Pharmacy

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3-Phenylpyrazino[1,2-a]indol-1(2H)-ones as dual cholinesterase and amyloid aggregation inhibitors
(University of Waterloo, 2017-05-10) Gujral, Sarbjeet Singh; Nekkar, Praveen
The year 2017, marks the 110th anniversary of the discovery of Alzheimer’s disease (AD)- a devastating neurodegenerative disease. Regardless of the significant advances made in the past century on the pathology of AD, the current pharmacotherapy options for AD remains woefully low and provide symptomatic relief only. Inhibitors of cholinesterase enzymes such as donepezil (Aricept®), rivastigmine (Exelon®) and galantamine (Razadyne®) which represents the primary class of agents used in the management of AD targets one of the many pathological routes of AD. Our study aims at discovering novel small hybrid molecules based on 3-phenylpyrazino[1,2-a]indol-1(2H)-one (PPI) ring system which can potentially exhibit multiple activities toward various factors involved in AD pathophysiology including (i) the inhibition of cholinesterase enzymes such as acetyl (AChE) and butyrylcholinesterases (BuChE); (ii) preventing the aggregation of the neurotoxic amyloid beta (Aβ) peptide and (iii) antioxidant properties. Initial modeling studies suggested that the tricyclic PPI template fits in the catalytic site of AChE and the C3 phenyl can orient toward the peripheral anionic subsite (PAS) in the AChE enzyme. In addition, C3-position provides opportunities to incorporate Aβ binding pharmacophores. With this goal, we synthesized the PPI compound library by coupling ethyl indole-2-carboxylates esters with 2-bromoacetophenones to obtain ethyl-1-(2-oxo-2-phenylethyl)-1H-indole-2-carboxylates which underwent an intramolecular cyclization in the presence of ammonium acetate to afford PPI derivatives (5a-n). The compounds were characterized by analytical methods including NMR and LCMS. The cholinesterase inhibition was evaluated using Ellman’s protocol by UV-Vis spectroscopy. The anti-Aβ-aggregation property was evaluated by fluorescence spectroscopy using thioflavin- T (ThT) assays. Antioxidant activity of the PPI derivatives was assessed using DPPH assay method. Transmission electron microscopic imaging (TEM imaging) were also performed to support the in vitro data obtained from ThT based fluorescence assays. The Discovery Studio (DS) software, Structure-Based-Design program (4.0) from BIOVIA Inc. was used to determine the binding interactions of the PPI derivatives for SAR optimization. Our results indicate that several compounds in the series exhibit dual cholinesterase inhibition properties; one such compound is 5h (3-(2-methoxyphenyl)pyrazino[1,2-a]indol-1(2H)-one) with IC50 AChE = 7.3 μM , IC50 BuChE = 1.9 μM. Compound 5h was found to be much more potent than reference agents donepezil and rivastigmine toward BuChE inhibition. Several other compounds such as 5d ( 3-(3,4-dimethoxyphenyl)pyrazino[1,2-a]indol-1(2H)-one) and 5h (- 3-(2-methoxyphenyl)pyrazino[1,2-a]indol-1(2H)-one exhibited excellent of Aβ40/42 inhibition (% inhibition of Aβ40 = 83.3% and 67.7% at 25 µM respectively, and % inhibition of Aβ42 = 90% and 94% at 25 µM respectively). Compound 5d and 5h were found to be more potent than curcumin and resveratrol towards Aβ42 inhibition. The PPI derivatives were also found to exhibit antioxidant activities. Unsubstituted PPI compound 5a exhibited good antioxidant activity (~33% DPPH radical scavenging at 50 μM), while, compound 5k (3-(4-hydroxy-3-methoxyphenyl)pyrazino[1,2-a]indol-1(2H)-one) exhibited excellent antioxidant activity (~ 84% DPPH radical scavenging at 50 μM). This proves the multi-targeted activities of PPI derivatives. Our results indicate that the fused tricyclic phenylpyrazino[1,2-a]indo-1(2H)-ones (PPI) represent a novel class of compounds which can be modified chemically to design and develop multi-targeting agents aimed at the cholinergic, amyloid cascade and oxidative stress hypothesis of AD.
