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.     

The antitumor agent mitoxantrone binds cooperatively to DNA: evidence for heterogeneity in DNA conformation

The equilibrium binding of the antitumor compound DHAQ, or mitoxantrone [1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9,10- anthracenedione], to various DNAs has been examined by optical titration and equilibrium dialysis methods. At low r (bound drug/DNA base pair) values, r less than 0.03, DHAQ binds, in a highly cooperative manner, to calf thymus and Micrococcus lysodeikticus DNAs. The binding isotherms for the interaction of DHAQ with Clostridium perfringens DNA and poly(dA-dT).poly(dA-dT) exhibit a small positive slope at low r values, suggestive of cooperative binding. In contrast, the binding of DHAQ to poly(dG-dC).poly(dG-dC) shows no evidence of cooperative binding even at very low r values. At higher r values (r greater than 0.05), the binding of DHAQ to all the DNAs studied is characterized by a neighbor-exclusion process. A model is proposed to account for the two modes of binding exhibited in the cooperative binding isotherms. The main feature of the proposed model is that local sequence and structural heterogeneity of the DNA give rise to sets of binding sites to which DHAQ binds in a highly cooperative manner, while the majority of the DNA sites bind DHAQ via a neighbor-exclusion process. This two-site model reproduces the observed binding isotherms and leads to the conclusion that DHAQ binds in clusters to selected regions of DNA. It is suggested that clustering may play a role in the physiological activity of drugs.     

A novel carbazole derivative, BMVC: a potential antitumor agent and fluorescence marker of cancer cells

We have investigated a novel compound, 3,6-bis[2-(1-methylpyridinium)vinyl]carbazole diiodide (BMVC), for inhibiting telomerase activity and distinguishing human lung H1299 and oral Ca9-22 cancer cells from lung IMR90 and skin Detroit-551 normal fibroblast cells. The telomeric repeat amplification protocol (TRAP) assay shows that the concentration of BMVC that inhibits 50% of the telomerase activity (IC50) is ca. 0.05 microM. On the other hand, the cell-viability assay indicates that the cytotoxicity was less than 15% to the H1299 and Ca9-22 cancer cells, and almost negligible to the MRC-5 and Detroit-551 normal cells after incubation with 0.5 microM BMVC for 72 h. The low concentration of 0.05 microM of BMVC can inhibit telomerase activity but does not have general toxic effects to normal cells, implying that BMVC is a promising telomerase inhibitor. Moreover, wide-field fluorescence images of 0.1 microM BMVC-treated cells show bright fluorescence spots in the nuclei of the most H1299 and Ca9-22 cancer cells. Interestingly, similar fluorescence spots are hardly observed in the nuclei of the IMR90 and Detroit-551 normal cells, implying that BMVC might be a useful marker to distinguish tumor cells and normal cells.     

Oxidative metabolism of the anti-cancer agent mitoxantrone by horseradish, lacto-and lignin peroxidase

Mitoxantrone (MH₂X), an anthraquinone-type anti-cancer agent used clinically in the treatment of human malignancies, is oxidatively activated by the peroxidase/H₂O₂ enzyme system. In contrast to the enzymatic mechanisms of drug oxidation, the chemical transformations of MH₂X are not well described. In this study, MH₂X metabolites, produced by the horseradish, lacto- or lignin peroxidase (respectively HRP, LPO and LIP)/H₂O₂ system, were investigated by steady-state spectrokinetic and HPLC-MS methods. At an equimolar mitoxantrone/H₂O₂ 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 (MH₂), previously identified in the urine of mitoxantrone-treated patients. As the reaction progressed, data gathered in this study suggests that further oxidation of the MH₂ side-chains occurred, yielding the mono- and dicarboxylic acid derivatives respectively. Based on the available data a further MH₂ derivative is proposed, in which the amino-alkyl side-chain(s) are cyclised. With increasing H₂O₂ concentrations, these novel MH₂ derivatives were oxidised to additional metabolites, whose spectral properties and MS data indicated a stepwise destruction of the MH₂ 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 MH₂ 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.     

A fluorescence assay for 4'-(9-acridinylamino)methanesulfon-m-anisidide, a new antitumor agent.

