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Ponatinib is a third-generation tyrosine kinase inhibitor approved for Chronic Myelogenous Leukemia and Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia and it is the only tyrosine kinase inhibitor able to bind T315I mutation of BCR-ABL1 (Breakpoint Cluster Region and Abelson1) kinase protein. However, the cardiotoxic adverse reactions related to Ponatinib treatment can result in serious health problems and discontinuation of the therapy. The underlying mechanisms of Ponatinib-induced cardiotoxicity are not known. This study hypothesized that Ponatinib downregulates leptin and serpine-1 expressions and inhibits angiogenesis through the adipokine-induced p38 MAPK signaling pathway in mouse hearts. To evaluate this proposed pathway C57BL/6J mice were divided into two groups: control and ponatinib. After 14 days of the injections, mice were sacrificed and the heart samples were collected for histological analysis and evaluation of mRNA and protein expression levels. The RNA sequence analysis of heart samples was used to detect the main angiogenic markers affected by the treatment. Further analysis was done by Western Blot, RT-PCR, and immunohistochemistry. The heart function was assessed by echocardiography. Overall, the data indicated that the angiogenic response was inhibited by Ponatinib treatment through leptin and serpine-1-mediated p38 MAPK pathway. The anti-angiogenic response is an important underlying pathological mechanism that could lead to disruption of heart function and the echocardiography data confirmed that ponatinib-treated mice showed impaired heart function. Our study suggested that the potential underlying mechanism of Ponatinib-induced cardiotoxicity can be explained by serpine-1 and leptin-mediated angiogenic pathways.

Abstract: Sunitinib (Sutent®) is a multitargeted, small molecule receptor tyrosine kinase inhibitor used as an anti-cancer drug. It has increased the overall survival rate of metastatic renal cell carcinoma patients as well as the survival time of patients with pancreatic neuroendocrine tumors. Although the clinical use of sunitinib is a significant leap forward in the therapy of those cancers, its induction of cardiac toxicity in a substantial fraction of patients remains a critical problem. Sunitinib may cause hypertension, arrhythmias, drop of the left ventricular ejection fraction and congestive heart failure, fatal in some cases. These side effects are a frequent reason for interruption of its use. The mechanism(s) underlying sunitinib cardiotoxicity are not fully understood. Similar to other receptor tyrosine kinase inhibitors, it binds to a large number of cellular kinases, thus it can affect multiple cellular processes. In vivo, the pattern of toxicity is complex and unpredictable, with symptomatic heart failure sometimes observed early during treatment. The pattern of events preceding the onset of symptomatic cardiac dysfunction during treatment is not established. This represents a significant problem for the clinical diagnosis of cardiovascular complications before they become symptomatic. The identification and early detection of those events would be highly-beneficial for the clinical management of anti-cancer therapy with sunitinib. Positron Emission Tomography (PET) is recognized for its ability to probe metabolic and functional aspects of myocardial function. Under the working concept that heart failure can occur early during sunitinib treatment, and may be sustained by early myocardial metabolic and structural alterations, we performed a study with the objective of assessing the use of PET for the early detection of sunitinib-induced ardiotoxicity. For this, we established a model of cardiotoxicity in C57BL/6 male mice given 80mg/Kg/day of sunitinib or water, orally for 4 weeks. General and cardiac toxicity were monitored by biochemical, microscopical (H&E, immunofluorescence and electron microscopy) as well as gene expression analyses and blood pressure measurements. PET scans were performed weekly using [superscript 11]C-acetate and [superscript 18]F-FDG to evaluated the myocardial blood flow (MBF), myocardial oxidative metabolism through the quantification of oxygen consumption (MVO[subscript 2]), glucose uptake (K[subscript i]), myocardial metabolic rate of glucose (MMRG) and the left ventricular ejection fractions (LVEF). We found that sunitibib was cardiotoxic as revealed by histopathology, immunostaining and electron microscopy. Signs of inflammation and tissue remodeling were found by gene expression analyses and collagen staining. No hypertension or renal damage were detected on the study. FDG-PET revealed an early decrease of the LVEF, indicative of cardiac dysfunction, which developed into grade-2 heart failure by the end of the study. However, no signs of alterations in cardiac metabolism were uncovered by FDG- or [superscript 11]C-acetate-PET. Our results hint that the onset of sunitinib-induced contractile dysfunction may occur in the absence of hypertension or overt metabolic damage and call for further studies with longer treatments to clearly mark the onset of metabolic cardiotoxicity. // Résumé: Le sunitinib est un inhibiteur de tyrosine kinase qui est utilisée comme agent anticancéreux. Bien que l'utilisation clinique du sunitinib représente une percée significative pour le traitement de certains cancers, ce médicament s’avère cardiotoxique chez plusieurs patients, une situation qui est problématique. Le sunitinib peut provoquer une hypertension, des arythmies, une chute de la fraction d'éjection ventriculaire gauche et une insuffisance cardiaque congestive qui peut être fatale. Le mécanisme responsable de la cardiotoxicité de sunitinib n’est pas encore bien compris. Comme plusieurs autres inhibiteurs