5-HT7 Receptor Neuroprotection against Excitotoxicity in the Hippocampus
(University of Waterloo, 2014-04-11) Vasefi, Seyedeh Maryam
Introduction and Objectives: The PDGFβ receptor and its ligand, PDGF-BB, are expressed throughout the central nervous system (CNS), including the hippocampas. Several reports confirm that PDGFβ receptors are neuroprotective against N-methyl-D-asparate (NMDA)-induced cell death in hippocampal neurons. NMDA receptor dysfunction is important for the expression of many symptoms of mental health disorders such as schizophrenia. The serotonin (5-HT) type 7 receptor was the most recent of the 5-HT receptor family to be identified and cloned. 5-HT receptors interact with several signaling systems in the CNS including receptors activated by the excitatory neurotransmitter glutamate such as the NMDA receptor. Although there is extensive interest in targeting the 5-HT7 receptor with novel therapeutic compounds, the function and signaling properties of 5-HT7 receptors in neurons remains poorly characterized. Methods: The SH-SY5Y neuroblastoma cell line, primary hippocampal cultures, and hippocampal slices were treated with 5-HT7 receptor agonists and antagonists. Western blotting was used to measure PDGFß receptor expression and phosphorylation as well as NMDA receptor subunit expression and phosphorylation levels. Real-time RT-PCR was used to measure mRNA level of PDGFß receptor in neuronal cultures. Cell death assays (MAP2, MTT) were used to measure the neuroprotective effects of 5-HT7 and PDGFß receptor activation. Results: My research involved elucidating the molecular mechanisms of neuroprotection after 5-HT7-induced PDGFß receptor upregulation. I demonstrated that 24 h treatment with the selective 5-HT7 receptor agonist, LP 12, increased not only the expression but also the activation of PDGFß receptors as measured by the phosphorylation of tyrosine 1021, the phospholipase Cγ binding site. Activation of the 5-HT7 receptor also selectively changed the expression and phosphorylation state of the NR2B subunit of the NMDA receptor. Activation of 5-HT7 receptors was neuroprotective against NMDA-induced toxicity in primary hippocampal neurons and this effect required PDGFß receptor kinase activity. Thus, long-term (24 h) activation of 5-HT7 receptors was neuroprotective via increasing the expression of a negative regulator of NMDA activity, the PDGFß receptor. In contrast, acute activation (5-30 min) of 5-HT7 receptor increased NMDA evoked current and altered NMDA receptor subunit phosphorylation in hippocampal neurons in a manner that was different from what we observed in our 24 h experiments. Conclusions: I identified two 5-HT7 receptor to NMDA receptor pathways: acute activation of the receptor increased NMDA-evoked currents whereas long-term 5-HT7 agonist treatment prevented NMDA-induced excitotoxicity in a PDGFß receptor-dependent manner. This research is significant in the ongoing advances for the treatment of mental heath disorders, such as schizophrenia and depression, that involve the 5-HT, glutamate, and neuronal growth factor systems.
Acceptance of Commercially Available Wearable Activity Trackers Among Adults Aged Over 50 and With Chronic Illness: A Mixed-Methods Evaluation
(JMIR Mhealth Uhealth., 2016-01-27) Mercer, Kathryn; Grindrod, Kelly; Schneider, Eric; Li, Melissa; Chilana, Parmit; Giangregorio, Lora M.
Background: Physical inactivity and sedentary behavior increase the risk of chronic illness and death. The newest generation of “wearable” activity trackers offers potential as a multifaceted intervention to help people become more active. Objective: To examine the usability and usefulness of wearable activity trackers for older adults living with chronic illness. Methods: We recruited a purposive sample of 32 participants over the age of 50, who had been previously diagnosed with a chronic illness, including vascular disease, diabetes, arthritis, and osteoporosis. Participants were between 52 and 84 years of age (mean 64); among the study participants, 23 (72%) were women and the mean body mass index was 31 kg/m2 . Participants tested 5 trackers, including a simple pedometer (Sportline or Mio) followed by 4 wearable activity trackers (Fitbit Zip, Misfit Shine, Jawbone Up 24, and Withings Pulse) in random order. Selected devices represented the range of wearable products and features available on the Canadian market in 2014. Participants wore each device for at least 3 days and evaluated it using a questionnaire developed from the Technology Acceptance Model. We used focus groups to explore participant experiences and a thematic analysis approach to data collection and analysis. Results: Our study resulted in 4 themes: (1) adoption within a comfort zone; (2) self-awareness and goal setting; (3) purposes of data tracking; and (4) future of wearable activity trackers as health care devices. Prior to enrolling, few participants were aware of wearable activity trackers. Most also had been asked by a physician to exercise more and cited this as a motivation for testing the devices. None of the participants planned to purchase the simple pedometer after the study, citing poor accuracy and data loss, whereas 73% (N=32) planned to purchase a wearable activity tracker. Preferences varied but 50% felt they would buy a Fitbit and 42% felt they would buy a Misfit, Jawbone, or Withings. The simple pedometer had a mean acceptance score of 56/95 compared with 63 for the Withings, 65 for the Misfit and Jawbone, and 68 for the Fitbit. To improve usability, older users may benefit from devices that have better compatibility with personal computers or less-expensive Android mobile phones and tablets, and have comprehensive paper-based user manuals and apps that interpret user data. Conclusions: For older adults living with chronic illness, wearable activity trackers are perceived as useful and acceptable. New users may need support to both set up the device and learn how to interpret their data.
