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Anthracycline Metabolism and Toxicity in Human Myocardium: Comparisons between Doxorubicin, Epirubicin, and a Novel Disaccharide Analogue with a Reduced Level of Formation and [4Fe-4S] Reactivity of Its Secondary Alcohol Metabolite Giorgio Minotti,*,† Sabrina Licata,‡ Antonella Saponiero,‡ Pierantonio Menna,† Antonio M. Calafiore,§ Gabriele Di Giammarco,§ Giovanni Liberi,§ Fabio Animati,| Amalia Cipollone,| Stefano Manzini,| and Carlo A. Maggi| Department of Drug Sciences, G. D’Annunzio University School of Pharmacy, and Department of Cardiac Surgery, G. D’Annunzio University School of Medicine, Chieti; Institute of Pharmacology, Catholic University School of Medicine, Rome; and Menarini Ricerche S. p. A., Pomezia (Rome), Italy Received July 11, 2000
Secondary alcohol metabolites have been proposed to mediate chronic cardiotoxicity induced by doxorubicin (DOX) and other anticancer anthracyclines. In this study, NADPH-supplemented human cardiac cytosol was found to reduce the carbonyl group in the side chain of the tetracyclic ring of DOX, producing the secondary alcohol metabolite doxorubicinol (DOXol). A decrease in the level of alcohol metabolite formation was observed by replacing DOX with epirubicin (EPI), a less cardiotoxic analogue characterized by an axial-to-equatorial epimerization of the hydroxyl group at C-4 in the amino sugar bound to the tetracyclic ring (daunosamine). A similar decrease was observed by replacing DOX with MEN 10755, a novel anthracycline with preclinical evidence of reduced cardiotoxicity. MEN 10755 is characterized by the lack of a methoxy group at C-4 in the tetracyclic ring and by intercalation of 2,6-dideoxy-L-fucose between daunosamine and the aglycone. Multiple comparisons with methoxy- or 4-demethoxyaglycones, and a number of mono- or disaccharide 4-demethoxyanthracyclines, showed that both the lack of the methoxy group and the presence of a disaccharide moiety limited alcohol metabolite formation by MEN 10755. Studies with enzymatically generated or purified anthracycline secondary alcohols also showed that the presence of a disaccharide moiety, but not the lack of a methoxy group, made the metabolite of MEN 10755 less reactive with the [4Fe-4S] cluster of cytoplasmic aconitase, as evidenced by its limited reoxidation to the parent carbonyl anthracycline and by a reduced level of delocalization of Fe(II) from the cluster. Collectively, these studies (i) characterize the different influence of methoxy and sugar substituents on the formation and [4Fe-4S] reactivity of anthracycline secondary alcohols, (ii) lend support to the role of alcohol metabolites in anthracycline-induced cardiotoxicity, as they demonstrate that the less cardiotoxic EPI and MEN 10755 share a reduction in the level of formation of such metabolites, and (iii) suggest that the cardiotoxicity of MEN 10755 might be further decreased by the reduced [4Fe-4S] reactivity of its alcohol metabolite.
Introduction The clinical use of the anticancer anthracycline doxorubicin (DOX)1 is limited by the possible development of cardiomyopathy and congestive heart failure upon chronic administration (1). Doxorubicin is composed of aglyconic and sugar moieties. The aglycone, called doxorubicinone, contains a quinone-hydroquinone tetracyclic ring and a short side chain with a carbonyl moiety at C-13 and a * To whom correspondence should be addressed: Department of Drug Sciences, G. D’Annunzio University School of Pharmacy, Via dei Vestini, 66013 Chieti, Italy. Phone: 011-39-0871-3555237. Fax: 01139-0871-3555315. E-mail:
[email protected]. † Department of Drug Sciences, G. D’Annunzio University School of Pharmacy. ‡ Institute of Pharmacology, Catholic University School of Medicine. § Department of Cardiac Surgery, G. D’Annunzio University School of Medicine. | Menarini Ricerche S. p. A., Pomezia (Rome).