des récepteurs de la tyrosine kinase, il se lie à un grand nombre de kinases et peut affecter de nombreux processus cellulaires. In vivo, les mécanismes responsables de la toxicité sont complexes et imprévisibles et une insuffisance cardiaque est parfois observée tôt pendant le traitement. La séquence des évènements menant à l'apparition d’une dysfonction cardiaque pendant le traitement n’est pas connue. Cela pose un problème important pour le diagnostic de complications cardiovasculaires avant qu'elles ne deviennent symptomatiques. Une identification précoce de ces événements néfastes serait très bénéfique pour le suivi du traitement au sunitinib. La tomographie d'émission par positrons (TEP) est une méthode reconnue pour l’évaluation du métabolisme et de la fonctionnalité du myocarde. Selon notre hypothèse de travail, une insuffisance cardiaque peut survenir rapidement pendant le traitement au sunitinib, elle est l’expression d’altérations structurelles et métaboliques au niveau du myocarde; ces modifications se produisent tôt pendant le traitement. Nous avons effectué une étude pour évaluer la faisabilité d’utiliser l’imagerie TEP pour la détection précoce de la cardiotoxicité induite par le sunitinib. La première étape a été de développer un modèle de cardiotoxicité chez des souris. L’induction de la cardiotoxicité s’est faite par administration orale pour une période de quatre semaines, soit de sunitinib 80mg/Kg/jour ou d'eau pour les souris contrôles. Le suivi inclut la mesure de la pression sanguine, l’évaluation des altérations biochimiques, l’expression de certains gènes et un examen histologique du myocarde. Un suivi par imagerie TEP a été effectué chaque semaine avec du [indice supérieur 11]C-acétate et du [indice supérieur 18]F-FDG afin d'évaluer le flux sanguin myocardique (MBF), le métabolisme oxydatif du myocarde incluant la consommation d'oxygène (MVO2), l'absorption du glucose (K[indice inférieur i]), le taux métabolique oxydatif du glucose (MMRG) ainsi que la fraction d'éjection ventriculaire gauche (FEVG). Les résultats que nous avons obtenus par histopathologie, immunocoloration et microscopie électronique montrent que notre modèle est capable d’induire une cardiotoxicité. Nous avons également observé des évidences d'inflammation et de remodelage tissulaire à partir de l’étude de l'expression de certains gènes et de l’analyse de l’accumulation de collagène. Nous n’avons pas observé d’hypertension ni de lésions rénales. La TEP avec [indice supérieur 18]FDG a montré une diminution rapide de la FEVG, une indication d’une dysfonction cardiaque qui a été classée comme insuffisance cardiaque de grade 2 à la fin de l'étude. Cependant, aucun signe de modifications du métabolisme cardiaque n’a été mis en évidence par TEP/[indice supérieur 18]FDG- ou TEP/[indice supérieur 11]C-acétate. Nos résultats laissent penser que l'apparition de la dysfonction contractile induite par sunitinib peut se produire en l'absence d'hypertension ou de dommages métaboliques manifestes. De nouvelles études avec des traitements plus longs permettraient peut être de mieux définir le début de la cardiotoxicité métabolique.

<p>Subteratogenic and other low-level chronic exposures to toxicant mixtures are an understudied threat to environmental and human health. It is especially important to understand the effects of these exposures for contaminants, such as polycyclic aromatic hydrocarbons (PAHs) a large group of more than 100 individual compounds, which are important environmental (including aquatic) contaminants. Aquatic sediments constitute a major sink for hydrophobic pollutants, and studies show PAHs can persist in sediments over time. Furthermore, estuarine systems (namely breeding grounds) are of particular concern, as they are highly impacted by a wide variety of pollutants, and estuarine fishes are often exposed to some of the highest levels of contaminants of any vertebrate taxon. Acute embryonic exposure to PAHs results in cardiac teratogenesis in fish, and early life exposure to certain individual PAHs and PAH mixtures cause heart alterations with decreased swimming capacity in adult fish. Consequently, the heart and cardiorespiratory system are thought to be targets of PAH mixture exposure. While many studies have investigated acute, teratogenic PAH exposures, few studies have longitudinally examined the impacts of subtle, subteratogenic PAH mixture exposures, which are arguably more broadly applicable to environmental contamination scenarios. The goal of this dissertation was to highlight the later-life consequences of early-life exposure to subteratogenic concentrations of a complex, environmentally relevant PAH mixture.</p><p>A unique population of Fundulus heteroclitus (the Atlantic killifish or mummichog, hereafter referred to as killifish), has adapted to creosote-based polycyclic aromatic hydrocarbons (PAHs) found at the Atlantic Wood Industries (AW) Superfund site in the southern branch of the Elizabeth River, VA, USA. This killifish population survives in a site heavily contaminated with a mixture of PAHs from former creosote operations. They have developed resistance to the acute toxicity and teratogenic effects caused by the mixture of PAHs in sediment from the site. The primary goal of this dissertation was to compare and contrast later-life outcomes of early-life, subteratogenic PAH mixture exposure in both the Atlantic Wood killifish (AW) and a naïve reference population of killifish from King’s Creek (KC; a relatively uncontaminated tributary of the Severn River, VA). Killifish from both populations were exposed to subteratogenic concentrations of a complex PAH-sediment extract, Elizabeth River Sediment Extract (ERSE), made by collecting sediment from the AW site. Fish were reared over a 5-month period in the