Helena Kaiserová

In vitro and in vivo examination of cardiac troponins as biochemical markers of drug-induced cardiotoxicity

Cardiac troponin T (cTnT) and troponin I (cTnI) are becoming acknowledged as useful biochemical markers of drug-induced cardiotoxicity. In this study we examined the release kinetics of cTnT and cTnI using an in vitro model of isolated rat neonatal ventricular cardiomyocytes (NVCM, 72h treatment with 0.1-3microM of daunorubicin) and compared it with data from a rabbit model of chronic anthracycline-induced cardiomyopathy in vivo (3mg/kg of daunorubicin weekly, 10 weeks). In cell-culture media, the cTnI and cTnT concentrations were concentration- and time-dependently increasing in response to daunorubicin exposure and were negatively exponentially related to cardiomyocyte viability. With 3microM daunorubicin, the relative increase of AUC of cTnT and cTnI was 2.4- and 5.3-fold higher than the increase of LDH activity, respectively. In rabbits, the daunorubicin-induced cardiomyopathy was associated with progressive increase of both cTnT and cTnI. Although the correlation between cTnT and cTnI cumulative release (AUCs) was found (R=0.81; P<0.01) and both cardiac troponins corresponded well with the echocardiographically-assessed systolic dysfunction (R=0.83 and 0.81 for cTnT and cTnI, respectively; P<0.001), the first significant increase in cTnI levels was observed earlier (at a cumulative daunorubicin dose of 200mg/m(2)) than with cTnT (350mg/m(2)). In conclusion, our study has confirmed cTnT and cTnI as very sensitive and specific markers of anthracycline-induced cardiotoxicity. The troponins can become not only the bridge between the clinical and experimental studies of drug-induced cardiotoxicity but also the linkage between the preclinical experiments in vitro and in vivo.

Protectors against doxorubicin-induced cardiotoxicity: Flavonoids

Doxorubicin is a widely used anthracycline anticancer agent. Its use may cause cardiomyopathy: in fact, the development of cumulative dose-related cardiotoxicity forms the major limitation of clinical doxorubicin use. We therefore searched for protective agents that combine iron-chelating and oxygen radical-scavenging properties. Moreover, any novel protector should not interfere with the cytostatic activity of doxorubicin. After extensive in vitro screening we found that flavonoids could serve this purpose. In particular 7-monohydroxyethylrutoside almost completely protected against the negative inotropic action of doxorubicin in the electrically paced mouse left atrium model. In vivo it gave full protection at 500 mg/kg intraperitoneally against the doxorubicin-induced ST-interval lengthening in the ECG. Moreover, this protector did not influence the antitumor effect of doxorubicin either in vitro using the human ovarian cell lines A2780 and OVCAR-3 and the human breast cancer cell line MCF-7 or in vivo in A2780 and OVCAR-3 subcutaneous xenografts in nude mice. Comparison of various iron chelators suggest that iron, in contrast to the general assumption, might not play a crucial role in the oxidative stress-induced toxicity of doxorubicin. Moreover, incubation of vascular endothelial cells with doxorubicin produced overexpression of adhesion molecules, which could be inhibited by 7-monohydroxyethylrutoside. From a study in human volunteers, we conclude that an intravenous dose of 1500 mg/m2 of 7-monohydroxyethylrutoside is feasible and is safe to be investigated as protection against doxorubicin-induced cardiotoxicity.

Iron is not involved in oxidative stress-mediated cytotoxicity of doxorubicin and bleomycin

The anticancer drugs doxorubicin and bleomycin are well‐known for their oxidative stress‐mediated side effects in heart and lung, respectively. It is frequently suggested that iron is involved in doxorubicin and bleomycin toxicity. We set out to elucidate whether iron chelation prevents the oxidative stress‐mediated toxicity of doxorubicin and bleomycin and whether it affects their antiproliferative/proapoptotic effects.

Anthracycline toxicity to cardiomyocytes or cancer cells is differently affected by iron chelation with salicylaldehyde isonicotinoyl hydrazone

The clinical utility of anthracycline antineoplastic drugs is limited by the risk of cardiotoxicity, which has been traditionally attributed to iron‐mediated production of reactive oxygen species (ROS).

Cardioprotective effects of a novel iron chelator - pyridoxal 2- chlorobenzoyl hydrazone - in the rabbit model of daunorubicin-induced cardiotoxicity.