Aging affects circadian clock and metabolism and modulates timing of medication
(Elsevier, 2021-04) Sadria, Mehrshad; Layton, Anita T.
Aging is associated with impairments in the circadian rhythms, and with energy deregulation that affects multiple metabolic pathways. The goal of this study is to unravel the complex interactions among aging, metabolism, and the circadian clock. We seek to identify key factors that inform the liver circadian clock of cellular energy status and to reveal the mechanisms by which variations in food intake may disrupt the clock. To address these questions, we develop a comprehensive mathematical model that represents the circadian pathway in the mouse liver, together with the insulin/IGF-1 pathway, mTORC1, AMPK, NAD+, and the NAD+ -consuming factor SIRT1. The model is age-specific and can simulate the liver of a young mouse or an aged mouse. Simulation results suggest that the reduced NAD+ and SIRT1 bioavailability may explain the shortened circadian period in aged rodents. Importantly, the model identifies the dosing schedules for maximizing the efficacy of anti-aging medications.
Alterations in Colorectal Cancer Cell-extracellular Matrix Interactions Upon Acquisition of Chemotherapy Resistance.
(University of Waterloo, 2015-12-16) Berg, Spencer; Blay, Jonathan
Adjuvant chemotherapy is an essential component in the treatment of advanced colorectal cancer (CRC). Unfortunately, many patients experience recurrence with aggressive, chemotherapy-resistant disease for which the prognosis is poor and treatment options are limited. Understanding the changes that cancer cells undergo during the acquisition of a drug-resistant phenotype is therefore of critical importance in improving CRC patient outcomes. We chose to study resistance to the CRC chemotherapy agent, irinotecan, by first creating a CRC cell line that is highly resistant to its active metabolite (SN-38). In particular, we were interested in how SN-38 resistant CRC cells (HT-29S) were altered from parental, drug-sensitive CRC cells (HT-29) in their relationship with a feature of their microenvironment, the extracellular matrix (ECM). We found that HT-29S cells form a matrix composed of the ECM glycoprotein fibronectin when cultured as 3-dimensional spheroids, whereas HT-29 cells do not. The increase in fibronectin matrix deposition coincided with an increased fibronectin adhesive capacity of the SN-38-resistant cells, likely due to an increased expression of the integrin α5 subunit, which together with integrin β1 forms the primary fibronectin receptor. Furthermore, we demonstrated an activation of a phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) pro-survival signalling pathway downstream of integrin α5β1 ligation to fibronectin. Inhibition of this signalling pathway with the PI3K inhibitor LY294002 sensitized HT-29S cells to SN-38, but did not alter the response of the parental cell line to the chemotherapy agent. Finally, we have determined that HT-29S cells appear to undergo an epithelial to mesenchymal transition during the acquisition of chemotherapy resistance, and that this transition may be responsible for the upregulation of integrin α5 in the resistant population.
Analysis of plasma chemokines and circulating tumour cells in colorectal and breast cancer patient peripheral blood
(University of Waterloo, 2020-01-22) Patel, Deep; Blay, Jonathan
Circulating tumour cells (CTCs) provide a prognostic value in solid tumours including colorectal and breast. Enumeration of tumour cells from blood is becoming a common practice in informing prognosis and may guide therapy decisions. Enumeration alone does not capture heterogeneity of tumours and varying functional abilities of the CTCs to interact with the secondary microenvironment. Characterizing the isolated CTCs and assessing their functional abilities can track molecular changes in the disease progress. As a step toward identifying functional features of CTCs that could aid in clinical decisions, this study was aimed at analyzing chemokine release profile in drug resistance and developing a CTC isolation technique based on extracellular matrix interactions. Cancer cells release different chemokines and express chemokine receptors which together work to direct cell infiltrates in the tumour microenvironment. This work examined changes in the profile of chemokine release using a model of drug resistance based on the colorectal cancer cell line HT29 and its counterpart HT29-R that is resistant to the late-stage chemotherapy drug irinotecan (SN-38). Following an initial screening of mRNA expression through PCR and qPCR, five of the chemokines (CCL2, CCL15, CXCL8, CXCL12, and CCL20) were analyzed further for their release patterns amongst cell lines and peripheral blood of healthy volunteers and stage IV colorectal and breast cancer patients. The release pattern of chemokines in patient samples differed from the results of the in vitro drug-resistance model. Specific tumour location, previous therapies, and genetic variability are all examples of the factors that may provide unique patterns and complicate modelling for chemokine release in late-stage cancer. A detailed analysis revealed an upward trend for midkine (NEGF2) when baseline and 12 months plasma samples were compared. Migration studies may further reveal the consequences of this expression profile. Migration assays were carried out with Transwell® chambers and HepG2 cells to partially mimic the hepatic microenvironment. Such studies can guide future functional studies for isolated CTCs. We next sought to investigate extracellular matrix protein interactions, which might depend on a changing chemokine milieu. We utilized cancer cells’ ability to adhere to extracellular matrix and created a platform to isolate CTCs from the peripheral blood samples. A total of 14 colorectal and 7 breast cancer patients donated blood samples. Adhesion assays were performed with a range of different ECM proteins. We identified an optimal ECM substratum composed of collagen and fibronectin at a mass coating ratio of 2:1. The isolated CTCs were identified through immunofluorescence with epithelial marker antibodies (EpCAM and pan-cytokeratin). Identification of CTCs was further confirmed by exclusion with a hematopoietic origin marker CD45. The captured number of cells ranged from 0 to 296, whereas the mean number was 26 and the median was 22 per patient sample (~8mL). This technique not only allows enumeration, but also isolates cells based on a functional approach. The isolated cells are successful in adhering to extracellular matrix proteins and can be further characterized through functional markers. Overall, this study addresses two unique functional features of CTCs – their expression of certain chemokines and their ability to interact with both fibronectin and collagen - that form the basis to provide future clinical utility. Such an approach will help to inform clinicians about the aggressive nature of an individual tumour and guide treatment decisions toward best prognostic outcomes.