primary alcohol at C-14; the sugar, called daunosamine, is attached by a glycosidic bond to C-7 of the tetracyclic ring, and consists of a 3-amino-2,3,6-trideoxy-L-fucosyl moiety (Figure 1). The mechanisms through which DOX induces cardiotoxicity are rather complex. One-electron 1 Abbreviations: DOX, doxorubicin [7-(3-amino-2,3,6-trideoxy-R-Llyxo-hexopyranosyl)doxorubicinone]; EPI, epirubicin [7-(3-amino-2,3,6trideoxy-R-L-arabino-hexopyranosyl)doxorubicinone]; MEN 10755, 7-[2,6dideoxy-4-O-(3-amino-2,3,6-trideoxy-R-L-lyxo-hexopyranosyl)-R-L-lyxohexopyranosyl]-4-demethoxydoxorubicinone; MEN 11463, 7-(2,6-dideoxyR-L-lyxo-hexopyranosyl)-4-demethoxydoxorubicinone; MEN 10959, 7-[2,6dideoxy-4-O-(2,6-dideoxy-R-lyxo-hexopyranosyl)-R-lyxo-hexopyranosyl]4-demethoxydoxorubicinone; MEN 11951, 7-[2,6-dideoxy-4-O-(3-amino2,3,6-trideoxy-R-arabino-hexopyranosyl)-R-lyxo-hexopyranosyl]-4demethoxydoxorubicinone; L-fucose, 2,6-dideoxy-R-lyxo-hexopyranose; daunosamine, 3-amino-2,3,6-trideoxy-R-lyxo-hexopyranose; epidaunosamine, 3-amino-2,3,6-trideoxy-R-arabino-hexopyranose; anthracyclineol, anthracycline secondary alcohol metabolite; (4-demethoxy)doxorubicinolone, (4-demethoxy)doxorubicinone secondary alcohol metabolite.
10.1021/tx000143z CCC: $19.00 © 2000 American Chemical Society Published on Web 11/30/2000
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Figure 1. Anthracyclines used in this study.
redox cycling of the quinone moiety and consequent generation of reactive oxygen species probably mediate acute, reversible arrhythmias and hypotension; however, several lines of evidence suggest that the chronic, irreversible cardiomyopathy may be mediated by a structurally distinct metabolite called doxorubicinol (DOXol) (2-9). This metabolite is formed by NADPH-dependent cytosolic enzymes, like aldo/keto or carbonyl reductases, which add two electrons to the side chain carbonyl group and convert it into a secondary alcohol (-CO-CH2OH f -CHOH-CH2OH) (10). A potential mechanism of DOXolinduced cardiotoxicity has been identified in its ability to delocalize Fe(II) from the [4Fe4S] cluster of cytoplasmic aconitase (4, 5). However, alcohol metabolites do not always contribute to the antitumor activity of the parent anthracyclines (11); in some cases, alcohol metabolites actually increase the resistance of cancer cells to anthracyclines, although the precise mechanisms of resistance have remained unclear (10, 12). The opposite effects of alcohol metabolites in cardiac or cancer cells predict that anthracyclines forming smaller amounts of such metabolites might prove to induce less severe cardiotoxicity while retaining equal or even improved anticancer efficacy. We have previously probed this hypothesis by comparing DOX to epirubicin (EPI), an analogue approved for clinical use on the basis of its characteristics of good antitumor activity and reduced cardiotoxicity (1). An axial-to-equatorial epimerization of the hydroxyl group at C-4 in the daunosamine moiety is the only difference between DOX and EPI (Figure 1). Such a minimal modification is often said to reduce the cardiotoxicity of EPI by facilitating its glucuronidation and clearance before it approaches toxic levels in the heart (1). However, we have shown that EPI is characterized also by a significant decrease in the level of formation of its alcohol metabolite EPIol in human myocardium (3). This observation lends support to the idea that a reduction in the level of alcohol metabolite formation may be a general characteristic of less cardiotoxic anthracyclines.
