Krzysztof Reszka

Enzymatic oxidative activation and transformation of the antitumor agent mitoxantrone.

Ambient temperature incubation of the anticancer agent mitoxantrone with horseradish peroxidase and hydrogen peroxide converts it into a hexahydronaphtho[2,3-f]quinoxaline-7,12-dione in which one side chain has cyclized to the chromophore. The structure of this cyclic metabolite was secured by independent synthesis. This peroxidative conversion of mitoxantrone, the progress of which can be followed spectrophotometrically, is accompanied by formation of a free radical species. The EPR characteristics, and dependence on pH of the latter, suggest it exists as a radical cation. The enzymatic oxidation of mitoxantrone is totally irreversible. The purified cyclic metabolite is a substrate for the peroxidase affording the unstable fully oxidized diimino compound and this reaction is fully reversible upon addition of ascorbate or other biological reductants. Admixture of the fully oxidized diimino product with the reduced cyclic metabolite generates the corresponding radical cation species by disproportionation-comproportionation processes. Independent kinetic studies confirm that reaction of the peroxidase with the cyclic metabolite proceeds more rapidly than with mitoxantrone itself. A derivative of mitoxantrone, in which the side-chain secondary amine functions are acylated, generates a radical cation upon treatment with the peroxidase-H2O2 system but does not cyclize subsequently. Derivatives without phenolic hydroxyls or those in which the phenolic hydroxyls are blocked also undergo peroxidative reaction. These observations suggest that initial peroxidative attack occurs at the aromatic nitrogens of mitoxantrone. The possible relevance of these results to the anticancer action of mitoxantrone and the implications for suppression of lipid peroxidation in vivo are discussed.

Evidence for a peroxidatic oxidation of norepinephrine, a catecholamine, by lactoperoxidase

The electron spin resonance-spin stabilization technique has been applied to identify the o-semiquinone intermediate produced during the lactoperoxidase-catalyzed oxidation of the catecholamine norepinephrine. The results of a rapid scan and spectrophotometric investigation of the reaction clearly indicate a normal peroxidatic pathway of catecholamine degradation.

Oxidation of the substituted catechols dihydroxyphenylalanine methyl ester and trihydroxyphenylalanine by lactoperoxidase and its compounds.

The reactions of native lactoperoxidase and its compound II with two substituted catechols have been investigated by ESR spin stabilization and spin trapping and by rapid scan and conventional spectrophotometric techniques. The catechols are Dopa methyl ester (dihydroxyphenylalanine methyl ester) and 6-hydroxy-Dopa (trihydroxyphenylalanine). o-Semiquinone radicals are formed in the anaerobic reaction of Dopa methyl ester with hydrogen peroxide catalyzed by native lactoperoxidase. The comparable anaerobic reaction of 6-hydroxy-Dopa appears to produce hydroxyl radicals in an unusual reaction. Compound II is reduced back to native lactoperoxidase by both catechols. The reaction between Dopa methyl ester and compound II undergoes an oscillation. The results on the overall lactoperoxidase cycle indicate two successive one-electron reductions of the peroxidase intermediates back to the native enzyme. The resulting free radical formation of o- and p-semiquinones and subsequent formation of stable quinones and Dopachromes is dependent upon the stereochemical arrangement of the catechol hydroxyl groups.

Interaction of the peroxidase-derived metabolite of mitoxantrone with nucleic acids: evidence for covalent binding of 14C-labeled drug

The antitumor agent mitoxantrone undergoes horseradish peroxidase-catalyzed oxidation by hydrogen peroxide to an identifiable cyclic metabolite which is a substituted hexahydronaphtho[2,3-f]-quinoxaline-7,12-dione. Binding of mitoxantrone to DNA inhibited enzymatic oxidation of the drug. The metabolite of mitoxantrone, derived from the action of the HRP/H2O2 system on the drug, bound non-covalently to DNA oligomers. Spectrophotometric analyses of such complexes showed formation of a new, blue-shifted, metachromatic absorption band which was observed when the DNA base pair to drug ratio was close to 1. Measurements of DNA unwinding angles suggest that the metabolite, in contrast to mitoxantrone, did not intercalate but rather bound externally to DNA. Experiments with 14C-labeled mitoxantrone confirmed that peroxidase-activated drug binds covalently to DNA.

PHOTOSENSITIZATION OF ANTICANCER AGENTS‐8. ONE‐ELECTRON REDUCTION OF MITOXANTRONE: AN EPR AND SPECTROPHOTOMETRIC STUDY

An efficient method of one‐electron reduction of the anticancer agent mitoxantrone is described. The method depends on illumination of a suitable photosensitizer absorbing blue light [acriflavine, anthrapyrazole, or Ru(bpy)+] in the presence of the drug and an electron donor, such as NAD(P)H, in deaerated solutions. An EPR spectrum, assigned to a semiquinone of mitoxantrone, is generated under these conditions and identified by spectral simulation. Decay of this species, attributed to a radical‐radical reaction, gives a second order rate constant of 1.7 102M‐1s‐1 in organic media [dimethylsulfoxide (DMSO)/pH 8 buffer, 1:1 vol/vol] but is more rapid (˜104 M‐1 s‐1) in aqueous media under comparable conditions. The considerably decreased lifetime of the mitoxantrone radical at pH 5 is attributed to an additional electron transfer, promoted by protonation of the radical, and/or to an accelerated recombination of neutral radicals, leading to an EPR‐silent species. Parallel spectrophotometric studies on the generation of the mitoxantrone reduced species by photosensitized reduction are described. The method offers convenient access to a key radical species involved in the metabolism and possible mode of action of this clinical anticancer agent.

