Caco-2 permeability

Avdeef, A., Artursson, P., NeuhofF, S., Lazorova, L., Grasjo, J., Tavelin, S. Caco-2 permeability of weakly basic drugs predicted with the double-sink PAM PA pKl method. Eur.J. Pharm. Sci. 2005, 24, 333-349. 

Since experimental determination of intestinal absorption is quite demanding, Caco-2 cell monolayers have been successfully used to model passive drug absorption. Several models for the prediction of Caco-2 permeability using PSA were developed, including those of van de Waterbeemd et al. [5] and Palm et al. [22] who found that relationships between Caco-2 permeability and PSA is stronger than with Clog D, Krarup et al. [23] who used dynamic PSA calculated for water accessible molecular surface and Bergstrom et al. [24]. 

Figure 7.8 Permeation of drugs through oil-soaked microfilters comparisons to Caco-2 permeabilities (dashed curves) [546] (a) oil-free (untreated hydrophilic) filters (b) unstirred water layer permeability versus log MW (c) octanol-soaked (impregnated) filters (d) isopropylmyristate-soaked filters.

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. 

Figure 7.59 Human jejunal permeabilities compared to Caco-2 permeabilities from several groups.

How Well Do Caco-2 Permeability Measurements Predict Human Jejunal Permeabilities ... 

In conclusion, the double-sink su m-P, PAMPA in vitro GIT assay seems to predict human absorption as well as in vivo human permeability measurements (see Figs. 7.66a,b) and in vitro Caco-2 permeability measurements (see Figs. 7.60 and 7.63), but at a lower cost and higher speed. 

Aungst, B. J. Nguyen, N. H. Bulgarelli, J. P. Oates-Lenz, K. The influence of donor and reservoir additives on Caco-2 permeability and secretory transport of HIV protease inhibitors and other lipophilic compounds, Pharm. Res. 17, 1175-1180 (2000). 

In addition, the calculation of many different ID, 2D and 3D descriptors is possible using a range of commercially available software packages, such as Sybyl, Cerius2, Tsar, Molconn-Z and Hybot. Several new descriptor sets are based on quantification of 3D molecular surface properties, and these have been explored for the prediction of, e.g., Caco-2 permeability and oral absorption. It is pointed out here that a number of these new descriptors are strongly correlated to the more traditional physico-chemical properties. 

Mandagere, A. K., T. N. Thompson, and K. K. Hwang. Graphical model for estimating oral bioavailability of drugs in humans and other species from their Caco-2 permeability and in vitro liver enzyme metabolic stability rates, J. Med. Chem. 2002, 45, 304-311... 

The trend in the industry has been to automate the Caco-2 permeability assay using semi- or fully automated procedures. With such a system it is possible to obtain a throughput in order of approximately 400-500 compounds per week. Automated Caco-2 assay systems are commercially available through Tecan/BD Bioscience and Bohdan Mettler Toledo. In addition, automated systems for maintenance of cell cultures are commercially available, while totally automated systems for both maintenance and culturing of cells grown on permeable filter supports are under development, e.g., by companies such as The Automation Partnership. 

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 our own studies to establish an in-house correlation between Caco-2 permeability and extent of drug absorption in humans, a set of 25 model drugs was used (Table 5.1). The importance of concentration and pH conditions were investigated and transport was studied both in apical to basolateral (absorptive) and basolateral to apical (secretory) directions. The apparent permeability coefficients were determined at concentrations of 10, 50, and 500 pM, and at two different settings of apical/basolateral pH values 6.5/7.4 and 7.4/7.4. The marker compounds represented a good diversity in molecular structure and transport properties and covered a range of low (<20%), moderate (20-80%) and high (>80%) extent of absorption in humans (Tab. 5.1). 

Fig. 15.4. Relationship between experimentally determined and PTSA-predicted Caco-2 permeability. PLS predicted permeability from PTSAs (predicted log Caco-2) is plotted versus experimentally obtained Caco-2 data (observed log Caco-2) [1 7]. The PTSAs used for the prediction were (in order of importance) PSA, fraction of total surface area that was polar...

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. 

The model interpretation is in good agreement with the known molecular factors influencing Caco-2 permeability. In addition - and this outlines the originality of the method - VolSurf allows the relevant 3D molecular properties to be quantified. Once the model is developed, as reported above, simple projection of the compound descriptors into it allows predictions to be made for new compounds. 

This model integrates existing in vitro data, such as Caco-2 permeability (Papp) and metabolic stability in liver S9 or microsomes, to estimate bioavailability as being either low, medium, or high. Oral bioavailability predictions for not only humans but also other species can be made by using the metabolic stability values of drugs in liver microsomal enzyme preparations from that species. A premise of this model is that metabolic clearance is more important than renal or biliary clearance in determining bioavailability. However, despite the lack of in vitro renal... 

It is important to recognize that the in vitro permeability obtained in cell mono-layers (such as Caco-2 models) should be considered as a qualitative rather than quantitative value. Especially poor are predictions of fraction dose absorbed for carrier-mediated drugs with low Caco-2 permeability and predictions of high fraction dose absorbed in humans [7, 20, 42, 48, 51]. However, it is possible to establish a reasonably good IVIVC correlation when passive diffusion is the dominating absorption mechanism. 

Fung, E.N. et al. 2003. Higher-throughput screening for Caco-2 permeability utilizing a multiple sprayer liquid chromatography/tandem mass spectrometry system. Rapid Commun. Mass Spectrom. 17 2147. 

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