GABA is a well established inhibitory neurotransmitter in the CNS, which has an opposing role to its precursor, glutamate, which is an excitatory neurotransmitter. In the CNS, both GABA and glutamate have multifunctional roles that are essential for normal brain functioning, which includes the regulation of cerebral blood flow. Both GABA and glutamate have been shown to induce pericyte-mediated changes in blood flow in the retinae and in the cerebellum, respectively. Pericytes are expressed throughout all mammalian tissue including the kidney, and they are renowned for their contractile nature and their ability to modulate capillary diameter. An increasing number of publications have suggested that both GABA and glutamate might also play a role in the regulation of renal function. All key enzymes associated with GABA/ glutamate metabolism have been localised to the kidney providing the necessary machinery for localised GABA/ glutamate synthesis and metabolism. Despite the collective evidence describing the presence of a GABA/ glutamate system in the kidney, the precise function of such a system requires further clarification. The work presented in this thesis is principally concerned with establishing the physiological role(s) of the GABA and glutamate system in the kidney. This thesis seeks to address this question using a live kidney slice model to investigate pericyte-mediated real-time changes in vasa recta diameter in response to GABA, glutamate and associated compounds. Confocal microscopy techniques were used to confirm the expression of key components in the GABA shunt pathway, in relation to the renal medulla. Data presented here, highlights a novel role for both GABA and glutamate, expressed in both vascular and tubular compartments in the renal medulla, to induce pericyte- mediated regulation of vasa recta diameter, and therefore medullary blood flow. The second aspect of this thesis focuses on determining whether functional GABA receptors exist within renal tissue, focusing specifically on their expression within the cortical collecting duct. Electrophysiological experimental data highlights that functional GABA receptors exist in a renal cell line, which serves to modulate solute transport. In conclusion, this thesis highlights that GABA is able to modulate both vascular and tubular aspects of renal function. While, glutamate, and its co- agonist, glycine, have an opposing effect to GABA, and serve to induce vasodilation. The results of this work highlight new key players that affect renal function, which may be significant in both health and disease.

Alkaptonuria (AKU) is an ultra-rare, autosomal recessive disorder of tyrosine catabolism due to mutations within the homogentisate 1,2-dioxygenase (HGD) gene. The resulting enzyme deficiency leads to accumulation of homogentisic acid (HGA) and deposition of melanin-like pigment polymers in the connective tissues of the body in a process called ochronosis. This leads to debilitating early onset osteoarthropathy, renal damage and aortic valve disease. As a multisystem disorder, AKU results in progressive and chronic pain and severe morbidity. Most management approaches for AKU are palliative and rely largely on analgesia and arthroplasty. Several therapeutic approaches have been tested with low degrees of clinical effectiveness. Nitisinone is a promising drug that blocks the enzyme catalysing the formation of HGA and thus lowers its plasma concentration. HGA lowering therapy has been widely used in another rare inborn error of metabolism, Hereditary Tyrosinemia type 1 (HT-1) for over 20 years. Nitisinone is highly efficacious in terms of its metabolic effect as it decreases HGA to very low levels, but there is limited toxicology data available for its use in AKU. There are also concerns relating to the adverse side effects of elevated tyrosine and potential neurotoxicity if treatment was implemented in children. The work presented within this thesis presents novel findings to inform the future licensing process for the use of nitisinone in AKU and investigates the safety of implementing treatment in younger patients. Nitisinone treatment had no detrimental effect on learning, memory or motor function in young AKU or wild type mice. The thesis also includes new data from mouse dosing studies concerning the correlation between plasma HGA and ochronotic pigmentation and reveals that plasma HGA must be lowered to a critical level before pigmentation is beneficially reduced. Finally, this thesis reports on the lability of the arteriovenous metabolome relating to AKU and initiates a discussion relating to the HPPA to HPLA excretory conversion pathway along with important considerations for collection, analysis and comparison of blood samples in future studies.

