Pharmacy - Doctoral Theses

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Preclinical characterisation of fingolimod as a potential therapeutic agent for stroke
(University College Cork, 2022-12) Diaz Diaz, Andrea C.; Waeber, Christian; Moore, Anne; Health Research Board
Stroke is the leading cause for death and disability worldwide, and the search for novel drug treatments has been affected by repeated clinical trial failures. One novel drug that has garnered promising results in preclinical and clinical stroke studies is fingolimod, an FDA approved drug for the treatment of multiple sclerosis. Even though there are several studies supporting the effectiveness of fingolimod for the treatment of stroke, the recommended characterisation based on the Stroke Treatment Academic Industry Roundtable (STAIR) guidelines is incomplete. Furthermore, the quality of the preclinical studies supporting fingolimod has been poor, thus rigorous studies are required to validate the effectiveness of fingolimod prior to evaluation in clinical trials. This thesis aimed to inform whether fingolimod is effective for the treatment of stroke in intracerebral haemorrhage and ischaemic stroke, and to inform whether fingolimod is a good candidate for evaluation in large randomised clinical trials. This goal was achieved by first using a model of intracerebral haemorrhage to evaluate the effect of administering fingolimod at 30 min, 24 and 48 h after stroke on lesion size and behaviour in a 14-day study on male and female mice. This was followed up by a series of studies using middle cerebral artery occlusion to cause a focal ischaemia. First an optimal dose of fingolimod was determined in a dose response study; then we evaluated the optimal drug dose in two animal model of common comorbidities associated with stroke, age and hyperlipidaemia; and the last study evaluated the effect of an extended treatment duration on stroke. For the ischaemic stroke studies we focused on lesion and behavioural measurements as primary outcomes, and secondary data was collected from daily scores, plus additional measures where necessary. The intracerebral haemorrhage study fingolimod treatment had no measurable effect on either lesion size or behavioural outcomes irrespective of sex, the only finding was that fingolimod treatment reduced mortality in female mice. The dose response study showed no difference in lesion size or behavioural outcomes between the two fingolimod doses (0.5 and 1.0 mg/kg) and control mice, the study did show that saline treated mice had a significantly larger atrophy compared to the lower dose of fingolimod. The lower dose was selected as optimal for further studies. The study evaluating the effect of 0.5 mg/kg fingolimod on stroke in aged mice showed that fingolimod-treated mice had a significantly larger atrophy and a significant improvement in the grid score 7 d after stroke compared to saline-treated mice. The study evaluating the effect of fingolimod on stroke in hyperlipidaemic mice showed that fingolimod-treated mice had a significantly reduced lesion size, without an effect on any other outcome measures. The final study evaluated two fingolimod treatment durations compared to saline controls, the study showed no difference between any of the outcome measures, with a trend towards improved behaviour outcomes in mice receiving 10 d of fingolimod treatment. Lastly, considering the fact that the results of these studies were inconclusive, we decided to pool the data of the ischaemic studies and evaluate whether fingolimod had an effect on the primary outcome measures in a heterogenous animal population. The pooled data showed that fingolimod treatment improved behaviour 7 d after stroke without an effect on lesion size or atrophy. The results of this thesis cast a doubt on the effectiveness of fingolimod and its suitability for translation into larger clinical trials. Furthermore, they highlight the need for thorough preclinical studies for promising drugs as well as the need for studies to meet the proposed STAIR guidelines prior to translation. Confirmatory studies, like those presented here, performed with measures intended to control for internal and external biases are all good measures to be implemented for future studies of novel and highly promising drugs for stroke treatment.
