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Thesis (M.S.)--Villanova University, 2006. / Chemistry Dept. Includes bibliographical references.

200 000 nouveaux cas de mélanome et 65 000 décès sont dus à ce cancer de la peau chaque année. Le mélanome représente donc un véritable problème de santé publique. Actuellement, plus de 50 % des patients sont toujours en échec thérapeutique malgré le développement des thérapies ciblées et des immunothérapies, et il est donc nécessaire de créer de nouvelles thérapies contre ce cancer.Dans notre laboratoire, nous avons montré que la Metformine, un antidiabétique, induit la mort des cellules de mélanomes par autophagie et apoptose. De plus, ce composé inhibe les capacités invasives de ces cellules. Un essai clinique a été effectué au CHU de Nice sur l’effet de la Metformine sur des mélanomes métastatiques mais les résultats ont été plutôt décevants. De ce fait, et en collaboration avec l’institut de chimie de Nice, notre équipe a développé des composés dérivés de la Metformine et a identifié le CRO15. Durant ma thèse, j’ai d’abord déterminé que CRO15 possédait diminuait la viabilité des cellules de mélanomes par différents mécanismes moléculaires. Tout d’abord, l’activation de la voie AMPK, comme ce qui est observée avec la Metformine, mais également l’inhibition de la kinase MELK, un oncogène très souvent surexprimé dans le cadre du mélanome. L’inhibition de MELK par CRO15 est responsable de l’activation de la voie p53. Ces deux voies, AMPK et p53, augmentent l’expression de REDD1 qui active alors les processus d’autophagie et d’apoptose pour induire la mort des cellules de mélanomes. Finalement, j’ai pu montrer que le CRO15 est également capable d’induire une diminution de la croissance tumorale de mélanomes dans différents modèles de souris. / Every year, 200 000 new cases of melanoma and 65 000 deaths occur as a result of skin cancer, making melanoma a real public health problem. Currently, more than 50 % of patients fail treatment despite new targeted therapies and immunotherapies being developed. As a result, there is an urgent need to develop new anti-melanoma compounds to treat this aggressive disease. In our laboratory, we shown Metformin, an antidiabetic, to induce melanoma cell death by autophagy and apoptosis. Furthermore, this compounds inhibits invasive capability of these cells. A clinical trial has been performed in collaboration with the Nice hospital looking at Metformin’s effects on metastatic melanoma treatment, however these results have been disappointing. In this context, and in collaboration with the chemical institute of Nice, our team developed new Metformin-derived compounds and identified CRO15 as a promising new lead. During my PhD, I determined that CRO15 decreases melanoma cells viability by different molecular mechanisms. First, just like Metformin, CRO15 activates AMPK pathway, however it also inhibits MELK kinase activity, a protein described as an oncogene in melanoma. This inhibition is responsible for p53 activation. The two pathways, AMPK and p53, lead to increased REDD1 expression which involves autophagy and apoptosis to induce melanoma cell death. Finally, I shown that CRO15 decreases melanoma tumoral volume in different mice models. These promising results highlight a novel compound that can now be investigated for its potential use in clinic.

