Caco2 cell permeability assays to measure drug absorption. Physicochemical qsar analysis of passive permeability across. The caco 2 assay models the epithelial cell layer permeability barrier in the small intestine. Automated caco2 assay automated caco2 permeability assay is performed using semi or fully automated procedures. If high permeability is inferred by means of an in vitro cell system, permeability independent of active transport should be proven as outlined in annex i, caco 2 cell permeability assay method considerations. Calculation of the apparent permeability coefficient and the efflux ratio 106 6.
Due to the small pore size of the tight junction, the caco2 system is less permeable to compounds that are absorbed paracellularly. Application of caco2 cell line in herbdrug interaction. The caco 2 cell line is a continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells, developed by the sloankettering institute for cancer research through research conducted by dr. Experimental results for caco2 ab and caco2 ab intrinsic assays. Preliminary bcs permeability classification using caco2 cell. In vitro intestinal permeability studies, pharmacokinetics. The caco2 model is employed in preclinical investigations to predict the likely gastrointestinal permeability of drugs because it expresses cytochrome p450 enzymes, transporters, microvilli and enterocytes of identical characteristics to the human small intestine. Determination of drug absorption parameters in caco2 cell. Caco 2 assay june 9, 2010 21 bovine serum albumin bsa no significant nonspecific binding to wells was observed in the caco 2 system addition of bsa did not improve aqueous recovery, but increased permeability liu t, j.
Caco2 wild type cell line 24 well assay ready plate catalog number mtoxp24 store at room temperature technical bulletin product description this product contains wild typecaco2 cells c2bbe1 subclone that have been differentiated for 14 days in a millicell multiwell insert 24 well assay ready plate see figure 1. Bovine serum albumin in caco 2 permeability testing and effect on p app values determined with highly protein bound compounds elke s. Caco2 cell lines in drug discovery an updated perspective kalyan kumar. Pdf caco2 cells are a human colon epithelial cancer cell line used as a model of human intestinal absorption of drugs and other compounds. Advanced experimental models and proteomics in nanomedicine and molecular medicine. Permeability data will vary quantitatively between laboratories, however there should be general agreement of the qualitative activity for different compounds in differentiated caco 2 cultures in their ability to demonstrate these transport functions. The xanthanchitosancurcumin nanofibers were incubated with caco 2 cells, and the cell viability, transepithelial transport and permeability properties across cell monolayers were investigated. In the caco 2 cell permeability assessment of drugs and herbal supplements, adequate expression of transport proteins can be utilized to predict the possible degree of bioavailability upon coadministration. Experimentally derived apparent permeabilities, papp, through cell monolayers such as caco2 and mdck are considered to be an invitro. The caco 2 cells isolated from human colorectal adenocarcinoma are widely used to screen for absorption rate of new compounds in the initial stage of drug discovery. Most transporters aforementioned can be expressed in caco 2 cell under appropriate culture media. The apparent permeabilities of caco2 cells to marketed drugs peerj. Thus, separating the apical compartment from the basolateral compartment which corresponds to the intestinal lumen side and the serosal side respectively is possible. Cyprotexs caco2 permeability assay uses an established method that measures the rate of flux of a compound across polarised caco2 cell monolayers and from which the data generated can be used to predict in vivo absorption of drugs.
Characterization of the human colon carcinoma cell line. Drug discell lines are the invitro models used for the drug permeability studies in. The caco 2 monolayer has been used extensively for the highthroughput screening of drug permeability and identification of substrates, inhibitors, and inducers of intestinal transporters. When cultured as a monolayer, caco2 cells differentiate to form tight junctions between cells to serve as a model of paracellular movement of. Drugpermeability and transporter assays in caco2 and mdck. Caco 2 permeability values, but are predicted by traditional pampa as low permeability. Typically, the substance to be studied is added to the apical side of a confluent monolayer of caco 2 cells and permeability is measured by monitoring the appearance of the test compound on the opposite side. Capa can provide a better understanding of how to improve the cell permeability of degraders and may help to. Permeability of 5fluorouracil and prodrugs in caco 2 cell monolayers. The results from caco 2 permeability assays should be discussed in the context of available data on human pharmacokinetics. The permeability of 6mc was evaluated by the caco2 cellular model in both the apicalbasal ab.
