Caco-2 cell lines

In-vitro models can provide preliminary insights into some pharmacodynamic aspects. For example, cultured Caco 2 cell lines (derived from a human colorectal carcinoma) may be used to simulate intestinal absorption behaviour, while cultured hepatic cell lines are available for metabolic studies. However, a comprehensive understanding of the pharmacokinetic effects vfill require the use of in-vivo animal studies, where the drug levels in various tissues can be measured after different dosages and time intervals. Radioactively labelled drugs (carbon-14) may be used to facilitate detection. Animal model studies of human biopharmaceutical products may be compromised by immune responses that would not be expected when actually treating human subjects. 

This in vitro approach thus has a great potential for studying the intestinal absorption processes of carotenoids and other pigments. It is important to note the existence of several clones isolated from the parent Caco-2 cell line that can be used for studying... 

Collett, A. Sims, E. Walker, D. He, Y.-L. Ayrton, J. Rowland, M. Warhurst, G., Comparison of HT29-18-C1 and Caco-2 cell lines as models for studying intestinal paracellular drug absorption, Pharm. Res. 13, 216-221 (1996). 

W Rubas, N Jezyk, GM Grass. Comparison of the permeability characteristics of a human colonic epithelial (Caco-2) cell line to colon of rabbit, monkey, and dog intestine and human drug absorption. Pharm Res 10 113-118, 1993. 

The Caco-2 cell line was isolated from a human colon carcinoma, and has been characterized as one of the best in vitro models of intestinal epithelium. Indeed, in contrast to other intestinal cell lines, Caco-2 cells are able to constitute a homogenous monolayer and to spontaneously differentiate into polarized cells, highly similar to human mature enterocytes, after approximately 2 weeks of culture. Furthermore, the Caco-2 cells present microvillosities at the apical side and have a high transmembrane resistivity, which confirms the fact that the cells are confluent and link to one another via gap junctions. Finally, they can absorb different compounds, express many enzymes involved in intestinal metabolic pathways (Pinto et al. 1983, Musto et al. 1995, Salvini et al. 2002), and give reproducible in vitro results consistent with results obtained in in vivo studies (Artursson and Karlsson 1991). 

Gres, M. C. et al. (1998). Correlation between oral drug absorption in humans, and apparent drug permeability in TC-7 cells, a human epithelial intestinal cell line Comparison with the parental Caco-2 cell line. Pharm. Res. 15(5) 726-733. 

Salvini, S. et al. (2002). Functional characterization of three clones of the human intestinal Caco-2 cell line for dietary lipid processing. Br. J. Nutr. 87(3) 211-217. 

Brodin, S. Frokjaer, M. Taub, and B. Steffansen. Dipeptide model prodrugs for the intestinal oligopeptide transporter. Affinity for and transport via hPepTl in the human intestinal Caco-2 cell line.,... 

D. D. Breimer. Specificity of doxorubicin versus rhodamine-123 in assessing P- glycoprotein functionality in the LLC-PK1, LLC-PK1 MDR1 and Caco-2 cell lines, Ear. J. Pharm. Sci. 2000, 11, 207-214... 

The main reasons for the popularity of the Caco-2 cell line are that the cells are easy to maintain in culture, and they develop unusually high degree of differentiation spontaneously under standard culture conditions. In fact, Caco-2 is the only human intestinal cell line that has been found so far spontaneously to undergo functional enterocytic differentiation. The cells exhibit a good reproducibility, robustness and functional properties of human intestinal epithelial cells. The model has proved capable of predicting the oral absorption of a variety of drug compounds [see references in 10]. 

Woodcock, S., Williamson, I., Hassan, I., MacKay, M., Isolation and characterization of clones from the Caco-2 cell line displaying increased taurocholic add transport, J. Cell Sd. 1991, 98, 323-332. 

Nakamura, T., Sakaeda, T., Ohmoto, N., Tamura, T., Aoyama, N. et al., Real-time quantitative polymerase chain reaction for MDR1, MRP1, MRP2, and CYP3A-mRNA levels in Caco-2 cell lines, human duodenal enterocytes, normal colorectal tissues, and colorectal adenocarcinomas, Drug Metab. Dispos. 2002, 30, 4—6. 

Caco-2 Cell line used to predict human oral absorption... 

The presence of a transporter can be assessed by comparing basolateral-to-apical with apical-to-basolateral transport of substrates in polarized cell monolayers. If P-gp is present, then basolateral-to-apical transport is enhanced and apical-to baso-lateral transport is reduced. Transport experiments are in general performed with radioactively labeled compounds. Several studies have been performed with Caco-2 cell lines (e.g. Ref. [85]). Since Caco-2 cells express a number of different transporters, the effects measured are most probably specific for the ensemble of transporters rather than for P-gp alone. P-gp-specific transport has been assayed across confluent cell layers formed by polarized kidney epithelial cells transfected with the MDR1 gene [86], Figure 20.11 shows experimental data obtained with these cell lines. A rank order for transport called substrate quality was determined for a number of compounds [86]. The substrate quality is a qualitative estimate, but nevertheless allows an investigation of the role of the air/water (or lipid/water) partition coefficient, log Kaw, for transport as seen in Fig. 20.11(A). For most of the compounds, a linear correlation is observed between substrate quality and log Kaw- However, four compounds are not transported at all despite their distinct lipophilicity. A plot of the substrate quality as a function of the potential of a... 

