The thesis describes a number of examples of the use of recently developed mass spectrometry experimental approaches to characterise biologically important mixtures. The recently introduced field of ambient ionisation mass spectrometry has been utilised in the rapid, sensitive, information rich characterisation of pharmaceutical formulations. Little, or no, sample treatment was required and the experiments were shown to provide detailed information on active ingredients in the presence of a number of other components. A number of ambient ionisation approaches including DART, DESI and DAPCI were compared and advantages and disadvantages of each approach outlined and discussed. The exciting technology of ion mobility has recently been commercially interfaced with mass spectrometry (IMMS). This has been utilised in a series of fundamental experiments that probe the interaction of varied cations with isomeric oligomers of carbohydrates. The approach enables conformational changes to be rapidly measured over a wide (500-6000 Da) mass range. Changes in conformations were observed for multiply cationised species which agree with previously measured solution phase measurements. The IMMS approach has also been used successfully to characterise a number of Nlinked glycans released from glycoproteins. The experiments enable isomeric structures to be differentiated and present an opportunity to develop a rapid, high information content screen. Estimated cross sectional measurements have been calculated and found to be in good agreement with those obtained from conventional drift cell approaches.

Background Rupture of the Achilles tendon occurs in over 11,000 people annually in the UK. Traditional management using cast immobilisation is being slowly replaced by immediate weight bearing rehabilitation, but currently there is no consensus regarding the exact protocol to be used. The aim of this thesis was to develop an immediate weight bearing rehabilitation protocol for patients who have sustained an acute rupture of their Achilles tendon to inform a definitive evaluation of its effectiveness. To achieve this aim a framework (by the Medical Research Council) for defining and developing interventions with several components was used to underpin the structure of this thesis. Pre-Clinical Development The first two objectives of this thesis were focussed on the ‘pre-clinical’ development phase. Firstly, a systematic review of the evidence base identified the components that define immediate weight bearing rehabilitation. Two of these were evaluated in controlled gait analysis studies to inform and develop a rationale for the intervention to establish what changes are expected and why. The key findings showed that rigid orthoses designs with a large degree of plantarflexion, increased heel pressures, reduced forefoot pressures and decreased the amount of time spent in the terminal stance and pre-swing phase of the gait cycle. Feasibility and Piloting The first clinical phases (feasibility/piloting) followed, which included testing procedures, establishing likely recruitment and follow up. Alongside this, a further systematic review was undertaken to identify what outcome measures are used in research for this injury to determine effectiveness. The Achilles tendon Total Rupture score was the only disease specific patient reported measure identified with supporting validation research. Further evaluation of its measurement properties found the score to be internally consistent, responsive and with good construct validity. Conclusions This thesis defines the rehabilitation components, proposes a theoretical framework and tests this in practice. The results will ensure that rehabilitation after an acute Achilles tendon rupture is based on a systematically developed protocol rather than ad hoc practice. This will now be used to inform future definitive research in this area.

Most drugs have a considerably low solubility in the environmentally friendly hydrofluoroalkane propellants (HFAs) currently used in pressurized metered dose inhalers (pMDIs). As a consequence, instability can occur from crystal growth and Ostwald ripening of the system altering the therapeutic performance of the pMDI. Understanding and being able to predict the behaviour of such drugs in the propellant will help in selecting the correct co-solvents and/or surfactants to increase the stability of such formulations. When anhydrous beclomethasone dipropionate (BDP) is suspended in tricholoromonofluoromethane (CFC-11) rapid crystal growth occurs, leading to the formation of a clathrate. Since chlorofluorocarbon (CFCs) propellants have been replaced by HFAs, many questions arose concerning the ability of BDP to form clathrates in the HFA and any stability issues that arise from such reformulation. Clathrates are crystalline compounds consisting of a lattice of one type of molecule that hosts a second type of “guest” molecule within its structure. Since the solid state chemistry can significantly alter the physical interactions within a suspension formulation, it is crucial to determine the most stable crystalline form in the presence of the propellant. Successful formation of BDP CFC-11 clathrates were observed in this work as well as positive outcomes in terms of reduction in the surface energy and the force of adhesion within a model pMDI formulation (even after processing i.e. size reduction). Following this, HFA-134a and 227-ae were selected to determine any potential clathrate formation and to monitor their stability within a pMDI formulation. The focus of this project was to determine the stability of BDP and budesonide in HFA propellants, as well as the appropriateness of each formulation for pMDI use. This project considered the potential use of complementary surface and solid-state analytical tools to provide fundamental understanding of clathrate formation and their physicochemical characteristics. A special interest in understanding the fundamentals behind the process of Ostwald ripening, a process that affects drug particle size and their related stability and hence ultimately dose consistency was also considered. Atomic force microscopy (AFM) was used in order to determine its applicability in studying Ostwald ripening and surface activity of the different APIs in model propellant. Furthermore, the effects of a range of parameters that included storage time, co-solvents and surfactants on Ostwald ripening were taken into account. The work presented in this thesis has demonstrated that the formation of a propellant clathrate is favourable for APIs that to improve formulation stability through a reduction in particle surface energy. However, isolation and full characterisation of such HFA clathrates remains challenging due to their decreased stability when removed from the high pressure media of the pMDI device. This thesis shows that a combination of co-solvent and surfactant provides an effective reduction in instabilities due to Ostwald ripening of the pMDI formulation and a better control of particle size of the APIs within the formulation. This work provides a platform for future formulation development for pMDIs.

