Pharmacology and Therapeutics - Doctoral Theses

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    Characterisation of the role of mitochondrial dysfunction and meta-inflammation as a shared pathogenic network in gestational diabetes mellitus
    (University College Cork, 2023-05-02) McElwain, Colm; McCarthy, Cathal; McCarthy, Fergus; Health Research Board
    Background Gestational diabetes mellitus (GDM) is one of the most prevalent obstetric complications, with a growing incidence resulting from upward trends in global obesity and metabolic syndrome. GDM pathology is considered a state of exaggerated insulin resistance in pregnancy, mediated by pancreatic beta cell insufficiency, adipose dysregulation and systemic meta-inflammation. Although the pathophysiology of GDM is complex, adipose tissue dysfunction is an established cause of metabolic dysfunction and systemic insulin resistance. Furthermore, the placental microenvironment is highly sensitive to GDM physiology and there is significant evidence that the hyperglycaemic state of GDM drives placental dysfunction, in part mediated by exaggerated oxidative stress and inflammation. This thesis aims to characterise the role of systemic and local tissue mitochondrial dysfunction and meta-inflammation in promoting GDM pathology. Methods In this study, the inflammatory and metabolic profiles of omental visceral adipose tissue (VAT), placental tissue and fasting maternal blood were extensively studied to identifiy pathways which may drive insulin resistance in GDM and contribute to both acute and chronic adverse maternal and fetal outcomes. Samples were collected at term pregnancy from nulliparous women with normal glucose tolerant pregnancies and women diagnosed with GDM. Various immune-endocrine mediators were investigated in VAT, placental tissue and the maternal circulation including markers of mitochondrial dysfunction, monocyte/macrophage population frequency, hormones, inflammatory cytokines and insulin signalling transduction. Results Firstly, distinct phenotypes of GDM were confirmed, relating to both insulin-deficient and insulin-resistant pathologies. Insulin-deficient GDM participants had lower body mass indexes (BMIs) and were significantly hypoinsulinaemic compared to insulin-resistant GDM participants, who had elevated BMIs with substantial VAT dysfunction, including adipocyte hypoplasia and dysregulated insulin signalling capacity. The placental microenvironment was also negatively affected in GDM, with a potent pro-inflammatory secretome including increased release of IL-6, TNF-α and IL-18. This inflammatory phenotype was mirrored in the maternal circulation, confirming that systemic low-grade inflammation is present in term GDM physiology and is, in part, facilitated by placental signalling. Mitochondrial dysfunction was also confirmed in GDM participants, characterised by exaggerated mitochondrial superoxide production in placental macrophages and elevated circulating levels of cell-free mitochondrial DNA. Ex vivo mitochondrial antioxidant therapy attenuated excessive placental inflammation in GDM, proposing a promising therapeutic avenue for alleviating systemic inflammation in GDM patients. Conclusion In this thesis, we have provided substantial evidence of metabolic dysfunction and inflammation in the maternal circulation, omental VAT and placental tissue of women with GDM. This includes evidence of defects in adipocyte expansion and impaired insulin signal transduction, in addition to placental and systemic mitochondrial dysfunction and inflammation, which may ultimately orchestrate GDM pathophysiology. These findings identify novel pathological pathways in GDM, which may be further delineated to establish their potential as therapeutic targets to alleviate adverse maternal outcomes.