A sensitive fluorescent method is described for the detection of 4′-(9-acridinylamino)-methanesulfon-m-anisidide (AMSA) in serum. The assay is based on the alkaline hydrolysis of AMSA into 9(10H)-acridone. While AMSA has negligible fluorescence, 9(10H)-acridone fluoresces brilliantly (excitation 266 nm, emission 470 nm). The assay is shown to be linear from 0.01 to 1.0 μm. In addition, the assay is shown to be useful, in conjunction with an ethyl acetate extraction, in distinguishing serum levels of parent AMSA from metabolized or protein-bound AMSA.     

Interaction of the antineoplastic agent mitoxantrone with double-stranded nucleic acids

The binding of mitoxantrone with double-helical nucleic acids was investigated by the methods of isothermal microcalorimetry, circular dichroism and absorption at the ionic strength mu = 0.11 and 0.011 M NaCl at temperature region of 30 divided by 60 degrees C. The investigation shows, that at mu = 0.11 M NaCl mitoxantrone interacts with double-helical nucleic acids in one way only. For such conditions using spectrophotometric titration data Scatchard plots for the binding of mitoxantrone with double-helical nucleic acids were constructed. The calculations show that the saturation stoichiometry is one mitoxantrone molecule per 2 divided by 3 base pairs DNA and 6 divided by 8 base pairs RNA. The dependence of binding constant from GC-content is observed. It is shown that the binding enthalpy of mitoxantrone with DNA and RNA increases linearly and reaches -(3.0 +/- 0.5) kkal per 1 mol mitoxantrone. It is shown that a binding mitoxantrone with double-helical nucleic acids, besides the intercalation of rings, a determinate contribution in the binding is given also by electrostatic interaction of side chains mitoxantrone with nucleic acids.     

DNA interaction with the antitumor agent thiophosphamide

DNA interaction with an alkylating antitumor drug N,N',N"-triethylenethiophosphoramide (thiotepa) in water-salt solutions at 37 degrees C has been studied by UV-spectroscopy, heat denaturation and electron microscopy methods. Changes of the DNA melting curve parameters provide information on the kinetics of alkylation. The dependence of the alkylation rate on DNA and thiotepa concentrations shows that the alkylation reaction is biomolecular. The increase of sodium chloride concentration from 10(-3) to 10(-1) M is accompanied by a drastic decrease of the alkylation rate. Thiotepa binding results in destabilization of the DNA secondary structure and formation of cross-links. An increased amount of bounded thiotepa results in DNA denaturation; prolonged alkylation causes breaks in the sugar-phosphate backbone. The results of the work are discussed in connection with the literature data on DNA interaction with thiotepa in vivo.     

High field 1H-NMR analysis of the 1:1 intercalation complex of the antitumor agent mitoxantrone and the DNA duplex [d(CpGpCpG)

Complete 1H-nmr assignment has been achieved of the stoichiometric 1:1 complex of the antitumor agent mitoxantrone with the duplex oligomer [d(CpGpCpG)]2. The techniques used included 2D-COSY, 1D-NOE and 2D-HH-INADEQUATE. Comparisons of 1H and 13C chemical shift changes upon addition of drug suggest symmetrical intercalative binding to the center of the tetramer. NOE difference measurements and 31P studies suggest binding of the terminal OH groups of the side chains to the central phosphate groups such that the methylene groups are proximate to C(3)6, C(3)6 and G(4)8 base protons all in the major groove. The data suggest that the side chains bind to the neighboring base pairs from the intercalation site. This is in accord with independent evidence of G,C base preference for binding from spectroscopic and electron microscopy studies.     

Features of the interaction of antitumor mitoxantrone compounds with sarcoma 45 tumor DNA]

The interaction of antitumoral drug mitoxantrone with DNA of the tumor sarcoma 45 and healthy animals liver has been investigated according to the character of changes on the absorption spectra at binding at 30 degrees C and 0.11 M NaCl. The investigation shows that the interaction of mitoxantrone with DNA of sarcoma 45 differs from that with DNA of healthy animals liver. The calculations show that the saturation stoichiometry by both DNA is one mitoxantrone molecule per 2.5 base pairs with the binding constant k = 4 x 10(5) M-1 (for binding mitoxantrone with liver DNA) and k = 3 x 10(6) M-1 (with tumor DNA). Possible reason of such a difference is discussed on the basis of structural peculiarities of tumor DNA.     

