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The antitumor anthracycline, doxorubicin (DOX), can cause heart failure (HF) upon cumulative administration. Lowering the cumulative dose of DOX proved useful to minimize HF risk, and, yet, there is a growing concern that HF might occur after doses that were thought to be safe. Clinical trials that prospectively address such concerns are lacking. Because HF risk correlates with cardiac exposure to DOX, cumulative doses associated with HF risk were re-explored by modeling the accumulation of anthracycline pools in human myocardium. Ex vivo myocardial samples were used in vitro to simulate DOX rapid infusions. The accumulation of anthracycline pools was measured and incorporated into equations from which a risk versus dose curve was obtained. The experimental curve identified a 5% risk dose that was congruent with a previously reported clinical value (380 versus 400 mg/m 2 , respectively); however, 1-2% risk occurred after lower doses than reported. Simulations of gain-of-function polymorphism of carbonyl reductase 3, which converts DOX to its poorly diffusible alcohol metabolite, doxorubicinol (DOXOL), expanded anthracycline pools and caused 5% or 1-2% risk doses to decrease to 330 or 180-230 mg DOX/m 2 , respectively. These data show there is no safe dose of DOX. Diminishing cardiac exposure to circulating DOX may represent a cardioprotective strategy. We show that DOX slow infusions or liposomal DOX, which reduce cardiac exposure to DOX, caused formation of smaller anthracycline pools, did not generate DOXOL, increased the 5% risk dose to 750-800 mg/m 2 , and prevented HF risk aggravation by carbonyl reductase polymorphism., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

This is a retrospective highlight on the publication by Ibsen and coworkers: Localized In Vivo Activation of a Photoactivatable Doxorubicin Prodrug in Deep Tumor Tissue, which appeared in a preceding issue of Photochem. Photobiol. (2013, 89:698-708). The authors describe the synthesis and properties of a novel doxorubicin (DOX) prodrug, DOX-PCB, which contains a photocleavable linker group. Systemic administration of the prodrug to a tumor-bearing animal followed by LED/fiber optic 365 nm light delivery allowed active DOX to be released site specifically in the tumor area. This elegant and timely study provides compelling evidence that photocleavable DOX-PCB can eliminate many of the toxic side effects of DOX that have plagued clinical use of this highly effective antitumor drug for many years., (© 2013 The American Society of Photobiology.)

Cardiotoxicity from the antitumor anthracycline doxorubicin correlates with doxorubicin cardiac levels, redox activation to superoxide anion (O(2)(.-)) and hydrogen peroxide (H(2)O(2)), and formation of the long-lived secondary alcohol metabolite doxorubicinol. Cardiotoxicity may first manifest during salvage therapy with other drugs, such as the anthracenedione mitoxantrone. Minimal evidence for cardiotoxicity in anthracycline-pretreated patients with refractory-relapsed non-Hodgkin lymphoma was observed with the novel anthracenedione pixantrone. We characterized whether pixantrone and mitoxantrone caused different effects on doxorubicin levels, redox activation, and doxorubicinol formation. Pixantrone and mitoxantrone were probed in a validated ex vivo human myocardial strip model that was either doxorubicin-naïve or preliminarily subjected to doxorubicin loading and washouts to mimic doxorubicin treatment and elimination in the clinical setting. In doxorubicin-naïve strips, pixantrone showed higher uptake than mitoxantrone; however, neither drug formed O(2)(.-) or H(2)O(2). In doxorubicin-pretreated strips, neither pixantrone nor mitoxantrone altered the distribution and clearance of residual doxorubicin. Mitoxantrone showed an unchanged uptake and lacked effects on doxorubicin levels, but synergized with doxorubicin to form more O(2)(.-) and H(2)O(2), as evidenced by O(2)(.-)-dependent inactivation of mitochondrial aconitase or mitoxantrone oxidation by H(2)O(2)-activated peroxidases. In contrast, pixantrone uptake was reduced by prior doxorubicin exposure; moreover, pixantrone lacked redox synergism with doxorubicin, and formed an N-dealkylated product that inhibited metabolism of residual doxorubicin to doxorubicinol. Redox inactivity and inhibition of doxorubicinol formation correlate with the cardiac safety of pixantrone in doxorubicin-pretreated patients. Redox inactivity in the face of high cardiac uptake suggests that pixantrone might also be safe in doxorubicin-naïve patients.

