Fabio Animati

Chronic cardiotoxicity of anticancer anthracyclines in the rat: role of secondary metabolites and reduced toxicity by a novel anthracycline with impaired metabolite formation and reactivity

The anticancer anthracycline doxorubicin (DOX) causes cardiomyopathy upon chronic administration. There is controversy about whether DOX acts directly or after conversion to its secondary alcohol metabolite DOXol. Here, the role of secondary alcohol metabolites was evaluated by treating rats with cumulative doses of DOX or analogues – like epirubicin (EPI) and the novel disaccharide anthracycline MEN 10755 – which were previously shown to form less alcohol metabolites than DOX when assessed in vitro. DOX induced electrocardiographic and haemodynamic alterations, like elongation of QαT or SαT intervals and suppression of isoprenaline‐induced dP/dt increases, which developed in a time‐dependent manner and were accompanied by cardiomegaly, histologic lesions and mortality. EPI caused less progressive or severe effects, whereas MEN 10755 caused essentially no effect. DOX and EPI exhibited comparable levels of cardiac uptake, but EPI formed ∼60% lower amounts of its alcohol metabolite EPIol at 4 and 13 weeks after treatment suspension (P<0.001 vs DOX). MEN 10755 exhibited the lowest levels of cardiac uptake; hence, it converted to its alcohol metabolite MEN 10755ol ∼40% less efficiently than did EPI to EPIol at either 4 or 13 weeks. Cardiotoxicity did not correlate with myocardial levels of DOX or EPI or MEN 10755, but correlated with those of DOXol or EPIol or MEN 10755ol (P=0.008, 0.029 and 0.017, respectively). DOX and EPI inactivated cytoplasmic aconitase, an enzyme containing an Fe–S cluster liable to disassembly induced by anthracycline secondary alcohol metabolites. DOX caused greater inactivation of aconitase than EPI, a finding consistent with the higher formation of DOXol vs EPIol. MEN 10755 did not inactivate aconitase, which was because of both reduced formation and impaired reactivity of MEN 10755ol toward the Fe–S cluster. Aconitase inactivation correlated (P<0.01) with the different levels of cardiotoxicity induced by DOX or EPI or MEN 10755. These results show that (i) secondary alcohol metabolites are important determinants of anthracycline‐induced cardiotoxicity, and (ii) MEN 10755 is less cardiotoxic than DOX or EPI, a behaviour attributable to impaired formation and reactivity of its alcohol metabolite.

Configurational requirements of the sugar moiety for the pharmacological activity of anthracycline disaccharides.

The amino sugar is recognized to be a critical determinant of the activity of anthracycline monosaccharides related to doxorubicin and daunorubicin. In an attempt to improve the pharmacological properties of such agents, novel anthracycline disaccharides have been designed in which the amino sugar, daunosamine, is separated from the aglycone by another carbohydrate moiety. In the present study, we examined the influence of the orientation of the second sugar residue on drug biochemical and biological properties in a series of closely related analogs. This structure-activity relationship study showed that the substitution of the daunosamine for the disaccharide moiety dramatically reduced the cytotoxic potency of the drug in the 4-methoxy series (daunorubicin analogs). In contrast, in the 4-demethoxy series (idarubicin analogs), the C-4 axial, but not the equatorial, configuration conferred a cytotoxic potency and antitumor activity comparable to that of doxorubicin. The configuration also influenced the drugs ability to stimulate topoisomerase II alpha-mediated DNA cleavage. Indeed, the glycosides with the equatorial orientation were ineffective as topoisomerase II poisons, whereas the compounds with axial orientation were active, although the daunorubicin analog exhibited a lower activity than the idarubicin analog. It is conceivable that the axial orientation allows an optimal interaction of the drug with the DNA-enzyme complex only in the absence of the methoxy group. Our results are consistent with a critical role of the sugar moiety in drug interaction with the target enzyme in the ternary complex.