Iron chelation is the only pharmacological intervention against anthracycline cardiotoxicity whose effectiveness has been well documented both experimentally and clinically. In this study, we aimed to assess whether pyridoxal 2-chlorobenzoyl hydrazone (o-108, a strong iron chelator) can provide effective protection against daunorubicin (DAU)-induced chronic cardiotoxicity in rabbits. First, using the HL-60 leukemic cell line, it was shown that o-108 has no potential to blunt the antiproliferative efficacy of DAU. Instead, o-108 itself moderately inhibited cell proliferation. In vivo, chronic DAU treatment (3 mg/kg weekly for 10 weeks) induced mortality (33%), left ventricular (LV) dysfunction, a troponin T rise, and typical morphological LV damage. In contrast, all animals treated with 10 mg/kg o-108 before DAU survived without a significant drop in the LV ejection fraction (63.2 ± 0.5 versus 59.2 ± 1.0%, beginning versus end, not significant), and their cardiac contractility (dP/dtmax) was significantly higher than in the DAU-only group (1131 ± 125 versus 783 ± 53 kPa/s, p < 0.05), which corresponded with histologically assessed lower extent and intensity of myocardial damage. Although higher o-108 dose (25 mg/kg) was well tolerated when administered alone, in combination with DAU it led to rather paradoxical and mostly negative results regarding both cardioprotection and overall mortality. In conclusion, we show that shielding of free intracellular iron using a potent lipophilic iron chelator is able to offer a meaningful protection against chronic anthracycline cardiotoxicity. However, this approach lost its potential with the higher chelator dose, which suggests that iron might play more complex role in the pathogenesis of this disease than previously assumed.

New iron chelators in anthracycline-induced cardiotoxicity

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.

PYRIDOXAL ISONICOTINOYL HYDRAZONE (PIH) AND ITS ANALOGS AS PROTECTANTS AGAINST ANTHRACYCLINE-INDUCED CARDIOTOXICITY

The risk of cardiotoxicity is the main drawback of anthracycline antibiotics. However, these drugs remain among the most effective and frequently used anti cancer drugs. In this study we aimed to assess the cardioprotective effects of aroylhydrazone iron (FE) chelators: pyridoxal isonicotinoyl hydrazone (PIH) and its two analogs: salicyladehyde isonicotinoyl hydrazone (SIH) and pyridoxal o-chlorbenzoyl hydrazone (o-108). In rabbits, chronic treatment with daunorubicin (DAU) (3 mg/kg weekly for 10 weeks) induced mortality (33%) as well as left ventricular (LV) dysfunction. Co-administrations of PIH (25 mg/kg, i.p.), SIH hydrochloride [1 mg/kg, iv] as well as o-108 (10 mg/kg, i.p.), fully prevented premature deaths and most of the DAU-induced functional impairments were significantly suppressed. However, when 2- to 2.5-fold higher doses of the chelators were used, they led to rather paradoxical and mostly negative results regarding both cardioprotection and overall mortality.

Inhibition study of rabbit liver cytosolic reductases involved in daunorubicin toxication

Anthracycline cardiotoxicity represents the most unfavorable side effect of these highly efficient anticancer drugs. Several biotransformation enzymes have been described to contribute to their cardiotoxicity. Besides the activities of CYP450 isoforms which lead to the generation of reactive oxygen species (ROS), the cytosolic reductases have attracted attention nowadays. The reductases known to metabolize anthracyclines to C13-hydroxyanthracyclines are carbonyl reductase (CR, 1.1.1.184) and the aldo-keto reductases (AKR1C2, 1.3.1.20; AKR1A1, 1.1.1.2). Their participation in the formation of the toxic C13-hydroxymetabolite has been investigated in rabbit using diagnostic inhibitors of CR and AKR1C2. The kinetics and the type of reductase inhibition exerted by the two inhibitors have been described and it was found that CR was the main daunorubicin reductase at both optimal and physiological pH with the kinetic parameters for daunorubicin reduction of and The IC50 values for quercitrin and flufenamic acid were 5.45 ± 1.37 μM and 3.68 ± 1.58 μM, respectively. The inhibition was uncompetitive for both inhibitors and irreversible in the case of flufenamic acid.