Pharmacy
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This is the collection for the University of Waterloo's School of Pharmacy, which includes papers submitted at the conclusion of pharmacy residencies administrated by the School of Pharmacy.
Research outputs are organized by type (eg. Master Thesis, Article, Conference Paper).
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Item Isoform-specific Roles of Prolyl-Hydroxylases in the Regulation of β-cell Insulin Secretion during Diet-Induced Obesity in Males(University of Waterloo, 2024-06-13) Jentz, EmelienType 2 diabetes affects approximately 480 million individuals worldwide and is associated with impaired tissue insulin sensitivity and β-cell dysfunction. Although there has been much research into nutrient-regulated insulin secretion and the progression to β-cell dysfunction in type 2 diabetes, the story is still incomplete. Hypoxia-inducible factor prolyl 4-hydroxylases (PHDs) are α-ketoglutarate dioxygenases commonly known to regulate hypoxia-inducible factor-1α (HIF-1α). Unique expression profiles of PHD1, PHD2 and PHD3 isozymes suggest isoform-specific roles in α-ketoglutarate-sustained insulin secretion. Our laboratory recently showed a role for β-cell PHD1 and PHD3 in insulin secretion, and previous research suggests that PHD2 may play a role in obesity-induced metabolic dysfunction. This thesis focuses on possible roles that β-cell PHDs may play in moderating the interrelationship between defective nutrient-sustained insulin secretion and obesity-induced β-cell dysfunction. We placed β-cell-specific PHD1, PHD2 or PHD3 knockout mice on a high-fat diet to explore the roles of PHD isoforms in regulating β-cell function under diet-induced obesity. β-cell-specific PHD1 knockout mice did not display any unique obesity-induced metabolic phenotypes compared to high-fat diet-fed control mice. β-cell-specific PHD3 knockout mice on the high-fat diet experienced increased weight gain compared to high-fat diet-fed control mice. However, despite increased fasting blood glucose levels, they showed no exacerbated impairments to in vivo glucose homeostasis and plasma lipid profiles. β-cell-specific PHD2 knockout mice resisted high-fat diet-induced obesity and showed improved in vivo glucose homeostasis combined with minor alterations in their plasma lipid profile. The lack of obesity-induced metabolic dysfunction in β-cell-specific PHD2 knockout mice could be explained by enhanced β-cell mass and ex vivo glucose-stimulated insulin secretion. Overall, β-cell-specific PHD2 knockout mice have ameliorated glucose homeostasis and β-cell function during obesity, potentially due to PHD2’s role in discouraging HIF-1α stability during metabolic stress.Item USER EXPERIENCE WITH MEDICATION ADHERENCE TECHNOLOGY: DETERMINING USABILITY BY CAPABILITIES(University of Waterloo, 2024-05-27) Baby, BincyBackground: As populations worldwide grow older, the prevalence of chronic conditions and the complexity of managing multiple medications significantly increase. This challenge is further complicated by a range of barriers older adults face, including physical limitations, cognitive impairments, sensory issues, motivational challenges, and non-supportive environments. Such barriers can lead to a decline in capacity to self-manage medications, resulting in poor adherence to prescribed medication regimens, which in turn can cause increased hospitalizations and a decrease in quality of life. Medication Adherence Technologies (MATech), which range from simple electronic devices to more complex smart devices with connectivity and real-time monitoring capabilities, are recognized as one of the solutions to these challenges. However, the design and features of these technologies vary significantly, influencing how they are used by different users. Usability varies widely; some older adults may find certain features of these devices challenging to use due to their barriers. Hence, it is crucial to ensure that MATech are accessible and user-friendly for all older adults, regardless of their individual challenges. This study aims to identify the most suitable MATech for older adults with various physical, cognitive, sensory, motivational, and environmental limitations, tailored to their unique needs and abilities. Objectives: The primary objectives of this study were to evaluate the usability and user experience (UX) of thirteen MATech devices among older adults facing various barriers to medication self-management and to gather comprehensive feedback on the usability and features of these technologies. Secondary objectives included determining how different barriers affect the usability outcomes of these technologies and identifying design features that best meet the needs of this demographic to enhance their independence and well-being. Methods: The study used a mixed-methods approach to evaluate the usability of MATech. Eighty older adults, aged 60 and older, were recruited through convenience, purposive, and snowball sampling methods from various settings across Ontario, including academic and residential facilities. Data collection was conducted in three steps after obtaining informed consent from the participants. The first step involved measuring barriers to medication self-management using various scales such as the Self-Medication Assessment Tool (SMAT) for physical, cognitive, and vision barriers; the Whisper Test for hearing barriers; the Self-Efficacy for Medication Adherence Scale (SEAMS) for motivational barriers; and the Martin and Park Environmental Demands (MPED) Questionnaire for environmental barriers. The second step involved usability and user experience testing of three smart devices and ten electronic devices, to measure various performance-based metrics (task success rate, total task completion time, efficiency, error rate) and perception-based usability metrics (System Usability Scale (SUS) score, NASA-TLX workload score, Single Ease of Use Question (SEQ), and Subjective Mental Effort Question (SMEQ)). The third step consisted of in-depth qualitative interviews to explore feedback regarding the features of various MATech tested. Quantitative data were statistically analyzed using descriptive statistics and univariate and multivariate regression to assess usability across various devices, while qualitative responses were analyzed using inductive thematic analysis. Results: Quantitative Results: Cognitive impairments were identified in 20% of participants, physical limitations in 33.75%, hearing impairment (both ears) in 60%, and vision impairments in 11.25%. Backward stepwise multivariate regression analysis identified critical predictors for task success rates, including 'SEAMS score' (p<0.001) which measures motivational barrier positively influencing outcomes, whereas 'Low vision score' negatively affected success rates (p<0.001). Moreover, Old 'age' (p<0.001) and 'number of subtasks for product' (p<0.001) notably extended the total task completion times, and 'physical score' (p<0.001) increased error rates, suggesting necessary improvements in MATech design for better usability. While no predictors significantly impacted the SUS scores, the NASA TLX identified 'old age', 'vision impairment', and the ‘number of products tested’ as significant factors in perceived task load, particularly noting that using multiple products increased task load considerably, underscoring their profound impact on user experience and workload management. Predictive models were also developed to determine each participant's ability to successfully complete subtasks. For example, the model for a participant characterized by significant cognitive, physical, hearing, motivational and environmental impairments, but with high vision capacity, indicated high success probabilities for visually intensive subtasks such as "scroll the screen options" (92%) and "locate and touch an icon on a screen" (87%). Conversely, tasks requiring more physical interaction like "flip device" showed much lower success probabilities (45%). Qualitative Findings: Five themes were identified: (1) the practicality of device design, (2) the impact of technological complexity, (3) the necessity for inclusivity in device functionality, which includes considerations for impairments, security, and privacy, (4) the influence of socio-economic and environmental factors, and (5) the importance of feedback for iterative design. Discussion: The findings from this study underscore the critical importance of designing MATech that are not only functional but also tailored to the unique needs of older adults who face multiple barriers to effective medication management. Key findings from the regression analyses highlighted the importance of addressing physical and sensory impairments in MATech design, as these significantly influence user performance and error rates. Additionally, factors such as age and the complexity of device operations significantly influence usability and workload, suggesting the need for simpler, more intuitive designs that minimize cognitive and physical strain. Overall, the research emphasizes the need for a user-centered design approach in developing MATech, emphasizing simplicity, accessibility, and personalization to better support older adults in managing their medications effectively. This approach not only aids in improving medication adherence but also contributes to the broader goal of facilitating a more independent, quality life for older adults.Item Arylbenzamide and Arylcarboxamide Derivatives as Modulators of Amyloid-Beta Aggregation(University of Waterloo, 