Caco-2 cell monolayers, drug permeability

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]. 

The identification and characterization of cell culture systems (e.g., Caco-2-cells) that mimic in vivo biological barriers (e.g., intestinal mucosa) have afforded pharmaceutical scientists the opportunity to rapidly and efficiently assess the permeability of drugs through these barriers in vitro. The results generated from these types of in vitro studies are generally expressed as effective permeability coefficients (Pe). If Pe is properly corrected to account for the barrier effects of the filter (PF) and the aqueous boundary layer (PAbl) as previously described in Section II.C, the results provide the permeability coefficient for the cell monolayer... 

Fig. 4.1. Relationship between the absorbed vivo perfusion of the human jejunum (B) (data fraction (FA) of structurally diverse sets of compiled from publications by Lennernas orally administered drugs and permeability laboratory [63, 115]). Sigmoidal relationships coefficients obtained in Caco-2 cell monolayers were obtained between FA and the (A) (data compiled from publications by permeability coefficients in both models.

Here, we briefly describe the automated Caco-2 assay used at the research site in AstraZeneca R D Molndal. The solubility of the test compounds is measured (or theoretically predicted) before they are run in the Caco-2 assay. In order to be able to make correct determinations of the permeability coefficient, the substance must be dissolved when added to cell monolayer in the transport experiment. Compounds with insufficient solubility are therefore not tested. We generally apply a test concentration of 10 pM, but in specific projects or under certain circumstances a concentration of only 1 pM is applied. The test compounds are first prepared in DM SO solution (1 mM) on a parent plate and are then diluted in transport buffer to give a final drug concentration of 10 pM (solution containing 1% DMSO) when added to the cell monolayers. 

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. 

Fig. 5.3. Relationship between the oral fraction basolateral Papp values were determined at absorbed in humans and apparent permeability drug concentrations of 10-500 pM at pH 6.5/ coefficients obtained in Caco-2 cell monoalyers 7.4 or 7.4/7.4 on the apical/basolateral sides of at two different pH conditions. Mean apical to the cell monolayers.

Molecules with a large molecular weight or size are confined to the transcellular route and its requirements related to the hydrophobicity of the molecule. The transcellular pathway has been evaluated for many years and is thought to be the main route of absorption of many drugs, both with respect to carrier-mediated transport and passive diffusion. The most well-known requirement for the passive part of this route is hydrophobicity, and a relationship between permeability coefficients across cell monolayers such as the Caco-2 versus log P and log D 7.4 or 6.5 have been established [102, 117]. However, this relationship appears to be nonlinear and reaches a plateau at around log P of 2, while higher lipophilicities result in reduced permeability [102, 117, 118]. Because of this, much more attention has recently been paid towards molecular descriptors other than lipophilicity [86, 119-125] (see section 5.5.6.). The relative contribution between the para-cellular and transcellular components has also been evaluated using Caco-2 cells, and for a variety of compounds with different charges [110, 112] and sizes [112] (see Section 5.4.5). 

Passive diffusion through the lipid bilayer of the epithelium can be described using the partition coefficient between octanol/water (log P) and A log P (the difference between the partition into octanol/water and heptane/ethylene glycol or heptane/ octanol) [157, 158], The lipophilicity of the drug (log P) (or rather log D at a certain pH) can easily be either measured or calculated, and is therefore generally used as a predictor of drug permeability. Recently, a method using artificial membrane permeation (PAMPA) has also been found to describe the passive diffusion in a similar manner to the Caco-2 cell monolayers [159]. 

There are several cell monolayer models that are frequently used for the evaluation of drug permeability and absorption potential (Table 18.1). For a more detailed discussion, please refer to Chap. 8. Caco-2 cells (adenocarcinoma cells derived from colon) are the most extensively characterized and frequently used of the available cell lines [5-9], A unique feature of Caco-2 cells is that they undergo spontaneous enterocyte differentiation in cell culture. Unlike intestinal enterocytes, Caco-2 cells are immortalized and replicate rapidly into confluent monolayers. When the cells reach confluency during culture on a semi-porous membrane, they start to polarize and form tight junctions, creating an ideal system for permeability and transport studies. During the past decade, use of... 

Motz SA, Schaefer UF, Balbach S, Eichinger T, Lehr CM (2006) Permeability assessment for solid oral drug formulations based on Caco-2 monolayer in combination with a flow through dissolution cell. Eur J Pharm Biopharm 66 (2) 286-295... 

