<p>The ability to manipulate the coordination chemistry of metal ions has significant ramifications for the study and treatment of metal-related health concerns, including iron overload, UV skin damage, and microbial infection among many other conditions. To address this concern, chelating agents that change their metal binding characteristics in response to external stimuli have been synthesized and characterized by several spectroscopic and chromatographic analytical methods. The primary stimuli of interest for this work are light and hydrogen peroxide.</p><p>Herein we report the previously unrecognized photochemistry of aroylhydrazone metal chelator ((E)-N&#8242;-[1-(2-hydroxyphenyl)ethyliden]isonicotinoylhydrazide) (HAPI) and its relation to HAPI metal binding properties. Based on promising initial results, a series of HAPI analogues was prepared to probe the structure-function relationships of aroylhydrazone photochemistry. These efforts elucidate the tunable nature of several aroylhydrazone photoswitching properties.</p><p>Ongoing efforts in this laboratory seek to develop compounds called prochelators that exhibit a switch from low to high metal binding affinity upon activation by a stimulus of interest. In this context, we present new strategies to install multiple desired functions into a single structure. The prochelator 2-((E)-1-(2-isonicotinoylhydrazono)ethyl)phenyl (E)-3-(2,4-dihydroxyphenyl)acrylate (PC-HAPI) is masked with a photolabile trans-cinnamic acid protecting group that releases umbelliferone, a UV-absorbing, antioxidant coumarin along with a chelating agent upon UV irradiation. In addition to the antioxidant effects of the coumarin, the released chelator (HAPI) inhibits metal-catalyzed production of damaging reactive oxygen species. Finally a peroxide-sensitive prochelator quinolin-8-yl (Z)-3-(4-hydroxy-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)acrylate (BCQ) has been prepared using a novel synthetic route for functionalized cis-cinnamate esters. BCQ uses a novel masking strategy to trigger a 90-fold increase in fluorescence emission, along with the release of a desired chelator, in the presence of hydrogen peroxide.</p> / Dissertation

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Emily Zsáková Supervisor: Assoc. Prof. Přemysl Mladěnka, PharmD., Ph.D. Title of diploma thesis: Interference of reducing agents with the hematoxylin assay for determination of cupric chelation Copper is an essential trace element of living organisms. Disorders of its homeostasis affect various pathological conditions. In excessive amounts, it is toxic due to its ability to form free radicals. Vitamin C, trolox (a water soluble form of vitamin E), glutathione and hydroxylamine possess reducing properties that protect our body from oxidative tissue damage. Oxidized glutathione (GSSG) was also tested for comparison. The object of study was to test the chelating ability of these substances. A simple and fast spectrophotometric method was used. The combination of reducing properties of copper and chelating agents could lead to an improvement in chelation therapy either in heavy metal poisoning, treatment of Wilson's disease, or other diseases. We found that all substances were chelators/copper-chelating agents in vitro in a slightly competitive environment, using hematoxylin as an indicator. However, by using the bathocuproine assays to verify chelating properties, none of the substances...

Poor rehydration properties of skim milk powder (SMP) can impact processing efficiency and functionality in finished product applications. Rehydration can be split into four stages: wettability, sinkability, dispersibility, and solubility. Previous work has suggested that chelator addition during SMP manufacture leads to higher solubility compared to SMP without chelators. This study focuses on the addition of ethylene diamine tetraacetic acid (EDTA) or sodium citrate dihydrate (SCD) at concentrations of 5, 10, and 15 mM to skim milk prior to evaporating and spray drying. The objective of this investigation was to determine the effects of the chelator additions on each SMP rehydration property (wettability, sinkability, dispersibility, and solubility) during reconstitution to 10% total solids. SCD 15 mM, SCD 10 mM, and SCD 5 mM did not have a significant effect on wettability as measured by IDF method (p-value 0.3234, 0.6376, and 1.0000, respectively). However, SCD 15 mM, SCD 10 mM, and SCD 5 mM had higher levels of solubility as measured by particle size analysis of reconstituted 10%TS samples (p-value