Background Glioblastoma Multiforme (GBM) is the most commonly occurring tumour of the central nervous system (CNS). Currently GBM is considered an incurable malignancy with patients experiencing abysmal life expectancies. Lack of progress in the discovery of novel treatments has led to the repurposing of existing licenced medication as a possible alternative option. Metformin is from the biguanide family of drugs and is the most common medication used in the treatment of type 2 diabetes. Clinical studies have reported that, in type 2 diabetic patients, metformin might reduce cancer incidence and severity. Currently, metformin is being assessed in clinical trials as a treatment for a range of cancer types including GBM. The antineoplastic mechanisms utilized by metformin and other biguanides have not been fully elucidated. Methods The effects of metformin were evaluated, alone and in combination with other agents, on a panel of GBM cell cultures. Functional analysis of metformin mechanism of action was assessed through measurement of apoptosis, depolarisation of the mitochondria membrane, caspase pathway activation, cell cycle progression and the expression levels of micoRNAs. Results Analysis of fourteen GBM cell cultures showed a cytotoxic response to metformin that was significantly linked to the P53 status (p=0.0024). In combination drug testing, one of the four drugs showed a synergistic pairing with metformin. The kinase inhibitor sorafenib, showed synergism (CI ≤ 1) in eight GBM cell cultures. Flow cytometry of metformin treated GBM cells showed no significant increase (p > 0.005) in apoptotic cell populations. Caspase 3/7 levels showed no significant increase post metformin treatment (p > 0.005). Metformin caused depolarisation of the mitochondrial membrane in six GBM cell cultures. Four microRNAs were shown to have expression levels changes post-metformin treatment. Upregulation of expression was identified in miR-140, miR-192, let-7c. Downregulation was identified in miR-222. Conclusions Metformin was shown to have cytotoxic effect on a GBM cell cultures and has potential as GBM therapeutic agent and possible treatment synergy with sorafenib. The significance of P53 status to metformin sensitivity may suggest that its use should be directed to a sub-set of GBM patients. Mechanism for cell death by metformin was shown not to rely on apoptotic pathways but caspase 3/7 independent depolarization of mitochondrial cell membranes and cell cycle arrest. Investigations into autophagy may help to further define the pathways metformin is utilising to promote cell death. The impact of metformin on the expression profile of miR-222, miR-192 and let-7c is in line with clinical studies of other cancer types. This shows possible insight into the cancer independent actions of metformin. The interplay recorded between glucose availability and cell death indicates a possible key factor in the utilisation of metformin as a therapeutic agent. This finding may warrant the addition of dietary control regimes in clinical trials to maximise metformin efficacy. This work highlights the strong potential for biguanides in the development of new drug treatments and in expanding our knowledge of cancer metabolism.

In this modern age, society has become much more aware of the danger bacteria can have on people's health. Personal and household antimicrobial formulations are commonly used within the home to lower the levels of harmful bacteria such as E. Coli, Salmonella and Pseudomonas. The active which kills the bacteria within the formulation is described as a biocide. This research looks at the often neglected potential of cationic polyelectrolyte as a biocide, firstly within solution and secondly in creating an antimicrobial surface. The solution properties and antimicrobial activity for a range of commercially available cationic polyelectrolytes (polymeric quaternary ammonium compounds (QAC) and biguanides) of differing molecular weights were investigated. All polyelectrolytes were observed to have some level of antimicrobial activity. The second phase of this research investigated polyelectrolyte/surfactant/water mixture of similar charge (cationic). Two QAC surfactants were investigated: Alkyl (C12 70%; C14 30%) dimethyl benzyl ammonium chloride (BAC) and Didecyldimethylammonium chloride (DDQ). At a critical concentration, these mixtures segregatively phase separate into a surfactant rich upper phase and polyelectrolyte rich lower phase. This phase separation phenomenon was investigated in respect of surfactant and polyelectrolyte type as well as polyelectrolyte molecular weight. Surfactant type was observed to be the dominant factor in determining the onset of phase separation and by mixing different ratios of surfactants the ability to tune this phase separation concentration was shown. Dilute solutions of these mixtures well below their respective phase separation concentration were then deposited onto glass substrates via a drop cast or inkjet printer method. The surfactant/polyelectrolyte film composites left after drop evaporation ranged from an amorphous film to nodular like structures. The ability to order/structure actives onto a surface could alter active adhesion and surface roughness properties of the film. This change in surface property could consequently affect antimicrobial performance.

