ItemStudies in the synthesis and impurity profiling of pharmacologically active benzodifurans(University College Cork, 2022) O'Connor, Richard Eric; Keating, J J; Higher Education AuthoritySubstituted 2,3-dihydrobenzofuran derivatives as well as their benzofuran and benzodifuranyl analogues are versatile heterocycles that are increasing in prominence as key building blocks in organic chemistry. In particular, benzodifuran-containing structures are gaining significant interest in medicinal chemistry as pharmacophores and in industrial chemistry as scaffolds for organic electroluminescent devices, organic field-effect transistors, solar cell sensitizers and semiconducting polymers. The synthesis of pharmacologically active benzodifurans are complex multi-step processes and each individual step has the potential for considerable side-product/impurity formation. This project focused on synthetic strategies to benzofuran and benzodifuran derivatives in addition to their hydrogenated and substituted analogues. The chosen synthetic routes to target molecules further focused on the propensity of synthetic steps to produce impurities, modifiable variables to modulate impurity formation and the impurity profile of each synthetic route explored. Organic process impurities identified were isolated and fully characterised where possible, in addition to intended products. ItemDeprescribing for frail older adults residing in long-term care facilities: a mixed-methods evaluation(University College Cork, 2023) Heinrich, Clara H.; Donovan, Maria; Mccarthy, Suzanne; Mchugh, Sheena; School of Pharmacy, University of SydneyIntroduction Globally, the population is ageing and the demand for long-term care (LTC) is increasing. Older adults residing in LTC have a higher degree of physical frailty compared to their community-dwelling counterparts. The physiological changes associated with ageing and frailty can increase the risk of experiencing an adverse event from a medication. Polypharmacy and potentially inappropriate medications (PIMs) are even more of a concern in frail older adults as frailty increases the risk of adverse outcomes. Deprescribing has been shown to reduce PIMs for older adults residing in LTC however, deprescribing is not universally implemented. The aim of this thesis is to develop and test an evidence- and theory-based implementation strategy to support HCPs engage with deprescribing in routine practice for frail older adults residing in LTC. Methods A mixed-methods approach was utilised for this thesis, informed by the Medical Research Council’s framework for designing and evaluating complex interventions. Firstly, the ‘best-fit’ framework method was used to synthesise the qualitative evidence, using the Theoretical Domains Framework. The resulting ‘best-fit’ framework was used to inform a semi-structured interview study of HCPs working in LTC to identify context-specific deprescribing barriers/enablers. A retrospective chart review was conducted in two long-term care facilities (LTCFs) using Screening Tool of Older Persons Prescriptions in Frail adults with limited life expectancy version 2 (STOPPFrail v2) to identify PIMs. Results supported the design of a deprescribing implementation strategy. The implementation strategy design consisted of 3 phases; a mapping process to identify behaviour change techniques (BCTs) which could form a strategy, a modified online Delphi survey to select feasible BCTs and a roundtable discussion to prioritise barriers/enablers and tailor the proposed strategies for deprescribing in LTC. A feasibility study of the resulting strategy was conducted in two public LTCFs focusing on implementation outcomes whilst collecting intervention data. Results The qualitative evidence synthesis and interview study summarised the deprescribing barriers/enablers in LTC. Barriers included insufficient resources, lack of co-ordination between healthcare settings and negative social influences. Deprescribing enablers included interprofessional support and patient social influence. The retrospective chart review study identified that lack of a documented indication for prescribed medications was the most prevalent PIM criterion, with antihypertensives the largest drug class potentially inappropriately prescribed. The final deprescribing implementation strategy consisted of an education-enhanced 3-monthly multidisciplinary team deprescribing review, led by a nurse, conducted at the LTC site targeting antihypertensives, named the DEFERAL strategy. Of HCPs involved in the feasibility study, the DEFERAL strategy was unanimously considered acceptable and appropriate, with 90% agreeing that it was feasible. Qualitative feedback outlined site-specific modifications for future