The pharmacokinetics of pegylated liposomal doxorubicin (PLD) were investigated in 17 women undergoing intraoperative hyperthermic intraperitoneal chemotherapy (HIPEC) for advanced ovarian cancer and peritoneal carcinomatosis. HIPEC was performed immediately after completing debulking surgery, which included a number of peritonectomy procedures. PLD was injected and allowed to equilibrate in peritoneal cavity filled with 4 liters of physiological solution and stabilized at 42°C; next, the outflow line was opened and perfusion proceeded for 1 h. PLD was stable in peritoneal perfusate and plasma. During HIPEC, PLD peritoneal perfusate/plasma gradients averaged ∼600 or ≥1000 for peak concentration or area under the curve. After HIPEC, PLD plasma levels remained stable or decreased. Biopsy samples of residual normal peritoneum or ovarian carcinomatosis were collected at the end of HIPEC and were shown to contain free doxorubicin. Correlating PLD decrements in peritoneal perfusate with plasma exposure to PLD or peritoneal deposition of free doxorubicin showed that the former occurred during preperfusional equilibration of PLD in peritoneal cavity, whereas the latter occurred during 1 h of perfusion. Plasma exposure to PLD correlated negatively with the number of peritonectomy procedures performed during surgery, whereas peritoneal deposition of free doxorubicin correlated positively. Taken together, these results show that PLD administered by intraoperative HIPEC undergoes limited systemic diffusion and releases active free doxorubicin in peritoneum exposed to ovarian carcinomatosis. PLD pharmacokinetics seem to be influenced by peritonectomy procedures.

Anthracycline-related cardiotoxicity correlates with cardiac anthracycline accumulation and bioactivation to secondary alcohol metabolites or reactive oxygen species (ROS), such as superoxide anion (O₂·⁻) and hydrogen peroxide H₂O₂). We reported that in an ex vivo human myocardial strip model, 3 or 10 μM amrubicin [(7S,9S)-9-acetyl-9-amino-7-[(2-deoxy-β-D-erythro-pentopyranosyl)oxy]-7,8,9,10-tetrahydro-6,11-dihydroxy-5,12-napthacenedione hydrochloride] accumulated to a lower level compared with equimolar doxorubicin or epirubicin (J Pharmacol Exp Ther 341:464-473, 2012). We have characterized how amrubicin converted to ROS or secondary alcohol metabolite in comparison with doxorubicin (that formed both toxic species) or epirubicin (that lacked ROS formation and showed an impaired conversion to alcohol metabolite). Amrubicin and doxorubicin partitioned to mitochondria and caused similar elevations of H₂O₂, but the mechanisms of H₂O₂ formation were different. Amrubicin produced H₂O₂ by enzymatic reduction-oxidation of its quinone moiety, whereas doxorubicin acted by inducing mitochondrial uncoupling. Moreover, mitochondrial aconitase assays showed that 3 μM amrubicin caused an O₂·⁻-dependent reversible inactivation, whereas doxorubicin always caused an irreversible inactivation. Low concentrations of amrubicin therefore proved similar to epirubicin in sparing mitochondrial aconitase from irreversible inactivation. The soluble fraction of human myocardial strips converted doxorubicin and epirubicin to secondary alcohol metabolites that irreversibly inactivated cytoplasmic aconitase; in contrast, strips exposed to amrubicin failed to generate its secondary alcohol metabolite, amrubicinol, and only occasionally exhibited an irreversible inactivation of cytoplasmic aconitase. This was caused by competing pathways that favored formation and complete or near-to-complete elimination of 9-deaminoamrubicinol. These results characterize amrubicin metabolic advantages over doxorubicin and epirubicin, which may correlate with amrubicin cardiac safety in preclinical or clinical settings.