Personal profiling is a dynamic learning process designed to enable individuals to identify their strengths, needs and aspirations in order to set strategic and behavioural goals. This single-site case study seeks to explore undergraduate occupational therapy students' attitudes and experiences of the process of personal profiling in the context of early professional development. The profiling system aims to support students through the process of integrating their University-based and practice-based learning experiences for personal and professional development. The investigation was carried out in two phases, a pilot study and main study, using an inclusive approach for inquiry. From the initial research question several sub-themes emerged during the pilot study which informed the overarching design of the main study. Students' attitudes towards the existing system were examined using a cohort study approach which employed a survey design of quantitative and qualitative data-gathering questionnaires. Attitudes and experiences of students were further explored through semi-structured interviews. A cross-sectional approach which included a sample of students from each year was adopted. In addition questionnaire data was collected from tutors and practice educators who play a significant role in students' professional education. Findings indicate students generally appear to have a positive attitude to personal profiling. The value is experienced at varying psychological levels, depending on students' willingness, motivation to engage with the process and psychological preparedness. A model of levels of engagement in profiling is proposed. Students' report the process of profiling was a challenging, psychologically messy and uncertain process yet worthwhile in the overall pursuit of professional development. Tutors perceived it as an enabling process in the students' learning. The conclusion drawn is that students have the intellectual capability and potential to benefit from personal profiling. Students appear to self-consciously recognise, articulate and acknowledge the value of personal profiling in facilitating early professional development.

Poly(lactide-co-glycolide) (PLG) biodegradable microparticles were evaluated as an oral delivery system for immunisation against equine influenza virus. Model particulates (fluorescent polystyrene and gold labelled polystyrene) were used to characterise the route and mechanism of intestinal uptake. Peyer's patches were found to be the site of particulate intestinal absorption characterised by phagocytosis at the M cell surface. Microparticles were found within intercellular compartments indicating a paracellular route for the transit of microparticles from lumen to lymph. A quantitative method of counting the number of microparticles passing into the lymph in both the superior mesenteric and thoracic lymph ducts indicated levels of intestinal uptake high enough to deliver vaccines via this route. PLG microparticles encapsulating equine influenza virus were prepared by the process of solvent evaporation. The immune responses were evaluated in mice after either systemic or oral immunisation of Balb\c mice using formalin treated equine influenza (Prague 56 H7N7) either encapsulated in PLG biodegradable microparticles or free in solution. Using the single-radial- immunodiffusion test, the haemagglutinin integrity of the microencapsulated influenza was found to be preserved during the microencapsulation process. When administered systemically the microencapsulated virus induced raised levels of anti-influenza IgG antibody in serum that were comparable with those obtained with the virus in solution. Oral administration of the microencapsulated virus induced raised levels of anti-influenza IgA antibody in saliva as well as levels of anti-influenza IgG comparable to those obtained after parenteral injection. Raised levels (compared to preimmune levels) of anti-influenza antibodies in both the systemic circulation and mucosal secretions indicates that orally administered microencapsulalated equine influenza virus represents a practical method of immunisation against influenza.

