Exploring the effects of topoisomerase II inhibitor XK469 on anthracycline cardiotoxicity and DNA damage.

Anthracyclines, such as doxorubicin (adriamycin), daunorubicin, or epirubicin, rank among the most effective agents in classical anticancer chemotherapy. However, cardiotoxicity remains the main limitation of their clinical use. Topoisomerase IIß has recently been identified as a plausible target of anthracyclines in cardiomyocytes. We examined the putative topoisomerase IIß selective agent XK469 as a potential cardioprotective and designed several new analogs. In our experiments, XK469 inhibited both topoisomerase isoforms (α and ß) and did not induce topoisomerase II covalent complexes in isolated cardiomyocytes and HL-60, but induced proteasomal degradation of topoisomerase II in these cell types. The cardioprotective potential of XK469 was studied on rat neonatal cardiomyocytes, where dexrazoxane (ICRF-187), the only clinically approved cardioprotective, was effective. Initially, XK469 prevented daunorubicin-induced toxicity and p53 phosphorylation in cardiomyocytes. However, it only partially prevented the phosphorylation of H2AX and did not affect DNA damage measured by Comet Assay. It also did not compromise the daunorubicin antiproliferative effect in HL-60 leukemic cells. When administered to rabbits to evaluate its cardioprotective potential in vivo, XK469 failed to prevent the daunorubicin-induced cardiac toxicity in either acute or chronic settings. In the following in vitro analysis, we found that prolonged and continuous exposure of rat neonatal cardiomyocytes to XK469 led to significant toxicity. In conclusion, this study provides important evidence on the effects of XK469 and its combination with daunorubicin in clinically relevant doses in cardiomyocytes. Despite its promising characteristics, long-term treatments and in vivo experiments have not confirmed its cardioprotective potential.

Hydrophobicity-enhanced ferritin nanoparticles for efficient encapsulation and targeted delivery of hydrophobic drugs to tumor cells.

Ferritin, a naturally occurring iron storage protein, has gained significant attention as a drug delivery platform due to its inherent biocompatibility and capacity to encapsulate therapeutic agents. In this study, we successfully genetically engineered human H ferritin by incorporating 4 or 6 tryptophan residues per subunit, strategically oriented towards the inner cavity of the nanoparticle. This modification aimed to enhance the encapsulation of hydrophobic drugs into the ferritin cage. Comprehensive characterization of the mutants revealed that only the variant carrying four tryptophan substitutions per subunit retained the ability to disassemble and reassemble properly. As a proof of concept, we evaluated the loading capacity of this mutant with ellipticine, a natural hydrophobic indole alkaloid with multimodal anticancer activity. Our data demonstrated that this specific mutant exhibited significantly higher efficiency in loading ellipticine compared to human H ferritin. Furthermore, to evaluate the versatility of this hydrophobicity-enhanced ferritin nanoparticle as a drug carrier, we conducted a comparative study by also encapsulating doxorubicin, a commonly used anticancer drug. Subsequently, we tested both ellipticine and doxorubicin-loaded nanoparticles on a promyelocytic leukemia cell line, demonstrating efficient uptake by these cells and resulting in the expected cytotoxic effect.

Examination of diverse iron-chelating agents for the protection of differentiated PC12 cells against oxidative injury induced by 6-hydroxydopamine and dopamine.

