Pharmacology and Therapeutics - Theses
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ItemThe impact of fetal and neonatal nutrition on adult obesity and cardiovascular regulation in animal models(University of Melbourne, 2009)
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ItemRoles and regulation of glutamate transporters in neurodegenerative diseases(University of Melbourne, 2008)
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ItemCharacterisation of a novel knockin mouse model of childhood absence epilepsy(University of Melbourne, 2007)
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ItemNeuropeptide Y and feeding regulation in mice(University of Melbourne, 2006)
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ItemThe comparative cardiovascular and non-cardiovascular actions of cannabidiol: an important Cannabis therapeutic agent( 2021)Cannabidiol is a major non-intoxicating Cannabis compound that is prescribed for the treatment of seizures in children with rare treatment-resistant epilepsy, and has been suggested to have extensive therapeutic potential including in the cardiovascular system. However, the mechanisms underlying the cardiovascular effects of cannabidiol remain elusive. Therefore the goal of this project was to further the understanding of the cardiovascular effects of cannabidiol and the interaction of cannabidiol with the cannabinoid receptors. More specifically, the project aimed to examine the local vascular and haemodynamic effects of cannabidiol, and the potential mechanisms involved in cannabidiol-specific cardiovascular effects. This study also aimed to pharmacologically characterise the cannabinoid receptor type 1 (CB1) activity of cannabidiol using the rat vas deferens bioassay. As previous studies using the isolated vas deferens bioassay have been performed in modified physiological salt solution (PSS) without Mg2+, this study sought to standardise the assay by examining the effect of varying Mg2+ concentrations on neurotransmitter-induced contractions in the vas deferens. To examine the vascular effects of cannabidiol, wire myography was used to quantify clinically used concentrations of cannabidiol (0.3-3 uM) on the effects of various contractile agents in rat isolated small resistance arteries. The effects of cannabidiol in small resistance arteries were compared with larger conduit arteries, given the morphological and functional differences between different artery types. The potential vascular mechanisms of action of cannabidiol in small resistance arteries were further investigated by pretreating arteries with various inhibitors prior to the treatment with cannabidiol and subsequent contractions. The vascular actions of cannabidiol were further compared with its actions on the contraction of various isolated non-vascular tissues – bronchial, urogenital, cardiac and skeletal muscles, using organ baths. To study the haemodynamic actions of cannabidiol, rats were anaesthetised and intravenously administered with cannabidiol. Evaluation of the acute cardiovascular actions of cannabidiol included measurements of heart rate and mean arterial pressure. Carotid and mesenteric vascular conductance and hindpaw cutaneous blood flow were measured to assess the effects of cannabidiol on haemodynamics. The potential haemodynamic mechanisms of action of cannabidiol were investigated by intravenously pretreating rats with various inhibitors prior to cannabidiol administration. Experiments on the vas deferens bioassay were performed in organ baths. The vas deferens bioassay was standardised by evaluating the influence of varying Mg2+ concentrations (0-3 mM) in the PSS on neurotransmitter-induced contractions. The CB1 receptor activity of cannabidiol was pharmacologically characterised by evaluating the ability of cannabidiol to alter CB1 receptor agonist-induced modulation of electrically evoked contractions in the standardised vas deferens bioassay. Cannabidiol, at clinically relevant concentrations, potently inhibited the contractions of small resistance arteries to multiple agents while being devoid of activity in large conduit arteries. The sensitivity of the small resistance arteries to cannabidiol involves CGRP and voltage-operated calcium channels. Non-vascular smooth muscles were generally unaffected by cannabidiol even at 10-100 times the therapeutic plasma level. Intravenously administered cannabidiol 10-30 mg/kg caused acute bradycardia and hypotension. Furthermore, cannabidiol modulated vascular tone by increasing vascular conductance and cutaneous blood flow. Electrically-induced contractions of vas deferens tissues were altered under non-physiological conditions (Mg2+ 0 mM in PSS). Cannabidiol antagonised CB1 receptor-mediated inhibition of contraction of the rat vas deferens in a manner consistent with simple competitive antagonism. The resulting potency of cannabidiol as a competitive antagonist of CB1 agonists was consistent with its binding affinity at the CB1 receptor. In conclusion, this project has broadened the understanding of the cardiovascular effects of cannabidiol by demonstrating that cannabidiol has potent vascular effects that are selective for small resistance arteries, which are the main contributors to peripheral vascular resistance and subsequent blood pressure regulation. The sensitivity of the small resistance arteries to cannabidiol involves CGRP and voltage-operated calcium channels. The vascular effects were confirmed in vivo, as cannabidiol transiently increased carotid vascular conductance and hindpaw cutaneous blood flow. This project has also contributed to the understanding of the pharmacological interactions of cannabidiol and CB1 receptors. Given that altering Mg2+ concentration in the PSS altered vas deferens responses, the vas deferens should be studied under physiological conditions (Mg2+ 1.2 mM). The CB1 antagonist activity of cannabidiol may have implications for neurotransmission modulation when plasma concentrations are raised above 1 uM.
