Cardiovasc Toxicol (2007) 7:145–150 DOI 10.1007/s12012-007-0020-6
ORIGINAL PAPER
New iron chelators in anthracycline-induced cardiotoxicity Helena Kaiserova´ Æ Toma´sˇ Sˇimu˚nek Æ Martin Sˇteˇrba Æ Gertjan J. M. den Hartog Æ Ladislava Schro¨terova´ Æ Olga Popelova´ Æ Vladimı´r Gersˇl Æ Eva Kvasnicˇkova´ Æ Aalt Bast
Published online: 19 April 2007 Humana Press Inc. 2007
Abstract The use of anthracycline anticancer drugs is limited by a cumulative, dose-dependent cardiac toxicity. Iron chelation has long been considered as a promising strategy to limit this unfavorable side effect, either by restoring the disturbed cellular iron homeostasis or by removing redox-active iron, which may promote anthracycline-induced oxidative stress. Aroylhydrazone lipophilic iron chelators have shown promising results in the rabbit model of daunorubicin-induced cardiomyopathy as well as in cellular models. The lack of interference with the antiproliferative effects of the anthracyclines also favors their use in clinical settings. The dose, however, should be carefully titrated to prevent iron depletion, which apparently also applies for other strong iron chelators. We have
shown that a mere ability of a compound to chelate iron is not the sole determinant of a good cardioprotector and the protective potential does not directly correlate with the ability of the chelators to prevent hydroxyl radical formation. These findings, however, do not weaken the role of iron in doxorubicin cardiotoxicity as such, they rather appeal for further investigations into the molecular mechanisms how anthracyclines interact with iron and how iron chelation may interfere with these processes. Keywords Doxorubicin Daunorubicin Iron homeostasis Aroylhydrazone chelators Oxidative stress
Iron and the organism––two faces of iron H. Kaiserova´ T. Sˇimu˚nek E. Kvasnicˇkova´ Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kra´love´, Charles University in Prague, Hradec Kra´love´, Czech Republic M. Sˇteˇrba O. Popelova´ V. Gersˇl Department of Pharmacology, Faculty of Medicine in Hradec Kra´love´, Charles University in Prague, Hradec Kra´love´, Czech Republic L. Schro¨terova´ Department of Medical Biology and Genetics, Faculty of Medicine in Hradec Kra´love´, Charles University in Prague, Hradec Kra´love´, Czech Republic G. J. M. den Hartog A. Bast Department of Pharmacology and Toxicology, Faculty of Medicine, University of Maastricht, Maastricht, The Netherlands H. Kaiserova´ (&) Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, Prague 6 166 10, Czech Republic e-mail:
[email protected]
Iron is a trace element essential for virtually all known organisms. It is necessary for cell proliferation, energy production, metabolism of xenobiotics and it also participates in immune reactions. Most of the iron is firmly incorporated inside the metalloproteins (e.g., hemoglobin, myoglobin, cytochromes) because in its ‘‘free’’ form it readily redox cycles between its ferrous (Fe2+) and ferric (Fe3+) oxidation states and can catalyze production of potentially harmful reactive oxygen species (ROS). The organisms therefore developed well-controlled systems of regulating the iron homeostasis in order to avoid both iron deficiency and iron overload. Cytosolic labile iron pool, which is apparently iron in transit between the iron transporter transferin and the iron storage protein ferritin, is sensed by iron regulatory proteins (IRPs). These posttranscriptionally control the expression of transferin receptor and ferritin [1]. It has been shown that both iron excess and iron deficiency lead to oxidative stress, most probably due to the mitochondrial dysfunction [2]. Iron
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overload-associated diseases in general also induce iron deposits in heart and parenchymal organs such as liver, which eventually lead to various functional alterations.
