Cytochrome P450 enzymes (CYPs) are of great interest to pharmaceutical industry due to their major role in drug metabolism and drug-drug interactions in man. As CYPs also act on endogenous substrates and are regulated by nuclear receptors involved in biochemical pathways, their profiles are likely to affect endogenous metabolic profiles. The work in this thesis aimed to establish baseline rat liver CYP levels and assess relationships between CYP profiles and corresponding endogenous metabolic profiles, before introducing CYP inducers in subsequent projects to identify early biomarkers of CYP induction. An efficient and reproducible protocol was first developed and optimised for liver untargeted metabolic profiling by ultra performance liquid chromatography-mass spectrometry (UPLC-MS). Comparison of technical and biological variation for this liver protocol then demonstrated that sample preparation and UPLC-MS variability was mostly small compared to inter-animal variability. Subsequently, an in vivo study was designed including 20 male and 20 female Wistar rats. Liver mRNA of 81 CYPs was quantified, of which 23 exhibited significant gender differences with a gender ratio > 2. Clear gender differences were also observed in serum, urine and liver UPLC-MS metabolic profiles, e.g for metabolites belonging to steroid, triglyceride and phospholipid families. Multivariate models constructed to investigate relationships between CYP and metabolic profiles mainly highlighted the latent variable “gender”. Separate investigations for each gender yielded good prediction of CYP mRNA profiles from male liver aqueous metabolic profiles and female urine metabolic profiles. For prediction of CYP activities, the best models were obtained from serum metabolic profiles. In conclusion, this work improved our knowledge of rat basal CYP and metabolic profiles, and provides a strong basis for subsequent studies on CYP inducers. Importantly, strategies developed for UPLC-MS sample preparation, study design and data pre-processing/analysis are now routinely employed in our laboratory and have applications in many metabolic profiling studies.

The human brain is a complex biological network consisting of spatially separated but functionally integrated regions. Study of functional connectivity gained immense popularity in recent years providing new insight into the mechanisms underlying complex functions and the fundamental organisation of the brain. This has led to the emergence of new techniques for investigating connectivity, such as the application of pattern recognition techniques and the investigation of network dynamics. While highly promising, the application of these new techniques to pharmacological imaging data has not yet been fully explored. In this thesis we apply pattern recognition techniques to functional connectivity measures obtained for pharmacological imaging data to discriminate patterns of whole brain connectivity. Furthermore, we demonstrate that consideration of functional connectivity dynamics provides additional insight into the effect of pharmacological interventions. Specifically, we explore the effects of the N-methyl-D-aspartate receptor antagonist, ketamine, on the connectivity within the human brain. We argue that the investigation of connectivity is a more appropriate tool for the investigation of this compound due to the highly distributed pattern of effects, as compared to traditional approaches investigating amplitude effects. We demonstrate the applicability of pattern recognition techniques for the discrimination pharmacological states using measures of regional connectivity over the whole brain, using network interactions and through the inspection of network dynamics. We expand upon traditional approaches in our investigation, introducing a new approach to investigate network effects and temporal dynamics of connectivity organisation.

