Pharmacy - Doctoral Theses
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Item The design of cyclodextrins for delivery of siRNA - a structure-activity relationship(University College Cork, 2024) Kont, Ayse; O'Driscoll, Caitriona M.; Griffin, Brendan T.; Science Foundation Ireland; Jazz Pharmaceuticals; Advanced Materials and Bioengineering ResearchPreviously non-viral delivery of therapeutic nucleic acids (NAs) has been achieved for the treatment of liver disease and in the case of the COVID-19 vaccine. The delivery vector in both applications was a lipid-base nanoparticle (LNP). To expand the therapeutic application of NAs to treat more complex chronic diseases, such as cancer, delivery systems with wider biodistribution capable of going beyond the vaccine and the liver are required. This thesis aims to investigate the potential of modified cyclodextrins (CDs) as alternative biomaterials for siRNA delivery and to identify the optimum functional groups to maximise safety and efficacy. To help reduce the overall cationic charge and the potential for in vivo toxicity a co-formulation approach using a blend of an anionic and cationic amphiphilic CDs was investigated. The co-formulation was characterised and a reduction in the positive charge was achieved. The NPs were evaluated in vitro in HL-60, a leukaemia cell line, and results indicate that endosomal escape was a limiting factor to gene silencing with the siRNA. Structural modification of amphiphilic cationic CDs was investigated as a second approach to enhance the efficacy of CD NPs. The structural changes included varying the terminal amine, the linker, and the CD type, β versus γ. Primary amine proved to be more successful compared to tertiary amine in β- and γ-CDs. However, neither CD type was superior to the other, containing the primary amines. The exhaustive derivatisation of the secondary side of γ-CDs increased charge density and led to better transfection efficiency compared to O2-modified γ-CDs. Finally, the exchange of the linker group from triazole to thiopropyl increased the efficiency further in primary amine O2- and O3-substituted γ-CD. The optimum cellular uptake and gene silencing, in a lung cancer cell line (A549), was achieved with an O2- and O3-substituted γ-CD with a thiopropyl-linked primary amine. Finally, the potential ability of CD polymers to deliver siRNA was studied. Two cationic β-CD-polymers one functionalised with a primary amine and the other with a quaternary ammonium were used to formulate NPs containing siRNA. Both polymers formed NPs with sizes in the range of 150 to 200 nm. The primary amine functionalised polymer was taken up into the cells (A549) and produced 40% gene silencing. In contrast, the quaternary ammonium polymer failed to show any cellular uptake. The superior delivery effect achieved with the primary amine functional group agreed with the previous results from the monomer CD. In conclusion, modulation of the physicochemical characteristics of siRNA-NPs was achieved by changing the chemistry of the incorporated CD. The chemical structure significantly influenced the degree of gene knockdown. The primary amine showed superior efficiency in both monomeric amphiphilic cationic CDs and polymeric cationic CDs. Results indicate that further functionalisation of the CD is possible, and the potential exists to fine-tune the structure to achieve more specific biodistribution.Item Exploring the potential impact of integrating pharmacists into general practice in Ireland(University College Cork, 2024) Hurley, Eoin; Byrne, Stephen; Dalton, Kieran; Foley, Tony; Walsh, Elaine; Irish Research CouncilIntroduction Globally, due to advances in medicine and public health, populations are living longer. Consequently, the prevalence of multimorbidity and polypharmacy is rising. General practitioners’ (GPs’) workloads are therefore increasing, further compounded by: initiatives to increase the capacity of primary care, the underserved needs of older frail adults in nursing homes, and workforce shortages. In response to growing pressures, pharmacists have been integrated into general practices and affiliated settings in several jurisdictions, most notably in the United Kingdom (UK). Despite evidence showing that pharmacists in general practices improve patient outcomes, they have not been integrated into general practices in many countries. Key gaps in the literature that may be contributing to this include: 1) limited research focusing on GPs’ perceptions of integrating pharmacists into practices, 2) little evaluation of the role of a general practice-based pharmacist in optimising frail older adults’ medications, and 3) economic evidence for the viability of pharmacists in practices outside of the cost savings attributed to deprescribed medications. Therefore, the overarching aims of this thesis were to: i) identify