Model Of Intestinal Absorption Research Articles

Investigation of the absorption of resveratrol oligomers in the Caco-2 cellular model of intestinal absorption

Resveratrol oligomers are biologically active polyphenols found in wine. No information about the bioavailability of these polyphenols is available. In order to discover if the resveratrol oligomers can pass the intestinal barrier, transport of the dimer ε-viniferin and the tetramer hopeaphenol was studied in the Caco-2 transwell system. A flux through the cell monolayer could neither be observed for ε-viniferin nor for hopeaphenol (apparent permeability coefficient (Papp)<1×10−6cms−1). In contrast, resveratrol showed a Papp of 11.9×10−6cms−1. Nevertheless, about 16–30% of the oligomers were found in the lysed cellular fraction. This leads to the conclusion that the intestinal absorption rate of the two resveratrol oligomers, ε-viniferin and hopeaphenol, is low and negligible when compared to resveratrol. Therefore, it is unlikely that the oligomers could elicit a systemic biological effect after dietary intake. However, the compounds may act locally on the intestinal epithelium.

In vitro absorption studies of chlorogenic acids from coffee using the Ussing chamber model

Hydroxycinnamates are secondary plant metabolites and ubiquitous constituents of plant materials, including many foods and beverages. Hydroxycinnamates are most often conjugated with d-quinic acid, giving rise to the substance class of chlorogenic acids (CGAs). Coffee beans contain high levels of CGAs and are therefore considered the richest dietary source of this substance class. CGAs have been shown to have several beneficial health properties, including antioxidant activity, anticarcinogenic and antidiabetic potential. However, their positive effects on human health highly depend on their bioavailability. In the present study, we investigated the intestinal absorption of hydroxycinnamates using the human colon carcinoma cell line Caco-2, serving as an in vitro model for intestinal absorption. Physiological conditions were modeled using the dynamic Ussing chamber model to simulate apical/basolateral compartments of the mucosa. The test substances (5-O-caffeoylquinic acid (5-CQA), caffeoylquinic acid lactones (CQLs), caffeic (CA) and ferulic acid (FA)) were added individually to the apical compartment of the chamber to physiological concentrations. Structure-dependent absorption was observed, resulting in higher absorption efficiency for FA (12.5±1.0%; 100μM) than CA (0.33±0.13%; 100μM). A low absorption rate for 5-CQA (0.1±0.08%; 100μM) was in line with the existing literature. CQA and CQL-isomers showed comparable absorption efficiencies at 100μM (0.07±0.02%). Concentration-dependent absorption of the more hydrophobic CQLs, as well as their precursor 5-CQA, was found to be non-saturable, indicating a passive diffusion process. Overall, our results indicated that the absorption rates of hydroxycinnamates are substantially influenced by their physicochemical properties.

Applications of a 7-day Caco-2 cell model in drug discovery and development

Oral delivery is the preferred route of administration and therefore good absorption after oral dosing is a prerequisite for a compound to be successful in the clinic. The prediction of oral bioavailability from in vitro permeability assays is thus a valuable tool during drug discovery and development. Caco-2 cell monolayers mimic the human intestinal epithelium in many aspects. These monolayers form tight junctions between cells and have been widely used as a model of human intestinal absorption. Caco-2 cells also express a variety of transporter proteins although the transformed nature of the cells results in unpredictable differentiation markers, transport properties and enzyme expression. Thus various modifications of the Caco-2 assay are used in laboratories across the globe. The purpose of this paper is to provide an overview of a time and resource saving 7-day Caco-2 assay protocol. We also discuss the impact of various experimental conditions on permeability measurements and its applications during lead optimization in early discovery and for clinical candidate characterization, specifically for prediction of absorption in human, at a later stage in drug development.

