Light-Induced Structural Rearrangements of Axitinib and Their Role in Phototoxic Responses.

Axitinib (AXT) is a second-generation tyrosine kinase inhibitor widely used in advanced renal cell carcinoma and currently explored in light-modulated therapies due to its E/Z photoisomerizable scaffold. Despite its clinical relevance and UVA absorption, the photobiological consequences of its light-induced structural rearrangements remain largely unexplored. Here, we elucidate the mechanistic basis of AXT phototoxicity by integrating photophysical characterization with cellular studies while distinguishing the roles of its (E)- and (Z)-isomers. Steady-state photolysis reveals that (E)-AXT undergoes photodegradation in aqueous media but preferentially isomerizes to (Z)-AXT in lipophilic environments, which is accelerated in the presence of human serum albumin (HSA). Conversely, (Z)-AXT displays medium-dependent photostability, being stabilized by HSA and reverting to the (E)-isomer in aqueous solution. Spectroscopic studies, including laser flash photolysis, support a mechanism involving tautomerization through excited-state intramolecular proton transfer and transient radical intermediates, consistent with predominant Type I photosensitization. Notably, (Z)-AXT is favored in lipophilic and protein-rich environments, likely through intramolecular hydrogen-bond stabilization of its 2H-tautomer. Cellular studies in keratinocytes revealed two complementary phototoxic pathways: photoinduced conversion of cytotoxic (Z)-AXT from (E)-AXT and the intrinsic phototoxicity of (Z)-AXT under irradiation. Although both isomers cause similar protein oxidation, (Z)-AXT is the main contributor due to its higher stability with HSA. While alkaline comet assays detected no direct DNA strand breaks, γ-H2AX immunostaining revealed light-induced DNA double-strand breaks. Overall, these findings identify (Z)-AXT as a previously unrecognized phototoxic species and establish a framework linking isomerization, microenvironmental stabilization, and Type I photosensitization in kinase inhibitors.