PDF(Pubmed)
Abstract：
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.
摘要：
目前的药物开发倾向于复杂的化学分子，被称为“超越五规则”(bRo5)化合物，通常表现出具有挑战性的物理化学性质。由于技术限制，难以测量这些化合物的Caco-2渗透性，包括回收率和检测灵敏度差。我们实施了一种新的检测方法,通过优化的孵化和分析，测量接近平衡的渗透率。在该设置中,可实现bRo5化合物的渗透性的适当表征。这种平衡的Caco-2测定法在数据有效性方面得到了验证，复合回收率，以及体外与体内人体吸收的相关性。与标准测定法相比，它在预测参考化合物的人体吸收分数（fa）方面表现出可比的性能。平衡测定还成功地表征了超过90％的分析化合物的渗透性，其中大多数是bRo5（68%）。这些化合物不能使用标准测定法测量。渗透性和流出比(ER)对于大量内部bRo5化合物的体内吸收是高度预测的。参考截止点启用了正确的高分类，中度,低吸收。这种优化的平衡Caco-2测定填补了bRo5化学空间中高通量细胞渗透性方法的空白。