Background: Anthracycline-containing regimens have demonstrated significant disease-free and overall survival benefits in the adjuvant setting and also provide palliative benefit in metastatic disease. . Over the past two decades, an increasing proportion of patients have been exposed to adjuvant anthracyclines with concomitant reduction in their use for palliation, as a result of concerns regarding efficacy and cumulative anthracycline-associated cardiotoxicity, as well as the availability of other systemic chemotherapeutic options. This report reflects the consensus view of a meeting of oncologists, pharmacologists and cardiologists held in Florence, Italy, on April 30, 2010. The objectives of the meeting were to review the role and limits of conventional anthracyclines in the treatment of breast cancer, to provide recommendations for the use of novel anthracycline formulations, such as non-pegylated liposomal doxorubicin (NPLD), and to identify potential future indications for NPLD that warrant further research.

Cardiotoxicity limits the clinical use of doxorubicin (DOX) and other quinone-hydroquinone antitumor anthracyclines. One-electron reduction of the quinone moiety is followed by the formation of reactive oxygen species (ROS) that have been proposed to induce cardiotoxicity through an oxidative stress; conversely, one-electron oxidation of the hydroquinone moiety by hydrogen peroxide (H(2)O(2)) and oxyferrous myoglobin (Mb(II)O(2)) is followed by an anthracycline degradation process that has been proposed to limit cardiotoxicity. We previously reported that tert-butoxycarbonyl-alanine (t-BA) impeded DOX oxidation/degradation by H(2)O(2)/Mb(II)O(2) in a cell-free system; accordingly, t-BA increased the levels of DOX, its conversion to ROS, and its concentration-related toxicity in cardiomyocytes. To re-explore methodological and toxicological aspects of anthracycline degradation, we used 4'-epidoxorubicin (EPI), an anthracycline analogue that is very similar to DOX but undergoes protonation-sequestration in cytoplasmic acidic organelles. t-BA lacked an effect on H9c2 cardiomyocytes exposed to EPI; however, blocking the protonation-sequestration mechanism with the vacuolar H(+)-ATPase inhibitor, bafilomycin A1 (BFL), enabled t-BA to increase the cellular levels of EPI, its conversion to ROS, and its concentration-related toxicity. This suggested that t-BA was specific enough to increase the cellular levels and toxicity of only those anthracyclines that were liable to oxidation/degradation by H(2)O(2)/Mb(II)O(2). By exposing cardiomyocytes to nontoxic concentrations of DOX or EPI and by increasing their cellular levels by means of appropriate combinations with t-BA, BFL, or t-BA+BFL, we nonetheless found that the loss of cardiomyocyte viability correlated with the accumulation of undegraded anthrayclines but not with their ability to form ROS or to induce lipid peroxidation. This suggested that an accumulation of undegraded anthracyclines might induce cardiotoxicity also by mechanisms independent of ROS and oxidative stress. Thus, EPI proved useful to refine the value of t-BA in the studies of anthracycline degradation and to reappraise the role of anthracycline degradation in cardiotoxicity.