Among the great breakthroughs in nanoscience and nanotechnology is the emergence of synthetic polymers that demonstrate biological activity and thus can be exploited for biomedical applications, extending from agents in therapeutics to drug delivery and tissue engineering. A key factor in the fabrication of such polymeric materials is the ability to tune and control their properties. To this end, an insight into the mode of interactions with biological systems is imperative. Computer simulations have proved to be a valuable tool that can compliment experiments and provide -otherwise inaccessible- information. In the context of this thesis, different aspects of the polymeric biological activity were investigated by studying two polymeric materials suitable for different types of applications, aiming to clarify yet undisclosed mechanisms that govern the polymers' behaviour either in solution or in conjunction with model lipid membranes. The first part of the thesis is dedicated to a nonionic amphiphilic copolymer known as Pluronic L64 that is considered as a candidate for the design of novel hybrid polymer-lipid vesicles that will act as carriers for drugs or genes. The hybrid bilayers are subjected to mechanical stress and their properties are compared to those of pure lipid bilayers. The simulations showed that the hybrid membranes can sustain increased surface tension prior to rupture, are stiffer, thicker and the polymers can induce higher lipid tail packing and also reduce the lipid mobility, rendering the membranes more ordered and less fluid. At high values of lateral pressure, which leads to pore formation, the copolymer chains decelerate the pore growth. The examination of the defect formation mechanism reveals that the hydrophilic PEO segment plays the most vital role. The same systems were also observed in varying temperatures and the impact of the inserted polymers on the phase behaviour was investigated. The data suggested that the polymers change the nature of the phase transition from a discontinuous to a continuous one. The hybrid membranes transform between the ordered and the disordered phase in a continuous manner and not at a critical melting temperature. Interestingly, the effect of polymers is different at the low and high temperature regions, as proved by the analysis of the mechanical, structural and dynamic membrane properties. The second part is focused on the study of polyhexamethylene biguanide (PHMB), a biguanide-based polyelectrolyte, that possesses remarkable biocidal properties. Even though PHMB's activity is known, the specific mode of action against bacterial membranes is still puzzling. Our work revealed that the polyelectrolyte assumes a counterintuitive behaviour in aqueous solution tending to self-organise into ordered compact structures, despite the repulsive electrostatic interactions of its positively charged segments. The formed nano-objects are thermodynamically stable, as was confirmed by free energy calculations and could be linked to PHMB's antibacterial mechanism. These findings pave the way for further computational and experimental exploration of these fascinating and promising materials that could lead to the design of novel smart biologically active nanoparticles.

Le taux de mortalité dû au cancer est en hausse d’année en année. Le cancer du pancréas est l’un des plus mortels. Avec un taux de survie inférieur à 20% un an suivant le diagnostic, il y a une urgence pour développer de nouvelles molécules pour cibler cette maladie. La metformine, un biguanide utilisé cliniquement en tant qu’agent antidiabétique, s’est avérée à avoir des propriétés anticancéreuses. Les patients souffrant de diabète de type 2 prenant la metformine comme traitement sont moins à risque de développer plusieurs cancers dont celui du pancréas. Cependant, la metformine n’étant pas biodisponible, les doses à administrer seraient trop élevées pour la considérer comme thérapie anticancéreuse. Le groupe de recherche Schmitzer a synthétisé de nouveaux analogues de type biguanide plus lipophiles dans le but d’améliorer leur biodisponibilité. Le phényléthynylbenzyle biguanide (PEBB) est un des analogues présentant des propriétés antiprolifératives environ 800 fois plus puissantes que la metformine contre des cellules du cancer du pancréas. L’hexylbiguanide s’est aussi démarqué par sa spécificité pour les cellules cancéreuses et sa faible toxicité pour les cellules saines. Étant de bons candidats, des études in vivo ont été faites sur des souris en leur administrant le PEBB et l’hexylbiguanide afin d’obtenir des informations sur l’absorption et la distribution des composés. Pour ce faire, une méthode par LC-MS en mode multiple reaction monitoring (MRM) a été développée afin de quantifier différents analogues de biguanides dans le plasma de souris. De plus, une méthode par MALDI-TOF a été développée afin de localiser et quantifier les analogues dans les tissus par imagerie couplée à la spectrométrie de masse (IMS). Les expériences