MEN 10755 is a novel anthracycline characterized by the lack of a methoxy group at C-4 in the tetracyclic ring of the aglycone and by intercalation of 2,6-dideoxy-Lfucose between the aglycone and daunosamine (Figure 1). Comparisons between MEN 10755 and DOX in mice and rats have provided preliminary evidence that MEN 10755 may exhibit a broader spectrum of activity against human tumor xenografts while inducing less severe cardiotoxicity (13, 14). On the basis of this information, we exploited MEN 10755 to obtain further evidence that good antitumor activity and reduced cardiotoxicity may coincide in anthracyclines forming fewer alcohol metabolites than DOX. We therefore determined whether MEN 10755 produces fewer alcohol metabolites than DOX, how it compares to EPI, and which structural determinants may be important in these settings. The experiments were performed in cytosolic fractions of human myocardium obtained during bypass surgery. This model avoids potential pitfalls due to the high variability of anthracycline metabolism in laboratory animals, and provides an ethically acceptable system for predicting the formation and mechanisms of toxicity of secondary alcohol metabolites in the human heart (3-6).
Experimental Procedures Chemicals. DOX and EPI were obtained through the courtesy of A. Suarato (Chemistry Department, Pharmacia-Upjohn, Milan, Italy). MEN 10755 and all other MEN-coded anthracyclines were synthesized at the Chemistry Department of Menarini Ricerche S. p. A. (Pomezia, Rome, Italy). Alcohol metabolites and aglycones were prepared by NaBH4 reduction of the side chain carbonyl group and thermoacid hydrolysis of the glycosidic bond, respectively (3, 15). Ammonium sulfate (ultrapure grade, 90% for all the anthracyclines that were tested; the lowest detection limit was 0.01 nmol/mg of protein. Cluster Iron Delocalization. Iron delocalization from the [4Fe-4S] cluster of cytoplasmic aconitase was studied under enzymatic or nonenzymatic conditions. Enzymatic experiments were carried out in 1 mL incubations containing iron-loaded cytosol (1 mg of protein), anthracyclines (100 µM), and ferrozine (0.25 mM) in 0.3 M NaCl (pH 7.0) at 37 °C. The aconitase substrate-intermediate cis-aconitate (100 µM) was included to facilitate reactions of alcohol metabolites with the Fea of [4Fe4S] clusters (4). Reactions were started by adding NADPH (100 µM) to promote the formation of secondary alcohol metabolites, and delocalized Fe(II) was detected spectrally by monitoring the formation of ferrozine-Fe(II) complexes (4). Nonenzymatic experiments were performed by replacing NADPH and anthra-
Minotti et al.
Figure 2. Alcohol metabolite formation in human cardiac cytosol, as determined by comparisons among DOX, EPI, and MEN 10755. Anthracycline alcohol metabolites were assessed in NADPH-supplemented iron-depleted human cardiac cytosol, as described in Experimental Procedures. Values are those determined at 4 h and are means ( SE of three experiments. An asterisk indicates p < 0.05 vs DOX. cylines with purified secondary alcohol metabolites (3.5 µM). Suppression of Fe(II) delocalization by the aconitase pseudosubstrate-inhibitor D,L-fluorocitrate (100 µM) confirmed that that the [4Fe-4S] cluster of cytoplasmic aconitase was the source of iron released by anthracycline alcohols (4). Other Assays and Conditions. Proteins were quantified by the bicinchoninic acid method (16). Data were expressed as arithmetic means ( SE and analyzed by paired or unpaired Student’s t tests, as appropriate; differences were considered significant when p < 0.05. Other conditions are described in the figure legends and table footnotes.