laboratory, during which they were examined for a variety of molecular, physiological and behavioral responses. </p><p>The central aims of my dissertation were to determine alterations to embryonic gene expression, larval swimming activity, adult behavior, heart structure, enzyme activity, and swimming/cardiorespiratory performance following subteratogenic exposure to ERSE. I hypothesized that subteratogenic exposure to ERSE would impair cardiac ontogenic processes in a way that would be detectable via gene expression in embryos, and that the misregulation of cardiac genes would help to explain activity changes, behavioral deficits, and later-life swimming deficiencies. I also hypothesized that fish heart structure would be altered. In addition, I hypothesized that the AW killifish population would be resistant to developmental exposures and perform normally in later life challenges. To investigate these hypotheses, a series of experiments were carried out in PAH-adapted killifish from Elizabeth River and in reference killifish. As an ancillary project to the primary aims of the dissertation, I examined the toxicity of weaker aryl hydrocarbon receptor (AHR) agonists in combination with fluoranthene (FL), an inhibitor of cytochrome P4501A1 (CYP1A1). This side project was conducted in both Danio rerio (zebrafish) and the KC and AW killifish.</p><p>Embryonic gene expression was measured in both killifish populations over an ERSE dose response with multiple time points (12, 24, 48, and 144 hours post exposure). Genes known to play critical roles in cardiac structure/development, cardiac function, and angiogenesis were elevated, indicating cardiac damage and activation of cardiovascular repair mechanisms. These data helped to inform later-life swimming performance and cardiac histology studies. Behavior was assessed during light and dark cycles in larvae of both populations following developmental exposure to ERSE. While KC killifish showed activity differences following exposure, AW killifish showed no significant changes even at concentrations that would cause overt cardiac toxicity in KC killifish. Juvenile behavior experiments demonstrated hyperactivity following ERSE exposure in KC killifish, but no significant behavioral changes in AW killifish. Adult swimming performance via prolonged critical swimming capacity (Ucrit) demonstrated performance costs in the AW killifish. Furthermore, swimming performance decline was observed in KC killifish following exposure to increasing dilutions of ERSE. Lastly, cardiac histology suggested that early-life exposure to ERSE could result in cardiac structural alteration and extravasation of blood into the pericardial cavity.</p><p>Responses to AHR agonists resulted in a ranking of relative potency for agonists, and determined which agonists, when combined with FL, caused cardiac teratogenesis. These experiments showed interesting species differences for zebrafish and killifish. To probe mechanisms responsible for cardiotoxicity, a CYP1A-morpholino and a AHR2-morpholino were used to mimic FL effects or attempt to rescue cardiac deformities respectively. Findings suggested that the cardiac toxicity elicited by weak agonist + FL exposure was likely driven by AHR-independent mechanisms. These studies stand in contrast to previous research from our lab showing that moderate AHR agonist + FL caused cardiac toxicity that can be partially rescued by AHR-morpholino knockdown.</p><p>My findings will form better characterization of mechanisms of PAH toxicity, and advance our understanding of how subteratogenic mixtures of PAHs exert their toxic action in naïve killifish. Furthermore, these studies will provide a framework for investigating how subteratogenic exposures to PAH mixtures can impact aquatic organismal health and performance. Most importantly, these experiments have the potential to help inform risk assessment in fish, mammals, and potentially humans. Ultimately, this research will help protect populations exposed to subtle PAH-contamination.</p> / Dissertation

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Introduction The discovery of Doxorubicin in the 1960s has drastically improved the survival rates of cancer patients, however, its success is limited by dose-dependent cardiotoxicity. While much of the literature has focused on acute cardiotoxicity which is minor and generally reversible, chronic cardiotoxicity poses a serious threat to cancer survivors since it can lead to dilative cardiomyopathy, congestive heart failure and even death. The mechanisms that contribute to cardiotoxicity are still a matter of controversy, however, oxidative stress-induced myocardial damage and apoptosis are thought to be the major role players. Reperfusion injury, also characterized by oxidative stress and apoptosis, occurs as a result of restoring blood flow to an ischemic heart. Fortunately, pre- and post-conditioning are techniques employed to minimize this damage and are thought to do so by activating the reperfusion injury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) pathways. The RISK pathway involves the pro-survival kinases, Erk1/2 and Akt, while the SAFE pathway, triggered by TNF-α, involves Jak2 and STAT3. Since both reperfusion injury and Doxorubicin-induced cardiotoxicity share similar characteristics, this study aimed to determine whether the RISK and SAFE pathways are activated in response to long-term Doxorubicin treatment. Furthermore, this study aimed to determine whether TNF-α is produced during treatment, since its role in Doxorubicin-induced cardiotoxicity is still relatively unknown. Methods H9c2 cardiomyocytes and differentiated C2C12 myotubes were treated daily with increasing concentrations of Doxorubicin for a total of 120 hours. Cell viability, apoptosis and necrosis