Comparison of mdckmdr1 and caco 2 cell based permeability assays for antimalarial drug screening and drug investigations. Intestinal permeability and transport of apigenin across. Drugtransporter mediated interactions between anthelminthic. Caco 2 cells are derived from human adenocarcinoma colon cells and spontaneously differentiate when grown on porous polyethylene terephthalate membranes petp in a 12 well format to form monolayers of polarized cells possessing function similar to. Caco 2 cells are a widely used model to assess passive permeability and mdr1, bcrp, and mrp2 mediated active efflux of compounds. Caco 2 permeability assay suitability demonstration. Pdf determination of drug permeability and prediction of drug. Thus, cultivation conditions can be expected to select for the growth of subpopulations of cells resulting in a cellular model system with properties that may differ from the original cell line. Caco 2 cells are most commonly used not as individual cells, but as a confluent monolayer on a cell culture insert filter e.
Moreover, to develop relatively universal predictive models, we used a large set of caco2 permeability data from a variety of sources. From table 4, it can be found that even for the same compound, the experimental logpapp values from different sources exist obvious variation. Caco2 cell monolayer is a suitable model to assay the permeability of drugs in vitro, and is used to predict the bioavailability in vivo 8. Caco2 cells are not used as individual cells but form confluent monolayer on the insert membrane. Nov 17, 2015 the apparent permeability of caco 2 cells to marketed drugs is poorly correlated with either simple biophysical properties, the extent of molecular similarity to endogenous metabolites endogenites, or any specific substructural properties. In vitro permeability enhancement of curcumin across caco. Setup instruments tissue culture co2 incubator with humidity control liquid handler orbital shaker evom epithelial voltohmmeter fitted with planar. Caco 2 cell lines are the principle cell type for studies of nanoparticle translocation. While the caco 2 assay is the gold standard for in vitro permeability assessment, interpretation of the results from such assays needs careful attention due to high labtolab variability 6, 8. Biouptake and bioabsorption via caco2 permeability assay. Data sheet caco2 permeability concept life sciences. Caco2 cells, passage 5570, were cultured on transwell cell culture supports and permeability assays were performed on day 21. Caco 2 permeability assay caco 2 permeability goal to measure directional caco 2 permeability of test compounds in cultured caco 2 monolayer.
The whole data set includes 100 drugs or druglike molecules. Assessing the cell permeability of bivalent chemical. The apparent permeabilities of caco2 cells to marketed. Permeability, transport, and metabolism of solutes in caco2. Permeability, transport, and metabolism of solutes in caco. Permeability measurements in caco 2 and mdckmdr1 cells for tested antimalarial compounds. When cultured in this format, the cells differentiate to form a polarized epithelial cell monolayer that provides a physical and biochemical barrier to the passage of ions and small molecules. Elucidation of the impact of cell culture conditions of caco. Experimental results for caco2 ab and caco 2 ab intrinsic assays. They also express cell membrane transporters on the apical surface, such as pglycoprotein, bcrp and mrp2. Caco 2 cells are not used as individual cells but form confluent monolayer on the insert membrane. Development of in vitro pharmacokinetic screens using caco. Elucidation of the impact of cell culture conditions of. Caco2 cells develop microvilli on their apical surface that resemble the morphology of gi epithelial cells that line the intestinal villi.
Caco2 cell lines are the principle cell type for studies of nanoparticle translocation. The human colon carcinoma cell line caco 2 has been shown to acquire many of the features of absorptive intestinal cells when grown as a confluent monolayer. Pdf permeability coefficients across monolayers of the human colon carcinoma cell line caco2, cultured on permeable supports, are commonly used to. Indeed, some authors report in caco 2 a low paracellular permeability and an ease of. Predicting apparent passive permeability of caco2 and mdck cell. Research on predicting caco 2 permeability from structures of compounds using quantitative structure property relationship qspr modeling. Due to these features, drug permeability in caco 2 cells correlates well with human oral absorption, making caco 2 an ideal in vitro permeability model. This study used a boxbehnken experimental design to optimise the experimental conditions in the caco2 assay for a series of phydroxybenzoate ester compounds log p 1. Caco2 permeability assay to investigate intestinal permeability.
Caco 2 cell permeability assays to measure drug absorption richard b van breemen university of illinois college of pharmacy, department of medicinal chemistry and pharmacognosy, 833 s. Our ultimate goal is to find a set of simple descriptors to estimate caco2 permeability as high throughput fashion. The caco2 cell line is derived from a human colon carcinoma. There is a slightly clearer relationship between caco2 permeability and a drugs total polar surface area, but again the relationship is fairly weak r 2 0.
