Pharmacy - Doctoral Theses
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Item Novel, bioinspired 3D bone models to simulate prostate cancer bone metastases(University College Cork, 2024) Dozzo, Annachiara; Ryan, Katie; O'Driscoll, Caitriona M.Prostate cancer (PC) ranks first amongst the types of cancer affecting the male population. There is a lack of effective drug treatments for patients diagnosed with metastatic spread of PC (mPC) to the bone and their prognosis is poor. The development of 3D, in vitro, models offers exciting opportunities to recapitulate the native tissue environment in vitro, to better understand diseases such as cancer, to reduce the overreliance on animal models in drug development and to develop more effective drug treatments. However, the number of 3D scaffold models of bone is limited, and many lack structural relevance to the hierarchy characterising the native bone tissue. While very few have been used to model mPC spread to the bone. The work presented in this aims to address these limitations by providing several scaffolds for deployment as models of mPC in the bone. The first models were produced using conventional techniques (CO2 foaming/porogen leaching), while additive manufacture (AM) methods including fused deposition modelling (FDM) and stereolithography (SLA) were used to produce advanced models based on a novel and versatile CAD design (iMARS), which was conceived as part of the work in the thesis. The different scaffolds proposed were physically and mechanically characterised and used as testbeds for drug studies with docetaxel to investigate their clinical correlation capabilities. Additive manufacture of scaffolds according to the novel and versatile iMARS design enabled reproducible scaffold production. In general, it was shown that scaffold type, the methodology applied to produce it and the blend of materials chosen, impacted the physicochemical properties of the scaffolds and influenced the behaviour of osteoblast cells, hFOB 1.19 in mono- or co-culture with prostate cancer cells, PC-3, and the cellular response to chemotherapeutics. The use of the biodegradable, PLGA copolymer either blended or coated with hydroxyapatite and/or collagen altered the scaffold’s microenvironmental cues, and enhanced the malignant profile of PC-3 cells at the expense of hFOB 1.19 cells contributing to the development of clinically relevant niches of mPC in the bone.Item Siponimod as a novel therapeutic for ocular neovascular diseases: biological characterization and development of controlled release systems(University College Cork, 2024) Alshaikh, Rasha A.; Waeber, Christian; Ryan, Katie; Irish Research Council for Science, Engineering and TechnologyBackground: Neovascular ocular diseases, including age-related macular degeneration, diabetic retinopathy, and retinal vein occlusion, are characterized by pathological processes such as angiogenesis, oedema, inflammation, cell death, and fibrosis. Current therapeutic approaches predominantly target vascular endothelial growth factor (VEGF), one of the primary mediators of pathological angiogenesis. However, while effective, VEGF blockers are costly macromolecules requiring frequent intravitreal administration and are associated with various adverse effects and high treatment resistance rates. Consequently, identifying small molecular weight angiogenesis inhibitors with alternative mechanisms of action and developing sustained, less invasive delivery systems for intravitreal administration are needed. In this thesis, I explored the potential of siponimod (a sphingosine 1-phosphate receptor modulator approved for multiple sclerosis) as an inhibitor of ocular angiogenesis in vitro and in vivo. Subsequently, upon demonstrating its potential efficacy, I characterized siponimod's ocular pharmacokinetics (PK) and toxicity in vivo, along with its solubility and stability profiles. Leveraging siponimod's small molecular weight and lipophilic nature, we investigated the ability of electrospinning to sustain the release of siponimod and improve the current standard of monthly intravitreal injection. Then, we explored the potential of improving the current treatment standard of diabetic retinopathy and diabetic macula oedema by cocrystallisation of siponimod and fluocinolone acetonide. If successful, these cocrystals can concomitantly address the components of angiogenesis and inflammation in these diseases. Methods: The effect of siponimod on angiogenesis in vitro was confirmed using endothelial cells and various functional assays, including growth factor-induced cell proliferation and migration. Siponimod’s impact on the integrity of the retinal endothelial barrier under stress conditions was also investigated using a combination of functional assays and immunofluorescence. Furthermore, the effect of siponimod on ocular neovascularisation in vivo was assessed using a suture-induced corneal neovascularisation