Role of iron in anthracycline cardiotoxicity––rationale for iron chelation Although there is usually no systemic iron overload in anthracycline-treated patients, it has been shown that the anthracyclines disturb cellular iron homeostasis [3]. The effects of the anthracyclines on cardiac and tumor cells are complex (Fig. 1) and not all of them are cell-type specific. Regarding the iron, these effects can be divided into iron-dependent and iron-independent. The most prominent characteristic of the anthracyclines is their ability to induce oxidative stress. Some ROS can be formed without presence of iron but their generation can also be iron-catalyzed (Fenton reaction, Haber–Weiss reaction) bringing to life highly reactive and damaging hydroxyl radicals (HO). Anthracyclines can also directly complexate with iron in a process that leads to intramolecular redox cycling and further ROS production [4]. Oxidative stress induces damage to both cardiac and neoplastic cells. The higher susceptibility of heart tissue to oxidative stimuli is
Fe (-) ,
RO S
(-)
Impairmentof calciumtrafficking Inflammation Endothelialdysfunction(vasculardamage)
) (+ Fe
OS ,R
( -)
Accumulationof Fein ferritin Decreasein IRP-IRE binding
Fe(-), ROS (+)
Oxidative stress
ANT
Cardiac toxicity Superoxide H2O2 DOX Semiquinone C7-DOX aglycone Hydroxyl radical
Fe(+), ROS (+) Fe (-) ,
RO S
(-
usually explained by its poor antioxidant defences and/or abundance of mitochondria, which are both important source, and target of ROS. In tumor cells, the effect of ROS is likely to be overwhelmed by other effects such as topoisomerase II inhibition or DNA intercalation. As suggested previously, not all interactions of anthracyclines with iron are oxidative stress-associated. It has been reported that in vitro anthracyclines decreased the binding of IRPs to the so-called iron responsive elements (IRE) of mRNA, thus modifying expression of the proteins that are critical for maintaining optimal intracellular iron levels [3]. Other authors have shown that doxorubicin induced an accumulation of iron in ferritin and prevented its mobilization, which might possibly result in relative iron depletion [5]. It is, however, not known whether and to which extent these effects contribute to anthracycline cardiotoxicity in a clinical setting. Using an in vivo rabbit model of chronic anthracycline-induced heart failure, no changes of myocardial iron content were observed either in animals treated for 10 weeks with daunorubicin or in the group where dexrazoxane was co-administered [6]. The concept of iron chelation as a means to prevent anthracycline cardiotoxicity was born with the discovery of the cardioprotective effects of dexrazoxane (ICRF-187). This bisdioxopiperazine compound is metabolized into a metal-chelating EDTA-like product (ADR 925), and its cardioprotective action was therefore assumed to be due to the chelation of redox-active iron and thus reducing doxorubicin-induced oxidative stress [7].
Topoisomerase II inhibition Intercalation Directmembraneeffects
Antitumor activity
Fig. 1 A simplified scheme of the proposed interactions of anthracyclines (ANT) with tumor and cardiac cells. The effects of ANT are either iron-dependent or iron-independent. ANT induce oxidative stress. Reactive oxygen species (ROS) can be formed either in the absence (semiquinone radical, C7-aglycone radical, superoxide, H2O2) or presence (HO• radical) of iron. ANT can also form ANTFe(III) complexes that also participate in ROS formation. ROS may induce damage to both cardiac and neoplastic cells. The major mechanisms that lead to cancerous cell death are, most probably, inhibition of topoisomerase II and DNA intercalation––the effects independent on oxidative stress as well as iron. Other effects that do not depend on ROS are thought to contribute predominantly to the cardiotoxicity of the anthracyclines: dysregulation of iron homeostasis via interaction with iron regulatory protein and inhibition of iron mobilization from ferritin are examples of iron-mediated effects, whereas inflammation, endothelial dysfunction, and calcium homeostasis impairment are both iron and ROS independent
Development of new iron chelators as protectors against anthracycline cardiotoxicity Today, dexrazoxane serves as the basic and reference drug to prevent cardiotoxicity of doxorubicin. Despite its high effectiveness, its use in clinical practice suffers from several disadvantages: bone-marrow toxicity, low bioavailability (need for i.v. application), high costs and possible antagonism with the antitumor effect of the anthracyclines. Therefore, alternative chelators of iron with lower toxicity, higher specificity for iron, oral availability and no interference with the antiproliferative effects of the anthracyclines are being studied. Iron has six coordination sites and, to make it redoxinactive, it is necessary to shield all these sites. We can distinguish between the chelators according to the number of free electron pairs that can be used to form a coordination bond––thus we recognize the bidentate, tridentate, and hexadentate chelators. In the case of hexadentate chelators (e.g., deferoxamine, DFO), a single molecule of a chelator occupies all six coordination sites of iron. When bidentate or tridentate ligands are employed, the