The model mimics processes such as transcellular transport, paracellular transport, and some aspects of efflux and active transport. The caco 2 cell model has the advantages of simplicity and reproducibility. We explored the properties of a catenary model that includes the basolateral b, apical a, and cellular compartments via simulations under linear and nonlinear conditions to understand the asymmetric observations arising from transporters, enzymes, and permeability in caco 2 cells. In this study, a database of 66 caco2 permeability. The caco 2 permeability assay utilizes a caco 2 cell layer grown on a supportive membrane surface that separates two compartments. Calculation of the apparent permeability coefficient and the efflux ratio. Solubility and intestinal permeability are the mostimportant factors determining oral absorption of drugs. February evaluation of the intestinal permeability of rosemary rosmarinus officinalis l. Quantitative analysis of labtolab variability in caco2. Caco 2 permeability assay to investigate intestinal permeability. This is a pdf file of an unedited manuscript that has been accepted for publication. This cell line in culture forms monolayers with an enterocytelike phenotype and exhibits properties associated with the physical and metabolic barrier.
The apparent permeability of caco2 cells to marketed drugs is poorly correlated with either simple biophysical properties, the extent of molecular similarity to endogenous metabolites endogenites, or any specific substructural properties. Pampa plate system with high caco 2 and human absorption predictability. Isbn 9789512708437 isbn 9789512706358 pdf issn 12350478 abstract oral administration is the most convenient and popular route for drug administration. Caco2 cells are a human colon epithelial cancer cell line used as a model of human intestinal absorption of drugs and other compounds. The objective of this study is to evaluate the caco 2 permeability and possible pgp efflux transport of a series of heterocyclic drugs.
Improvement of the caco 2 permeability model by genetic and hydrodynamic modifications. Borchardt department of pharmaceutical chemistry, university of kansas, lawrence, kansas, and drug delivery systems research, the upjohn company, kalamazoo, michigan caco 2 cells develop morphologic characteristics of. Evaluation of optimized caco2 permeability measurements oksana tsinman, konstantin tsinman,and alex avdeef pion inc. Caco2 permeability, pglycoprotein transport ratios and. Cyprotexs caco 2 permeability assay uses an established method that measures the rate of flux of a compound across polarised caco 2 cell monolayers and from which the data generated can be used to predict in vivo absorption of drugs. Furthermore, using the pgp efflux transport data of the caco 2 experiments it is examined whether pgp efflux influences brain penetration in rats in vivo. This should be kept in mind when the modelling accuracy is assessed. Drug interactions between antiretroviral drugs arvs and anthelminthic drugs, ivermectin ivm and praziquantel pzq were assessed by investigating their permeation through the caco2 cell monolayers in a transwell. An hplcuv method for the measurement of permeability of. Combining the permeability data derived from caco 2 and mdck cell monolayers in one dataset is commonly done in the literature but goes along with some uncertainties, which are generally smaller than one order of magnitude 9,14,21,22. However, the quantitative link between in vitro permeability and apparent in vivo absorption kinetics, based on drug appearance in plasma, is poorly understood.
Estimation of caco 2 cell permeability using calculated molecular descriptors. The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. Optimisation of the caco2 permeability assay using. Comparison of function and relative transporter protein. Evaluation of the intestinal permeability of rosemary. Caco2 permeability assay caco 2 permeability goal to measure directional caco2 permeability of test compounds in cultured caco2 monolayer.
Some sticky compounds are trapped in the artificial membrane, resulting in high mass retention values. Us fda recognizes caco 2 to measure permeability as part of the bioequivalence waiver process. Caco2 screening as well as the correlation between caco2 permeability and absorption in humans. Caco 2 cell monolayers are used to determine an apparent permeability coefficient, which allows the ranking of compounds as highly, moderately, or poorly permeable relative to well characterised reference standards. Optimized conditions for prediction of intestinal drug permeability using caco2 cells. Apr 25, 2017 advanced experimental models and proteomics in nanomedicine and molecular medicine. Pdf permeability of 5fluorouracil and prodrugs in caco. Comparison of mdckmdr1 and caco2 cell based permeability.
Predicting caco2 permeability using support vector. Caco 2 cells also express a variety of transport systems including dipeptide transporters and pglycoproteins pgp. Pdf caco2 cell permeability assays to measure drug. Estimating human drug oral absorption kinetics from caco2. The impact of anthelminthics on the transport of arvs was determined by assessing the apical to basolateral ap bl passive and basolateral to apical bl ap efflux. Caco2 cell line is frequently used as a simplified in vitro model of intestinal absorption.