2024-03-11) Zhao, YushengAlzheimer’s disease (AD) is a complex neurodegenerative disease with increasing incidence and prevalence globally. The current AD therapies based on small molecules offer only symptomatic relief and are not curative therapies. The recently launched anti-amyloid monoclonal antibodies hold promise although these are new to the market and their long-term benefits and potential disease-modifying effects are unknown. The global increases in the aging population and increasing life span mandate the need to understand the mechanisms of AD and discover effective and safe therapies. Over the past several decades, few hypotheses have been proposed to explain the pathophysiology of AD, among which the amyloid beta (Aβ) cascade is now considered as one of the initiating factors that drives the progression and other pathological factors of AD. The aggregation of Aβ into oligomers and fibrils together with its downstream signaling pathway are neurotoxic. Thus, small molecule modulators that could reduce the overall toxic burden of Aβ aggregates are thought to be beneficial in treating AD. In this thesis, a library of 72 small molecule derivatives were designed based on the chemical structure of chalcone and curcumin, two bioactive natural compounds that are able to modulate Aβ aggregation and reduce their neurotoxicity. The derivatives reported in this thesis encompass four different templates, namely, N-benzyl (Chapter 2), N-phenethyl (Chapter 3), N-benzyloxy (Chapter 4), and N-phenyl (Chapter 5) benzamide and carboxamides. These compounds were synthesized by coupling the amine substrates with either acid halides or carboxylic acids to obtain the target compounds in 72-93.6% yields. A number of biophysical and biochemical experiments were carried out to determine the ability of these small molecules to modulate the aggregation properties of Aβ42. The experiments carried out include i) thioflavin T based fluorescence aggregation kinetics experiments; ii) transmission electron microscopy studies; iii) 8-anilino-1-naphthalenesulfonic acid based fluorescence spectroscopy; iv) antioxidant assay by fluorescence spectroscopy; iv) cell viability studies in mouse hippocampal HT22 neuronal cells and Aβ42-induced neurotoxicity assay; v) fluorescence microscopy studies to assess the neurotoxicity using Proteostat dye, and vi) computational modelling studies to determine the interactions of small molecules with Aβ42 aggregates. From this library, 51 aggregation inhibitors were identified (inhibition of Aβ42 ranging from 7-53.1% at 25 µM). These derivatives were able to provide significant neuroprotection from Aβ42-induced cytotoxicity in mouse hippocampal HT22 cells (cell viability ranging from 80.8-96.8% versus 38.7% for Aβ42-treated control). Molecular docking studies indicate that these derivatives were able to interact with the hydrophobic domains of the Aβ42 oligomer and fibril through hydrophobic interactions. In a striking and unusual finding, 8 derivatives were identified as Aβ42 aggregation promotors with the ability to promote the aggregation by 1.2-5.1 folds. Two lead promotors 14b (N-benzylbenzofuran-2-carboxamide) and 14c (N-benzylbenzo[b]thiophene-2-carboxamide) were identified. These two compounds were able to rescue HT22 cells from Aβ42-induced cytotoxicity (cell viability 73.8% and 73.9% for 14b and 14c versus 19.7% for Aβ42-treated control). These two compounds have the ability to increase the surface hydrophobicity of Aβ42 aggregates and promote fibrillogenesis. Molecular docking studies suggested that Aβ42 aggregates might undergo conformational change upon binding and thus transit to much more stable and less toxic/nontoxic fibrils. Further structure-activity relationship study indicated that the hydroxy- and methoxy-disubstituted phenyl moiety was required to possess Aβ42 inhibition activity, where the presence of bicyclic aromatic rings such as benzofuran and benzothiophene, and 4-methoxyphenyl moiety is required for pro-aggregation activity. The results show that these benzamides and carboxamides possessing N-benzyl, N-phenethyl, N-benzyloxy, and N-phenyl templates hold promise in the design and development of novel small molecules as Aβ42 aggregation modulators. Remarkably 14b (N-benzylbenzofuran-2-carboxamide) and 14c (N-benzylbenzo[b]thiophene-2-carboxamide) were able to accelerate Aβ42 aggregation and remodel the aggregation pathway to form less toxic/nontoxic aggregates suggesting their application as novel chemical tools to understand the mechanisms of Aβ42 aggregation cascade.Item Enhancing the Ministring DNA (msDNA) Purification Using PI-Sce1 Homing Endonuclease/CRISPR-Cas3 Recombinant System(University of Waterloo, 2024-02-05) Fernando, MeriumIn the generation of msDNA the recombinant E. coli cells are transformed by a msDNA generating precursor plasmid, whereupon expression of the Tel protelomerase enzyme, acting on the pal target sequence present in the precursor plasmid, generated linear covalently closed (LCC) msDNA. However, the in vivo recombinant platform to produce msDNA results in a mixture of plasmids including unprocessed precursor plasmid, unwanted LCC bacterial backbone, and their topological isoforms, which interferes with the purification of the target species. For larger scale synthesis, the plasmid extract needs to be pretreated with commercially available restriction enzymes before being purified through chromatographic columns. Meanwhile, at the laboratory scale, msDNA is purified from agarose gels based on their size. These purification processes are time-consuming and inefficient and therefore, there is a need to optimize the process. To address this issue, we developed two in vivo recombinant systems for digesting the unwanted prokaryotic backbone and unprocessed precursor plasmid. These systems are the PI-SceI homing endonuclease enzyme system and the clustered regularly interspaced short palindromic repeats-Cas3 (CRISPR-Cas3) system. Homing endonucleases are highly specific DNA cleaving enzymes. The homing endonuclease PI-SceI, encoding gene vma from Saccharomyces cerevisiae was successfully integrated into the tel integrated bacterial chromosome via site-specific recombination using conditional replication and integration (CRIM) plasmid. The double integrants, both vma and tel integrated recombinant bacteria, were transformed with msDNA synthesizing precursor plasmids and induced the msDNA synthesis and vma gene overexpression. Even though the double integrants were able to overexpress the homing endonuclease enzyme and digest the precursor plasmid, they were not able to synthesize msDNA. Therefore, the Tel protelomerase enzyme was expressed episomally inside the vma integrated recombinant bacteria. This vma gene is under the control of an inducible PBAD promoter. In the presence of L-arabinose in the media, the Tel protelomerase enzyme was episomally expressed and synthesized msDNA by acting on the precursor plasmid. Subsequently, the overexpressed PI-SceI homing endonuclease enzyme digested the undesired byproducts of msDNA synthesis as expected. Introducing homing endonuclease enzyme recognition sequences into the Tel protelomerase enzyme-expressing plasmid will further improve the purification process. The other recombinant system that was developed is the utilization of the CRISPR-Cas3 system which is naturally present in W3110 E. coli K-12 bacteria. A pre-crRNA targeting the origin of replication (ori) of the msDNA synthesizing precursor plasmid was successfully designed and cloned into the low copy number plasmid. The pre-crRNA expressing gene cassette was placed under the control of the PBAD promoter. Upon overexpression, crRNA was synthesized inside the W3110 E. coli K-12 bacteria. The crRNA bound to the expressed CRISPR-Cas3 protein cascade of the bacteria, guided the effector complex to the target sequence and successfully digested the targeted precursor plasmid. Even though the W3110 tel+ recombinant bacteria synthesized msDNA in a pre-crRNA expressing background, an efficient degradation of the unwanted by-products of msDNA synthesis was not observed. This could be due to the disruption of the CRISPR locus of W3110 tel+ recombinant bacteria. Episomal expression of the CRISPR-Cas genes inside W3110 tel+ recombinant bacteria will enhance the digestion of the non-msDNA species.Item Development of Modular Polymeric NPs for Drug Delivery Using Amine-Reactive Chemistry(University of Waterloo, 2024-01-26) Wong, CalvinCancer remains one of the leading causes of death worldwide and very often requires chemotherapy treatment. Despite advances in chemotherapy treatments, some cancers remain difficult to treat due to tumour type, location, and in some cases, the development of drug resistances. In order to tackle cancer more effectively, researchers have explored and developed novel chemotherapy agents. However, many of these agents suffer from low bioavailability or prohibitively high toxicity to the body. Nanotechnology-based drug delivery systems aim to assist in protection and site-specific delivery of these potential anti-cancer agents, increasing their effectiveness and lowering toxic effects. Polymeric NP delivery systems can encapsulate drugs and be coated with functional groups or moieties to enhance various properties such as targeting. In this project, poly(lactic-co-glycolic) acid (PLGA ) NPs were synthesized to encapsulate curcumin (CUR) via single emulsion method. CUR, the principal constituent of Curcuma longa, commonly known as turmeric, has been explored for its anti-cancer potential, but is severely limited by its hydrophobicity and