Marcel Bertran Bally

The gold standard for treating non-resectable, late-stage squamous cell carcinomas in the head and neck regions is through a combined modality approach of radiation therapy (RT) and chemotherapy. Despite its clinical success, a large percentage of patients suffer significant toxicities, which ultimately impact their compliance and quality-of-life. To remedy this, a drug delivery platform utilizing nanoparticles (NPs) could be employed where the NP would sequester the drug during circulation and would preferentially release its payload at the site of the tumor due to an external stimulus, such as RT. The protein, Zein, was selected as the NP platform as it is biocompatible, formulated using non-toxic reagents, and approved by the FDA. While Zein is able to make NPs due to the protein’s intrinsic properties, challenges regarding the reproducibility as well as its stability in physiological conditions are still being investigated. To overcome these limitations, the synthesis method of formulating Zein NPs was first investigated. It was found that Zein NPs synthesized using microfluidics produced consistent formulations. Moreover, the size and polydispersity of the formulations could predictively be modulated and selected in accordance with their intended application. Additionally, Zein NPs when combined with gold NPs demonstrated triggered release in preliminary studies using RT. To make the Zein NPs suitable for in vivo use, the chemical modification of Zein with polyethylene glycol (PEG) was investigated. PEG conjugation was investigated both pre- and post- NP formation and it was discovered that pre- functionalization of the Zein was a more robust method in forming PEG-Zein NPs. When injected intravenously into animals, both high and low PEG containing PEG-Zein NP formulations displayed no acute or chronic toxicities at the doses given (70 mg/kg). Despite the significant potential of PEG-Zein NPs uncovered, multi-dose administration of PEG-Zein NPs caused significant clinical signs upon the second administration (e.g. labored breathing, visible pain, and even death). Therefore, further considerations will be necessary to devise an optimal strategy for PEG-Zein NPs, such as priming of the immune system before PEG-Zein NP administration (e.g. the use of prophylactic therapy) and/or selecting dosing schedules which align with the constraints of the technology.

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For more than 30 years, treatment of acute myeloid leukemia (AML) has remained largely unchanged and reliant on chemotherapeutic drug combinations. Despite the approval of novel targeted therapies, broad-spectrum chemotherapy drugs have been used to address the genetic heterogeneity of AML. One class of broad-spectrum compounds called flavonoids has shown promise in the treatment of AML. Of the flavonoids, quercetin and flavopiridol were selected for further investigations. Both quercetin and flavopiridol are hindered therapeutically by their poor solubility in aqueous solutions and tendency to bind serum proteins. To overcome these limitations, quercetin and flavopiridol were reformulated in copper-containing liposomes using the flavonoids’ metal-binding properties. Both formulations attained significant increases in apparent solubility (>100-fold). When injected intravenously into animals, both formulations exhibited an increased plasma area under the curve (AUC)) with no acute or chronic toxicities at the doses given. Given quercetin’s general lack of therapeutic activity as a single agent (IC50 of >10µM when tested against cancer cell lines), the research focused on the formulation of flavopiridol. In subcutaneous AML models, the flavopiridol formulation exhibited enhanced activity compared to a low pH free flavopiridol formulation. Despite the significant therapeutic potential uncovered in this thesis, liposomal flavorpidol’s potential is likely best achieved as part of a combination treatment. A combination regimen called FLAM (sequential treatment of flavopiridol, cytarabine, and mitoxantrone) inspired the development of a combination product consisting of liposomal flavopiridol and liposomal mitoxantrone. Free flavopiridol and mitoxantrone combined to produce significant synergistic activity in vitro. However, when administered in vivo, the combination of liposomal flavopiridol and liposomal mitoxantrone (referred to as enFlaM) resulted in an unexpected increase in toxicity and limited the dose of the combination that could be used in vivo. Although the enFlaM combination exhibited therapeutic effects at reduced doses, the effects were significantly less than what could be achieved with a well-tolerated dose of the single agent. Despite that further modifications will be necessary to devise an optimal combination scheme for the administration of enFlaM, the liposome-based Metaplex technology demonstrated the abilities to reformulate poorly soluble drugs to overcome limitations that would otherwise hinder their clinical utilities.

