v angličtině Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Michela Morkusová Supervisor: Přemysl Mladěnka, Pharm.D., Ph.D. Title of diploma thesis: Chelation ability of experimentally used iron chelators Iron is a vital element, playing many important functions in the body. Its presence in too low or too high quantities is harmful. The body has no excretion route for iron, and in certain circumstances, excess iron can accumulate in body tissues. Iron is present in the body in excess particularly in individuals undergoing frequent blood transfusions or having genetically based diseases associated with iron overload (hemochromatosis). Iron excess is medically treated by administration of iron chelators. Such substances can find wider therapeutic use as well, e.g. in individuals with acute myocardial infarction or with tumours. This Thesis was aimed at determining the stoichiometric ratio in the complexes of two experimental chelators, salicylaldehyde isonicotinoyl hydrazone (SIH) and ethylenediaminetetraacetic acid (EDTA), with iron(II) and iron(III) ions at four different pathophysiologically significant pH levels (4.5, 5.5, 6.8 and 7.5). Spectrophotometry was used as the analytical method. In the processing stage, various new...

[pt] A partir dos anos 70 constatou-se a formação de uma massa aderida às paredes do canal radicular, após a instrumentação, que foi denominada smear layer. Esta camada é composta de material orgânico e inorgânico, tecido pulpar e bactérias, e contribui para obliterar os túbulos dentinários. A remoção desta camada melhora a adaptação entre o material obturador e as paredes dentinárias, elimina bactérias, limpa e alarga os túbulos. Essa remoção é realizada por soluções quelantes, mas a substância e o tempo ideais permanecem desconhecidos. Os quelantes atuam removendo Cálcio da microestrutura dentinária. No presente estudo, foi analisado o poder quelante de MTAD, HEBP e EDTA sobre a dentina coronária, avaliando a fração de área tubular aberta (AreaP) e a massa de Cálcio quelado (MNC (Ca)). Três hipóteses foram propostas e testadas: Não existe diferença entre os quelantes quanto a AreaP (H1) e quanto a MNC(Ca) (H2); existe correlação entre AreaP e MNC(Ca) (H3). Através de Microscopia Óptica Co-localizada (MCL) foram capturadas imagens de vários campos de cada amostra, em diferentes tempos de ataque ácido. Uma seqüência de processamento e análise de imagens foi utilizada para medir AreaP versus tempo, para cada quelante. Esta análise foi complementada qualitativamente com Microscopia Eletrônica de Varredura (MEV). As medidas de MNC(Ca) foram realizadas por Espectroscopia de Absorção Atômica (AA) sobre as soluções quelantes utilizadas no experimento de MCL. H1 foi negada, com MTAD sendo o mais potente e HEBP o menos potente dos quelantes. Os resultados de MEV corroboram os resultados de MCL. Os resultados de AA apresentaram grande dispersão, mas indicam que H2 é falsa. H3 foi rejeitada. / [en] In the 70's it was discovered that after instrumentation of the root canal there was the formation of a smear layer, attached to the walls of the canal. This layer is composed of organic and inorganic material, pulp tissue and bacteria, and contributes to the obliteration of the dentine tubules. The removal of this layer improves the adaptation of the filling material to the dentine walls, eliminates bacteria, cleans and enlarges the tubules. Smear layer removal is achieved through the use of chelators but the ideal substance and application time remain unknown. Chelators act through the removal of Calcium from the dentine microstructure. In the present study, the chelating power of MTAD, HEBP and EDTA on coronal dentine was analyzed through the measurement of the area fraction of open tubules (AreaP) and of the chelated Calcium mass (MNC(Ca)). Three hypotheses were proposed and tested: There is no difference between chelators regarding AreaP (H1) and regarding MNC(Ca) (H2); there is correlation between AreaP and MNC(Ca) (H3). Images of several fields of each sample, for different chelation times, were acquired through Co-Site Optical Microscopy (CSOM). An image processing and analysis sequence was employed to measure AreaP versus time, for each chelator. This analysis was qualitatively complemented by Scanning Electron Microscopy (SEM). Measurements of MNC(Ca) were obtained by Atomic Absorption Spectroscopy (AA) from the chelator solutions used during the CSOM experiments. H1 was denied, with MTAD found to be the most powerful chelator while HEBP was the weakest. SEM results corroborate these findings. AA results showed wide dispersion but indicate that H2 is false. H3 was rejected.