Results and Discussion Alcohol Metabolite Formation. Anthracycline secondary alcohol metabolites would go underestimated or even undetected if they reacted with [4Fe-4S] clusters prior to their assay (4, 6); therefore, we used iron-depleted human cardiac cytosol in which clusters had been disassembled by treatment with dithiothreitol at pH 8.9. Reconstitution of this cytosol with NADPH and anthracyclines resulted in formation and detection of sizable amounts of alcohol metabolites; however, EPI formed significantly less metabolite than DOX, and a similar reduction was exhibited by MEN 10755 (Figure 2). To characterize the determinants of alcohol metabolite formation, we compared DOX and EPI to their aglycone doxorubicinone; similar experiments were performed by comparing MEN 10755 to its aglycone 4-demethoxydoxorubicinone and other mono- or disaccharide 4-demethoxyanthracyclines. Results from these experiments are reported in Table 1. Doxorubicinone was a very good substrate for carbonyl reduction, but the yield of the alcohol metabolite decreased upon attachment of daunosamine which formed DOX. Replacement of daunosamine with epidaunosamine, i.e., a daunosamine moiety bearing axial-to-equatorial epimerization of OH at C-4, converted DOX to EPI and made the anthracycline molecule even less susceptible to alcohol metabolite formation. The presence and epimerization of a daunosamine moiety were therefore identified as factors decreasing the level of carbonyl reduction in doxorubicinone-centered anthracyclines such as DOX or EPI. 4-Demethoxydoxorubicinone was dramatically less susceptible than doxorubicinone to carbonyl reduction, yielding less alcohol metabolite than either DOX or EPI. The attachment of 2,6-dideoxyL-fucose produced the monosaccharide MEN 11463, and restored the extent of alcohol metabolite formation to ∼70% of the yield of DOXol. The lack of the methoxy group at C-4 in the aglycone and the attachment of 2,6-
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Table 1. Determinants of Alcohol Metabolite Formation by Doxorubicinone- or 4-Demethoxydoxorubicinone-Centered Anthracyclinesa aglycone doxorubicinone
4-demethoxydoxorubicinone
sugar
anthracycline
alcohol metabolite (nmol/mg of protein)
none daunosamine epidaunosamine none 2,6-dideoxy-L-fucose 2,6-dideoxy-4-O-(daunosaminyl)-L-fucose 2,6-dideoxy-4-O-(epidaunosaminyl)-L-fucose 2,6-dideoxy-4-O-(2,6-dideoxy-L-fucosyl)-L-fucose
doxorubicinone DOX EPI 4-demethoxydoxorubicinone MEN 11463 MEN 10755 MEN 11951 MEN 10959
2.7 ( 0.2 1.5 ( 0.3 0.5 ( 0.1b 0.3 ( 0.1c 1.1 ( 0.2 0.6 ( 0.1b 0.3 ( 0.04d 0.3 ( 0.03d
a Experimental conditions were as described in the legend of Figure 2, with the exception that doxorubicinone and 4-demethoxydoxorubicinone were dissolved in 20-25 µL of ethanol; similar aliquots of ethanol were included in other incubations to permit direct comparisons. Values are those determined at 4 h and are means ( SE of three experiments. See Figure 1 for the structures of MEN 11463, MEN 11951, and MEN 10959. b p < 0.05 vs DOX. c p < 0.001 vs doxorubicinone. d p < 0.05 vs MEN 10755.
dideoxy-L-fucose were therefore identified as factors decreasing and increasing the level of anthracycline carbonyl reduction, respectively. MEN 10755 was obtained by attaching daunosamine to 2,6-dideoxy-L-fucose in MEN 11463. Under such conditions, the level of alcohol metabolite formation decreased back to the same levels as observed in the case of EPI. An even greater decrease in the level of metabolite formation was observed by replacing the daunosamine residue of MEN 10755 with epidaunosamine, producing the analogue MEN 11951. This finding showed that daunosamine epimerization could decrease the level of alcohol metabolite formation also in 4-demethoxydoxorubicinone-centered anthracyclines. However, a similar decrease in the extent of metabolite formation was also obtained by replacing daunosamine with 2,6-dideoxy-L-fucose to produce the bis-2,6-dideoxy-L-fucosyl analogue MEN 10959. Inasmuch as 2,6-dideoxy-L-fucose per se favored carbonyl reduction in the monosaccharide MEN 11463, a decrease in the level of alcohol metabolite formation after duplication of the same sugar in MEN 10959 clearly indicated that the presence of a disaccharide moiety was an independent determinant of a reduced level of metabolism of the 4-demethoxyanthracyclines tested in this study, regardless of the chemical structure or epimerizations of the individual sugar moieties. Collectively, these