High chronic fructose consumption has been linked to many diseases. Sugar metabolites, especially glyoxal and methylglyoxal can form advanced glycation products, which contribute to the pathology of diabetic complications. Our objective was to study the metabolism of these metabolites and the associated protein carbonyation and cytotoxicity in isolated hepatocytes. In addition, the effect of oxidative stress on the metabolism of these toxins was also investigated. Methylglyoxal and glyoxal can induce protein carbonylation, which contributes to hepatocyte toxicity. Methylglyoxal, but not glyoxal, was detoxified mainly by the glyoxalase system. Both toxins can be metabolized by mitochondrial aldehyde dehydrogenase. The detoxification of glyoxal was impaired under oxidative stress conditions (i.e. increased hydrogen peroxide level). Glyoxal was found to be a common autoxidation product from glyceraldehyde, hydroxypyruvate and glycolaldehyde. Glyoxal and the reactive oxygen species formation during the autoxidation process contributed to the hepatocyte toxicity of glyceraldehyde, hydroxypyruvate and glycolaldehyde.

High chronic fructose consumption has been linked to many diseases. Sugar metabolites, especially glyoxal and methylglyoxal can form advanced glycation products, which contribute to the pathology of diabetic complications. Our objective was to study the metabolism of these metabolites and the associated protein carbonyation and cytotoxicity in isolated hepatocytes. In addition, the effect of oxidative stress on the metabolism of these toxins was also investigated. Methylglyoxal and glyoxal can induce protein carbonylation, which contributes to hepatocyte toxicity. Methylglyoxal, but not glyoxal, was detoxified mainly by the glyoxalase system. Both toxins can be metabolized by mitochondrial aldehyde dehydrogenase. The detoxification of glyoxal was impaired under oxidative stress conditions (i.e. increased hydrogen peroxide level). Glyoxal was found to be a common autoxidation product from glyceraldehyde, hydroxypyruvate and glycolaldehyde. Glyoxal and the reactive oxygen species formation during the autoxidation process contributed to the hepatocyte toxicity of glyceraldehyde, hydroxypyruvate and glycolaldehyde.

Doctor of Philosophy / Department of Chemistry / Stefan H. Bossmann / Pancreatic cancer is the fourth leading cause of cancer-related deaths in the USA. Asymptomatic early cancer stages and late diagnosis leads to very low survival rates of pancreatic cancers, compared to other cancers. Treatment options for advanced pancreatic cancer are limited to chemotherapy and/or radiation therapy, as surgical removal of the cancerous tissue becomes impossible at later stages. Therefore, there's a critical need for innovative and improved chemotherapeutic treatment of (late) pancreatic cancers. It is mandatory for successful treatment strategies to overcome the drug resistance associated with pancreatic cancers. Nanotechnology based drug formulations have been providing promising alternatives in cancer treatment due to their selective targeting and accumulation in tumor vasculature, which can be used for efficient delivery of chemotherapeutic agents to tumors and metastases. The research of my thesis is following the principle approach to high therapeutic efficacy that has been first described by Dr. Helmut Ringsdorf in 1975. However, I have extended the use of the Ringsdorf model from polymeric to nanoparticle-based drug carriers by exploring an iron / iron oxide nanoparticle based drug delivery system. A series of drug delivery systems have been synthesized by varying the total numbers and the ratio of the tumor homing peptide sequence CGKRK and the chemotherapeutic drug doxorubicin at the surfaces of Fe/Fe₃O₄-nanoparticles. The cytotoxicity of these nanoformulations was tested against murine pancreatic cancer cell lines (Pan02) to assess their therapeutic capabilities for effective treatments of pancreatic cancers. Healthy mouse fibroblast cells (STO) were also tested for comparison, because an effective chemotherapeutic drug has to be selective towards cancer cells. Optimal Experimental Design methodology was applied to identify the nanoformulation with the highest therapeutic activity. A statistical analysis method known as response surface methodology was carried out to evaluate the in-vitro cytotoxicity data, and to determine whether the chosen experimental parameters truly express the optimized conditions of the nanoparticle based drug delivery system. The overall goal was to optimize the therapeutic efficacy in nanoparticle-based pancreatic cancer treatment. Based on the statistical data, the most effective iron/iron oxide nanoparticle-based drug delivery system has been identified. Its Fe/Fe₃O₄ core has a diameter of 20 nm. The surface of this nanoparticle is loaded with the homing sequence CGKRK (139-142 peptide molecules per nanoparticle surface) and the chemotherapeutic agent doxorubicin (156-159 molecules per surface), This nanoplatform is a promising candidate for the nanoparticle-based chemotherapy of pancreatic cancer.