were assessed using the MTT, Caspase-Glo® 3/7 and lactate dehydrogenase assays respectively. TNF-α production was measured using Quantikine® ELISA kits and various assays were used to assess oxidative stress, anti-oxidant capacity and anti-oxidant status. The protein expression of the RISK and SAFE pathways were analysed by western blotting using both phospho-specific and total antibodies. Results and Discussion Treatment with Doxorubicin caused a time- and dose-dependent decrease in cell viability in both cell lines and this was accompanied by an increase in apoptosis. In the H9c2 cardiomyocytes, treatment with 0.2 μM Doxorubicin yielded significant levels of TNF-α after 120 hours and we can speculate that these low levels partially protected the cells from the toxic effects of Doxorubicin by activating the SAFE pathway, since both Jak2 and STAT3 were phosphorylated at this concentration. Treatment with 1 μM Doxorubicin caused a larger and biphasic pattern of TNF-α release, which may have then contributed to the decrease in cell viability, since the SAFE pathway was not activated at this concentration. Akt was phosphorylated during the first 72 hours of treatment with the low dose of Doxorubicin, but chronic treatment prevented this phosphorylation. While Erk1/2 was not phosphorylated at all at the low dose of Doxorubicin, neither Akt nor Erk1/2 was phosphorylated at the high dose and their inhibition may contribute to the cardiotoxic effects of Doxorubicin. In the C2C12 myotubes, a significant amount of TNF-α was produced after 120 hours of treatment with the low dose of Doxorubicin. Treatment with the high dose of Doxorubicin induced significant TNF-α production at every time point. While STAT3 was phosphorylated at the serine residue after treatment with the low dose of Doxorubicin, treatment with the high dose induced phosphorylation at the tyrosine residue in a time-dependent manner. p-Jak2 expression was significantly down-regulated at both concentrations of Doxorubicin, suggesting that STAT3 proteins can by-pass activation by Jak2. The Erk1/2 leg of the RISK pathway was also not activated for the majority of the treatment period, however, p-Akt expression was increased at the low concentration of Doxorubicin relative to total Akt expression. Conclusion These observations indicate that treatment with Doxorubicin causes a severe, dose-dependent loss in viability which is likely to mediated by high concentrations of TNF-α (induced by high concentrations of Doxorubicin) and down-regulation of protective signaling pathways. TNF-α may confer partial protection at low concentrations by activating the SAFE pathway. However, activation of the SAFE pathway could not provide sufficient protection from Doxorubicin, most probably because the RISK pathway was not simultaneously activated. Our results also clearly highlight the differences between acute and chronic treatment since a single high dose of Doxorubicin produced vastly different responses to cumulative treatment with a low dose. Before one can extrapolate these results into the clinical setting, further research is required to provide a better understanding of the RISK and SAFE pathways and whether stimulation thereof will provide a protective effect. In addition, although our study has shown that TNF-α is produced in response to Doxorubicin treatment, its true role, whether beneficial or detrimental, remains to be determined. / AFRIKAANSE OPSOMMING: Inleiding Die ontdekking van Doksorubisien (DOKS) in die 1960’s het die oorlewingsyfer van kankerpasiënte drasties verhoog, maar DOKS-gebruik gaan egter ook gepaard met dosis-afhanklike kardiotoksisiteit. Terwyl die literatuur grootliks fokus op akute kardiotoksisiteit, wat minimaal en algemeen omkeerbaar is, hou kroniese kardiotoksisiteit ‘n ernistige bedreiging vir kankeroorlewendes in, aangesien dit kan lei tot dilatiewe kardiomiopatie, kongestiewe hartversaking, en selfs dood. Die spesfikieke meganismes wat bydrae tot kardiotoksisiteit is tans steeds onbekend, maar oksidatiewe stres-geinduseerde miokardiale skade en apoptose word beskou as hoof bydraende faktore. Reperfussie skade, ook gekaraktiseer deur die teenwoordigheid van oksidatiewe stres en apoptose, kom voor as gevolg van die herstel van bloedtoevoer na ‘n isgemiese hart. Om die skade te minimaliseer word voor- en nakondisionerings tegnieke geïmplimenteer wat die RSHK (Reperfussie Skade Herwinnings Kinase) en OAFV (Oorlewerings Aktiverings Faktor Versterkings)-weë aktiveer. Die RSHK weg maak gebruik van pro-oorlewings kinases Erk1/2 en Akt, terwyl die TNF-α geaktiveerde OAFV weg Jak2 en STAT3 betrek. Aangesien beide reperfussie skade en DOKS-geinduseerde kardiotoksisiteit soortgelyke eienskappe deel, is die doel van hierdie studie om vas te stel of die RSHK en OAFV-weë geaktiveer word in langtermyn DOKS behandeling. Boonop is nog ‘n doel van hierdie studie om vas te stel of TNF-α geproduseer word tydens behandeling, aangesien die rol daarvan in DOKS-geinduseerde kardiotoksisiteit steeds onbekend is. Metodes H9c2 kardiomiosiet en gedifferensieerde C2C12 miobuise was daagliks behandel met toenemende konsentrasies van Dox vir 120 ure. Die effekte van DOKS op sel lewensvatbaarheid, apoptose en nekrose is onderskeidelik ondersoek deur middel van die MTT, Caspase-Glo® 3/7 en LDH toetse. TNF-α produksie is bepaal deur van die Quantikine® toets gebruik te maak, en verskeie metodes is gebuik om die oksidatiewe stres, anti-oksidantkapasiteit en anti-oksidantstatus te bepaal. Die proteïenuitdrukking van die RSHK (Erk1/2 en Akt) en OAFV (Jak2 en STAT3) weë was ontleed deur middel van westerse afklattingstegniek deur van beide fosfospesifieke en totale teenliggaampies gebruik te maak. Resultate en Bespreking Behandeling met DOKS het ‘n tyd en dosis-afhanklike afname in sel