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Maria Carmen Catapano, MSc. Supervisor: Assoc. Prof. Přemysl Mladěnka, PharmD., Ph.D. Co-supervisor: Assoc. Prof. Laura Mercolini, Ph.D. Title of Doctoral Thesis: Screening of novel chelators of microbiogenic metals Iron, copper and zinc are microbiogenic elements which play crucial roles in a series of physiological processes in human organism. Homeostasis of these transition metals is strictly regulated since, among others: a) free or loosely bound iron or copper can catalyse the production of hydroxyl radical; b) lack of zinc but also of the previously mentioned metals is associated with significant impairments. Hereditary hemochromatosis, transfusion-induced secondary iron overload and Wilson's disease are known as pathological conditions associated with metal overload in the organism. Chelator agents have vital relevance for the treatment of these impairments. There are also numerous diseases with homeostatic imbalances in iron, copper and or zinc: neurodegenerative diseases, cardiovascular diseases, cancer and diabetes mellitus. Different chelating compounds have been examined for the treatment of these impairments. The aim of this doctoral thesis was to perform a screening of metal...

Doctor of Philosophy / Department of Chemistry / Stefan H. Bossmann / Recent development of a variety of superparamagnetic and ferromagnetic iron/iron oxide (Fe/Fe₃O₄) nanoparticles with different surface chemistry have been widely studied for numerous biological applications such as drug delivery, as diagnostics, hyperthermia and magnetic resonance imaging. The wide applications of Fe/Fe₃O₄ nanoparticles are possible since they exhibit favorable properties as high magnetization ability, are smaller than 100 nm in size, they can be coated with several ligands which allow drug delivery at a specific site and are biocompatible. By using Fe/Fe₃O₄ nanoparticles as drug delivery agents treatment costs and side effects can be reduced, however treatment efficacy can be increased. We have demonstrated that Fe/Fe₃O₄ nanoparticles can be utilized in different methods depending on their properties, to be used as therapeutic agents for cancer treatment. In one method we have taken advantage of the Fe/Fe₃O₄ nanoparticles magnetic ability to produce hyperthermia (heat) in cancer cells when subjected to an alternative magnetic field. Here we use the cell based delivery system since the size of the nanoparticles are small they can be taken up by monocyte/ macrophage like cells for systemic transportation to the inflamed cancer cite. The hyperthermia study was conducted in mice with pancreatic cancer. This study demonstrated that the life expectancy of the mice increased by 31%. In the next method we took the advantage of the surface chemistry of the Fe/Fe₃O₄ nanoparticles and changed it with dopamine-peptide and dopamine-thiosemicarbazone ligands. The advantage of the peptide is to deliver the nanoparticle to its target site and the thiosemicarbazone analogue is used as an iron chelator that would initiate apoptosis in cancer cells. This nanoplatform was tested in 4T1 breast cancer cell line and normal fibroblast cell line and demonstrated that it was effective towards the cancer cell line than the normal cell line at a ratio of 5:1 of thiosemicarbazone analogue : dopamine on the nanoparticle. However further studies are needed to be done to clarify the effectiveness of this nanosystem.