L’utilisation de nanovecteurs pour la livraison contrôlée de principes actifs est un concept commun de nous jours. Les systèmes de livraison actuels présentent encore cependant des limites au niveau du taux de relargage des principes actifs ainsi que de la stabilité des transporteurs. Les systèmes composés à la fois de nanovecteurs (liposomes, microgels et nanogels) et d’hydrogels peuvent cependant permettre de résoudre ces problèmes. Dans cette étude, nous avons développé un système de livraison contrôlé se basant sur l’incorporation d’un nanovecteur dans une matrice hydrogel dans le but de combler les lacunes des systèmes se basant sur un vecteur uniquement. Une telle combinaison pourrait permettre un contrôle accru du relargage par stabilisation réciproque. Plus spécifiquement, nous avons développé un hydrogel structuré intégrant des liposomes, microgels et nanogels séparément chargés en principes actifs modèles potentiellement relargués de manière contrôlé. Ce contrôle a été obtenu par la modification de différents paramètres tels que la température ainsi que la composition et la concentration en nanovecteurs. Nous avons comparé la capacité de chargement et la cinétique de relargage de la sulforhodamine B et de la rhodamine 6G en utilisant des liposomes de DOPC et DPPC à différents ratios, des nanogels de chitosan/acide hyaluronique et des microgels de N-isopropylacrylamide (NIPAM) à différents ratios d’acide méthacrylique, incorporés dans un hydrogel modèle d’acrylamide. Les liposomes présentaient des capacités de chargement modérés avec un relargage prolongé sur plus de dix jours alors que les nanogels présentaient des capacités de chargement plus élevées mais une cinétique de relargage plus rapide avec un épuisement de la cargaison en deux jours. Comparativement, les microgels relarguaient complétement leur contenu en un jour. Malgré une cinétique de relargage plus rapide, les microgels ont démontré la possibilité de contrôler finement le chargement en principe actif. Ce contrôle peut être atteint par la modification des propriétés structurelles ou en changeant le milieu d’incubation, comme l’a montré la corrélation avec les isothermes de Langmuir. Chaque système développé a démontré un potentiel contrôle du taux de relargage, ce qui en fait des candidats pour des investigations futures. / Controlled delivery of active compounds using nanoscale carriers is nowadays a common concept, but there are still limitations in current delivery systems related to active compound release rate and nanocarriers stability. To address these limitations, delivery systems can be made to incorporate both nanocarriers (liposomes, microgels and nanogels) and hydrogels. In this study, we have developed controlled delivery systems by combining different carriers in order to overcome deficiencies observed in systems using only one type of carrier. Such a combination could lead to an enhanced controlled release delivery system through synergistic stabilization. More specifically, we created a structured hydrogel embedded with either liposomes, microgels, or nanogels, each loaded with model active compounds that would be released in a controlled fashion by manipulating the temperature of release medium and nanocarriers composition and concentration. We compared drug loading and release kinetics of sulforhodamine B from liposomes (composed of DOPC and DPPC at different ratios) and nanogels (chitosan/hyaluronic acid) embedded in acrylamide hydrogels. We also compared drug loading and release kinetics of rhodamine 6G from microgels of N-isopropylacrylamide (NIPAM) with different ratios of methacrylic acid embedded in acrylamide hydrogel. Liposomes demonstrated a moderate drug loading capacity with sustained release for over ten days, while nanogels showed high drug loading but faster release kinetics, exhausting their contents within two days. Comparatively, microgels completely released their content within a day. Despite their faster release kinetics, microgels have shown the capacity to be finely tuned for efficient drug loading. The Langmuir isotherms indicated that it can be achieved by altering their structural properties or by changing their incubation medium. Each developed system has demonstrated a potential in controlling the release rate, which makes them candidates for further investigations in the future.