[pt] Com os métodos de instrumentação usados atualmente na terapia endodôntica ocorre a formação de uma camada residual composta de matéria orgânica e inorgânica que freqüentemente pode reter bactérias e seus subprodutos. Os métodos disponíveis para a remoção do smear layer incluem o ataque químico, técnicas ultra-sônicas e a laser, sendo que nenhum desses métodos se mostra totalmente eficaz, não existindo um consenso universal a respeito. A ação do EDTA, do EDTAC e do ácido cítrico sobre a dentina radicular foi analisada neste estudo por dois métodos. Primeiramente, foi realizada uma análise da microdureza radicular antes e depois da aplicação dos quelantes. Em seguida, utilizou-se microscopia de força atômica (AFM) para mapear e caracterizar as alterações microestruturais ocorridas na dentina durante o processo de quelação dos íons de cálcio. No caso da microscopia, realizou-se experimentos em que a ação quelante foi observada em tempo real, in situ, através do uso de um porta-amostras especial - uma célula de líquido - que permitiu observar a superfície das amostras durante o condicionamento ácido. Seqüências de imagens foram capturadas durante o processo e uma rotina de processamento e análise digital de imagens (PADI) foi criada para avaliar qualitativa e quantitativamente o efeito das substâncias quelantes. O desenvolvimento desta metodologia de microscopia in situ e análise digital é uma das principais contribuições da presente dissertação. / [en] Instrumentation methods currently used in endodontic therapy create a residual layer composed of organic and inorganic material that frequently retains bacteria and their subproducts. The available methods to remove this smear layer include chemical etching, ultrasonic and laser techniques but none of these is completely efficient and there is no universal consensus regarding this subject. In the present study the effect of EDTA, EDTAC and citric acid upon radicular dentin was analyzed by two methods. Dentin microhardness was measured before and after etching. Atomic force microscopy was used to map and characterize dentinary microstructural changes throughout the chelation process. Real time, in situ, observations of the process were performed through the use of a special sample holder - a liquid cell - that allowed the observation of the samples surfaces during etching. Image sequences were acquired and an image processing and analysis procedure was developed to evaluate, both qualitatively and quantitatively, the effect of the 3 chelators. The development of this methodology employing in situ microscopy and digital image analysis is one of the main contributions of the present dissertation.

