Pharmacy - Masters by Research Theses

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    Novel models for understanding traumatic stress
    (University College Cork, 2022-09-30) Lannon, Adam; Moloney, Rachel; Cryan, John; Brain and Behavior Research Foundation
    Undergoing trauma, be it physical, psychological, or observed, can induce pathological alterations leading to disorders such as Post-Traumatic Stress Disorder (PTSD). PTSD is highlighted by negative cognitive alterations, behavioural changes, and interruptions in arousal and sociability. PTSD is comorbid with disorders such as anxiety and depression, gastrointestinal disturbances, pain, and the gut microbiome is hypothesized to play a role in this trauma-related disorder. Direct experience of traumatic events is the most common method of generating traumatic-stress related pathologies, however indirect exposure through witnessing another endure a traumatic event can also lead to PTSD-like symptoms. This method of traumatic transference is called secondary traumatic stress (STS). While PTSD and STS are clinically relevant, and ever-growing in importance due to the recent COVID-19 pandemic, there is still a lot to be learned about their molecular underpinning, mechanisms, and biomarkers. In order to appropriately investigate these neurobiological features of traumatic stress, valid and effective animal models are absolutely essential. Utilizing the most appropriate animal models for the representation of neuropathologies is essential for extracting critical information in the process of developing novel therapeutic options. In chapter 2, we aim to develop the knowledge of secondary traumatic stress, we investigated whether a novel observational model, combining visceral pain, a common comorbidity of traumatic stress related disorders, and observed stress could result in a suitable phenotype. Utilizing colo-rectal distension (CRD) to induce visceral pain behaviours, we had rodents observe another rodent undergoing this procedure. These observer rodents then underwent the CRD themselves 24 hours later in order to assess whether they had visceral hypersensitivity. Indeed, it was seen that observer animals had hyperalgesia measured in visceral pain threshold and total behaviours, an impacted HPA axis, and altered neuronal activation in key brain regions. Our results suggest that this novel model was effective in producing secondary traumatic stress-like phenotypes, and would be well suited for further research into the social transference of pain and developing therapeutic options for traumatic-stress induced disorders and visceral pain comorbidity. In chapter 3, we look at Single prolonged stress (SPS), which is a well-validated and commonly used model however there are ethical concerns that limit its widespread use. The classical SPS model involves a 2-hour restraint, immediately followed by a 20-minute forced swim, a 15-minute rest and culminates with diethyl ether exposure until loss of consciousness. Recent focus on ethical standards and interests in refining animal models has led to concerns in the usage of diethyl ether, leading us to investigate whether the model would still be effective using isoflurane as a replacement for diethyl ether. Our findings suggest that this model is effective in recapitulating a key PTSD phenotype in the contextual fear conditioning paradigm. Impaired fear learning has been repeatedly found to be a key component of PTSD phenomenology, and our model induced significantly impaired fear learning in stress rats. Further to this, we found that SPS with isoflurane caused significant reduction in learned helplessness in rodents, paired with time specific changes in corticosterone concentration. Anxiety-like behaviours also appear to be implicated by this model, with Isoflurane exposure leading to reduced anxiety-like behaviour, suggesting its potential as an adequate PTSD model. The encouraging results from these two models of traumatic stress provide a significant interest in further studies using them. With the future intention of developing novel and effective therapeutics for undermedicated sufferers of these disorders, the hope is that these models can help provide valuable insights into the mechanisms of action behind the pathologies, illuminating potential therapeutic avenues.
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    The evaluation of estradiol and indomethacin for incorporation into microneedle formulations
    (University College Cork, 2022) Lalchandani, Akash Parsram; Crean, Abina; Faisal, Waleed; Enterprise Ireland
    The objective of this thesis is the technical evaluation of the suitability of drug substances, estradiol and indomethacin, for incorporation into a patented dissolvable microneedle (DMN) platform technology for intradermal drug delivery. Estradiol and indomethacin were chosen based on their physicochemical properties (melting point, glass transition temperature, amorphous solid state after melt-cooling) which were favourable for manufacturing microneedles using the melt DMN technology. Oral dosage forms of both drugs, and transdermal patches of estradiol are available in the market. Currently no indomethacin transdermal patch formulation is available. This project aims to assess the feasibility of developing dosage forms of both drugs for microneedle-assisted, intradermal delivery. The research presented, initially provides a background to the drivers and challenges related to the development of microneedle (MN) drug combination products for inclusion in the mainstream pharmaceutical and medical device market. The thesis then focuses on the experimental studies evaluating the feasibility of developing DMN from the drug substances of interest, estradiol and indomethacin. Melt-cooled samples of both drugs were also prepared to simulate the processing stress of the DMN manufacturing process. Melt-cooled samples were evaluated for solid-state form by differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD), and chemical composition by high performance liquid chromatography. Microneedle formulations of both drugs were produced using a previously developed manufacturing process and their mechanical strength and ability to penetrate pig skin analysed. A stability study was conducted to evaluate chemical and solid-state stability of the melt-cooled samples at 3 storage conditions: condition A (25°C/60% RH), condition B (40°C/ 75% RH), and condition C (2°C to 8°C in nitrogen filled vials) over a 3 month time period. The melt-cooled solid form of both drugs post melting was found to be chemically stable over the period of 3 months when stored at all 3 storage conditions studied. The amorphous melt-cooled samples of both drugs showed greatest stability at 2°C to 8°C in nitrogen. However, DSC and PXRD results identified crystallisation of the melt-cooled estradiol and indomethacin samples to when stored at 25°C/60% RH and 40°C/ 75% RH over 3 months. This loss in amorphous form was paramount for indomethacin when stored at 40°C/ 75% RH. These results indicated the need for storage and packaging precautions for MN of both drugs to prevent solid-state alterations upon storage which could impact on MN mechanical strength and dissolution in the skin. The MNs formed from both drugs, using the melt dissolvable microneedle technology, were found to be physically strong and capable of penetrating ex-vivo pig ear skin. The research presented illustrated that estradiol and indomethacin DMN can be manufactured using a melt DMN technology, but packaging and storage precautions are required to stabilise the drug solid-state form.
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    The impact of variation in freezing and thawing process parameters on the critical quality attributes of a monoclonal antibody
    (University College Cork, 2019-09-23) Day, Neil; Crean, Abina; Vucen, Sonja
    Therapeutic proteins or biopharmaceuticals have been playing an ever-increasing role in the treatment of human diseases over last 40 years. One of the main challenges with manufacture of these proteins is the stabilization of both the finished product and its processing intermediates during storage. Freezing and frozen storage is widely applied to improve stability of the bulk drug substance. The process of freezing a protein results in stresses that can cause protein degradation and subsequent aggregation. The aim of this project was to evaluate the effect of parameters involved with freezing and thawing of a formulated monoclonal antibody solution in polycarbonate bottles and to assess the scalability of these experiments to the respective full-scale commercial process. Initial experiments were performed to characterise the formulation and develop analytical methods that can detect change in unfolding and aggregation of a fully human IgG1 monoclonal antibody (‘Protein Y’). A designed set of experiments were then executed to understand the effect of parameters involved in freezing and thawing steps of a formulated therapeutic protein solution on protein aggregation and perturbations in tertiary structure. Results showed that the processing parameters studied caused significant variation in freeze and thaw process times, with the factors causing slower rates of freeze and thaw also shown to cause changes in the tertiary structure of Protein Y. Despite the observed changes in tertiary structure, the effect on aggregation was less pronounced, with only a significant change noted for the polydispersity index (PdI), as measured by dynamic light scattering (DLS).