Permeation and metabolism of Alternaria mycotoxins with perylene quinone structure in cultured Caco-2 cells

The absorption of four Alternaria toxins with perylene quinone structures, i.e. altertoxin (ATX) I and II, alteichin (ALTCH) and stemphyltoxin (STTX) III, has been determined in the Caco-2 cell Transwell system, which represents a widely accepted in vitro model for human intestinal absorption and metabolism. The cells were incubated with the four mycotoxins on the apical side, and the concentration of the toxins in the incubation media of both chambers and in the cell lysate were determined by liquid chromatography coupled with diode array detection and mass spectrometry (LC-DAD-MS) analysis. ATX I and ALTCH were not metabolised in Caco-2 cells, but ATX II and STTX III were partly biotransformed by reductive de-epoxidation to the metabolites ATX I and ALTCH, respectively. Based on the apparent permeability coefficients (Papp), the following ranking order for the permeation into the basolateral compartment was obtained: ATX I > ALTCH >> ATX II > STTX III. Total recovery of the four toxins decreased in the same order. It is assumed that the losses of STTX III, ATX II and ALTCH in Caco-2 cells are caused by covalent binding to cell components due to the epoxide group and/or the α,β-unsaturated carbonyl group present in these toxins. We conclude from this study that ATX I and ALTCH are well absorbed from the intestinal lumen into the portal blood in vivo. For ATX II and STTX III, intestinal absorption of the parent toxins is very low, but these toxins are partly metabolised to ATX I and ALTCH, respectively, in the intestinal epithelium and absorbed as such.

Vitamin E Secretion by Caco-2 Monolayers to APOA1, but Not to HDL, Is Vitamer Selective1,2

The aim of this study was to characterize the pathways of basolateral secretion of common dietary tocopherols from polarized Caco-2 monolayers, a model of intestinal absorption. Given differences in structure and physical properties, we hypothesized that secretion may differ between different forms of vitamin E, thus potentially contribute to the selectivity seen in vivo. Monolayers were incubated apically and simultaneously with 10 μmol/L α-, γ-, and δ-tocopherol (1:1:1) in lipid micelles. Treatment with the microsomal triglyceride transfer protein inhibitor BMS201038 revealed that the triglyceride-rich particle secretory pathway (apolipoprotein B–dependent pathway) accounted for ∼80% of total tocopherol secretion, without selectivity among the three forms of vitamin E. Apolipoprotein B–independent secretion of tocopherols (and cholesterol) was greatly enhanced by the liver X receptor agonist T0901317. T0901317 induced ATP-binding cassette transporter A1 (ABCA1) protein expression and basolateral secretion of tocopherols to apolipoprotein A1. ABCA1-dependent secretion demonstrated vitamer selectivity such that efficiency of secretion of α- and γ-tocopherols exceeded that of δ-tocopherol. Basal addition of HDL stimulated vitamin E secretion but without selectivity among the three forms, whereas LDL had no effect. Basal addition of scavenger receptor class B member I (SR-BI) blocking antibody, which inhibits the interaction between SR-BI and HDL, increased basal accumulation of all tocopherols, demonstrating a role for SR-BI in cellular re-uptake of secreted vitamin E. These findings demonstrated that vitamin E and cholesterol utilize common pathways of secretion and that secretion via the ABCA1 pathway favors certain forms of vitamin E.

The effect of probiotic Escherichia coli strain Nissle 1917 lipopolysaccharide on the 5-aminosalicylic acid transepithelial transport across Caco-2 cell monolayers

The object of this study was to investigate the effect of probiotic Escherichia coli strain Nissle 1917 (EcN) (i) EcN lipopolysaccharide (EcN LPS) and (ii) bacteria-free supernatant of EcN suspension (EcN supernatant) on in vitro transepithelial transport of mesalazine (5-aminosalicylic acid, 5-ASA), the most commonly prescribed anti-inflammatory drug in inflammatory bowel disease (IBD). Effect of co-administered EcN LPS (100 µg/ml) or EcN supernatant (50 µg/ml) on the 5-ASA transport (300 µmol/l) was studied using the Caco-2 monolayer (a human colon carcinoma cell line) as a model of human intestinal absorption. Permeability characteristics for absorptive and secretory transport of parent drug and its intracellularly-formed metabolite were determined. The quantification of 5-ASA and its main metabolite N-acetyl-5-amino-salicylic acid (N-Ac-5-ASA) was performed by high performance liquid chromatography. Obtained results suggest that neither EcN LPS nor EcN supernatant had effect on the total 5-ASA transport (secretory flux greater than absorptive flux) and on the transport of intracellularly formed N-Ac-5-ASA (preferentially transported in the secretory direction). The percent cumulative transport of the total 5-ASA alone or in combination with EcN LPS or EcN supernatant did not exceed 1%.