Secondary alcohol metabolites and reactive oxygen species mediate cardiomyopathy induced by cumulative doses of antitumor anthracyclines, such as doxorubicin and epirubicin. Epirubicin exhibits a defective conversion to both toxic species, thereby inducing cardiotoxicity at doses higher than equiactive to doxorubicin; however, the gain in cardiac tolerability seems to be marginal compared with the magnitude of the metabolic defects of epirubicin. Cardiomyopathy may occur independent of toxic metabolites if a given anthracycline tends to accumulate in the heart; therefore, we characterized whether epirubicin showed an unusual accumulation in human myocardial strips incubated in plasma. Epirubicin exhibited a higher uptake and reached myocardial levels 2 times higher than those of doxorubicin. Epirubicin also showed a unique metabolization to doxorubicinolone, the product of epirubicin deglycosidation and carbonyl reduction. In diffusing from the strips to plasma, doxorubicinolone caused membrane permeation effects that augmented epirubicin elimination. Experiments with purified doxorubicinolone showed that the efflux of 1 mol doxorubicinolone promoted the concomitant elimination of as many as approximately 40 mol epirubicin. Doxorubicinolone could also diffuse from plasma back to the strips, causing a permeation effect that promoted epirubicin reuptake; however, this reverse process was slower and less potent. On balance, doxorubicinolone efflux diminished the epirubicin to doxorubicin accumulation ratio to approximately 1.5. These results suggest that the cardiac tolerability of epirubicin is limited by its accumulation in the heart and that such accumulation would be even higher in the absence of doxorubicinolone formation and efflux. These results may also serve guidelines for developing noncardiotoxic anthracyclines.

Antitumor therapy with the anthracycline doxorubicin is limited by a severe cardiotoxicity, which seems to correlate with the cardiac levels of doxorubicin and its metabolization to reactive oxygen species. Previous biochemical studies showed that hydrogen peroxide-activated myoglobin caused an oxidative degradation of doxorubicin; however, a pharmacological evaluation of this metabolic pathway was precluded by the lack of safe and specific inhibitors of doxorubicin degradation. We found that tert-butoxycarbonyl-alanine inhibited doxorubicin degradation induced in vitro by hydrogen peroxide-activated oxyferrous myoglobin. When assessed in H9c2 cardiomyocytes, tert-butoxycarbonyl-alanine neither stimulated the cellular uptake of doxorubicin nor diminished its efflux; moreover, tert-butoxycarbonyl-alanine did not cause toxicity per se nor did it augment the toxicity induced by hydrogen peroxide or chemical agents that increased the cellular levels of reactive oxygen species. Nonetheless, tert-butoxycarbonyl-alanine increased the cellular levels of doxorubicin, its conversion to reactive oxygen species, and its concentration-related toxicity. tert-Butoxycarbonyl-alanine also aggravated the toxicity of a degradation-prone anthracycline analog, daunorubicin, but it caused a minor effect on the toxicity of a degradation-resistant analog, aclarubicin. These results suggested that tert-butoxycarbonyl-alanine increased the cellular levels and toxicity of doxorubicin by inhibiting its oxidative degradation to harmless products. Accordingly, doxorubicin samples that had been oxidized in vitro with hydrogen peroxide and oxyferrous myoglobin lacked toxicity to cardiomyocytes. The effects of tert-butoxycarbonyl-alanine were most evident at 0.1 to 1 microM doxorubicin, which may be relevant to clinical conditions. These studies identify an oxidative degradation of doxorubicin as a possible salvage mechanism for diminishing its levels and toxicity in cardiomyocytes.

The antitumor anthracycline doxorubicin induces a dose-related cardiotoxicity that correlates with the myocardial levels of its secondary alcohol metabolite doxorubicinol. Combining doxorubicin with taxanes such as paclitaxel or docetaxel may aggravate cardiotoxicity, presumably because the taxanes cause an allosteric-like stimulation of cytoplasmic aldehyde reductases that convert doxorubicin to doxorubicinol in the heart. A less severe aggravation of cardiotoxicity was observed on combining taxanes with epirubicin, a closely related analog of doxorubicin; therefore, we characterized whether the cardiac tolerability of epirubicin-taxane therapies could be due to a defective taxane stimulation of the conversion of epirubicin to its secondary alcohol metabolite epirubicinol. Comparisons between doxorubicin and epirubicin in isolated human heart cytosol showed that epirubicin exhibited a lower V(max)/K(m) value for reaction with aldehyde reductases and a defective stimulation of epirubicinol formation by paclitaxel or docetaxel. A similar pattern occurred in the soluble fraction of human myocardial strips incubated in plasma with anthracyclines and paclitaxel or docetaxel, formulated in their clinical vehicles Cremophor EL or polysorbate 80. Doxorubicin, but not epirubicin, was also able to generate reactive oxygen species in the membrane fraction of myocardial strips; however, the levels of doxorubicin-derived reactive oxygen species were not further augmented by paclitaxel. These results support the notion that taxanes might aggravate the cardiotoxicity of doxorubicin through a specific stimulation of doxorubicinol formation. The failure of paclitaxel or docetaxel to stimulate epirubicinol formation therefore uncovers an important determinant of the improved cardiac tolerability of epirubicin-taxane combinations.

