Caco-2 cell

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. 

Fujikawa M, Ano R, Nakao K, Shimizu R and Akamatsu M. Relationships between structure and high-throughput screening permeability of diverse drugs with artificial membranes application to prediction of Caco-2 cell permeability. Bioorg Med Chem 2005 13 4721-32. 

Artursson P and Karlsson J. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells. Biochem Biophys Res Commun 1991 175 880-5. 

Snyder NJ, Tabas LB, Berry DM, Duckworth DC, Spry DO and Dantzig AH. Structure-activity relationship of carbacephalosporins and cephalosporins antibacterial activity and interaction with the intestinal proton-dependent dipeptide transport carrier of Caco-2 cells. Antimicrob Agents Chemother 1997 41 1649-57. 

MANNA c, GALLETTi p, MAISTO G, cucciOLLA V, d angelo s, zappia V (2000) Transport mechanism and metabolism of olive oil hydroxytyrosol in Caco-2 cells. FEBS Lett. 470 341-4. 

LIU Y and HU m (2002) Absorption and metabolism of flavonoids in the caco-2 cell culture model and a perfused rat intestinal model. Drug Metab Dispos. 30 (4) 370-77. 

In culture, the human colon carcinoma cell hne Caco-2 spontaneously differentiates at confluency into polarized cells with enterocyte-like characteristics. The principle of this approach consists of following the passage of the compound of interest from the apical or lumen-like sides to the basolateral or lymph-hke sides of Caco-2 cells, thus following the absorption of the compound per se. One obhgate step for fat-soluble nutrients such as carotenoids to cross the intestinal barrier is their incorporation into CMs assembled in the enterocytes. Under normal cell culture conditions, Caco-2 cells are unable to form CMs. When supplemented with taurocholate and oleic acid, Caco-2 cells were reported to assemble and secrete CMs. ... 

In this in vitro system, the presence of serum in cell culture medium is not necessary, but the type of transwell is important (the total amount of H-triglycerides secreted was two-fold higher when using 3 pm versus 1 pm pore size transwells), and oleic acid supplementation is required for the formation and secretion of CMs as well as the transport of 3-carotene through Caco-2 cells. Finally, the presence of Tween 40 does not affect CM synthesis and secretion in this in vitro cell culture system. Thus, CMs secreted by Caco-2 cells were characterized as particles rich in newly synthesized H-triglycerides (90% of total secreted) containing apolipoprotein B (30% of total secreted) and H-phospholipids (20% of total secreted) and with an average diameter of 60 nm. These characteristics are close to those of CMs secreted in vivo by enterocytes. ... 

In contrast to previous in vivo models, this in vitro model provides the possibility of dissociating experimentally two important processes of intestinal absorption cellular uptake and secretion. Under conditions mimicking the postprandial state (taurocholate/oleic acid supplementation), differentiated Caco-2 cells were able to (1) take up carotenoids at the apical sides and incorporate them into CMs and (2) secrete them at the basolateral sides associated with CM fractions. Using this approach, the extent of absorption of P-carotene through Caco-2 cell monolayers after 16 hr of incubation was 11.2%, a value falling within the in vivo range (9 to 22%). ° - Of the total amount of P-carotene secreted, 78% was associated with the two CM fractions and 10% with the VLDL fraction. ... 

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... 

FIGURE 3.2.1 In vitro digestion/Caco-2 cell model combination approach to assess carotenoid bioavailability. LCM = large chylomicrons. SCM = small chylomicrons. VLDL = very low density lipoproteins. 

S In Vitro Digestion/Caco-2 Cell Model Combination Approach... 

The configuration of the molecule can also be another factor affecting the degree of micellarization of a compound in the lumen. For instance, cis isomers of P-carotene present a greater solubilization in mixed micelles in vitr(f and in the duodenal micellar phase in vivo than all-trans P-carotene. Despite their higher efficiency of micellarization, cis isomers of p-carotene are less absorbed by Caco-2 cells and also in vivo than the all-trans forms. 

The hydrolysis of zeaxanthin esters by a carboxyl ester lipase indeed enhanced both the incorporation of zeaxanthin in the micellar phase and uptake of zeaxanthin by Caco-2 cells. As mentioned earher, carotenoids can also be linked to proteins by specific bindings in nature and these carotenoid-protein complexes may slow the digestion process and thus make their assimilation by the human body more difficult than the assimilation of free carotenoids. Anthocyanins are usually found in a glycosylated form that can be acetylated and the linked sugars are mostly glucose, galactose, rhamnose, and arabinose. 

Caco-2 cells and ezetimibe, a potent inhibitor of chloresterol absorption in humans, it was reported that (1) carotenoid transport was inhibited by ezetimibe up to 50% and the extent of that inhibition diminished with increasing polarity of the carotenoid molecule, (2) the inhibitory effects of ezetimibe and the antibody against SR-BI on P-carotene transport were additive, and (3) ezetimibe may interact physically with cholesterol transporters as previously suggested - and also down-regulate the gene expression of three surface receptors, SR-BI, NPCILI, and ABCAl. 

Luchoomun, J. and Hussain, M.M., Assembly and secretion of CM by differentiated Caco-2 cells. Nascent triglycerides and preformed phospholipids are preferentially used for hpoprotein assembly, J. Biol. Chem., 274, 19565, 1999. 

During, A., Albaugh, G., and Smith, J.C., Characterization of 3-carotene 15,15-dioxygenase activity in TC7 clone of human intestinal cell line Caco-2 cells, Biochem. Biophys. Res. Commun., 249, 467, 1998. 

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. 

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

Ferruzzi, M.G., Failla, M.L., and Schwartz, S.J., Assessment of degradation and intestinal cell uptake of carotenoids and chlorophyll derivatives from spinach puree using an in vitro digestion and Caco-2 cell model, J. Agric. Food Chem., 49, 2082, 2001. 

Liu, C.S., Glahn, R.P., and Liu R.H., Assessment of carotenoid bioavailability of whole foods using a Caco-2 cell culture model coupled with an in vitro digestion, J. Agric. Food Chem., 52, 4330, 2004. 

Garrett, D.A., Failla, M.L., and Sarama, R.J., Estimation of carotenoid bioavailability from fresh stir-fried vegetables using an in vitro digestion/Caco-2 cell culture model, J. Nutr. Biochem., 11, 574, 2000. 

Fahy, D.M., O Callaghan, Y.C., and O Brien, N.M., Phytosterols lack of cytotoxicity but interference with 3-carotene uptake in Caco-2 cells in culture, FoodAddit. Con-tarn., 21, 42, 2004. 

During, A., Dawson, H.D., and Harrison, E.H., Carotenoid transport is decreased and expression of the lipid transporters SR-Bl, NPCILI, and ABCAl is down-regulated in Caco-2 cells treated with ezetimibe, J. Nutr., 135, 2305, 2005. 

When screening for absorption by passive membrane permeability, artificial membranes have the advantage of offering a highly reproducible and high-throughput system. Artificial membranes have been compared to Caco-2 cells and for passive... 

Camenisch, G., Folkers, G., Van de Waterbeemd, H. Comparison of passive drug transport through Caco-2 cells and artificial membranes. Int. J. Pharm. 1997, 147, 61-70. 

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