the key factors affecting pharmacist integration into practices through extensive theory-informed engagement with GPs, ii) evaluate the clinical outcomes of a pharmacist-led intervention with frail older adults in general practice-affiliated nursing homes, and iii) to determine the economic viability of the intervention by calculating the costs avoided as a result of the pharmacist-led intervention. Methods Firstly, a qualitative evidence synthesis (QES) was conducted to capture GPs’ views regarding pharmacists working in general practices. Secondly, a qualitative interview study informed by the Theoretical Domains Framework (TDF), was undertaken to explore GPs’ perceptions of working with pharmacists in general practices and identify the behavioural determinants of them doing so. Then, based on the QES and interview study, a mixed-methods survey study was carried out to establish consensus on key issues identified and to determine characteristics of GPs or practices that may be particularly suited to general practice-based pharmacists. Parallel to this work, evaluation of the clinical outcomes from a pharmacist-led intervention in general practices and affiliated nursing homes was conducted; this involved pharmacist-led medication reviews guided by Screening Tool of Older Persons Prescriptions in Frail adults with limited life expectancy (STOPPFrail) that were carried out on frail older adults. Finally, the economic viability of the pharmacist-led intervention was determined by calculating the cost savings associated with avoided adverse drug events (ADEs) and direct cost savings due to the cost of deprescribed medications. Results Firstly, a conceptual model was developed based on the QES’ findings that described GPs’ views of pharmacists working in general practices, which could be used to develop or optimise pharmacist services in general practice. The QES demonstrated that little research had been conducted that sought GPs’ perceptions of pharmacists working in general practices prior to pharmacist integration. The TDF-informed qualitative interview study that followed identified the predominant behavioural determinants of GPs integrating pharmacists into their practices and found that GPs were broadly optimistic about pharmacists working in GP practices and the potential outcomes of them doing so. However, GPs were concerned about the impact of pharmacists on GPs’ and other practice staffs’ workloads, funding the salary of a general practice pharmacist, and training for pharmacists to work in practices. In the subsequent cross-sectional survey study of GPs (n = 152), the majority (78%) indicated that they would participate in a hypothetical pilot study of having a practice-based pharmacist. Certain roles for pharmacists in practices (e.g. medication reviews) had higher levels of agreement (≥90% agreement) from GPs than other roles (<50% agreement) like independent prescribing. Again, concern was identified amongst GPs concerning the potential impact on workloads, indemnification of pharmacists, and the potential weakening of patient-GP relationships. Evaluation of pharmacist-led application of STOPPFrail to nursing home residents (n = 99), found the most frequently identified potentially inappropriate medications (PIMs) were medications without a clear clinical indication (29.6%) and vitamin D (16.9%). Of the 349 clinically relevant deprescribing recommendations made by the pharmacist, 55% were implemented by GPs. There were significant post-review decreases in the number of prescribed medications, modified medication appropriateness index (MMAI), drug burden index (DBI), and anticholinergic cognitive burden (ACB) which remained significant at six months (p<0.05). There were no significant differences in falls, emergency department visits, non-elective hospitalisations, or quality of life (QoL). Finally, the cost avoidance evaluation of the intervention showed the intervention to be associated with significant cost savings (€24,827 - 251,903). The cost-benefit ratio remained positive in all scenarios examined in sensitivity analyses (1:11.8 - 1:67.6). Conclusion This thesis has made a significant original contribution to knowledge; it has identified key barriers and enablers to integrating pharmacists into practices and shown that there are clear benefits to doing so for patients, GPs, and the wider healthcare system. The embedding of theory into this research has laid the groundwork for future research which seeks to integrate pharmacists into the general practice setting in a theoretically-informed manner, ultimately with the goal of increasing expertise and support within the general practice setting to optimise medications and improve health outcomes for patients.Item Application of fluorescence spectroscopy for the in-vial investigation of protein solutions(University College Cork, 