Labile redox-active iron ions have been implicated in various neurodegenerative disorders, including the Parkinson's disease (PD). Iron chelation has been successfully used in clinical practice to manage iron overload in diseases such as thalassemia major; however, the use of conventional iron chelators in pathological states without systemic iron overload remains at the preclinical investigative level and is complicated by the risk of adverse outcomes due to systemic iron depletion. In this study, we examined three clinically-used chelators, namely, desferrioxamine, deferiprone and deferasirox and compared them with experimental agent salicylaldehyde isonicotinoyl hydrazone (SIH) and its boronate-masked prochelator BSIH for protection of differentiated PC12 cells against the toxicity of catecholamines 6-hydroxydopamine and dopamine and their oxidation products. All the assayed chelating agents were able to significantly reduce the catecholamine toxicity in a dose-dependent manner. Whereas hydrophilic chelator desferrioxamine exerted protection only at high and clinically unachievable concentrations, deferiprone and deferasirox significantly reduced the catecholamine neurotoxicity at concentrations that are within their plasma levels following standard dosage. SIH was the most effective iron chelator to protect the cells with the lowest own toxicity of all the assayed conventional chelators. This favorable feature was even more pronounced in prochelator BSIH that does not chelate iron unless its protective group is cleaved in disease-specific oxidative stress conditions. Hence, this study demonstrated that while iron chelation may have general neuroprotective potential against catecholamine auto-oxidation and toxicity, SIH and BSIH represent promising lead molecules and warrant further studies in more complex animal models.

Primary prevention of chronic anthracycline cardiotoxicity with ACE inhibitor is temporarily effective in rabbits, but benefits wane in post-treatment follow-up.

Angiotensin-converting enzyme inhibitors (ACEis) have been used to treat anthracycline (ANT)-induced cardiac dysfunction, and they appear beneficial for secondary prevention in high-risk patients. However, it remains unclear whether they truly prevent ANT-induced cardiac damage and provide long-lasting cardioprotection. The present study aimed to examine the cardioprotective effects of perindopril on chronic ANT cardiotoxicity in a rabbit model previously validated with the cardioprotective agent dexrazoxane (DEX) with focus on post-treatment follow-up (FU). Chronic cardiotoxicity was induced by daunorubicin (DAU; 3 mg/kg/week for 10 weeks). Perindopril (0.05 mg/kg/day) was administered before and throughout chronic DAU treatment. After the completion of treatment, significant benefits were observed in perindopril co-treated animals, particularly full prevention of DAU-induced mortality and prevention or significant reductions in cardiac dysfunction, plasma cardiac troponin T (cTnT) 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 (HF)-related mortality. Continued perindopril treatment in the FU period did not reverse this trend but prevented HF-related mortality and reduced the severity of the progression of cardiac damage. These findings contrasted with the robust long-lasting protection observed previously for DEX in the same model. Hence, in the present study, perindopril provided only temporary control of ANT cardiotoxicity development, which may be associated with the lack of effects on ANT-induced and topoisomerase II ß (TOP2B)-dependent DNA damage responses in the heart.

Clinically Translatable Prevention of Anthracycline Cardiotoxicity by Dexrazoxane Is Mediated by Topoisomerase II Beta and Not Metal Chelation.

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.

Prodrug of ICRF-193 provides promising protective effects against chronic anthracycline cardiotoxicity in a rabbit model in vivo.

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 (DEX) 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. The present study used a rabbit model of chronic ANT cardiotoxicity to examine another bisdioxopiperazine compound called GK-667 (meso-(butane-2,3-diylbis(2,6-dioxopiperazine-4,1-diyl))bis(methylene)-bis(2-aminoacetate) hydrochloride), a water-soluble prodrug of ICRF-193 (meso-4,4'-(butan-2,3-diyl)bis(piperazine-2,6-dione)), as a potential cardioprotectant. The cardiotoxicity was induced by DAU (3 mg/kg, intravenously, weekly, 10 weeks), and GK-667 (1 or 5 mg/kg, intravenously) 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 tumor suppressor p53 (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 IIß (TOP2B) 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 (DAUol) in rabbits in vivo. Hence, GK-667 merits further investigation as a promising drug candidate for cardioprotection against chronic ANT cardiotoxicity.

Structure-Activity Relationship Study of Dexrazoxane Analogues Reveals ICRF-193 as the Most Potent Bisdioxopiperazine against Anthracycline Toxicity to Cardiomyocytes Due to Its Strong Topoisomerase IIß Interactions.