Cell monolayers grown on permeable culture inserts form confluent mono-layers with barrier properties and can be used for drug absorption experiments. The most well-known cell line for the in vitro determination of intestinal drug permeability is the human colon adenocarcinoma Caco-2 [20, 21], The utility of the Caco-2 cell line is due to its spontaneous differentiation to enterocytes under conventional cell culture conditions upon reaching confluency on a porous membrane to resemble the intestinal epithelium. This cell model displays small intestinal carriers, brush borders, villous cell model, tight junctions, and high resistance [22], Caco-2 cells express active transport systems, brush border enzymes, and phase I and II enzymes [22-24], Permeability models... 

Other cell lines used in permeability studies include the T84 human colonic adenocarcinoma colonic crypt cell model. This line has a reduced carrier expression, secrets mucus, and has very high resistance [31, 32], The IEC cell line is a rat fetal intestinal epithelium cell with higher permeabilities than Caco-2 cells [33], LLC PKi is a pig kidney epithelial cell line with low expression of efflux systems, but expression systems for transport proteins [32], 2/4/A1 cells are a conditionally immortalized rat fetal intestinal epithelium line with crypt cell-like morphology and temperature-sensitive differentiation [34], They form differentiated monolayers with tight junctions, increased brush border enzymes when grown on extracellular matrices with laminin. Transport of drugs with LP in 2/4/A1 monolayers was comparable to that in the human jejunum and up to 300 times faster than that in Caco-2 monolayers. In contrast, the permeability of HP drugs was comparable in both cell lines [34],... 

Compared to in vivo studies, the Caco-2 model substantially increases the speed at which absorption potential can be estimated and reduces the amount of drug substance needed. However, manually performed assays are still too slow and labor intensive compared to biological high-throughput screening assays. Caco-2 cells take about 3 weeks to form monolayers of fully differentiated cells. At this point, Caco-2 monolayers are used to evaluate absorption potential under a variety of permeability protocols. In order to further expedite the process of absorption potential assessment, efforts have been made to increase the throughput of Caco-2 transport experiments. 

VolSurf was also successfully applied in the literature to predict absorption properties [156] from experimental drug permeability data of 55 compounds [165] in Caco-2 cells (human intestinal epithelial cell line derived from a colorectal carcinoma) and MDCK cell monolayers (Madin-Darby canine kidney). In this interesting case, it was shown that models including counterions for charged molecules clearly show significantly better quality and overall performance. The final model was also able to correctly predict, to a great extent, the relative ranking of molecules from another Caco-2 permeability study by Yazdanian et al. ]166]. 

Wang, Z. Hop, C. E. Leung, K. H. Pang, J. Determination of in vitro permeability of drug candidates through a caco-2 cell monolayer by liquid chromatography/tandem mass spectrometry. J Mass Spectrom 2000, 35, 71-76. 

The paracellular permeation pathway in the intestinal cell monolayer models is often limited. Therefore these models are not suitable for predicting permeability of paracellularly absorbed compounds. The average pore radius in Caco-2 cells (<6 A) is more representative of the colon than the small intestine (8-13 A)and paracellular transport can be up to 100-fold lower in Caco-2 cells than in the small intestine. Investigation of a rat intestinal cell line 2/4/Al, which forms polarized cell mono-layers and has an average pore radius (9 A) more representative of the small intestine, showed improved prediction of oral absorption for incompletely absorbed drugs [24, 25]. 

One of the main in vitro permeability assays used in the pharmaceutical industry has been for many years the Caco-2 monolayer. Therefore, most of the in silica models developed to predict permeability were based on Caco-2 data. Hou and Johnson produced a couple of reviews that comprehensibly summarizes the recent efforts using Caco-2 permeability data [92, 94]. All those models are designed to predict the influx or apparent permeability of drugs in the same direction as intestinal absorption occurs, that is, from the apical to the basal side of the cell line, regardless of the extent of active transport involved in the permeation process. 

Camenisch, G., et al. 1998. Estimation of permeability by passive diffusion through Caco-2 cell monolayers using the drugs lipophilicity and molecular weight. Eur J Pharm Sci 6 317. 

Batrakova, E.V., et al. 1999. Pluronic P85 increases permeability of a broad spectrum of drugs in polarized BBMEC and Caco-2 cell monolayers. Pharm Res 16 1366. 

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