sensitivity to light and water. The PLGA NPs were coated with oligomeric chitosan (COS) and RGD peptide (peptide consisting of Arg-Gly-Asp) using amine-reactive chemistry (NHS and EDC). Both COS and RGD had been previously shown to accumulate and target many different types of cancer cells. NPs were characterised based on size distribution, zeta potential, and binding efficiency of RGD peptide. They were also evaluated on encapsulation efficiency, and stability, of CUR within the NPs. OVCAR-3 cancer cells were treated with COS and RGD-coated PLGA NPs loaded with Coumarin-6 dye for fluorescent imaging of cell uptake. They were also treated with CUR-loaded NPs to determine cytotoxicity and effectiveness of delivery. The NPs exhibited size distribution and zeta potential within expected values, though binding efficiency of RGD was low. CUR-loaded NPs showed significant increase in cytotoxicity over free (unencapsulated) CUR, and void (empty) NPs, suggesting successful delivery of CUR as an anti-cancer agent; the performance of COS and RGD coated NPs over bare PLGA NPs was inconclusive, however. Optimization will be required to improve formulation during the coating steps. Further investigation may be required into alternative binding chemistry, such as click chemistry.Item Probing the interactions that drive RNA binding and self-association of hnRNPA1 implicated in neurodegeneration(University of Waterloo, 2024-01-17) Fatima, Syeda SakinaThe heteronuclear ribonucleoprotein A1 (hnRNPA1 or A1) is associated with the pathology of different diseases, including neurological disorders and cancers. In particular, the aggregation and dysfunction of A1 has been identified as a critical driver for neurodegeneration in Multiple Sclerosis (MS). Structurally, A1 includes a low-complexity domain (LCD) and two RNA-recognition motifs (RRMs), and their interdomain coordination may play a crucial role in A1 aggregation. Previous studies propose that RNA-inhibitors or nucleoside analogs that bind to RRMs can potentially prevent A1 self-association. Therefore molecular-level understanding on the RNA recognition by A1 RRMs remains of scientific interest. Although several crystal structures of RNA-bound RRM complexes have been reported in the literature, there are still open questions about which RRM RNA prefers to bind and why only specific RNA sequences tend to bind A1. This thesis aims at probing the structures, dynamics and nucleotide interactions with A1’s RRMs using a combination of advanced computational methods. Our research to-date has revealed that adenine and guanine in RNAs (or DNAs), and the key residues from the interdomain linker connecting the two RRM domains contribute significantly for RNA binding to A1 RRMs. Further research will seek to address the impact of RNA length on its binding and how RNA specificities vary between the RRMs. Critical residues for RNA-binding have been identified and their molecular-level insights on their nucleotide preferences have been evaluated. As a final addition, the full-length A1 protein for which a crystal structure in the PDB does not exist, is modeled, to analyze the interactions that occur between the RRMs and the LCD domain that could promote A1’s aggregation. Both of A1’s known isoforms, isoform A (320aa) and isoform B (372aa) have been modeled and studied, with and without RNA bound to them. Our data suggests that interplay between the LCD and the RRMs may block exposure of critical RNA-binding residues to the environment when RNA is not already bound to the protein. Taken together, this thesis elaborates on full protein dynamics and nucleotide-protein interactions that may be helpful in designing therapeutics. Nucleotide-based therapies or nucleoside analogs in particular, can be designed based on specific interactions outlined in this thesis.Item Medication Review in Cognitive Impairment and Dementia: Comparison of Instruments(University of Waterloo, 2023-12-19) Sharma, RishabhBackground: - Cognitive impairment (CI) and dementia are significant concerns in older adults in Canada. Drug-related problems (DRPs) are common and can cause up to 30% of hospitalizations in older individuals, including adverse drug reactions, drug interactions, potentially inappropriate medication (PIM) use, and medication adherence. Prescribing in older patients with multiple morbidities, especially with dementia, is a complex process that demands regular review of medications to provide quality care to dementia patients. Objective: - The primary objective was to compare the mean number of DRPs using the Medication Review in Cognitive Impairment and Dementia (MedRevCiD) Checklist to the Medication Appropriateness Index (MAI) criteria in older adults with CI and/or dementia. The secondary objective was to identify which explicit tool, Beers Criteria 2023, or the Screening Tool of Older People Potentially Inappropriate Prescriptions (STOPP) Criteria 2023, identified more PIM use among older adults with CI or dementia. Methods: - A cross-sectional study was carried out with older adults receiving care for CI or dementia. Forty-four patients from the Multi-specialty Interprofessional Team-based (MINT) memory clinic were recruited to participate in the study. The researcher employed two distinct tools, namely the MAI and the MedRevCiD Checklist, to conduct a medication review. PIMs were identified utilizing the Beers Criteria 2023 and the STOPP Criteria 2023. The Wilcoxon signed-rank test was used to assess whether there is a significant difference in the mean number of DRPs identified by the MedRevCiD versus MAI. Bivariate logistic regression analysis was employed to identify potential factors associated with DRP and PIM use. Results: - A total of 134 DRPs were identified in 44 patients per the MedRevCiD checklist. The average number of DRPs identified was 3.05, with a standard deviation (SD) of 4.0 DRPs per person. Notably, over half of the DRPs (53%, n= 71) identified fell into domain 6 of the MedRevCiD checklist (optimizing medication use). In comparison, 81 DRPs were identified in 44 patients per MAI criteria (mean: 1.84 per person, SD 2.9) DRPs per person. The majority of the DRPs identified using MAI criteria (44.4%, n= 36) were from clinically significant drug-disease/condition interactions. There was a significant difference in the mean number of DRPs between the two instruments (Z= -4.735, p-value <0.001). In this study, at least one PIM was used by 47.7% (n= 21) and 27.2% (n= 12) of participants based on Beers and STOPP criteria, respectively. Binary logistic regression revealed a statistically significant association between the number of comorbidities (P= 0.002), number of medications per day (P= 0.032) with DRP use as per MAI criteria. For each additional comorbidity, there was 1.86 times higher odds of experiencing DRPs and 1.20 times higher odds of having DRPs according to MAI criteria. Individuals with nine or more comorbidities had 8.4 times higher odds of being prescribed PIMs (p = 0.027, 95% confidence interval (CI): 1.27 – 55.39); given the wide range of the confidence interval it is essential to note that there was considerable uncertainty about the strength of the association. Discussion: - The findings of this study provided insights into the higher prevalence of DRPs among older adults with CI or dementia. The MedRevCiD Checklist emerged as a valuable tool, demonstrating a heightened ability to uncover DRPs in this population. This underscores the importance of utilizing tools tailored to the unique needs of individuals with dementia when assessing DRPs. Furthermore, identifying PIMs using Beers and STOPP criteria highlights the significance of addressing PIMs in this demographic. This study adds valuable insights to the progressing comprehension of medication complexities in older adults facing CI and/or dementia.Item Serious Games on the Lived Experience of Dementia as Learning Tools in Pharmacy Education(University of Waterloo, 2023-10-23) Eskandari, YasamanDementia is a stigmatized and often ‘invisible’ condition which requires clinicians to have a nuanced understanding of the lived experience to build trust and provide better quality of care. Pharmacists are at the frontline of care for patients who may have dementia and there is a need for effective and engaging learning opportunities to prepare them for caring for patients living with dementia. Serious games have gained popularity for their potential in facilitating safe and engaging learning opportunities. However, there are limited applications of serious games in clinical education on the topic of dementia and little transparency on the development process. The thesis work outlined in this project intends to explore how serious games can best facilitate a learning experience for senior pharmacy students to better their understanding of the lived experience of dementia. The primary objective was to develop a serious game with multi-stakeholder input. The secondary objective was to provide game design recommendations for development of serious games on this topic based on end-user play-testing experiences. During both the development and user-testing, qualitative methods were used to gather thorough feedback and understand individual play experiences, namely semi-structured interviews and the think-aloud protocol. To develop a serious game, the game design framework for medical education was adapted in this project, which involved three stages: preparation and design, development, and formative evaluation. In the first stage, a clinician and a systems design expert were consulted to develop the first