Caco standardization

A i reaction (CaO) + S (C02) - S (CaCO ) The entropy of each solid is its standard entropy, S °, but the entropy of the gas must be corrected for the deviation of pressure from standard conditions. Equation gives the molar entropy of carbon dioxide as a ftmction of its partial pressure S (CO2) — S ° (CO2) - i In p(pc02) Now substitute the corrected entropy of CO2 into the equation for A ... 

The main reasons for the popularity of the Caco-2 cell line are that the cells are easy to maintain in culture, and they develop unusually high degree of differentiation spontaneously under standard culture conditions. In fact, Caco-2 is the only human intestinal cell line that has been found so far spontaneously to undergo functional enterocytic differentiation. The cells exhibit a good reproducibility, robustness and functional properties of human intestinal epithelial cells. The model has proved capable of predicting the oral absorption of a variety of drug compounds [see references in 10]. 

Culture protocols have been published which describes an accelerated differentiation process where monolayers are ready to be used after 3-7 days of culture [90-92]. One of these systems, the so-called BD BioCoat Intestinal Epithelium Differentiation Environment, is commercially available through BD Bioscience. This system is described to produce monolayers of a quality that are comparable with the typical Caco-2 cells with respect to permeability for drugs transported transcellularly. The paracellular barrier function is however low, as indicated by high mannitol permeability and low TER. The functional capacity for active uptake and efflux is not as thoroughly characterized as for the standard Caco-2 mono-layers. 

These suggested factors for standardization of the Caco-2 model might also be reasons for the variability in published permeability values obtained from different laboratories [86]. Clearly, data from different publications or laboratories should not be mixed without prior harmonization of the experimental protocols. 

Many academic and industrial laboratories have shown that the drug permeability measured in Caco-2 cell monolayers can be used to predict the oral absorption of drugs in humans. Various datasets have therefore been used to establish correlations between Caco-2 permeability and the fraction absorbed orally in humans [85, 86, 96]. Taken together, these studies show good predictability, though with a relatively wide variation in the appearance of correlation profiles between different laboratories [86]. These studies emphasize the need to establish correlations and standardization procedures in each laboratory. 

In general, the first choice is to use the standard screening model, e.g., Caco-2 cell monolayers and to ... 

When comparing different experimental sources of Caco-2 permeability data, quantitative comparison is almost impossible, due to intervariations of experimental procedures between laboratories, although standard protocols and reference molecules are often used. 

In order to avoid a lack of consistent literature-based data, the Caco-2 permeability values of our selected compounds were transformed according to the following scheme the majority of compounds with Papp < 4 x 10 6 cm s 1 were classified as poorly absorbed and assigned a score of —1 compounds with Papp > 8 x 10 6 cm s 1 were classified as well-absorbed and assigned a score of +1. Different assumptions were made in special cases, when the experimental protocols were different or no internal standard compounds were used. 

MDCK Madin-Darby canine kidney (MDCK) cells have received attention as an alternative to Caco-2 cells for permeability measurements. When grown under standard culture conditions, MDCK cells develop tight junctions and form monolayers of polarized cells. The main advantage over Caco-2 cells is the shorter culture time to confluence (3-5 days). The transep-ithelial electrical resistance of MDCK cells is lower than that of Caco-2 cells and thus, closer to the TEER of the small intestine in vivo. The permeability coefficients of hydrophilic compounds are usually lower in Caco-2 cells than in MDCK cells, which is consistent with the lower TEER values for MDCK cell monolayers. The nonhuman (canine) and nonintestinal (renal) origin of MDCK cells is considered as a disadvantage. They have low expression levels of transporter proteins and low metabolic activity [34], MDCK cells that are stably transfected with P-gp/MDRl are often proposed as an alternative for Caco-2 cells to study bidirectional transport of compounds and, more... 

Figure 11 Effect of AMP and standard dispersant on gloss for 70/30 Clay/CaCO, coating...

Koljonen, M., Hakala, K.S., Antola-Satila, T., Laitinen, L., Kostiainen, R., Kotiaho, T., Kaukonen, AM. and Hirvonen, J. (2006). Evaluation of cocktail approach to standardize Caco-2 permeability experiements. Eur. J. Pharm. Biopharm., 64, 379-387. 

Fig. 4.4 Relationship between PSAd vacuum of nine compounds and their Caco-2 cell monolayer permeability coefficients. The insert shows the correlation between log Pc and PSAd vacuum The dashed line describes the resulting linear correlation (H216/44 is excluded, open circle). Error bars represent standard deviation of Pc and range of PSAdiVacuum. (Reprinted from Fig. 2 of ref. 54 with permission from the American Chemical Society.)...

However, the sensibility of the Caco-2 monolayers to the different standard operation procedures used for its culture increases the inter-laboratory variability and complicates the combination of data coming from different laboratories and the interpretation of results. Animal in vivo models reproduce the real situation better, as those models include all the physiological variables but, of course, have a low throughput capacity and are not adequate for screening a high number of compounds. 

As can be seen, PAM PA measurements can predict fluoroquinolone in vivo data remarkably well, as well as the standard benchmark, Caco-2. Accordingly, they can be complementary tools, quick in approaching the potential of absorption of a potential molecule in the early stages of a discovery project. 

The thermodynamic criterion for the equilibria CaCO, ) = Ca0(,) + C02 (,) is AG ° = -RT n Kp, where AG° is the change in Gibbs free energy of the reactants and products in their standard state, R is the gas constant, and Kp is the equilibrium constant. For this equilibria, A p = pco, for pressure in units of atmospheres. Values for AG are tabulated in the form AG° = a+ bT combining these expressions yields an exponential relationship between the partial pressure of CO2 and temperature for the above equilibria. Complete derivations and discussion of these equations may be found in physical chemistry textbooks such as references [13] and [14]. 

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