Improving the Accuracy of Permeability Data to Gain Predictive Power: Assessing Sources of Variability in Assays Using Cell Monolayers.

The ability to predict the rate of permeation of new compounds across biological membranes is of high importance for their success as drugs, as it determines their efficacy, pharmacokinetics, and safety profile. In vitro permeability assays using Caco-2 monolayers are commonly employed to assess permeability across the intestinal epithelium, with an extensive number of apparent permeability coefficient (Papp) values available in the literature and a significant fraction collected in databases. The compilation of these Papp values for large datasets allows for the application of artificial intelligence tools for establishing quantitative structure-permeability relationships (QSPRs) to predict the permeability of new compounds from their structural properties. One of the main challenges that hinders the development of accurate predictions is the existence of multiple Papp values for the same compound, mostly caused by differences in the experimental protocols employed. This review addresses the magnitude of the variability within and between laboratories to interpret its impact on QSPR modelling, systematically and quantitatively assessing the most common sources of variability. This review emphasizes the importance of compiling consistent Papp data and suggests strategies that may be used to obtain such data, contributing to the establishment of robust QSPRs with enhanced predictive power.

Caco-2 Monolayer as a Model of the Intestinal Barrier: Permeability of Magnesium Salts.

Magnesium, an essential mineral for various physiological functions, is subject to tight regulation within the body. Understanding its absorption across epithelial cell monolayers is crucial for optimizing dietary magnesium intake and therapeutic strategies. The Caco-2 monolayer model, widely recognized for its relevance to the human intestinal epithelium, provides a suitable platform for this investigation. This protocol covers the step-by-step procedures for the cultivation of Caco-2 monolayer preparation of transwell systems. It provides guidance on the setup of magnesium transport experiments, which involve the application of magnesium salts to the apical side of the Caco-2 monolayer and monitoring their transport to the basolateral side.

Effect of Coffee on the Bioavailability of Sterols.

Absorption at the intestinal epithelium is a major determinant of cholesterol levels in the organism, influencing the entry of dietary cholesterol and the excretion of endogenous cholesterol. Several strategies are currently being followed to reduce cholesterol absorption, using both pharmacological agents or food ingredients with hypocholesterolemic properties. Coffee has recently been shown to affect cholesterol bioaccessibility, although it has not been shown if this translates into a decrease on cholesterol bioavailability. In this work, coffee obtained with different commercial roasting (light and dark) and grinding (finer and coarser) was evaluated regarding their effect on cholesterol absorption through Caco-2 monolayers, mimicking the intestinal epithelium. The fluorescent dehydroergosterol was used as a sterol model, which was shown to permeate Caco-2 monolayers with a low-to-moderate permeability coefficient depending on its concentration. In the presence of coffee extracts, a 50% decrease of the sterol permeability coefficient was observed, showing their potential to affect sterol bioavailability. This was attributed to an increased sterol precipitation and its deposition on the apical epithelial surface. A higher hypocholesterolemic effect was observed for the dark roasting and finer grinding, showing that the modulation of these technological processing parameters may produce coffees with optimized hypocholesterolemic activity.

Permeability-enhancing effects of three laurate-disaccharide monoesters across isolated rat intestinal mucosae.

Laurate (C12)-sucrose esters are established intestinal epithelial permeation enhancers (PEs) with potential for use in oral delivery. Most studies have examined blends of ester rather than specific monoesters, with little variation on the sugar moiety. To investigate the influence of varying the sugar moiety on monoester performance, we compared three monoesters: C12-sucrose, C12-lactose, and C12-trehalose. The assays were: critical micellar concentration (CMC) in Krebs-Henseleit buffer, MTS and lactate dehydrogenase assays in Caco-2 cells, transepithelial electrical resistance (TEER) and apparent permeability coefficient (Papp) of [14C] mannitol across isolated rat intestinal mucosae, and tissue histology. For CMC, the rank order was C12-trehalose (0.21 mM) < C12-sucrose (0.34 mM) < C12-lactose (0.43 mM). Exposure to Caco-2 cells for 120 min produced TC50 values in the MTS assay from 0.1 to 0.4 mM. Each ester produced a concentration-dependent decrease in TEER across rat mucosae with 80% reduction seen with 8 mM in 5 min, but C12-trehalose was less potent. C12-sucrose and C12-lactose increased the Papp of [14C] mannitol across mucosae with similar potency and efficacy, whereas C12-trehalose was not as potent or efficacious, even though it still increased flux. In the presence of the three esters, gross intestinal histology was unaffected except at 8 mM for C12-sucrose and C12-lactose. In conclusion, the three esters enhanced permeability likely via tight junction modulation in rat intestinal tissue. C12-trehalose was not quite as efficacious, but neither did it damage tissue to the same extent. All three can be considered as potential PEs to be included in oral formulations.