Cardiovasc Toxicol (2007) 7:145–150
147
ligand-iron ratio must be 3:1 or 2:1, respectively, to prevent the formation of redox-active partial complexes. It is believed that it is difficult to design new chelators that would be both hexadentate and possess a good bioavailability. Therefore, most of the newly synthesized compounds belong to the group of bidentate or tridentate chelators. Iron chelators are typically not designed directly for use in prevention of anthracycline cardiotoxicity but rather for the treatment of chronically iron-overloaded patients such as those with b-thalassemia. The chelators that have been previously tested as protectors against anthracycline cardiotoxicity are depicted in the Table 1. Studies with deferoxamine (DFO) gave mixed results. It was found that iron loading aggravated the doxorubicin cardiotoxicity both in vitro and in vivo and this effect could be prevented with DFO, however, in models with normal iron stores, the protection disappeared. In the study of Voest [8] the protective effect of deferoxamine was dose-dependent and disappeared with higher dose of the chelator. Deferiprone (L1) has been shown to protect isolated cardiomyocytes against doxorubicin toxicity [9] and an in vivo study in rats also suggested certain protection [10]. Surprisingly, the lipophilic (cell permeable) and strong chelator of irondeferasirox (ICL670) lacked any protective effect in isolated myocytes [11]. Apparently, the findings coming from different studies depend on many factors: chelator dose, model used, and/or parameters followed. A major limitation of most of the latter models is the fact that they reflect rather an acute toxicity, whereas it is the chronic (or delayed) cardiotoxicity that makes anthracyclines dangerous. In our laboratory we have developed and validated a rabbit model of chronic daunorubicin cardiotoxicity [12] and we use this model for in vivo evaluation of new lipophilic iron chelators of the aroylhydrazone class.
The ‘‘mother’’ compound of these tridentate ligands, pyridoxal isonicotinoyl hydrazone (PIH), was discovered in late 1970s by Ponka and colleagues. Many analogs have been derived by now and the compounds with enhanced antiproliferative properties are recently being developed as new anticancer drugs [13]. We are, on the contrary, interested in less toxic derivates that could be used in the prevention of anthracycline cardiotoxicity. The major drawback of these compounds seems to be their short plasma half-life, which might be in part due to the hydrolysis of the chelator [14, 15]. This disadvantage can be overcome for example by means of creating stable pro-chelators that are only activated at the site where oxidative stress occurs [16]. To date, we have evaluated the cardioprotective effects of the three aroylhydrazone analogs, PIH, SIH, and o-108 (Fig. 2). In our preliminary in vitro experiments we have found that SIH efficiently prevented oxidative stressinduced mitochondrial injury in H9c2 cardiomyoblast cells [17]. We also found out that both PIH and SIH were able to prevent daunorubicin and doxorubicin-induced loss of CYP450 activities in isolated hepatocytes [18]. Using two different cancer cell lines (HL60, A549) we have further confirmed that the antiproliferative effects of the anthracyclines are not hampered by any of these chelators [19, 20]. In the next step, the cardioprotective effects of these chelators have been evaluated in the rabbit model of chronic daunorubicin cardiomyopathy. Chronic daunorubicin treatment (3 mg/kg weekly for 10 weeks) induced mortality (33%), left ventricular (LV) dysfunction, cardiac troponin T (cTnT) plasma level rise as well as typical morphological LV damage. Coadministrations of PIH (25 mg/kg, i.p.) or SIH hydrochloride (1 mg/kg, i.v.), fully prevented premature deaths and the DAU-induced changes were less pronounced in
Table 1 Iron chelators as protectors against anthracycline cardiotoxicity Chelator
Study
Model
Protection (±)
Methodology
Deferoxamine (DFO)
Hershko [23]
Rat cardiomyocytes
+ (iron loaded cells)
LDH, contractility
Herman [24] Voest [8]
Spont. hypertensive rats Isolated mouse left atria
Saad [25]
Rats in vivo (acute toxicity)
– (normal cells) – + (200 lM DFO)
Histology Contractility
– (500 lM DFO) Deferriprone (L1)
Deferasirox (ICL670) Aroyl hydrazones (PIH analogs)
a
+
CK-MB, LDH, AST, GSH, MDA
