Mitoxantrone (MH(2)X), an
anthraquinone-type anti-
cancer agent used clinically in the treatment of human
malignancies, is oxidatively activated by the
peroxidase/H(2)O(2)
enzyme system. In contrast to the enzymatic mechanisms of
drug oxidation, the chemical transformations of MH(2)X are not well described. In this study, MH(2)X metabolites, produced by the horseradish, lacto- or
lignin peroxidase (respectively HRP, LPO and LIP)/H(2)O(2) system, were investigated by steady-state spectrokinetic and HPLC-MS methods. At an equimolar
mitoxantrone/H(2)O(2) ratio, the efficacy of the
enzyme-catalyzed oxidation of
mitoxantrone decreased in the following order: LPO > HRP > LIP, which accorded with the decreasing size of the substrate access channel in the
enzyme panel examined. In all cases, the central
drug oxidation product was the redox-active cyclic metabolite, hexahydronaphtho-[2,3-f]-
quinoxaline-7,12-dione (MH(2)), previously identified in the urine of
mitoxantrone-treated patients. As the reaction progressed, data gathered in this study suggests that further oxidation of the MH(2) side-chains occurred, yielding the mono- and dicarboxylic
acid derivatives respectively. Based on the available data a further MH(2) derivative is proposed, in which the amino-alkyl side-chain(s) are cyclised. With increasing H(2)O(2) concentrations, these novel MH(2) derivatives were oxidised to additional metabolites, whose spectral properties and MS data indicated a stepwise destruction of the MH(2) chromophore due to an oxidative cleavage of the
9,10-anthracenedione moiety. The novel metabolites extend the known sequence of
peroxidase-induced
mitoxantrone metabolism, and may contribute to the cytotoxic effects of the
drug in vivo. Based on the structural features of the proposed MH(2) oxidation products we elaborate on various biochemical mechanisms, which extend the understanding of
mitoxantrone's
pharmaceutical action and its clinical effectiveness with a particular focus on
peroxidase-expressing solid
tumors, such as
breast carcinoma.