Figure 1 below schematically shows transcellular p. A drug substance with respect to solubility and permeability can be classified in 4 categories. Caco 2 cells develop microvilli on their apical surface that resemble the morphology of gi. Mathematical model for determining drug absorption parameters in caco 2 cell monolayers 107 6. The cell lineage caco 2 is a model commonly used to reproduce the features of the bowel epithelium. As a majority of discovery projects are focused on the development of orally bioavailable drugs, the need for predictability and correlation. The apparent permeability of caco 2 cells to marketed drugs is poorly correlated with either simple biophysical properties, the extent of molecular similarity to endogenous metabolites endogenites, or any specific substructural properties. Analysis of glucosamine and chondroitin sulfate content in. Thus, we evaluated the permeability and cytotoxicity of 18 coumarins, with different substitution patterns involving oh, och 3 and ch 3 groups. Intestinal transcellular permeability p m, measured across cell lines such as caco 2 cells in vitro, is often used for assessing oral drug absorption potential in humans. Quinidine showed high permeability in caco 2 cells. Predicting apparent passive permeability of caco2 and mdck. The assay was designed to measure directional caco 2 permeability of test compounds a diverse collection of marketed drugs, drug failures and nondrugs was assembled from a broad variety of structural and target classes.
When run with iontox the caco 2 permeability assay is overseen by a diplomat of the american board of toxicology, run in replicants of 3 to 4, and follows the internationally recognized guidance documents on good in vitro method practices givmp and good cell culture practices gccp. Caco 2 cell lines in drug discovery an updated perspective kalyan kumar. The caco2 cell line is heterogeneous and contains cells with slightly different properties. Poor recovery is a commonly encountered issue in caco. The expression of multiple transporters in caco 2 makes it more difficult to clearly determine which transporter is involved. Mathematical model for determining drug absorption parameters in. Pdf caco2 cell permeability assays to measure drug absorption. Variability between caco2 permeability studies, because of. Caco 2 cells were inoculated in the millicell inserts at the density of 1. In this report, we use the largest data set of measured p caco 2, consisting of 157 structurally diverse compounds. The formation of a functional single monolayer on polycarbonate inserts is an important feature regarding the mimicking of in vivo conditions in the caco 2 permeability assay.
Different cell line models used in the drug permeability studies, their characteristics has been summarized emphasizing on caco2. Prediction of caco 2 phdependent permeability based on highquality in vitro training set alex avdeef and oksanatsinman, pion inc, 5 constitution way, woburn, ma 01801, usa email. Linear discriminant analysis lda was used to obtain quantitative models that discriminate higher absorption compounds from those with. Bovine serum albumin in caco2 permeability testing and. However, there is a strong debate regarding the value of caco 2 cell culture to mimick in vivo situation. To evaluate and optimize the use of caco 2 cell monolayers to predict thein vivo absorption of a broad range of compounds in man methods. A schematic representation of culture of caco 2 cells on a microporous filter caco 2 cells grow as a monolayer and differentiate on a semipermeable membrane. In vitro permeability across caco2 cells colonic can. The growth, maturation, and seeding of caco 2 cells have been previously described. Preliminary bcs permeability classification using caco 2 cell monolayers this nonglp assay is used to determine a preliminary bcs permeability classification by measuring the permeability of a test compound through caco 2 cell monolayers in both the apicaltobasolateral and basolateraltoapical direction. Prediction of caco2 phdependent permeability based on. Caco2 wild type cell line mtoxp24 technical bulletin. Under specific culture conditions, caco 2 cells are differentiated.
Thus, caco 2 permeability is a valuable index for assessing oral absorption of compounds, which, in turn, calls for the methods for predicting chemical caco 2 permeability. Automated caco2 assay systems are commercially available through tecan 12, bd bioscience and bohdan, mettler toledo. Evaluation of optimized caco 2 permeability measurements oksana tsinman, konstantin tsinman,and alex avdeef pion inc. Timo korjamo improvement of the caco2 permeability. After 24 h of incubation, the exposure of caco 2 cell monolayers to xchcu nanofibers resulted in a cell viability of 80%. The apparent permeabilities of caco2 cells to marketed drugs. Apparent permeability coefficient p app in caco 2 ab and b caco 2 ab intrinsic assays. In vitro permeability assays remain a valuable tool of screening scientists for lead compound optimization. The caco2 permeability coefficients for all compounds are shown in table 4. Permeability of chondroitin sulfate across caco 2 cell monolayers. Using this system it is possible to obtain a throughput of approximately 400500 compounds per week. Predicting caco2 permeability using support vector machine.
Caco 2 human colon adenocarcinoma cells assay is a widely used model in drug discovery for evaluationof compound permeability properties. In the current study, a novel absorptiondisposition kinetic. The caco2 bidirectional permeability assay is a useful tool in preclinical drug discovery to predict the likelihood of a compound being able to cross the barrier posed by the gastrointestinal. Permeability of rhynchophylline across human intestinal cell in vitro. The caco2 assay models the epithelial cell layer permeability barrier in the small intestine. As small changes in the culture conditions can lead to significant differences in cell phenotype 10, the.
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