The ultimate aim of the work described in this thesis was to (1) utilise PAMAM dendrimers as a tool to achieve differential transport across the intestinal mucosa and the blood brain barrier, where these dendrimers can be used to achieve oral bioavailability but avoid BBB penetration and CNS access and (2) to create cannabinoid-dendrimer conjugates that are active in their own right and whose penetration to the brain is prevented but whose intestinal activity is afforded for the treatment of IBD. Overall, the work described in this thesis has promoted a strategy whereby an active polymer (dendrimer)-drug conjugate could be formed that is active in its own right and where the polymer can serve to provide differential biological barrier transport which with regard to cannabinoid pharmacology obviates adverse CNS effects. The work in this thesis describes the design and synthesis of novel and active cannabinoid structures that should have commercial interest. These novel compounds served to further elucidate SAR in amino alkyl indole cannabinoids. SAR findings have revealed a site on these cannabinoids that can be functionally altered without loss of pharmacological activity. Additionally, studies in this thesis have led to the development of a novel radiolabelling strategy for anionic polymers that offers a number of distinct advantages over other approaches. Ultimately, a novel stable Dendrimer-cannabinoid conjugate has been synthesised but to date has not shown biological activity in the models utilised in this work.

An investigation into the pH stability of the BCNAs was also carried out and the parent analogue bearing a pentyphenyl side chain was found to be stable at a range of pHs. Finally, with the X-ray crystal structure of VZV thymidine kinase published we began a preliminary investigation into the possible interaction the BCNAs with this enzyme using molecular modelling

Muscle injuries are one of the most common sports related injuries. An audit published by The Football Association (FA) in 2004, suggested that 12% of all injuries were hamstring injuries, which are 2.5 times more common than quadriceps injuries (Woods et al., 2004). Recent figures published by Ekstrand et al suggested that, in a professional male football team of 25 players, about 5 hamstring injuries occur each season, equivalent to more than 80 lost football days (Ekstrand et al., 2011). In terms of professional elite athletes, shortened recovery time could mean continuing with training, increased game play and benefit to the team and club. Therefore, further research is needed to analyse the new techniques in treating muscle injuries. In 2008, an article in the British Journal of Sports Medicine titled "The early management of muscle strains in the elite athlete: Best practice in the world with a limited evidence", summarised that currently almost all our socalled knowledge has a basis of level 4 or level 5 (Orchard et al., 2008a). The panel of experts continued to highlight the importance of Dr. Mueller- Wohlfahrt's injection treatment regimen for treating muscle injuries. In brief, the treatment protocol involves multiple local injections and associated back injections with a mixture of a homeopathic and pharmacological cocktail (Wohlfahrt, 2008). Therefore, the biochemical property, pharmacodynamics and pharmacokinetics of each drug are altered and unpredictable. The only potential “active” substance in Dr. Mueller-Wohlfahrt’s cocktail could be a drug called “Actovegin” which is a licenced clinically used drug with a track record of over 60 years. Therefore in this PhD thesis, in order to avoid the unpredictable nature of poly-pharmacy as discussed above, only Actovegin will be investigated. In order to investigate the potential therapeutic effect or efficacy of Actovegin on muscle injury, basic muscle structures, histology and pathophysiology of the healing process were discussed. The biochemical 10 events following skeletal muscle injuries and repair are driven by cytokines, monocytes and leukocytes. The speed and quality of muscle healing are dependent on the inflammatory process. In order to alter the speed or quality of muscle repair, Actovegin must be able to modulate the inflammatory process. The in-vitro study in this PhD thesis was the first study to investigate the role of Actovegin in the inflammatory process and demonstrated significant results. It confirmed that Actovegin could modulate the inflammatory process by influencing the CD68+ and CD163+ macrophages and CD163+ THP-1 cells, which could influence the muscle healing process. Based on the findings from the in vitro studies and data from previous literature, a stand-alone single drug intramuscular Actovegin injection therapy regimen was developed to treat acute muscle injuries. The first clinical study using this stand-alone Actovegin treatment regimen was conducted in this PhD in professional footballer players and translated the in vitro findings to clinical practice, which confirmed that Actovegin could influence clinical outcome in treating acute muscle injuries. This thesis summarises the current evidence on Actovegin. Compared with conventional conservative RICE and NSAID therapy, Actovegin proposes an exciting and legal alternative for high performance athletes. From the studies, Actovegin injection therapy seems safe and well tolerated. Overall, this PhD has suggested that Actovegin has an active role in the treatment of muscle strain injuries biochemically and clinically.

