This thesis describes the results from a series of studies designed to evaluate the gastrointestinal transit of oral dosage forms. The transit of placebo pellet and tablet formulations was monitored using the technique of gamma scintigraphy. The formulations were radiolabelled with either technetium-99m or indium-lil. Four parameters, two physiological and two pharmaceutical, were selected for investigation. All the studies were conducted in healthy male volunteers. The first study examined the influence of the supine position on the gastric emptying of pellets in fasted and fed subjects. There was no marked difference between the supine and control gastric emptying data. As would be expected, food had a significant effect on gastric emptying. The influence of the time of day of administration on the gastrointestinal transit of pellets was investigated in fasted subjects. Transit of the pellets was not affected by their time of administration. The effect of the putative bioadhesive, polycarbophil, on the gastrointestinal transit of a pellet formulation was studied in fasted subjects. The pellets emptied from the stomach, rapidly and in an exponential manner. A set of studies was conducted to evaluate the transit of tablets in fed and fasted subjects. Tablet size did not affect gastric emptying, although there was an increase in the variability of gastric emptying with increasing tablet size. Food had a marked effect on gastric emptying. The rate of emptying was related to the energy content of the meal. Tablet size did not appear to be a determinant of transit through the ileocaecal sphincter. The colon transit and dispersion of the tablets was examined. Neither the ingestion of food nor defecation appeared to alter the rate of transit through the colon.

Hard gelatin capsules are manufactured from blends of limed ossein (LOG), acid ossein and acid pigskin gelatins, with LOGS usually forming the main portion of the blend. Unfortunately, the quality of the finished capsule cannot be predicted by routine quality control tests and 'poor' batches of LOG are encountered which form unsatisfactory capsules. In addition, differences between gelatin types are encountered in practice but the supporting literature is sparse and conflicting. This work examines some properties of gelatins pertinent to hard capsule manufacture in an attempt to relate these to gelatin type and known performance on capsule machines. Molecular mass distributions (MMD) were examined by polyacrylamide gel electrophoresis. Each different gelatin type possessed a characteristic shape of MMD which could be related to the source tissue and the treatment undergone during gelatin manufacture. MMDs, isoionic point determinations and an assessment of the content of protein impurities by ultraviolet spectrophotometry failed to resolve differences between 'good' and 'poor' LOG batches. The drying rates of freshly-cast gelatin films were studied under conditions of controlled humidity, temperature and air velocity. No significant differences between gelatin types were observed. Small variations in equilibrium moisture content were seen and were tentatively ascribed to the generation of water binding sites (free carboxyl groups) during gelatin manufacture. The mechanical properties of dry gelatin films were examined by tensile stress-strain and stress-relaxation measurements. Viscoelastic behaviour typical of a polymer in the glass transition region was observed, and significant differences in film fracture strain were observed but these were not related to gelatin type. Optical rotation measurements indicated similar orders of film crystallinity and determinations of frictional characteristics similarly revealed no varietal differences. Overall, few differences related to gelatin type were seen within the properties examined. There was no evidence in these studies to explain the unsatisfactory behaviour of 'poor' LOG batches in capsule manufacture. NB. This ethesis has been created by scanning the typescript original and may contain inaccuracies. In case of difficulty, please refer to the original text.