prototype. In the development stage, the prototype was reviewed by stakeholders including clinicians, people with lived experiences of dementia or care partners, and serious game researchers through semi-structured interviews, resulting in iterative improvements. Stakeholder feedback culminated in the development of a serious game with the goal of helping pharmacy students better understand the lived experience of dementia, in a digital, non-linear story format. During the final formative evaluation stage of game design, 11 senior pharmacy students, a pharmacy educator, and a social worker with expertise in dementia care tested the game. Their learning and play experiences were gauged through the semi-structured interview and think-aloud protocols. The qualitative data was analyzed using the framework method of analysis. Three factors were necessary for creating an engaging learning experience about dementia for senior pharmacy students. The first was facilitating understanding of dementia through an experiential approach using a realistic environment and authentic storytelling. The second was providing a problem-oriented experience by providing meaningful player interaction opportunities and creative freedom. Finally, novelty in the game format was necessary for an engaging experience. Future directions include recruiting more stakeholders and student participants with experiences relating to dementia, and utilizing these recommendations to improve on the game and assessing its impact on student empathy and confidence in caring for patients who have dementia.Item Machine learning modelling in predicting and optimizing PLGA nanoparticle encapsulation efficiency and therapeutic efficacy(University of Waterloo, 2023-09-20) Dong, SihanNanoparticles (NP) have become a promising drug delivery system in the past few decades in pharmaceutics for its diversity in encapsulating different types of drugs, including proteins/peptides, nucleic acids and small molecule drugs for the treatment of a variety of diseases. Application in cancer cell NP-based drug delivery has been a majority focus because of NP’s capability in delivering effective treatment while keeping side effects low. Often, series of chemical and biological assays need to be carried out to pursue certain research goals. However, NP fabrication process is rather time-consuming and costly, consisting of material selection, formation, purification, and characterization. As NP composition choices can directly influence the NP physicochemical properties and biological behaviors, it is crucial to find the optimized combination efficiently to achieve better NP performances. To ease the burden of conducting experiments manually, collaboration with artificial intelligence (AI) techniques is likely to be a promising choice. Machine learning (ML) as a sub-concept of AI has been a popular tool in many pharmaceutical sciences studies, such as prediction of protein molecular structures, drug discoveries, high throughput screening, and prediction of drug formulation compositions, etc. It has been of researchers’ great interests in implementing this emerging technique to a variety of tasks to speed up pharmaceutics development. In this study, we formulated 32 doxorubicin (DOX) or docetaxel (DTX)-loaded NPs to train and test ML-based Gaussian Processes (GP) models that can estimate the underlying relationships between four NP composition physicochemical properties (e.g., poly (lactic-co-glycolic) acid (PLGA) molecular weight (MW), PLGA lactic acid: glycolic acid (LA/GA) ratio, PLGA: drug weight ratio, and drug lipophilicity) and the corresponding drug EE% and therapeutic efficacy in ovarian cancer cells. No universal relationships between the predictor and response variables can be concluded. Three GP models including EE% model, DOX NP IC50 model, and DTX NP IC50 model were evaluated for their prediction accuracies that were measured by normalized-RMSE in testing sets. The normalized RMSE are 0.187, 0.296, and 0.206, respectively. The EE% model has the highest prediction accuracy that may be attributed to the larger training dataset compared to the other two models. Furthermore, a simplified Bayesian Optimization (BO) model was built to output a set of x variable values that can potentially help to find formulations that optimize the NP EE% and therapeutic efficacy. In EE% model, the suggested formulation is 2mg drug with lipophilicity of 2.12 being loaded in 94mg of 20001 Da, 1.17:1 (LA/GA) PLGA NP. In DOX NP IC50 model, the suggested formulation is 2 mg DOX-loaded 68mg of 39997 Da, 1.53:1 (LA/GA) PLGA NP. In DTX NP IC50 model, the suggested formulation is 2 mg DTX-loaded 90 mg of 20008 Da, 1.70:1 (LA/GA) PLGA NP.Item Evaluation of feasibility and potential impact of a clinical innovative software tool to support pharmacists to prescribe for minor ailments(University of Waterloo, 2023-09-05) Tasnim, HumayraBackground: The role of community pharmacists has shifted from dispensing prescribed medications to providing patient-focused clinical services such as Pharmacists Prescribing for Minor Ailments (PPMA). These services are expected to improve patient care. Pharmacists across provinces have been given some level of prescriptive authority since 2005. The PPMA service was first introduced in Alberta. Currently, nine out ten Canadian provinces have the authority for PPMA service. Ontario recently adopted PPMA, in January 2023. However, there are several barriers such as workload, time constraints and integration to workflow that can hinder pharmacists’ ability to implement PPMA into existing practice. Technological solutions such as computerized decision support system (CDSS) can help pharmacists and facilitate performing PPMA service. PharmAssess Diagnostics developed a CDSS that provides a digital software platform for pharmacists to provide clinical services such as minor ailment prescribing. Although previous research examined the acceptability and impact of CDSS in other professions, there is paucity of research about the feasibility and impact of implementing supporting software tools into the workflow of community pharmacies on adapting PPMA. Objective: The aim of this project is to evaluate the feasibility and potential impact of a clinical innovative software tool to support pharmacists for minor ailment prescribing. Method: This project followed a mixed method design. It included an anonymous online survey and semi-structured interview with community pharmacists/pharmacy student interns to explore their perspectives on the usability, acceptability, potential impact on integration in workflow and overall effect on workload while using the PharmAssess Diagnostics software for PPMA. In Ontario, pharmacists were recruited via the list of pharmacists in the Ontario College of Pharmacist (OCP) who agreed to be contacted for research purposes. Additionally, the co-op supervisors list from the School of Pharmacy University of Waterloo, and clients of the software company were contacted to be recruited. Demographic (age, gender) and professional characteristics (academic qualification, type of community pharmacy, current position in the pharmacy) were collected from participating pharmacists. Then, pharmacists’ perspectives were examined using a survey with Likert’s scale questions and were summarized. Descriptive statistics were used to describe the distribution of the responses. Semi-structured interviews were conducted with pharmacists/student interns who were further interested in providing their detailed perspectives with the use of this software tool in community pharmacy settings. The interviews were analyzed thematically. Then both forms of data were merged using the side-by-side approach to draw conclusion. Results: A total of 11 survey responses were collected. Pharmacists agreed that the software tool was usable (72%), acceptable (81%), had positive impact on workload (63%) and had positive impact on workflow (45%). Overall, 90% of participants stated that the average time per consultation using the software tool ranged between 5 to 15 minutes. Seven pharmacists were interviewed virtually. Three major themes emerged from the interviews, revealing usability of the software tool, facilitators, and barriers to the implementation of the software tool and impact of the software tool implementation into community pharmacy.Item Design and Characterization of Various Lipid, Polymer, and Gemini-Surfactant – Based Vectors and DNA as Non-Viral Delivery Systems for Gene Therapy Applications(University of Waterloo, 2023-08-31) Lall, Gurmeet KaurThe bulk of this thesis work is the result of a pharmaceutical collaboration, and it utilizes several proprietary systems. While Chapters 3 and 4 will provide an in-depth discussion of the results, the specific names, structures, and manufacturing processes of the systems cannot be disclosed. Genes are segments of DNA – the blueprint of all living organisms which contain instructions needed for organisms to grow, survive, and reproduce. However, genes that do not function properly can cause various diseases and genetic defects. Gene therapy has the potential to treat these diseases by correcting the underlying genetic problem. Recent success with the use of lipid nanoparticles have demonstrated that gene delivery by using synthetic vectors is effective and commercially achievable. However, other synthetic carriers, such as polymers and gemini surfactants (GS), continue to face challenges associated to low gene expression which limits their clinical application. The rational design approach, where individual vector components are