Pharmacy - Masters by Research Theses
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Item Engineering therapeutic messenger RNA for triple-negative breast cancer(University College Cork, 2024) Leahy, Mark; Krajewska, Malgorzata; Kowalski, Piotr; O'Connor, RosemaryBackground: Targeted therapies are revolutionising the landscape of cancer treatment, providing better therapeutic responses and minimising the adverse effects of conventional treatments such as radiation and chemotherapy. Human epidermal growth factor receptor 2 (HER2) is overexpressed in multiple cancer types and remains one of the therapeutic flagship targets in oncology. The HER2-targeting monoclonal antibody trastuzumab has dramatically improved the survival of patients with HER2-positive breast cancer. More recently developed antibody-drug conjugates, such as trastuzumab deruxtecan (T-DXd), have demonstrated unprecedented efficacy in HER2-overexpressing cancers. However, only a small subset of patients can benefit from HER2-targeted treatments (15-30% of breast cancers). Triple-negative breast cancer (TNBC) is characterised by the absence of HER2 and hormone receptors. TNBC is an aggressive subset of breast cancers for which few targeted therapies are available. It has recently been shown that artificial overexpression of HER2 could sensitise TNBC xenograft models to trastuzumab, however the clinical relevance of this strategy was limited by the use of viral vectors. Messenger RNA (mRNA) technology has been cemented as a safe and effective therapeutic modality, as evidenced by its success in SARS CoV-2 vaccination. Its potential to revolutionise cancer therapy is now becoming increasingly evident. We hypothesised that mRNA technology could be used to transiently overexpress HER2 in TNBCs to sensitise them to anti-HER2 therapeutics. Methods: Molecular cloning techniques were used to engineer a plasmid vector for in vitro transcription (IVT) of a truncated variant of HER2 (TrHER2), lacking the intracellular domain (ICD) to eliminate the potential for proliferative signalling. Modified nucleosides were incorporated into IVT reactions and mRNA was enzymatically capped and poly(A) tailed. TrHER2 and control (Luciferase and WtHER2) mRNA were delivered to TNBC cells using conventional transfection reagents. TrHER2 protein expression and HER2 signalling were measured by means of western immunoblot. TrHER2 localisation was assessed by immunofluorescent microscopy and flow cytometry. The impact of TrHER2 expression on migration and clonogenicity was measured by scratch and colony formation assays. Trypan blue and CellTiter-Fluor assays were used to assess the impact of TrHER2 expression on the efficacy of T-DXd and trastuzumab. Induction of apoptosis was determined by flow cytometry with Annexin V and propidium iodide stain. The effect of TrHER2 mRNA on trastuzumab function was determined by co-culture of TNBC cells with human immune cells. Results: TrHER2 plasmids were engineered and validated by sanger sequencing. IVT reactions with these plasmids produced pure, high yield TrHER2 mRNA. TrHER2 protein was shown to be approximately 100 kDa, lacking expression of the HER2 ICD. TrHER2 mRNA expression demonstrated cell surface localisation with a >110-fold increase in expression, exceeding that of WtHER2 mRNA (50-fold) at an equal dose. TrHER2 mRNA delivery did not active the HER2 signalling pathway, instead reducing cell migration and clonogenicity compared to WtHER2 mRNA. TrHER2 mRNA expression significantly increased the efficacy of T-DXd, causing an 80% reduction in viability (p<0.0001) by inducing DNA damage and apoptosis. TrHER2 mRNA also sensitised TNBC cells to antibody-dependent cell-based cytotoxicity by trastuzumab, causing an 37% reduction in viability compared to cells transfected with reporter mRNA (p<0.001). Significance: These findings demonstrate proof-of-concept for utilising mRNA-based overexpression as a strategy to enhance the sensitivity of TNBC cells to anti-HER2 therapies. Additionally, the results validate the safety profile of the engineered TrHER2 mRNA. The observed high efficacy suggests promising in vivo effectiveness. When paired with a suitable delivery system, this approach could have the potential for clinical translation.Item Development of an end-to-end monoclonal antibody production process(University College Cork, 2024) Pradeep, Rithwik; Allen, Evin; Tangney, MarkMonoclonal antibodies (mAbs) represent the dominant modality of biological therapeutics currently marketed. The manufacture of monoclonal antibodies consists of a series of processing steps including upstream cell culture, downstream purification and formulation and a final step of drug product manufacture. In this thesis, a small-scale end-to-end production process was developed for two monoclonal antibodies namely an anti-TNF Alpha mAb and cNISTmAb whose cell lines were sourced from commercial and public bodies respectively. A cell culture process for both cell lines was successfully developed including the creation of working cell banks (WCB). Titers were optimised, for both cell lines through a series of media, bioreactor and cell density modifications. In addition, the critical quality attributes of potency and purity were assessed via cell-based assays and SDS-PAGE respectively, that were designed and developed as part of this thesis. The potency of anti-TNF Alpha mAbs produced was assessed through two distinct in-vitro cell-based assays developed using HEK and L929 reporter cell lines and determined to be functional and more potent than a commercially available anti-TNF Alpha antibody The stability of cNISTmAb was also analysed across a range of temperature conditions from -20°C to 40°C after 50 days and also post-lyophilisation, with stability maintained at room temperature. Additional modifications of cNISTmAb were performed in an attempt to yield antibody fragments through pepsin digestion with partial success through pepsin digestion to yield F(ab’)2 fragments. This work demonstrates the successful production of two monoclonal antibodies and fragments but also sets the stage for further process improvement and enhanced characterisation to fully define and analyse an endto-end monoclonal antibody production systemItem Dissolvable microneedle manufacturing methods and the application of three-dimensional printing to pharmaceuticals(University College Cork, 2024) Wilkstein, Katerina; Moore, Anne; Vucen, Sonja; HEA AIVRTDissolvable microneedles are an intradermal drug delivery technology with exciting potential for widespread distribution of minimally invasive, self-administered medicines and vaccines. The most popular manufacturing method for medical dissolvable microneedles is micromoulding, a process which can have drawbacks of low material efficiency and high regulatory requirements. The UCC-patented “ImmuPatch” process is a micromoulding method which significantly improves on the material efficiency of traditional micromoulding. In this study, a specific method based upon the ImmuPatch process was developed for aseptic manual fabrication of two-layered dissolvable microneedles capable of incorporating at 2 μg protein dose. The developed method was not ideal, with high variability in the patches produced and significant challenges in verifying the protein dose. Fabrication of dissolvable microneedles is well positioned for the application of automation methods. One proposed option for the automation of dissolvable microneedle manufacture is additive manufacturing, which in addition to automation has the advantage of eliminating the regulatory challenges associated with micromoulding. A systematic literature review of methods applying additive manufacturing to the fabrication of solid dosage formats was conducted and identified processing parameters and format properties associated with different technologies. Additive manufacturing has been applied to micromould-free dissolvable microneedle fabrication in limited examples and should be explored further.Item Novel models for understanding traumatic stress(University College Cork, 2022-09-30) Lannon, Adam; Moloney, Rachel; Cryan, John; Brain and Behavior Research FoundationUndergoing 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.Item The evaluation of estradiol and indomethacin for incorporation into microneedle formulations(University College Cork, 2022) Lalchandani, Akash Parsram; Crean, Abina; Faisal, Waleed; Enterprise IrelandThe 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.