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Copper-based therapeutics (CBTs) are a promising class of drug candidates that have been shown to have anti-cancer activity. One of the major challenges associated with developing this emerging class of medicines, however, is their poor aqueous solubility (
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Novel treatments are urgently needed for patients with non-small cell lung cancer (NSCLC). Currently, these patients are almost always first treated with cisplatin (CDDP)-containing drug combinations. To identify therapeutic targets that could enhance CDDP activity, a genome-wide siRNA screen looking for synthetic lethal partners with low-dose CDDP was completed. These data were combined with results from a microarray study assessing differentially expressed genes in NSCLC cells exposed to low-dose CDDP. The results indicated that 151 genes were differentially expressed in cells exposed to low-dose CDDP. Nine up-regulated genes ranked within the top 10% of the siRNA screen based on a scoring method that considered minimal loss of cell viability from gene knockdown alone and significant enhancement of CDDP activity. Five genes were further validated and two (RRM2B and CABYR) were found to significantly improve the cytotoxic effects of CDDP. Pathways involved in repairing double-stranded DNA breaks and INO80 chromatin remodeling were enriched in both datasets. Analysis of the kinome subset of the siRNA screen also identified PAPSS1 (3’-phosphoadenosine 5’-phosphosulfate synthase 1) as a protein that when silenced sensitized NSCLC cells to CDDP. PAPSS1 produces the biologically active form of sulfate for sulfonation reactions. PAPSS1-silencing combined with low-dose CDDP reduced the clonogenicity of NSCLC cells by 98.7%, increased DNA damage, and induced G1/S phase cell cycle arrest. PAPSS1 suppression also sensitized NSCLC cells to radiation and topoisomerase I inhibitors. Sensitization was cancer cell specific. The extent of CDDP potentiation increased substantially when NSCLC cells were stressed by starvation or hypoxia. In NSCLC cell spheroids and zebrafish xenografts, PAPSS1 silencing in combination with CDDP decreased tumor size, while the same dose of CDDP combined with non-silencing controls led to increases in tumor size. In a subcutaneous tumour model, expression of PAPSS1-targeting shRNA in combination with a non-curative dose of CDDP enhanced activity compared to controls. Future studies are needed to identify small molecule inhibitors and proteins that interact with PAPSS1. These tools will be useful to fully understand the mechanisms by which chemosensitization occurs and such tool compounds may prove useful as therapeutics that would benefit NSCLC patients when first treated.

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This body of work describes a novel methodology for discovering and developing new cancer drugs based on therapeutic monoclonal antibodies. Such antibodies generally contain two sites where they bind to their target, but interesting improvements are often observed when the valence (number of target-binding sites) is increased above two. The methodology outlined in this dissertation involves using liposomes to prepare multivalent antibody-lipid nanoparticle formulations of different valence that can be utilized for preclinical drug development. As a proof-of-concept, we applied the methodology to rituximab, a therapeutic antibody used to treat lymphomas and leukemias. For the same dose of rituximab, multivalent rituximab-lipid nanoparticles with valences up to ~250 showed significantly elevated anticancer activity from enhanced complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and direct induction of apoptosis. A valence-dependent improvement in apoptosis in lymphoma cells was observed up to levels that were 21-fold higher than those observed after treatment with bivalent rituximab.We subsequently employed the different valences of multivalent rituximab to investigate its poorly defined direct mechanism of action. We uncovered a novel mechanism consisting of upregulation and activation of CD120a which led to ensuing apoptosis. Effector cells of the immune system were capable of hypercrosslinking rituximab on lymphoma cells and reproducing this mechanism, suggesting that it contributes to the in vivo cytotoxicity of regular bivalent rituximab therapy.The methodology described in this dissertation can therefore serve to identify antibodies that are more active as multivalent rather than bivalent molecules, define the optimal valence of such antibodies, and elucidate the mechanism of action of the new multivalent drugs. Furthermore, we illustrate that this information applies to other types of constructs with similar valences, enabling use of the methodology for advancing both liposomal and non-liposomal multivalent antibody formulations to preclinical maturity. Finally, this work suggests that every therapeutic antibody may have a different valence where it shows optimal therapeutic activity. For example, antibodies directed against targets that exert therapeutic effects upon clustering may show maximum activity at valences above two. This methodology can easily be applied to other antibodies in an effort to develop superior therapies against nearly any type of cancer.

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Platinum-refractory ovarian cancer is considered an incurable disease as current treatments are only palliative. Improvements in treatment will be realized as our understanding of the unique signaling pathways driving disease development increases and new therapeutics targeting these pathways are developed. It’s important to recognize, however, that at this time new molecularly targeted agents are not replacing the drugs being used to treat cancer; they are being used in combination with existing standards of care. In light of this, it is important to explore novel approaches using existing agents that are designed to achieve maximum therapeutic benefits in dosage forms that are well tolerated. Like most cancers, ovarian cancer is treated with a combination of drugs selected on the basis of complementary mechanisms of action and non-overlapping toxicities. Synergistic drug combinations can achieve therapeutic effects equal to that achieved with single agents, but at significantly lower and better tolerated doses. The factors that govern synergistic drug:drug interaction are, however, poorly understood and it is argued in this thesis that drug interactions favoring synergy will be influenced by drug exposure time. An effective method to enhance drug exposure time involves the use of drug carriers and a goal of this thesis was to develop an effective combination regimen against recurrent ovarian cancer using a novel lipid nanoparticle (LNP) formulation of topotecan and Doxil®; a LNP formulation of doxorubicin that has already been approved for the treatment of relapsed ovarian cancer. The LNP formulation of topotecan developed through this thesis research, referred to as TopophoreC™, was 2-to-3-fold more toxic than free topotecan, however this product candidate showed significantly better anti-tumor activity when compared to free topotecan administered at equivalent doses. Combinations of this LNP topotecan formulation with Doxil® were therapeutically superior to combinations of free topotecan and Doxil® as judged in two models of ovarian cancer. On the basis of these studies, it can be concluded that interaction between TopophoreC™ and Doxil® affect the pharmacokinetic behavior of Doxil® however the results provide proof of concept data to support the use of TopophoreC™ and Doxil® combination for treatment of recurrent ovarian cancer.