2024) ElKassas, Khaled; Crean, Abina; Vucen, Sonja; Science Foundation Ireland; Engineering and Physical Sciences Research CouncilTherapeutic protein formulations are subjected to various stressors during manufacturing, transport, and storage, causing destabilisation, in turn leading to deleterious effects such as immunogenic reactions or inefficacy upon administration. An essential part of this production and supply process is establishing protein formulation stability at all stages. Spectroscopy is a common laboratory tool for the characterisation of therapeutic proteins. Conventional techniques for determining protein denaturation are lab-based and require sample removal from the sealed primary packaging vial and sample destruction. To overcome the limitations of traditional techniques, the development of a fluorescence-based spectroscopic technique for “in-vial” protein analysis was a key thesis aim. A fluorescence excitation wavelength of 365 nm was found and confirmed to be transmissible through borosilicate glass vial walls. The excitation resulted in an intrinsic emission at 462 nm for a model protein, bovine serum albumin (BSA). Using a bespoke apparatus, the change in fluorescence signal with time was monitored over the reconstitution period of lyophilised protein formulations, without breach of the vial seal. First, we demonstrated how analysis of changes in fluorescence signal with time during reconstitution, using principal component analysis, could be used to determine an instrumentally quantified reconstitution time. At high protein concentrations (10% w/v BSA) the variability of the reconstitution time measurements was reduced from 80.4% relative standard deviation obtained via the pharmacopeial visual method to 8.2% for the instrumental method. Spectroscopic measurements resulted in longer measured reconstitution time compared to the visual method, possibly owing to the detection of subvisible particulates undergoing dissolution. In the second part of this work, the bespoke spectroscopic apparatus was modified with polarising filters to monitor protein aggregation and denaturation in-vial. Detector measurement of the emission wavelength (365 nm) allowed measurement of protein aggregation via light scattering. Fluorescence anisotropy (a measurement of optical polarizability at different angles) at 462 nm was used to determine protein conformational changes. Fluorescence scattering and anisotropy measurements for freshly prepared, aggregated, and denatured BSA solutions were compared to reference circular dichroism (CD) and size-exclusion chromatography (SEC) measurements. Anisotropy analysis of thermal unfolding showed strong correlations with secondary structure as quantified through a CD-neural networking model. Scattering correlated with large molecular weight aggregate measurements via SEC. A study was conducted to find the relationship between the fluorescent amino acids and the overall protein fluorescence for 4 different proteins. Using a multivariate technique, multivariate curve resolution to resolve 3D emission excitation spectra, the fluorescence of individual amino acid solutions and the total fluorescence of the proteins was correlated. Finally, a partial least squares regression (PLSR) model relating the anisotropy and scattering measurements to reference CD and SEC measurements was established from a sample set of standardised and stored samples. A total of 72 spectra were collected for the batch standardised protein solutions and the stored stability study samples. Samples were stored under real-world-like stress conditions (temperature, shaking, ambient light exposure). The fluorescence anisotropy and scattering results were included as the predictors vs. CD and SEC measurements as the responses. The PLSR showed strong correlations with an average R2 of 0.87 and a root mean PRESS of 0.487 at 9 latent variables. In conclusion, the combination of bespoke apparatus and 365 nm excitation was capable of monitoring protein stability in-vial successfully. The spectroscopic measurement of reconstitution time resulted in more precise measurements. The method overcomes the challenges present in the current pharmacopeial standard measurement as it can detect subvisible particulates and is independent of analyst capabilities and subjectivity. The protein solution stability monitoring provided a holistic non-destructive alternative to multi-instrument analysis while maintaining reasonable correlations with established reference stability indicators. This research provides a platform for a cost-effective portable solution to provide a top-level overview of biopharmaceutical product stability in-vial, from manufacture to the point of administration.Item Studies 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.Item Deprescribing 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.