In vitro and in vivo ADME of heterobifunctional degraders: a tailored approach to optimize DMPK properties of PROTACs©†

Abstract

Proteolysis-targeting chimeras (PROTACs©) have recently emerged as a promising new drug modality. Residing beyond the rule-of-five space, they pose challenges in terms of physicochemical properties. With this study, we contribute to enhancing the understanding of their early ADME characterization. For permeability assessment, transwell assays such as Caco-2 remain challenging. Although the addition of serum may reduce unspecific binding and improve recovery, the assay was not found predictive for absorption. As a surrogate, we propose to focus optimization on molecular descriptors and support a preferred space for oral PROTACs© with ≤3 H-bond donors (HBDs), molecular weight (MW) ≤950 Da and number of rotatable bonds ≤12. We have developed a predictive score serving as initial guidance for design and prioritization according to this property space. In addition, the reduction of exposed polar surface area, e.g. through shielding of HBDs, is a powerful approach to optimize permeability. Using standard small molecule-based methods for in vitro–in vivo extrapolation (IVIVE) of intrinsic clearance (CL_int) with experimentally determined hepatocyte CL_int and fraction unbound in plasma, and predicted fraction unbound in the incubation (f_u,inc), a systematic under-prediction from mouse hepatocytes was observed for PROTACs©. In line with our observation that the Kilford equation was not suitable for PROTAC© f_u,inc prediction, this bias could be overcome by using experimentally determined f_u,inc. Taken together, this study suggests a tailored in vitro DMPK discovery assay cascade and frontloading in vivo studies. It also underlines the need for inclusion of surrogate permeability descriptors and experimentally determined values for IVIVE of CL_int.