Antitumor therapy with the anthracycline doxorubicin is limited by a dose-related cardiotoxicity that is aggravated by a concomitant administration of the taxane paclitaxel. Previous limited studies with isolated human heart cytosol showed that paclitaxel was able to stimulate an NADPH-dependent reduction of doxorubicin to its toxic secondary alcohol metabolite doxorubicinol. Here we characterized that 0.25 to 2.5 microM paclitaxel caused allosteric effects that increased doxorubicinol formation in human heart cytosol, whereas 5 to 10 microM paclitaxel decreased doxorubicinol formation. The closely related taxane docetaxel caused similar effects. Basal or taxane-stimulated doxorubicinol formation was blunted by 2,7-difluorospirofluorene-9,5'-imidazolidine-2',4'-dione (AL1576), a specific inhibitor of aldehyde reductases. Doxorubicinol was measured also in the cytosol of human myocardial strips incubated in plasma and exposed to doxorubicin in the absence or presence of paclitaxel or docetaxel and their clinical vehicles Cremophor EL or polysorbate 80. Low concentrations of taxanes stimulated doxorubicinol formation, whereas high concentrations decreased it. Doxorubicinol formation reached its maximum on adding plasma with 6 microM paclitaxel or docetaxel; this corresponded to the partitioning of 1.5 to 2.5 microM taxanes in the cytosol of the strips. Taxane-stimulated doxorubicinol formation was not mediated by vehicles, nor was it caused by increased doxorubicin uptake or de novo protein synthesis; however, doxorubicinol formation was blunted by AL1576. These results show that allosteric interactions with cytoplasmic aldehyde reductases enable paclitaxel or docetaxel to stimulate doxorubicinol formation in human heart. This information serves metabolic insights into the risk of cardiotoxicity induced by doxorubicin-taxane therapies.

The cardiotoxicity induced by the anticancer anthracycline doxorubicin (DOX) is attributed to reactions between iron and reactive oxygen species (ROS) that lead to oxidative damage. We found that DOX forms ROS in H9c2 cardiomyocytes, as shown by dichlorodihydrofluorescein oxidation and the expression of stress-responsive genes such as catalase or aldose reductase. DOX also increased ferritin levels in these cells, particularly the H subunit. A considerable increase in ferritin mRNA levels showed that DOX acted at transcriptional level, but an additional potential mechanism was identified as the down-regulation of iron regulatory protein-2, post-transcriptional inhibitor of ferritin synthesis. Pretreatment with DOX protected H9c2 cells against the damage induced by subsequent exposure to ferric ammonium citrate, and experiments with (55)Fe revealed that the protection was due to the deposition of iron in ferritin. Cytoprotection was also observed when DOX was replaced by glucose/glucose oxidase, a source of H(2)O(2), thus suggesting that DOX increases ferritin synthesis through the action of ROS. This concept was supported by three more lines of evidence. (i) DOX-induced ferritin synthesis was blocked by N-acetylcysteine, a scavenger of ROS. (ii) Mitoxantrone, a ROS-forming analogue, similarly induced ferritin expression and protected the cells against iron toxicity. (iii) 5-Iminodaunorubicin, an analogue lacking ROS-forming activity, did not induce ferritin synthesis or protect the cells against iron toxicity. These results characterize a paradoxically beneficial link between anthracycline-derived ROS, increased ferritin synthesis, and resistance to iron-mediated damage. The role of iron and ROS in anthracycline-induced cardiotoxicity may, therefore, be more complex than previously believed.