Antibiotic resistance is recognised as an international health concern due to its potential to increase morbidity and mortality from illnesses that are currently treatable. Antibiotic prescribing by GPs in primary care has been shown to directly contribute to rates of antibiotic resistance. Many interventions have been introduced across European countries in an attempt to promote prudent use of antibiotics for respiratory tract infections in primary care. Exploring GPs' views and experiences of interventions to promote prudent antibiotic use can help to understand what is viewed as acceptable by GPs and what may encourage behaviour change. In addition, investigating these views across countries can highlight any similarities and differences between contexts and examine whether interventions which are acceptable, feasible and potentially effective in one context may be appropriate for implementation in others. The main aim of this research was to explore GPs' and experts' views and experiences of antibiotic prescribing and interventions to promote prudent antibiotic use in the management of RTIs. If professionals hold different views there is a need to develop interventions for each country, whereas if professionals hold similar views then an intervention which is acceptable in one country can be implemented in others. Two qualitative studies were carried out to explore GPs' and experts' views of antibiotic prescribing and interventions to promote prudent use across five European countries. The results of both studies revealed consistent views despite differences in context, indicating that both GPs and experts who develop interventions held similar beliefs about the acceptability and feasibility of different types of interventions. Secondly, a systematic review was undertaken which synthesised all qualitative work which had explored GPs' views on antibiotic prescribing or interventions to promote prudent use. The review incorporated studies from several countries and produced a model highlighting seven factors which influence GPs' prescribing decisions and aspects of interventions which could address these factors. The findings of this thesis reveal the barriers experienced by GPs in prudent antibiotic prescribing and suggest that it is suitable to develop an intervention to promote prudent antibiotic use for implementation at an international level.

No description available.

Clostridium sporogenes is part of a highly diverse group of Gram positive, spore forming, anaerobic bacteria. C. sporogenes can be used as a delivery vehicle for chemotherapeutics in cancer treatment due to the inactive spore form of C. sporogenes only germinating in the microenvironment of the hypoxic tumour. Cancer, despite large investment in treatment and diagnosis, still remains one of the leading causes of death in the world. As such, improvements on current treatments are necessary to improve patient prognosis. Utilising C. sporogenes could be a cheap and effective way to do this by utilising their germination properties to deliver anti-cancer therapeutics directly to the hypoxic regions of solid tumours. Through introducing Prodrug Converting Enzymes (PCEs) into C. sporogenes when the spores germinate in the tumour the production of the PCE will result in the breakdown of a Prodrug into a toxic product resulting in an anti-cancer effect. This system is known as Clostridial Directed Enzyme Prodrug Therapy (CDEPT). Previous iterations of this system incorporated a nitroreductase gene, used for the breakdown of the prodrug CB1954. During this project alternative prodrugs were investigated, in this case carboxypeptidase G2. Alternatives into the prodrug and enzyme system are also being investigated in a direct action therapy in the form of the monoclonal anti-VEGF. The aims of this project were to implement the genes and optimise the activity of the drugs if necessary. It was also necessary to confirm that the bacterium was a non-pathogenic group 1 bacterium through sequencing and annotation of the genome.

Polyadenylation is a universal step in the production of all metazoan mRNAs except histone mRNA. Despite being universal, previous experiments have implicated it in the regulation of inflammation. An inflammatory system using RAW 264.7 murine macrophage cells was established with bacterial lipopolysaccharide (LPS) used as a stimulus. After improving the poly(A) tail test (PAT) method of measuring poly(A) tail lengths, it was applied to inflammatory mRNAs during the inflammatory response. Poly(A) tail length was shown to vary over the course of the inflammatory response, and for Tnf, this was even true of initial poly(A) tail size, which is widely believed to be uniform for the majority of mRNAs. The adenosine analogue cordycepin (3’-deoxyadenosine) was shown to have anti-inflammatory effects on mRNA, in line with existing literature, and is likely to be the anti-inflammatory component of Cordyceps militaris ethanol extract. Inhibition of either import of cordycepin into cells or phosphorylation of cordycepin was sufficient to abolish its anti-inflammatory effects. Adenosine treatment led to repression of Il1b mRNA, but did not repress other mRNAs tested that were cordycepin-sensitive. This suggests that cordycepin does not simply act by mimicking the effect of adenosine, and that the two compounds have distinct modes of action. Inhibiting deamination of cordycepin potentiated its effects. We also observed that pre-mRNA levels of inflammatory genes were decreased by cordycepin treatment, indicative of effects on transcription. Other groups have reported that cordycepin interferes with NF-B signalling. As NF-B is an important transcription factor for the induction of inflammatory genes, this would provide a basis for explaining our observation that cordycepin represses at the transcriptional level. However, we did not observe any changes in NF-B signalling, with degradation of IB completely unimpeded by cordycepin treatment. Notably, cordycepin did shorten the Tnf poly(A) tail, and the observed inhibition of polyadenylation is consistent with observations that cordycepin led to decreased efficiencies of mRNA 3’ cleavage and transcription termination for Tnf. Such effects on polyadenylation and 3’ processing of mRNA were hypothesised to particularly affect unstable mRNAs that depend on longer poly(A) tails for avoiding decay and/or mRNAs with a high rate of transcription. However, comparison of microarray data to data from RNA-seq of RNA from 4-thiouridine labelling experiments showed that cordycepin-sensitivity did not correlate with mRNA stability or transcription rate. Long noncoding RNAs (lncRNAs) were found to be enriched in cordycepin-treated cells. If some of those lncRNAs have regulatory roles in inflammation, cordycepin’s effects may be mediated through them. Lastly, cordycepin significantly altered pain behaviour in a rat model of osteoarthritis (OA), supporting its continued use as a lead compound for exploration of new OA therapeutics.

