Staffan Tavelin

Prediction of the Oral Absorption of Low-Permeability Drugs Using Small Intestine-Like 2/4/A1 Cell Monolayers

AbstractPurpose. To characterize the paracellular route of 2/4/A1 monolayers and to compare the permeabilities of incompletely absorbed oral drugs in 2/4/A1 with those in Caco-2 monolayers. Methods. The cells were cultivated on permeable supports. The 2/4/A1 expression of genes associated with tight junctions was compared with that in the small intestine using RT-PCR. The aqueous pore radii were determined using paracellular marker molecules. The permeabilities of a series of incompletely absorbed drugs (defined as having a fraction absorbed 0 to 80%) after oral administration to humans were studied. Results. Occludin and claudin 1 and 3 were expressed in 2/4/A1. The pore radius of 2/4/A1 was 9.0 ± 0.2 Å, which is similar to that in the human small intestine, although the pore radius was smaller (3.7 ± 0.1 Å) in Caco-2. The relationship between permeability and fraction absorbed of 13 drugs was stronger in 2/4/A1 than in Caco-2. The relationships were used to predict the intestinal absorption of another seven drugs. The prediction was more accurate in 2/4/A1 (RMSE = 15.6%) than in Caco-2 (RMSE = 21.1%). Further, Spearmans rank coefficient between FA and permeability was higher in 2/4/A1. Conclusion. The improved 2/4/A1 cell culture model has a more in vivo-like permeability and predicted the oral absorption of incompletely absorbed drugs better than Caco-2 cells.

Exploring the quantitative relationship between the level of MDR1 transcript, protein and function using digoxin as a marker of MDR1-dependent drug efflux activity

A limited number of gene expression studies have investigated the quantitative relationships between the amount of transcript, level of protein or activity/function, with disparate conclusions regarding these relationships. Collectively these studies indicate that the relevance of quantitative transcript analysis as a predictor of phenotype has to be evaluated on a gene-by-gene or even a case-by-case basis. The purpose of this study was to define a suitable marker for MDR1-dependent drug efflux, and to quantitatively investigate the relationships between the amount of transcript, protein and drug efflux in the frequently used Caco-2 cell model. The substrate specificity of digoxin, a commonly used marker for MDR1, was investigated using transgenic MDCK II or LLC-PK1 cell lines expressing the efflux proteins MDR1, BCRP and MRP2, since these proteins are localised to the apical part of the enterocyte plasma membrane and exhibit comparatively high transcript levels in the human small intestine. Relationships between levels of transcript, protein and function were investigated quantitatively using real-time RT-PCR, ECL western blot analysis and basolateral-to-apical and apical-to-basolateral efflux ratios. Our results indicate that digoxin is a specific marker for MDR1-dependent drug efflux in the Caco-2 cell drug absorption model and that MDR1 transcript abundance is at least as valid as MDR1 protein abundance as a predictor of MDR1 efflux activity.

An Improved Cell Culture Model Based on 2/4/A1 Cell Monolayers for Studies of Intestinal Drug Transport: Characterization of Transport Routes

AbstractPurpose. To improve the viability of the 2/4/A1 cell culture model and to investigate different routes of drug transport in this cell line. Methods. Two approaches were taken to decrease apoptosis. First, rat intestinal 2/4/A1 cells were transfected to overexpress the antiapoptotic protein Bcl-2. Second, normal 2/4/A1 cells were cultivated under conditions that stimulate differentiation and limit apoptosis. The monolayer integrity was investigated by transepithelial electrical resistance, permeability, and microscopy. The expression of drug transporters was investigated by RT-PCR, and transport function was assessed using specific markers. Results. Normal 2/4/A1 cells died by apoptosis at 39°C. Bcl-2-expressing 2/4/A1 cells were viable but adopted a morphology of less-differentiated epithelial cells. Optimization of the culture conditions for 2/4/A1 cells inhibited cell death. The integrity was comparable to that of the human jejunum (50 Ω × cm2), making this approach preferable to Bcl-2 overexpression. Transcriptional analysis showed that some (e.g., MDR1), but not all (e.g., PepT1), transporters were found in 2/4/A1 cells. Studies using substrates for PepT1, P-gp, MRP2, and BCRP showed that none of the transporters were functional in 2/4/A1. Conclusions. The improved culture procedure will facilitate the use of 2/4/A1 cells. 2/4/A1 lack several transporters, which makes them a promising alternative to Caco-2 cells and artificial membranes in studies of passive drug transport.

Passive permeability and active transport models for the prediction of oral absorption

A variety of cell culture models, including Caco-2 cells, are used to provide better pharmacokinetic decision grounds in drug discovery. This article examines the properties of such cell culture models and discusses their roles during the various stages in the drug discovery process. The strengths and weaknesses of different models for the assessment of passive drug permeability, active drug transport, and for the prediction of oral drug absorption are reviewed. The performance of the cell culture models as compared to emerging alternatives is also discussed. Further, the influence of the quality of the data and data sets used in permeability and transport assessment is investigated. We conclude that cell culture models can be used both as qualitative tools in higher-throughput modes and as quantitative tools for the selection of druglike compounds with optimal permeability and transport properties in later stages of the drug discovery process.