selected based on favourable transfection efficiency properties, is often used in non-viral formulation development. Countless studies have used this approach to focus on designing efficient synthetic carriers. However, highly efficient non-viral gene therapeutics require the rational design of all components of the vector – namely the synthetic carrier and the DNA cargo. For example, to address the several challenges associated with the use of conventional circular covalently closed (CCC) plasmid DNA (pDNA) vectors, a new generation of DNA vectors have been under investigation, linear covalently closed (LCC) DNA minivectors. Although preliminary studies have shown that the in vitro transfection efficiency of formulations incorporating the LCC DNA minivectors is higher compared to formulations incorporating the CCC DNA counterpart, their overall transfection efficiencies still appear to be limited. Key to improving their efficiency is characterizing both the synthetic carrier and the DNA construct and to establish a relationship between molecular interactions, physicochemical properties, and transfection. This approach may optimize LCC DNA minivector-based formulations for future non-viral gene therapy applications. In collaboration with a pharmaceutical partner, we designed a study to assess the influence of DNA size and topology on physicochemical properties (i.e. size, polydispersity index (PDI), and zeta potential) and in vitro transfection efficiency. Cationic lipids (Lipofectamine 3000 and proprietary Lipid 2) complexed with either the proprietary LCC DNA minivectors or CCC pDNA in deionized water were prepared and evaluated. Comparable physicochemical properties were observed despite the differences in DNA size and topology. Sizes of all lipoplexes were below 325 nm and the positive zeta potentials appeared to increase linearly with increasing N+/P- ratio. Transfection efficiency was evaluated by expression of enhanced green florescence protein (eGFP) in HEK 293 and ARPE-19 cells in vitro, and cell viability was determined by propidium iodide staining. Statistically significant increases in transfection efficiency were observed with samples prepared with the LCC DNA minivectors and higher overall transfection was associated with Lipofectamine 3000/LCC DNA minivector lipoplexes as determined by one-way analysis of variance (ANOVA) and a dependent, two-tailed, two sample Student’s t-test (same results obtained by both methods). These findings reveal that a relationship between DNA size, topology, and transfection efficiency exists and should be considered for effective cationic lipid/LCC DNA minivector lipoplex design and formulation development. In an effort to improve the transfection efficiencies of LCC DNA minivectors for non-viral gene therapy, the relationship between physicochemical properties and transfection efficiency of polymer-LCC DNA minivectors were studied. Formulations comprising the proprietary Polymer 1 or proprietary Polymer 2 incorporating either LCC DNA minivectors or CCC pDNA in deionized water were prepared at several N+/P- ratios (2:1, 4:1, and 8:1) and their physicochemical properties (size, zeta potential, and PDI), transfection efficiency (eGFP expression), and cell viability (determined by propidium iodide staining) were evaluated. Higher transfection was achieved with polyplexes forming small (< 150 nm), uniformly distributed (PDI < 0.2) nanoparticles with an excess positive charge (zeta potential ~ 25 mV), presumably due to high cell interactions and internalization. The link to transfection efficiency found from this study may contribute to the rational design and optimization of future LCC DNA minivector-based polyplexes. To briefly provide insight into the low gene expression of GS-DOPE-LCC DNA minivector systems previously determined by our group, the mixing behaviour and interactions between DOPE and two GS (18-7-18 or 18-7NH-18) were characterized at pH 4 and pH 7 at various ratios using Langmuir monolayer methods. The mixing behaviours were derived from π–A curves by examining the excess free energy of mixing (ΔGE) which was calculated through the surface area additivity rule, while the intermolecular interactions of the mixtures were evaluated using the compressibility modulus (Cs-1). Synergistic interactions dominated in most binary mixtures, with the exception of XGS = 0.5 monolayers where a net repulsive force was dominant. This synergism may explain the low gene expression of GS-DOPE-LCC DNA minivectors, as it may attribute to low LCC DNA release from the vector. These findings may be used for future optimization.Item Piperine Derivatives as Modulators of Amyloid-Beta (Aβ) Aggregation(University of Waterloo, 2023-07-12) Wang, ShirleyAlzheimer’s Disease (AD) is a life-changing neurodegenerative disease that progresses in stages and currently has no effective cure. More than 597,000 Canadians suffer from AD. Symptoms of late-stage AD are severe and include memory loss, apathy, depression, and psychosis, resulting in large burdens for caregivers and the economy. A major factor leading to AD is the aggregation of a protein known as amyloid-β (Aβ). However, most pharmacotherapies for AD provide only symptomatic relief and target other pathways. The recently approved monoclonal antibodies (mAbs) are the only disease-modifying therapies targeting Aβ peptide aggregation in AD that are available in the US. Limitations of mAb therapies include high-cost for patients, high manufacturing costs and resource-intensive parenteral administration. In this regard, the current study focused on the development of novel anti-amyloid small molecules based on piperine, derived from the black pepper plant, that was reported to enhance cognition. Advantages of small molecule AD therapeutics include being highly scalable and applicable to non-invasive routes of drug administration. A library of 13 piperine derivatives were designed, synthesized, and evaluated as potential inhibitors of Aβ42 aggregation. The experiments carried out include i) developing chemistry reaction conditions to synthesize the target compounds, ii) compound characterization by analytical chemistry, iii) determining their inhibition activity toward Aβ42 aggregation by fluorescence aggregation kinetics studies and transmission electron microscopy, iv) computational modeling studies in the Aβ42 model to understand the binding interactions of lead compounds and v) cell viability studies in mouse hippocampal HT22 cells. The structure activity relationship (SAR) studies led to the identification of three piperine derivatives 4a (R = pyrrolidine), 4b (R = thiomorpholine) and 4c (R = morpholine) as the lead compounds with inhibition ranging from 35-48 % (at 10 µM). The results were also confirmed by electron microscopy studies which demonstrated the ability of these compounds to reduce Aβ42 aggregation. Molecular docking studies in the Aβ42 pentamer model show that the terminal acyl substituents play a major role in stabilizing the Aβ42 pentamer assembly by interacting at the amyloidogenic interface consisting of the C- and N-terminal residues. This prevents further self-assembly and aggregation. Cell culture assays in HT22 mouse hippocampal cells showed that the lead compounds 4a, 4b and 4c were not toxic (cell viability >90 %, at 25 µM). This is the first study that reports the discovery of novel piperine based compounds as direct inhibitors of Aβ42 aggregation. Overall, the results obtained from this thesis provide valuable insights in the design, development, and application of novel small molecules to study and treat AD.Item Investigating the Interactions of Fatty Acids, Amyloid Beta & SARS-CoV-2 Spike Protein Fragment(University of Waterloo, 2023-05-16) Toma, LilianThere is no cure for Alzheimer’s disease (AD), and the negative implications of having AD were further exacerbated in recent years, as patients with dementia are at the highest risk for mortality upon contracting COVID-19. The amyloid cascade theory postulates that AD is caused by toxic aggregates of amyloid beta (Aβ) peptide. The main objective of this project was to design, synthesize and evaluate a library of fatty acid derivatives based on docosahexaenoic acid (DHA), oleic acid (OA), eicosapentaenoic acid (EPA), linoleic acid (LNA), and α-linolenic acid (ALA) as inhibitors of Aβ42 aggregation. 