Pharmacy - Doctoral Theses
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Item A study on the impact of primary packaging interfaces on protein adsorption and stability(University College Cork, 2024) Downey, John D.; Ryan, Katie; Crean, Abina; Synthesis and Solid State Pharmaceutical Centre; Science Foundation Ireland; Engineering and Physical Sciences Research CouncilProtein-based therapies represent a transformative class of biotherapeutics, yet their stability remains a critical challenge. Therapeutic proteins are intrinsically unstable molecules owing to their structural complexity and amphiphilic properties and potential to adsorb to interfaces present during the product development lifecycle and shelf-life. Protein adsorption promotes unfolding and aggregation which can result in altered bioactivity of the therapeutic moiety or particle formation in solution. Consequently, there is an increased risk of deleterious effects such as lack of efficacy upon administration or an adverse immunogenic reaction. To overcome these challenges, rational formulation design and selection of compatible packaging materials can minimise protein adsorption. However, the excipients used in biological medicines have drawbacks such as poor chemical stability in aqueous solution and the potential to degrade packaging coatings. Moreover, owing to the stringent requirements for primary packaging materials, innovative packaging solutions have not received significant attention to date. The overall aim of this thesis is to explore the impact of packaging interfaces and formulation on the adsorption and stability of two model proteins, lysozyme and bovine serum albumin (BSA), and develop novel formulation and packaging mitigation strategies to minimise the adverse impacts arising from protein-packaging interactions. Chapter One introduces protein therapeutics, focusing on their higher order structure, the impact of various stresses on protein physicochemical stability and formulation requirements to maintain stability. It also considers primary packaging for liquid parenterals and the impact of the protein formulation on container/closure compatibility. Chapter Two reviews prior literature on protein adsorption-induced instability during fill pumping and storage. Topics include factors influencing protein adsorption behaviour, interfaces present during unit operations, pump and tubing types, primary packaging materials, excipient-packaging incompatibility, and formulation considerations. Chapters Three, Four and Five focus on investigating the gaps in the formulation and packaging approaches to reduce protein adsorption and instability identified in Chapter Two. Chapter Three principally explores the effect of buffer type and pH on lysozyme adsorption to borosilicate glass. Lysozyme adsorption to borosilicate was found to be mediated by electrostatic interactions owing to the ionisation of glass in aqueous solutions generating an anionic material interface. Buffer type did not have a significant impact on the quantity of adsorbed lysozyme, but it was found to influence the properties of the adsorbed protein layer. Chapter Four examines the effect of vial orientation (upright versus inverted storage) on BSA stability under different storage conditions over one week. Inverted storage in vials with polydimethylsiloxane-coated stoppers at 40°C under agitation resulted in greater protein unfolding and aggregation. Packaging and formulation mitigation strategies of altering the vial stopper to an ethylene tetrafluoroethylene coating and including leucine as an aggregation suppressor were shown to be unsuitable for maintaining BSA in its native conformation under these storage conditions. Chapter Five focuses on developing an innovative vial stopper coating to reduce protein instability where sustained contact with vial stoppers occurs. A copolymer, poly(MPC-co-BMA), comprised of 2-methacryloyloxyethyl phosphorylcholine and butyl methacrylate was used to deposit a thin film coating to bromobutyl rubber stoppers using an innovative aerosol-assisted cold plasma deposition technique. Plasma process parameters (flow rate, speed and voltage) were found to affect coating coverage as indicated by changes in stopper hydrophobicity. BSA compatibility with poly(MPC-co-BMA)-coated stoppers was found to be comparable to commercial polydimethylsiloxane and ethylene tetrafluoroethylene stopper coatings over a 3-month storage period. Chapter Six provides a general discussion of the research performed and presents the key findings, limitations, future work and conclusions of this thesis. In brief, this thesis investigates how packaging interfaces and formulations affect protein adsorption and stability, focusing on strategies to mitigate protein instability. This thesis found that for the lysozyme, adsorption can be controlled by the solution pH and that for BSA, interfacial stress plays a minor role while storage environments significantly impact stability. Additionally, aerosol-assisted, low-temperature plasma is a viable thin film coating deposition