In Vitro to in Vivo Extrapolation and Physiologically Based Modeling of Cytochrome P450 Mediated Metabolism in Beagle Dog Gut Wall and Liver

The beagle dog is a widely used in vivo model to guide clinical formulation development and to explore the potential for food effects. However, the results in dogs are often not directly translatable to humans. Consequently, a physiologically based modeling strategy has been proposed, using the dog as a validation step to verify model assumptions before making predictions in humans. One current weakness in this strategy is the lack of validated tools to incorporate gut wall metabolism into the dog model. In this study, in vitro to in vivo extrapolation factors for CYP2B11 and CYP3A12 mediated metabolism were established based on tissue enzyme abundance data reported earlier. Thereafter, physiologically based modeling of intestinal absorption in beagle dog was conducted in GastroPlus using V(max) and K(m) determined in recombinant enzymes as inputs for metabolic turnover. The predicted fraction of absorbed dose escaping the gut wall metabolism (F(g)) of all five reference compounds studied (domperidone, felodipine, nitrendipine, quinidine, and sildenafil) were within a two-fold range of the value estimated from in vivo data at single dose levels. However, further in vivo studies and analysis of the dose-dependent pharmacokinetics of felodipine and nitrendipine showed that more work is required for robust forecasting of nonlinearities. In conclusion, this study demonstrates an approach for prediction of the gut wall extraction of CYP substrates in the beagle dog, thus enhancing the value of dog studies as a component in a strategy for the prediction of human pharmacokinetics.

Permeation of roxarsone and its metabolites increases caco-2 cell proliferation.

The benzenearsonate, Roxarsone, has been used since 1944 as an antimicrobial, growth-promoting poultry feed additive. USGS and EPA report that Roxarsone (4-hydroxy-3-nitrobenzenearsonate) and metabolites, including AHBA (3-amino-4-hydroxybenzenearsonate), contaminate waterways at greater than 1100 tons annually. To assess human impact of these organic arsenic water contaminants, it was important to study their potential absorption. The human adenocarcinoma cell line, Caco-2, is a model for intestinal absorption. We found proliferative effects on Caco-2 cells at micromolar levels of these compounds, as monitored by [3H]-thymidine incorporation into DNA. Flow cytometry cell cycle analysis confirmed accumulation in S phase from 21% (control) to 36% (24 hour exposure to 10 μM AHBA). Confluent Caco-2 cells grown on collagen-coated Transwell plates were dosed on the apical side. After exposure, media from apical and basolateral sides were collected separately. Following removal of FBS by 30K centrifugal filtration, the benzenearsonates in the collected media were analyzed by HPLC. Analyses were at wavelengths in the ultraviolet/visible range where the absorbance values were linear with respect to concentration. Concentrations were calculated by comparison with analytically-prepared commercial standards. Results from cells dosed at 10 μM for 24 hours with AHBA, Roxarsone, or Acetarsone indicated 6% - 29% permeation occurring from apical to basolateral side, modeling absorption across intestinal epithelium to the circulatory system. Benzenearsonate feed additives are frequently applied in combination with antibiotics, raising additional health concerns. We conclude that micromolar levels of these benzenearsonates are adequate to stimulate Caco-2 cell proliferation.

Improving Oral Absorption Via Drug-Loaded Nanocarriers: Absorption Mechanisms, Intestinal Models and Rational Fabrication

Although it is acknowledged that the main impediment of orally administered therapeutic agents is their extensive and changeable pre-systemic metabolism, low absorption and instability in harsh environment of the gastrointestinal (GI) tract are also main influential factors, resulting into inadequate and erratic drug bioavailability. To overcome these shortcomings, nanotechnology has offered new promising strategies to prevent and treat a wide variety of diseases by employing different oral drug-carrier structures capable to enhance therapeutic effects and minimize the toxicity of healthy organs or cells. This review, in general, elucidates some considerable features of in vitro oral drug delivery in three different parts. The first one summarizes the main challenges for oral drug delivery and available absorption mechanisms. The second part embodies an in-depth discussion on the role of the intestinal absorption models used to predict permeability, cellular uptake or even toxicity of nanoparticles, resulting into the design of nanocarriers with optimum efficacy for oral delivery. The third section of the literature is devoted, more particularly, to nanocarriers developed for oral absorption in the past few years, including the behavior of nanovehicles upon oral administration with respect to membrane permeability, retention properties and stability, as well as methods which may lengthen residence time in the GI environment or improve drug absorption.

