Examinations of the antioxidative properties of the topically administered drug bufexamac reveal new insights into its mechanism of action

Abstract

The effect of bufexamac on UV-irradiation-induced lipid peroxidation was investigated. Linolenic acid was used as a model lipid. Bufexamac was shown to be capable of reducing the amount of lipid peroxidation. The quantification was carried out by the thiobarbituric acid assay. Irradiation experiments were also performed using HaCaT keratinocytes as a model system. The oxidative changes were quantified by DNA synthesis measurements and cell viability determinations. Bufexamac was found to act antioxidatively again. To investigate free radical involvement, electron paramagnetic resonance studies were carried out. The influence of bufexamac on the concentration of hydroxyl radicals generated by the Fenton system was examined using the spin trapping technique. Moreover, the hydroxamic acid's ability to react with stable radicals was checked. The quantification assay of 2,2-diphenyl-1-picrylhydrazyl hydrate showed no concentration changes of the stable radical caused by bufexamac. In the Fenton assay antioxidative effects were measured after the addition of the drug. The qualitative changes after irradiating bufexamac were studied at a molecular level by electrospray mass spectrometry. Multiple-stage mass spectrometry experiments enabled the establishment of fragmentation schemes. Phenolic degradation products were detected. The results suggest a new interpretation of the controversially debated mechanism of action of bufexamac and indicate possible reasons for its eczema provoking potential.