Appendix to "Characteristics, predictors, and reasons for regulatory body disciplinary action in health care: A scoping review"
(Journal of Medical Regulation, 2022-01) Foong-Reichert, Esther Ai-Leng; Fung, Ariane; Carter, Caitlin A.; Houle, Sherilyn; Grindrod, Kelly
In this appendix to the manuscript "Characteristics, predictors, and reasons for regulatory body disciplinary action in health care: A scoping review”, the studies included in the scoping review are summarized.
Arylbenzamide and Arylcarboxamide Derivatives as Modulators of Amyloid-Beta Aggregation
(University of Waterloo, 2024-03-11) Zhao, Yusheng; Nekkar Perampalli Rao, Praveen
Alzheimer’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.
Assessing Medication Adherence in Older Adults with Memory Concerns in a Primary Care-Based Memory Clinic
(University of Waterloo, 2015-10-02) Hudani, Zain
There is an increasing prevalence of cognitive impairment in the rapidly ageing population. Cognitive impairment has been shown to be a unique barrier to medication adherence in older adults coupled with an increased number of chronic conditions and prescription medications. There is a need to identify those who are non-adherent in order to reduce the repercussions related to non-adherence such as hospitalization and increased health-care costs. Although several instruments exist to assess adherence, not all can be applied to primary health based setting. Pharmacists practicing in novel primary care settings such as Center for Family Medicine’s Family Health Team’s Memory clinic measure adherence using pill count and pharmacy refill data, however, which method is more feasible remains unknown. Furthermore, it remains unknown whether the scores obtained using these instruments correlate. There is paucity in the current literature that addresses the aforementioned gaps. In addition, cognitively impaired older adults require assistance in managing medication, however, it remains unclear which adherence aids are being used by the caregivers of these patients seen at the memory clinic. Therefore, the overall objectives of this thesis were: 1) To map and describe the various aspects described in the current literature regarding medication adherence in older patients with memory concerns, and, 2) To determine the most feasible method of assessing adherence in an interdisciplinary, primary care based memory clinic setting.
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, Wajd; Gamble, John-Michael
Although 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.
A bacteria-responsive drug release platform for the local treatment of bacterial vaginosis
(University of Waterloo, 2022-07-25) Feng, Chuying; Ho, Emmanuel
Bacterial vaginosis (BV) is a common vaginal anaerobic dysbiosis affecting millions of women, with a high recurrence rate probably due to the stubborn existence of biofilms and Gardnerella vaginalis (G. vaginalis) as the primary pathogen. Antimicrobial therapy has been the standard treatment for BV for several decades, which includes oral and intravaginal therapy. However, the major drawback of using antibiotics extensively is drug resistance, leading to the ineffectiveness in killing bacterial pathogens. Stimuli-responsive systems are a promising solution to address bacterial resistance because they release drugs only in the presence of specific stimulus, e.g., changes in physical micro-environment and bacterial factors, which greatly avoids overdosing. In terms of treatment method, intravaginal formulations are superior to the conventional oral route with reduced side effects such as stomach irritation and more effective drug dose. This thesis focused on the development and characterization of a bacteria-responsive drug release platform that is aimed for intravaginal therapy to cure BV. Vaginolysin (VLY), a virulence factor produced specifically by G. vaginalis, was utilized as the stimulus to trigger drug release with the aim of achieving high specificity and improving antibacterial activity. Series of liposome formulations were developed, optimized, and characterized in terms of preparation method, purification method, encapsulation efficiency, size distribution and zeta potential. We demonstrated that centrifugation was the optimal method to separate liposomes from the model free drug (calcein) while keeping liposomes stable without much aggregation. Increasing the initial calcein concentration and total amount of lipids facilitated a higher encapsulation efficiency. All liposomes had an average diameter of around 300 nm with a polydispersity index (PDI) less than 0.3 and an average zeta potential of -60 mV. Second, we optimized bacteria culture and evaluated the responsive calcein release in the presence of G. vaginalis and Lactobacillus crispatus (L. crispatus) supernatants. All liposome formulations showed sustained calcein release (cumulative release: 46.7%-51.8%) in response to G. vaginalis supernatant and very little calcein release (cumulative release: 1.1%) in the presence of L. crispatus supernatant, indicating that the responsive drug release platform had good specificity. We then constructed an Escherichia coli (E. coli)-based recombinant protein expression system to express recombinant VLY which could be used as a standard to quantify VLY produced by G. vaginalis. After that, a series of optimizations in terms of host strain, cultivation conditions, cell lysis method, purification process optimization, etc. regarding recombinant VLY expression were carried out to promote soluble protein production. Unfortunately, we did not achieve the desired results as the recombinant VLY was expressed in the form of inclusion bodies. Overall, the bacteria-responsive drug release platform has great potential, since it will be the first time to realize controlled drug release activated by a specific pathogen for BV prevention and treatment. This on-demand therapy can be potentially used by industry to provide millions of women patients with more sexual and reproductive health care.
