Synthesis And Analysis Of Novel Analogues Of Dexrazoxane And Its Open-Ring Hydrolysis Product For Protection Against Anthracycline Cardiotoxicity In Vitro And In Vivo

Cardiotoxicity is a serious drawback of anthracycline anti-cancer drugs and dexrazoxane is the only cardioprotective agent with clinically established efficacy. Iron-mediated oxidative stress is traditionally believed to be the primary cause of anthracycline cardiotoxicity, and dexrazoxane-induced cardioprotection is attributed to iron chelating properties of its open ring metabolite, ADR-925, which may inhibit the oxidative injury. However, dexrazoxane is also a catalytic inhibitor of topoisomerase II (TOP2), and the role of oxidative stress in clinically relevant forms of cardiotoxicity has increasingly been questioned. In this study, novel analogues of dexrazoxane (MK-15, ES-5) and ADR-925 (KH-TA4, JR-159) were synthesized, and evaluated in vitro and in vivo. When examined in the leukemic cell line, HL-60, these novel analogues did not interfere with the anti-proliferative action of daunorubicin. In contrast to dexrazoxane, they had no anti-cancer effect on their own and the changes in the chemical structure resulted in a loss of TOP2 inhibitory activity. Although some of the novel compounds showed significant anti-oxidant and iron chelating properties in vitro, they did not protect isolated cardiomyocytes and rabbits from daunorubicin-induced cardiotoxicity and heart failure. Importantly, dexrazoxane has been found to be a relatively weak intracellular iron chelator and it failed to protect the isolated cardiomyocytes from model oxidative injury induced by hydrogen peroxide. However, in contrast to all novel analogues, dexrazoxane induced depletion of the TOP2 beta isoform. This isoform is typical for terminally differentiated cells and its genetic deletion has been reported to overcome anthracycline-induced cardiotoxicity. Hence, TOP2 beta, rather than (or along with) iron chelation, may be a promising target for effective cardioprotection induced by bisdioxopiperazine agents

Molecular Remodeling of Left and Right Ventricular Myocardium in Chronic Anthracycline Cardiotoxicity and Post-Treatment Follow Up

<div><p>Chronic anthracycline cardiotoxicity is a serious clinical issue with well characterized functional and histopathological hallmarks. However, molecular determinants of the toxic damage and associated myocardial remodeling remain to be established. Furthermore, details on the different propensity of the left and right ventricle (LV and RV, respectively) to the cardiotoxicity development are unknown. Hence, the aim of the investigation was to study molecular changes associated with remodeling of the LV and RV in chronic anthracycline cardiotoxicity and post-treatment follow up. The cardiotoxicity was induced in rabbits with daunorubicin (3 mg/kg/week for 10 weeks) and animals were sacrificed either at the end of the treatment or after an additional 10 weeks. Daunorubicin induced severe and irreversible cardiotoxicity associated with LV dysfunction and typical morphological alterations, whereas the myocardium of the RV showed only mild changes. Both ventricles also showed different expression of ANP after daunorubicin treatment. Daunorubicin impaired the expression of several sarcomeric proteins in the LV, which was not the case of the RV. In particular, a significant drop was found in titin and thick filament proteins at both mRNA and protein level and this might be connected with persistent LV down-regulation of GATA-4. In addition, the LV was more affected by treatment-induced perturbations in calcium handling proteins. LV cardiomyocytes showed marked up-regulation of desmin after the treatment and vimentin was mainly induced in LV fibroblasts, whereas only weaker changes were observed in the RV. Remodeling of extracellular matrix was almost exclusively found in the LV with particular induction of collagen I and IV. Hence, the present study describes profound molecular remodeling of myocytes, non-myocyte cells and extracellular matrix in response to chronic anthracycline treatment with marked asymmetry between LV and RV.</p></div

Expression of thin filament proteins in chronic anthracycline cardiotoxicity and post-treatment follow up.

<p>Gene expression of cardiac α-actin (<b>A</b>, <b>E</b>) and cardiac troponin T (<b>C</b>, <b>G</b>) in the left and right ventricular myocardium, respectively. Western blot analysis of protein levels of cardiac α-actin in the left (<b>B</b>) and right (<b>F</b>) ventricle. ELISA determined abundance of the cardiac troponin T in the left (<b>D</b>) and right (<b>H</b>) ventricular myocardium. Statistical significances (One Way ANOVA/One Way ANOVA on Ranks according to data character, <i>P</i><0.05) within each study period (*) or between the treatment and post-treatment period (#). C - control group, DAU - daunorubicin group.</p

Morphology of the left and right ventricle in chronic anthracycline cardiotoxicity and post-treatment follow up.

<p><b>A,</b> Left ventricle, <b>B,</b> right ventricle. Masson’s blue trichrome; collagen fibers stained in blue. Scale bar - 20 µm. FU - follow up, DAU - daunorubicin group, DAU(1) and DAU-FU(1) - typical (almost normal) morphology in the RV of DAU-treated animals without severe congestion of the lungs, DAU(2) and DAU-FU(2) - pathological changes in the RV of DAU-treated animals with severe left-sided heart failure accompanied by severe congestion of the lungs.</p

Expression of titin and cardiac transcriptional factors in chronic anthracycline cardiotoxicity and post-treatment follow up.