La dérégulation du métabolisme des cellules tumorales est un hallmark du cancer clairement établi. Pour assurer leur taux de proliferation élevé, les cellules cancéreuses adaptent leur métabolisme, ce qui leur permet de répondre à leurs nouveaux besoins énergétiques. Dans ce contexte, les cellules tumorales présentent des besoins en fer augmentés ainsi que de multiples perturbations du métabolisme du fer, ce qui les rend plus sensibles à la privation en fer. Cette vulnérabilité pourrait ainsi faire l’objet d’un ciblage thérapeutique. Dans les cancers du sein, de nouvelles approches thérapeutiques sont très attendues en particulier pour les cancers triple-négatifs qui développent fréquemment des résistances à la chimiothérapie et qui souffrent d'un manque de cibles thérapeutiques spécifiques. L’activité antitumorale des chélateurs de fer tels que le déférasirox (DFX) évalués en monothérapie a déjà été démontrée dans différents types de cancers mais ne semble pas être suffisamment efficace pour éradiquer les tumeurs. Dans cette étude, nous avons démontré que le DFX agit en synergie avec des molécules de chimiothérapies conventionnelles telles que la doxorubicine, le cisplatine et le carboplatine pour inhiber la prolifération cellulaire et induire l’apoptose et l’autophagie de lignées cellulaires mammaires de sous-type triple-négatif. De plus, la combinaison du DFX avec la doxorubicine et le cyclophosphamide permet de retarder voire d’éviter les récidives dans des xénogreffes de tumeurs mammaires triple-négatives (PDX) sans augmenter les effets secondaires de la chimiothérapie seule ni impacter les réserves en fer globales des souris. Au niveau moléculaire, nous avons montré que la synergie antitumorale du DFX et de la doxorubicine implique une inhibition des voies PI3K et NF-κB. Par ailleurs, étant donné que les patients présentant un cancer triple-négatif avec de faibles réserves en fer tumorales présentent un bon pronostic, nous pensons que la privation en fer au moyen de chélateurs de fer pourrait constituer une approche d’autant plus efficace pour augmenter l’efficacité des chimiothérapies conventionnelles dans le traitement de ces cancers. / Deregulation of tumor cell metabolism is a clearly established cancer hallmark. To ensure their high proliferation rate, cancer cells adapt their metabolism to meet their new energy needs. In this context, tumor cells display increased iron needs as well as multiple disturbances in their iron metabolism, making them more susceptible to iron deprivation. This vulnerability could be a therapeutic target. In breast cancers, the development of new therapeutic approaches is urgently needed for patients with triple negative tumors (TNBC) which frequently develop chemotherapies resistance and suffer from a lack of targeted therapies. The anticancer activity of iron chelators such as deferasirox (DFX) assessed in monotherapy has been demonstrated in different types of cancers. However, iron chelators do not appear to be effective enough to eradicate cancer. In this work, we demonstrated that DFX synergizes with standard chemotherapeutic agents such as doxorubicin, cisplatin and carboplatin to inhibit cell proliferation and induce apoptosis and autophagy in TNBC cell lines. Moreover, the combination of DFX with doxorubicin and cyclophosphamide allowed to delay or avoid recurrences in breast cancer patient-derived xenografts (PDX) without increasing the side-effects of chemotherapies alone or altering global iron storage of mice. At the molecular level, we showed that the antitumor synergy of DFX and doxorubicin involves a down-regulation of PI3K and NF-κB pathways. Furthermore, as TNBC patients with low iron storage in their tumor present a better prognosis, we thought that iron deprivation mediated by iron chelators may all the more increase the effectiveness of conventional chemotherapies for TNBC treatment.

Cancer remains one of the most feared diseases affecting the modern world. Second to heart disease, it is the largest cause of deaths, affecting one in three persons. Cancer cells are formed when normal, healthy cells become damage, losing their normal regulatory mechanism that control cell growth. There are many different types and progression of these cancer cells that determine the type of treatment a patient receives. The primary focus of this dissertation is to propose three studies of anticancer agents. In Chapter one, a CpG-lytic peptide conjugate was designed to target receptors on the cell membrane to concentrate the lytic peptide around the cells to cause triggered cell death, in the treatment of Myelodysplastic Syndromes (MDS). This conjugate act like monoclonal antibodies in that the molecular size is too large to enter the cell, therefore it targets the TLR9 receptors expressed extracellularly in precancer cells in MDS. Chapter two, focuses on the screening of anticancer agents used in targeted therapy. It provides a general scheme applied to the synthesis of a combinatorial library of primary amines used as small-molecule drugs coupled unto a solid support bead (Positional Scanning Library Method) to screen for biological effects on various types of cancers. Chapter three address the issue of radiotherapy treatments, one of the most widely used treatment of cancer. To improve the efficacy of conventional radiation therapy and reduce the cytotoxicity of healthy tissue, High-Dose Rate brachytherapy (HDR) may be used as a stand-alone treatment or after surgery to prevent the recurrence of cancer cells. To design and provide studies of these brachytherapy beads, a model was developed by coupling a chelating agent DOTA onto the surface of macrobeads that coordinated to Europium (III) in efforts to mimic the radiolabeling with a radioactive metal. These brachytherapy beads will be used to conduct in vitro studies in the treatment of local cancers with massive concentrations of radiation without damaging surrounding healthy tissue.