lewensvatbaarheid in beide sellyne veroorsaak, wat gepaard gegaan het met ‘n toename in apoptose. In die H9c2 kardiomiosiete, het ‘n lae DOKS dosisbehandeling (0.2 μM) betekenisvolle vlakke van TNF-α na 120 uur opgelewer en ons kan spekuleer dat hierdie lae vlakke gedeeltelik die selle van die toksiese effekte van DOKS deur die aktivering van die OAFV weg beskerm het omrede beide Jak2 en STAT3 by hierdie konsentrasie gefosforileer is. Die hoë DOKS dosis (1 μM) het ‘n groter en bifasiese patroon van TNF-α vrystelling vertoon, wat kon bydra tot die DOKS-geinduseerde afname in sel lewensvatbaarheid. Akt is gedurende die eerste 72 uur van behandeling gefosforileer met die lae DOKS dosis, maar kroniese behandeling het hierdie fosforilering verhoed. Terwyl Erk1/2 glad nie gefosforileer is by die lae DOKS dosis nie, is nie Akt of Erk1/2 by die hoë dosis gefosforileer nie, en kan hierdie inhibering bydrae tot die kardiotoksiese effekte van DOKS. In die C2C12 miobuise, is ‘n betekenisvolle hoeveelheid TNF-α na 120 uur van behandeling geproduseer by die lae DOKS dosis. Behandeling met die hoë DOKS dosis het betekenisvolle TNF-α produksie geinduseer by elke tydspunt. Terwyl STAT3 gefosforileer is by die serienresidu na behandeling met die lae DOKS dosis, het behandeling met die hoë dosis fosforilering by die tirosienresidu op ’n tydsafhanklike wyse plaasgevind. p-Jak2 uitdrukking was betekenisvol verminder by beide DOKS konsentrasies, wat aanduidend is dat die STAT3 proteïene nie geaktiveer hoef te word deur Jak2 nie. Die Erk1/2 been van die RSHK weg is ook nie geaktiveer gedurende die oorhoofse behandelingstydperk nie, alhoewel, p-Akt wel uitgedruk is by die lae konsentrasie van DOKS relatief tot die totale Akt uitdrukking. Gevolgtrekkings Die resultate van hierdie studie toon dat DOKS-behandeling tot ‘n dosis-afhanklike verlies in sel lewensvatbaarheid lei. Hierdie effek word waarskynlik bemiddel deur die teenwoordigheid van hoë konsentrasies TNF-α, en ook die afregulering van die beskermende seinweë. TNF-α kan moontlik gedeeltelike beskerming bied by lae konsentrasies deur aktivering van die OAFV weg. Die aktivering van die OAFV weg kon egter nie voldoende beskerming teen DOKS bied nie; moontlik as gevolg van die afwesigheid van die gelyktydige RSHK weg aktivering. Ons resultate vertoon die verskille tussen die akute en kronies behandeling aangesien ‘n enkele hoë-dosis van DOKS, in vergelyking met ‘n kumulatiewe lae-dosis, grootliks verskillende resultate opgelewer het. Voordat hierdie resultate klinies verder ondersoek kan word is verdere navorsing nodig om TNF-α en die RSHK en OAFV-weë beter te verstaan, en om vas te stel of stimulering van hierdie seinoordragpaaie ‘n beskermende effek teweeg sal bring.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Lucie Gavurová Supervisor: PharmDr. Anna Jirkovská, PhD. Title of diploma thesis: Antiproliferative and cardioprotective activity of novel dexrazoxane analogues Anthracycline antibiotics (such as daunorubicin, doxorubicin or epirubicin) forms the basis of anticancer therapy in many hematological malignancies and solid tumors. However, their clinical use is limited by adverse effects. The most serious of these effects is chronic form of anthracycline-induced cardiotoxicity. Dexrazoxane is the only one clinically approved cardioprotective agent against anthracycline cardiotoxicity so far. Despite its well-evidenced cardioprotective effects, dexrazoxane use is very limited due to its possible adverse effects. The the synthesis of novel analogs of might contribute to understanding of the relationship between structure and effects of dexrazoxane. Finally, this approach could lead to the synthesis of structure with better pharmacological properties. The aim of this diploma thesis was to assess the antiproliferative activity of novel analogues of dexrazoxane (JR-281B, JR-311, JR-306A, JR-306B, JR-232 and JR-312B), and the study of the influence on the antiproliferative effect of anthracyclines....

La doxorubicine (DOX) est un médicament très efficace utilisé en chimiothérapie pour traiter de nombreuses tumeurs. Cependant, son utilisation clinique est limitée par des effets secondaires cardiotoxiques sévères causés par une augmentation de production de radicaux libres de l'oxygène (RLO) et de l'apoptose. De surcroit, une chimiothérapie pourrait être indiquée alors qu'une ischémie cardiaque est déjà installée. Dans cette thèse, nous avons donc examiné au niveau du cœur isolé de lapin et des cardiomyocytes en culture; d'une part, la cardiotoxicité de la DOX dans les conditions de l'ischémie/reperfusion (IR) myocardique et d'autre part, la cardioprotection assurée dans ces conditions par un antioxydant naturel puissant, le safran (SAF).D'abord nous avons procédé à une extraction méthanol-Eau des stigmates du safran cultivé au Liban. Une analyse par HPLC des principaux constituants a été réalisée afin de s'assurer de la qualité des extraits, suivie par des tests (xanthine/xanthine oxydase et électrolyse) comme sources de RLO pour rechercher le pouvoir antioxydant de ces extraits.La toxicité du SAF a été évaluée sur des cœurs isolés de lapin selon la technique de Langendorff et la dose thérapeutique optimale établie est de 10 μg/mL. Ensuite, la toxicité aigüe de la DOX a été testée sur des cœurs isolés sains traités ou non par la DOX et le SAF en comparaison avec des cœurs soumis à l'électrolyse ou à une période d'ischémie globale. La toxicité chronique de la DOX a été évaluée chez des lapins traités pendant 21 jours avec des extraits de SAF (par voie orale) et la DOX (par voie ip). Les cœurs ont été par suite isolés et soumis à une ischémie globale. D'après les études cardiodynamiques, anatomopathologiques et biochimiques; il apparaît que les extraits de safran protègent le cœur de la toxicité aigüe et