Controlled destabilization of a liposomal drug delivery system enhances mitoxantrone antitumor activity.

Programmable fusogenic vesicles (PFVs) are lipid-based drug-delivery systems that exhibit time-dependent destabilization. The rate at which this destabilization occurs is determined by the exchange rate of a bilayer-stabilizing component, polyethylene glycol-phosphatidylethanolamine (PEG-PE) from the vesicle surface. This exchange rate is controlled, in turn, by the acyl chain composition of the PEG-PE. We describe in vitro and in vivo studies using PFVs as delivery vehicles for the anticancer drug mitoxantrone. We demonstrate that the PEG-PE acyl composition determined the rate at which PFVs are eliminated from plasma after intravenous administration, and the rate of mitoxantrone leakage from PFV. The nature of the PEG-PE component also determined the antitumor efficacy of mitoxantrone-loaded PFV in murine and human in murine and human xenograft tumor models. Increased circulation time and improved activity were obtained for PFV containing PEG-PE with an 18-carbon acyl chain length, as a result of slower vesicle destabilization.     

Synthesis of a lipid derivative of the antitumor agent methotrexate

A lipophilic methotrexate prodrug capable of incorporation into membranes of carrier liposomes was synthesized. The conjugate consists of a lipophilic rac-1,2-dioleoylglycerol anchor connected to methotrexate through beta(Ala)-N-carbonylmethylene linker, which should be located in the polar region of the lipid bilayer. The ester bond between the hydrophilic linker and the antitumor agent can be hydrolyzed by intracellular esterases. The liposomal formulation of the prodrug exhibited a cytotoxic activity in vitro. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.     

Unexpected beta adrenergic effects of the antitumor agent, cyclocytidine, on rat salivary glands.

In addition to its potent antileukemic properties, cyclocytidine has a sialogogue action that depends on stimulation of beta adrenergic ereceptors of salivary glands. Furthermore, when chronically administered (for 3 days), cyclocytidine caused enlargement of parotid and submaxillary glands and heart that resembled the hypertrophy caused by chronic isoproterenol administration. The salivas evoked by cyclocytidine also closely resembled those evoked by isoproterenol, and were extremely viscous, and high in K+, (121 plus or minus 5.6, for submaxillary, and 42 plus or minus 2.9, for parotid), low in flow rate (0.007 mg/min times mg) and parotid saliva contained high concentrations of amylase (805 plus or minus 33 mg/mg gland). Cyclocytidine also caused marked emptying of parotid gland amylase. The cyclocytidine-induced salivary flow and gland emptying of amylase were prevented for 90 min when propranolol (but not dibenzyline or atropine) was administered prior to injection of the cyclocytidine. In addition, when the superior cervical ganglion was acutely removed, administration of cyclocytidine elicited salivary flow from the denervated as well as the innervated glands. These findings suggest that cyclocytidine does not affect salivary glands through indirect central or ganglionic actions. Cyclocytidine action does not exclusively involve beta receptors, since even in the presence of propranolol, secretory flow was evident after 90 min but when dibenzyline was given with the propranolol, complete blockade of cyclocytidine-stimulated saliva was effected. The dominant effect is, however, a beta adrenergic one. The undesirable side effects of cyclocytidine (parotid pain, postural hypotension, and cardiac hypertrophy) probably stem chiefly from its beta adrenergic properties and might be eliminated (or at least modified) by administration of propranolol with the cyclocytidine.     

Covalent binding to glutathione of the DNA-alkylating antitumor agent, S23906-1.