<p>Cellular damage due to oxidative stress is implicated in a wide variety of conditions including degenerative diseases like Alzheimer's and Parkinson's Diseases. One source of oxidative stress is the interaction of redox-active metals such as copper and iron with hydrogen peroxide to produce hydroxyl radicals. Preventing metal-induced oxidative stress by metal chelation is one potential approach to treat some of these diseases, but there remain significant challenges in designing chelators that target damaging metals while not disturbing healthy metal ion distribution.</p><p>To overcome this challenge, prochelators that are responsive to conditions of oxidative stress have been introduced. By designing ligands that only bind metal ions in the presence of oxidants, damaging metals can be bound and removed while not perturbing the metals necessary for cell function. Masking the phenol of a chelator with a boronic ester creates a prochelator that has little to no affinity for metal ions until exposure to H₂O₂ converts the prochelator to the chelator, which is then available to bind metal ions. Described here is the development of boronate-based prochelators that react with H₂O₂ to produce chelating agents of variable denticity, ranging from 2 to 6.</p><p>Quinoline boronic acid pinanediol ester, or QBP, is a new bidentate prochelator introduced here that reacts with H₂O₂ with a rate of 0.22 M<super>-1</super>s<super>-1</super> to produce 8-hydroxyquinoline, a known metal-binding agent. Results in Chapter 2 show that QBP can be activated in vitro under conditions that mimic early Alzheimer's Disease pathology where copper, amyloid beta peptide, and ascorbic acid exacerbate formation of reactive oxygen species. QBP does not bind metal ions, nor does it disaggregate metal-promoted amyloid beta peptide aggregates. However, the released 8-hydroxyquinoline sequesters copper from amyloid beta and both diminishes further formation of reactive oxygen species and inhibits further aggregation of amyloid-beta.</p><p>The syntheses and crystal structures of hexadentate prochelators are described in Chapter 3, along with their rates of oxidation in response to hydrogen peroxide exposure and their ability to protect against hydroxyl radicals formed in vitro by iron (or copper), ascorbic acid, and hydrogen peroxide. The hexadentate chelators are based on a tripodal architecture in which three phenol moieties are linked via nitrogens on three alkyl arms to a central nitrogen to provide an N₃O₃ donor set for metal complexation. Of three prochelator/chelator pairs prepared, the pair (trenBsalam/trensalam) with amine linkages was deemed most suitable for potential biological studies. The prochelator trenBsalam oxidizes at a rate of 0.72 M<super>-1</super>s<super>-1</super> to produce the chelator trensalam in the presence of hydrogen peroxide. The transition metal coordination chemistry and metal ion affinities of trensalam were further studied in Chapter 4 by x-ray crystallography, UV/Vis spectroscopy and cyclic voltammetry.</p><p>The response of a series of bidentate prochelators to various oxidants, including hydrogen peroxide, superoxide, peroxynitrite and hypochlorite, was evaluated by UV/Vis spectroscopy in Chapter 5. Varying the diol that is appended to the boronic ester results in hydrogen peroxide oxidation rates ranging from 0.018 to 1.27 M<super>-1</super>s<super>-1</super>. Lastly, the stability of different boronic acid and diol combinations was probed by spectroscopic techniques and indicate that boronic esters formed with pinanediol form the most stable prochelators under physiological conditions.</p> / Dissertation