10 fatty acid derivatives were synthesized, characterized, and evaluated for Aβ42 aggregation inhibition activity using thioflavin T-based Aβ42 aggregation kinetics assays. The methyl ester derivatives were found to be the most promising inhibitors, with the LNA derivative methyl (9Z,12Z)-octadeca-9,12-dienoate (2a) being the most potent (61% inhibition at 25 μM). Transmission electron microscopy (TEM) experiments confirmed the anti-aggregation activity of 2a, and computational modeling studies suggest that the evaluated fatty acid derivatives bind in a narrow channel at the interface of the N- and C-termini in the Aβ42 pentamer model. Furthermore, the fatty acid derivatives were not toxic to HT22 mouse hippocampal cells (cell viability ~94–104% at 25 μM). Our secondary objective was to evaluate amyloidogenic peptide fragment FKNIDGYFKI derived from the SARS-CoV-2 spike protein for its ability to promote Aβ42 aggregation. Interestingly, the decapeptide was found to inhibit Aβ42 aggregation at all tested concentrations (~37–52%). In summary, thesis outcomes demonstrate that fatty acid derivatives and spike peptide fragment exhibit anti-Aβ42 activity by direct binding and have the potential to be used as novel pharmacological tools to study Aβ aggregation and to design novel therapies to treat AD.Item Pediatric Physiologically Based Pharmacokinetic (PBPK) Modeling to Advance Knowledge of Breastfeeding Infant Exposure to Maternal Medications(University of Waterloo, 2023-05-04) Yeung, Cindy Hoi TingWhile there are benefits of breastfeeding to the maternal-infant pair, mothers taking medication may decide not to breastfeed amid unclear risks of exposing the infant to the drug through milk. Uncertainty arises mainly due to the fact that lactating mothers and breastfeeding infants are excluded in the drug development process. In lieu of necessary data for decision making, existing resources include metrics to help estimate risk to the breastfed infant and informational resources that aim to gather all sparsely available information in databases to increase accessibility and empower healthcare providers with knowledge. Current metrics such as the relative infant dose, solely estimate the dose the infant would intake. Before better understanding the potential adverse events an infant might experience (response), a step further to understand exposure is paramount. Yet, the availability of exposure information is difficult to ascertain due to the lack of critical information on the pharmacokinetics (PK; movement of drugs in the body describing dose to exposure) of drug secretion into breast milk, and the resultant levels or exposure of the drug in infant plasma. Physiologically based pharmacokinetic (PBPK) modeling is a promising tool to fill in the gap of scant maternal medication exposure information in breastfeeding infants. PBPK models use a simulation-based approach to model drug kinetics in an organism using knowledge of anatomy and physiology and the physicochemical properties of the drug. Pediatric PBPK models can be developed with minimal a priori data in children because these models rely on a mechanistic understanding of the disposition of the drug typically learned from rich adult data. Thus, despite the lack of available data on drug PK in infants, pediatric PBPK modeling can be used to simulate virtual breastfeeding infant populations to predict exposure given proper estimated doses. The aim of this thesis is to use PBPK modeling to produce a novel risk metric that advances the knowledge of breastfeeding infant exposure to maternal medications. The objectives are to (1) create and apply a workflow incorporating pediatric PBPK modeling to develop the novel metric with infants breastfed from mothers taking lamotrigine, cannabidiol (CBD), and ezetimibe, (2) identify potential maternal factors that may impact concentrations of drugs in milk for incorporation into the workflow established in objective 1 for CBD, and (3) optimize the utility of the novel metric for use in clinical practice. To arrive at the first objective, a literature review was used to develop a model to describe the weight-normalized volume of intake infants typically receive. The model was then used in combination with literature (lamotrigine) or collaborator collected (CBD and ezetimibe) drug concentrations in breast milk to estimate infant daily doses. The doses were then given to virtual breastfeeding infants created through developed and evaluated pediatric PBPK models. For the second objective, linear regression was used to identify influential maternal factors on CBD milk concentrations and breastfeeding exposure predictions. Finally, qualitative interviews were conducted with healthcare providers to ascertain perspectives on the novel metric for use in practice. Through this work, a milk intake model described weight-normalized milk intake with a maximum of 152.6 mg/kg/day at 19.7 days postnatal age. The greatest risk for breastfeeding infant exposure to maternal medications occurred during the 2-4 week postnatal age window. Pediatric PBPK models were developed for lamotrigine, CBD, and ezetimibe. For CBD, literature in vitro data informed the identity and percent contributions of metabolizing enzymes to clearance. These contributions were ascertained as UGT1A7 4%, UGT1A9 16%, UGT2B7 10%, CYP3A4 38%, CYP2C19 21%, and CYP2C9 11%. This information was used to populate the CBD pediatric PBPK model. Results from the linear regression analysis with maternal factors, including administration type, dose-frequency of use, and time after last dose of CBD, revealed that oil or pipe and joint/blunt or edible administrations produced the highest and lowest CBD concentrations in milk, respectively. Overall, the three PBPK models were able to adequately predict exposures of the drug administered in children. A novel risk metric termed the upper area under the curve ratio (UAR) was developed to describe the 95th percentile of breastfed infant AUC divided by the median therapeutic AUC of adults or children for approved indications. Across all ages (0-1 years old), the UAR ranged from 0.18-0.44, 0.00022-0.0044, and 0.0015-0.0026 for lamotrigine, CBD, and ezetimibe, respectively. From the qualitative interviews with 28 healthcare providers, six main themes emerged: (1) Current Practice Approaches, (2) Advantages of Existing Resources, (3) Disadvantages of Existing Resources, (4) Advantages of the UAR, (5) Disadvantages of the UAR, and (6) Strategies to Improve the UAR. Multiple strategies to improve the UAR, such as combining the UAR with another resource and providing guidance to interpret the UAR were attained. The work in this thesis developed the UAR to account for the relative exposure of breastfeeding infants to maternal medications and identify potential outliers who may be most vulnerable. Through healthcare provider interviews, it was evident that the UAR confers benefits over existing metrics and can be optimized for use in practice. With the workflow applied to further drugs, the UAR has the potential to improve our understanding of drug exposures in breastfeeding infants and be used by healthcare providers in their advising.Item The associations between insulin, hypoglycemia, and dementia: Combating threats to internal validity in a series of population-based cohort studies(University of Waterloo, 2023-05-01) Alkabbani, WajdAlthough the association between type 2 diabetes and dementia is recognized, findings from the epidemiology literature on the effect of insulin and one of its side effects, hypoglycemia, are less clear. The currently available observational studies assessing these associations suffer from a wide range of methodological limitations that diminish their internal validity and lead to contradictory evidence. The aim of this thesis is to implement design and analysis techniques to combat bias and confounding in previous studies and to further extend knowledge on the risk of dementia associated with four interconnected diabetes-related exposures, each assessed in a separate study: 1) severe hypoglycemia, 2) age of severe hypoglycemia, 3) insulin use, and 4) the mediating effect of severe hypoglycemia from insulin use. Herein, a series of cohort studies were conducted using population-based health administrative data (1996-2018) from British Columbia, Canada housed by Population Data BC. First, we identified individuals newly diagnosed with type 2 diabetes between 01 January 1998 and 31 December 2016. Each cohort was then designed based on the research question, wherein exposure was defined accordingly. For studies 1 and 2, the exposure of interest was severe hypoglycemia compared to no hypoglycemia. For studies 3 and 4, the exposure of interest was insulin initiation compared to initiating a non-insulin class. For all cohorts, the outcome of interest was all-cause dementia. Confounding adjustment techniques including inverse probability of treatment weighting (IPTW) were used in all studies. In each study, a wide range of sensitivity analyses were conducted to ensure the robustness of results. Findings from study 1 confirm the previously reported higher risk of all-cause dementia with severe hypoglycemia after implementing exposure density sampling, a lag period, and IPTW (HR 1.83; 95% CI 1.31-2.57). Findings from study 2 show that the increased risk of dementia observed in study 1 is consistent whether hypoglycemia occurs in midlife (HR 2.85; 95% CI 1.72-4.72) or late life (HR 2.38; 95% CI 1.83-3.11). Conversely, findings from study 3 negate existing evidence and do not show an increased risk of dementia associated with insulin use (HR 1.14; 95% CI 0.81-1.60). Lastly, findings from study 4 indicate a potential role of severe hypoglycemia as a mediator of the association between insulin and dementia (Natural Indirect Effect HR 1.04; 95% CI 1.01-1.08). Collectively these studies provide further insight on the complex associations between insulin, hypoglycemia, and the risk of all-cause dementia to inform both clinicians and patients with type 2 diabetes on the need to prevent hypoglycemia. Importantly, these studies showcase the need for robust methodology when conducting observational studies for type 2 diabetes-related exposures.Item Pharmacokinetic and economic implications when switching between hemophilia A treatments(University of Waterloo, 2023-04-18) Yu, JackyHemophilia is a bleeding disorder in which the blood is unable to form clots, and is also classified as severe (defined as having an endogenous factor VIII [FVIII] concentration < 1 IU/dL, or 1%), moderate (1-5%) or mild (5-50%). Those with severe hemophilia may spontaneously bleed without physical trauma. If not treated appropriately, people with hemophilia may develop hemophilic arthropathy, a joint disease commonly showing signs of bleeding in the knees, ankles, or elbows, which not only