La transglutaminase tissulaire est une enzyme dépendante du calcium qui catalyse la formation de liens isopeptidiques, entre les chaînes latérales de résidus glutamine et lysine, permettant, par le fait même, la réticulation des protéines dans les systèmes biologiques. Elle joue un rôle, entre autres, dans l’endocytose, la régulation du développement des cellules, et même dans l’apoptose. Néanmoins, une dérégulation de l’activité biologique de cette enzyme peut entrainer différentes pathologies, comme la formation de cataractes, de plaques amyloïdes dans la maladie d’Alzheimer, ou encore peut mener au développement de la maladie céliaque. C’est pourquoi une meilleure connaissance du mécanisme d’action de cette enzyme et la possibilité de réguler son action à l’aide de substrats ou d’inhibiteurs sont nécessaires. Dans cette optique, une méthode d’expression et de purification de la transglutaminase humaine a été développée, permettant de travailler directement avec la cible pharmacologique désirée. De plus, une étude du mode d’inhibition et de liaison d’une classe d’inhibiteurs réversibles précédemment découverte dans le groupe, soit la famille des trans-cinnamoyles, a permis d’identifier que la puissance de ces molécules est influencée par la présence du calcium et qu’une inhibition dépendante du temps est observée, en lien avec un potentiel équilibre conformationnel lent de la transglutaminase. D’un autre côté, la susceptibilité à une attaque nucléophile par des thiols de cette classe de molécule rend leur potentiel pharmacologique grandement diminué, et c’est pourquoi une nouvelle famille de molécules a été identifiée, basée sur un squelette ynone, avec une valeur d’IC50 très prometteuse de 2,6 μM, en faisant un des meilleurs inhibiteurs réversibles de la transglutaminase développés à ce jour. Finalement, une stratégie de photomarquage jumelée à une analyse de spectrométrie de masse en tandem a été développée pour la découverte du site de liaison du substrat dérivé de la lysine, dans le but de mieux comprendre le mécanisme complexe de cette enzyme. / Tissue transglutaminase is a calcium-dependent enzyme that catalyzes the formation of isopeptide bonds between the side chains of glutamine and lysine residues, thereby resulting in the crosslinking of proteins in biological systems. It plays a role, among others, in endocytosis, the regulation of cell growth, and even in apoptosis. However, a deregulation of the biological activity of this enzyme can result in various pathologies, such as cataract formation, amyloid plaque formation in Alzheimer’s disease, or the development of celiac disease. Therefore, a better understanding of the mechanism of action of this enzyme and the ability to regulate its action using inhibitors or substrates is necessary. In this context, a method of expression and purification of human transglutaminase has been developed, allowing one to work directly with the desired pharmacological target. In addition, a study of the mode of inhibition and binding mode of a reversible inhibitor class previously discovered in the group, the family of trans-cinnamoyl derivatives, revealed that the potency of these molecules is influenced by the presence of calcium and a time-dependent inhibition is observed, related to a putative slow conformational equilibrium of transglutaminase. On the other hand, the susceptibility of this class of molecules to nucleophilic attack by thiols greatly diminishes their pharmacological potential, and that is why a new family of molecules has been identified, based on a ynone skeleton, with a very promising IC50 value of 2.6 μM, making this molecule one of the best transglutaminase reversible inhibitors developed to date. Finally, a photolabelling strategy combined with a tandem mass spectrometry analysis has been developed for the discovery of the binding site of the lysine derivative substrate, in order to better understand the complex mechanism of this enzyme.