Le projet de thèse concerne l’étude des réseaux de régulation en lien avec l’homéostasie des ions métalliques chez la bactérie à Gram-positif Bacillus subtilis. Les ions métalliques tels que Fe(II), Mn(II) et Zn(II) sont essentiels dans un grand nombre de processus cellulaires entant que cofacteur d’enzymes ou en tant qu’élément structural dans les protéines. Cependant, à trop hautes concentrations, ils peuvent avoir des effets toxiques en endommageant la membrane cellulaire et l’ADN ainsi qu’en inactivant la fonction de certaines protéines. De plus, en condition aérobie, B. subtilis produit du peroxyde d’hydrogène H₂O₂ qui réagit avec les ions Fe(II) pour produire des radicaux libres hautement toxiques pour la cellule. La régulation de l’homéostasie des ions métalliques doit donc être parfaitement régulée et coordonnée avec les autres processus cellulaires. J’essaie de comprendre de façon globale, via des approches expérimentales à grande échelle, les interconnexions entre les régulateurs de l’homéostasie des ions métalliques et autres voies métabolique dans Bacillus subtilis. / The present doctoral thesis concerns the study of the regulatory network linked tometal ions homeostasis in the Gram+ Bacillus subtilis. Metal ions such as Fe(II), Mn(II) andZn(II) are essential for many metabolic processes, since they function as enzyme cofactors andstructural ligands of proteins. Changes in ions availability can alter activity of enzymes of thecarbon metabolism and lead to changes in gene expression. In addition, the modulation of metalion homeostasis is intimately linked with the oxidative stress response: during aerobic growth,hydrogen peroxide is generated and it rapidly reacts with ferrous iron to form ROS molecules.Hence, regulation of metal ions uptake/efflux has to be finely regulated and coordinated with othercellular processes. With the present project, I aim to understand at system’s level how Bacillussubtilis integrates the control of metal ions homeostasis with other metabolic processes.

Mercury is an environmental contaminant that preferentially accumulates in the kidney. It has been previously shown using proton-induced X-ray emission analysis that mercury (HgCl2) accumulated in precision-cut rabbit renal cortical slices. In this study, the efficacy of seven chelating agents for the removal of Hg from renal dices has been examined. Rabbits were injected with HgCl2 (10 mg/kg) and 3 h later kidneys were sliced, or renal slices were exposed in vitro to a mildly toxic concentration of HgCl2 (5 x 10-5 M, 4 h). The slices were then treated in vitro with 10 mM concentrations of EDTA, lipoic acid (LA), penicillamine (PA), glutathione (GSH), 1,4-dithiothreitol (DTT), DMSA, or DMPS. DMPS proved to be the most effective in mobilizing Hg from in vivo or in vitro HgCl2-exposed renal tissue (>85% of control after 3 h incubation). Relative efficacies for the seven agents were DMPS > DMSA, PA > DTT, GSH > LA, EDTA. The use of renal slices appears to be a useful in vitro tool for assessing the efficacy of chelating agents on mobilizing accumulated Hg from renal tissue.

The effects of varying concentrations of two chelators: EDTA, citric acid (CA), and phosphorus on the accumulation of arsenic in soil by two perennial grasses, Leersia oryzoides and (rice cut-grass) Festuca arundinacea (tall fescue). Each experiment was run for eight weeks, after which plants were harvested, oven-dried, digested in acid and analyzed using an ICP-OES. Phosphorus soil amendments were applied as 0, 15, 30, 60, 120 mg P/kg soil. For the first chelator experiment, CA and EDTA concentrations of 0, 2.5, and 5.0 mmol/kg soil were added in pulse form to the soil. In the second chelator experiment, concentrations of CA were added in 0, 2.0, 4.0 mmol/kg soil and EDTA was added as 0, 0.25, 0.50 mmol/kg soil. Both plant species accumulated enough arsenic in the root and shoot tissues to be considered at hyperaccumulator species. Citric acid produced comparable results with EDTA, and is considerable much safer for the environment than any synthetic chelator. Certain soil amendments (citric acid and phosphorus) and hyperaccumulator species used in this study warrant further research in the field.

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