chronique de la doxorubicine et assurent une protection optimale lors de la reperfusion. De plus, le SAF a inhibé l'apoptose induite par IR et DOX en activant la voie de survie AKT/mTOR, en augmentant le niveau de Troponine T et en inhibant l'expression de p-P38 tel que montré par les analyses de Western blot. Le safran contribuerai donc à améliorer nettement le retour des fonctions myocardiques vers la normale suite à l'IR et au traitement par la DOX.Les effets de la DOX et du SAF ont aussi été étudiés dans les cardiomyocytes H9c2 soumis à une IR simulée. Les effets des différents traitements ont été évalués par les tests MTT, LDH, Western blot, TMRM et caspases. Nos données prouvent que DOX et IR diminuent significativement la viabilité cellulaire ; inhibent la voie RISK ; réduisent les protéines contractiles ; activent les caspases 3 et provoquent la perte du potentiel de membrane mitochondrial. Ces effets ont été remarquablement inhibés par le traitement avec le SAF à la reperfusion. Les résultats suggèrent que le SAF a un effet protecteur contre la DOX et l'IR par un mécanisme impliquant la réduction de l'apoptose cardiaque, des RLO et du stress oxydatif. La cardioprotection du SAF est médiée par l'activation des voies de survie AKT/P70S6K et ERK ; augmentation de l'expression des protéines contractiles ; inhibition de la perméabilité des pores de transition mitochondrial et diminution de l'activité des caspases 3. En outre, nous avons montré que l'effet protecteur du SAF serait attribué à la crocine, son métabolite actif principal. Pris ensemble, nos résultats indiquent que la DOX exacerbe les lésions myocardiques de l'IR. Le traitement par le SAF à la reperfusion pourrait exercer des effets cardioprotecteurs contre la toxicité induite par la DOX via son activité antioxydante. Ainsi, nos travaux sur le safran se révèlent fort intéressants dans le développement d'une stratégie thérapeutique antioxydante potentielle afin de limiter la toxicité cardiaque de la DOX, ouvrant la voie à de futurs essais cliniques. / Doxorubicin (DOX) is a highly active chemotherapeutic drug used to treat many tumors. However, its clinical use is limited by severe cardiotoxic side effects. Previous studies have shown that DOX-Induced cardiotoxicity is mainly mediated through increased oxidative stress and apoptosis. In addition, DOX could be indicated in case of pre-Established myocardial ischemia. In this thesis we investigated, in isolated rabbit heart and cultured cardiomyocytes, the cardiotoxic effects of DOX in conditions of ischemia/reperfusion (IR). We also studied whether saffron (SAF), a natural compound that showed potent antioxidant activity, could exert cardioprotection against DOX and IR toxicity.First, the extraction of saffron stigmas grown in Lebanon was performed using methanol-Water mixture. HPLC analysis of the main components (safranal and crocines) was used to ensure the quality of the extracts; followed by tests (xanthine/xanthine oxidase and electrolysis) as sources of free radicals to determine the antioxidant activity of the sample.The toxicity of SAF was evaluated on isolated rabbit hearts according to Langendorff technique and the optimal therapeutic dose established is 10 μg/mL. Then, the acute toxicity of DOX was tested on isolated hearts treated with or without DOX and SAF compared with hearts subjected to electrolysis or a period of global ischemia (I 30 min/R 40 min). The chronic toxicity of DOX was evaluated in rabbits treated for 21 days with SAF extracts (orally) and DOX (ip). The hearts were afterward isolated and subjected to global ischemia (I 30 min/R 30 min). According to cardiodynamic, pathological and biochemical studies, it appears that saffron extracts protect the heart from the acute and chronic toxicity of doxorubicin, and ensure optimal protection during reperfusion. Furthermore, SAF inhibited IR and DOX-Induced apoptosis by activating the AKT/mTOR survival pathway, increasing Troponine T and inhibiting p-P38 expression as shown by Western blot analysis. Saffron could therefore significantly improve myocardial function to normal state following IR and DOX treatment.The effects of DOX and SAF were also investigated in H9c2 cardiomyocytes subjected to simulated IR injury. We evaluated the effects of the treatments by MTT assay, LDH release measurements and Western blot analysis. Apoptosis was assessed by Hoechst 33258 staining, TMRM probes and caspase activity. The results proved that DOX exacerbates cardiac cell injury when an underlying IR heart condition is present. Our data showed that DOX and IR significantly decreased cardiac cells viability; inhibited RISK cardioprotective pathway; reduced contractile proteins (α-Actinine, Troponine C and MLC); increased caspase 3 expression and induced loss of mitochondrial membrane potential. These effects were remarkably inhibited by treatment with SAF (10 μg/mL) at reperfusion. Our data suggested that SAF has a protective effect against DOX and IR induced cardiac apoptosis through a mechanism involving scavenging free radicals and reducing oxidative stress. SAF cardioprotection is mediated by activating survival pathways including AKT/P70S6K and ERK; increasing the expression of contractile proteins; inhibiting of mitochondrial permeability transition pore and decreasing caspase 3 activity. In addition, studies showed that protective activities of SAF belong to crocin, its major active metabolite.Taken together, our findings indicate that DOX exacerbates IR myocardial injury. The SAF treatment at reperfusion could exert cardioprotective effects against DOX induced toxicity by its antioxidant activity. Thereby, saffron offers a potential novel antioxidant therapeutic strategy to counteract doxorubicin cardiotoxicity, paving the way for future clinical trials.