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ItemMRGPRX2 and its role in drug-induced anaphylaxis( 2021)Drug-induced anaphylaxis is a growing concern with both the better characterised IgE-dependent mechanism and less well-established IgE-independent mechanisms being implicated. IgE-independent anaphylaxis has recently been suggested to be associated with direct mast cell activation via the newly identified G protein-coupled receptor, MRGPRX2. While the clinical relevance of the receptor is not yet well understood, a range of polybasic agonists, including therapeutic drugs like neuromuscular blocking agents (NMBAs), have been found to be agonists at the receptor. Presumably, mast cells in all humans express MRGPRX2, therefore the reason why only certain individuals are susceptible to sometimes extreme adverse reactions induced by these drugs remains unclear. In this project, we aimed to better characterise the role and activation of MRGPRX2 by: 1) Investigating if MRGPRX2-mediated mast cell activation can be halted with chemical antagonists; 2) Examining if receptor activation is subject to biased agonism so that some compounds more effectively trigger mast cell activation; 3) Determining if MRGPRX2-mediated anaphylaxis can be predicted through the identification of insightful biomarkers. To achieve this, we quantified mast cell activation through calcium mobilization, degranulation, and cytokine CCL2 release, using an in vitro human mast cell model (LAD2). We also examined clinical samples from patients who experienced an anaphylactic reaction during surgery to identify possible predictive biomarkers, whether genomic or serum components. Firstly, we generated a novel CRISPR-Cas9 MRGPRX2 knock down LAD2 model to verify the activity of known and proposed MRGPRX2 agonists at the receptor. Following this, we observed that chemical antagonism of some MRGPRX2 agonists using sugammadex (counter ligand of rocuronium) and heparin (counter ligand of protamine), when administered after stimulation, does not affect the release of mediators contributing to the immediate symptoms of anaphylaxis. However, these agents were shown to inhibit release of the late phase mediator, cytokine CCL2, which may perhaps be associated with the more delayed effects of anaphylaxis. Secondly, we demonstrated evidence in LAD2 cells that some NMBAs known to cause perioperative anaphylaxis (rocuronium and atracurium) are paradoxically biased against degranulation and cytokine release compared to calcium mobilization. Another agonist, substance P, was shown to be a full unbiased agonist, while protamine was identified as a partial yet unbiased agonist. Lastly, we have identified a novel double polymorphic form of MRGPRX2 in a patient who experienced anaphylaxis. We have also shown that sera from two anaphylactic patients had the ability to activate resting LAD2 mast cells. Whilst these findings were not broadly predictive of anaphylaxis across the cohort they may contribute in part to susceptibility to anaphylaxis in those individual patients. The lack of efficacy of post hoc chemical antagonism of MRGPRX2 stimuli on mast cell degranulation is not unexpected given the rapidity of the degranulation process. However, the inhibition of the more slowly released cytokine mediators may have at least some theoretical benefit in patient recovery from anaphylaxis, although further work is needed to establish the role of cytokines in anaphylaxis. In addition, that certain NMBAs were biased against MRGPRX2-induced degranulation suggests against this mechanism underpinning NMBA-induced anaphylaxis. This, however, assumes that the LAD2 mast cell model is broadly representative of human mast cells, but it is possible that patients susceptible to MRGPRX2-dependent anaphylaxis may have ‘overreactive’ mast cells where NMBAs trigger a full degranulatory response. Our ambitious attempts to identify a genetic or serum biomarker that defined patients who experienced anaphylaxis were not however fruitful. Broader genomic, transcriptomic, and proteomic studies on a larger, more clearly characterised patient cohort are necessary to identify useful biomarkers that help firstly establish and perhaps then predict drug-induced, MRGPRX2-dependent anaphylaxis. In summary, the findings presented in this thesis lay important groundwork for the better understanding of MRGPRX2, and can be effectively utilised in future research to define its role in drug-induced anaphylaxis.
