Caco limitations

The presence of a time limitation suggests that there must be a kinetically controlled interference, possibly arising from a competing chemical reaction. In this case the interference is the possible precipitation of CaCOs. 

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). 

As with all new techniques that are rapidly embraced by the scientific community, the initial enthusiasm and in some cases uncritical use of Caco-2 cells unravelled the limitations of this in vitro model [7] and other, similar models e.g. [9]. A period of critical evaluation followed and today the majority of researchers using these models are aware not only of their advantages, but also of their limitations. In the following, we will briefly discuss the research opportunities that Caco-2 cells offer and also give a few examples of the successful application of these cell cultures in drug discovery. We will also discuss the limitations of Caco-2 cells and discuss new research that addresses these limitations. 

Limitations of Caco-2 Cells in Predicting Intestinal Drug Transport... 

Another limitation is that there is no quantitative relationship between active drug transport in the cell culture models and in vivo e.g. [92, 93]. The reason may be that the expression level of the transporter in Caco-2 cells is not comparable to that in vivo or that there is a difference in effective surface area (see Section 4.3.2.2 below). One solution to this problem is to determine the apparent transport constants, Km and Vmax, for each transporter and subsequently, to determine a scaling factor. However, this is not readily done. In addition these studies are further complicated by the lack of specific substrates. For example, there are almost no specific substrates for the drug efflux transporters [18]. Therefore, other epithelial... 

Delie, F. and W. Rubas. A human colonic cell line sharing similarities with enterocytes as a model to examine oral absorption advantages and limitations of the Caco-2 model, Crit. Rev. Ther. Drug Carrier Syst. 1997, 34, 221-286... 

Another limitation of the Caco-2 monolayers is their colonic origin and tight paracellular pathway, which tend to lead to underestimations in permeability to paracellularly transported compounds [97]. This is likely to be correct for small compounds (MW < 150) - i.e., compounds smaller than normal drugs - but it remains to be seen to what extent the Caco-2 model gives false-negative predictions of the fraction absorbed for polar drugs of normal size in humans where para-... 

The paracellular pathway, between the epithelial cells, is both size- (MW, volume) and charge-dependent [60, 109, 110]. In general, compounds that are limited to paracellular transport are not efficiently absorbed due to the small available absorptive area and the restriction by tight junctions. The molecular weight cut-off seems to be around 400 g mol-1 and 300 g mol-1 for the small and large intestine respectively, and 300 g mol-1 for the Caco-2 cell monolayers [60], which shows the more colonic nature of the Caco-2 monolayer model. Compounds with a... 

The oral administration of large proteins and peptides is limited due to their low membrane permeability. These compounds are mainly restricted to the para-cellular pathway, but because of their polar characteristics and their size the pore of the tight junctional system is also highly restrictive. An additional transcellular pathway has therefore been suggested for these peptides, i.e., the transcytotic pathway, which involves a receptor-mediated endocytosis in Caco-2 cells [126],... 

N. L., Drugs absorption limited by P-glycoprotein-mediated secretory drug transport in human intestinal epithelial Caco-2 cell layers, Pharm. Res. 1993, 10, 743-749. 

Some drugs with low intrinsic permeability achieve acceptable oral bioavailability because they are substrates for uptake transporters, which normally function in nutrient uptake. The most prominent example is the peptide transporter, PepTl, which is active toward peptidomimetic antibiotics such as cephalexin, the antiviral agent valacyclovir [24] and other drugs. PepTl is natively expressed in Caco-2 cells, and adenovirus transduction has been used to increase PepTl expression levels [25]. However, the expression of PepTl was not polarized in this system and this expressed system appears to be of limited value as an improved screening model. PepTl has also been expressed in Chinese hamster ovary cells and a variety of other mammalian systems [26, 27]. 

The use of Caco-2 cell monolayers has gained in popularity as an in vivo human absorption surrogate moreover, the monolayers are generally accepted as a primary absorption screening tool by several pharmaceutical companies [10]. However, Caco-2 cell permeability measurements exhibit certain limitations due to the mechanisms involved. Both passive and active pathways exist active transport tends to increase the absorption across the cells and, since Caco-2 cells overexpress the P-glycoprotein (P-gp) efflux pump, the absorption of some compounds across these cells may be underestimated. 

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. 

Other Limitations The small intestine is a very dynamic environment the pH of the medium changes, the intestinal content is propelled by muscular contractions, food and xenobiotics are being digested by different enzymes and after absorption of compounds by the enterocytes, these compounds reach the blood vessels to be transported throughout the body. In contrast to the in vivo situation, the Caco-2 model is a static model consisting of one single cell type which is unable to secrete mucus. The unstirred water layer is thicker than the one in vivo and for some compounds it is difficult to work under sink conditions. 

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