Caco-2 studies

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]. 

In the presence of 10 24 mM sodium caprate on Caco-2 monolayers a dilatation of the tight junctions was observed (Anderberg et al. 1993). Based on Caco-2 studies Tomita et al. (1995) and Lindmark et al. (1998) proposed the activation of phospholipase C, followed by an increase in inositol triphosphate and intracellular calcium concentration leading to a contraction of calmodulin-dependent... 

The parallel artificial membrane permeability assay (PAMPA) is a recent development in the area of artificial membranes that appears to offer considerable potential. Measuring the flux values (membrane permeation levels) of a range of test compounds by PAMPA and relating these values to the flux curves obtained in Caco-2 studies have shown good correlations, indicating that the PAMPA assay could be a good alternative to Caco-2 cells for the measurement of passively diffusing compounds. 

No model can predict human absorption 100% of the time, but Caco-2 has a relatively good track record for at least binning compounds. Along with the early information advanced Caco-2 studies can provide information on transport mechanism, gut wall metabolism and toxicity, and its utility in establishing structure property relationships, this makes it a valuable addition to the armamentarium of drug discovery. 

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... 

Another relatively new lipophilicity scale proposed for use in ADME studies is based on MEKC [106]. A further variant is called BMC and uses mobile phases of Brij35 [polyoxyethylene(23)lauryl ether] [129]. Similarly, the retention factors of 16 P-blockers obtained with micellar chromatography with sodium dodecyl sulfate as micelle-forming agent correlates well with permeability coefficients in Caco-2 monolayers and apparent permeability coefficients in rat intestinal segments [130]. 

Calculated molecular descriptors including H-bond parameters were used for QSAR studies on different types of permeabiUty. For example, the new H-bond descriptor characterizing the total H-bond ability of a compound, was successfully appUed to model Caco-2 cell permeability of 17 drugs [30]. A similar study on human jejunal in vivo permeabiUty of 22 structurally diverse compounds is described in Ref. [62]. An exceUent one-parameter correlation of human red ceU basal permeabiUty (BP) was obtained using the H-bond donor strength [63] ... 

Yamashita et al. [82] also studied the effect of BSA on transport properties in Caco-2 assays. They observed that the permeability of highly lipophilic molecules could be rate limited by the process of desorption off the cell surface into the receiving solution, due to high membrane retention and very low water solubility. They recommended using serum proteins in the acceptor compartment when lipophilic molecules are assayed (which is a common circumstance in discovery settings). 

Measurements of Pe in fixed-pH solutions but at various different stirring speeds need to be made. The double-reciprocal analysis, HPe versus 1/v , for Caco-2 permeability measurements in the Transwell (Corning Costar) system produced a linear plot for x- 0.8 [514]. The intercept yields the membrane permeability for the particular pH value in the study the slope determines the k constant. From the analysis of testosterone transport, for the stirring speed of 25 rpm (planar rotating shaker), the thickness of each UWL (assuming symmetric geometry) was calculated to be 465 pm at 150 rpm, haq= 110 pm [514], Karlsson and Artursson [512] found x = 1.0 to best represent their stirring-based analysis of the UWL permeability. 

Artursson, R, Epithelial transport of drugs in cell culture. I A model for studying the passive diffusion of drugs over intestinal absorptive (Caco-2) cells, J. Pharm. Sci. 79, 476-482 (1990). 

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). 

The identification and characterization of cell culture systems (e.g., Caco-2-cells) that mimic in vivo biological barriers (e.g., intestinal mucosa) have afforded pharmaceutical scientists the opportunity to rapidly and efficiently assess the permeability of drugs through these barriers in vitro. The results generated from these types of in vitro studies are generally expressed as effective permeability coefficients (Pe). If Pe is properly corrected to account for the barrier effects of the filter (PF) and the aqueous boundary layer (PAbl) as previously described in Section II.C, the results provide the permeability coefficient for the cell monolayer... 

This type of information about a homologous series of drug candidates, when considered in light of the propensity of these compounds to undergo first-pass metabolism and/or liver clearance, allows pharmaceutical scientists to make more intelligent decisions about which compounds to move into animal studies. In addition, when an in vitro-in vivo correlation can be demonstrated for a series of compounds, the results of Caco-2 experiments can be used as a guide by medicinal chemists to make structural modifications to optimize oral bioavailability. 

Despite the feasibility of using cultured RPE cells for studies similar to those performed using Caco-2 cells, the role of the RPE in carotenoid uptake and dynamic regulation has only just begun to be investigated. As carotenoids are carried in blood by lipoproteins, lipoprotein-rich serum seems to be the most appropriate vehicle for carotenoid delivery to cultured RPE cells. Indeed, recent studies comparing carotenoid delivery from fetal calf serum and from organic solvents showed that delivery in the presence of serum was superior to tetrahydrofuran (Shafaa et al., 2007). 

Under linear concentration conditions (for a P-C concentration range of 0.12-6pM) at 16h incubation and under cell culture conditions mimicking the in vivo postprandial state, the extent of absorption of all-trans P-C through Caco-2 cell monolayers was 11% a value similar to that reported from different human studies. In humans, the bioavailability of a single dose of P-C... 

The first study was conducted to determine whether carotenoids and cholesterol share common pathways (transporters) for their intestinal absorption (During et al., 2005). Differentiated Caco-2 cells on membranes were incubated (16 h) with a carotenoid (1 pmol/L) with or without ezetimibe (EZ Zetia, an inhibitor of cholesterol transport), and with or without antibodies against the receptors, cluster determinant 36 (CD36) and scavenger receptor class B, type I (SR-BI). Carotenoid transport in Caco-2 cells (cellular uptake + secretion) was decreased by EZ (lOmg/L) as follows P-C and a-C (50% inhibition) P-cryptoxanthin and LYC (20%) LUT ZEA (1 1) (7%). EZ reduced cholesterol transport by 31%, but not retinol transport. P-Carotene transport was also inhibited by anti-SR-BI, but not by anti-CD36. The inhibitory effects of EZ and anti-SR-BI on P-C transport... 

The Human Caco-2 Intestinal Cell Model A Valuable Tool for Studying Carotenoid... 

THE HUMAN CACO-2 INTESTINAL CELL MODEL A VALUABLE TOOL FOR STUDYING CAROTENOID ABSORPTION... 

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). 

In summary, Caco-2 cells studies strongly suggest that carotenoids interact with each other at the level of cellular uptake by the enterocyte. This phenomenon has been explained by the fact that the uptake of several carotenoids involves, at least in part, the same intestinal membrane transporter the scavenger receptor class B type ISR-BI (Reboul et al. 2005, van Bennekum et al. 2005, Moussa et al. 2008). 

In conclusion, the Caco-2 cell monolayer model has given original data on the competition effect of several nutrients on carotenoid uptake. Most of these data have been confirmed in several in vivo studies, including clinical studies, confirming that this model is a valuable tool to study competition effects on carotenoid absorption. 

Carotenoids are highly lipophilic an active area of research concerns how carotenoids interact with and affect membrane systems (see Chapters 2 and 10). Also, the lipid solubility of these compounds has important implications for carotenoid intestinal absorption (see Chapter 17) models such as the Caco-2 cell model are being used to conduct detailed studies of carotenoid absorption/ competition for absorption (Chapter 18). The lipid solubility of these carotenoids also leads to the aggregation of carotenoids (see Chapter 3). Carotenoids aggregate both in natural and artificial systems, with implications for carotenoid excited states (see Chapter 8). This has implications for a new indication for carotenoids, namely, serving as potential materials for harnessing solar energy. 

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