Abstract
The expression levels of genes involved in drug and nutrient absorption were evaluated in the Madin-Darby Canine Kidney (MDCK) in vitro drug absorption model. MDCK cells were grown on plastic surfaces (for 3 days) or on Transwell® membranes (for 3, 5, 7, and 9 days). The expression profile of genes including ABC transporters, SLC transporters, and cytochrome P450 (CYP) enzymes was determined using the Affymetrix® Canine GeneChip®. Expression of genes whose probe sets passed a stringent confirmation process was examined. Expression of a few transporter (MDR1, PEPT1 and PEPT2) genes in MDCK cells was confirmed by RT-PCR. The overall gene expression profile was strongly influenced by the type of support the cells were grown on. After 3 days of growth, expression of 28% of the genes was statistically different (1.5-fold cutoff, p < 0.05) between the cells grown on plastic and Transwell® membranes. When cells were differentiated on Transwell® membranes, large changes in gene expression profile were observed during the early stages, which then stabilized after 5–7 days. Only a small number of genes encoding drug absorption related SLC, ABC, and CYP were detected in MDCK cells, and most of them exhibited low hybridization signals. Results from this study provide valuable reference information on endogenous gene expression in MDCK cells that could assist in design of drug-transporter and/or drug-enzyme interaction studies, and help interpret the contributions of various transporters and metabolic enzymes in studies with MDCK cells.
Highlights
Cell-based in vitro drug permeation models are very attractive because they are amenable to high-throughput screening and can be implemented at a relatively low cost compared to the in vivo models [1]
Madin-Darby Canine Kidney (MDCK) cells grown on the Transwell® membranes resulted in much denser monolayers than the cells grown on plastic plates for the same period (7 days)
To study the gene expression profile of MDCK cells during the differentiation process, MDCK cells grown on the Transwell® membranes were harvested at various time points, including before and after TEER values were stabilized
Summary
Cell-based in vitro drug permeation models are very attractive because they are amenable to high-throughput screening and can be implemented at a relatively low cost compared to the in vivo models [1]. Caco-2 in vitro cell culture model is a well-established and widely used model to characterize the human intestinal permeability of compounds. Studies have shown that Caco-2 permeability of the compounds that are absorbed passively correlated with their human intestinal permeability; this is not the case for compounds that depend on membrane transporters for absorption across intestinal barriers [2]. Β-lactam antibiotics and angiotensin-converting enzyme (ACE) inhibitors are well-absorbed in vivo, but not in the Caco-2 model, presumably due to lower expression of the transporters, such as PepT1 in Caco-2 cells than in human small intestine [1,2]. To overcome the issues mentioned above, some investigators have suggested using alternative in vitro cell culture models [1,4,5]
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