Clinical use of the anticancer anthracyclines doxorubicin (DOX) and daunorubicin (DNR) is limited by development of cardiotoxicity upon chronic administration. Secondary alcohol metabolites, formed after two-equivalent reduction of a carbonyl group in the side chain of DOX or DNR, have been implicated as potential mediators of chronic cardiotoxicity. In the present study we characterized how human heart converted DOX or DNR to their alcohol metabolites DOXol or DNRol. Experiments were carried out using post-mortem myocardial samples obtained by ethically-acceptable procedures, and results showed that DOXol and DNRol were formed by flavin-independent cytoplasmic reductases which shared common features like pH-dependence and requirement for NADPH, but not NADH, as a source of reducing equivalents. However, studies performed with inhibitors exhibiting absolute or mixed specificity toward best known cytoplasmic reductases revealed that DOX and DNR were metabolized to DOXol or DNRol through the action of distinct enzymes. Whereas DOX was converted to DOXol by aldehyde-type reductase(s) belonging to the superfamily of aldo-keto reductases, DNR was converted to DNRol by carbonyl reductase(s) belonging to the superfamily of short-chain dehydrogenase/reductases. This pattern changed in cardiac cytosol derived from rabbit, a laboratory animal often exploited to reproduce cardiotoxicity induced by anthracyclines and to develop protectants for use in cancer patients. In fact, only carbonyl reductases were involved in metabolizing DOX and DNR in rabbit cardiac cytosol, although with different K(m) and V(max). Collectively, these results demonstrate that human myocardium convert DOX and DNR to DOXol or DNRol by virtue of different reductases, an information which may be of value to prevent alcohol metabolite formation during the course of anthracycline-based anticancer therapy. These results also raise caution on the preclinical value of animal models of anthracycline cardiotoxicity, as they demonstrate that the metabolic routes leading to DOXol in a laboratory animal may not be the same as those occurring in patients.

Lipid peroxidation has been proposed to mediate cardiotoxicity induced by doxorubicin (DOX) and other anticancer anthracyclines; however, there have been reports showing that DOX can also inhibit lipid peroxidation. Here we characterized the effects of DOX on the oxo-ferryl moiety [Fe(IV)=O, Mb(IV)] of H(2)O(2)-activated myoglobin, a lipid oxidant likely formed in the heart during treatment with DOX. Mb(IV) was formed in vitro by reacting 100 microM H(2)O(2) with 50 microM horse heart metmyoglobin (Mb(III)). Spectral studies showed that DOX reduced Mb(IV) to Mb(III), half-maximal regeneration of Mb(III) occurring at approximately 18 microM DOX. Comparisons between DOX, its aglycone doxorubicinone, and other approved or investigational anthracyclines or model compounds (daunorubicin, idarubicin, aclarubicin, and naphthazarin), showed that DOX reduced Mb(IV) through the hydroquinone moiety of its tetracyclic ring. DOX inhibited Mb(IV)-dependent peroxidation of arachidonic acid, suppressing the formation of thiobarbituric acid-reactive substances with an IC(50) of approximately 18 microM. Lipid peroxidation was inhibited also by the hydroquinone-containing daunorubicin and idarubicin but not by the hydroquinone-deficient aclarubicin; moreover, neither simple hydroquinone nor other known Mb(IV) reductants (ascorbate, glutathione, and ergothioneine) reached measurable IC(50)s in a micromolar range. DOX-dependent inhibition of lipid peroxidation correlated with its ability to reduce Mb(IV) to Mb(III) in competition with arachidonic acid (r = 0.83, P = 0.029); it did not correlate with its ability to scavenge other free radical species [like e.g., peroxyl radicals generated through the thermal decomposition of 2,2'-azo-bis(2-amidinopropane)]. DOX reduced Mb(IV) and inhibited lipid peroxidation also when H(2)O(2), Mb(III) and arachidonic acid were reacted in cytosol of human myocardial biopsies, a model developed to predict the cardiotoxic mode of action of DOX in patients. These results illustrate "antioxidant" properties of DOX, mediated by reduction of Mb(IV) to Mb(III), and cast doubts on lipid peroxidation as a causative mechanism of anthracycline-induced cardiotoxicity.