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ItemVirus-induced Glucocorticoid Insensitivity in the Airways( 2020)Chronic respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD) pose a major public health burden. Hospitalization, mortality and increased health care costs owing to these diseases are amongst the consequences of exacerbations. Exacerbations are usually elicited by respiratory infections, allergens, environmental pollutants or occupational sensitizers. These factors may work in either an additive or synergistic manner. Regular management using inhaled glucocorticoids (GCs) has an unquestioned benefit in alleviating exacerbations unless the respiratory viruses were the trigger. Due to the few alternative anti-inflammatory therapies available, restoring glucocorticoid (GC) efficacy is indispensable. Among the respiratory viruses implicated in asthma/COPD exacerbations is respiratory syncytial virus (RSV); the prime respiratory virus associated with bronchiolitis and pneumonia in infants, young children, immunocompromised adults and the elderly worldwide. Despite the fact that RSV-induced pathogenesis is in part inflammatory, the beneficial effect of GCs in alleviating the symptoms or altering the course of illness is still debatable. Several reports suggested that RSV infection has a detrimental effect on GC signalling through mechanisms that are not yet defined. Being the prime locus for viral infection and replication and the crucial target for glucocorticoid therapy as well, airway epithelium has been suggested as a key cell type in which virus-induced glucocorticoid resistance may occur. The airway epithelium releases a surplus of cytokines and chemokines leading to airway inflammation and airway structural changes seen in asthma. Among these inflammatory mediators is the transforming growth factor beta1 (TGF-b1). Our laboratory has identified TGF-b1 as the key mediator of glucocorticoid insensitivity in bronchial epithelial cells where it ablates glucocorticoid activity at pathophysiologically relevant picomolar concentrations. We, therefore, investigated the possible contribution of this key cytokine to RSV-induced GC resistance. Data presented within this thesis has provided the first evidence that TGF-b1 drives RSV-induced GC-insensitivity in bronchial epithelium transformed with Ad12-SV40 2B (BEAS-2B) cell line. However, blanket inhibition of TGF-b1 using TGF-b receptor (activin-like kinase 5 (ALK5/ TGF-bR1 kinase) selective inhibitor, SB431542 compromises its beneficial pleiotropic effects leading to serious adverse effects. Our group has recently identified casein kinase 1delta /epsilon (CK1d/e) as a downstream effector of TGFb-induced remodelling and steroid resistance. Our data also implicated CK1d/e in RSV triggered impairment of GC responsiveness where pharmacological inhibition of CK1d/e by PF670462 has inhibited the expression and activity of TGF-b1 and subsequently prevented the GC impairment provoked by RSV infection in our in vitro model. Moreover, PF670462 has significantly subdued RSV-induced expression of pro-inflammatory gene suggesting the potential for additional beneficial anti-inflammatory activities mediated by PF670462. This potential was further confirmed in vivo, where we have provided evidence of an interaction between PF670462 and dexamethasone to achieve greater control of the pulmonary inflammation following RSV infection. Moreover, we evaluated the contribution of different epithelial pattern recognition receptors (PRRs) involved in RSV sensing in the apparent resistance using a wide array of PRRs ligands. Among the ligands tested was polyinosinic:polycytidylic acid (poly I:C); a synthetic analogue of double-stranded RNA (dsRNA) that is frequently used as a viral mimic. Our results showed that that viral pathogen-associated molecular patterns (PAMPs), most importantly double-stranded RNA (dsRNA), activates toll-like receptor 3 (TLR3), activates toll-like receptor 3 (TLR3) that in turn signals through the extracellular signal-regulated kinase 1/2 (ERK1/2)-dependent pathway to promote TGF-b activity hence mediating GC resistance in airway epithelial cells. Genetic approaches established that targeting TLR3 may restore the responsiveness to glucocorticoids. In conclusion, we demonstrated that RSV impairs glucocorticoid sensitivity in airways epithelium partly through TLR3 activation with subsequent release of TGF-b1, a potent mediator of glucocorticoid resistance. We also implicated CK1d/e in the apparent resistance. This thesis provided novel insights into mechanisms of glucocorticoid insensitivity in the airway epithelium. These findings offered two novel anchor points; CK1d/e and TLR3, for a potential treatment for the prevention/treatment of GC insensitivity occurring during RSV infection-induced bronchiolitis or asthma/ COPD exacerbations.