Chronic pain is a condition that is thought to affect roughly 8 million people in the UK. It is classified as pain that persists for more than 6 months. Chronic pain is commonly associated with depression, insomnia, anxiety and poor quality of life. Many treatments for chronic pain are accompanied by numerous debilitating side-effects, this in combination with insufficient pain relief means that approximately 50% of patients will discontinue their treatment. Most sufferers choose to live with the pain rather than deal with numerous adverse-effects. There is a great need for new therapeutics that are specifically designed to target the underlying mechanisms of chronic pain, therefore providing safer and more effective treatments. One such mechanism is the down-regulation of strychnine-sensitive glycine receptors (SSGRs) localised in the dorsal horn. Glycinergic activity is known to be inhibitory and artificial stimulation can produce analgesia. Positive allosteric modulators acting on α1 SSGRs may able to compensate for the inhibitory glycinergic activity that is reduced in chronic pain. Previous work within the group lead into the identification of propofol analogues designed to be novel positive allosteric modulators of α1 SSGRs. Work presented in this thesis describes the generation and optimisation of these analogues with a focus of drug metabolism and pharmacokinetics. The hit to lead process has resulted in the development of a lead compound that is highly potent at the target, has excellent pharmacokinetic and safety profiles and is able to produce high levels of analgesia in an animal model of neuropathic pain.

The main hypothesis of this research project was that optimisation of treatment based on pharmacokinetic principles is on its own a powerful approach in improvement of treatment outcomes. This work therefore focused on optimisation of treatment of cancer based on principles of pharmacokinetics using two main approaches 1) lipophilic prodrug approach to specifically target the intestinal lymphatic system following oral administration and 2) identification of orally bioavailable candidate anticancer agents and biopharmaceutical development to increase the bioavailability for sufficient systemic exposure to the drug. The first approach was the prodrug derivatisation to take advantage of the physiological process of intestinal lymphatic transport in order to deliver anticancer agents to the mesenteric lymph nodes. Similar prodrug approaches have been researched by other groups but the main focus previously was on increasing the overall bioavailability where they mostly used long-chain or triglyceride mimetic prodrug moieties. However, in this project, through a series of stability and chylomicron association studies, it was revealed that activated ester prodrugs are the most suitable forms for yielding high concentrations of active drugs in the mesenteric lymph nodes. It was remarkable that using this novel approach significantly higher concentrations of the active drugs were achievable in the intestinal lymphatics without affecting the systemic exposure. The second approach taken in this PhD project was achieving sufficient systemic exposure of anticancer agents by identification of orally bioavailable candidate and improvement of oral bioavailability by biopharmaceutical development. The candidates with promising pharmacokinetic properties were rank-ordered by application of a rational drug discovery and development approach of integrated in vitro-in silico assessments. Following in vivo confirmation studies, oral bioavailability was further enhanced for a compound that exhibited a double-peak phenomenon. The results of the two approaches indicate that pharmacokinetic optimisation can be useful in development of anticancer agents to improve the treatment outcomes of cancer.