One-electron quinone reduction and two-electron carbonyl reduction convert the anticancer anthracycline doxorubicin to reactive oxygen species (ROS) or a secondary alcohol metabolite that contributes to inducing a severe form of cardiotoxicity. The closely related analogue epirubicin induces less cardiotoxicity, but the determinants of its different behavior have not been elucidated. We developed a translational model of the human heart and characterized whether epirubicin exhibited a defective conversion to ROS and secondary alcohol metabolites. Small myocardial samples from cardiac surgery patients were reconstituted in plasma that contained clinically relevant concentrations of doxorubicin or epirubicin. In this model only doxorubicin formed ROS, as detected by fluorescent probes or aconitase inactivation. Experiments with cell-free systems and confocal laser scanning microscopy studies of H9c2 cardiomyocytes suggested that epirubicin could not form ROS because of its protonation-dependent sequestration in cytoplasmic acidic organelles and the consequent limited localization to mitochondrial one-electron quinone reductases. Accordingly, blocking the protonation-sequestration mechanism with the vacuolar H+-ATPase inhibitor bafilomycin A1 relocalized epirubicin to mitochondria and increased its conversion to ROS in human myocardial samples. Epirubicin also formed ∼60% less alcohol metabolites than doxorubicin, but this was caused primarily by its higher Km and lower Vmax values for two-electron carbonyl reduction by aldo/keto-reductases of human cardiac cytosol. Thus, vesicular sequestration and impaired efficiency of electron addition have separate roles in determining a defective bioactivation of epirubicin to ROS or secondary alcohol metabolites in the human heart. These results uncover the molecular determinants of the reduced cardiotoxicity of epirubicin and serve mechanism-based guidelines to improving antitumor therapies. [ABSTRACT FROM AUTHOR]

We show that the pseudoperoxidase activity of ferrylmyoglobin (MbTM) promotes oxidative degradation of doxorubicin (DOX), an anticancer anthracycline known to induce severe cardiotoxicity. MbIV, formed in vitro by reacting horse heart MbIII with H2O2, caused disappearance of the spectrum of DOX at 477 nm and appearance of UV-absorbing chromophores that indicated opening and degradation of its tetracyclic ring. Electron spray ionization mass spectrometry analyses of DOXfMbIV ultrafiltrates showed that DOX degradation resulted in formation of 3-methoxyphthalic acid, the product of oxidative modifications of its methoxy-substituted ring D. Other methoxy-substituted anthracyclines similarly released 3-methoxyphthalic acid after oxidation by MbIV, whereas demethoxy analogs released simple phthalic acid. Kinetic and stoichiometric analyses of reactions between DOX and MbIII/H2O2 or hemin/H2O2 showed that the porphyrin radical of MbIV-compound I and the iron-oxo moiety of MbIV-compound II were sequentially involved in oxidizing DOX; however, oxidation by compound I formed more 3-methoxyphthalic acid than oxidation by compound II. Sizeable amounts of 3-methoxyphthalic acid were formed in the heart of mice treated with DOX, in human myocardial biopsies exposed to DOX in vitro, and in human cardiac cytosol that oxidized DOX after activation of its endogenous myoglobin by H2O2. Importantly, H9c2 cardiomyocytes were damaged by low concentrations of DOX but could tolerate concentrations of 3-methoxyphthalic acid higher than those measured in murine or human myocardium. These results unravel a novel function for MbIV in the oxidative degradation of anthracyclines to phthalic acids and suggest that this may serve a salvage pathway against cardiotoxicity. [ABSTRACT FROM AUTHOR]