Automation, drug transporter

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. 

Receptors or enzymes are the preferred dmg targets since binding and enzyme inhibitor assays are standard in a research laboratory, they can be established easily and offer the possibility of automation. Transport proteins, ion channels and transcription factors are less attractive targets for drug screening programs, because automation is costly and not always possible. In addition, the requirement for highly sophisticated equipment and expertise is decisive. 

Some of the advantages of cell monolayer models include the ability to use human instead of animal cell types as well as the ability to perform cellular uptake and bidirectional cell transport studies for evaluation of absorptive and secretory processes. The potential for automation to achieve higher throughput in the early drug discovery setting is an added attraction. Regardless of the cell type used, the utility of these models in transport studies is based on the correlation between permeability properties determined in these models and those obtained in vivo, such as fraction of dose absorbed (Fig. 3). To date, numerous laboratories have established a correlation between apparent permeability coefficients (P pf) from Caco-2 or MDCK cells and in vivo fraction absorbed of drugs in solution [58—62]. Construction of correlation plots for known compounds or reference markers then provides an opportunity for interpolation of fraction absorbed for NMEs for which in vivo fraction absorbed is unknown. 

---