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    Preclinical characterisation of fingolimod as a potential therapeutic agent for stroke
    (University College Cork, 2022-12) Diaz Diaz, Andrea C.; Waeber, Christian; Moore, Anne; Health Research Board
    Stroke is the leading cause for death and disability worldwide, and the search for novel drug treatments has been affected by repeated clinical trial failures. One novel drug that has garnered promising results in preclinical and clinical stroke studies is fingolimod, an FDA approved drug for the treatment of multiple sclerosis. Even though there are several studies supporting the effectiveness of fingolimod for the treatment of stroke, the recommended characterisation based on the Stroke Treatment Academic Industry Roundtable (STAIR) guidelines is incomplete. Furthermore, the quality of the preclinical studies supporting fingolimod has been poor, thus rigorous studies are required to validate the effectiveness of fingolimod prior to evaluation in clinical trials. This thesis aimed to inform whether fingolimod is effective for the treatment of stroke in intracerebral haemorrhage and ischaemic stroke, and to inform whether fingolimod is a good candidate for evaluation in large randomised clinical trials. This goal was achieved by first using a model of intracerebral haemorrhage to evaluate the effect of administering fingolimod at 30 min, 24 and 48 h after stroke on lesion size and behaviour in a 14-day study on male and female mice. This was followed up by a series of studies using middle cerebral artery occlusion to cause a focal ischaemia. First an optimal dose of fingolimod was determined in a dose response study; then we evaluated the optimal drug dose in two animal model of common comorbidities associated with stroke, age and hyperlipidaemia; and the last study evaluated the effect of an extended treatment duration on stroke. For the ischaemic stroke studies we focused on lesion and behavioural measurements as primary outcomes, and secondary data was collected from daily scores, plus additional measures where necessary. The intracerebral haemorrhage study fingolimod treatment had no measurable effect on either lesion size or behavioural outcomes irrespective of sex, the only finding was that fingolimod treatment reduced mortality in female mice. The dose response study showed no difference in lesion size or behavioural outcomes between the two fingolimod doses (0.5 and 1.0 mg/kg) and control mice, the study did show that saline treated mice had a significantly larger atrophy compared to the lower dose of fingolimod. The lower dose was selected as optimal for further studies. The study evaluating the effect of 0.5 mg/kg fingolimod on stroke in aged mice showed that fingolimod-treated mice had a significantly larger atrophy and a significant improvement in the grid score 7 d after stroke compared to saline-treated mice. The study evaluating the effect of fingolimod on stroke in hyperlipidaemic mice showed that fingolimod-treated mice had a significantly reduced lesion size, without an effect on any other outcome measures. The final study evaluated two fingolimod treatment durations compared to saline controls, the study showed no difference between any of the outcome measures, with a trend towards improved behaviour outcomes in mice receiving 10 d of fingolimod treatment. Lastly, considering the fact that the results of these studies were inconclusive, we decided to pool the data of the ischaemic studies and evaluate whether fingolimod had an effect on the primary outcome measures in a heterogenous animal population. The pooled data showed that fingolimod treatment improved behaviour 7 d after stroke without an effect on lesion size or atrophy. The results of this thesis cast a doubt on the effectiveness of fingolimod and its suitability for translation into larger clinical trials. Furthermore, they highlight the need for thorough preclinical studies for promising drugs as well as the need for studies to meet the proposed STAIR guidelines prior to translation. Confirmatory studies, like those presented here, performed with measures intended to control for internal and external biases are all good measures to be implemented for future studies of novel and highly promising drugs for stroke treatment.
