Cell Culture-Based Models Caco

As primary cultures of enterocytes fail to form a polarized epithelial monolayer and therefore, do not display an apical and basolateral surface, continuously growing (tumor) cell cultures can be used to investigate the permeation and 

Cell line Species or origin Specific characteristics 

Caco-2 Human colon adenocarcinoma Well-established cell model Differentiates spontaneously and expresses some relevant efflux transporters (e.g., P-gp, MRP1-2, BCRP) Interlaboratory differences 

MDCK Dog kidney epithelial cells Polarized cells with low intrinsic expression of transporters Suitable cell fine for transfections 

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Both active and passive transport occur simultaneously, and their quantitative roles differ at different concentration gradients. At low substrate concentrations, active transport plays a major role, whilst above the concentration of saturation passive diffusion is the major transport process. This very simple rule can be studied in an experimental system using cell culture-based models, and the concentration dependency of the transport of a compound as well as asymmetric transport over the membrane are two factors used to evaluate the presence and influence of transporters. Previous data have indicated that the permeability of actively absorbed compounds may be underestimated in the Caco-2 model due to a lack of (or low) expression of some uptake transporters. However, many data which show a lack of influence of transporters are usually derived from experiments... 

We define this permeability as apparent, to emphasize that there are important but hidden assumptions made in its derivation. This equation is popularly (if not nearly exclusively) used in culture cell in vitro models, such as Caco-2. The sink condition is maintained by periodically moving a detachable donor well to successive acceptor wells over time. At the end of the total permeation time f, the mass of solute is determined in each of the acceptor wells, and the mole sum mA (t) is used in Eq. (7.10). Another variant of this analysis is based on evaluating the slope in the early part of the appearance curve (e.g., solid curves in Fig. 7.14) ... 

The enthusiasm for using Caco-2 cells and other epithelial cell cultures in studies of drug transport processes has been explained by the ease with which new information can be derived from these fairly simple in vitro models [7]. For instance, drug transport studies in Caco-2 cells grown on permeable supports are easy to perform under controlled conditions. This makes it possible to extract information about specific transport processes that would be difficult to obtain in more complex models such as those based on whole tissues from experimental animals. Much of our knowledge about active and passive transport mechanisms in epithelia has therefore been obtained from Caco-2 cells and other epithelial cell cultures [10-15]. This has been possible since Caco-2 cells are unusually well differentiated. In many respects they are therefore functionally similar to the human small intestinal enterocyte, despite the fact that they originate from a human colorectal carcinoma [16, 17]. 

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. 

The pH in the cellular interstitial space and blood compartment is known to be about 7.4. The pH present in the G1 tract will not only have an impact on the ionization of drugs, and thus on their partitioning capacity, but it will also affect the pH-dependent functionality of various carriers located in the intestinal mucosa (Tsuji and Tamai, 1996). Many of these carriers are also expressed and functionally active in the Caco-2 cell culture model (Hidalgo and Li, 1996 Ogi-hara et al., 1999 Friedrichsen et al., 2002 Putman et al., 2002a,b). Therefore, the pH of the apical medium can have a critical effect on ionization and transport of drugs, ft has for instance been shown that the absorption of w eak bases, like beta-blockers, are absorbed better in the ileum region, where the pH is around... 

Our experience with trying to build computational models based on experimental permeability screening in Caco-2 cell culture illustrates the problem introduced by multiple mechanisms. We found that deviation from a single mechanism could arise either in the assay per se or could arise from the compounds that were screened in the assay. One aspect of the multiple mechanism problem is the presence of active multiple biological transport mechanisms for both enhancing and reducing absorption in cell culture assays. This issue is well documented and is outside the scope of this chapter. 

Since the widely accepted in vitro permeability model in the pharmaceutical industry is based on the use of cultured cells, such as Caco-2 or MDCK, it was appropriate to analyze the regression correlation coefficients based on the comparisons of Caco-2 log Pe and the log Pe values based on the human jejunal measurements [56]. 

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