<p>Gene expression of titin in the left (<b>A</b>) and right (<b>E</b>) ventricular myocardium. Quantification of titin isoforms (top T1 band and bottom T2 band) in the left (<b>B</b>) and right (<b>F</b>) ventricular myocardium. SYPRO Ruby stained 2% polyacrylamide gel strengthen with 0.75% agarose. Gene expression of cardiac transcriptional factors GATA-4 (<b>C</b>, <b>G</b>) and cardiac ankyrin repeat protein (CARP, Ankrd1, <b>D</b>, <b>H</b>) in the left and right ventricular myocardium, respectively. Statistical significances (One Way ANOVA/One Way ANOVA on Ranks according to data character, <i>P</i><0.05) within each study period (*) or between the treatment and post-treatment period (#) and within each study period in abundance of T1 titin isoform (<b>1</b>). C - control group, DAU - daunorubicin group.</p

Expression of vimentin in chronic anthracycline cardiotoxicity and post-treatment follow up.

<p>Expression of vimentin at the mRNA (<b>A</b>, <b>C</b>) and protein (<b>B</b>, <b>D</b>) level in the left and right ventricular myocardium, respectively. Statistical significances (One Way ANOVA/One Way ANOVA on Ranks according to data character, <i>P</i><0.05) within each period (*) or between the treatment and post-treatment period (#). Immunohistochemical detection of vimentin (brown) in the left (<b>E</b>) and right (<b>F</b>) ventricular myocardium. Nuclei counterstained with Gill’s hematoxylin. Scale bar - 20 µm. C - control group, DAU - daunorubicin group, FU - follow up.</p

Expression of light and heavy myosin chains in chronic anthracycline cardiotoxicity and post-treatment follow up.

<p>Gene expression of cardiac myosin light chain isoform 1 (<b>A</b>, <b>D</b>) and isoform 2 (<b>B</b>, <b>E</b>) in the left and right ventricular myocardium, respectively. Western blot analysis of myosin light chain isoform 2 in the left (<b>C</b>) and right (<b>F</b>) ventricular myocardium. Gene expression of myosin heavy chain isoform α (<b>G</b>, <b>J</b>) and isoform β (<b>H</b>, <b>K</b>) in the left and right ventricular myocardium, respectively. Quantification of myosin heavy chains in the left (<b>I</b>) and right (<b>L</b>) ventricular myocardium. SYPRO Ruby stained 2.8% polyacrylamide gel strengthen with 0.75% agarose. Statistical significances (One Way ANOVA/One Way ANOVA on Ranks according to data character, <i>P</i><0.05) within each study period (*) or between the treatment and post-treatment period (#). C - control group, DAU - daunorubicin group.</p

Markers of cardiac damage and myocardial dysfunction in chronic anthracycline cardiotoxicity and post-treatment follow.

<p>Expression of atrial natriuretic peptide in the left (<b>A</b>) and right (<b>B</b>) ventricle. <b>C</b>, Area under curve of plasma concentrations of cardiac troponin T determined by hypersensitive ELISA. Statistical significances (One Way ANOVA on Ranks, <i>P</i><0.05) within each study period (*). C - control group, DAU - daunorubicin group.</p

Extracellular matrix remodeling in chronic anthracycline cardiotoxicity and post-treatment follow up.

<p>Gene expression of collagen I (<b>A</b>, <b>F</b>), collagen III (<b>B</b>, <b>G</b>), collagen IV (<b>C</b>, <b>H</b>) and collagen VI (<b>D</b>, <b>I</b>) in the left and right ventricular myocardium, respectively. Biochemical analysis of collagenous proteins estimated by hydroxyproline determination in the left (<b>E</b>) and right (<b>J</b>) ventricular myocardium. Statistical significances (One Way ANOVA/One Way ANOVA on Ranks according to data character, <i>P</i><0.05) within each study period (*). Detection of collagen fibers (particularly of collagen type I) using Picrosirius red staining in the left (<b>K</b>) and right (<b>L</b>) ventricular myocardium. Scale bar - 20 µm. C - control group, DAU - daunorubicin group, FU - follow up.</p

Expression of desmin in chronic anthracycline cardiotoxicity and post-treatment follow up.

<p>Expression of desmin at the mRNA (<b>A</b>, <b>C</b>) and protein (<b>B</b>, <b>D</b>) level in the left and right ventricular myocardium, respectively. Statistical significances (One Way ANOVA/One Way ANOVA on Ranks according to data character, <i>P</i><0.05) within each study period (*) or between the treatment and post-treatment period (#). Immunohistochemical detection of desmin (brown) in the left (<b>E</b>) and right (<b>F</b>) ventricular myocardium. Nuclei counterstained with Gill’s hematoxylin. Scale bar - 20 µm. C - control group, DAU - daunorubicin group, FU - follow up.</p