consideration. Conclusion This thesis has designed and feasibility tested a theory- and stakeholder-informed implementation strategy to support HCPs to engage with deprescribing PIMs prescribed to frail older adults residing in public LTCFs in Ireland. The DEFERAL strategy helped to address a gap reported in the deprescribing literature by incorporating concepts from behavioural and implementation science to target barriers and enablers inherent with the LTC setting and translate deprescribing into routine practice. The strategy was considered acceptable, appropriate and majorly feasible, with modifications reported for consideration in future piloting and effectiveness research. ItemSmart drug delivery systems for siRNA therapeutics in prostate cancer immunotherapy(University College Cork, 2023) Sun, Yao; O'Driscoll, Caitriona M.; Synthesis and Solid State Pharmaceutical Centre; Science Foundation IrelandProstate cancer (PCa) as an immunosuppressive cancer remains a serious condition threatening the health of men due to the complicated nature of the tumour microenvironment (TME). Conventional treatments of PCa face challenges including poor prognosis and tumour resistance, therefore new therapeutic strategies are urgently needed. Tumour-associated macrophages (TAMs) are a potential therapeutic target in cancer immunotherapy. Colony stimulating factor-1/colony stimulating factor-1 receptor (CSF-1/CSF-1R) pathway plays a crucial role in the polarization of the immunosuppressive TAMs, M2 macrophages. Downregulation of CSF-1R expression by small interfering RNA (siRNA), a double-stranded non-coding RNA, is known to reprogram the immunosuppressive TAMs, M2 macrophages, to the immunostimulatory phenotype, M1 macrophages. Sialic acid (SA) is a ligand for Siglec-1 (CD169) which is overexpressed on M2 macrophages with little expression in other phenotypes. M2 macrophage-targeting peptide (M2pep) is an artificially designed peptide and could specifically target M2 macrophages, the sequence of which was selected by subtractive phage biopanning. Beta-cyclodextrins (CDs) are cyclic oligosaccharides derived from starch, consisting of at 7 glucose subunits linked by α-1,4 glycosidic bonds, the primary and secondary sides of the ring structure are hydrophilic and the cavity is hydrophobic. Cationic CDs have been used for delivery of therapeutic nucleic acids including siRNA as non-viral vectors. Therefore, in this thesis, 2 different CD-based nanoparticles (NPs) with PEGylated SA or PEGylated M2pep as the ligand was designed and characterized for the delivery of CSF-1R to M2 macrophages in both human and mouse cells to study the macrophage reprogramming efficacy. In the meanwhile, a Transwell model was built for co-culture of M2 macrophages and PCa cells to test the anti-PCa effectiveness in vitro. In vitro results show that the sizes of three NPs were around 250 nm, and the surface charges were 11 ~ 29 mV, the poly dispersity index (PDI) was lower than 0.30. All CD-based formulations were stable in both salt- and serum-containing media, and did not present any cytotoxicity in either human or mouse macrophages. Both two NPs achieved M2 macrophage reprogramming, and post-transfected cells expressed M1 macrophage markers. The co-culture results show that the formulations caused the apoptosis of PCa cells due to the M2 macrophage reprogramming. In the in vitro studies, the CD.siRNA.DSPE-PEG-M2pep formulation presented the best CSF-1R gene silencing efficacy, superior M2 macrophage reprogramming and anti-tumour efficacy. Therefore, the CD.siRNA.DSPE-PEG-M2pep formulation was selected for assessment in vivo. A PCa-bearing mouse model was built in C57 BL/6 mice by subcutaneous injection of PCa cells and used to study the targeting and anti-PCa efficacy of CD.siRNA.DSPE-PEG-M2pep in vivo. The NPs increased M2 macrophage targeting in vivo, promoting the release of M1 factors and simultaneously downregulating the levels of M2 factors through TAM reprogramming. The subsequent remodelling of the TME resulted in a reduction in tumour growth in the PCa mouse model, mainly mediated through the recruitment of cytotoxic T cells. In summary, the CD-based targeted siRNA NPs successfully reprogrammed the immunosuppressive M2 macrophages thus promoting PCa immunotherapy and providing an alternative strategy for PCa treatment worthy of further investigation. ItemPreclinical 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 BoardStroke 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. ItemThe role of regulatory T cells in stroke recovery(University College Cork, 2022-06-16) Malone, Kyle; Waeber, Christian; Moore, Anne; Irish Research CouncilBackground: 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.