Natural P-gp inhibitor EGCG improves the acteoside absorption in Caco-2 cell monolayers and increases the oral bioavailability of acteoside in rats.

Acteoside is one of the most widespread phenylethanoid glycosides with pharmacological activities, including antioxidant, neuroprotective property, etc. However, its bioavailability is poor due to the low absorption and P-gp efflux. This study aimed to select food derived P-gp inhibitors for promoting the acteoside absorption and investigate whether the inhibitors could increase the bioavailability and stability of acteoside. Results showed that EGCG and quercetin significantly decreased the BL-to-AP efflux and promoted the AP-to-BL influx of acteoside across Caco-2 monolayers with optimum concentrations of 320 µM EGCG or 240 µM quercetin adding to 320 µM acteoside. EGCG increased the bioavailability of acteoside to 1.43-fold, but quercetin had no such effect. Further study showed that EGCG and quercetin had no effects on the storage and digestion stability of acteoside. This work revealed that EGCG could improve the acteoside absorption across the Caco-2 monolayers and enhance the bioavailability of acteoside in rats.

The inhibitory role of purinergic P2Y receptor on Mg2+ transport across intestinal epithelium-like Caco-2 monolayer.

The mechanism of proton pump inhibitors (PPIs) suppressing intestinal Mg2+ uptake is unknown. The present study aimed to investigate the role of purinergic P2Y receptors in the regulation of Mg2+ absorption in normal and omeprazole-treated intestinal epithelium-like Caco-2 monolayers. Omeprazole suppressed Mg2+ transport across Caco-2 monolayers. An agonist of the P2Y2 receptor, but not the P2Y4 or P2Y6 receptor, suppressed Mg2+ transport across control and omeprazole-treated monolayers. Omeprazole enhanced P2Y2 receptor expression in Caco-2 cells. Forskolin and P2Y2 receptor agonist markedly enhanced apical HCO3- secretion by control and omeprazole-treated monolayers. The P2Y2 receptor agonist suppressed Mg2+ transport and stimulated apical HCO3- secretion through the Gq-protein coupled-phospholipase C (PLC) dependent pathway. Antagonists of cystic fibrosis transmembrane conductance regulator (CFTR) and Na+-HCO3- cotransporter-1 (NBCe1) could nullify the inhibitory effect of P2Y2 receptor agonist on Mg2+ transport across control and omeprazole-treated Caco-2 monolayers. Our results propose an inhibitory role of P2Y2 on intestinal Mg2+ absorption.

SEDDS for intestinal absorption of insulin: Application of Caco-2 and Caco-2/HT29 co-culture monolayers and intra-jejunal instillation in rats.

To face the challenges of oral delivery of peptide and protein (P/P) drugs, self-emulsifying drug delivery systems (SEDDSs) containing monoacyl phosphatidylcholine (MAPC), Labrasol (LAB) and medium-chain (MC) monoglycerides as permeation enhancers (PEs) were evaluated for their effect on intestinal absorption of insulin. In this study, insulin was complexed with phosphatidylcholine (SPC) to form an insulin-SPC complex (ins-SPC) with increased lipophilicity. The following three SEDDSs: MCT(MAPC) (MC triglycerides and MAPC included), MCT(RH40) (MC triglycerides and Kolliphor® RH40 included) and LCT(MAPC) (long-chain triglycerides and MAPC included) were loading with ins-SPC (4% or 8% w/w of SPC). Three SEDDSs generated emulsions with droplet sizes between 50 and 470 nm and with zeta potentials between -5 to -25 mV in a simulated intestinal medium. Mucus-secreting Caco-2/HT29-MTX-E12 co-culture and Caco-2 monolayers were used as in vitro cell transport models to investigate insulin permeability. In comparison to insulin HBSS solution, MCT(MAPC) significantly increased the insulin permeability across co-culture and Caco-2 monolayers (2.0-2.5 × 10-7 cm/s). In an intra-jejunal (i.j.) instillation model in rats, MCT(RH40) significantly decreased the rat blood glucose after 0.5 h by 17.0 ±â€¯2.5% and for MCT(MAPC), it was 23.6 ±â€¯10.6%. Furthermore, a lipase inhibitor orlistat was incorporated into MCT(MAPC) to evaluate the effect of lipid digestion on insulin absorption. Results indicated that the incorporation of orlistat did not significantly alter the in vivo insulin absorption. Overall, the SEDDS MCT(MAPC) composed of natural PEs (MAPC and MC glycerides) and synthetic PE (LAB) significantly increased the intestinal absorption of insulin upon i.j. instillation. Although it is not possible to conclude if a single PE is dominating the intestinal absorption of insulin, MCT(MAPC) seems to have the potential for oral insulin delivery.