The benzoacronycine derivative, S23906-1, was characterized recently as a novel potent antitumor agent through alkylation of the N2 position of guanines in DNA. We show here that its reactivity towards DNA can be modulated by glutathione (GSH). The formation of covalent adducts between GSH and S23906-1 was evidenced by EI-MS, and the use of different GSH derivatives, amino acids and dipeptides revealed that the cysteine thiol group is absolutely required for complex formation because glutathione disulfide (GSSG) and other S-blocked derivatives failed to react covalently with S23906-1. Gel shift assays and fluorescence measurements indicated that the binding of S23906-1 to DNA and to GSH are mutually exclusive. Binding of S23906-1 to an excess of GSH prevents DNA alkylation. Additional EI-MS measurements performed with the mixed diester, S28053-1, showed that the acetate leaving group at the C1 position is the main reactive site in the drug: a reaction scheme common to GSH and guanines is presented. At the cellular level, the presence of GSH slightly reduces the cytotoxic potential of S23906-1 towards KB-3-1 epidermoid carcinoma cells. The GSH-induced threefold reduction of the cytotoxicity of S23906-1 is attributed to the reduced formation of lethal drug-DNA covalent complexes in cells. Treatment of the cells with buthionine sulfoximine, an inhibitor of GSH biosynthesis, facilitates the formation of drug-DNA adducts and promotes the cytotoxic activity. This study identifies GSH as a reactant for the antitumor drug, S23906-1, and illustrates a pathway by which GSH may modulate the cellular sensitivity to this DNA alkylating agent. The results presented here, using GSH as a biological nucleophile, fully support our initial hypothesis that DNA alkylation is the major mechanism of action of the promising anticancer drug S23906-1.     

Selective immunomodulation by the antineoplastic agent mitoxantrone. I. Suppression of B lymphocyte function

Novantrone mitoxantrone, an antineoplastic agent with antiproliferative properties, is under investigation as an immunomodulating agent. The impact of mitoxantrone treatment on B lymphocyte reactivity is presented here. Administered i.p. in H2O at a dose of 0.5 mg/kg, daily for 14 days, mitoxantrone abrogated both the in vivo antibody response (to ovalbumin) and the in vitro plaque-forming cell (PFC) response (to SRC). In addition to the effects on thymus-dependent reactivity, PFC responses to the thymus-independent antigens TNP-LPS and TNP-Ficoll were also inhibited when tested in vivo or in vitro. B cells were identified as a target for the suppressive activity of mitoxantrone by using T cell-replacing factor to reconstitute the in vitro anti-SRC PFC response of a T lymphocyte-depleted spleen cell preparation. LPS-induced B cell mitogenesis was largely inhibited by mitoxantrone treatment. However, depletion of Sephadex G-10-adherent cells significantly restored the proliferative response. Flow cytometric analysis revealed a dramatic decrease in splenic B lymphocyte content. Therefore, mitoxantrone exerted a potent suppressive influence on the humoral immune system through a direct reduction in B cell number augmented by macrophage-mediated inhibition of B cell proliferation.     

Interaction of the antitumor agent vanadocene dichloride with phosphate buffered saline

EPR study has shown that in aqueous solution the anticancer agent vanadocene dichloride (Cp₂VCl₂) interacts with phosphates contained in phosphate buffered saline (PBS). Chelate complex Cp₂VO₂PO₂H (|A_iso(V)|=189.0 MHz, |a_iso(P)|=81.5 MHz and g_iso=1.9862) is only one paramagnetic compound formed at the range about the physiological pH (6.3-7.8). Its structure was validated by comparison of observed and theoretical calculated HFC tensor (at the density functional level of theory).     

Reactions of the antitumor agent carminic acid and derivatives with DNA

The antitumor agent carminic acid 1a does not bind to DNA but nicks it slowly, more rapidly when reduced in situ, and still more rapidly when prereduced at the quinone moiety. The nicking process requires oxygen and is selectively inhibited by (i) superoxide dismutase, (ii) catalase, and (iii) free radical scavengers indicating the involvement of $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}\text{, H}{}_2\text{O}{}_2$, and OH^., respectively. The intermediacy of OH^. was supported by spin trapping with N-t-butyl-α-phenylnitrone and epr of the radical produced via the carminic acid semiquinone. The single strand scission of DNA by carminic acid requires two adjacent hydroquinone moieties in the chromophore since reduced methyl tetra-O-methylcarminate 1b is without effect although it binds weakly to DNA. Polarographic redox potentials for the reversible (2e, 2H⁺) reduction of 1a and 1b are ?0.736 ± 0.003 V and ?0.56 ± 0.010 V against SCE, respectively. The fact that daunorubicin and adriamycin produce more extensive DNA strand scission than carminic acid under comparable conditions of prereduction and on a molar basis is largely attributed to the assistance of intercalative binding afforded in the case of the anthracyclines.     