Bacteriophage interactions with mammalian tissue: Therapeutic applications
(Elsevier, 2019-05) Huh, Haein; Wong, Shirley; St. Jean, Jesse; Slavcev, Roderick
The human body is a large reservoir for bacterial viruses known as bacteriophages (phages), which participate in dynamic interactions with their bacterial and human hosts that ultimately affect human health. The current growing interest in human resident phages is paralleled by new uses of phages, including the design of engineered phages for therapeutic applications. Despite the increasing number of clinical trials being conducted, the understanding of the interaction of phages and mammalian cells and tissues is still largely unknown. The presence of phages in compartments within the body previously considered purely sterile, suggests that phages possess a unique capability of bypassing anatomical and physiological barriers characterized by varying degrees of selectivity and permeability. This review will discuss the direct evidence of the accumulation of bacteriophages in various tissues, focusing on the unique capability of phages to traverse relatively impermeable barriers in mammals and its relevance to its current applications in therapy.
Behavior Change Techniques Present in Wearable Activity Trackers: A Critical Analysis
(JMIR mHealth and uHealth, 2016-04-27) Mercer, Kathryn; Burns, Catherine; Giangregorio, Lora M.; Li, Melissa; Grindrod, Kelly
Background: Wearable activity trackers are promising as interventions that offer guidance and support for increasing physical activity and health-focused tracking. Most adults do not meet their recommended daily activity guidelines, and wearable fitness trackers are increasingly cited as having great potential to improve the physical activity levels of adults. Objective: The objective of this study was to use the Coventry, Aberdeen, and London-Refined (CALO-RE) taxonomy to examine if the design of wearable activity trackers incorporates behavior change techniques (BCTs). A secondary objective was to critically analyze whether the BCTs present relate to known drivers of behavior change, such as self-efficacy, with the intention of extending applicability to older adults in addition to the overall population. Methods: Wearing each device for a period of 1 week, two independent raters used CALO-RE taxonomy to code the BCTs of the seven wearable activity trackers available in Canada as of March 2014. These included Fitbit Flex, Misfit Shine, Withings Pulse, Jawbone UP24, Spark Activity Tracker by SparkPeople, Nike+ FuelBand SE, and Polar Loop. We calculated interrater reliability using Cohen's kappa. Results: The average number of BCTs identified was 16.3/40. Withings Pulse had the highest number of BCTs and Misfit Shine had the lowest. Most techniques centered around self-monitoring and self-regulation, all of which have been associated with improved physical activity in older adults. Techniques related to planning and providing instructions were scarce. Conclusions: Overall, wearable activity trackers contain several BCTs that have been shown to increase physical activity in older adults. Although more research and development must be done to fully understand the potential of wearables as health interventions, the current wearable trackers offer significant potential with regard to BCTs relevant to uptake by all populations, including older adults.
Bone health in adults with epilepsy
(University of Waterloo, 2017-08-30) Fernandez, Haya; Tejal, Patel
Epilepsy has been associated with an increased fracture risk by various studies in the literature. This is attributed to the decreased bone mineral density (BMD) linked to the anti–epileptic drugs (AEDs) that are used to control seizures. As increasing age is also associated with decreasing BMD, older adults with epilepsy are at particular risk for fracture. There is no consensus on managing this risk and many epilepsy care guidelines do not address the risk at all. This is understandable considering the lack of adequate research on counteractive measures in this specific population. Moreover, though a prevailing theory explains the observed decreased BMD to be a result of AEDs reducing active vitamin D levels, the efficacy of vitamin D supplementation as a protective measure does not have robust evidence nor is there an agreed upon treatment regimen. The proposed project will aim to fill the gaps in the research and establish groundwork for future research on prevention and treatment by: examining the existing literature on vitamin D supplementation for bone health in adults with epilepsy; investigating the prevalence of osteoprotective behaviours and their impact on fracture occurrence in older Canadian adults with and without epilepsy; and determining the prevalence of vitamin D supplementation in the older adult Canadian epileptic population.