Subcellular distribution of a nitroxide spin-labeled netropsin in living KB cells: Electron paramagnetic resonance and sequence specificity studies

A nitroxide spin-labeled netropsin was studied by EPR spectroscopy with respect to its uptake and localization in living KB cells. Whereas the drug was taken up readily, there was relatively little drug in the cytoplasm, but a significant concentration of the drug in the cell nucleus. The EPR signal in the latter site corresponded to a relatively freely rotating radical. The drug exhibited good intracellular stability up to 25 hr. While a delta Tm of 24 degrees between the spin-labeled netropsin and calf thymus DNA confirmed strong binding, the absence of any DNA elongation by viscometry was consistent with nonintercalative exterior binding which was confirmed to be minor groove specific by binding of the agent to T4 DNA with a delta Tm of 17.5 degrees. The sequence specificity of the DNA binding of the spin-labeled drug was confirmed by methidiumpropyl-EDTA (MPE) footprinting on a fragment of pBR322 DNA to be very similar to that of the parent netropsin, i.e. selective for AT-rich sites, with minor differences of protection afforded by introduction of the nitroxide label.

PHOTOSENSITIZATION BY ANTITUMOR AGENTS—1. PRODUCTION OF SINGLET OXYGEN DURING IRRADIATION OF ANTHRAPYRAZOLES WITH VISIBLE LIGHT

Abstract— Oxygen consumption, photoinduced by visible light, and sensitized by novel anthrapyrazole antitumor agents has been observed. Generation of singlet oxygen upon irradiation of ethanolic solutions of the drugs with visible light (480–520 nm) was demonstrated using a specific 1O2 acceptor, 2.5‐dimethylfuran and a quencher, sodium azide. An electron paramagnetic resonance method was employed to measure the rate of oxygen consumption. Significant differences were found in the sensitizing properties among the anthrapyrazoles studied. Intramolecular hydrogen bonding within the chro‐mophore is one of the structural factors that determine the efficacy of a given anthrapyrazole in 1O2 generation

PHOTOSENSITIZATION BY ANTITUMORAGENTS–6. PRODUCTION OF SUPEROXIDE RADICAL AND HYDROGEN PEROXIDE DURING ILLUMINATION OF DIAMINOANTHRACENEDIONES IN THE PRESENCE OF NADH IN AQUEOUS SOLUTIONS: AN EPR STUDY

Abstract— Photosensitizing capabilities of anthracenedione anticancer agents to oxidize NADH in aqueous solutions have been studied by EPR and spin trapping techniques. It is demonstrated that 1,4‐diamino substituted anthraquinones, like mitoxantrone and ametantrone, do not photosensitize NADH oxidation while 1,5‐ and l,8‐bis[[(diethylamino)ethyl]amino]anthraquinones do, undergoing simultaneous one‐electron reduction to their semiquinone radical forms upon illumination with visible light. In aerated aqueous solutions the reaction leads to the production of superoxide ion and hydrogen peroxide.

Photosensitization by antitumor agents 3: spectroscopic evidence for superoxide and hydroxyl radical production by anthrapyrazole-sensitized oxidation of NADH.

EPR and spin-trapping techniques were employed to study the oxidation of the dihydronicotinamide adenine dinucleotide (NADH) photosensitized by an anthrapyrazole-antitumor agent. The superoxide radical was detected as a DMPO adduct upon illumination of the system with visible light. Photoinduced generation of hydroxyl radicals is demonstrated by detection of DMPO adducts of OH scavengers, such as ethyl alcohol, sodium formate, and sodium azide. The dependence of the production of these spin adducts on the presence of catalase implies the involvement of hydrogen peroxide in that process. The production of hydrogen peroxide is demonstrated independently during oxygen consumption measurements with the Clark electrode technique.

Photosensitization by selected anticancer agents

Novel anticancer anthrapyrazoles and anthracenediones are available as alternatives to the cardiotoxic clinical agents, doxorubicin and daunorubicin. Certain representatives of these new classes of compounds possess photosensitizing properties. The structural features influencing the photophysical parameters of these agents are discussed. Photosensitizing reactions involving singlet oxygen production, free radical formation, decomposition of hydrogen peroxide and organic hydroperoxides, oxidation of certain biochemical electron donors, DNA damage and killing of human leukemic cells in vitro in the presence of photoactive anthrapyrazoles, anthracenediones and anthracyclines are described.