Antraciclinas estão entre os mais eficazes quimioterápicos contra o cancer. São fármacos glycosídicos compostos pelo carboidrato daunosamina ligado a uma aglicona hidróxi antraquinona, e atuam por intercalação ao DNA, geração de estresse oxidative e envenenamento de topoisomerase II. Apesar de sua utilidade terapêutica, multirresistência e cardiotoxicidade grave são importantes limitações decorrentes do tratamento com antraciclinas, estimulando a descoberta de novos análogos, por exemplo através de glicodiversificação. Este trabalho objetivou explorar azido glicosídeos, a serem combinados com agliconas de antraciclinas para gerar novos glicosídeos. Em uma estratégia semi-sintética, daunorrubicinona e doxorrubicinona protegida foram glicosiladas com doadores 2-azido glucosídicos e -galactosídicos, além de glicais. Uma varredura de metodologias de glicosilação envolveu cloretos, imidatos e tioglicosídeos, sendo os promotores com melhores rendimentos HgO/HgBr2 (4-52%) e TMSOTf (38-41%); para glucais e galactais, catalisadores de Au(I) and Cu(I) forneceram moderados rendimentos (15-46%), mas o sistema mais eficiente foi o organocatalisador de tiouréia e ácido fosfórico (18-95%). A clivagem dos grupos de proteção foi desafiadora, dificultando e atrasando a obtenção dos glicosídeos livres. Mediante desproteção, os glicosídeos obtidos incluíram glucosídeo 49 (13%), 2-azido glucosídeo 51 (34%), 2-desóxi glucosídeo 58 (11%) e 2-desóxi galactosídeo 61 (85%), todos com o esqueleto de daunorrubicina. Em ensaios de proliferação celular, os glicosídeos 61? e 61? foram testados em linhagens de células tumorais humanas HeLa, MDA-MB-231 e MCF-7 e um modelo de células sadias (HDF), com IC50 na faixa de 27.1 a 74.6 ?M para o anômero ?, e superior a 250 ?M para o anômero ?. Estudos preliminares com cardiomiócitos humanos derivados de células-tronco induzidas foram inconclusivos para estabelecer um modelo experimental de toxicidade cardíaca. / Anthracyclines are ranked among the most effective chemotherapeutics against cancer. They are glycoside drugs comprised by the aminosugar daunosamine linked to a hydroxyanthraquinone aglycone, and act by DNA-intercalation, oxidative stress generation and topoisomerase II poisoning. Regardless of their therapeutic value, multidrug resistance and severe cardiotoxicity are important limitations arising from anthracycline treatment, prompting the discovery of novel analogues, for instance through glycodiversification. This work aimed to exploit azido glycosides, to be combined with anthracycline aglycone and generate novel glycosides. In a semi-synthesis approach, both daunorubicinone and protected doxorubicinone were glycosylated with conveniently functionalised 2-azido glucosyl and galactosyl donors, as well as glycals. A screening of glycosylation protocols involved glycosyl chlorides, imidates and thioglycosides with the most successful promoters being HgO/HgBr2 (4-52% yield) and TMSOTf (38-41%); for glucals and galactals, Au(I) and Cu(I) catalysts gave moderate yields (15-46%), but thiourea-phosphoric acid was the most efficient catalyst system (18-95%). Cleavage of protecting groups proved challenging, hampering and delaying the obtention of free glycosides. Upon deprotection, the glycosides obtained included glucoside 49 (13%), 2-azido glucoside 51 (34%), 2-deoxyglucoside 58 (11%), and 2-deoxygalactoside 61 (85%), all with the daunorubicin scaffold. In cell proliferation assays, glycosides 61? and 61? were tested against human cancer cell lines HeLa, MDA-MB-231 and MCF-7 and a model of healthy cells (HDF), with IC50 in the range of 27.1 to 74.6 ?M for the ? anomer, and higher than 250 ?M for the ? anomer. Preliminary studies with human cardiomyocytes derived from induced pluripotent stem cells were inconclusive to establish a cardiac toxicity experimental model.

INTRODUÇÃO: Cardiotoxicidade é frequentemente associada ao uso crônico de doxorubicina (DOX) podendo levar a cardiomiopatia e insuficiência cardíaca. A identificação de miRNAs cardiotoxicidade-específicos e seu potencial como biomarcadores poderia fornecer uma ferramenta prognostica valiosa e uma potencial área de intervenção. METODOLOGIA: Este é um sub-estudo do ensaio clínico prospectivo \"Efeito do Carvedilol na Prevenção da Cardiotoxicidade Induzida por Quimioterapia\" (ensaio CECCY) no qual incluiu 56 pacientes do sexo feminino (idade 49.9±3.3) provenientes do braço placebo. Os pacientes incluídos foram submetidos à quimioterapia com DOX seguido por taxanos. Troponina cardiaca I (cTnI), fração de ejeção do ventrículo esquerdo (FEVE) e microRNAs foram mensurados periodicamente. RESULTADOS: Os níveis circulantes de miR-1, -133b, -146a e -423-5p aumentaram significativamente durante o tratamento (18.6, 11.5, 10.6 e 12.1-vezes respectivamente; p < 0.001) enquanto miR-208a e -208b foram indetectáveis. cTnI aumentou de 6.6 ± 0.3 para 46.7 ± 5.5 pg/ml (p < 0.001) enquanto FEVE tendeu a diminuir de 65.3±0.5 para 63.8±0.9 (p=0.053) após 12 meses; deis pacientes (17.9%) desenvolveram cardiotoxicidade. miR-1 foi associado a mudanças na FEVE (r2=0.363, p < 0.001) enquanto miR-1 e -133b foram associados a cTnI (r2 = 0.675 e 0.758; p < 0.001). Além disso, miR-1 antecipou a cardiotoxicidade e mostrou uma area sobre a curva maior que cTnI para discriminar pacientes que desenvolveram cardiotoxicidade daqueles que não desenvolveram (AUC = 0.849 e 456, p<0.001 e 0.663, respectivamente). CONCLUSÃO: Nossos dados sugerem miR-1 como um potencial novo biomarcador de cardiotoxicidade induzida por DOX em pacientes com câncer de mama. Estes resultados podem levar a novas estratégias de detecção precoce do risco de lesão cardíaca induzida por DOX bem como a introdução de uma nova área para intervenção / INTRODUCTION: Cardiotoxicity is frequently associated with the chronic use of doxorubicin (DOX) and may lead to cardiomyopathy and heart failure. Identification of cardiotoxicity-specific miRNA biomarkers could provide clinicians with a valuable prognosis tool and a potential area for intervention. METHODS: This is an ancillary study from the prospective trial \"Carvedilol Effect in Preventing Chemotherapy-Induced Cardiotoxicity.