réalisées ont permis de suivre les composés dans le plasma et d’établir une cinétique d’absorption révélant que le PEBB atteint sa concentration plasmatique maximale environ à 1h après l’administration et que le composé est éliminé de la circulation sanguine à 80% au bout de 4h. Dans le cas de l’hexylbiguanide, la concentration plasmatique maximale est atteinte environ 30 minutes après l’administration pour être éliminé à plus de 90% après 4h. De plus, les études d’IMS ont révélé que le PEBB se distribue principalement dans le foie et légèrement dans les tumeurs. Aucune accumulation à long terme dans le foie n’a été observée, ce qui signifie que les risques de dommages hépatiques sont faibles. Les deux méthodes développées sont des méthodes puissantes IV et reproductibles afin de quantifier les différents types de biguanides dans les liquides biologiques ainsi que dans les tissus. / The death rate of cancer is increasing every year. Pancreatic cancer is one of the deadliest. With a survival rate of less than 20% one year post diagnosis, there is an emergency to develop new molecules to target this disease. Metformin, a biguanide clinically used as an antidiabetic agent, has been shown to have anticancer properties as well. Patients with type 2 diabetes taking metformin are less likely to develop several cancers including pancreatic cancer. However, due to the poor bioavailability of metformin, the doses would be too high to consider it as an anticancer treatment. The Schmitzer group has synthesized new biguanide analogues that are more lipophilic and thus more bioavailable. Phenylethynylbenzyl biguanide (PEBB) is one of the analogues with about 800 times more effective antiproliferative properties than metformin against pancreatic cancer cells. Hexylbiguanide also stood out for its specificity for cancer cells and its low toxicity for normal cells. In vivo studies were performed on mice by administering PEBB and hexylbiguanide to study the absorption and distribution of the compounds. For this aim, a LC-MS method was developed using Multiple Reaction Monitoring (MRM) mode to quantify different biguanide analogues in mice plasma. Complementarily, a MALDI-TOF method was developed to localize and quantify the analogues in tissues by imaging coupled to mass spectrometry (IMS). The experiments performed allowed to follow the compounds in plasma to establish absorption kinetics. These experiments revealed that PEBB reaches its maximum plasma concentration at 1h after administration and the compound is eliminated from the bloodstream at 80% after 6h. For hexylbiguanide, the maximum plasma concentration is reached about 30 minutes after administration and more than 90% is eliminated after 4 hours. In addition, IMS studies have shown that PEBB is distributed mainly in the liver and slightly in tumors. No accumulation in the liver was observed, which suggests that the risk of liver damage is low. These two methods are powerful and reproducible methods to quantify the different types of biguanides in biological fluids and tissues.

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Submitted by (edna.saturno@ufrpe.br) on 2016-10-05T12:25:20Z No. of bitstreams: 1 Ana Janaina Jeanine Martins de Lemos.pdf: 2611080 bytes, checksum: d1d2c6ec413abbc4cb5450d5df0a4be4 (MD5) / Made available in DSpace on 2016-10-05T12:25:20Z (GMT). No. of bitstreams: 1 Ana Janaina Jeanine Martins de Lemos.pdf: 2611080 bytes, checksum: d1d2c6ec413abbc4cb5450d5df0a4be4 (MD5) Previous issue date: 2013-10-22 / The polycystic ovary syndrome (PCOS) is endocrine disorder in women of reproductive age extremely common and heterogeneous clinical features. In cases of patients with ovarian polycystic wishing to treat infertility, metformin hydrochloride is the drug most widely used and considered safer compared to the other treatments. When diagnosing the syndrome, it has been observed that women have a high prevalence of nonalcoholic fatty liver disease, steatosis, fibrosis, abnormal liverenzyme factors and increased indicators of oxidative stress in plasma, raising concern among doctors about the disease in the liver. Furthermore, recent studies have linked oxidative stress in the pathogenesis of diseases which cause infertility, for this reason, research has suggested the use of antioxidants such as melatonin, due to liver protecting effect in the pharmacological treatment or supplementation to combat these types of disease. However, there are no reports in the literature about association of metformin hydrochloride and melatonin for the treatment of PCOS. The objective of this work was to investigate the effects of combination therapy of these drugs in rats induced to PCOS on to reproductive and liver. Thus, we used 75 albino rats were divided randomly into five groups of 15 animals. All rats except those in group I were subjected to induction of PCOS by constant illumination, and the rats of groups III, IV and V received treatment with the drug melatonin, metformin hydrochloride