%0 Journal Article
%T Conquering the beyond Rule of Five Space with an Optimized High-Throughput Caco-2 Assay to Close Gaps in Absorption Prediction.
%A Muschong P
%A Awwad K
%A Price E
%A Mezler M
%A Weinheimer M
%J Pharmaceutics
%V 16
%N 7
%D 2024 Jun 22
%M 39065543
%F 6.525
%R 10.3390/pharmaceutics16070846
%X Current drug development tends towards complex chemical molecules, referred to as "beyond rule of five" (bRo5) compounds, which often exhibit challenging physicochemical properties. Measuring Caco-2 permeability of those compounds is difficult due to technical limitations, including poor recovery and detection sensitivity. We implemented a novel assay, with optimized incubation and analytics, to measure permeability close to equilibrium. In this setup an appropriate characterization of permeability for bRo5 compounds is achievable. This equilibrated Caco-2 assay was verified with respect to data validity, compound recovery, and in vitro to in vivo correlation for human absorption. Compared to a standard assay, it demonstrated comparable performance in predicting the human fraction absorbed (fa) for reference compounds. The equilibrated assay also successfully characterized the permeability of more than 90% of the compounds analyzed, the majority of which were bRo5 (68%). These compounds could not be measured using the standard assay. Permeability and efflux ratio (ER) were highly predictive for in vivo absorption for a large set of internal bRo5 compounds. Reference cut-offs enabled the correct classification of high, moderate, and low absorption. This optimized equilibrated Caco-2 assay closes the gap for a high-throughput cellular permeability method in the bRo5 chemical space.