In order to achieve drug delivery via the respiratory route, an understanding of particulate interactions is of vital importance. For successful delivery to the distal airways, an aerodynamic diameter of less than 5 μm must be achieved. However, particles of this size presents a difficult formulation challenge, due to the inherent cohesiveness between particles and adhesion to the device, due to the high surface to volume ratio of such small particles, causing the particles to clump together. This tendency will thereby cause a reduction in dispersion, aerosolisation and device efficiency; for this reason dry powder inhalers (DPIs) invariably fail to achieve a fine particle fraction efficiency above 15%. There are a wide variety of factors which affect particulate interactions including; surface roughness, surface chemistry, particle size or shape and particle mechanical properties. However, these factors are highly interrelated and so previous attempts to investigate their effect on particle adhesion generally have difficulty isolating the impact of each factor. For instance, investigating the effect of morphology on particulate interactions invariably utilise destructive techniques to alter the roughness, which is likely to alter other factors like surface energy and provide limited control for optimisation. With the rise of 3D printing (additive manufacturing) there is now the capability to produce sub- micron morphologies, and so a bottom-up approach to studying the effect of morphology on particulate interactions can be achieved. The aims of this thesis are therefore twofold. Firstly, to identify, optimise and evaluate a suitable additive manufacturing technique to produce well-defined micron scale morphologies appropriate for furthering the understanding of the importance of morphology on particle adhesion. This is a scale which is at least two orders of magnitude improvement on current state of the art 3D inkjet printers. Secondly, to measure the effect on particle adhesion and deposition to these morphologies, both on an individual particle and on a bulk powder basis, allowing elucidation and understanding of the effect of surface roughness on particle adhesion, with a specific focus on respiratory drug delivery. Printing well defined geometries of an appropriate micron scale size range for particle adhesion testing has been achieved, using two photon polymerisation (TPP). TPP is a novel 3D printing technique which as its name suggests involves the curing of usually acrylate containing polymer resins by the absorption of two infra-red photons in the focus of the laser beam. TPP has been shown to produce a sub-diffraction limit lateral resolution of 120 nm. By optimising the printer parameters and experimentation with differing structure fill and input settings the creation of a well- defined curve on a micron scale was achieved. The initial test morphologies comprised of a ridge with a semi-circular top with a diameter of 1 μm, which were shown to be reproducibly printed. These morphologies were then varied in a controllable fashion with varying ridge height and spacing between the ridges. A uniform and consistent surface chemistry was created using a plasma polymerised hexane (ppHex) coating. In order to evaluate particulate interactions relevant to pulmonary drug delivery both an understanding of the effect of morphology on both individual particle adhesion and bulk powder deposition in a fluid environment is needed. Individual particle-surface adhesion was achieved by testing the TPP structures against three particle types using single particle colloidal probe microscopy (polystyrene beads diameter 10 μm and 5 μm and a lactose particle designed for inhalation formulations). The analysis of this data provides evidence of a clear trend between particle contact area and adhesion recorded both on the ppHex control and the TPP coated morphologies. The TPP morphologies are shown to locally reduce the overall adhesion, in comparison to the flat substrate. The ridge height is also seen to have a significant effect on particle adhesion, with 5 μm < 3 μm < 1 μm for the polystyrene beads, but 3 μm < 5 μm < 1 μm for the Respitose SV003 lactose particle for all ridge spacings. Varying the ridge spacing produced two differing trends in adhesion to the polystyrene beads. If the particle was unable to penetrate the valleys of the roughness, for the 1 μm high ridges, a significant effect on particle adhesion was seen with 3 μm < 1 μm for the polystyrene beads. In contrast, the 3 μm and 5 μm high ridges showed the opposite trend when the particle is unable to descend between the ridges with 1 μm < 3 μm < 8 μm for the polystyrene beads. Investigation of the bulk powder deposition of the particles on the TPP structures and any subsequent re-entrainment in a fluid environment was then achieved using a novel methodology developed during the course of this work. This combines the use of a standard next generation impactor, which generally is used to separate out a respiratory formulation based on aerodynamic diameter, with the TPP substrates. This shows that ridge height has a significant effect on particle adhesion with 3 μm < 1 μm < 5 μm. In contrast, the different spacings of the ridges were not shown to produce a significant difference in particle deposition. This is likely due to the conflicting effect of asperity spacing on the processes of particle deposition and re-entrainment. This thesis therefore highlights the capability of TPP, to produce well-defined micron scale structures with varying morphologies. It then shows that these can be successfully utilised to provide valuable insight into the effect of surface morphology on particle- surface interactions, specifically; adhesion, deposition and re-entrainment.

