Caco-2 cell selection

During, A. et al.. Carotenoid uptake and secretion by Caco-2 cells 3-carotene isomer selectivity and carotenoid interactions, J. Lipid. Res., 43, 1086, 2002. 

The importance of drug ionization using cell-based methods such as Caco-2 in the in vitro prediction of in vivo absorption was discussed [45]. It was observed that when the apical pH used in Caco-2 studies was lowered from 7.4 to 6.0 a better correlation was obtained with in vivo data, demonstrating that careful selection of experimental conditions in vitro is crucial to produce a reUable model. Studies with Caco-2 monolayers also suggested that the ionic species might contribute considerably to overall drug transport [46]. 

The P-C isomer selectivity seems to be tissue-specific a preferential uptake of the all-trans isomer was shown in hepatic stellate HSC-T6 cells and in cell-free system from rat liver microsomes, but not in endothelial EAHY cells or U937 monocyte-macrophages (During et al., 2002). When Caco-2 cells were incubated with only 9-cis P-C, all -trans P-C did not increase in cells or in the basolateral medium, indicating that there is no cis-trans isomerization occurring in intestinal cells. Thus, the isomerization of 9-cis P-C observed in vivo (You et al., 1996) could take place in the... 

The applicability range of any model should be limited to molecules having a similar mechanism of transport. Therefore, we have selected from the literature only those compounds with well-characterized Caco-2 cell permeability and excluded compounds with a high efflux ratio. Known P-gp substrates and actively transported compounds were also excluded from the list. 

Summing up, the selected Caco-2 data contain qualitative permeability measurements for 450 related (but chemically diverse) compounds that had been either collected from the literature or measured experimentally in laboratories connected with our group. Penetrating compounds were indicated by a score of +1, whereas a score of —1 was attributed to compounds having little (if any) ability to penetrate the epithelial cells. Passive permeation was used as a basic assumption of the model. 

Despite the availability of other cell lines, Caco-2 cells remain the most widely used intestinal cell culture model at present. This model has provided valuable information necessary for lead optimization in the drug discovery process. However, it is important to understand that compounds with high permeability in this model are typically well absorbed, whereas compounds with low solubility and low permeability in this model may not necessarily be poorly absorbed in vivo. Although this type of positive selection limits the usefulness in providing a structure-permeability relationship, the Caco-2 model has the most effect in drug discovery when the screen is implemented early and in conjunction with other types of in vitro and in vivo permeability/absorption screens. 

This permeability barrier shows selectivity in that small hydrophobic molecules can partition into and diffuse across the lipid bilayer of the cell membrane, whereas small hydrophilic molecules can only diffuse between cells (i.e., through the intercellular junctions). In addition, the presence of uptake and efflux transporters complicates our ability to predict intestinal permeability based on physicochemical properties alone because transporters may increase or decrease absorptive flux. The complexity of the permeability process makes it difficult to elucidate permeability pathways in complex biological model systems such as animals and tissues. For this reason, cultured cells in general, and Caco-2 cells in particular, have been used extensively to investigate the role of specific permeability pathways in drug absorption. 

Complementary use of PAMPA and Caco-2 cells for evaluation of absorption potential. PAMPA measurements are used to discard compounds with clear absorption problems whereas Caco-2 cells would be used to evaluate mechanisms of permeation or reasons for low permeation. It is highly unlikely that PAMPA measurements would be used to select compounds to be tested in vivo. 

LC/MS/MS techniques with selective and sensitive detection methods make it possible to quantitatively analyze samples from Caco-2 cell and PAMPA buffer matrices. A high-throughput permeability screen with robust LC/MS technology can quickly generate information about structure-permeability relationships that are extremely valuable in the lead optimization phase for the selection of pre-clinical candidates with favorable oral bioavailability properties. 

In summary, CACO-2 cells express many enzymes characteristic for intestinal cells involved in drag metabolism. The major drug metabolising enzyme, CYP 3A, is active only in selected clones pointing to polyclonal origin of CACO-2 and the necessity to characterize CACO-2 cells extensively for growth and experimental conditions used for given experiments. 

Jumarie Cand Malo C (1991) CACO-2 cells cultured in serum-free medium as a model for the study of enterocytic differentiation in vitro. J Cell Physiol 149 24-33 Karlsson J, Ungell AL, Artursson P (1994) Effect of an oral rehydration solution on paracellular drug transport in intestinal epithelial cells and tissues assessment of charge and tissue selectivity. Pharm Res 11 248... 

TEER, normalized to surface, was 188-221 Ohm/cm2 in CACO-2 cells and 78-125 Ohm/cm2 in colon (Rubas et al. 1996). The presence of villi and crypts in vivo with a higher surface and a different cellular composition (goblet cells, M cells, higher permeability in crypts) compared to a cell mono-layer might lead to higher permeability. Tanaka et al. (1995) compared permeability of FITC-Dextran (MW 4000) in CACO-2 cells and rat jejunum and colon the permeability was 10 fold decreased in CACO-2 compared to jejunum and 5 fold lower than in colon. TEER in CACO-2 was 470 Ohm x cm2 in this study, 40 Ohm x cm2 in rat jejunum and 80 Ohm x cm2 in rat colon. Permeability of standard markers like PEG was compared between CACO-2 cells and colon (Artursson et al. 1993). The conclusion for cellular studies is to select cells with acceptable TEER and permeability values and to perform quality assurance tests on a regular basis. 

Prognosis of a compounds permeability should be made stressing limitations of the model. There is no bioavailability prognosis from in vitro data - a cellular assay can provide only permeability potential through a biological membrane. The membrane, in most cases CACO-2 cells, is very similar to what we observe in vivo in the small intestine and resembles many characteristics to in vivo enterocytes. CACO-2 cells can be used for prediction of different pathways across intestinal cells. Best correlation occurs for passive transcellular route of diffusion. Passive paracellular pathway is less permeable in CACO-2 and correlations are rather qualitative than quantitative for that pathway. CACO-2 cells are an accepted model for identification of compounds with permeability problems, for ranking of compounds and selection of best compounds within a series. Carrier-mediated transport can be studied as well using careful characterization of transporters in the cell batch or clone as a prerequisite for transporter studies. 

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