Caco passive transport

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]. 

As the Caco-2 cell model contains most of the important transporters, it can be used for the study not only of passive transport mechanisms but also of mechanisms involving transporters. This is a major advantage of the Caco-2 model when... 

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... 

Discrimination of efflux, active or passive transport is already feasible by suitable in vitro experiments. For instance, the PAMPA assay detects passive transport only, while Caco-2 cells include transporters. A comparison between transport in PAMPA and Caco-2 cells by a calibration plot reveals compounds with greater or less transport in Caco-2 cells than in PAMPA. These compounds should be tested in uptake and efflux transport assays in order to gain deeper insight into absorption fate. 

Garberg et al. compared a number of in vitro cell models of the BBB with in vivo data (including primary cow and human brain endothelial cells co-cultured with astrocytes, MDCK, MDCK-MDRl, Caco-2, ECV304/C6, MBEC4, SV-ARBEC cocultured with astrocytes). The best correlation, although poor, was seen with cow brain endothelial cells (r 0.43) and MDCK (r 0.46). The correlation was improved with Caco-2 when only passively transported compounds were included in the analysis (r = 0.86), BBEC showing a similar correlation [39]. 

Permeability can be assessed by pharmacokinetic studies (for example, mass balance studies), or intestinal permeability methods, e.g. intestinal perfusion in humans, animal models, Caco 2 cell lines or other suitable, validated cell lines. In vivo or in situ animal models or in vitro models (cell lines) are only considered appropriate by HHS-FDA for passively transported drugs. It should be noted that all of these measurements assess the fraction absorbed (as opposed to the bioavailability, which can be reduced substantially by first-pass metabolism). 

PAMPA-biomimetic-Caco-2-comparison Several in vitro assays have been developed to evaluate the Gl absorption of compounds. Our aim was to compare three of these methods (/) the BAMPA method, which offers a HT, noncellular approach to the measurement of passive transport ( ) the traditional Caco-2 cell assay, the use of which as a HT tool is limited by the long cell differentiation time (21 days) and (// ) The BioCoat HTS Caco-2 assay system, which reduces Caco-2 cell differentiation to three days. The transport of known compounds (such as cephalexin, propranolol, or chlorothiazide) was studied at pH 7.4 and 6.5 in BAMPA and both Caco-2 cell models. Permeability data obtained was correlated to known values of human absorption. Best correlations (f= 0.9) were obtained at pH 6.5 for BAMPA and at pH 7.4 for the Caco-2 cells grown for 21 days. The Caco-2 BioCoat HTS Caco-2 assay system does not seem to be adequate for the prediction of absorption. The overall results indicate that BAMPA and the 21 -day Caco-2 system can be complementary for an accurate prediction of human intestinal absorption. 

The use of the Caco-2 cell line assays provide more physiologically relevant data than the PAMPA assays since they express transporters so that both active and passive transport can be determined. Caco-2 cell monolayers are contained in a 96-well plate format and test compounds can be incubated to model and test absorption [85],... 

Figure 9.10 Relationship between permeability coefficients obtained from Caco-2 and PAMPA screenings. Circles represent passively transported compounds, diamonds are actively transported, and triangles are compounds subject to efflux. (Reprinted from with permission from Fujikawa, M., et al. Relationship 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 732, copyright 2005, Elsevier).

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. 

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). 

Ho NFH, PS Burton, RA Conradi, CL Barsuhn. (1995). A biophysical model of passive and polarized active transport processes in Caco-2 cells Approaches to uncoupling apical and basolateral membrane events in the intact cell. J Pharm Sci 84 21-27. 

Pade, V., Stavchansky, S., Estimation of the relative contribution of the transcellular and paracellular pathway to the transport of passively absorbed drugs in the Caco-2 cell culture model, Pharm. Res. 1997, 34, 1210-1215. 

Initially the good relationship between the passive drug transport across Caco-2 cells and the absorbed fraction after oral administration to humans [5] may be... 

Some laboratories have found an alternative to the short-term cultures by using cell lines other than Caco-2 cells. The most popular of these is Madin-Darby canine kidney (MDCK) cells, an epithelial cell line from the dog kidney. MDCK cells have been suggested to perform as well as Caco-2 cells in studies of passive drug permeability [56]. These cells have also been used to optimise the conditions for studies of low-solubility drugs [53]. However, as noted previously, the active transport processes of this cell line can be quite different to those of Caco-2 cells [28-30], Another cell line that only requires short-term culture is 2/4/A1, which is a conditionally immortalised rat intestinal epithelial cell line [86]. The 2/4/A1 cell line is discussed in Section 4.3.2.2 below. 

As for active drug transport, there is no quantitative relationship between passive drug permeability in Caco-2 cells in vitro and drug transport in the human small... 

X. Liu, and L. Z. Benet. Contributions of saturable active secretion, passive transcellular, and paracellular diffusion to the overall transport of furosemide across adenocarcinoma (Caco-2) cells,/. Pharm. Sci. 2002,... 

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