impairs movement, but significantly impacts life expectancy and quality of life. The current treatment of hemophilia involves prophylactic administration of factor concentrates. Although prophylactic treatment has improved health outcomes compared to on-demand treatment, there are still some challenges regarding the use of clotting factor concentrates. Generally, clotting factor concentrates are dosed based on international units per weight, but this one size fits all dosing mechanism fails to account for pharmacokinetic variability within this population. Appropriate dosing regimens vary by patient and treatment with hemophilia A patients using FVIII concentrates should be individualized from a therapeutic and economic standpoint. Population pharmacokinetic (PopPK) models were used as the basis for individualizing dosing regimens for patients with hemophilia while on factor concentrates. PopPK models are built using PK data from multiple participants to quantify the relationships between covariates such as age and weight to PK parameters as well as define variability between and within participants for these same PK parameters. To determine individual PK using only a few clotting factor activity levels and patient covariates, PopPK models are leveraged for Bayesian forecasting. People with hemophilia who have few sampling points are still available to be assessed using prior knowledge available from the patient population. While PopPK models may be helpful in hemophilia, this poses a concern when switching between factor concentrates. Factor concentrate switches may be prompted by the availability of new, improved concentrates by termination of national contracts resulting in a discontinuation of drug coverage, hypersensitivity to their current drug formulation, or adverse drug reactions. The lack of PK-tailored guidance when switching from one product to another may result in a period of time where treatment may increase the risk of inappropriate dosing, leading to either an increased risk of bleeds or waste of expensive factor concentrate. The work presented in this dissertation uses knowledge of an individual’s PK on a prior factor concentrate to better predict an individual’s PK on a new factor concentrate using data available from the Web-Accessible Population Pharmacokinetic Service – Haemophilia (WAPPS-Hemo). Emicizumab is a bispecific, recombinant, monoclonal antibody that bridges activated factor IX and X, mimics and partially restores the function of clotting FVIII in people with hemophilia A without inhibitors and was approved as routine prophylaxis by Health Canada in 2019. While emicizumab has its advantages over factor concentrates, such as decreased frequency of administration, and subcutaneous route of administration versus intravenous injections, the drug label recommends that any unused solution from a vial must be discarded, thereby wasting expensive resources. The use of a PopPK model of emicizumab was used to explore the implications of dosing based on vial size. This dissertation concludes that administering the entire vial of emicizumab and reducing the frequency may result in a reduction of vials used annually and consequently potential cost-savings. With high treatment costs and the approval of emicizumab, understanding the pharmacoeconomics of hemophilia is imperative for healthcare systems for reimbursement approval and contributing to commercial success and decision making as to whether emicizumab should be covered or not. Given the lifelong burden of the disease, the high cost of treatment in hemophilia, and the approval of emicizumab, a drug that may change the landscape of how hemophilia is treated, a cost-utility analysis studying the cost and quality-of-life of different prophylactic treatment regimens was presented in this dissertation, concluding that emicizumab is more effective and may be less costly than FVIII for patients with hemophilia A in Canada, conditional on drug cost assumptions. The method for estimating individual PK using PopPK models developed described in this dissertation may have a high impact for patients with hemophilia taking factor concentrates, who benefit from a safer individualized dosing regimen when switching between factor concentrates, potentially reducing adverse events or medication wastage. For patients with hemophilia on emicizumab, the simulations conducted exploring the use of emicizumab dosed based on vial size may have significant economic implications in cost-savings and provide a more practical dosing regimen. Finally, the economic evaluation conducted in the Canadian healthcare landscape may provide the healthcare system insight regarding the health and economic effects of using emicizumab compared to factor concentrates.Item In-solution and physical state properties of polymer and lipid excipients and their effects on the supersaturation evolution of poorly water-soluble drugs(University of Waterloo, 2023-04-14) Huang, JiahaoSolubility has become an increasing issue for the pharmaceutical industry, as the advances in combinatorial chemistry and high throughput screening methods have resulted in an increase in the molecular weight and lipophilicity of new chemical entities. There is a constant need for the development of formulation strategies to address these challenges for the oral delivery of poorly soluble drugs. The supersaturated drug delivery system (SDDS) based on amorphous solid dispersion (ASD) is a novel strategy to increase solubility and dissolution of hydrophobic drug compounds in order to achieve an improved oral absorption. The kinetic solubility profile of an ASD formulation in aqueous environment depends on the buildup and maintenance of supersaturated drug solution, which are determined by parameters including drug amorphization state in ASD carrier, drug release from the carrier matrix, liquid-liquid phase separation (LLPS) and nucleation of supersaturated drug solution, and crystal growth rate of drug molecules. These factors are closely correlated to the solid-state and in-solution properties of ASD carrier materials. This thesis aims to investigate the effects of polymer and lipid materials on the LLPS, recrystallization profile, drug-carrier miscibility and dissolution behaviors of two model drugs, indomethacin (IDM) and apixaban (APX), in an effort to benefit both the investigation of fundamental mechanisms of drug supersaturation and the development of pharmaceutical excipients into ASD carriers. Firstly, the LLPS behaviors of both drugs in the presence of polymer and lipid carriers were successfully characterized using the double wavelength UV extinction method. The LLPS behaviors of both drugs were depended on one or more of the following mechanisms: in-solution hydrogen bonding, drug aggregation facilitated by amphiphilic agents, micelle/vesicle incorporation, and ionization induced solubility enhancement. Polymer or lipid carrier can modify the LLPS behaviours of different drugs in completely different ways depending upon the nature of the drugs. The methodology is expected to serve the LLPS investigations of more types of drugs and excipients. Our work suggests that the effects of excipients on drug LLPS must be analyzed on a case by case basis, given the fact that an ASD carrier can behave markedly differently for different drug compounds. Secondly, the supersaturation effects of polymer and lipid ASD carriers on IDM and APX under non-sink dissolution conditions were revealed using a solvent-shift method and compared with the identified LLPS properties. Results showed that the supersaturation behaviors of both drugs were influenced by ASD carriers by mechanisms that include in-solution hydrogen bonding, pH-induced solubility change, solubilization, and reduced crystal growth rate. The recrystallization behaviors of both drugs did not necessarily align with the corresponding LLPS results, as LLPS properties were considered to influence the early stage of drug recrystallization. By comparing LLPS and supersaturation result for certain drug-excipient pairs, we also suggested that the different principles of characterization methods may induce variations in the characterization results. Therefore, multiple characterization methods should be combined to obtain accurate in-solution properties of drugs and excipients. Thirdly, we successfully characterized the drug-excipient miscibility for IDM and APX under a solid state, aiming to analyze the amorphization and loading state of both drugs in different carriers. Meanwhile, the feasibility of using fluorescence techniques to evaluate drug-carrier miscibility was testified by various drug-excipient pairs. Spectral parameters including peak shape, peak intensity and peak wavelength were used to analyze drug-excipient miscibility and physical state of IDM and APX in ASD systems. Distribution of fluorescence intensity and birefringence properties of fluorescence microscopy were also used to analyze the drug-excipient miscibility. Our work suggests that the fluorescence spectroscopy and fluorescence microscopy must be combined to obtain the accurate drug amorphization state in ASD systems, as fluorescence spectroscopy alone may be ineffective in identifying the amorphous drug aggregates in ASD systems. The drug amorphization and loading states were used to analyze the drug release behavior that was closely associated with supersaturation buildup of an ASD formulation. Lastly, ASDs for IDM and APX were prepared with polymers, lipids, and their combinations as carriers. The dissolution behaviors of prepared samples were tested under non-sink conditions where