Cationic Gelatin/Pluronic-based Nanoparticles as Novel Non-Viral Delivery Systems for Gene Therapy
(University of Waterloo, 2018-08-09) Madkhali, Osama; Wettig, Shawn
A delivery system is essential to protect the DNA from degradation because of the extracellular and intracellular barriers. Two main types of delivery systems have been used to deliver genes into the body: viral and non-viral vectors. Although viral vectors are still superior over non-viral vectors, non-viral types are a better and safer alternative due to the safety concern of viral vectors. The low transfection efficiency of non-viral vectors remains a challenge due to the barriers that must be tackled. An effective non-viral vector must fulfill many conditions in order to be able to handle these barriers. The vector should be biocompatible, biodegradable, able to interact with DNA and cell membrane, able to successfully escape the endosome, and finally, capable of entering the nucleus to express the required protein. Cationic polymers are one of the most effective non-viral delivery systems. Cationic polymer/DNA (also called polyplexes) are able to interact with the DNA through electrostatic interaction, which results in DNA complexation and condensation. Gelatin is a natural polymer that can be easily modified by increasing the positive charge to effectively interact with the DNA and the cell membrane. In this project, gelatin nanoparticles were prepared using the two-step desolvation method, and were modified with cholamine as a cationic agent. Pluronic block copolymers were subsequently added to protect the cationic gelatin/DNA from degradation, and to increase the circulation time. The interactions of gelatin/Pluronic/DNA with the model membranes DPPC-CHOL and POPC-CHOL were studied using Langmuir’s monolayer study, as well as Brewster’s angle microscopy at the air/water interface. Transfection efficiency and cell viability were then evaluated at COS-7 cells. The results revealed that gelatin nanoparticles were successfully modified, at which point the positive charge increased from +11 to +32. Additionally, cationic gelatin (CG) was able to interact with and neutralize the negative charge of the DNA. CG/Pluronic/DNA complexes was characterized by size and zeta potential, showing a small particle size and a positive charge. The interaction of CG/Pluronic/DNA complex with the model membranes demonstrated a fluidization effect, especially with CG and Pluronics, whereas DNA showed an ineffective and negligible condensation effect. With respect to transfection efficiency, CG results were poor compared to positive control jetPEI®, with no improvement after adding Pluronics. Gemini surfactant (GS) was also used in the transfection experiments in hope that it might improve the transfection efficiency of cationic gelatin. However, CG/GS showed some aggregation, and the positive charge decreased by increasing the CG, which resulted in lower transfection efficiency. Cell viability of the cells containing cationic gelatin was very high (similar to non-treated cells), which was confirmed by the safety of the gelatin, even after modification. More investigational studies and optimizations are required to understand the low transfection efficiency of cationic gelatin. These studies will help design more effective delivery vectors, either with gelatin or with any other non-viral system.
Cell Density-Dependent Changes in the Localization of the CD26 Protein in Colorectal Cancer Cells in Response to Flavonoid Treatment
(University of Waterloo, 2019-05-24) Diaconu, Bogdan; Blay, Jonathan
Colorectal cancer is the 3rd most common cancer worldwide and this rate of incidence is largely attributable to lifestyle factors such as the diet. A group of plant secondary metabolites called flavonoids has been found to exert various anticancer activities in colorectal cancer cell lines and is indeed thought to function similarly in vivo. The cell-surface enzymes CD26, CD38, and CD73 are present in colorectal cancers and their presence and activities in this context have the potential to be modulated by extracellular factors such as dietary flavonoids. The levels of CD26 protein in particular have been previously found to increase at the cell surface in HT-29 colorectal cancer cells following treatment with the flavonoid apigenin. Another extracellular factor which may alter the response of cancer cells is cell density. In this work, the role of apigenin was investigated first on the mRNA transcription of CD26, CD38, and CD73 in HT-29 cells cultured to increasing degrees of confluence. CD26 was identified as the most promising target because it revealed the greatest degree of mRNA variability. Next, the amount of CD26 mRNA and protein was quantified in HT-29 cells treated with apigenin and the related flavonoids genistein, kaempferol, and luteolin. Finally, the localization of CD26 protein was observed in these cells following flavonoid treatment. This investigation showed no consistent changes in either mRNA expression or whole cell protein abundance for CD26. However, there was a distinct change in cellular localization of CD26 in response to apigenin and genistein and this was seen particularly in colorectal cancer cells at low levels of confluence. The relocation of the CD26 protein may depend on particular features of the flavonoid structure. Furthermore this effect appears be modulated by a change in cell confluence. Therefore this study provides new insights with respect to the role of flavonoids in regulating CD26 in colorectal cancer cells.
