Synthesis, in vitro characterization and biopharmaceutical evaluation of a novel phosphate prodrug of sorafenib

Sorafenib is a multi-kinase inhibitor approved for several cancers with poor aqueous solubility, resulting in low oral bioavailability (38-49 %) despite its high permeability. As a BCS Class II compound, its absorption is solubility-/dissolution-limited. Prodrug strategies, such as phosphate esterification, can improve solubility and promote oral bioavailability upon enzymatic cleavage by intestinal alkaline phosphatase (IALP), abundantly expressed at the intestinal brush border membrane and present in the intestinal lumen. This cleavage is hypothesized to lead to a transient supersaturation of the parent drug, enhancing absorption unless (premature) precipitation prevails. Besides traditional in vitro models like Caco-2 cells, recently microdialysis-sampling has been introduced to study ALP-mediated biomimetic conversion of oral phosphate prodrugs in vitro. Microdialysis enables real-time, non-destructive sampling of molecularly dissolved drug, which allows to study the complex inter-related dynamics under (simulated) human gastrointestinal conditions. In this study, a novel and new phosphate prodrug of sorafenib (fossorafenib) was synthesized and its physico-chemical and biopharmaceutical properties were evaluated. Fossorafenib exhibited ∼600-fold higher aqueous solubility than sorafenib in HBSS pH 7.4 and was rapidly cleaved by externally added alkaline phosphatase (ALP), yielding supersaturated sorafenib. Combined cleavage & permeability studies across Caco-2 cell monolayers demonstrated rapid bioconversion of fossorafenib in the apical compartment, with permeation of both the converted parent drug but, surprisingly, also the prodrug. Microdialysis enabled real-time monitoring of the biomimetic conversion of fossorafenib without the requirement for enzyme inactivation, thereby allowing for unprecedented mechanistic insights into the interplay between micellar solubilization and enzymatic cleavage. Microdialysis results suggest that micellar incorporation of fossorafenib into mixed micelles of bile salts and phospholipids can hinder enzymatic cleavage by intestinal ALP, thereby potentially limiting the extent of prodrug activation in the intestinal environment. Taken together with the unexpected ability of the prodrug to cross biological/biomimetic barriers, these findings suggest that fossorafenib may not follow the conventional behavior of classical phosphate-ester oral prodrugs in vivo, highlighting the need for further investigation into its unique biopharmaceutical profile.