Improving Oral Absorption Via Drug-Loaded Nanocarriers: Absorption Mechanisms, Intestinal Models and Rational Fabrication

Although it is acknowledged that the main impediment of orally administered therapeutic agents is their extensive and changeable pre-systemic metabolism, low absorption and instability in harsh environment of the gastrointestinal (GI) tract are also main influential factors, resulting into inadequate and erratic drug bioavailability. To overcome these shortcomings, nanotechnology has offered new promising strategies to prevent and treat a wide variety of diseases by employing different oral drug-carrier structures capable to enhance therapeutic effects and minimize the toxicity of healthy organs or cells. This review, in general, elucidates some considerable features of in vitro oral drug delivery in three different parts. The first one summarizes the main challenges for oral drug delivery and available absorption mechanisms. The second part embodies an in-depth discussion on the role of the intestinal absorption models used to predict permeability, cellular uptake or even toxicity of nanoparticles, resulting into the design of nanocarriers with optimum efficacy for oral delivery. The third section of the literature is devoted, more particularly, to nanocarriers developed for oral absorption in the past few years, including the behavior of nanovehicles upon oral administration with respect to membrane permeability, retention properties and stability, as well as methods which may lengthen residence time in the GI environment or improve drug absorption.

Study on compound compatibility of Jinglingzi powder based on absorption-metabolism model

To establish an absorption-metabolic model suitable for studying the complex traditional Chinese medicine (TCM) system, with the classic Jinlingzi Powder formula as the example, in order to explore the correlation among absorption behavior and absorption-metabolism behavior of different Jinlingzi Powder formulas and their compound compatibility. An absorption-metabolic model suitable for TCM study was established according to in vivo characteristics of drugs, to combine the intestinal absorption model with the liver microsomal metabolism model. A quantitative analysis was conduced for absorbable components of Jinlingzi Powder and its absorption-metabolism components by HPLC. The model could be used for studies on the absorption-metabolism process of TCM. Among the 15 main components which were derived from Jinlingzi Powder extracts, 10 could be absorbed by intestinal tract. A new component peak was shown after metabolism with the A-M model. The absorbable components of Jinlingzi Powder were related to its compatibility. Toosendan was found to be the major factor impacting the main component-absorption ratio (Ar) and absorption-metabolism ratio (Mr), followed by Rhizoma Corydalis. The absorption-metabolism model suitable for studying the complex traditional Chinese medicine system was established and used for the study on compound compatibility of Jinglingzi Powder. The compatibility of the formula has an impact on absorbable component ratio of Jinlingzi Powder, which helps interpret the theory of formula compatibility from the angle of in vitro compound pharmacokinetics (the difference between absorption and metabolism). Toosendan is the main factor impacting overall absorption and absorption-metabolism, while Rhizoma Corydalis is the minor factor.

The impact of training set data distributions for modelling of passive intestinal absorption

This study presents regression and classification models to predict human intestinal absorption of 645 drug and drug like compounds using percentage human intestinal values from the published dataset by Hou et al. (2007c). The problem with this dataset and other datasets in the literature is there are more highly than poorly absorbed compounds. Any models developed using these datasets will be biased towards highly absorbed compounds and not applicable for use in industry where now more compounds are likely to be poorly absorbed. The study compared two training sets, TS1, a balanced (50:50) distribution of highly and poorly absorbed compounds created by under-sampling the majority high absorption compounds, with TS2, a randomly selected training set with biased distribution towards highly absorbed compounds. The regression results indicate that the best models were those developed using the balanced dataset (TS1). Also for classification, TS1 led to the most accurate models and the highest specificity value of 0.949. In comparison, TS2 led to the highest sensitivity with a value of 0.939. Thus, under-sampling the majority class of the highly absorbed compounds leads to a balanced training set (TS1) that can achieve more applicable in silico regression and classification models for the use in the industry.