Impairment of myocardial contractility by anticancer anthracyclines: role of secondary alcohol metabolites and evidence of reduced toxicity by a novel disaccharide analogue

The anticancer anthracycline doxorubicin (DOX) causes cardiotoxicity. Enzymatic reduction of a side chain carbonyl group converts DOX to a secondary alcohol metabolite that has been implicated in cardiotoxicity. We therefore monitored negative inotropism, assessed as inhibition of post‐rest contractions, in rat right ventricle strips exposed to DOX or to analogues forming fewer amounts of their alcohol metabolites (epirubicin, EPI, and the novel disaccharide anthracycline MEN 10755). Thirty μM EPI exhibited higher uptake than equimolar DOX, but formed comparable amounts of alcohol metabolite due to its resistance to carbonyl reduction. MEN 10755 exhibited also an impaired uptake, and consequently formed the lowest levels of alcohol metabolite. Accordingly, DOX and EPI inhibited post‐rest contractions by ∼40 – 50%, whereas MEN 10755 inhibited by ∼6%. One hundred μM EPI exhibited the same uptake as equimolar DOX, but formed ∼50% less alcohol metabolite. One hundred μM MEN 10755 still exhibited the lowest uptake, forming ∼60% less alcohol metabolite than EPI. Under these conditions DOX inhibited post‐rest contractions by 88%. EPI and MEN 10755 were ∼18% (P<0.05) or ∼80% (P<0.001) less inhibitory than DOX, respectively. The negative inotropism of 30 – 100 μM DOX, EPI, or MEN 10755 correlated with cellular levels of both alcohol metabolites (r=0.88, P<0.0001) and carbonyl anthracyclines (r=0.79, P<0.0001). Nonetheless, multiple comparisons showed that alcohol metabolites were ∼20 – 40 times more effective than carbonyl anthracyclines in inhibiting contractility. The negative inotropism of MEN 10755 was therefore increased by chemical procedures, like side chain valeryl esterification, that facilitated its uptake and conversion to alcohol metabolite but not its retention in a carbonyl form. These results demonstrate that secondary alcohol metabolites are important mediators of cardiotoxicity. A combination of reduced uptake and limited conversion to alcohol metabolite formation might therefore render MEN 10755 more cardiac tolerable than DOX and EPI.

New anthracycline disaccharides. Synthesis of L-daunosaminyl-α(1 →4)-2-deoxy-L-rhamnosyl and of L-daunosaminyl-α(1 →4)-2-deoxy-L-fucosyl daunorubicin analogues

The synthesis of the new disaccharide anthracyclines 20, 21, 24 and 25, where the daunosamine moiety is separated from the aglycone by either a rhamnose or a fucose residue, performed following a convergent procedure, gives insight into the configurational requirement of the first sugar residue and opens the way to a new class of antitumour anthracyclines.

Oxidative Degradation of Cardiotoxic Anticancer Anthracyclines to Phthalic Acids NOVEL FUNCTION FOR FERRYLMYOGLOBIN

We show that the pseudoperoxidase activity of ferrylmyoglobin (MbIV) 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 DOX/MbIV 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.

Binding of Epstein-Barr virus nuclear antigen 1 to DNA : inhibition by distamycin and two novel distamycin analogues

Modulation of the interaction between cellular or viral transcription factors and target DNA sequences may represent a potential experimental strategy to control proliferation of neoplastic cells as well as virus DNA replication. Distamycin represents a likely candidate to mediate such modulation by pharmacological means. In order to obtain more detailed information on structure-activity relationships of these compounds, we have analysed the effects of distamycin and two distamycin analogues on the binding of a recombinant protein, the Epstein-Barr virus nuclear antigen 1 (EBNA-1) to its target sequence of Epstein-Barr virus, containing the 12 bp palindromic consensus TAGCATATGCTA. The sequence selectivity in the binding of distamycin to DNA was evaluated by footprinting experiments, while the effects of distamycins on DNA-protein interactions was analysed by means of electrophoretic mobility shift assay. The data presented in this paper suggest that distamycin and its analogues differentially inhibit the interaction between DNA-binding proteins and target DNA sequences.

Synthesis, biological evaluation, and molecular modeling studies of rebeccamycin analogues modified in the carbohydrate moiety.