experiments showed that (i) EPI and MEN 10755 shared a reduction in the level of formation of an alcohol metabolite as compared to DOX and (ii) the metabolism of MEN 10755 was limited by both the lack of the methoxy group at C-4 in the aglycone and attachment of a disaccharide moiety. [4Fe-4S] Reactivity of Anthracycline Alcohol Metabolites. Elegant in vitro studies studies by Zweier (17) and Myers et al. (18) have shown that DOX binds lowmolecular weight Fe(III) and forms drug-metal complexes that undergo self-reduction to the ferrous form, eventually producing oxygen free radicals. The quinonehydroquinone moieties of the tetracyclic ring and the C-14 primary alcohol of the side chain have been envisioned as the chemical residues that enable DOX to bind and reduce iron, respectively (19). Whether these mechanisms contribute to the development of anthracyclineinduced cardiotoxicity has nonetheless remained uncertain, mainly because of very limited information about the existence of low-molecular weight Fe(III) in the cell (20), or on the actual stability of anthracycline-iron complexes under in vivo conditions of protonation and hydrolysis (21). As an alternative mechanism of ironmediated toxicity, we have proposed that anthracyclines target the [4Fe-4S] cluster of cytoplasmic aconitase (4,
5). The role of this enzyme in energy metabolism is uncertain or irrelevant, especially if it is compared to the established function of the mitochondrial aconitase in the Krebs cycle. However, reversible processes of cluster assembly and disassembly may confer to cytoplasmic aconitase a unique role in the post-transcriptional regulation of iron uptake and storage proteins as well as of the mitochondrial aconitase and presumably of succinate dehydrogenase (5, 22). Inappropriate delocalization of cluster iron may therefore disrupt several homeostatic processes in cardiomyocytes, possibly synergizing other mechanisms of anthracycline toxicity in a unifying pathway leading to cardiomyopathy (reviewed in ref 5). We have previously shown that neither anthracyclines per se nor their secondary alcohol metabolites are able to chelate iron ions coordinated in the [4Fe-4S] cluster of cytoplasmic aconitase (4); however, secondary alcohol metabolites have redox reactivity with the fourth iron atom (Fea) of the cluster, and may release it in a lowmolecular weight Fe(II) form while oxidizing back to the parent carbonyl anthracyclines (4). Both cluster iron delocalization and alcohol metabolite oxidation are limited by the presence of the aconitase-substrate intermediate cis-aconitate, which is needed to facilitate redox and sterical interactions of alcohol metabolites with Fea (4). Once these limiting interactions have occurred, the process of delocalization extends to the remaining iron centers of the cluster (Feb1-3), and the Fe-S motif undergoes disassembly (4). In the study presented here, alcohol metabolite-induced cluster iron delocalization was monitored by incubating NADPH, anthracyclines, and cis-aconitate in iron-loaded human cardiac cytosol in which [4Fe-4S] clusters had been reconstituted by treatment with cysteine and ferrous ammonium sulfate. As shown in Figure 3, EPI delocalized less Fe(II) than DOX, consistent with the reduced level or formation of EPIol versus DOXol. The extent of iron delocalization decreased even more significantly when EPI was replaced with MEN 10755, but this finding was not consistent with the observation that MEN 10755 and EPI produced similar levels of MEN 10755ol and EPIol (cf. Figure 2 and Table 1). We therefore hypothesized that MEN 10755 was characterized not only by a reduced level of formation of MEN 10755ol but also by an altered reactivity of MEN 10755ol with [4Fe-4S] clusters. To probe this hypothesis and to identify the structural determinants of [4Fe-4S] reactivity of anthracycline alcohol metabolites, we reconstituted iron-loaded human cardiac cytosol with the metabolites of both glycosidic and aglyconic (4-demethoxy)anthracyclines. Results from these experiments are
