Automated Caco-2 Assay

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. 

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. 

Very few published data exist on the evaluation of automated systems, though one report has been made of an automated absorption assay using Caco-2 cells cultured on both sides of polycarbonate membranes [93], The concept of culturing cells on the lower sides of the membranes was investigated as a means of improving the opportunity to study transport in the secretory basolateral to apical direction. However, this approach resulted in increased variability and impaired active transport properties of the cell monolayers, and was therefore not recommended. 

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Partly due to the limited throughput of Caco-2 permeability measurements, the structure-activity evaluation of compounds tested in the hit-to-lead phase is done with minimal permeability information, at best. Given the importance of membrane permeability in drug absorption, early consideration of the permeability characteristics of hit compounds would enhance the drug-like quality, and ultimately the probability of success, of selected lead candidates. To incorporate permeability information into the hit-to-lead phase of the drug discovery process it is necessary that permeability measurements be made quickly and with small amounts of material. Thus, efforts have been made to automate and miniaturize the Caco-2 permeability assay. 

The kitchen of a fast food restaurant is characterized by islands of automation, with well defined subprocesses focused on producing a certain kind of output, coordinated by a crew chief The principal advantage of a fast food restaurant is consistency and fast delivery. The dedicated subunits are designed to perform a certain type of process (assay) at a high rate with very little room for change. Economically, this model is difficult to sustain unless each assay type has sufficient demand to justify the existence of dedicated space, equipment and personnel. It is also not as efficient as a secondary screening model. For assays that are routinely, but not always, requested then this model is very appropriate (e.g., CACO-2 permeability, microsomal stability). However, for the more costly and complex assays that are requested less often, the cost of dedicated people and equipment is hard to justify and as a result the assay has to come off the menu. This is why most fast food restaurants have a relatively limited menu, including mostly foods that are simple to prepare. 

Caco-2 cells are currently used at all levels of pharmaceutical research and development. Automation technologies allow tissue culture labs to easily maintain a large number of Caco-2 cells as well as to perform numerous permeability studies without the introduction of many common human errors. Combinatorial chemistry provides vast arrays of compounds, and Caco-2 assays can be used to assess potential permeability and metabolic issues before much money is invested in the candidate.6... 

These automated assays can be used for high-throughput ADME screening in early drug discovery. The double-sink PAMPA permeability assay mimics in vivo conditions by the use of a chemical sink in the acceptor wells and pH gradient in the donor wells. The use of the pION gut-box integrated on the deck has shortened the PAMPA assay incubation time to 30 minutes. The permeability coefficient and rank order correlate well with data obtained using the in vitro Caco-2 assay and in vivo permeability properties measured in rat intestinal perfusions. 

Due to the high cost of cell culture, Caco-2 assays are usually used as a follow-up to PAMPA in ADME screening [78], and as a result, the sample burden for bioanalysis is not as heavy as for some first-hne assays, such as metabolic stability. There have been a number of reports in the literature that use automated optimization and single LC-MS/MS for sample analysis for Caco-2 assay support [46,79-81]. Nevertheless, Caco-2 samples pose a unique bioanalytical challenge. Unlike plasma or microsomal samples rich in proteins that help solubilize compounds and prevent adsorptive loss, Caco-2 samples are essentially aqueous buffer samples with very little protein. As a result, compounds with low solubility and/ or adsorption problems tend to exhibit poor recoveries in the assay due to precipitation and adsorptive losses [82,83]. An effective solution to this problem is the use of organic solvent to catch compounds immediately after incubation, but prior to analysis, in order to maintain solubility and prevent adsorptive loss to container surfaces. Another approach involves the addition of some protein such as bovine serum albumin (BSA) to the assay buffer system, thus reducing compound loss/ precipitation and improving recoveries [84]. 

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