Labile complexes facilitate cadmium uptake by Caco-2 cells

The Free Ion Activity Model (FIAM) predicts that metal uptake in biota is related to the free ion activity in the external solution and that metal complexes do not contribute. However, studies with plants have shown that labile metal complexes enhance metal bioavailability when the uptake is rate-limited by transport of the free ion in solution to the uptake site. Here, the role of labile complexes of Cd on metal bioavailability was assessed using Caco-2 cells, the cell model for intestinal absorption. At low Cd2+ concentration (1nM), the CdCln2−n complexes contributed to the uptake almost to the same extent as the free ion. At large Cd2+ concentration (10μM), the contribution of the complexes was much smaller. At constant Cd2+ concentration, Cd intake in the cells from solutions containing synthetic ligands such as EDTA increased as the dissociation rate of the cadmium complexes increased, and correlated well with the Cd diffusion flux in solution measured with the Diffusive Gradient in Thin Films technique (DGT). The Cd intake fluxes in the cells were well predicted assuming that the specific uptake is limited by diffusion of the free Cd2+ ion to the cell surface. Our results underline that speciation of Cd has a major effect on its uptake by intestinal cells, but the availability is not simply related to the free ion concentration. Labile complexes of Cd enhance metal bioavailability in these cells, likely by alleviating diffusive limitations.

Consensus hologram QSAR modeling for the prediction of human intestinal absorption

Consistent in silico models for ADME properties are useful tools in early drug discovery. Here, we report the hologram QSAR modeling of human intestinal absorption using a dataset of 638 compounds with experimental data associated. The final validated models are consistent and robust for the consensus prediction of this important pharmacokinetic property and are suitable for virtual screening applications.

Study on absorption kinetics of betulic acid in rat's intestines

To establish a HPLC-DAD method for determining concentrations of betulic acid and phenol red in intestinal circulation liquid, and probe into the absorption kinetic characteristics of betulic acid at different intestine segments in rats and the effect of different drug concentrations on absorption. The rat intestinal absorption model was established to detect the impact of absorption site, drug concentration and pH value on drug absorption. Within the range from 75-125 mg x L(-1), the absorption rate and the quality concentration of betulic acid had a linear relation, with Ka value keeping unchanged. The absorption rate for each intestinal segment showed no remarkable difference, with Ka values in duodenum, jejunum, ileum and colon being (0.151 +/- 0.0049), (0.159 +/- 0.0056), (0.156 +/- 6.0083), (0.149 +/- 0.0041) h(-1), respectively. Betulic acid is proved to be well absorbed in intestines marked by no specific absorption site in the intestine. The absorption mechanism of the drug conforms to passive transport mechanism and first-order kinetics. The bioavailability of betulic acid preparation can be increased by enhancing the dissolution rate and the solubility.

Bioavailability of vitamin B12 in cows' milk

The natural source of vitamin B₁₂ in human diets comes from animal products. For example, one glass (250 ml) of milk provides approximately 50 % of the RDA (2·4 μg/d). It was hypothesised that the provision of vitamin B₁₂ from milk is more efficiently absorbed than the synthetic form used in vitamin supplements. Pigs (n 10) were used as a model for intestinal absorption of vitamin B₁₂ in humans to compare the net fluxes of vitamin B₁₂ across the portal-drained viscera (PDV; an indicator of intestinal absorption) after ingestion of meals complemented with conventional and vitamin B₁₂-enriched (via injections to cows) milk (raw, pasteurised or microfiltrated) or with equivalent amounts of cyanocobalamin, the synthetic form used in supplements or unsupplemented. Net flux of vitamin B₁₂ across PDV after the ingestion of milk was positive, though not influenced by milk enrichment (P>0·3) or technological processes (P = 0·8) and was greater than after ingestion of equivalent amounts of cyanocobalamin (cyanocobalamin v. all milk, P ≤ 0·003). In fact, net fluxes of this vitamin were not different from 0 after either cyanocobalamin or the meal devoid of vitamin B₁₂ (unsupplemented v. cyanocobalamin, P = 0·7). The cumulative PDV fluxes during the 24 h following ingestion of meals complemented with milk varied from 5·5 to 6·8 μg. These values correspond to an efficiency of intestinal absorption of vitamin B₁₂ from milk varying between 8 and 10 %. Therefore, vitamin B₁₂, which is abundant in cows' milk, is also substantially more available than the most commonly used synthetic form of this vitamin.