A radiomarcação de biomoléculas específicas com o tecnécio-99m 99mTc utilizando agentes quelantes bifuncionais é um campo em crescimento na Medicina Nuclear. Em especial, a classe de peptídeos regulatórios, como a Neurotensina, participa de processos fisiológicos essenciais no organismo, como o crescimento tumoral. O objetivo do presente trabalho foi o estudo comparativo da influência dos agentes quelantes bifuncionais 6-hidrazinonicotinamida (HYNIC) e S-acetil-mercaptoacetiltriglicina (MAG3), no comportamento in vitro e in vivo do análogo duplamente estabilizado da Neurotensina(8-13) radiomarcado com 99mTc, em células tumorais de mama da linhagem MDA-MB-231. Um elevado rendimento radioquímico (> 97%) e estabilidade frente aos agentes transquelantes foi observado para ambos análogos radiomarcados. Foram também obtidos comportamentos similares in vitro, no que diz respeito à porcentagem de ligação às proteinas plasmáticas (aproximadamente 22%), estabilidade metabólica, ligação aos receptores celulares (intervalo nM) e taxas de internalização/externalização para ambos radiocomplexos. A maior lipofilicidade encontrada para o análogo radiomarcado via MAG3 refletiu nas principais diferenças nos estudos de biodistribuição. A degradação do análogo radiomarcado via HYNIC nos estudos de estabilidade metabólica in vivo aos 90 min levou a menor retenção tumoral (0,44±0,02% DI/g), e consequentemente, às menores razões tumor/órgãos não-alvos (< 5%). Embora a superioridade do traçador marcado via MAG3 tenha sido comprovada no presente estudo, um redesenho estrutural objetivando contornar a alta captação no trato gastrointestinal deve ser realizada a fim de que sua potencial aplicabilidade não seja comprometida. / The radiolabeling of receptor specific biomolecules with 99mTc using bifunctional chelator agents represents a growing field in Nuclear Medicine, specially, regarding regulatory peptides, such as Neurotensin, which are important in several essential physiological functions, particularly in tumor growth. The aim of the study was the comparative radiolabeling evaluation of the double-stabilized NT(8-13) analog with 99mTc, via the bifunctional chelating agents 6- hydrazinonicotinamide (HYNIC) and S-acetyl-mercaptoacetyltriglycine (MAG3) in MDA-MB-231 breast cancer cell line. High radiochemical yields (> 97%) and stability toward transchelant agents was observed for both radiolabeled analogs. Also, comparable in vitro behaviour regarding the percentage of plasma protein binding (nearby 22%), metabolic stability, receptor binding affinity (nM range), and internalization/externalization rates were obtained. The greater lipophilicity found for the analog radiolabeled via MAG3, reflected in the major differences in biodistribution studies. The in vivo metabolic stability studies suggested that the degradation observed in the later time point (90 min) for the conjugate radiolabeled via HYNIC, leads not only to lower tumor uptake accumulation (0,44±0,02% ID/g), but also to lower tumor-to-non-tumor ratios (< 5%). Although the superiority of the tracer radiolabeled via MAG3 had been confirmed in the present study, a strucutural re-design aiming the reduction of the high gastrointestinal uptake must be done in order to guarantee the potential applicability of MAG3-radiocomplex.