Characterising changes in adhesion and enzyme activity related to drug resistance in colon cancer cells
(University of Waterloo, 2018-12-21) Dekker, Heather; Blay, Jonathan
Metastatic colorectal cancer is often fatal, and drug resistance to chemotherapeutic agents is one of the primary contributing factors to this lethality. Drug resistance arises from exposure to chemotherapies, and it can be mediated through a variety of mechanisms. One of these mechanisms is alteration of enzymes within the cancer cells to affect the processing or removal of the drug. Carboxylesterase is an example of an enzyme that converts irinotecan, a drug used in metastatic colorectal cancer treatments, into the active metabolite SN-38. Carboxylesterase enzymes are found in high quantities in both the liver and intestinal cells. The presence of carboxylesterase in intestinal and liver cells is an important consideration in the processing of colorectal cancer treatments. Glutathione S-transferase is another enzyme that has been implicated in drug resistance because of its ability to conjugate reduced glutathione to xenobiotic substances, facilitating their removal. Additionally, drug resistance can affect the behaviours of cells. Drug-resistant cells can exhibit changes in their motility and aggressiveness compared to drug-sensitive cells. In this study I investigated cellular behavioural changes in SN-38-resistant colon cancer cells compared to their SN-38-sensitive counterparts. In addition to behavioural changes, I also sought to determine if elevations in carboxylesterase and glutathione S-transferase enzymes were contributing to the drug resistance in these colon cancer cells.
Characterization of Bacteriophage λ Displaying Epidermal Growth Factor in the Uptake, Infiltration and Formation of HT29 Spheroids
(University of Waterloo, 2019-07-31) Huh, Haein; Slavcev, Roderick; Blay, Jonathan
Solid tumours are characterized by a complex structure comprised of extracellular matrix, neoplastic cells and stromal cells, each presenting a barrier to conventional anticancer chemotherapy as well as carrier-mediated drug delivery. Poor penetration of therapeutics into the interstitial tumour microenvironment remains a challenge, with drugs accumulating primarily in the regions of tumours that are situated closer to blood vessels. Bacteriophages do not infect eukaryotic cells, yet they have been shown to penetrate mucosal barriers, including multiple layers of epithelial cells and endothelium. In this work, we used NIH3T3 fibroblast and HT29 colon adenocarcinoma multicellular spheroids to represent the stroma and parenchyma of solid tumours and then to examine infiltration of bacteriophages as a means of delivering therapeutic cargo. By using phage display technology to decorate the surface of λ bacteriophage with epidermal growth factor (EGF), we compared the cell-phage interactions between wildtype phages and EGF-displaying phages, including the assessment of phages’ capacity to traverse the tumour interstitium, to be subjected to cell internalization and their effects on spheroid growth. Both wildtype and EGF-displaying λ phages were observed to adhere to the HT29 and NIH3T3 spheroids and to internalize into cells as early as 30 min following administration. EGF-phage treatment also slowed HT29 spheroid growth – demonstrating a delay in initial aggregation and formation of loosely-organized structures that led to much smaller spheroid formation in the latter stages of growth. These results support the potential for the therapeutic employment of bacteriophages as nanocarriers for targeted delivery.
Characterization of counterion effects of gemini surfactants and in vitro studies of transfection efficiency for gene therapy in epithelial ovarian cancer.
