Bisdioxopiperazine agent dexrazoxane (ICRF-187) has been the only effective and approved drug for prevention of chronic anthracycline cardiotoxicity. However, the structure-activity relationships (SAR) of its cardioprotective effects remain obscure owing to limited investigation of its derivatives/analogues and uncertainties about its mechanism of action. To fill these knowledge gaps, we tested the hypothesis that dexrazoxane derivatives exert cardioprotection via metal chelation and/or modulation of topoisomerase II beta (Top2B) activity in chronic anthracycline cardiotoxicity. Dexrazoxane was alkylated in positions that should not interfere with the metal-chelating mechanism of cardioprotective action; that is, on dioxopiperazine imides or directly on the dioxopiperazine ring. The protective effects of these agents were assessed in vitro in neonatal cardiomyocytes. All studied modifications of dexrazoxane molecule, including simple methylation, were found to abolish the cardioprotective effects. As this challenged the prevailing mechanistic concept and previously reported data, the two closest derivatives (GK-627 and GK-580) were thoroughly scrutinized in vivo using a rabbit model of chronic anthracycline cardiotoxicity. In contrast to dexrazoxane, both compounds failed to protect the heart as demonstrated by mortality, cardiac dysfunction, and myocardial damage parameters, although the pharmacokinetics and metal-chelating properties of their metabolites were comparable to those of dexrazoxane. The loss of cardiac protection was shown to correlate with their abated potential to inhibit and deplete Top2B both in vitro and in vivo. These findings suggest a very tight SAR between bisdioxopiperazine derivatives and their cardioprotective effects, and support Top2B as a pivotal upstream druggable target for effective cardioprotection against anthracycline cardiotoxicity.

Background: Anthracycline-induced heart failure has been traditionally attributed to direct iron-catalyzed oxidative damage. Dexrazoxane (DEX)—the only drug approved for its prevention—has been believed to protect the heart via its iron-chelating metabolite ADR-925. However, direct evidence is lacking, and recently proposed TOP2B (topoisomerase II beta) hypothesis challenged the original concept. Methods: Pharmacokinetically guided study of the cardioprotective effects of clinically used DEX and its chelating metabolite ADR-925 (administered exogenously) was performed together with mechanistic experiments. The cardiotoxicity was induced by daunorubicin in neonatal ventricular cardiomyocytes in vitro and in a chronic rabbit model in vivo (n=50). Results: Intracellular concentrations of ADR-925 in neonatal ventricular cardiomyocytes and rabbit hearts after treatment with exogenous ADR-925 were similar or exceeded those observed after treatment with the parent DEX. However, ADR-925 did not protect neonatal ventricular cardiomyocytes against anthracycline toxicity, whereas DEX exhibited significant protective effects (10–100 µmol/L; P <0.001). Unlike DEX, ADR-925 also had no significant impact on daunorubicin-induced mortality, blood congestion, and biochemical and functional markers of cardiac dysfunction in vivo (eg, end point left ventricular fractional shortening was 32.3±14.7%, 33.5±4.8%, 42.7±1.0%, and 41.5±1.1% for the daunorubicin, ADR-925 [120 mg/kg]+daunorubicin, DEX [60 mg/kg]+daunorubicin, and control groups, respectively; P <0.05). DEX, but not ADR-925, inhibited and depleted TOP2B and prevented daunorubicin-induced genotoxic damage. TOP2B dependency of the cardioprotective effects was probed and supported by experiments with diastereomers of a new DEX derivative. Conclusions: This study strongly supports a new mechanistic paradigm that attributes clinically effective cardioprotection against anthracycline cardiotoxicity to interactions with TOP2B but not metal chelation and protection against direct oxidative damage.