The aim of this project is to compare pharmaceutical particles made using a Nektar supercritical fluid technology technique called solution enhanced dispersion by supercritical fluids (SEDSTM) to those made using more traditional techniques. This involves a comparison of not only the surface properties of both types of particles, but also the interparticulate interactions. The majority of the work has involved the use of the atomic force microscope (AFM) as both a tool for imaging and for the acquisition of localized force measurements. The first experimental chapter of this work describes a method developed in order to image the contacting asperities of a particle. The AFM has the potential to provide useful information regarding single particle interactions to complement data generated from bulk techniques. In this chapter, the AFM artefact of tip imaging was used to produce 3D images of the asperities of particles of micronised and SEDSTM salbutamol sulphate, an anti-asthma drug, contacting a model surface of highly orientated pyrolytic graphite (HOPG). These data were recorded in a model propellant environment, used in order to simulate the environment that would be found in pressurised metered dose inhalers, such as those used by asthmatics. From the images generated the contacting area was estimated to be 1.1x10-3 mm2 for the micronised material, and 1.4x10-3 for the SEDSTM material. The work of adhesion for both of the materials was also calculated, and the values of 19.0 mJm-2 and 4.0 mJm-2 were obtained for the micronised and SEDSTM samples respectively. This supported available data that indicated the SEDS material had a lower surface energy than the micronised drug, and that it is possible to make comparisons between different modified AFM probes. The second chapter develops this work so that it can be applied to an air environment, which is applicable to more pharmaceutical systems. Here, force measurements were again performed using AFM, with the same drug samples studied in the first chapter, except a controlled relative humidity (RH) environment was used, so that the variation in adhesion with increasing RH could be studied. Two types of measurement were undertaken. The first involved the use of blank AFM tips on compressed disks of drug material, and the second involved the use of drug particles mounted onto AFM tips on both HOPG and compressed disks of drug. With the blank AFM tip and particle modified AFM tip on HOPG work it was observed that the SEDSTM materials showed a peak in adhesion force at 22% RH while the micronised salbutamol showed a peak at 44% RH. From this, a three-scenario model of linking morphology of contact to adhesion was developed to explain the observed peaks in adhesion. In addition, the surface energies of each of the two samples were calculated using the force measurements acquired against HOPG and compressed disks of material and compared. The micronised material was found to have a higher surface energy than the SEDSTM material (10.8 mJm-2 cf 5 mJm-2) when data acquired against HOPG was used. However, when data acquired using the compressed disks of drug were used, the SEDSTM had a higher surface energy than the micronised (29.9 mJm-2 cf 22.6 mJm-2). This higher value was attributed to different surface roughness effects found with the compressed disks. The third chapter uses the techniques and models developed in the previous chapters to examine the effect of polymorphism on surface energy, structure and particulate interactions. Three polymorphs of the drug sulphathiazole (forms I, II and IV) were formed using the SEDSTM technique, one of which (form I) was formed using two different solvents: methanol and acetone. Force measurements were performed using the AFM at controlled humidity using particles of each of the polymorphs mounted onto AFM tips against substrates of HOPG and the polymorph under analysis. This data was then related to the model developed in the previous chapter, and calculations were undertaken to assess the different surface energies of each of the four samples. For some of the samples it was observed that peaks were again occurring in the data, at 22% RH for polymorphs I-methanol and III, and 44% for polymorph IV. No peak was seen for polymorph I-acetone. These peaks were then related to the surface energy calculated for each of the polymorphs, as polymorphs I-methanol and III were found to have lower surface energy (0.99 mJm-2 and 1.17 mJm-2 respectively) than polymorphs IV and I-acetone (20.33 mJm-2 and 309 mJm-2). The fourth chapter examines the application of AFM to an industrial problem. When using the SEDSTM process to manufacture insulin, it was observed that the SEDSTM material had poorer flow properties than that of the unprocessed material. Using the AFM as both an imaging and force measurement tool, this chapter explores the application of imaging and the adhesion models and surface energy calculations previously developed to understand this problem. The AFM images showed the presence of highly aggregated particles of SEDSTM insulin, compared to the unprocessed insulin that appeared to be more crystalline. When force measurements were performed against both HOPG and particles of the material under analysis, non of the unprocessed, and only one of the SEDSTM particle tips prepared displayed the peak behaviour seen with previous measurements, and instead displayed a continual increase in adhesion force with humidity. In addition, when the surface energy was calculated, the SEDSTM material was found to be higher than the unprocessed insulin (77.5 mJm-2 cf 2.4 mJm-2). The increase in adhesion force was related to the particles agglomerating together, due to the presence of a higher surface energy and high amorphous content of the particles. The final experimental chapter uses techniques that compliment AFM analysis to examine another industrial problem. The SEDSTM process can be used to co-formulate drugs with other materials such as polymers. In this chapter, the drug pregabalin has been co-formulated with lipid in order to produce a coating around the drug to mask taste. The use of AFM as an imaging tool, and the additional techniques of X-ray photoelectron spectrometry (XPS) and time-of-flight secondary ion mass spectroscopy (ToF-SIMS) have been used to generate an understanding of surface structure and chemistry of this heterogeneous system. The AFM images showed no areas of surface heterogeneous behaviour, although the largest scan size was only 5 mm x 5 mm. However both the XPS and ToF-SIMS spectra, which samples far larger areas (up to 75 mm x 75 mm) showed the presence of lipid and drug molecules. It was concluded that the lipid was not forming a uniform layer around the drug molecule, but was instead forming large patches that were beyond the resolution of the AFM. This work aims therefore to provide a fundamental study of the application of AFM to real pharmaceutical systems. In particular models are developed which allow not only ranking of particle interactions but the quantification of factors such as surface energy and work of adhesion. Finally the significance of the morphology of the inter-particulate contact has been explored at the nanoscale.