LLPS and recrystallization were expected to occur. The dissolution and supersaturation parameters were analyzed based on previously investigated properties including LLPS, solubilization, recrystallization, and drug amorphization state. For both IDM and APX, dissolution parameters were greatly influenced by carrier type in different manners. A given type of carrier could provide different effects when it was used to load different drugs. Preparation methods were found to bring inconsistent changes to different formulations. The disintegration, dissolution and dispersion of carrier materials were critical factors to determine the expression of carriers’ ability to generate and maintain supersaturated drug solutions. The correlations between carriers’ effect on crystalline solubility, LLPS, recrystallization, and dissolution parameters could only be established when drug carriers could rapidly and completely dissolve or disperse in dissolution media. The methodology and implications provided by this study could be used to investigate the in-solution and physical-state properties of more types of poorly soluble drugs and potential ASD carriers, in order to benefit the development of ASD formulations.Item Clinical Pharmacy Services in Ambulatory Oncology: An Environmental Scan of the Canadian Practice Landscape(University of Waterloo, 2023-02-21) Tibensky, Bryanna; Hutton, Lauren; Wentzell, Jason; LeBlanc, Michael; Edwards, Scott; McFarlane, ThomasBackground: Canadian clinical pharmacy key performance indicators (cpKPIs) have been developed for inpatient hospital practice but are not established for ambulatory oncology. This study represents the first step in developing cpKPIs for ambulatory oncology. Objectives: To describe the current landscape of pharmacy services in ambulatory oncology in Canada and to identify perspectives related to the development and implementation of cpKPIs in this practice setting. Methods: In this national cross-sectional study, a web-based questionnaire was distributed to pharmacists working in ambulatory oncology settings. Potential participants who self-identified as pharmacists practising in an ambulatory oncology setting were eligible. Survey questions focused on participants’ demographic characteristics, oncology pharmacy services provided, metrics captured, and pharmacists’ perceptions of cpKPIs. All data were analyzed using descriptive statistics. Results: A total of 44 responses were received, with most respondents practising in community hospitals in British Columbia, Ontario, and Atlantic Canada. The services most commonly provided were chemotherapy order verification, laboratory monitoring, identification and resolution of drug therapy problems, and counselling on anticancer medications. Twenty-six of the 44 respondents (59%) indicated that performance metrics or patient outcomes were tracked at their respective institutions, with none being universally captured. Overall, 43 (98%) of the respondents favoured the development of cpKPIs for ambulatory oncology practice. Conclusions: Despite growing patient care needs in ambulatory oncology, there is significant heterogeneity in the scope of pharmacy services offered and the outcomes used to qualify their impact within this setting across Canada. This study demonstrates a clear need for national consensus cpKPIs to inform pharmacy resource utilization and patient-centred quality improvement initiatives.Item Evaluation of a microlearning module on hypertension in Canada(University of Waterloo, 2023-02-20) Waked, Khrystine; Nagge, Jeff; Grindrod, KellyObjectives: To use a microlearning platform to evaluate pharmacists’ knowledge and behaviour related to hypertension. Methods: Pharmacy 5in5 is an online microlearning platform designed to help pharmacy professionals develop skills and acquire a deeper understanding of a variety of clinical and professional practice topics. Each topic includes a fact-based quiz, six case-based quizzes, six reflection questions, animated videos, infographics and flash cards. In April 2019, a hypertension module was released to engage pharmacists in managing hypertension and improving current practice behaviours. The module was developed using Canadian and American hypertension guidelines, landmark trials, and tertiary drug resources. The hypertension module was accessible on the Pharmacy5in5 website. Preliminary data were collected over 8 weeks between April-June 2019. User performance was analyzed with a binomial regression model. Results: Overall, 600 users accessed the Pharmacy 5in5 platform in the first two months, 81% of which were in Ontario. Licensed pharmacists performed significantly better than other registered users (β = −0.56, t (3362) =−2.15, p = 0.03), with users working in hospitals performing the best. The quiz resulting in the highest average focused on proper techniques for home blood pressure monitoring (mean score of 87%; SD 17%), while the quiz resulting in the lowest average focused on choosing medication therapy (mean score of 59%; SD 22%). In the reflection questions for behaviour in the last three months, 68% of users reported counselling a patient on home blood pressure monitoring and 62% had educated patients on their blood pressure targets, but only 18% had contacted a prescriber to optimize pharmacotherapy. Conclusion: The Pharmacy5in5 hypertension module is an engaging method of continuing education which can be used to rapidly reach many healthcare professionals in a short time. More education is needed to support pharmacists in working with prescribers to proactively optimize hypertension therapy.Item The Interplay Between the Neuronal Plasma Membrane and Cell Signaling in Alzheimer’s Disease(University of Waterloo, 2023-03-10) Robinson, MorganThe lack of understanding in the molecular and cellular mechanisms of Alzheimer’s disease (AD) has hindered efforts towards finding treatments that effectively modify disease trajectory. Therapeutic development for AD has focused on targeting amyloid-β (Aβ) pathology, long thought to be the cause of AD pathogenesis, but these have failed in clinical trials. Aβ is a sticky aggregation-prone protein that disrupts membrane structure and interferes with specific receptors in the brain, impairing synaptic plasticity, an important process for learning and memory, and eventually causes cell death. The interplay between disruption of the neuronal membrane and neuronal receptors in AD overlaps with inflammation and oxidative stress in a feedback loop that makes it difficult to ascertain the causes and effects of AD. More recent genetic and epidemiology data indicates that lipid metabolism is critical in AD pathogenesis, underscoring the need to understand how brain lipid composition (especially cholesterol) in brain affects amyloid toxicity. In the first part of this work the relevant background literature of lipid mediated mechanisms of AD is discussed and an overview of methods used herein are provided. In the second part, the results of biomedical nanotechnology experiments where atomic force microscopy (AFM) was used to study interactions of supported lipid bilayers (SLBs) with melatonin and Aβ at the molecular level. Chapter 3 shows the characterization of biophysical changes that melatonin induces in SLBs of DOPC/DPPC/Cholesterol by AFM and atomic force spectroscopy (AFS). Overall, AFM imaging revealed that melatonin increases disordered domain coverage, reduces bilayer thickness and indentation depth, increases membrane fluidity, and decreases membrane adhesion, though large variability was observed. In Chapter 4, for the first-time contact mode high-speed AFM (HS-AFM) was shown to be able to image lipid membranes of different compositions. HS-AFM was used to capture large areas of membranes comparing the effects of Aβ monomers and oligomers to different phase separated lipid bilayers composed of low and high cholesterol showing different interaction mechanisms. In the third part of this thesis the influence of membrane composition and amyloid toxicity on HT22 neuronal cell viability, cholesterol metabolism, morphology, and receptor tyrosine kinase (RTK) signaling pathways was elucidated. Beginning in Chapter 4, cholesterol oxidase assays and AFM verify cell cholesterol content reduction and Aβ structure, respectively. There was no effect of Aβ on cholesterol recovery and cell viability studies show that cholesterol depletion was modestly protective against both Aβ monomers and oligomers. In Chapter 5, the cholesterol-dependent effects of Aβ monomers and oligomers on HT22 cell morphology by phase contrast optical microscopy and atomic force microscopy (AFM) reveal apoptotic and necrotic populations of HT22 cells exposed to Aβ and that that membrane cholesterol depletion prevents these changes in morphology. In Chapter 7, the effects of cholesterol and Aβ on baseline Tyrosine Receptor Kinase B (TrkB) receptors and PDGF receptor-α (PDGFRα) signaling, reveal that RTK signaling is cholesterol-dependent and that high concentration Aβ oligomers increase the likelihood of RTK impairment, but there was no statistically significant effect of Aβ on PDGFRα signaling. This work provides experimental evidence that membrane cholesterol is not strongly involved in the mechanisms of Aβ toxicity in HT22 cells, but its reductions may be mildly protective. RTK signaling in HT22 cells is impaired by Aβ but is not involved in the protective mechanisms of cholesterol depletion. Aβ disrupts membrane biophysical structure and receptor signaling pathways triggering metabolic dysfunction and both apoptotic and necrotic cell death mechanisms.