The role of regulatory T cells in stroke recovery
(University College Cork, 2022-06-16) Malone, Kyle; Waeber, Christian; Moore, Anne; Irish Research Council
Background: Acute ischaemic stroke is a major cause of mortality worldwide. Despite the search for new therapies, tissue plasminogen activator remains the only FDA-approved medication. The immune system is involved in all stages of stroke, from risk factors to pathogenesis and tissue repair. The presence of a lymphocyte subpopulation termed regulatory T cells (Tregs) appears to correlate with improved disease outcome. However, the impact of stroke on Tregs, and the contribution of these cells to stroke recovery remains under debate. Likewise, attempts at developing Treg-targeted immunotherapies have been hampered by high cost, toxicity, and the stability of expanded Tregs. The aim of this thesis was to explore the role Tregs play in ischaemic stroke recovery and evaluate the neuroprotective and immunomodulatory effects of two potential Treg-targeted stroke immunotherapies, namely fingolimod and recombinant pregnancy-specific glycoprotein-1 (rPSG1-Fc). Methods: The effect of fingolimod on Tregs in a mouse model of permanent brain ischaemia was first investigated using a combination of flow cytometry and immunohistochemistry. Next, the impact of fingolimod on Treg suppressive function was characterised via Treg suppression assay. Following this, the immunomodulatory and neuroprotective properties of rPSG1-Fc in permanent brain ischaemia were determined. Finally, the changes in Treg frequency and function in the peripheral blood of mild stroke patients were quantified. Results: Fingolimod increased peripheral Treg frequency in the post-ischaemic mouse. Fingolimod augmented brain infiltration of FoxP3+ T cells, possibly via CCR8 signalling. Fingolimod also enhanced the secretion of IFN-γ, IL-17, and IL-10 from CD4+ T cells. Likewise, fingolimod promoted both suppressive and effector T cell function. rPSG1-Fc improved neurobehavioural recovery in mice post-brain ischaemia, possibly via increased CD4+IL-10+ T cells. Finally, an increased Treg frequency and an increased expression of functional markers of suppression (CTLA-4, PD-1) was observed in stroke patients at 24 hours post-ischaemia, and specifically among proliferating Tregs. However, by 7 days, the expression of PD-1/CTLA-4 among proliferating Treg frequency had returned to baseline. Conclusions: This thesis has made a number of novel insights. A positive impact of fingolimod on both Treg frequency and function post-ischaemia was revealed. The observed dual effect of fingolimod on regulatory and pro-inflammatory T cell function may explain why the drug fails to consistently improve experimental stroke outcome. The immunomodulatory and neuroprotective effects of rPSG1-Fc post-stroke were also characterised for the first time. Finally, the impact of clinical stroke on Treg frequency and phenotype was comprehensively quantified. Overall, this thesis shows Tregs may not play a major role in the early stages of recovery of mild stroke, but therapies manipulating them can still promote functional recovery in a mouse model. It provides a basis for further study on Tregs in ischaemic stroke. It also illustrates rigorous methods by which researchers should test future Treg-targeted stroke immunotherapies.
Computational pharmaceutics approaches to inform drug developability: focus on lipid-based formulations
(University College Cork, 2021-10-04) Bennett-Lenane, Harriet; Griffin, Brendan T.; O'Shea, Joseph; O'Driscoll, Caitriona M.; Irish Research Council
Purpose: Declining productivity in the face of increasing numbers of poorly water-soluble drugs has fast-tracked necessity for predictive tools which assess the delivery potential of bio-enabling formulations. However, there is a perceived risk associated with early-stage selection of bio-enabling formulations. Computational pharmaceutics is a growing area of research interest to support structured guidance in formulation strategy. Using data-driven modelling, a streamlined roadmap of computational possibilities for development scientists is possible. Accordingly, the aim of this thesis was to examine the application of machine learning (ML) computational modelling to inform candidate developability. Via prediction of both quality target product profile characteristics and formulation performance indicators for lipid-based formulations (LBF). In recognition of the fact that computational models will not entirely circumvent need for manual screening, a further aim was to explore if analysis of landrace pig gastrointestinal fluids could facilitate increased bio-predictive performance of in vitro tools for LBFs. Methods: Data-driven computational models using various ML algorithms, with both classification and regression outputs, were developed to predict food effect on bioavailability, solubility ratio (SR) upon self-emulsifying drug delivery system (SEDDS) dispersion and apparent degree of supersaturation (aDS) ratio in supersaturated LBFs (sLBF). Model performance was validated using test sets or comparisons to ex vivo results. Gastrointestinal fluids from the landrace pig were collected in the fasted state, fed state and post placebo SEDDS administration. Ex vivo solubility analysis and microscopic imaging were completed using these fluids, where in vitro biorelevant dispersion screening with SEDDS using various dilution conditions was compared to the ex vivo results. Results: Firstly, this thesis demonstrated the applicability of ML for the prediction of a quality target product profile characteristic of interest in early development, namely food effect on bioavailability. Secondly, this thesis has advanced computational pharmaceutics to inform drug developability. Computational predictions of solubility gain upon SEDDS dispersion informed a biopharmaceutical dose number in intestinal fluids, which can be incorporated within the developability classification system (DCS) framework to inform drug developability. Thirdly, in recognition of the use of supersaturated LBFs to overcome dose loading limitations, this thesis has demonstrated how ML algorithms can predict the maximum dose loading upon thermal induced supersaturation. Moreover, increased understanding of the fate of SEDDS upon oral administration furthered the utility of the pig pre-clinical model, validated accuracy of in silico predictions and aided development of a bio-predictive in vitro screening tool for LBFs. Ultimately, it was demonstrated that the computational and in vitro tools developed in this thesis can be embedded within a wider refined drug substance to drug product development framework. Conclusion: This thesis highlighted how pharmaceutics datasets are amenable to ML. The ability of computational pharmaceutics to facilitate structured formulation decisions was demonstrated. As model development aided increased understanding of the investigated phenomena through their relationship to drug properties, this thesis identified the significant potential to be gained from early analysis of drug properties. Additionally, utility of the landrace pig model to inform increasingly bio-predictive in vitro screening tools was established. The proposed refining of the drug substance to drug product development framework demonstrated the significance of both computationally informed and experimentally confirmed aspects of drug developability decision-making.