Development of a Non-Aqueous Dispersion to Improve Intestinal Epithelial Flux of Poorly Permeable Macromolecules.

Intestinal permeation enhancers (PEs) offer an attractive strategy to enable oral peptide administration. However, optimal presentation of peptide and PE from solid-dosage forms is offset by slow dissolution rates in the small intestine, which reduces the likelihood that the PE can reach the threshold concentration for sufficient permeability enhancement. The purpose of this study was to design a PE-based liquid dispersion that can improve intestinal permeation of macromolecules across Caco-2 monolayers and isolated rat/human intestinal mucosae mounted in Ussing chambers. An enhancer screen in monolayers based on permeability (TEER, Papp [14C]-mannitol) and cytotoxicity (MTT assay) initially identified methyl 10-hydroxydecanoate (10-OHC10CH3) as a candidate. 10-OHC10CH3 (20 mM) increased the Papp of fluorescent dextran of 4 kDa (FD4) (167-fold), 10 kDa (FD10) (429-fold), and 40 kDa (FD40) (520-fold) across monolayers. Blends of 10-OHC10CH3 with low molecular weight PEGs (0.2-1 kDa) formed liquid dispersions in which enhancement capacity across monolayers of 10-OHC10CH3 was increased over 10-OHC10CH3 alone in the order PEG200 < PEG400 < PEG600 < PEG1000. Finally, a 1:5 ratio of 10-OHC10CH3 (10-20 mM)/PEG600 (50-100 mM) increased the Papp of [14C]-mannitol across rat and human intestinal mucosae. This study highlights the potential future role for non-aqueous, PE-based liquid dispersions in oral delivery of macromolecules.

Intestinal absorption mechanism of saikosaponin d in vitro and in vivo / 中国药科大学学报

@#In order to reveal the intestinal absorption mechanism of saikosaponin d (SSd) in vitro and in vivo, the current research investigated the effects of different experimental conditions (time, concentration, temperature, pH, intestinal segments), transporter inhibitors, paracellular pathway enhancer, metabolic enzyme inhibitors on the intestinal absorption of SSd, in Caco-2 monolayers and a single pass perfusion model in rats.The results showed that the apparent permeability coefficient (Papp) and effective permeability coefficient (Peff) of SSd were 4.75 × 10-7 - 6.38 × 10-7 cm/s and 0.19 × 10-4- 0.27 × 10-4 cm/s, respectively, indicating that it was a low permeability compound, and that the transmembrane transport of SSd was concentration-dependent (0.5-5 μmol/L) and time-dependent (0-180 min).Ileum was the main absorption site for SSd. Experimental results based on Caco-2 monolayers showed that the P-gp inhibitor and paracellular permeability enhancer significantly increased the absorption of SSd (P < 0.05), which was consistent with the results obtained in rats. Inhibitors of OATPs and OCTs showed different results in vitro and in vivo, which may be related to the lower expression of them in jejunum.In summary, the intestinal absorption of SSd occurs through a carrier-mediated and energy-dependent transport, as well as passive diffusion, and P-glycoprotein plays an important role in the active transport of SSd.

Sodium caprate-induced increases in intestinal permeability and epithelial damage are prevented by misoprostol.

Epithelial damage caused by intestinal permeation enhancers is a source of debate concerning safety. The medium chain fatty acid, sodium caprate (C10), causes reversible membrane perturbation at high dose levels required for efficacy in vivo, so the aim was to model it in vitro. Exposure of Caco-2 monolayers to 8.5mM C10 for 60min followed by incubation in fresh buffer led to (i) recovery in epithelial permeability (i.e. transepithelial electrical resistance (TEER) and apparent permeability coefficient (Papp) of [(14)C]-mannitol), (ii) recovery of cell viability parameters (monolayer morphology, plasma membrane potential, mitochondrial membrane potential, and intracellular calcium) and (iii) reduction in mRNA expression associated with inflammation (IL-8). Pre-incubation of monolayers with a mucosal prostaglandin cytoprotectant was attempted in order to further decipher the mechanism of C10. Misoprostol (100nM), inhibited C10-induced changes in monolayer parameters, an effect that was partially attenuated by the EP1 receptor antagonist, SC51322. In rat isolated intestinal tissue mucosae and in situ loop instillations, C10-induced respective increases in the [(14)C]-mannitol Papp and the AUC of FITC-dextran 4000 (FD-4) were similarly inhibited by misoprostol, with accompanying morphological damage spared. These data support a temporary membrane perturbation effect of C10, which is linked to its capacity to mainly increase paracellular flux, but which can be prevented by pre-exposure to misoprostol.

