Addition of Optimized Bovine Serum Albumin Level in a High-Throughput Caco-2 Assay Enabled Accurate Permeability Assessment for Lipophilic Compounds.

The Caco-2 permeability assay is a well-accepted in vitro model to evaluate compounds' potential for oral absorption at early discovery. However, for many lipophilic compounds, no meaningful Caco-2 data could be generated due to their low solubility in assay buffer and/or poor recovery from the assay. In our previous study, we reported an organic catch approach to improve compound recovery. To further reduce compound loss and increase solubility in aqueous buffer, we explored the addition of bovine serum albumin (BSA). However, in contrast to the commonly used BSA level at 4%, a lower level of BSA was selected in an effort to minimize the potential risk of missing the identification of efflux substrates, and to avoid the extensive sample cleanup needed for 4% BSA. Through a systematic evaluation, it was found that 0.5% BSA was effective in enhancing compound solubility and reducing nonspecific binding, which allowed reliable assessment of the permeability and efflux potential for lipophilic compounds. Also, with an optimized sample handling process, no extra sample cleanup was required before liquid chromatography-mass spectrometry (LC-MS) analysis. The implementation of this assay has enabled accurate permeability assessment for compounds that had poor solubility and/or poor mass balance under the non-BSA assay conditions.