technique for pharmaceutical packaging but requires further research to determine the long-term stability of the coating in the presence of liquid protein formulations.Item Oral liquid antibiotic formulations with the focus on organoleptic properties that impact paediatric acceptability(University College Cork, 2025) Elgammal, Ayat; Crean, Abina; Bermingham, Margaret; Science Foundation Ireland; European Regional Development FundPalatability, ‘the overall appreciation of a drug in relation to its smell, taste, aftertaste, texture and appearance’, is a key element influencing paediatric medicine acceptability. Nonadherence to antibiotic treatment because of poor palatability can cause disease recurrence and may contribute to increasing rates of antimicrobial resistance. While the relationship between a medicine’s acceptability and taste has been widely explored, the relationship between a medicine’s odour and its acceptability has received less investigation. Initially, a questionnaire study was conducted to explore General Practitioners’ (GPs’) and pharmacists’ experiences of prescribing and dispensing oral liquid antibiotics for children respectively in Ireland. Study findings highlight that palatability issues caused GPs and pharmacists to switch oral liquid antibiotics to ensure treatment adherence despite their preference to follow the guidelines and select a first-choice antibiotic. Flucloxacillin was highlighted as the least palatable oral liquid antibiotic for children with a malodour. Subsequently, an interview study was conducted to obtain a deeper and richer understanding of the experience, attitudes, and challenges faced by GPs and pharmacists. Both professions reported that in some circumstances such as the type of infection, lack of alternatives, and allergies they had to select an unpalatable antibiotic despite knowing that it has poor palatability. Both professions reported that they had never started the palatability discussion with parents to avoid negotiation because of the lack of alternatives. The study findings highlighted the importance of proactive dialogue discussing antibiotic palatability with parents and carers during prescribing and prior to dispensing. A human sensory study was conducted to assess the odour acceptability of a range of oral flucloxacillin formulations by healthy adult volunteers. The odour of the commercial flucloxacillin solution and capsule formulations were rated acceptable. The commercial solution odour was described as fruity, and aromatic, while the commercial capsule was described as odourless. An unformulated flucloxacillin aqueous solution, and flucloxacillin formulated as minitablets, were deemed to have an unacceptable odour. Their odour was described as pungent, musty, plastic, and rotten. However, the odour of the flucloxacillin minitablet formulation, with silica incorporated as an odour suppressant, was described as odourless and deemed to be acceptable. Finally, Gas chromatography - mass spectrometry (GC-MS) was used to identify the Volatile Organic Compounds (VOCs) associated with flucloxacillin’s malodour. Two sampling techniques, Head Space (HS) and Solid Phase Microextraction (SPME), were investigated to detect VOCs in the headspace above flucloxacillin samples. The direct HS sampling detected two volatile compounds in flucloxacillin powder of which decane has a particular malodour that could contribute to the overall malodour of flucloxacillin. SPME analysis detected a greater number of VOCs of which octane, decane, undecane, toluene, and styrene are associated with a malodour that can contribute to the overall malodour of flucloxacillin. Interestingly, while the malodour of flucloxacillin was initially thought to be related to its sulfur content, dimethyl disulfide was the only volatile sulfur compound detected and only detected in the aqueous flucloxacillin solution headspace. Dimethyl disulfide can result from degradation of ethynthiol which is one of the flucloxacillin degradation-related VOC impurities. In conclusion, poor palatability caused GPs and pharmacists to switch oral liquid antibiotics to ensure treatment adherence. Flucloxacillin was identified as a poorly palatable antibiotic with a malodour. Human volunteers associated the odour of unformulated aqueous flucloxacillin solution and minitablets with the descriptors musty, pungent, plastic, and rotten and unacceptable. The odour of a commercial solution was described as acceptable, fruity and aromatic whereas, commercial capsules and minitablets with silica were deemed acceptable and odourless. GC-MS using SPME analysis was used to identify the chemical origin of the malodour described in the human sensory study. A number of volatile compounds which could contribute to the overall flucloxacillin malodour were detected. However, future work is needed to quantify the volatile sulfur compounds (VSCs) associated with flucloxacillin’s overall odour and inform an odour suppression formulation strategy based on component chemistry.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.