Isolation and structure of the active component of the antitumor agent blastolysin

Active principle of blastolysin, comprising ca. 40% of the total weight of Lactobacillus bulgaricus cell wall preparation, has been isolated and structurally studied by chemical and spectral methods. The substance a glycopeptide with Mr aa. 10,000, consists of two tetrasaccharide moieties connected by oligopeptide bridges; glycerophosphate, glucose, and galactose moieties are also attached to N-acetylmuramyl residue of the peptidoglucan core. Tetrasaccharide-containing muramylpeptides were shown to be responsible for the antitumour activity.     

Interaction between double helix DNA fragments and the new antitumor agent sabarubicin,Men10755

Among the disaccharide derivatives of the antitumor anthracycline doxorubicin, sabarubicin (Men10755) is more active and less cytotoxic than doxorubicin. It showed a strong in vivo antitumor activity in all preclinical models examined, in conjunction with a better tolerability, and is now in phase II clinical trials.The interaction of sabarubicin and Men10749 (a similar disaccharide with a different configuration at C-4′ of the proximal sugar) with the hexanucleotides d(CGTACG)₂ and d(CGATCG)₂ was studied by a combined use of 2D-¹H and ³¹P NMR techniques. Both ¹H and ³¹P chemical shifts of imino protons and phosphates allowed to established the intercalation sites between the CG base pairs, as it occurs for other anthracyclines of the series. The dissociation rate constants (k_off) of the slow step of the intercalation process were measured for Men10755 and Men10749, by NMR NOE-exchange experiments. The increase of k_off , with respect of doxorubicin, showed that the intercalation process is significantly faster for both drugs, leading to an average residence time for sabarubicin into d(CGTACG)₂ sixfold shorter than for doxorubicin. This could give account of both higher cytoplasmic/nuclear ratio and lower cellular uptake of sabarubicin in comparison with doxorubicin and accordingly of the lower cytotoxicity of these disaccharide analogues.A relevant number of NOE interactions allowed the structure of the complexes in solution to be derived through restrained MD calculations. NMR-DOSY experiments were performed with several drug/oligonucleotide mixtures in order to determine the structure and the dimension of the aggregates.     

Characterization of reductant-induced, tryptophan fluorescence changes in cytochrome oxidase

We have measured the steady-state tryptophan fluorescence spectrum of cytochrome oxidase in its oxidized and fully reduced states. Reduction of the oxidized enzyme by sodium dithionite causes an apparent shift in the fluorescence emission maximum from 328 nm, in the oxidized enzyme, to 348 nm, in the reduced enzyme. This spectroscopic change has been observed previously and assigned to a redox-linked, conformational change in cytochrome oxidase [Copeland, R. A., Smith, P. A., & Chan, S. I. (1987) Biochemistry 26, 7311-7316]. When dithionite-reduced enzyme sits in an open cuvette, the enzyme returns to the oxidized state, and the fluorescence maximum shifts back to 328 nm. However, the time course of the fluorescence change does not follow the redox state of the enzyme, monitored spectrophotometrically at 445,605, and 820 nm, but follows the disappearance of dithionite, which absorbs at 315 nm. Moreover, when the fluorescence emission spectrum of the dithionite-reduced enzyme is corrected for the absorbance due to dithionite, the fluorescence maximum is found 2 nm blue shifted, relative to that of the oxidized enzyme, at 326 nm. This dithionite-induced, red-shifted steady-state tryptophan fluorescence is also seen with the non-heme-containing enzyme carboxypeptidase A. The tryptophan emission spectrum of untreated carboxypeptidase A is at 332 nm, whereas in the presence of dithionite the emission spectrum of carboxypeptidase A is at 350 nm. When corrected for the absorbance of dithionite, the tryptophan emission maximum is at 332 nm. We have also used the photoreductant 3,10-dimethyl-5-deazaisoalloxazine (deazaflavin) to reduce cytochrome oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)