\" (CECCY trial) which included 56 female patients (49.9±3.3 age) from placebo arm. Enrolled patients were treated with DOX followed by taxanes. Cardiac troponin I (cTnI), left ventricle ejection fraction (LVEF) and miRNAs were measured periodically. RESULTS: Circulating levels of miR-1, -133b, -146a and -423-5p increased along the treatment (18.6, 11.5, 10.6 and 12.1-fold respectively; p < 0.001); miR-208a and -208b were undetectable. cTnI increased from 6.6±0.3 to 46.7 ± 5.5 pg/ml (p < 0.001) while LVEF tended to decrease from 65.3±0.5 to 63.8±0.9 (p=0.053) over 12 months; ten patients (17.9%) developed cardiotoxicity. miR-1 was associated to changes in LVEF (r2=0.363, p < 0.001) while miR-1 and -133b were associated to cTnI (r2 = 0.675 and 0.758; p < 0.001). Furthermore, miR-1 anticipated cardiotoxicity and showed greater area under the curve than cTnI to discriminate between patients who did and did not developed cardiotoxicity (AUC = 0.849 and 456, p < 0.001 and 0.663, respectively). CONCLUSION: Our data suggest circulating miR-1 as a potential new biomarker of DOX-induced cardiotoxicity in breast cancer patients. These results may lead to new earlier strategies to detect drug-induced cardiac injury risk before it develops to an irreversible stage or introduce new area for intervention

La doxorubicine (DXR) est l’un des médicaments les plus efficaces en chimiothérapie, mais sonapplication clinique est limitée par ses effets cardiotoxiques. Malgré des décennies de recherche, sesmécanismes pathogéniques ne sont toujours pas entièrement compris. Il s’ensuit qu’aucun traitementsatisfaisant, curatif ou préventif, n’existe. Dans cette étude, nous recherchons les mécanismes designalisation cellulaire impliqués. Deux modèles expérimentaux de toxicités, aigue d’une part (coeurisolé et perfusé de rat avec la DXR), et chronique d’autre part (rat traité à la DXR), ont permis deréaliser une étude ciblée (sur les voies de signalisation énergétiques) et deux études systémiques(phosphoprotéomique et transcriptomique). Les résultats combinés de ces travaux ont montré que laDXR modifiait le niveau de phosphorylation (activation) ou l’expression génique de protéinesimpliquées dans trois domaines fonctionnels distincts : métabolisme énergétique, réponses au stress,et structure/fonction du sarcomère. (i) Métabolisme énergétique : nous avons confirmé la surprenanteinhibition de l’AMPK, probablement provoquée par un contrôle négatif exercé par des partenaires designalisation (Akt et ERK), plutôt que par une modification des kinases activatrices en amont. Nousavons également montré l’augmentation du niveau de phosphorylation de la PDH, ce qui, en inhibantl’enzyme, ralentit le cycle de Krebs. Cependant, nous avons également observé un phénomènecompensatoire de surexpression de gènes codant pour des enzymes de la glycolyse et du cycle deKrebs ; (ii) Réponses au stress : dans nos modèles, la DXR génère des stress énergétique,génotoxique et oxydatif. Cependant, seuls quelques mécanismes compensatoires sont activés (lesvoies de signalisation de DNA-PK–Akt–GSK3, diverses chaperonnes). Les autres semblent êtreinhibées suggérant que l’amoindricement des réponses au stress serait un des mécanismes de lacardiotoxicité de la DXR; (iii) Structure/fonction du sarcomère: L’augmentation de la phosphorylationde la desmine ainsi que la réduction du nombre de transcrits codant pour des protéines essentiellesau développement cardiaque normal pourraient être la cause de la désorganisation du réseaumyofibrillaire. En conclusion, ces résultats révèlent potentiellement de nouveaux mécanismes de lacardiotoxicité induite par la DXR et permettent d’envisager de nouvelles cibles moléculaires pour ledéveloppement de stratégies protectrices. / Doxorubicin (DXR) is an efficient anticancer drug, the use of which is limited by seriouscardiotoxicity. Despite decades of research, its pathogenic mechanisms are not fully understood, andefficient preventive or curative strategies are not available. Here we address the question whethermechanisms in cardiac cell signaling contribute to the toxicity phenotype. Using experimental modelsfor acute (DXR-perfused, isolated rat hearts) or chronic toxicity (rats injected with DXR), we conducteda targeted study (focusing on energy signaling pathways) and two non-biased studies(phosphoproteomics and transcriptomics). The combined data reveal DXR-induced alterations inphosphorylation (activation) status or gene expression of proteins in mainly three functional domains:energy metabolism, stress responses, and sarcomere structure. (i) Energy metabolism: We confirm aparadox inhibition of AMPK signaling, that is rather due to inhibitory cross-talk with related signalingpartners (Akt, ERK) than impaired AMPK upstream signaling. We also show, among others, theincrease of inhibitory phosphorylation of pyruvate dehydrogenase, slowing down Krebs cycle, but alsoa compensating upregulation of glycolysis and Krebs cycle enzyme transcripts. (ii) Stress-responses:In our models, DXR generates energetic, oxidative and genotoxic stress, but only some compensatorystress responses are activated (DNA-PK–Akt–GSK3 pathway, chaperones). Many others seem to beinhibited, suggesting a blunted response to stress as component of DXR toxicity. (iii) Sarcomerestructure/function: We detect increased phosphorylation of desmin and reduced transcripts essentialfor e.g. normal heart development as potential causes for a disorganized myofibrillar network. Inconclusion, these results reveal some novel potential mechanisms of DXR-induced cardiotoxicity andsuggest new targets for protective strategies.