In Silico Predictions of ADME-Tox Properties: Drug Absorption

The accurate prediction of the in-vivo pharmacokinetics of a new potential drug compound based on only its virtual structure is the ultimate goal of in-silico ADME-Tox property modeling. A comprehensive review is made on recent studies concerning the A (absorption) in ADME-Tox, i.e. the in-silico modeling of both Caco-2 permeability and human intestinal absorption. The data sets used, the descriptors selected to build the models, the variable selection methods, the modeling techniques and the performed model validation are critically discussed. It was concluded that reliable models which improve the success rate of compound selection and drug development are still lacking. Limiting the quality of the models are, for instance, inappropriate compilation of data sets, lack of an appropriate outlier detection and unrepresentativeness of training and test sets for the data population. The definition of some best practices or guidelines for the different steps of the modeling procedure might improve the predictions and make the procedure uniform, i.e. "standard tools" in drug development would become available.

Black lipid membranes as a model for intestinal absorption of drugs.

Black lipid membranes were generated in isotonic buffer (pH 4-5 and pH 6-5) from egg phosphatidylcholine and intestinal lipid, and the permeability to salicylamide, salicylic acid, p-aminobenzoic acid and tryptophan of these membranes was studied. Electrical resistance of intestinal lipid membranes was higher than that of phosphatidylcholine membranes. The presence of cholesterol produced an increase in the electrical resistance of black lipid membranes and a small decrease in the permeability of membranes to drugs. The permeability coefficient of salicylamide, an uncharged drug, was much larger than the coefficients of the charged drugs examined. The values for salicylic acid and p-aminobenzoic acid were much larger than comparable values predicted from their partition coefficients. Intestinal lipid membranes were more permeable to acidic drugs than phosphatidylcholine membranes. It is suggested that phospholipids and other lipid components of the small intestine may play an important role in the membrane permeability to acidic drugs. This method may be of interest in studying the complex processes of drug absorption from intestine.

Induction of drug efflux protein expression by venlafaxine but not desvenlafaxine

Venlafaxine and its metabolite desvenlafaxine are serotonin-norepinephrine reuptake inhibitors currently prescribed for the treatment of depression. Previously, it was reported that venlafaxine is an inducer of MDR1, the gene responsible for P-glycoprotein (P-gp). The present study expanded upon these findings by examining the effect of venlafaxine and desvenlafaxine on the expression of both P-gp and the breast cancer resistance protein (BCRP) in human brain endothelial cells (HBMEC), an in vitro model of the blood-brain barrier (BBB). The HBMEC were treated for 1 h with various concentrations (500 nM to 50 µM) of venlafaxine and desvenlafaxine. Western blot analysis revealed treatment with venlafaxine significantly induced the expression of P-gp (2-fold) and BCRP (1.75-fold) in a dose-dependent manner, while treatment with desvenlafaxine had no effect on drug efflux transporter expression. To determine the functional significance of this effect, the permeability of a known drug efflux probe, rhodamine 123, across the BBB model and Caco-2 cells, a model of intestinal absorption, were examined. Treatment with venlafaxine (1-50 µM) for 1 h significantly reduced the apical-to-basolateral permeability of R123 across the BBB model (30%) and Caco-2 cell monolayers (25%), indicative of increased drug efflux transporter expression at the apical membrane. Conversely, desvenlafaxine had no effect on R123 permeability in either cellular model. These studies indicate that venlafaxine, but not desvenlafaxine is an inducer of drug efflux transporter expression, which consequently increases the potential for clinical drug-drug interactions. Therefore, based on these preliminary results, caution should be taken when prescribing venlafaxine with other P-gp substrates.

Caco‐2 Cells as a Model for Intestinal Absorption

The Caco-2 cell system, a well characterized intestinal in vitro model, makes it possible to evaluate the ability of chemicals to cross the intestinal barrier, as well as to study their transport mechanisms. Permeability values estimated with this model correlate well with human in vivo absorption data for many drugs and chemicals. As a consequence, the use of the Caco-2 cellular model as a permeability assay to predict oral absorption in humans is growing, and its importance is increasing as a screening tool in drug-discovery strategies for the prediction of intestinal drug permeability. Strict observance of the experimental conditions, coupled with careful control of the cell-culture procedures, are crucial for obtaining a meaningful correlation with in vivo data. Because of the intrinsic variability of this absorption model, it is important to highlight that each experimental system must be calibrated with internal reference compounds, to permit data sharing between different laboratories.