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Introduction: Despite the use of treatments, tumour recurrence in glioblastoma (GBM) patients is inevitable, partly because of the blood-brain barrier and the poor perfusion of the tumour vasculature, which act as two major obstacles to effective drug delivery. In order to address the latter, the capacity of liposomal formulations of irinotecan (Irinophore CTM; IrCTM), doxorubicin (Caelyx®) and vincristine to improve vascular function through normalization of GBM vasculature was assessed. In the following studies, the effect of IrCTM on the pharmacokinetics of irinotecan and its therapeutic efficacy in an orthotopic GBM model was compared to administration of the free form of the drug. In addition, siRNA-based therapy was explored as a potential strategy to enhance the efficacy of chemotherapeutics such as irinotecan. In one series of studies, the impact of cationic liposomes used for in vitro ILK (Integrin-Linked Kinase)-targeting siRNA delivery was compared to electroporation. Following successful identification of the most efficacious siRNA delivery method, EGFR and Rictor were selected as therapeutic targets because these proteins are involved in two of the most common molecular pathways reported to be dysregulated in GBM. The therapeutic potential of the combined silencing of EGFR and Rictor was assessed in in vitro and in vivo models of GBM. Results and conclusion: It was found that IrCTM, Caelyx® and liposomal vincristine induce vascular normalization in GBM tumours. It was also demonstrated that IrCTM increases exposure of the brain to irinotecan and its active metabolite SN-38 and improves survival of GBM tumour-bearing animals compared to treatment with free irinotecan. In vitro siRNA transfection using cationic lipids was found to alter the ILK downregulation time course compared to electroporation and to induce changes in pathway signaling that occurred independently of ILK silencing. Combined silencing of EGFR and Rictor reduced cell migration and increased cell sensitivity to chemotherapeutics in vitro. In vivo, dual silencing of EGFR and Rictor led to GBM tumour eradication. In parallel, GBM cell lines expressing red fluorescent proteins were developed as a tool for orthotopic GBM tumour imaging in live animals. These studies demonstrate the potential of siRNA-based therapy targeting EGFR and Rictor to act in combination with optimized chemotherapy agents such as IrCTM to improve treatment outcome in GBM.

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Substantial preclinical evidence indicates that inhibition of Integrin Linked-Kinase (ILK) correlates with cytotoxic/cytostatic cellular effects, delayed tumour growth in animal models of cancer and inhibition of angiogenesis. It is increasingly evident that optimal therapeutic benefits obtained using ILK targeting strategies will only be achieved in combination settings. For this reason the therapeutic potential of the ILK small molecule inhibitor, QLT0267, alone or in combination with chemotherapies commonly used to treat breast cancer patients was investigated. The results suggested that the combination of QLT0267 and docetaxel (Dt) interacted synergistically when assessing metabolic activity as a therapeutic endpoint. Further endpoint analysis in cell lines with low Her2/neu levels revealed that the QLT0267/Dt combinations resulted in a 3- fold decrease in concentration of QLT0267 required to achieve 50% inhibition of P-AKT. For Her2/neu positive cell lines the QLT0267/Dt combination was antagonistic. In vivo studies using breast cancer cells (LCC6) implanted orthotopically demonstrated that treatment with QLT0267/Dt engendered improved therapeutic effects. Using luciferase positive LCC6 cells metastatic, orthotopic and ascites tumour models were characterized. The results suggested that the orthotopic LCC6 tumour model was most sensitive to docetaxel. Using the more docetaxel treatment refractory LCC6 model (disseminated disease) it was shown that QLT0267 could not sensitize the tumours to Dt treatment. These data suggest that clinical benefits of QLT0267/Dt in patients with breast cancer would most likely be observed used in the adjuvant or neoadjuvant setting. Finally, preliminary studies indicate that the effects of QLT0267 were influenced by Her2/neu expression. To understand how, six Her2/neu positive breast cancer cell lines were evaluated following treatment with QLT0267. These cell lines demonstrated suppression (32 to 87%) of total Her2/neu protein. Attenuation of ILK activity or expression was associated with decreases in YB-1 protein and transcript levels and decreased YB-1 promoter activity. YB-1 is a known transcriptional regulator of Her2/neu expression. ILK inhibition also engendered suppression in TWIST (a regulator of YB-1 expression) protein expression. Taken together, these data indicate that ILK regulates the expression of Her2/neu through TWIST and YB-1, lending support to the use of ILK inhibitors in the treatment of aggressive Her2/neu positive tumours.

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No abstract available.