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Biologics are an increasing class of pharmaceuticals that possess many therapeutic benefits over typical, small‐molecule drugs, such as; high specificity, reduced frequency of off‐target effects, and the ability to mimic the body’s own physiological system. Despite these advantages, biologics still suffer from limitations due in part to their inherent protein composition, including; rapid circulatory degradation, renal clearance, and immunogenicity. Consequently, methods to modify biologics have been sought that can ameliorate these limitations yet still maintain their efficacy. PEGylation, defined as the conjugation of polyethylene glycol (PEG) to biologics, is one such modification. The coupling of PEG increases the overall molecular weight (MW) of the biologic, resulting in reduced renal clearance, whilst simultaneously acting as a shield to protect against proteolytic degradation. Both of these effects extend the half‐life of coupled biologics and improve their bioavailability. Furthermore, the extended residence times of PEGylated agents leads to an increase in efficacy in comparison to the non‐PEGylated counterpart; since the therapeutic moiety has more opportunities to elicit a response. The shielding effect of PEG is also reported to reduce the immunogenicity of biologics through preventing immune recognition of antigenic epitopes present on the biologic’s surface. The benefits of PEGylation have been well validated and there are a number of PEGylated agents clinically available, with more in various stages of development. Despite this, however, there is still little known concerning the disposition, metabolism and biological fate of PEGylated proteins. Furthermore, whilst PEGylation can reduce the immunogenicity of a protein, this has not precluded the onset of a new immunogenicity raised against the PEG moiety itself, nor indeed has PEGylation been shown to universally reduce the immunogenicity of coupled proteins. Anti‐drug antibodies (ADAs) and hypersensitivity reactions have been reported in both animal studies and patients receiving PEGylated agents. Furthermore, these adverse drug reactions (ADRs) occur against both the PEG and protein moieties of the conjugate. Consequently, there is a real need to understand the fundamental mechanics behind the metabolism, disposition and biological fate of PEGylated biologics, as well as defining the effect of PEGylation on the immune recognition, processing and presentation of the coupled protein. The bioanalysis of PEGylated proteins is hindered by inherent difficulties associated with the PEG moiety. PEG is transparent, non‐fluorescent, contains no ultra‐violet (UV) chromophore, is polydisperse, and is not easily ionised; making analysis by spectroscopy and mass spectrometry difficult. The utility of radiolabeling is also limited due to issues arising from placement of the radiolabel within the conjugate. Consequently, alternative analytical tools are required for the comprehensive bioanalysis of PEGylated proteins. In light of these issues the studies described in this thesis aimed to develop methodologies that can: 1) provide quantitative information concerning the biological fate and disposition of both the PEG and protein moieties of a PEGylated protein, and 2) define the effect of PEGylation on the processes involved in generating an immune response. The first investigation in this thesis involved developing and optimising gel‐based methodology and 1H nuclear magnetic resonance (NMR) spectroscopy to monitor the kinetics, disposition and biological fate of a model PEGylated protein, 40KPEG‐insulin, in a rodent disposition study. Male Wistar rats were intravenously administered a single dose of 40KPEG‐insulin and maintained over a period of 28 days. Plasma and urine samples were collected almost daily and liver and kidneys harvested on days 14 and 28. 1H NMR and gelbased analysis, incorporating western blotting for both PEG and insulin, and a barium iodide (BaI2) stain for PEG, revealed that PEG persists in both biological tissues and fluids across 28 day days. However, the anti‐insulin western blots revealed that the insulin moiety was either metabolically cleaved or sequentially degraded from PEG to the extent that no immunodetectable insulin was detected by day 7 in urine, or by day 14 in plasma, and could not be detected at all in liver and kidney tissue on days 14 and 28. However, an in vitro plasma stability study found the insulin moiety of 40KPEG‐insulin to be stable in plasma over 7 days, indicating that the loss of insulin signal observed in vivo must be occurring following cellular internalisation; potentially allowing for the liberated protein to be immunologically processed. The second investigation in this thesis concerned the effect of PEGylation on in vitro cellular internalisation. A range of different PEG MWs were incubated with dendritic cells (DCs) – a model antigen presenting cell (APC) – and internalisation was assessed by flow cytometry and fluorescence microscopy. These data revealed that DC internalise PEG regardless of MW, suggesting that PEGylation, in terms of MW, may have little effect on the internalisation of a PEGylated biologic by APC – over the clinically relevant PEG MW range used in this study. Thirdly, the effect of PEGylation was assessed on the lysosomal and proteasomal pathways of antigen processing and presentation, events en route to producing an immune response which occur after cellular internalisation. Insulin was conjugated to a range of different MW PEGs and incubated with either lysosomes or proteasomes. PEG was shown to be stable to lysosomal proteolysis over a period of 7 days; however insulin was completely degraded within 2 hours. When coupled to PEG, the insulin moiety was again degraded completely within 2 hours regardless of PEG MW. No PEG MW effect was observed when analysing the peptide repertoires generated between the PEGylated insulin conjugates. Indeed, it was shown that PEGylation provides only local protection against lysosomal degradation at the site of attachment. However, when degraded by proteasomes a PEG MW‐dependent effect was observed. Over 48 hours, insulin was nearly degraded to completion when conjugated to 20 kDa PEG. But when coupled to either 30 or 40 kDa PEG, only ~50% of the insulin moiety was degraded. When analysing the peptide repertoires, again PEG, regardless of MW, provided partial protection at the site of attachment. However, there was much more variation in the peptides generated between each PEGylated insulin. In conclusion, the methods developed in this thesis represent facile, inexpensive analytical tools to comprehensively analyse the disposition, kinetics and biological fate of both the PEG and protein moieties of PEGylated proteins. Furthermore, the investigation presented in this thesis was the first of its kind to demonstrate that cleavage and/or degradation of a PEGylated protein can occur in vivo. Consequently, the methods described in this thesis could be easily used to provide a measure of the pharmacokinetics and tissue retention of PEGylated biologics in man. When analysing the effect of PEGylation on the processing of PEGylated proteins, there was shown to be little difference when processed via the lysosomal pathway of antigen processing. However, based on the peptide repertoires generated PEG did provide local protection against degradation at the site of attachment – suggesting that site‐specific PEGylation, tailored for individual biologics, may provide a viable route to reduce the immunogenicity of the attached protein. Future investigation is warranted to further elucidate the potential effects of PEGylation on immunological events occurring downstream of lysosomal/proteasomal processing.