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ItemInvestigating olfactory deficits as a preclinical marker of Parkinson's disease( 2020)Parkinson’s disease is the fastest growing neurological condition in the world, expected to affect more than 12 million people globally by 2040. Despite centuries of ground-breaking research, the diagnosis of Parkinson’s disease is unreliable and there are no disease-modifying therapeutics. This may be attributable to a focus on the movement disorders associated with disease, which present only after substantial neurodegeneration has occurred. This results in a late, inaccurate diagnosis which is problematic for the development and utilisation of neuroprotective therapies which need to be administered early. Although there has been recognition of non-motor aspects of Parkinson’s disease as early as 1817 by James Parkinson himself, there is no standardised utility of non-motor symptoms in the identification and monitoring of disease. The concurrence of non-motor symptoms in early Parkinson’s disease has great promise in identifying people at risk of developing the clinical disease, especially in those with REM sleep behaviour disorder, however these assays still require further development. The most prevalent non-motor symptom is a loss in the sense of smell (hyposmia) and understanding the pathobiology underlying this phenomenon may help to inform future diagnostic approaches through the development of novel biomarkers of disease. As such, the aim of this thesis was to test the utility of concurrent non-motor symptoms in a clinical setting, as well as investigate the underlying biology of the olfactory system in Parkinson’s disease using post-mortem human olfactory bulbs and tau knockout mice. The concurrence of non-motor symptoms was investigated in a cohort of participants presenting with multiple non-motor symptoms, including REM sleep behaviour disorder, hyposmia, and anxiety. Upon positron emission tomography scanning, it was demonstrated that the participants with concurrent non-motor symptoms had reduced vesicular monoamine transporter 2 binding in the caudate nucleus and the putamen in a degenerative pattern like that of Parkinson’s disease. Although these findings demonstrate the utility of non-motor symptoms to identify people with early nigrostriatal degeneration, understanding the underlying pathological changes will allow the development of more sensitive diagnostic tools. As such, human post-mortem olfactory bulbs from subjects with pathologically confirmed Parkinson’s disease and neurological controls were examined for changes in systems associated with Parkinson's disease, including protein clearance, the dopaminergic system, and metal homeostasis. The accumulation of monomeric alpha-synuclein was observed within the bulbs as determined by immunoblot and there were protein changes indicative of an environment under oxidative stress. Characterisation of the dopaminergic system demonstrated perturbations in catechol-O-methyltransferase-mediated dopamine breakdown. Finally, there was a loss of metal homeostasis as demonstrated by an accumulation of key metals, including iron, sodium, zinc, and lead. These findings warranted further investigation in an in vivo model. Tau knockout mice have been reported as an age-dependent model of Parkinson’s disease, and the next study of this thesis demonstrated that these mice develop an olfactory impairment at seven months of age, approximately five months before they develop a motor impairment, aligned with the presentation of symptoms in Parkinson’s disease. The mechanism(s) underlying the olfactory deficits of these animals was characterised to determine the congruency between the mice and the post-mortem tissue. Firstly, it was found that this olfactory deficit correlates with an accumulation of alpha-synuclein and autophagic impairment, in the olfactory bulb. Similar to the human bulbs, young tau knockout mice appear to have a deficit in catechol-O-methyltransferase dopamine breakdown as well as an accumulation of bulbar metals. These findings were further implicated in the functional olfactory deficit as treatment with the dopamine 2 receptor antagonist haloperidol, and the iron/copper chelator PBT434, resulted in a rescue of the hyposmic phenotype in young tau knockout mice. These findings implicate a loss of tau function as a contributor to disease pathways. This was further investigated using the mutant tau overexpressing mice, the rTg4510s, and like the tau knockout mice these animals demonstrate an age-dependent hyposmia. Together these studies have enabled the identification of potential mechanisms of olfactory impairment in Parkinson’s disease that warrant further scrutiny, including dopamine mis-metabolism and metal accumulation. Findings in the tau knockout mice add face validity to the model and support the utility of this animal in non-motor studies. These findings may help aid in the development of novel diagnostic approaches, as well as patient stratification strategies to overcome the current hurdles in clinical trials of neuroprotective therapies in Parkinson’s disease.