Applied cancer research is heavily focused on the development of diagnostic tools with high sensitivity and specificity that are able to accurately detect the presence and anatomical location of neoplastic cells, as well as therapeutic strategies that are effective at curing or controlling the disease while being minimally invasive and having negligible side effects. Recently, much effort has been placed on the development of nanoparticles as diagnostic imaging and therapeutic agents, and several of these nanoplatforms have been successfully adopted in both the research and clinical arenas. This thesis describes the development of a nanoparticulate liposome system for use in a number of applications including multimodality imaging with computed tomography (CT) and magnetic resonance (MR), longitudinal vascular imaging, image-based biodistribution assessment, and CT detection of neoplastic and inflammatory lesions. Extensive in vitro and in vivo characterization was performed to determine the physico-chemical properties of the liposome agent, including its size, morphology, stability and agent loading, as well as its pharmacokinetics, biodistribution, tumor targeting and imaging performance. Emphasis was placed on the in vivo CT-based quantification of liposome accumulation and clearance from healthy and tumor tissues in a VX2 carcinoma rabbit model, gaining insight not only on the spatial but also the temporal biodistribution of the agent. The thesis concludes with a report that describes the performance of liposomes and CT imaging to detect and localize tumor and inflammatory lesions as compared to that of 18F-fluorodeoxyglucose (FDG) – positron emission tomography (PET). The outcome of the study suggests that liposome-CT could be employed as a competitive method for whole body image-based disease detection and localization. Overall, this work demonstrated that this liposome agent along with quantitative imaging systems and analysis tools, has the potential to positively impact cancer treatment outcome through improved diagnosis and staging, as well as enable personalization of treatment delivery via target delineation. However, in order to prove clinical benefit, steps must be taken to advance this agent through the regulatory stages and obtain approval for its use in humans. Ultimately, the clinical adoption of this multifunctional agent may offer improvements for disease detection, spatial delineation and therapy guidance.

Ovarian cancer has the highest mortality rate of all gynecological malignancies, due to inadequate treatment strategies and poor early diagnosis. Intraperitoneal (IP) chemotherapy administered on an intermittent schedule has been pursued for ovarian cancer treatment. However, local toxicities and complications associated with indwelling IP catheters required to deliver the chemotherapeutics have been documented. Furthermore, shortening or completely removing treatment-free periods between each chemotherapy cycle has shown improved efficacy compared to intermittent chemotherapy. The focus of this thesis was to develop and characterize a biocompatible and biodegradable IP injectable depot sustained drug delivery system as a new treatment strategy for ovarian cancer. A polymer-lipid injectable formulation (PoLigel) was developed and used for sustained docetaxel (DTX) delivery. The PoLigel resulted in homogeneous DTX peritoneal distribution and sustained plasma levels in healthy mice, which was in contrast to Taxotere®, the clinically used formulation of DTX. Sustained plasma, tissue, tumor and ascites DTX concentrations were observed in mice bearing IP SKOV3 tumors or ID8 ascites over a 3 week period following IP administration of the PoLigel. The intratumoral distribution and tumor penetration of DTX in subcutaneous (SC) and IP SKOV3 tumors were characterized. DTX distributed more towards the tumor core and diffused 1.5 fold further from blood vessels of the IP tumors compared to the SC tumors. The high efficacy observed in the IP SKOV3 and ID8 models and the SC SKOV3 model was attributed to favorable drug distribution at the whole-body, peritoneal and intratumoral levels in combination with local and systemic sustained drug exposure. Sustained chemotherapy with DTX alone and in combination with a drug efflux transporter inhibitor was investigated in multidrug resistant (MDR) ovarian cancer. In vitro, combination delivery via the PoLigel resulted in more apoptosis, greater intracellular accumulation of DTX, and lower DTX efflux in MDR ovarian cancer cells. Sustained combination chemotherapy was more than twice as efficacious as intermittent Taxotere® treatment in MDR ovarian cancer. Significant anti-tumor efficacy was also observed in the MDR model following sustained DTX chemotherapy compared to intermittent Taxotere®. Overall, results presented here encourage the clinical investigation of IP sustained chemotherapy for ovarian cancer treatment.

