2 h). No statistically significant differences were observed for AUC0-t values of both formulations and the free drug. Still, Cmax for the Lau/Lab/Cap SEDDs formulation was 2-fold higher than for the free drug. These findings suggest that SEDDS can increase resveratrol solubility and reduce its metabolization, resulting in an overall improvement of its oral pharmacokinetics profile.

Hydrolysis and Transport of Egg White-Derived Peptides in Caco-2 Cell Monolayers and Everted Rat Sacs.

The objective of this paper was to investigate the hydrolysis and transepithelial transport of egg white peptides in Caco-2 cell monolayers and everted rat sacs. Results showed that egg white peptides had higher permeability but lower degradation in Caco-2 cell monolayers than found for everted rat sacs. Peptides LGAKDSTRT, DGSRQPVDN, VNDLQGKTS, and GKKDPVLKD were identified from not only the basolateral (BL) side of Caco-2 cell monolayers but also the serous side of everted rat sacs, suggesting that these four peptides could be transported intact in both model systems. In addition, there were 24 peptides identified from the apical (AP) side of Caco-2 cell monolayers and the mucosal side of everted rat sacs, indicating potential resistance to hydrolysis by brush border membrane peptidases. Among these, peptides IRDLLER, YAEERYP, and IRNVLQPS were demonstrated as having dipeptidyl peptidase IV (DPP-IV) inhibitory activities with IC50 values of 186.23 ± 15.25, 340.62 ± 4.73, and 598.28 ± 15.12 μM ( P < 0.05), respectively. Furthermore, molecular docking revealed that the DPP-IV inhibitory peptides were predicted to form hydrogen-bonds, π-π bonds, and charge interactions with the activity sites, especially the amino acid residues located in the S2 pocket of DPP-IV, potentially contributing to their DPP-IV inhibitory activities.

In Silico Assessment of ADME Properties: Advances in Caco-2 Cell Monolayer Permeability Modeling.

One of the main goals of in silico Caco-2 cell permeability models is to identify those drug substances with high intestinal absorption in human (HIA). For more than a decade, several in silico Caco-2 models have been made, applying a wide range of modeling techniques; nevertheless, their capacity for intestinal absorption extrapolation is still doubtful. There are three main problems related to the modest capacity of obtained models, including the existence of inter- and/or intra-laboratory variability of recollected data, the influence of the metabolism mechanism, and the inconsistent in vitro-in vivo correlation (IVIVC) of Caco-2 cell permeability. This review paper intends to sum up the recent advances and limitations of current modeling approaches, and revealed some possible solutions to improve the applicability of in silico Caco-2 permeability models for absorption property profiling, taking into account the above-mentioned issues.

Cryptosporidium parvumdisrupts intestinal epithelial barrier function via altering expression of key tight junction and adherens junction proteins

Infection with the protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a widespread diarrhoeal disease. Impaired intestinal epithelial barrier function and increased permeability are most commonly associated with diarrhoeal diseases caused by enteric infections. However, studies on barrier disruption and underlying mechanisms in cryptosporidiosis are extremely limited. Epithelial tight junctions (TJs) and adherens junctions (AJs) are important in maintaining barrier integrity. Therefore, we examined the effects of CP infection on paracellular permeability and on the expression of the major TJ and AJ proteins utilising in vitro, ex vivo, and in vivo models. CP infection (0.5×106 oocysts/well in Transwell inserts, 24hr) increased paracellular permeability (FITC-dextran flux) in Caco-2 cell monolayers and substantially decreased the protein levels of occludin, claudin 4, and E-cadherin. Claudin 3, zonula occludens-1 (ZO1) and α-catenin were also significantly decreased, whereas claudins 1 and 2 and β-catenin were not altered. Substantial downregulation of occludin, claudin 4, and E-cadherin was also observed in response to CP infection ex vivo in mouse enteroid-derived monolayers and in vivo in the ileal and jejunal mocosa of C57BL/6 mice. The mRNA levels of these proteins were also significantly decreased in CP-infected mouse ileum and jejunum but were unaltered in Caco-2 cells. Further, bafilomycin-A, an inhibitor of lysosomal proton pump, partially abrogated CP effects on occludin expression in Caco-2 cells, suggesting a potential role of posttranslational mechanisms, such as induction of protein degradation pathways, in mediating the effects of the parasite. Our studies suggest that disruption of barrier function via downregulation of specific key components of TJ and AJ could be a major mechanism underlying CP infection-induced diarrhoea.

Predicting apparent passive permeability of Caco-2 and MDCK cell-monolayers: A mechanistic model.

Experimentally derived apparent permeabilities, Papp, through cell monolayers such as Caco-2 and MDCK are considered to be an in-vitro gold standard for assessing the uptake efficiency of drugs. Here, we present a mechanistic model that describes ‘passive’ Papp values (i.e., neglecting active transport) by accounting for the different resistances solutes encounter when permeating a cell monolayer. We described three parallel permeation pathways, namely a cytosolic-, paracellular-, and lateral route, each of which consists of a number of serial resistances. These resistances were accounted for via a mechanistic depiction of the underlying processes that are largely based on literature work. For the present Papp dataset, about as much chemicals are dominated by the cytosolic route as were dominated by the paracellular route, while the lateral route was negligible. For the cytosolic route by far the most chemicals found their main resistance in the various water layers and not in the membrane. Although correlations within the subclasses of chemicals dominated by a specific permeation route were rather poor, we could overall satisfyingly predict Papp for 151 chemicals at a pH of 7.4 (R2 = 0.77, RMSE = 0.48). For a specific evaluation of the intrinsic membrane permeability, Pm, a second experimental dataset based on experiments with black lipid membranes, BLM, was evaluated. Pm could be predicted for 37 chemicals with R2 = 0.91 and RMSE = 0.64 log units.

Pulmonary absorption – estimation of effective pulmonary permeability and tissue retention of ten drugs using an ex vivo rat model and computational analysis

Permeation of inhaled drugs across the pulmonary epithelium can regulate the rate and extent of local drug absorption and hence the pulmonary tissue concentration. Therefore, understanding pulmonary epithelial transport could be important for successful design of novel inhaled medicines. To enhance understanding of pulmonary epithelial transport, drug transport data were generated for a set of inhaled compounds (n = 10) in the single-pass, isolated perfused rat lung model. A compartmental in silico model was used to estimate pulmonary permeability and tissue retention. The theoretical model was also used to re-analyze previously obtained historical drug transport data from the isolated perfused lung (n = 10) with re-circulating buffer. This was performed to evaluate the re-circulating model for assessing tissue retention measurements and to increase the number of data points. The tissue retention was an important parameter to estimate to be able to describe the drug transport profiles accurately of most of the investigated compounds. A relationship between the pulmonary permeability and the intrinsic (carrier-mediated transport inhibited) permeability of Caco-2 cell monolayers (n = 1–6) was also established. This correlation (R2 = 0.76, p < .0001) suggests that intrinsic Caco-2 permeability measurements could offer early predictions of the passive transcellular permeability of lung epithelium to candidate drugs. Although, for some compounds a deviation from the correlation suggests that other transport mechanisms may coexist. The compartmental in silico model was successful in describing the pulmonary drug transport profiles of the investigated compounds and has potential for further development to investigate the effects of formulations with different features on the pulmonary overall absorption rate.