It has been widely reported that cell culture dimension and microenvironment influence cell proliferation, differentiation, and gene expression, which lead to different interactions between drug delivery systems and cells. The development in evaluation of drug delivery systems has reached the stage where investigations are now concentrating on intracellular uptake and subcellular localization of drug delivery systems.This thesis investigates the use of three-dimensional (3-D) tissue culture models to study how nanoparticles (NPs) may behave in vivo. Poly (glycerol-adipate) (PGA) NPs can degrade into glycerol and adipate, which are not having toxic and anyundesirable local or systemic effects in the host. Following on the initial physicochemical characterization of PGA NPs loaded with drug and fluorescent dyes, investigations moved on to the biological studies of NPs in various cell culture model, e.g. monolayer culture, 3-D culture models, and brain tumour invasion model. Particle size, surface charge, and hydrophobicity are important features affecting the amount of particles taken up by cells and intracellular localisation of particles. Thus, the physicochemical properties of drug and fluorescent dye loaded PGA NPs were assessed by Photon Correlation Spectroscopy, Laser Doppler Anemometry, and drug/fluorescent dye loading studies. These studies indicated that physicochemical properties of drug, fluorescent dyes and PGA polymer could influence drug /fluorescent dye loading, which results in different particle size and surface charge of PGA NPs. Quantitative and qualitative investigations into the influence of cell culture dimension on uptake of NPs by cells, both by confocal fluorescence microscopy and flow cytometry, revealed that DAOY cells took up NPs more effectively when in 3-D spherical aggregate culture than in 2-D monolayer culture while uptake of NPs by normal brain cells was lower in 3-D cell culture than that seen in 2-D monolayer culture. This resulted in intracellular fluorescence intensity about 6 times higher in DAOY aggregates than normal brain cell aggregates while in monolayer culture mixed brain cells took up 2 times as many NP as the DAOY cells. The results from studies of NPs migrating through aggregates and tissue slices also indicated that penetration ofNPs in 3-D culture models was affected by the structure of the interstitial compartment and composition of extracellular matrix. Microscopic investigation of the histology of a co-culture invasion model of DAOY aggregates and a organotypic brain slice confirmed that DAOY cells massively invaded into cerebellum slices after a 4-day co-culture while the invasion of DAOY cells were limited within cerebral cortex slices even after a 6-day co-culture. Selective uptake of NPs by host cells and brain tumour cells were also assessed in this 3-D brain tumour invasion model. It showed that most NPs were taken up by DAOY cells instead of brain cells.

Aims of study The overall aim of this study was to investigate factors affecting service delivery within a national pharmacy chain, from the perspective of pharmacists and consumers, using asthma services as an example. Data were collected to explore the current environment and opportunities available to pharmacy, the factors affecting service delivery, and to identify recommendations for future service models. The impact of the design and route of service implementation were studied through two different types of asthma services. Methods The brief intervention in asthma was designed centrally and implemented nationally, whilst the asthma service was designed and implemented locally by a group of pharmacists. A triangulation of qualitative and quantitative methods were used throughout this study, including an omnibus survey, audits, mystery customer research, customer and pharmacist interviews, and a review of the dispensing data. Results A total of 81 facilitators, 45 barriers and 23 motivators were identified. In addition to extending those factors that had been previously recognised within the literature, new factors were also identified. Firstly, the route and design of service implementation to promote local ownership and responsibility for delivery of services was found to be a key factor, as was having flexibility in the length and content of service delivery. Clear and visible benefits to the pharmacists delivering the service, the customers accessing the service, and the pharmacy organisation were also found to play an important role in the delivery of services. Conclusions This is the first large scale study of its kind to look at all the factors involved from the perspective of both customers and pharmacists, and many of the facilitators and barriers identified extend beyond those provided within the current literature. The motivators identified within the previous studies have been from the perspective of pharmacists only. This study has looked at the perspective of not only pharmacists, but also the motivators to customers and the service provider. Based on all the factors identified throughout this study, a number of recommendations have been made for future service delivery.