A radiomarcação de biomoléculas específicas com o tecnécio-99m 99mTc utilizando agentes quelantes bifuncionais é um campo em crescimento na Medicina Nuclear. Em especial, a classe de peptídeos regulatórios, como a Neurotensina, participa de processos fisiológicos essenciais no organismo, como o crescimento tumoral. O objetivo do presente trabalho foi o estudo comparativo da influência dos agentes quelantes bifuncionais 6-hidrazinonicotinamida (HYNIC) e S-acetil-mercaptoacetiltriglicina (MAG3), no comportamento in vitro e in vivo do análogo duplamente estabilizado da Neurotensina(8-13) radiomarcado com 99mTc, em células tumorais de mama da linhagem MDA-MB-231. Um elevado rendimento radioquímico (> 97%) e estabilidade frente aos agentes transquelantes foi observado para ambos análogos radiomarcados. Foram também obtidos comportamentos similares in vitro, no que diz respeito à porcentagem de ligação às proteinas plasmáticas (aproximadamente 22%), estabilidade metabólica, ligação aos receptores celulares (intervalo nM) e taxas de internalização/externalização para ambos radiocomplexos. A maior lipofilicidade encontrada para o análogo radiomarcado via MAG3 refletiu nas principais diferenças nos estudos de biodistribuição. A degradação do análogo radiomarcado via HYNIC nos estudos de estabilidade metabólica in vivo aos 90 min levou a menor retenção tumoral (0,44±0,02% DI/g), e consequentemente, às menores razões tumor/órgãos não-alvos (< 5%). Embora a superioridade do traçador marcado via MAG3 tenha sido comprovada no presente estudo, um redesenho estrutural objetivando contornar a alta captação no trato gastrointestinal deve ser realizada a fim de que sua potencial aplicabilidade não seja comprometida. / The radiolabeling of receptor specific biomolecules with 99mTc using bifunctional chelator agents represents a growing field in Nuclear Medicine, specially, regarding regulatory peptides, such as Neurotensin, which are important in several essential physiological functions, particularly in tumor growth. The aim of the study was the comparative radiolabeling evaluation of the double-stabilized NT(8-13) analog with 99mTc, via the bifunctional chelating agents 6- hydrazinonicotinamide (HYNIC) and S-acetyl-mercaptoacetyltriglycine (MAG3) in MDA-MB-231 breast cancer cell line. High radiochemical yields (> 97%) and stability toward transchelant agents was observed for both radiolabeled analogs. Also, comparable in vitro behaviour regarding the percentage of plasma protein binding (nearby 22%), metabolic stability, receptor binding affinity (nM range), and internalization/externalization rates were obtained. The greater lipophilicity found for the analog radiolabeled via MAG3, reflected in the major differences in biodistribution studies. The in vivo metabolic stability studies suggested that the degradation observed in the later time point (90 min) for the conjugate radiolabeled via HYNIC, leads not only to lower tumor uptake accumulation (0,44±0,02% ID/g), but also to lower tumor-to-non-tumor ratios (< 5%). Although the superiority of the tracer radiolabeled via MAG3 had been confirmed in the present study, a strucutural re-design aiming the reduction of the high gastrointestinal uptake must be done in order to guarantee the potential applicability of MAG3-radiocomplex.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Lucia Hanuščinová Supervisor: Assoc. Prof. Přemysl Mladěnka, PharmDr., PhD. Title of diploma thesis: Ability of copper chelators to interact with iron and zinc Copper plays in the human organism a role of an element with indispensable significance, whose biological influence and effects depend on its quantity. With elevated concentrations in the human body, copper becomes toxic, resulting in pathological conditions. The most well-known diseases is the Wilson's disease, whose treatment consists of oral administration of chelators, i.e. chemical compounds, which are capable of binding copper ions in various proportions and eliminating them from the organism. Chelation therapy is currently the first choice after confirmation of the diagnosis. Chelation toxicity results from several factors, e.g. inhibition of copper dependent enzymes or low selectivity to metals. And precisely the selectivity of chelators is being discussed in this diploma thesis. An ideal chelator should not interact with any of the other physiological ions, that are necessary for the proper functioning of the organism. Five of the most frequently therapeutically or experimentally used substances /trientine, D-penicillamine,...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Andrea Kladivová Consultant: Václav Tvrdý, MSc. Supervisor: Assoc. Prof. Přemysl Mladěnka, PharmD., Ph.D. Title of diploma thesis: Chelation of copper ions by thiol containing chelators Copper is an essential trace element that is important for many physiological functions. On the other hand, disruption of copper homeostasis associated with its elevated level is dangerous for the organism, because of the formation of free radicals. Copper chelators can represent an effective tool in the treatment of such conditions. Due to the ability of a thiol group to bind metal ions and form a chelate, thiol containing chelators are promising compounds for the reduction of copper levels. Within this diploma thesis, four compounds containing a thiol group were tested for the ability to chelate copper ions. Spectrophotometric measurement was used for this determination. It is a simple, fast but precise method for determination of the chelation properties in vitro. In addition, the ability of these compounds to reduce cupric ions was examined. When using the basic hematoxylin method, all the tested substances proved they can chelate copper. Their efficacy was practically identical except for the...

Iron is an essential trace element required for many processes within a cell, including DNA synthesis and cell cycle progression. Moreover, it is critical for cellular respiration in mitochondria. Due to their proliferative nature, cancer cells are dependent on iron, and depleting this element via iron chelators results in the inhibition of ribonucleotide reductase, leading to cell cycle arrest and apoptosis of cancer cells. Recently, an alternative mechanism for the effect of iron chelators have been proposed, including induction of N-myc downstream regulated gene 1 (NDRG1) expression and its inhibitory effect on c-MET, EGFR, and NF-κB pathways, which can act as oncogenes in a certain context. NDRG1 is a tumour suppressor gene, which is downregulated in many cancers and its downregulation correlates with cancer progression, poor differentiation, and higher metastatic potential. It has been shown that NDRG1 expression can be regulated by intracellular iron - a decrease in intracellular iron leads to upregulation of NDRG1 at mRNA and protein level via the HIF-1-dependent mechanism by inhibiting prolyl hydroxylases. Recently, we have conceived the concept of mitochondrially targeted chelators as an effective anti-cancer agent and in this work, we focused on the evaluation of mitochondrially targeted...