The bisdioxopiperazine topoisomerase IIβ inhibitor ICRF-193 has been previously identified as a more potent analog of dexrazoxane (ICRF-187), a drug used in clinical practice against anthracycline cardiotoxicity. However, the poor aqueous solubility of ICRF-193 has precluded its further in vivo development as a cardioprotective agent. To overcome this issue, water-soluble prodrugs of ICRF-193 were prepared, their abilities to release ICRF-193 were investigated using a novel UHPLC-MS/MS assay, and their cytoprotective effects against anthracycline cardiotoxicity were tested in vitro in neonatal ventricular cardiomyocytes (NVCMs). Based on the obtained results, the bis(2-aminoacetoxymethyl)-type prodrug GK-667 was selected for advanced investigations due to its straightforward synthesis, sufficient solubility, low cytotoxicity and favorable ICRF-193 release. Upon administration of GK-667 to NVCMs, the released ICRF-193 penetrated well into the cells, reached sufficient intracellular concentrations and provided effective cytoprotection against anthracycline toxicity. The pharmacokinetics of the prodrug, ICRF-193 and its rings-opened metabolite was estimated in vivo after administration of GK-667 to rabbits. The plasma concentrations of ICRF-193 reached were found to be adequate to achieve cardioprotective effects in vivo. Hence, GK-667 was demonstrated to be a pharmaceutically acceptable prodrug of ICRF-193 and a promising drug candidate for further evaluation as a potential cardioprotectant against chronic anthracycline toxicity.

The anthracycline (ANT) anticancer drugs such as doxorubicin or daunorubicin (DAU) can cause serious myocardial injury and chronic cardiac dysfunction in cancer survivors. A bisdioxopiperazine agent dexrazoxane has been developed as a cardioprotective drug to prevent these adverse events, but it is uncertain whether it is the best representative of the class. This study used a rabbit model of chronic ANT cardiotoxicity to examine another bisdioxopiperazine compound called GK-667, a water-soluble prodrug of ICRF-193, as a potential cardioprotectant. The cardiotoxicity was induced by DAU (3 mg/kg, i.v. weekly, 10 weeks), and GK-667 (1 or 5 mg/kg, i.v.) was administered before each DAU dose. The treatment with GK-667 was well tolerated and provided full protection against DAU-induced mortality and left ventricular (LV) dysfunction (determined by echocardiography and LV catheterization). Markers of cardiac damage/dysfunction revealed minor cardiac damage in the group co-treated with GK-667 in the lower dose, whereas almost full protection was achieved with the higher dose. This was associated with similar prevention of DAU-induced dysregulation of redox and calcium homeostasis proteins. GK-667 dose-dependently prevented p53-mediated DNA damage response in the LV myocardium not only in the chronic experiment but also after single DAU administration. These effects appear essential for cardioprotection, presumably because of the topoisomerase IIβ inhibition provided by its active metabolite ICRF-193. In addition, GK-667 administration did not alter the plasma pharmacokinetics of DAU and its main metabolite daunorubicinol in rabbits in vivo. Hence, GK-667 merits further investigation as a promising drug candidate for cardioprotection against chronic ANT cardiotoxicity.

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Angiotensin-converting enzyme inhibitors (ACEis) have been used to treat anthracycline-induced cardiac dysfunction, and they appear beneficial for secondary prevention in high-risk patients. However, it remains unclear whether they truly prevent anthracycline-induced cardiac damage and provide long-lasting cardioprotection. This study aimed to examine the cardioprotective effects of perindopril on chronic anthracycline cardiotoxicity in a rabbit model previously validated with the cardioprotective agent dexrazoxane with focus on post-treatment follow-up (FU). Chronic cardiotoxicity was induced by daunorubicin (3 mg/kg/week for 10 weeks). Perindopril (0.05 mg/kg/day) was administered before and throughout chronic daunorubicin treatment. After the completion of treatment, significant benefits were observed in perindopril co-treated animals, particularly full prevention of daunorubicin-induced mortality and prevention or significant reductions in cardiac dysfunction, plasma cardiac troponin T levels, morphological damage, and most of the myocardial molecular alterations. However, these benefits significantly waned during 3 weeks of drug-free FU, which was not salvageable by administering a higher perindopril dose. In the longer (10-week) FU period, further worsening of left ventricular function and morphological damage occurred together with heart failure-related mortality. Continued perindopril treatment in the FU period did not reverse this trend but prevented heart failure-related mortality and reduced the severity of the progression of cardiac damage. These findings contrasted with the robust long-lasting protection observed previously for dexrazoxane in the same model. Hence, in this study, perindopril provided only temporary control of anthracycline cardiotoxicity development, which may be associated with the lack of effects on anthracycline-induced and topoisomerase II beta-dependent DNA damage responses in the heart.