(University of Waterloo, 2015-01-23) Islam, Muhammad Shahidul
Gene therapy has emerged as a promising strategy for the treatment or prevention of many acquired or genetic diseases that are considered incurable at the present time. Although viral and non-viral vector approaches are the major techniques employed for somatic gene transfer, non-viral vectors (cationic liposomes, dendrimers, chitosans, polymers & surfactants) have attracted great interest recently, due to their unique properties. A number of non-viral carriers have been extensively investigated and developed in recent years for targeted drug delivery or gene therapy in various pre-clinical/clinical trials. Despite this, the quest for new non-viral carriers with improved transfection and low toxicity is still proceeding, driven by a need to overcome safety concerns associated with viral vectors. Of the non-viral vectors, an intriguing class of building blocks which has elicited extensive interest are the third generation di-cationic surfactants: a class of bis-surfactants called “gemini surfactants (GSs)”. The interest is due to their unique self-assembly, hundredfold lower CMC (compared to their monomeric counterparts), thousand-fold improved surface activity, and ability to form a rich array of aggregate morphologies. In this project, the effect of various inorganic and organic counterions on micellization was studied and analyzed at air–water surfaces as well as in bulk solutions. Additionally, the size & zeta potential of the nanoparticles, and the in vitro transfection efficiency studies in human ovarian cancer cell lines were also analysed to investigate the dominant influence of the anions on the aggregation behavior and DNA delivery efficiency of eight surfactants of the ethanediyl-α,ω-bis-(dimethylhexadecyl-ammonium) type, [C16H33(CH3)2-N-(CH2)2-N-(CH3)2C16H33].2X– referred to as gemini 16-2-16; where X refers to the counterion were studied. Counterions of chloride (Cl–), bromide (Br–), ½ malate (C4H4O5– –), ½ tartrate (C4H4O6– –), adenosine mono phosphate, AMP (C10H13N5O7P–), guanosine mono phosphate, GMP (C10H13N5O8P–), cytidine mono phosphate, CMP (C9H13N3O8P–), and uridine mono phosphate, UMP (C9H12N2O9P–) were investigated and were classified into three different categories depending on their nature: (1) small inorganic counterions [chloride (Cl–), and bromide (Br–)] taken from the Hofmeister series were studied to focus on the effect of ion type; (2) Hydroxy-alkyl di-carboxylate counterions [malate (C4H4O5– –), and tartrate (C4H4O6– –)] were studied to focus on the effect of the hydrophilicity of counterions; and (3) heterocyclic ring containing nucleotide mono phosphate counterions were included to focus on mainly self-assembly and other parameters. We demonstrate the influence of different anions associated with this 16-2-16 series of gemini by analyzing the effect of counterions on the micellization and aggregation behavior of these gemini surfactants, characterized by determination of the critical micelle concentration (CMC), degree of micelle ionization (α), and free energy of micellization (ΔGM) and are discussed in terms of the hydrophilicity of anions, counterion hydration, interfacial packing of ions, and ionic morphology. Our results clearly revealed that a counterion effect on micellization and aggregate morphology, attributed to the balancing and controlling forces of the counterions to the surfactant itself. Hydrogen bonding among the –OH groups of the counterions (where applicable) and water molecules, as well as the strong hydrophobic interaction among the hydrocarbon side chains is postulated to be the main origins for the unique aggregation behaviors of these gemini surfactants. These amphiphiles can form both micelles and vesicles spontaneously with a micelle-to-vesicle transition at a concentration above the respective CMC. Furthermore, the size & zeta potential characterizations along with the in vitro transfection data manifest the significant impact of counterions on the GSs as therapeutic drug delivery carrier. Our transfection efficiency (TE) data also demonstrated that the surface charge density of the particles formed by the GSs is the predominant factor for cellular uptake and consequent TE of the respective GSs.
Characterization of gemini nanoparticle assembly by fluorescence correlation spectroscopy
(University of Waterloo, 2014-01-23) Dong, Chilbert
Research in the field of non-viral gene delivery has demonstrated that a deeper understanding of the fundamental processes of nanoparticle assembly is required in order to improve their efficacy. While gemini nanoparticles (gemini NPs) and other non-viral delivery systems have been vigorously characterized using several techniques, our knowledge is still incomplete. The first objective of this study was the development of new methodology using fluorescence correlation spectroscopy (FCS) to investigate the stages of gemini NPs assembly. It was demonstrated that by labeling the plasmid, different stages of gemini NP assembly from the gemini-plasmid pre-complex (GP) to the final gemini nanoparticle (or gemini-plasmid-lipid complex; GPL), could be studied. Based on diffusion coefficients and particle numbers extrapolated from the autocorrelation function (ACF), FCS was able to determine that each phase of assembly had distinct characteristics. The FCS study using 12-3-12 gemini surfactant showed that both the diffusion coefficient and particle number of GPs (0.98±0.31 x 10-12 m2/s) was significantly lower than the final GPL (3.11±0.41 x 10-12 m2/s). Based on the Stokes-Einstein equation the particle size was calculated to be 300-500 nm for GP and 200-300 nm for GPLs. The raw intensity histograms showed that both GPs and GPLs are composed of multiple plasmids. Furthermore the study showed that the final GPLs contain fewer plasmids compared to the intermediate GP. FCS results were validated by using existing characterization methods including dynamic light scattering (DLS), zeta potential and dye exclusion assays. The second objective involved the detailed characterization of gemini NP. Nine different gemini surfactants and two different phospholipids were used in a systematic study to assess the effect of gemini surfactant and lipid structure on the final morphology of gemini NP. The study revealed that gemini surfactant structure had a strong effect on structure of GP intermediates, but addition of phospholipids resulted in the formation of uniform gemini NPs. Based on the results of this study a new model for GP and GPL assembly is proposed based on the formation of supramolecular aggregates of gemini-plasmids, governed by gemini surfactant chemical structure, and dispersed by phospholipids to form GPLs.

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