A novel concentration dependent amino acid ion pair strategy to mediate drug permeation using indomethacin as a model insoluble drug.

Assessment of oral drug bioavailability is an important parameter for new chemical entities (NCEs) in drug development cycle. After evaluating the pharmacological response of these new molecules, the following critical stage is to investigate their in vitro permeability. Despite the great success achieved by prodrugs, covalent linking the drug molecule with a hydrophobic moiety might result in a new entity that might be toxic or ineffective. Therefore, an alternative that would improve the drug uptake without affecting the efficacy of the drug molecule would be advantageous. The aim of the current study is to investigate the effect of ion-pairing on the permeability profile of a model drug: indomethacin (IND) to understand the mechanism behind the permeability improvement across Caco-2 monolayers. Arginine and lysine formed ion-pairs with IND at various molar ratios 1:1, 1:2, 1:4 and 1:8 as reflected by the double reciprocal graphs. The partitioning capacities of the IND were evaluated using octanol/water partitioning studies and the apparent permeabilities (Papp) were measured across Caco-2 monolayers for the different formulations. Partitioning studies reflected the high hydrophobicity of IND (LogP=3) which dropped upon increasing the concentrations of arginine/lysine in the ion pairs. Nevertheless, the prepared ion pairs improved IND permeability especially after 60 min of the start of the experiment. Coupling partitioning and permeability results suggest a decrease in the passive transcellular uptake due to the drop in IND portioning capacities and a possible involvement of active carriers. Future work will investigate which transport gene might be involved in the absorption of the ion paired formulations using molecular biology technologies.

Combined probiotic bacteria promotes intestinal epithelial barrier function in interleukin-10-gene-deficient mice.

AIM: To investigate the protective effects of combinations of probiotic (Bifico) on interleukin (IL)-10-gene-deficient (IL-10 KO) mice and Caco-2 cell monolayers. METHODS: IL-10 KO mice were used to assess the benefits of Bifico in vivo. IL-10 KO and control mice received approximately 1.5 × 10(8) cfu/d of Bifico for 4 wk. Colons were then removed and analyzed for epithelial barrier function by Ussing Chamber, while an ELISA was used to evaluate proinflammatory cytokines. The colon epithelial cell line, Caco-2, was used to test the benefit of Bifico in vitro. Enteroinvasive Escherichia coli (EIEC) and the probiotic mixture Bifico, or single probiotic strains, were applied to cultured Caco-2 monolayers. Barrier function was determined by measuring transepithelial electrical resistance and tight junction protein expression. RESULTS: Treatment of IL-10 KO mice with Bifico partially restored body weight, colon length, and epithelial barrier integrity to wild-type levels. In addition, IL-10 KO mice receiving Bifico treatment had reduced mucosal secretion of tumor necrosis factor-α and interferon-γ, and attenuated colonic disease. Moreover, treatment of Caco-2 monolayers with Bifico or single-strain probiotics in vitro inhibited EIEC invasion and reduced the secretion of proinflammatory cytokines. CONCLUSION: Bifico reduced colon inflammation in IL-10 KO mice, and promoted and improved epithelial-barrier function, enhanced resistance to EIEC invasion, and decreased proinflammatory cytokine secretion.

κ-carrageenan induces the disruption of intestinal epithelial Caco-2 monolayers by promoting the interaction between intestinal epithelial cells and immune cells.

κ-carrageenan (κ-CGN) is an important food additive that has been demonstrated to induce colitis in animal models. In the present study, the effects of κ-CGN were assessed using an in vitro co-culture system that contained intestinal epithelial Caco-2 cells and activated macrophage-like THP-1 cells. The results demonstrated that in single cultures of Caco-2 and THP-1 cells treated with κ-CGN, the cytotoxicity and the secretion levels of IL-1ß, IL-6 and TNF-α were low. In the co-culture system, however, κ-CGN treatment resulted in apoptosis and reduced the transepithelial electrical resistance of the Caco-2 cell monolayers. The secretion levels of TNF-α, IL-1ß and IL-6 from the two cell types were increased significantly by κ-CGN treatment. Furthermore, pretreatment of the co-culture system with anti-TNF receptor 1 antibody suppressed the κ-CGN-induced apoptosis and attenuated the changes in the levels of IL-6 and IL-1ß in the Caco-2 monolayers. This study indicated that κ-CGN-induced TNF-α secretion is the main contributor to cellular damage in Caco-2 monolayers exposed to κ-CGN.