Opportunities to measure and improve antimicrobial stewardship in the Irish hospital setting
(University College Cork, 2022-02-20) O'Riordan, Frank; Fleming, Aoife; Shiely, Frances; Byrne, Stephen
Background: Antimicrobial resistance (AMR) is one of the most significant threats to public health and increasing levels of resistance among gram negative organisms, and levels of vancomycin-resistant Enterococcus faecium (VRE) are of concern across Europe and in Ireland. Antimicrobial stewardship (AMS) programmes are well established in the Irish hospital setting but it is important that they continue to respond and evolve to the threat of AMR. This can be achieved by investigating new interventions and adopting new diagnostics methods to support hospital AMS programmes and other opportunities to measure and improve AMS programmes. Aim: The overall aim of this research thesis was to investigate opportunities to measure and improve the delivery of AMS programmes in the Irish hospital setting. Methods: A randomised feasibility study was conducted to investigate the implementation of Procalcitonin (PCT) testing in patients with a respiratory tract infection (RTI) as an intervention to support the AMS programme in an Irish hospital setting. An in-depth qualitative process evaluation (PE) of the PCT intervention was conducted to examine the implementation process and to determine the barriers and facilitators to the implementation of PCT as an AMS intervention. The Consolidated Framework for Implementation Research (CFIR) was used to guide data collection, analysis, and interpretation. A systematic review of quality indicators (QIs) for hospital AMS programmes was undertaken, with a critical appraisal of their methodological qualities using the Appraisal of Indicators through Research and Evaluation (AIRE) instrument. A time series analysis (TSA) of antimicrobial consumption (AC) data and AMR data was conducted to investigate trends, and possible relationships between AC and AMR. Results: The feasibility study determined that the introduction of PCT testing in patients with a RTI had a positive impact on antimicrobial prescribing resulting in a significant decrease in duration of antimicrobial prescriptions and decreased length of hospital stay. The qualitative PE identified positive elements of the implementation process along with modifications to improve the delivery of the intervention. The contextual factors which can act as barriers and facilitators to the implementation of AMS interventions were identified and included the concepts of fear, risk and the influence of respiratory clinicians on AMS interventions. The systematic review collated an extensive range of QIs for AMS programmes in the hospital setting which predominantly consisted of process (e.g. infection diagnosis) and structural indicators (e.g. AMS governance), with few outcome indicators developed, a major deficiency in this area. There was limited reporting of information about validation and piloting of QIs sets in practice which is an essential element of the QI development process. The TSA analysis of AC and AMR rates found decreasing or relatively stable rates of AMR in Enterobacterales and VRE isolates but increasing incidence of carbapenem resistance which must be addressed as part of the local AMS programme. Conclusion: This thesis presents a comprehensive and detailed body of research investigating AMS opportunities in the Irish hospital setting. The results of this research indicate that AMS programmes will need to continue to evolve, and dedicated resources will be required to support research in AMS practice. This will include optimising the use of new diagnostic tools such as PCT to support physicians in making antimicrobial prescribing decisions; the incorporation of QIs to assess and improve AMS programmes; and the implementation of in-depth surveillance analysis of AC and AMR using TSA. These measures have the potential to make substantial contributions to hospital AMS measures locally and internationally.
Synthesis and evaluation of novel quorum sensing inhibitors
(University College Cork, 2021-07-01) Lyons, Thérèse; O'Sullivan, Tim; Gahan, Cormac G.; Future University Egypt; University College Cork
This thesis details the synthesis of a range of furanones and the subsequent evaluation of their quorum sensing inhibitory effects. Chapter One focuses on the role of quorum sensing in bacteria and fungi. It explores the potential use of quorum sensing inhibitors as an alternative to, or in combination with antibiotics during the treatment of microbial infections. This chapter is also comprised of a review which details the structure activity relationships of halogenated furanones which have previously been evaluated in the literature. Synthetic routes to a number of these analogues are also detailed. In Chapter Two a concise preparation of gem-dibromofuranones is presented. A total of 33 novel compounds were subsequently generated via a variety of different chemistries, namely Suzuki and Sonogashira couplings. The synthesis of chlorine-, iodine- and fluorine-containing furanones is described in Chapter Three. The reactivity of various dihalofuranones is compared via palladium-catalysed Suzuki couplings, which proceed with high stereoselectivity. While modified Ramirez olefination conditions provided access to bromofluorofuranone analogues, in the end they were unstable. Chapter Four describes our ultimately unsuccessful attempt at coupling a bromofuranone with L-cysteine. Nevertheless, we were able to successfully couple the N-Boc-protected-methyl ester of L-cysteine with a tribromofuranone. The biological activity of the novel compounds generated throughout this project is detailed in Chapter Five. The furanones were subjected to AI-2 bioluminescence assays in University College Cork, anti-biofilm assays at Universitat Autònoma de Barcelona in Spain and the Cellular Microbiology Research Centre in Borstal Germany and anti-microbial assays at CO-ADD, Australia. A number of novel furanones displayed very promising activity against several different strains of bacteria and fungi including P. aeruginosa, V. harveyi, S. enterica and S. aureus. Avenues for further study on this project are detailed in Chapter Six. Full experimental procedures, including spectroscopic and analytical data, for all compounds synthesised throughout this work are outlined in Chapter Seven.

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