model in albino rabbits. Then, siponimod’s ocular PK and toxicity were investigated after intravitreal injection in albino rabbits and the PK parameters of the drug were computed. This was followed by a preformulation study to determine the drug's solubility and stability under stress conditions. A microfibrous PLGA implant carrying siponimod was developed using electrospinning. The implant was characterised using solid-state characterisation techniques and drug-polymer interaction, and the drug release from the implant in vitro was investigated. The implant stability under stress conditions and the ability of the released drug to produce a therapeutic effect was also confirmed. Finally, novel siponimod fluocinolone acetonide cocrystals were produced for dual drug delivery in diabetic retinopathy and macular oedema. Cocrystals were characterised using solid-state characterisation techniques. Their dissolution rate, saturation solubility, and stability under stress conditions were also evaluated. Then, PLGA microparticles loaded with cocrystals were produced using electrospraying. Their drug loading, size, morphology, drug release and ability to produce dual angiogenesis and inflammation inhibition over 100 days were investigated. Results: In vitro data showed that siponimod is a potent angiogenesis inhibitor and protects endothelial barrier function. The drug inhibited endothelial cell migration towards different growth factors, increased retinal endothelial barrier integrity, and reduced TNF-α-induced barrier disruption. These actions were mediated by sphingosine 1-phosphate receptor-1 modulation. Furthermore, siponimod, tested in two different doses, completely abolished the progression of suture-induced corneal neovascularisation in albino rabbits and reduced corneal epithelial thinning. Siponimod’s half-life after intravitreal injection of low and high doses was 2.8 h and 3.9 h, respectively. No signs of retinal toxicity were observed after intravitreal administration. Siponimod showed a significant increase in solubility in porcine vitreous compared to aqueous saline and exhibited temperature-dependent degradation in aqueous solution. An electrospun bead-free microfibrous PLGA implant carrying siponimod was successfully produced under optimised conditions. Siponimod showed a uniform distribution within the electrospun fibres as a stabilised, amorphous, solid dispersion with a significant drug-polymer interaction. Siponimod dispersion and drug-polymer interactions formed smooth fibres devoid of porous structures. This inherent lack of porosity, coupled with the drug’s hydrophobic dispersion, afforded resistance to water penetration. This results in a slow Higuchi-type diffusion of siponimod, with approximately 30% of the drug load being released over 90 days. The released drug maintained a pharmacological effect in vitro comparable to that of a freshly prepared drug solution, indicating the stability of the drug cargo. Moreover, the implant retained physical and chemical stabilities under stress conditions for 3 months. Finally, siponimod and fluocinolone acetonide cocrystals exhibited higher physical stability than the parent siponimod, maintaining their crystalline pattern under stress conditions. The cocrystals also showed a lower dissolution rate under sink conditions compared to siponimod. Electrosprayed cocrystal-loaded PLGA microparticles released ~ 70% of the drug load over 100 days. The released drugs successfully and simultaneously inhibited retinal endothelial cell migration and IL-6 production. This indicates the superior efficacy of the microparticles in the dual inhibition of angiogenesis and inflammation, crucial aspects in managing diabetic retinopathy and macular oedema. Conclusion: The efficacy of siponimod in vitro and in vivo supports its therapeutic potential in ocular neovascular diseases. The drug has a small molecular weight, lipophilic nature, short ocular half-life and is prone to thermal degradation in solution. Siponimod was formulated in a stabilising, sustained-release microfibrous electrospun PLGA implant. The drug interaction with PLGA provided a unique opportunity to sustain drug release from the electrospun fibres, thereby reducing the frequency of intravitreal injection, improving patient adherence, and representing a potential alternative to anti-VEGF treatments in AMD and DR. Such drug-polymer interactions can provide a low-cost, effective method for sustaining small molecules for ocular applications. Finally, siponimod-fluocinolone acetonide cocrystals in PLGA microparticles offer a promising new approach to treating AMD and DR. By providing sustained drug release and targeting both angiogenesis and inflammation; this formulation has the potential to reduce the frequency of intravitreal injections, improve patient adherence, and offer a viable alternative to single treatments.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.