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The photobiological damage that certain drugs or their metabolites can photosensitize in proteins is generally associated with the nature of the excited species that are generated upon interaction with UVA light. In this regard, the photoinduced damage of the anticancer drug gefitinib (GFT) and its two main photoactive metabolites GFT-M1 and GFT-M2 in cellular milieu was recently investigated. With this background, the photophysical properties of both the drug and its metabolites have now been studied in the presence of the two main transport proteins of human plasma, i.e., serum albumin (HSA) and α1-acid glycoprotein (HAG) upon UVA light excitation. In general, the observed photobehavior was strongly affected by the confined environment provided by the protein. Thus, GFT-M1 (which exhibits the highest phototoxicity) showed the highest fluorescence yield arising from long-lived HSA-bound phenolate-like excited species. Conversely, locally excited (LE) states were formed within HAG, resulting in lower fluorescence yields. The reserve was true for GFT-M2, which despite being also a phenol, led mainly to formation of LE states within HSA, and phenolate-like species (with a minor contribution of LE) inside HAG. Finally, the parent drug GFT, which is known to form LE states within HSA, exhibited a parallel behavior in the two proteins. In addition, determination of the association constants by both absorption and emission spectroscopy revealed that the two metabolites bind stronger to HSA than the parent drug, whereas smaller differences were observed for HAG. This was further confirmed by studying the competing interactions between GFT or its metabolites with the two proteins using fluorescence measurements. These above experimental findings were satisfactorily correlated with the results obtained by means of molecular dynamics (MD) simulations, which revealed the high affinity binding sites, the strength of interactions and the involved amino acid residues. In general, the differences observed in the photobehavior of the drug and its two photoactive metabolites in protein media are consistent with their relative photosensitizing potentials.

Gefitinib (GFT) is a selective epidermal growth factor receptor (EGFR) inhibitor clinically used for the treatment of patients with non-small cell lung cancer. Bioactivation by mainly Phase I hepatic metabolism leads to chemically reactive metabolites such as O-Demethyl gefitinib (DMT-GFT), 4-Defluoro-4-hydroxy gefitinib (DF-GFT), and O-Demorpholinopropyl gefitinib (DMOR-GFT), which display an enhanced UV-light absorption. In this context, the aim of the present study is to investigate the capability of gefitinib metabolites to induce photosensitivity disorders and to elucidate the involved mechanisms. According to the neutral red uptake (NRU) phototoxicity test, only DF-GFT metabolite can be considered non-phototoxic to cells with a photoirritation factor (PIF) close to 1. Moreover, DMOR-GFT is markedly more phototoxic than the parent drug (PIF = 48), whereas DMT-GFT is much less phototoxic (PIF = 7). Using the thiobarbituric acid reactive substances (TBARS) method as an indicator of lipid photoperoxidation, only DMOR-GFT has demonstrated the ability to photosensitize this process, resulting in a significant amount of TBARS (similar to ketoprofen, which was used as the positive control). Protein photooxidation monitored by 2,4-dinitrophenylhydrazine (DNPH) derivatization method is mainly mediated by GFT and, to a lesser extent, by DMOR-GFT; in contrast, protein oxidation associated with DMT-GFT is nearly negligible. Interestingly, the damage to cellular DNA as revealed by the comet assay, indicates that DMT-GFT has the highest photogenotoxic potential; moreover, the DNA damage induced by this metabolite is hardly repaired by the cells after a time recovery of 18 h. This could ultimately result in mutagenic and carcinogenic effects. These results could aid oncologists when prescribing TKIs to cancer patients and, thus, establish the conditions of use and recommend photoprotection guidelines.