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    The role of regulatory T cells in stroke recovery
    (University College Cork, 2022-06-16) Malone, Kyle; Waeber, Christian; Moore, Anne; Irish Research Council
    Background: Acute ischaemic stroke is a major cause of mortality worldwide. Despite the search for new therapies, tissue plasminogen activator remains the only FDA-approved medication. The immune system is involved in all stages of stroke, from risk factors to pathogenesis and tissue repair. The presence of a lymphocyte subpopulation termed regulatory T cells (Tregs) appears to correlate with improved disease outcome. However, the impact of stroke on Tregs, and the contribution of these cells to stroke recovery remains under debate. Likewise, attempts at developing Treg-targeted immunotherapies have been hampered by high cost, toxicity, and the stability of expanded Tregs. The aim of this thesis was to explore the role Tregs play in ischaemic stroke recovery and evaluate the neuroprotective and immunomodulatory effects of two potential Treg-targeted stroke immunotherapies, namely fingolimod and recombinant pregnancy-specific glycoprotein-1 (rPSG1-Fc). Methods: The effect of fingolimod on Tregs in a mouse model of permanent brain ischaemia was first investigated using a combination of flow cytometry and immunohistochemistry. Next, the impact of fingolimod on Treg suppressive function was characterised via Treg suppression assay. Following this, the immunomodulatory and neuroprotective properties of rPSG1-Fc in permanent brain ischaemia were determined. Finally, the changes in Treg frequency and function in the peripheral blood of mild stroke patients were quantified. Results: Fingolimod increased peripheral Treg frequency in the post-ischaemic mouse. Fingolimod augmented brain infiltration of FoxP3+ T cells, possibly via CCR8 signalling. Fingolimod also enhanced the secretion of IFN-γ, IL-17, and IL-10 from CD4+ T cells. Likewise, fingolimod promoted both suppressive and effector T cell function. rPSG1-Fc improved neurobehavioural recovery in mice post-brain ischaemia, possibly via increased CD4+IL-10+ T cells. Finally, an increased Treg frequency and an increased expression of functional markers of suppression (CTLA-4, PD-1) was observed in stroke patients at 24 hours post-ischaemia, and specifically among proliferating Tregs. However, by 7 days, the expression of PD-1/CTLA-4 among proliferating Treg frequency had returned to baseline. Conclusions: This thesis has made a number of novel insights. A positive impact of fingolimod on both Treg frequency and function post-ischaemia was revealed. The observed dual effect of fingolimod on regulatory and pro-inflammatory T cell function may explain why the drug fails to consistently improve experimental stroke outcome. The immunomodulatory and neuroprotective effects of rPSG1-Fc post-stroke were also characterised for the first time. Finally, the impact of clinical stroke on Treg frequency and phenotype was comprehensively quantified. Overall, this thesis shows Tregs may not play a major role in the early stages of recovery of mild stroke, but therapies manipulating them can still promote functional recovery in a mouse model. It provides a basis for further study on Tregs in ischaemic stroke. It also illustrates rigorous methods by which researchers should test future Treg-targeted stroke immunotherapies.
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    A study of magnesium stearate behaviour in pharmaceutical blends and tablets employing Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS)
    (University College Cork, 2019) Peddapatla, Raghu V. G.; Crean, Abina; Sousa Gallagher, Maria J; Enterprise Ireland; Science Foundation Ireland
    Magnesium Stearate (MgSt) is the most commonly used lubricant in pharmaceutical industries. Effects of MgSt on the final product have been extensively studied in batch processing. In recent times pharmaceutical companies have been increasingly interested in continuous processing, where the relative effects of material properties and process parameters on blend behaviour during continuous processing has gained significant attention. It is important to assess the behaviour of materials and it is a challenging feat to monitor behaviour of very cohesive materials like MgSt in continuous processing. The main aims of this thesis were, to investigate the role of MgSt supplier variability during continuous feeding through a loss in weight feeder (LIW) and to investigate the capability of Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS) to discriminate between blends and tablets with variable MgSt distribution. Initially, the variability among four different grades of MgSt samples from two different suppliers was studied. The variability among the samples was evident (chapter-3) and the effect of this variability on continuous feeding performance of MgSt samples was studied (Chapter-4). Bulk density of the samples dictated the feed factor achieved for the MgSt samples, when fed through K-Tron MT12 feeder (Chapter-4). Higher variability in the feed rate RSD was noticed for the MgSt samples, when fed at lower feed rate of 0.15 kg/hr