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The aim of this work was to assess the ability of the static and dynamic (incorporating the time-course of the inhibitor) prediction models to predict drug-drug interactions (DDIs) using a population-based ADME simulator (Simcyp). This analysis focused on fluconazole, ketoconazole, itraconazole, fluoxetine and fluvoxamine, as CYP inhibitors. The rationale for their selection was an abundance of reported DDI studies, involving a wide range of victim drugs. Preliminary analysis focused on the individual victim drug and inhibitor parameters that are utilised in the DDI prediction models. The victim drug properties included in the DDI prediction models are calculated intrinsically in the Simcyp simulator from in vitro data; these values were compared to estimates obtained by different in vivo methods. Estimations of the fraction metabolised by CYP enzymes were generally consistent with <20% difference between all methods for 15/23 victim drugs. No relationship was observed per CYP enzyme or per inhibitor utilised for phenocopying methods. Estimates of fraction of drug escaping metabolism in the gut were variable across methods with up to 60% coefficient of variation in the case of saquinavir. In vitro assessment of potential liver uptake of the inhibitors was identified for further investigation due to inconsistency in available literature data and sensitivity of the model to this parameter. Extent of liver uptake of selected inhibitors was assessed via comparison of clearance obtained in hepatocytes and microsomes (conventional depletion assay) and values obtained by the conventional depletion and media loss assays in hepatocytes. Clearance was determined at a low concentration (0.1μM) and both rat and human hepatocytes and microsomes were used. The clearance ratios ranged from no difference to >1500 (fluvoxamine from the media loss assay in human hepatocytes). No consistency was observed between methods and human or rat source for any of the inhibitors investigated; therefore, the inclusion of liver uptake into the prediction of DDIs for the current inhibitors was not supported. A database was collated from literature reports of DDIs involving the above named CYP inhibitors (n=97) and used to assess the inclusion of the time-course of inhibition into DDI prediction using the Simcyp simulator. In addition, the impact of active metabolites, dosing time and the ability to predict inter-individual variability in DDI magnitude were investigated using the dynamic prediction model. Simulations comprised of 10 trials with matching population demographics and dosage regimen to the in vivo studies. The predictive utility of the static and dynamic models was assessed relative to the inhibitor or victim drug investigated; both models were employed within Simcyp for consistency in parameters. Use of the dynamic and static models resulted in comparable prediction success, with 67 and 70% of DDIs predicted within two-fold, respectively. Over 60% of strong DDIs (>five-fold AUC increase) were under-predicted by both models, particularly for fluoxetine and fluvoxamine. Incorporation of the itraconazole metabolite into the dynamic model resulted in increased prediction accuracy of strong DDIs (80% within two-fold); no difference was observed for the inclusion of the fluoxetine metabolite. Predicted inter-individual variability in the DDI magnitude was also assessed in healthy, patient and genotyped subjects using a subset of clinical interactions (n=24). Mixed prediction success was observed and the importance of reliable clinical data was highlighted. The differences observed with the dose staggering and the incorporation of active metabolite highlight the importance of these variables in DDI prediction. Finally, the traditional 'two-fold limits' as a measure of the prediction success were reassessed, in particular at AUC ratios approaching one. New limits proposed are applicable for both inhibition and induction DDIs and allow incorporation of the variability in pharmacokinetics of the victim drug when required. DDI predictions were refined using in vitro clearance data for the inhibitors, and assessed using the new predictive measure.

The thesis investigates establishing a hypertussive model of cough primarily in the rabbit and with comparative experiments conducted in the guinea pig. These models were then used to investigate the effectiveness of various antitussives such as codeine and levodropropizine, as well as, putative antitussives such as anticholinergics, PDE inhibitors, bronchodilators and drugs affecting targets on sensory nerves. “Hypertussive” is a poorly recognised term and it is defined in the context of this thesis to describe an inappropriately frequent and/or loud cough response when compared to normative cough responses for the same given dose and route of a given tussive stimuli. A novel model of hypertussive cough responses was established and validated in the rabbit and guinea pig using ozone as a sensitising agent. Primary measures include cough frequency, cough magnitude, time to first cough and cough duration. In further experiments lung function parameters such as dynamic compliance and total lung resistance, and total and differential cell counts, as well as pilot experiments involving analyzing categories of cough “sounds” were measured. The thesis was also concerned with the measurement and classification of cough events and in particular the discrimination of cough events from sneeze events. Two commercially available systems and ad hoc approaches were used to evaluate how best to describe, count and classify the cough response and qualitative and quantitative judgement have been made to assess a best approach. In summary, the data in this thesis suggests that ozone is a particularly effective acutely-acting non-allergic sensitising agent capable of shifting the dose response curve of the cough response to citric acid leftward by 0.5 to 1 log units. Sensitization of the cough reflex overcame desensitization of rabbits and guinea pigs to citric acid, allowing cross-over designs to be employed. Ozone appears to act via sensitization of the peripheral airway sensory input, but I found no evidence that this was via an action on Transient Receptor Potential Ankyrin 1 (TRPA1), which has previously been suggested to be an important target for ozone. Codeine and levodropropizine were effective against hypertussive responses, but did not block the normotussive cough. Anticholinergic drugs were not effective against ozone sensitised cough nor normotussive cough responses in the rabbit, but significantly inhibited sensitised cough responses and normotussive cough responses in guinea pigs. However, salbutamol demonstrated a similar treatment profile to the anticholinergic drugs implying that bronchodilation is an important mechanism to reduce the cough response in guinea pigs. Thus, these data suggest that drug candidates that cause bronchodilation may falsely identify as antitussives in the guinea pig model. Phosphodiesterase inhibitors were effective at blocking the infiltration of leukocytes in both guinea pigs and rabbits, but did not effect the acutely sensitised cough, suggesting that in this model ozone is inducing hypertussive responses independently of leukocyte infiltration.