P-glycoprotein (P-gp – MDR-1), a 170 kDa glycosylated membrane bound protein, is a member of the ATP-binding cassette transporter family. The potential for P-gp to reduce drug absorption across lung epithelia is of significant interest; this is particularly so, given P-gp’s broad substrate specificity mediating efflux transport of a range of structurally unrelated substrates. Within lung, Pgp expression is evident in bronchial and alveolar epithelia with functional characterisation of P-gp transport capacity within lung epithelial cells currently restricted to respective in-vitro cell culture models. The aims of this project were to establish the relative mRNA expression of several ATP Binding Cassette (ABC), Solute Carrier (SLC) and Solute Carrier Organic Anion (SLCO) drug transporters within rat lung samples through use of RT-PCR; expression suggesting the potential to serve as targets for pulmonary drug delivery. Further, validation of an Isolated Perfused Rat Lung preparation for use in assessment of drug transport across the lung was conducted. In order to assess the functional significance of the ABC drug transporter, P-glycoprotein, on drugs instilled intra-tracheally to the IPRL set-up, use of the P-gp substrates; Rhodamine 123 (Rh123), digoxin, and flunisolide and the P-gp inhibitor, GF120918 was employed. Further, use of kinetic modelling was employed to establish pharmacokinetic parameters involved. Using the IPRL, the P-gp dependent pulmonary absorption of the P-gp substrate, Rh123, was demonstrated. Dose-dependent absorption, consistent with a saturable component in the molecule’s pulmonary absorption, was demonstrated. Further, the absorption of low dose Rh123 was promoted by the presence of the highly selective P-gp inhibitor GF120918, consistent with a functional role of P-gp mediated efflux within an intact lung; an efflux process which may limit the pulmonary absorption of a lung administered molecule. Further studies using this system and extending the range of molecules studied will provide greater understanding of the quantitative significance of P-gp in limiting pulmonary absorption across lung epithelium.

Cannabinoids show potential as new treatments for inflammatory bowel disease (IBD),exerting several favourable effects in the gut, including anti-inflammatory and antimotilityeffects. The main difficulty with cannabinoids is their psychotropic side effects,but access to the brain may be prevented by conjugating the cannabinoid to a bulkygroup such as a dendrimer. The aims of this thesis were to investigate the mechanism by which cannabinoids reduce gut motility and to investigate whether cannabinoids protect the intestine from inflammatory-damage. A further aim was to determine whether cannabinoids remain pharmacologically active when conjugated to a dendrimer. All cannabinoids used (apart from arachidonoylcyclopropylamide and (-)WIN 55,212-2) caused a concentration-dependent reduction in the size of electrically-stimulated contractions in the guinea-pig ileum. The responses were not blocked by CB1, CB2, CBe(putative endothelial cannabinoid receptor) or GPR55 antagonists, suggesting that none of these receptors were involved in mediating cannabinoid responses. PSN 375963 reduced carbachol-induced contraction, suggesting that the GPR119 may be present on ileum smooth muscle. (+)WIN 55,212-2 was shown to protect the guinea-pig ileum from hydrogen peroxide-induced damage but this protection was not blocked by CB1, CB2, CBe or GPR55 antagonists, suggesting that the protective effects were not mediated through these receptors. Conjugation of JWH007 to a spacer (GA003) abolished activity in the guinea-pig ileum and the conjugation of JWH007 to a spacer and dendrimer (GA006) was found to be toxic in the macrophage assay. These studies show that cannabinoid-mediated inhibition of guinea-pig ileum contractions is not mediated through the CB1, CB2, CBe or GPR55 receptor. These receptors were not involved in the (+)WIN 55,212-2 mediated protection against hydrogen peroxide-induced damage in the ileum. The approach of attaching a dendrimer to JWH007 to prevent central nervous system (CNS) penetration does not appear to be a feasible approach because the cannabinoid-dendrimer was unexpectedly cytotoxic.