A new series of indolocarbazole glycosides containing disaccharides were synthesized and their in vitro antiproliferative activity was evaluated against three human cancer cell lines (A2780, H460, and GLC4). Cytotoxicity appeared to be remarkably affected by the regio‐ and stereochemical features of the disaccharide moiety. In vivo antitumor activity of the compounds studied, two of which having IC50<100 nm, was determined using ovarian cancer cell line A2780 xenografted on nude mice. One compound showed an efficacy similar to that of the reference compound edotecarin, though with a lower long‐lasting activity. The topoisomerase I inhibitory properties of some compounds were also examined. Molecular dynamics simulations of the ternary topoisomerase I–DNA–ligand complexes were performed to analyze the structural features of topoisomerase I poisoning with this class of indolocarbazoles. A plausible explanation of their biological behavior was provided. These theoretical results were compared with the recently published crystal structure of an indolocarbazole monosaccharide bound to the covalent human topoisomerase I–DNA complex.

A comparative study of cellular and molecular pharmacology of doxorubicin and MEN 10755, a disaccharide analogue.

MEN 10755 is a disaccharide anthracycline endowed with a broader spectrum of antitumour activity than doxorubicin (DOX). To investigate the cellular and molecular basis of its action, cytotoxic activity, drug uptake, subcellular localisation, induction of DNA damage, and apoptosis were assessed in the human A2780 ovarian carcinoma cell line. Experiments with radiolabelled anthracyclines indicated that MEN 10755 exhibited reduced cellular accumulation and a different subcellular distribution (higher cytoplasmic/nuclear ratio) than DOX. In spite of the lower nuclear concentration, MEN 10755 was as potent as DOX in eliciting DNA single- and double-strand breaks, G2/M cell arrest, and apoptosis. Sequencing of drug-induced topoisomerase II cleavage sites showed a common DNA cleavage pattern for MEN 10755 and DOX. Cleavage sites were always characterised by the presence of adenine in -1 position. However, the extent of DNA cleavage stimulation induced by MEN 10755 was greater than that produced by DOX. Reversibility studies showed that MEN 10755-stimulated DNA cleavage sites were more persistent than those induced by DOX, thus suggesting a more stable interaction of the drug in the ternary complex. As a whole, the study indicated that the cellular pharmacokinetics of MEN 10755 substantially differs from that of DOX, showing a lower uptake and a different subcellular disposition. In spite of the apparently unfavourable cellular pharmacokinetics, MEN 10755 was still as potent as DOX in inducing topoisomerase-mediated DNA damage. Although the extent and persistence of protein-associated DNA breaks may contribute to the cytotoxic effects, the drugs efficacy as apoptosis inducer and antitumour agent could not be adequately explained on the basis of DNA damage mediated by the known target (i.e. topoisomerase II), thus supporting additional cellular effects that may be relevant in cellular response.

Distamycin analogues with improved sequence-specific DNA binding activities

In the present study we have investigated the effect of unprecedented chemical modifications introduced in the distamycin molecule, with the aim of assessing their ability to interfere with sequence-specific DNA-protein interactions in vitro. By using an electrophoretic mobility shift assay, we have been able to identify novel distamycin analogues with improved displacing abilities on the binding of octamer nuclear factors to their target DNA sequence. While variations in the number of pyrrole rings and/or reversion of an internal amide bond result in distamycin-like compounds with identical or very similar properties, the reversion of the formamido into a carboxyamido group or its replacement with the charged formimidoyl moiety significantly improves the ability of the resulting novel distamycin derivatives to compete with OCT-1 (octamer 1 nuclear factor) for its target DNA sequence. Tissue-specific octamer-dependent in vitro transcription is similarly affected by these chemical modifications, suggesting that the ability of distamycins to bind octamer sequences has a direct influence on the functional state of octamer-containing promoters. These data represent an initial, successful attempt to rationalize the design of DNA binding drugs, using distamycins as a model.

Synthesis and biological evaluation of rebeccamycin analogues modified at the imide moiety.

Glycosylated indolocarbazoles related to the antibiotic rebeccamycin represent an important class of antitumour drugs. In the course of our structure-activity relationship studies, new rebeccamycin analogues modified at the imide moiety were synthesised. The antiproliferative activity of the compounds was evaluated on three human cancer cell lines, A2780 (ovarian cancer), H460 (lung cancer), and GLC4 (small-cell lung cancer). The in vitro cytotoxicity of compounds 2 and 4, characterised respectively by a 1,3-dioxolan and (1,3-dioxolan-4-yl)methylene groups linked to the imide moiety, was higher than the reference compound, edotecarin. The effect of compound 2 in inducing tumour regression in the A2780 xenograft model was also investigated.