Barnabe [9]
Rat cardiomyocytes
+
LDH
Link [10]
Rats in vivo
+
LDH, contractility, mitochondrial function
Hasinoff [11] Sˇimu˚nek [21]
Rat cardiomyocytes
–
Rabbits in vivo
± (PIH)
Sˇteˇrba [20]
Rabbits in vivo
+ (o-108)
LDH a
Significantly increased survival of the animals, cardioprotection did not reach statistical significance
Mortality, histology, biochemistry, functional parameters Mortality, histology, biochemistry, functional parameters
148
Cardiovasc Toxicol (2007) 7:145–150 Table 2 Survival of rabbits, left ventricular contractility (dP/dtmax index) and left ventricular ejection fraction (LVEF) after the 10-week repeated administration of daunorubicin (DAU; 3 mg/kg i.v. weekly) Survival (%)
LVEF (%)
dP/dtmax (kPa/s)
Control
100
61.7 ± 0.9
1345 ± 61
DAU
67
47 ± 2a,b
783 ± 53
PIH 25 mg + DAU
100
52 ± 3
872 ± 128
b
PIH 50 mg + DAU
43
48 ± 10
815 ± 364
b
Group
b
c
o-108 10 mg + DAU
100
59 ± 1
o-108 25 mg + DAU
50
45 ± 4b
1131 ± 125
691 ± 101b
SIH 1 mg + DAU SIH 2.5 mg + DAU
100 67
60 ± 2 54 ± 1a
1185 ± 80c 811 ± 144b
Statistical significance (P < 0.05) Paired t-test: aPaired comparison with the initial values within each group; ANOVA: bComparison with control group, cComparison with daunorubicin group
most functional, biochemical as well as morphological parameters. The best results were achieved with o-108 (10 mg/kg, i.p.). All animals survived without a significant drop in the LV ejection fraction (63.2 ± 0.5% vs. 59.2 ± 1.0%, beginning vs. end, n.s.) and their cardiac contractility (LV dP/dtmax) was significantly higher than in the daunorubicin-only group (1131 ± 125 vs. 783 ± 53 kPa/s, P < 0.05). These findings well corresponded with lower extent and intensity of myocardial damage as assessed by histology [21, 20]. Although higher doses of the chelators (PIH––50 mg/kg; SIH––2.5 mg/kg; o-108–– 25 mg/kg) were well tolerated when administered alone, in a combination with DAU they surprisingly not only did not provide additional protection, but on the contrary they hampered virtually all their beneficial effects regarding both cardioprotection and overall mortality (Table 2).
Dequenching with SIH
1000
Fluorescence intensity (AU)
Fig. 2 Structures of the three aroylhydrazone iron chelators investigated. PIH, pyridoxal isonicotinoyl hydrazone; SIH, salicylaldehyde isonicotinoyl hydrazone; o-108, pyridoxal 2-chlorobenzoyl hydrazone
chelator are we dealing with. It seems that the ability of a compound to chelate iron (i.e., ‘‘strength of the chelator’’) does not correlate with its cardioprotective potential. To investigate this, we have compared iron-chelation properties of the five diverse chelators of iron: two aroylhydrazones (PIH and SIH), two clinically used agents (DFO and dexrazoxane) and the flavonoid with iron chelating properties––monohydroxyethylrutoside (monoHER). We have employed the calcein fluorescence assay, where the ability of a chelator to displace Fe3+ from calcein-Fe3+complex was measured as a function of time (Fig. 3). The results
PIH 800 SIH 600 DFO 400 Dexrazoxane 200
monoHER
0 0
200
400
600
Time (s) How much iron can we chelate? Balancing at the edge of iron depletion The puzzling dose-dependency of the chelators in our latter experiments suggests that iron chelation therapy has to be designed deliberately, knowing exactly what type of iron
Fig. 3 Displacement of iron from its complexes with calcein. Ferrous-ammonium sulfate was pre-reacted with a fluorescent probe calcein for 1 h (quenching of fluorescence), after which the chelators at 5 lM concentration (final) were added and the rate of calcein dequenching was monitored. Dequenching reactions were followed by addition of SIH (5 lM, final) in order to obtain maximal fluorescence
Cardiovasc Toxicol (2007) 7:145–150
showed that PIH and SIH displaced iron from the calceiniron complex rapidly and completely, whereas DFO was slower, monoHER was even weaker chelator and dexrazoxane did not chelate iron at all in this assay. This is not surprising because it is known that dexrazoxane only chelates iron after its intracellular hydrolysis. Based on its reported association constants toward iron, iron-chelating capacity of activated dexrazoxane can be expected to be somewhat lower than that of DFO. The high clinical cardioprotective efficiency of dexrazoxane is in contrast with its relatively weak iron chelating capacity. Moreover it is known that ADR-925 does not specifically bind iron but also other metal ions. MonoHER serves as another example of a compound that has been shown to possess cardioprotective potential [22] in spite of its rather weak ironchelation ability. It appears that, indeed, the compounds with moderate iron chelating properties rather than the strongest chelators of iron are particularly interesting for investigation as protectors against anthracycline cardiotoxicity.