Glucocorticoids are widely used as a clinical treatment mainly due to their potent anti-inflammatory abilities, however some patients develop glucocorticoid resistance and high levels of steroid are required to maintain effective treatment. Use of prolonged high glucocorticoid levels is associated with several severe side effects. ARID1a mutations are frequently identified across numerous cancers and have been linked to glucocorticoid resistance. ARID1a is a subunit of the SWI/SNF complex important for binding the glucocorticoid receptor (GR) and for interactions of the complex with DNA. Chromatin remodelling by the SWI/SNF complex is a vital component of genomic GR signalling. Chromatin is dynamically opened and closed at GR binding sites in target genes to regulate transcription. Loss of the full length ARID1a is thought to impact upon GR dependent gene regulation. In this study transcriptional profiling using next-generation RNA sequencing enables the assessment of GR dependent gene transcription in the absence of the full length ARID1a and Chromatin immunoprecipitation is used to assess differences in GR and RNA Polymerase II binding. In the first part of this thesis we compare two cell lines, SKOV3 and HeLa, which contain a truncated or full length ARID1a protein respectively. Here we show a limited response to glucocorticoids in SKOV3 cells and reveal greatly diminished GR binding at a regulatory site known to require remodelling. In the second part, ARID1a knock-down and overexpression of the ARIDla C-terminal are assessed. In these chapters we identify novel 'roles of GR with ARID1a in cell cycle regulation. This thesis highlights several molecular mechanisms through which the loss of ARID1a can impair GR regulation and disproves previous hypotheses that ARID1a is essential for large-scale regulation of robustly glucocorticoid responsive genes. This research may form the foundations for further research leading to a better understanding of how to approach treatment of cancers with ARID1a mutations.

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The calcium-sensing receptor (CaSR) has a well-established role in regulating Ca2+ homeostasis by controlling the release of parathyroid hormone from the parathyroid gland. CaSRs are also expressed in the vasculature where they regulate vascular tone, although the underlying cellular mechanisms are poorly understood. This thesis investigates the functional role and cellular mechanisms of CaSRs regulating vascular tone. Wire myography studies, nitric oxide imaging, western blotting, immunocytochemistry, proximity ligation assays, and patch-clamp electrophysiology experiments were performed using vessel segments and isolated endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) from rabbit and mouse mesenteric arteries. Initial experiments revealed that stimulating CaSRs by increasing the extracellular Ca2+ concentration induces endothelium-dependent vasorelaxations of precontracted arteries, mediated predominantly via nitric oxide (NO) signalling, which stimulates BKca channels in VSMCs. In vessels lacking a functional endothelium, CaSR activation mediates vasoconstrictions. CaSR expression was also demonstrated in isolated ECs and VSMCs. Further experiments revealed that CaSR-evoked NO synthesis and NO-mediated vasorelaxations involve the activation of a heteromeric cation channel composed of transient receptor potential vallinoid 4 (TRPV4) and transient receptor potential canonical 1 (TRPC1) proteins expressed in ECs. As a result, CaSR-induced vasorelaxations mediated by NO are impaired in TRPCT/_ mice lacking this channel. Additional studies revealed that stimulation of CaSRs also evokes vasorelaxations via the activation of IKca channels in ECs, and although cation channels are involved in mediating CaSR-IKca signalling, these channels are different to those involved in activating NO production. CaSR-evoked vasorelaxations were also revealed to be confined to larger diameter mesenteric artery branches. Finally, the vascular actions of synthetic CaSR modulators were examined as these have previously been insufficiently understood. The current study reveals that the negative CaSR modulators calhex-231 and NPS 2143, and the positive CaSR modulator calindol impair vascular reactivity via direct inhibition of voltage-dependent calcium channels, in a CaSR-independent manner.

No description available.