and the drug combination, respectively. After confirmation of PCOS and treatments, we analyzed the effects of plasma biochemical and hormonal treatments, and rats were mated and monitored during pregnancy for analysis of implantation sites, ovarian, weight gain, reproductive viability and levels of oxidative stress. At the end of the experiment was conducted histopathology, and immunohistochemistry staining of liver of rats and analysis of the puppies. Pharmacological treatments decreased the time allowed copulation, increased plasma levels of progesterone, the number and weight of pups and reduced plasma levels of estrogen and endometrial content of collagen fibers. In the liver, treatment with metformin hydrochloride, melatonin and more significantly the association of these drugs reduced plasma levels of the liver enzyme alanine trasaminase, nitric oxide and total glutathione, leading to results similar to the control group, were also observed similarities between those of control rats and rats that received a combination of drugs for the content of hepatic polysaccharide, and of proinflammatory cytokines. Thus, we conclude that the association of metformin with melatonin provides the best results compared to the other treatments, in order to regulate hormonal and histochemical the reproductive system and increase the chance of conception resembling the control group, and the liver, the combination of drugs works more effectively against liver toxicity produced by PCOS, favoring normalization of biochemical parameters and oxidative stress during pregnancy, compared to therapies only these drugs. / A síndrome do ovário policístico (SOP) é um distúrbio endócrino feminino extremamente comum na idade reprodutiva e apresenta quadro clínico bastante heterogêneo, entre eles os cistos foliculares ovarianos aumentados, hiperandrogenismo, infertilidade resistência à insulina, e dislipidemia. Em casos de pacientes com policistose ovariana que desejam tratar a infertilidade, o cloridrato de metformina é a droga mais utilizada e considerada mais segura em comparação com os demais tratamentos. Ao diagnosticar a síndrome, tem-se observado que mulheres apresentam grande prevalência a doença hepática gordurosa não alcoólica, esteatose, fibrose, alterações hepático-enzimáticas e aumento do estresse oxidativo plasmático, elevando a preocupação dos médicos quanto às patologias hepáticas. Além disso, recentes estudos têm relacionado o estresse oxidativo na patogênese de doenças que causam a infertilidade, por esta razão, pesquisas tem sugerido a utilização de antioxidantes como a melatonina como suplemento a outras drogas. No entanto, não há relatos na literatura sobre a associação do cloridrato de metformina e melatonina para o tratamento da policistose ovariana. Assim, o objetivo deste trabalho foi investigar os efeitos da combinação terapêutica desses fármacos em ratas induzidas à SOP sobre os aspectos reprodutivos e hepáticos. Para tanto, foram utilizadas 75 ratas albinas, divididas aleatoriamente em cinco grupos de 15 animais. Todas as ratas, exceto as do grupo I, foram submetidas à indução da SOP pela iluminação constante, e as ratas dos grupos III, IV e V receberam tratamentos com os fármacos melatonina, cloridrato de metformina e a associação destes, respectivamente. Após a confirmação da SOP e tratamentos, foram analisados os efeitos plasmáticos bioquímicos e hormonais dos tratamentos, e as ratas foram acasaladas para análise dos sítios de implantação e acompanhamento gestacional dos níveis de estresse oxidativo. Ao final do experimento foi analisada a histopatologia e imunohistoquímica do fígado das ratas. Os tratamentos farmacológicos diminuíram o número de dias para confirmação do acasalamento, aumentaram os níveis séricos de progesterona, bem como o número e o peso dos filhotes. Reduziram os níveis plasmáticos de estrógeno e teor de fibras colágenas. No fígado, os tratamentos com cloridrato de metformina, melatonina, e mais significativamente a associação destes fármacos, reduziram os níveis plasmáticos da enzima hepática alanina transaminase, do óxido nítrico e da glutationa total, levando a resultados semelhantes aos animais do grupo controle, mais uma vez, as ratas que receberam associação dos fármacos quanto assemelhou-se ao grupo controle quanto ao conteúdo hepático de polissacarídeos, e aos de citocinas próinflamatórias. Desta forma, pôde-se concluir que a associação do cloridrato de metformina com melatonina proporciona os melhores resultados em comparação aos demais tratamentos, de forma a regular hormonal e histoquimicamente o sistema reprodutor e aumentar as chances de concepção de forma semelhante ao grupo controle. No fígado a associação dos fármacos atuou de forma mais eficaz contra a toxicidade hepática produzida pela SOP experimental, favorecendo uma normalização dos parâmetros bioquímicos e estresse oxidativo durante a gestação, quando comparada às monoterapias destas drogas.

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