The endocannabinoid system (ECS) has been identified in the urinary bladder of humans and rats and is purported to play a modulatory role in detrusor overactivity (DO). Clinical studies demonstrated that cannabis improves urgency in patients with neurogenic DO. However, how endocannabinoids are regulated in vivo as well as the pathophysiological relevance of deregulation of the system in the aetiopathogenesis of DO remain unexplored. This thesis explores the clinical relevance of the ECS in the aetiopathogenesis of DO, by evaluating the effects of cannabinoid agonists on bladder contractions in vitro and in vivo. Cannabinoid receptor (CBR) expression was localised in human and rat bladders. In vitro, ACEA (CB1 agonist) had both pre- and postsynaptic effects on bladder contraction, while GP1a (CB2 agonist) only had postsynaptic effect. CB1 receptor was co-localised with ChAT (choline acetyltransferase enzyme) in human detrusor but not in the urothelium, while CB2 co-localised with ChAT in both urothelium and detrusor. qRT-PCR showed that CB1 receptor was significantly upregulated by 2.8-fold in the urothelium of DO patients and downregulated by 3.2-fold in the detrusor of DO patients compared to normal. Rat cystometry experiments demonstrated CP55,940 (CBR non-selective agonist) significantly increased micturition interval (MI) and bladder capacity (BC) by 52±5.5% and induced a 25 ± 2.9% decrease in maximal pressure (MP). Addition of CP55,940 to rats with induced bladder irritation, showed an increase in MI by 78% ± 16.7% and a 22% ± 5.8% reduction in MP. In conclusion, cannabinoid receptors are involved in normal micturition at both peripheral and CNS sites, data supported by in vitro studies where CB1 reduced neuronal activity and where both cannabinoid receptors modulated bladder contractility. CB1 receptors expressed in afferent fibre endings in the urothelium could influence bladder function. CB1 receptor agonists may be useful for future treatment of DO as there is altered CB1 receptor expression in these bladders.

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The present research involved the implementation / evaluation of several approaches to optimising antibiotic use and infection control. The first study reported within this thesis aimed at assessing the impact of an antibiotic policy on reducing high-risk antibiotic usage in hospital practice and the associated incidence rates of MRSA and C. difficile infection (CD!). There was a significant decrease in high-risk antibiotic use within the three studied hospitals as a result of the policy. MRSA and CD! incidence rates were monitored in one of the hospitals and both rates decreased significantly. The next study reported in the thesis focused on the influence of antibiotic prescribing in primary care on the incidence rates of ESBL-producing bacteria detected in patients who were hospitalised. It was clearly shown that previous exposure in primary care to fluoroquinolones and cephalosporins (second and third generation) were independently associated with ESBL detection in hospital. Moving to the wound infection area, an interrupted timeseries analysis was performed to examine the impact of infection control practices on rates of post-caesarean surgical site infection (SSI). In this work, there was a significant drop in the incidence rate of SSIs after the introduction of the main intervention in the study (use of ChloraPrep® skin disinfectant). In the final experimental chapter, the aim was to evaluate changes in the incidence of central venous catheter-related bloodstream infection (CRBSI when switching from mechanical-valve needleless connector use to the use of split-septum needle less connectors in an intensive care unit. A reduction in the CRBSI incidence rate was identified after this switch took place. Overall, the work presented in this thesis adds evidence which supports guidelines on controlling antibiotic use and healthcare acquired infections. The findings will help inform future new services and practices that will enhance healthcare quality and patient safety.

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