and for samples (Ligamed MF-2- V and Ligamed MF-2-V-BI) with similar properties. Post feeding characterisation of MgSt samples was performed to identify any effect of feeding on particulate properties. A reduction in particle size due to feeding of the samples was noted and these samples when included in tablet blends, showed a delayed drug release, which was more prominent in tablets with fed MgSt of Alfa Aesar and Ligamed MF2-V samples. Ligamed MF-3-V was least effected by feeding and when fed samples were included in formulations a very slight delay in drug release was noted compared to other tablets with other MgSt samples (Chapter-4). A novel technology, BARDS was employed for the first time to analyse the excipients, tablet blends and tablets (Chapter-5 and Chapter-6). Analysis of unlubricated and lubricated blends using BARDS, clearly discriminated between the blends, resulting inan extended acoustic response for lubricated blends. K-Tron MT12 feeder, was used to feed the unlubricated and lubricated blends at three different feed rates (0.2238 kg/hr, 0.5594 kg/hr and 1.006 kg/hr), anticipating lubricated blend with varied degrees of blend lubrication. When analysed using BARDS, unlubricated blend fed at increasing feed rates showed similar acoustic response, whereas lubricated blend showed extended acoustic response, which was dependent on the feed rate (Chapter-5). Gas elimination rate constant was used to determine the degree of lubrication within blends. The degree of overlubrication was further confirmed by the standard wetting techniques and tabletability of the blends (Chapter-5). Tablets were produced from unlubricated and lubricated blends at a compression pressure range of 30 MPa to 234 MPa. The influence of compression pressure and tablet properties, on the BARDS acoustic response was investigated in Chapter-6. The yield pressures calculated from the Heckel analysis was 98 MPa and 102 MPa for unlubricated and lubricated tablets. A significant change in the BARDS acoustic response was noticed for tablets produced above and below yield pressure for both blends. Lubricated tablets produced at higher compression pressures, showed delamination, which was identified by BARDS. Slow gas elimination rate constant was observed for lubricated tablets compared to unlubricated tablets.
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    Examining potential causes of neuronal dysfunction in Alzheimer’s disease
    (University College Cork, 2019) Jaisimha, Anirudh Vinay; Boland, Barry
    Alzheimer’s disease (AD) is a debilitating neurodegenerative disease that is characterised by a number of intraneuronal hallmarks, which include the accumulation of autophagic vacuoles (AVs) within dystrophic neurites, and neurofibrillary tangles (NFTs) composed of both truncated and full-length forms of tau protein. Work presented in this thesis, outlines findings from three lines of investigation that set out to determine potential causative factors that contribute to AD-related neurodegeneration. For Aim 1, I investigated the role of impaired lysosomal digestion as a cause of AV accumulation in AD. Having developed a novel assay that utilised the detection of specific truncated forms of amyloid precursor protein C-terminal fragments (APP-CTFs), which preferentially accumulate when lysosomal digestion is impaired, findings from post-mortem human brain tissue at different Braak stages of AD (0 – VI), indicate that the accumulation of AVs in the AD brain is not caused by an impairment in lysosomal digestion. For Aim 2, I investigated the role of altered glucose availability as a cause of tau hyperphosphorylation in AD. To determine if excessive or insufficient amounts of glucose availability to neurons is a direct cause of tau hyperphosphorylation in the AD brain, I utilised a primary rat neuron culture system, to determine if hyperglycaemic or hypoglycaemic stress could lead to tau hyperphosphorylation. Despite finding high basal amounts of the AD-related tau phospho-epitope (PHF1), in both primary neurons and mouse brain, I did not report any change in levels of phospho-tau under glucose altering conditions, suggesting these changes are not directly responsible for inducing tau hyperphosphorylation in AD. For Aim 3, I investigated the role of dysfunctional neuron-glial interactions as a cause of truncation tau in AD. Having identified truncated forms of tau as early as Braak stage II in post-mortem human brain tissue, I subsequently found that neurons grown in co-cultures with glial cells, develop truncated forms of tau after two weeks in culture, which correlated with the progressive proliferation of astrocytes and microglia. I also found that certain excitatory stimuli, in particular glutamate and zinc, produced a rapid but transient increase in truncated tau, which was prevented by kynurenic acid (KynA). Concluding thoughts from all three investigations suggest that dysfunctional neuron-glial interactions are likely to occur early in AD pathogenesis and the therapeutic targeting of autonomous (neuronal) or non-autonomous (glial-mediated) factors that contribute to dysregulated neuronal excitation may prove to be beneficial in treating AD.