Iron and anthracycline-induced oxidative stress Traditionally, the cardioprotective effects of iron chelators have been attributed to their ability to decrease oxidative stress via chelation of iron that could participate in Fentontype ROS reactions. A question arises here, whether preventing the formation of hydroxyl radicals (by means of iron chelation) can be regarded as a major pathway how to prevent or decrease the cardiotoxicity of anthracyclines. We have compared the effects of various iron chelators on the prevention of hydrogen peroxide/Fe2+-induced oxidative stress and doxorubicin-induced oxidative stress. Their ability to prevent hydroxyl radical formation was first investigated by means of EPR spectroscopy and it was found that their efficiency decreased in this order: PIH = SIH > dexrazoxane > DFO > monoHER [19]. When a more complex cellular model was used (A549, human lung adenocarcinoma cells), it turned out that only DFO and SIH were able to prevent hydrogen peroxide/Fe2+-induced oxidative damage as monitored by LDH release, TBARS formation, and cellular GSH levels. In contrast, when oxidative stress was induced with doxorubicin, DFO and SIH were not effective at all, whereas dexrazoxane and monoHER significantly decreased the doxorubicin-induced oxidative damage [19] (Table 3). These results clearly demonstrate that iron-promoted hydroxyl radical formation is not a major determinant of doxorubicin-induced oxidative injury. Therefore, we assume that the role of iron in anthracycline cardiotoxicity is more complex and further studies should focus more on oxidative stress-independent
149 Table 3 Comparison of the ability of various iron chelators to ameliorate DOX and H2O2/Fe2+-induced oxidative toxicity in the A549 cells DOX-induced toxicity
H202/Fe2+-induced toxicity
LDH
LDH
TBARS
GSH
TBARS
GSH
Dexrazoxane +
–
+
–
–
–
DFO
–
–
–
+
–
+
monoHER PIH
+ –
– –
+ –
– –
– –
– –
SIH
–
–
–
+
+
+
LDH, lactate dehydrogenase activity in culture media (cytotoxicity marker); TBARS, thiobarbituric-acid reactive substances (lipid peroxidation marker); GSH, total glutathione (marker of cellular antioxidant status)
mechanisms, by which the anthracyclines interfere with iron.
Iron chelation in anthracycline cardiotoxicity: conclusions and future perspectives Iron chelation remains one of the most successful pharmacological strategies in terms of reducing cardiotoxicity of the anthracyclines. New iron chelators are continuously being developed in order to overcome adverse effects of the only clinically approved cardioprotector at this moment––dexrazoxane. We have demonstrated, that three aroylhydrazone iron chelators (PIH, SIH, and o-108), when administered at optimal dose, are capable to significantly abate the chronic anthracycline-induced cardiomyopathy. The promise of PIH and its analogs has been, however, weakened by our finding that the therapeutic range of these compounds is rather narrow. We have demonstrated that iron-chelating capacity of the chelator does not correlate with its cardioprotective efficiency, probably due to the chelator-induced iron depletion. Weak to moderate iron chelators should be preferred in further development or, in cases when stronger iron chelators are supposed to be used, the dose should be carefully titrated to achieve cardioprotection without critical iron depletion. We have challenged the classic concept of iron-promoted hydroxyl radical formation as a primary pathway of anthracycline-induced oxidative stress. These findings, however, do not weaken the role of iron in doxorubicin cardiotoxicity as such, they rather appeal for further investigations into the mechanisms how anthracyclines and iron interact and how iron chelation may interfere with these processes. Acknowledgments ˇR 97/2005, GAC MSM0021620820.
The authors were supported by grants GAUK 305/05/P156, and a Research Project
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