Cardioprotective activity of dexrazoxane (ICRF-187), the only clinically approved drug against anthracycline-induced cardiotoxicity, has traditionally been attributed to its iron-chelating metabolite. However, recent experimental evidence suggested that the inhibition and/or depletion of topoisomerase IIß (TOP2B) by dexrazoxane could be cardioprotective. Hence, we evaluated a series of dexrazoxane analogues and found that their cardioprotective activity strongly correlated with their interaction with TOP2B in cardiomyocytes, but was independent of their iron chelation ability. Very tight structure-activity relationships were demonstrated on stereoisomeric forms of 4,4'-(butane-2,3-diyl)bis(piperazine-2,6-dione). In contrast to its rac-form 12, meso-derivative 11 (ICRF-193) showed a favorable binding mode to topoisomerase II in silico, inhibited and depleted TOP2B in cardiomyocytes more efficiently than dexrazoxane, and showed the highest cardioprotective efficiency. Importantly, the observed ICRF-193 cardioprotection did not interfere with the antiproliferative activity of anthracycline. Hence, this study identifies ICRF-193 as the new lead compound in the development of efficient cardioprotective agents.

Development of water-soluble prodrugs of the bisdioxopiperazine topoisomerase IIß inhibitor ICRF-193 as potential cardioprotective agents against anthracycline cardiotoxicity.

The bisdioxopiperazine topoisomerase IIß 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.

Investigation of Structure-Activity Relationships of Dexrazoxane Analogs Reveals Topoisomerase IIß Interaction as a Prerequisite for Effective Protection against Anthracycline Cardiotoxicity.

Bisdioxopiperazine agent dexrazoxane (ICRF-187) has been the only effective and approved drug for prevention of chronic anthracycline cardiotoxicity. However, the structure-activity relationships (SARs) of its cardioprotective effects remain obscure owing to limited investigation of its derivatives/analogs 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 IIß (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. Because this challenged the prevailing mechanistic concept and previously reported data, the two closest derivatives [(±)-4,4'-(propane-1,2-diyl)bis(1-methylpiperazine-2,6-dione) and 4-(2-(3,5-dioxopiperazin-1-yl)ethyl)-3-methylpiperazine-2,6-dione] 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. SIGNIFICANCE STATEMENT: This study has revealed the previously unexpected tight structure-activity relationships of cardioprotective effects in derivatives of dexrazoxane, which is the only drug approved for the prevention of cardiomyopathy and heart failure induced by anthracycline anticancer drugs. The data presented in this study also strongly argue against the importance of metal-chelating mechanisms for the induction of this effect and support the viability of topoisomerase IIß as an upstream druggable target for effective and clinically translatable cardioprotection.

In vitro and in vivo investigation of cardiotoxicity associated with anticancer proteasome inhibitors and their combination with anthracycline.

Although proteasome inhibitors (PIs) are modern targeted anticancer drugs, they have been associated with a certain risk of cardiotoxicity and heart failure (HF). Recently, PIs have been combined with anthracyclines (ANTs) to further boost their anticancer efficacy. However, this raised concerns regarding cardiac safety, which were further supported by several in vitro studies on immature cardiomyocytes. In the present study, we investigated the toxicity of clinically used PIs alone (bortezomib (BTZ), carfilzomib (CFZ)) as well as their combinations with an ANT (daunorubicin (DAU)) in both neonatal and adult ventricular cardiomyocytes (NVCMs and AVCMs) and in a chronic rabbit model of DAU-induced HF. Using NVCMs, we found significant cytotoxicity of both PIs around their maximum plasma concentration (cmax) as well as significant augmentation of DAU cytotoxicity. In AVCMs, BTZ did not induce significant cytotoxicity in therapeutic concentrations, whereas the toxicity of CFZ was significant and more profound. Importantly, neither PI significantly augmented the cardiotoxicity of DAU despite even more profound proteasome-inhibitory activity in AVCMs compared with NVCMs. Furthermore, in young adult rabbits, no significant augmentation of chronic ANT cardiotoxicity was noted with respect to any functional, morphological, biochemical or molecular parameter under study, despite significant inhibition of myocardial proteasome activity. Our experimental data show that combination of PIs with ANTs is not accompanied by an exaggerated risk of cardiotoxicity and HF in young adult animal cardiomyocytes and hearts.