Pullulan has been chemically modified by the incorporation of various hydrophobic molecules, to produce two types of derivatives (hydrophobically modified carboxymethyl pullulans (HMCMPs), and crosslinked carboxymethyl pullulan). The foregoing modifications were performed in two steps from the parent pullulan. The first step involved carboxylation of pullulan with sodium chloroacetate yielding carboxymethyl pullulan (CMP). The second step involved the medication of CMP by coupling different amine functionalized molecules onto the carboxylic groups of CMP, using the coupling agent dicyclohexylcarbodiimide. The amines used in this modification step were: hexadecylamine, decylamine, cadaverine (1-5 diaminopentane), and three jeffamine® (polyoxypropylenediamine) compounds of differing molecular weights (230, 400 and 2000). Results concluded that all six pullulan derivatives show an increase in reduced viscosity to varying degrees, compared to the parent pullulan. Characterization of pullulan and the derivatives concluded that the correct structures have been synthesised. Gel permeation chromatography confirmed that four pullulan derivatives had been crosslinked (due to doubled molecular weight terms), and a further two have increased in molecular weights, with no increase in polydispersity indices. Isothermal titration calorimetry experiments were initially performed on model systems (two different ß-cyclodextrins, and benzoic acid and eletriptan hydrobromide), and then on pullulan and the derivatives with eletriptan hydrobromide. These experiments probed the nature and extent of drug binding interactions. Results concluded that pullulan derivatives showed exothermic drug binding interactions with the named drug, with the exception of jeffamine 2000 crosslinked CMP, which exhibited endothermic interactions with the titrated drug. Pullulans excellent film forming capabilities may lead these polymers to a novel oral dosage form containing active (dissolving films).

Latrophilin1 (LPHN1) is a presynaptic adhesion G protein-coupled receptor involved in the control of spontaneous exocytosis of neurotransmitters. The effects of LPHN1 activation on exocytosis have been described on several model systems, such as cultured hippocampal neurons and neuromuscular junction (NMJ) using its well known agonist LTXN4C, and include a massive increase in exocytosis characterised by periods of intense release (bursts) interspersed with periods of moderate activity (interburst intervals). However, the molecular mechanisms underlying these effects were yet to be determined. Based on previous observations that LPHN1 is associated to G proteins, and that its activation leads to activation of PLC and increased IP3, we hypothesize that LPHN1 controls exocytosis via the Gαq protein pathway, whose activation ultimately results in the release of Ca2+ from IP3-sensitive Ca2+ stores. Using a pharmacological approach and the current clamp method at the mouse NMJ, we first used LPHN1 KO preparations to study the role of LPHN1 on spontaneous exocytosis in resting conditions, and to show that LPHN1 is the only receptor mediating the effects observed upon stimulation by LTXN4C. Then, we interfered with several molecules involved in the Gαq pathway to test their involvement in LPHN1 activation, and we investigated the role of store-operated (SOCCs) and voltage-gated (VGCCs) Ca2+ channels in mediating the Ca2+ influx that is necessary for the development of LPHN1 effects. Our results support the hypothesis LPHN1 is involved in the regulation of spontaneous exocytosis at rest and that it is the receptor mediating the increased exocytosis following stimulation by LTXN4C; the suggestions that Gαq and its intracellular pathway mediate the effects of LPHN1 activation on spontaneous exocytosis, and that SOCCs and VGCCs (particularly Cav2.1) mediate the Ca2+ influx necessary for the development of LPHN1 effects are also supported by our findings. Altogether, this work uncovered the mechanisms by which G protein-coupled receptors, in this case LPHN1, can regulate the rate of spontaneous neurotransmitter release at the mouse NMJ.