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ItemSleep/wake disruptions in a neurodegenerative tauopathy mouse model: Role of tau and therapeutic potential of orexin receptor antagonists( 2020)Sleep disturbances and neurodegenerative diseases are prevalent in an aging population and represent a major health concern. Mounting evidence highlights a bidirectional relationship between neurodegenerative disease and sleep disturbances, whereby initial pathology disrupts sleep/wake regulation which subsequently exacerbates pathology, leading to a detrimental cycle that accelerates disease progression and cognitive decline. Hence, targeting sleep is a promising therapeutic option to slow disease progression and functional decline. The exact mechanisms underlying this bidirectional relationship, however, are incompletely understood, especially with respect to the processes that initiate sleep/wake disturbances. Initial research focused on sleep and amyloid beta in animal models of Alzheimer’s disease (AD). More recently, attention has turned to pathological tau variants, yet there are few studies investigating the impact of sleep in models of tauopathies, or vice versa. The aim of this thesis was to investigate the role that mutant tau expression plays in mediating sleep/wake disruptions and cognitive deficits, primarily using the inducible/conditional rTg4510 tau transgenic mouse model, which overexpresses mutant human tau, containing the P301L MAPT mutation, in the forebrain. Given the key role of the orexin neuropeptide system in sleep/wakefulness, initial experiments characterised orexin receptor mRNA expression in rTg4510 and tau knockout (KO) mouse brains using in situ hybridisation. Both strains exhibited a specific and comparable decrease in orexin receptor 1 (OX1R) mRNA expression in the sleep/wake regulating locus coeruleus (LC). These data indicate that orexinergic input to the LC is modulated under conditions of altered/lost tau function, linking tau to molecular changes in the ascending arousal system. Functional and behavioural studies were then carried out to assess the cognitive, neurodegenerative and sleep/wake phenotype of rTg4510 mice. Tau transgene expression was suppressed in rTg4510 mice by administering doxycycline (Dox) in the diet from 4.5-6.5 months of age. Control rTg4510 mice displayed a markedly disrupted sleep/wake phenotype manifesting primarily as a hyperarousal phenotype during the active phase, and was exacerbated in female rTg4510 mice. rTg4510 mice exhibited elevated tau and phosphorylated tau levels in the hippocampus and cortex, accelerated brain atrophy, white matter deficits, substantial spatial recall deficits and locomotor hyperactivity. Suppression of the tau transgene with Dox prevented or attenuated all measured outcomes, with the noted exception of the sleep/wake phenotype. Thus, tau pathology may preferentially target the arousal-regulating nuclei in the brain, which may be less responsive to reducing hyperphosphorylated tau accumulation. The bidirectional relationship between sleep and tau pathology was further probed in rTg4510 mice, by enhancing sleep with orexin receptor antagonist hypnotics. Correcting REM sleep deficits with the dual orexin receptor antagonist, suvorexant, did not ameliorate the rTg4510 phenotype. In contrast, the selective orexin receptor 2 (OX2R) antagonist, MK-1064, effectively promoted sleep and attenuated the hyperarousal phenotype of rTg4510 mice and partially improved spatial memory recall, but only in male and not female rTg4510 mice. These data revealed striking sex differences in the sleep/wake phenotype of rTg4510 mice, which pointed to a tau- and sex-dependent enhanced hyperarousal drive, and potential modulation in OX2R signalling, in female rTg4510 mice. Together these studies have enabled the identification of key, converging factors underlying cognitive restoration in this mouse model. These included reducing hyperphosphorylated tau accumulation, the rescue of long white matter tracts and/or promoting sleep, in particular slow delta wave sleep. Accessing these features by enhancing sleep may thus provide potentially viable, effective therapeutic strategies for tauopathies. Noted sex differences may have implications for the application of OX2R selective antagonists in a sex-hormone dependent manner.
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ItemDevelopmental differences in the regulation and functional capacity of ABC efflux transporters at brain barrier interfaces( 2019)The safety and potential risks of many medications used in treating pregnant women and newborn children are not well defined. This is, in part, due to limited knowledge of how readily drugs transfer into integral organs such as the brain early in development. The present Thesis investigated the expression, regulation and function of ATP-binding cassette (ABC) efflux transporters at brain, cerebrospinal fluid (CSF) and placental interfaces of the Sprague Dawley rat, highlighting age-dependent differences. The expression of eight main ABC efflux transporters (abcb1a/b, abcg2, abcc1-5) was described in the rat brain, choroid plexus and placenta, revealing distinct developmental profiles. Immunostaining of the interface between the ventricular CSF and the brain identified PGP and BCRP transporters at this interface in the newborn rat but not in the adult. Drugs (rhodamine-123, digoxin, cimetidine, paracetamol) entered the developing brain more than the adult brain. Chronic drug exposure (diallyl sulfide, digoxin, paracetamol) caused an up-regulation of efflux transporter expression and functional capacity at brain barriers in adult but not fetal (E19) or newborn (P4) rats. Fetal rats exposed chronically to paracetamol via the dam had increased transfer into the brain compared to acute treatment, with this result being dose-dependent. Transcriptomic analysis revealed that chronic paracetamol exposure caused a large inflammatory profile in the placenta, indicating potential toxicity of paracetamol use in pregnancy over extended periods. The results in this Thesis suggest that drug transport into the rat brain is higher during development for acute and chronic conditions and indicate aspects of a treatment regime that may need to be considered differently for the safe prescription of medications to patients of different ages.