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or epimerizations in this moiety produced further reduction in the level of metabolite formation but not in [4Fe4S] reactivity, as indicated by the experiments with the analogues MEN 10959 and MEN 11951. Collectively, these results identified the presence of a disaccharide moiety as a factor decreasing the [4Fe-4S] reactivity of anthracycline alcohol metabolites, thus explaining how MEN 10755 delocalized less Fe(II) than EPI despite the comparable formation of MEN 10755ol and EPIol. Figure 3. Anthracycline-dependent release of Fe(II) from [4Fe4S] clusters, as determined by comparison among DOX, EPI, and MEN 10755. Cluster iron delocalization was monitored in NADPH- and cis-aconitate-supplemented iron-loaded human cardiac cytosol, as described in Experimental Procedures. Values are those determined at 4 h and are means ( SE of three to five experiments. An asterisk indicates p < 0.001 for EPI vs DOX. Two asterisks indicate p < 0.01 for MEN 10755 vs EPI. Table 2. [4Fe-4S] Reactivity of Anthracycline Secondary Alcohol Metabolitesa nmol/mg of protein
alcohol metabolite
Fe(II) delocalization
oxidation to carbonyl anthracycline
doxorubicinolone DOXol EPIol 4-demethoxydoxorubicinolone MEN 11463ol MEN 10755ol MEN 10959ol MEN 11951ol
3.2 ( 0.4 2.8 ( 0.2 2.9 ( 0.3 2.6 ( 0.3 2.7 ( 0.2 1.5 ( 0.1b 1.4 ( 0.4b 1.7 ( 0.2b
1.5 ( 0.2 1.3 ( 0.2 1.4 ( 0.4 1.2 ( 0.4 1.5 ( 0.4 0.22 ( 0.04b 0.27 ( 0.05b 0.18 ( 0.02b
a The [4Fe-4S] reactivity of anthracycline secondary alcohol metabolites was determined in iron-loaded human cardiac cytosol by assaying for Fe(II) delocalization, as described in Experimental Procedures. Reoxidation of alcohol metabolites to carbonyl anthracyclines was assessed by organic extraction of the incubation mixtures followed by two-dimensional TLC and fluorescence detection, as also described in Experimental Procedures. Values are those determined at 1 h and are means of three to five experiments. b p < 0.01 vs other anthracyclines.
reported in Table 2. Doxorubicinolone, DOXol, and EPIol exhibited equal reactivity with [4Fe-4S] clusters, as evidenced by comparable delocalization of Fe(II) and reoxidation to the parent carbonyl anthracyclines. 4-Demethoxydoxorubicinolone and the monosaccharide MEN 11463ol had the same [4Fe-4S] reactivity as DOXol or EPIol, but a significant reduction in the levels of both Fe(II) delocalization and alcohol metabolite oxidation was observed in the case of disaccharides MEN 10755ol, MEN 10959ol, and MEN 11951ol. When compared to the previous results in Table 1, these findings highlighted multiple dissociations between the determinants of alcohol metabolite formation and [4Fe-4S] reactivity. In the case of doxorubicinone-centered anthracyclines, like DOX and EPI, the presence and epimerization of a daunosamine moiety clearly limited the formation of alcohol metabolites but not their intrinsic reactivity with [4Fe4S] motifs. In the case of 4-demethoxydoxorubicinonecentered anthracyclines, the formation of alcohol metabolites was impaired or facilitated by the lack of a methoxy group at C-4 in the aglycone or by attachment of 2,6-dideoxyfucose, respectively; however, neither modification could influence the [4Fe-4S] reactivity of alcohol metabolites. With regard to the role of the disaccharide moiety, it was evident that the presence of 2,6-dideoxy4-O-(daunosaminyl)-L-fucose limited both the formation and [4Fe-4S] reactivity of MEN 10755ol; substitutions
Conclusions We have demonstrated that the novel anthracycline MEN 10755 is identical to EPI in producing less alcohol metabolite than DOX in human cardiac cytosol. Because MEN 10755 and EPI are less cardiotoxic than DOX in preclinical or clinical settings, these findings lend support to the hypothesis that alcohol metabolites may contribute to the development of anthracycline-induced cardiomyopathy, perhaps by targetting the [4Fe-4S] cluster of cytoplasmic aconitase. In this respect, our results provide novel evidence that MEN 10755 is characterized also by a peculiar decrease in the [4Fe-4S] reactivity of its secondary alcohol metabolite. This factor may represent an additional determinant of reduced cardiotoxicity, and raises the possibility that MEN 10755 might prove to be even less cardiotoxic than EPI. Because of its good activity and tolerability in animal models, MEN 10755 has already entered clinical studies that will assess its safety and efficacy. Here we have described some biochemical properties of MEN 10755 in human myocardium. This information may serve mechanism-based guidelines for interpreting clinical studies that compare the cardiotoxicity of MEN 10755 and other approved or investigational anthracyclines.
Acknowledgment. This work was supported in part by MURST COFIN ’98 and ’99 and by CNR Contracts 98.03091.CT04 and 99.02531.CT04 (to G.M.).
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