The pharmaceutical industry has experienced an increase in the amount of development candidates with low aqueous solubility and accordingly poor bioavailability. In order for this problem to be solved, amorphisation is thought to be the most favourable solution. The amorphous state is higher in free energy thus higher in solubility when compared to the crystalline form. Milling and specially cryomilling is a very unique technique for providing of the crystalline to amorphous transformation since there are no heat or solvents involved. Phthalic acid, isophthalic acid and terephthalic acid, individual and pair mixtures, are crystalline organic non medicinal compounds, which have been used for the first time as model compounds to investigate whether cryogenic milling can induce crystal to amorphous transformation and if the preparation of pair mixtures could affect the recrystallization rate of the subjected materials or not. The materials were cryomilled and analysed by DSC, XRPD, and FTIR. It was found that only terephthalic acid become amorphous after cryomilling, and even after the cryomilled sample been stored for three weeks DSC thermogram still detects recrystallization exothermic along with the XRPD pattern, which shows a very broad peaks indicative of particle size reduction. Pair mixtures were also studied and analysed by DSC and XRPD. Phthalic acid/isophthalic acid, isophthalic acid/terephthalic acid, phthalic acid/terephthalic acid were cryomilled together and mixed physically after been cryomilled separately. XRPD results show that unlike the cryomilled separately mixtures, phthalic acid/ isophthalic acid, isophthalic acid/terephthalic acid, terephthalic acid/phthalic acid cryomilled together samples produces a synergistic effect in which the Bragg peaks of both phthalic acid and isophthalic acid are suppressed. It appears that co cryomilling of these pair mixtures together resulted in the production of a new material that could potentially either be two-component single phase (nano-sized co-crystal), or a new polymorphic form of either phthalic acid, isophthalic acid or terephthalic acid. Single-component of aspirin (ASP), paracetamol (PCM) and caffeine (CAFF), along with multi-component systems of paracetamol/aspirin, paracetamol/caffeine and aspirin/caffeine were milled at room temperature and by a cryomill. The milled samples were analysed using DSC, XRPD and FTIR. It was noted that there are no clear indications of crystal to amorphous transformation in all three materials. When milling aspirin at room temperature a marked reduction in the melting point was observed. Generally, a reduction in the melting point is either attributed to particle size effects, polymorphism, impurities and decomposition. In this case, the decrease in the melting point was only noticed when aspirin was milled at room temperature, so it is possible that the heat generated during the milling process resulted in chemical decomposition of aspirin to salicylic acid. Anhydrous caffeine is acknowledged to have two polymorphic forms, Form II which is considered to be stable at room temperature until ~145 °C. Form I is stable from ~145 °C to its melting point ~ 236 °C. This polymorphic transformation was detected by DSC, XRPD and hot stage microscope and it was noticed only with the as received and the room temperature milled samples. Cryomilled caffeine data showed only the presence of Form I. On the other hand, for the cryomilled multi-component systems DSC and hot stage microscope images confirmed the eutectic formation with a composition of 45:55% w/w (PCM:ASP), 50:50% (ASP:CAFF) AND 50:50% (PCM:CAFF). The obtained data were compared with room temperature milled and the theoretical values resulted from Van Laar equation. Solid pharmaceuticals represent heterogeneous systems that typically consist of one or more active pharmaceutical ingredients (APIs) and a number of excipients. Multi-component systems from mixing aspirin, paracetamol and caffeine with different excipients, which included sucrose, lactose monohydrate, xylitol and trehalose dihydrate were prepared by the use of a cryomill and were analyesd by DSC and XRPD. It was found from the XRPD data that mixing both sucrose and lactose monohydrate respectively with ASP, PCM and CAFF would produce more of a synergistic effect than xylitol and trehalose dihydrate. Cryomilling caffeine/sucrose and caffeine/lactose resulted in a production of a new XRPD trace that cannot be described in terms of a linear combination of caffeine, sucrose and lactose monohydrate. A new material was therefore formed as a result of cryomilling which has not been reported before.