An estimated 2% of the human genome encodes for proteolytic enzymes. It is becoming increasingly apparent that serine proteases have diverse and critical roles in many physiological and pathophysiological processes. Studies investigating the role of serine proteases in the kidney have focussed primarily on their pro-inflammatory effects and their ability to cleave and activate the epithelial sodium channel (ENaC). P2X receptors, which are structurally very similar to ENaC and have been identified throughout the nephron and in the renal vasculature, are thought to contribute to the regulation of tubular transport mechanism and renal haemodynamics, as well as be involved in several renal pathologies. It is shown here that the serine protease, trypsin, has a significant inhibitory effect on recombinant human P2X3 and P2X7 receptor activity. Moreover, it is shown that trypsin may also have an inhibitory effect on purinergic signalling in the mouse cortical collecting duct. In addition, the single channel activity of P2X receptors expressed on the apical membrane of renal collecting duct epithelial cells is described for the first time. Finally, a novel use of the live-tissue slice method is described and the first direct evidence showing trypsin causes significant morphological changes in renal tubules and medullary microvessels in situ is provided. Collectively, data presented here provides evidence to suggest that serine proteases may contribute to several aspects of renal function that have not previously been explored.

Influenza viruses represent an important public health burden since they cause annual epidemics associated with severe illness and mortality in high-risk populations. Additionally, zoonotic influenza virus infections have potential to produce intermittent pandemics, which have led to millions of deaths globally. However, strategies to prevent influenza severity and spread can be implemented. It is known that antibodies against the haemagglutinin play a key role in protection from influenza virus infection, thus both seasonal and pandemic influenza vaccines aim to elicit such antibodies. Generally, they are directed against haemagglutinin globular head epitopes and are able to neutralize closely related influenza strains, but recently antibodies able to neutralize multiple influenza strains and subtypes have also been described. The discovery of these antibodies, primarily directed against the haemagglutinin stalk, has generated interest in understanding how they are generated and how widespread they are in the human population. Furthermore, eliciting such antibodies has become the aim of many ‘universal’ vaccine approaches. However, the study of these cross-reactive antibodies using classical serological assays is problematic since the current assays have been shown to be relatively insensitive in detecting them. The main objective of this thesis was to study the presence and breadth of cross-reactive neutralizing responses in human populations. To overcome the limitations of current serological tests in detecting these responses, lentiviral pseudotype particles bearing the haemagglutinins of different influenza A subtypes and influenza B strains were used as surrogate antigens in neutralization assays. After the generation of these novel tools and the establishment of appropriate controls, pseudotype particle neutralization assays were employed to investigate cross-reactive antibody responses in pre- and post-vaccination sera. Next, the use of chimeric haemagglutinins, in which the globular head was substituted with the head of a different subtype, was incorporated into the pseudotype system. This allowed the differentiation between haemagglutinin head-directed and stalk-directed antibody responses. The ability to efficiently detect broadly neutralizing antibody responses, including those directed against the haemagglutinin stalk, shows that pseudotype particles are tools that should be further characterised and implemented to be used in sero-epidemiological studies and for ‘universal’ vaccine immunogenicity studies.

Xanthine oxidoreductase (XOR) is one of the main purine catabolising enzymes which converts hypoxanthine into xanthine and further into uric acid. The enzyme has a homodimeric structure and contains two FeS centres, one FAD molecule and one molybdenum atom per monomer. Recent evidence clearly demonstrated that XOR activity is highly increased in human hematopoietic cells of myeloid lineage during their pathogen-induced and endogenously generated biological responses. The integrative signalling role and especially involvement of XOR in cross-talk of metabolic and signalling machinery of human leukocytes remains poorly understood. We have demonstrated that XOD is activated in human myeloid cells in response to pro-inflammatory and growth factor stimulation. Hypoxia-inducible factor 1 (HIF-1) and activator protein 1 (AP1) transcription complexes were found responsible for maintaining XOR catalytic activity and protein levels. Importantly, the mammalian target of rapamycin (mTOR), a major myeloid cell translation regulator, appeared to be essential for XOR activation. Specific inhibition of XOR led to an increase in intracellular AMP levels triggering downregulation of mTOR activation. Taken together, these results show that XOD is not only activated by pro-inflammatory stimuli or SCF (growth factors), but also plays a crucial role in maintaining mTOR-dependent translational control during the biological responses of hematopoietic cells of myeloid lineage. Findings reported in this thesis open a new field in human myeloid cell research and translational medicine. XOR is an easily accessible therapeutic target, which could be pharmacologically corrected using non-toxic drugs.