P-gp substrate 22 substrates and 31 nonsubstrates 115 substrates and 157 nonsubstrates 61% substrates and 81% nonsubstrates correctly predicted. Overall accuracy 72.4% Transport Caco-2 cell line Size, shape (e.g. molecular surface and glo-bularity), hydrophilic and H-bonding related descriptors correlated positively with P-gp activity, log P0/w not significant [54]... 

The permeability of the drug substance can be determined by different approaches such as pharmacokinetic studies in humans (fraction absorbed or mass balance studies) or intestinal permeability studies (in vivo intestinal perfusion studies in humans or suitable animal models or in vitro permeation studies using excised intestinal tissue or epithelial cell culture monolayers like CaCo-2 cell line). In order to avoid misclassification of a drug subject to efflux transporters such as P-glycoprotein, functional expression of such proteins should be investigated. Low- and high-permeability model... 

Sambuy Y, De Angelis I, Ranaldi G, Scarino ML, Stammati A, Zucco F (2005) The Caco-2 cell line as a model of the intestinal barrier Influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biol Toxicol 21 1-26. 

Sandri G, Rossi S, Ferrari F, Bonferoni MC, Zerrouk N, Caramella C (2004) Mucoadhesive and penetration enhancement properties of three different grades of hyaluronic acid using porcine buccal and vaginal tissue, Caco-2 cell lines and rat jejunum. J Pharm Pharmacol 56 1083-1090. 

At the beginning of the 1990s, the general acceptance of Caco-2 cell monolayers as a model of the small intestinal mucosa caused a significant push toward the field of permeability testing [2, 43], For detailed information about the Caco-2 cell line, the reader is referred to Chap. 8. As a consequence, the pharmaceutical community became more aware that permeation through intestinal epithelia is oftentimes more sophisticated than mere diffusion through a lipophilic membrane. 

Ranaldi G, Marigliano I, Vespignani I, Perozzi G, Sambuy Y (2002) The effect of chitosan and other polycations on tight junction permeability in the human intestinal Caco-2 cell line. J Nutr Biochem 13 157-167... 

Cell monolayers grown on permeable culture inserts form confluent mono-layers with barrier properties and can be used for drug absorption experiments. The most well-known cell line for the in vitro determination of intestinal drug permeability is the human colon adenocarcinoma Caco-2 [20, 21], The utility of the Caco-2 cell line is due to its spontaneous differentiation to enterocytes under conventional cell culture conditions upon reaching confluency on a porous membrane to resemble the intestinal epithelium. This cell model displays small intestinal carriers, brush borders, villous cell model, tight junctions, and high resistance [22], Caco-2 cells express active transport systems, brush border enzymes, and phase I and II enzymes [22-24], Permeability models... 

To meet the need of conducting HTS for ADME-Tox properties, many slow and expensive in vivo ADME assays are now being replaced by in vitro cell models. For intestinal absorption, Caco-2 cell lines and Madin Darby canine kidney (MDCK) cell lines are widely used to predict the absorption rate of candidate drug compounds across the intestinal epithelial cell barrier. A number of models for Caco-2 cell permeability and MDCK cell permeability have been reported that predict the oral absorption properties of drugs, mostly limited to small organic molecules. Caco-2 and MDCK permeability are related to "A" and "D" in the ADME-Tox. 

The known variability associated to the behavior of Caco-2 cell lines, together with the need to find a more robust and easy to grow type of culture led the pharmaceutical industry to substitute this system by, for example, MDCK cell cultures. So in silico modelers have also turned towards this new kind of in vitro cell data, although to a lesser extent than with Caco-2 systems. Perhaps the only notable example of the use of MDCK data is Refsgaard s categorical model, where a dataset of Caco-2 permeability values was enriched with MDCK permeability data [99]. 

The most recent example of in silico efflux modeling has been based on Caco-2 permeability measured in the basal to apical direction [100]. This model can be very effective at ruling out compounds that most likely will show low in vivo intestinal absorption - however it carmot indicate which efflux pump(s) is/are responsible for that, making it more difficult for designers to circumvent the problem. Johnson [92] also included in his review an excellent summary of QSAR models and rules of thumb developed for P-gp substrates and inhibitors. These models are normally based on efflux ratios from MDCK/MDRl or Caco-2 cell lines - in the latter case it is important to notice that the data is combined with inhibition values from the calcein-AM assay, as the observed efflux might not be exclusively due to P-gp. 

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