Title: “A comparative study of the impact of sustained and intermittent docetaxel chemotherapy in brain in a mouse model” Ji Zhang Master of Science Graduate Department of Pharmaceutical Sciences, University of Toronto November, 2011 Abstract A subset of patients suffers cognitive impairment during or long after chemotherapy. This may result from chemotherapeutic agents crossing the blood brain barrier (BBB). This thesis examined the effects of docetaxel (DTX) on brain toxicity, and the effects of different dosing schedules on brain DTX concentrations and neurotoxicity. Examination of DTX treated mice (total dose of 32mg/kg) revealed appreciable amounts of DTX crossed the BBB after either intermittent (four weekly doses) or sustained (one injection of DTX-PoLigel) administration despite differences in peak drug concentrations and overall exposure profiles. Measurements of autophagy and astrocytes activation not only provided evidence of DTX caused neurotoxicity in the central nervous system, but also revealed a link between dosing schedule and neurotoxicity. Furthermore, the discovery suggested connections between DTX brain exposure, diverse biological events (such as BBB permeability and reactive oxygen species activity), and the microenvironment at synapse-neuron junctions, which should be further explored.

Title: “A comparative study of the impact of sustained and intermittent docetaxel chemotherapy in brain in a mouse model” Ji Zhang Master of Science Graduate Department of Pharmaceutical Sciences, University of Toronto November, 2011 Abstract A subset of patients suffers cognitive impairment during or long after chemotherapy. This may result from chemotherapeutic agents crossing the blood brain barrier (BBB). This thesis examined the effects of docetaxel (DTX) on brain toxicity, and the effects of different dosing schedules on brain DTX concentrations and neurotoxicity. Examination of DTX treated mice (total dose of 32mg/kg) revealed appreciable amounts of DTX crossed the BBB after either intermittent (four weekly doses) or sustained (one injection of DTX-PoLigel) administration despite differences in peak drug concentrations and overall exposure profiles. Measurements of autophagy and astrocytes activation not only provided evidence of DTX caused neurotoxicity in the central nervous system, but also revealed a link between dosing schedule and neurotoxicity. Furthermore, the discovery suggested connections between DTX brain exposure, diverse biological events (such as BBB permeability and reactive oxygen species activity), and the microenvironment at synapse-neuron junctions, which should be further explored.

Ovarian cancer has the highest mortality rate of all gynecological malignancies, due to inadequate treatment strategies and poor early diagnosis. Intraperitoneal (IP) chemotherapy administered on an intermittent schedule has been pursued for ovarian cancer treatment. However, local toxicities and complications associated with indwelling IP catheters required to deliver the chemotherapeutics have been documented. Furthermore, shortening or completely removing treatment-free periods between each chemotherapy cycle has shown improved efficacy compared to intermittent chemotherapy. The focus of this thesis was to develop and characterize a biocompatible and biodegradable IP injectable depot sustained drug delivery system as a new treatment strategy for ovarian cancer. A polymer-lipid injectable formulation (PoLigel) was developed and used for sustained docetaxel (DTX) delivery. The PoLigel resulted in homogeneous DTX peritoneal distribution and sustained plasma levels in healthy mice, which was in contrast to Taxotere®, the clinically used formulation of DTX. Sustained plasma, tissue, tumor and ascites DTX concentrations were observed in mice bearing IP SKOV3 tumors or ID8 ascites over a 3 week period following IP administration of the PoLigel. The intratumoral distribution and tumor penetration of DTX in subcutaneous (SC) and IP SKOV3 tumors were characterized. DTX distributed more towards the tumor core and diffused 1.5 fold further from blood vessels of the IP tumors compared to the SC tumors. The high efficacy observed in the IP SKOV3 and ID8 models and the SC SKOV3 model was attributed to favorable drug distribution at the whole-body, peritoneal and intratumoral levels in combination with local and systemic sustained drug exposure. Sustained chemotherapy with DTX alone and in combination with a drug efflux transporter inhibitor was investigated in multidrug resistant (MDR) ovarian cancer. In vitro, combination delivery via the PoLigel resulted in more apoptosis, greater intracellular accumulation of DTX, and lower DTX efflux in MDR ovarian cancer cells. Sustained combination chemotherapy was more than twice as efficacious as intermittent Taxotere® treatment in MDR ovarian cancer. Significant anti-tumor efficacy was also observed in the MDR model following sustained DTX chemotherapy compared to intermittent Taxotere®. Overall, results presented here encourage the clinical investigation of IP sustained chemotherapy for ovarian cancer treatment.