Dissertations / Theses on the topic 'Materia medica and therapeutics'

For the past two decades, the development of protein therapeutics has significantly expanded with numerous biopharmaceutical and biosimilar products entering the medicine market every year, and even more queuing in the pipeline globally. Biologics are very complex molecules and therefore extremely sensitive to minor changes in the manufacturing process, which can result in heterogeneity and affect the stability, potency and immunogenicity of the final product. Public health organisations, such as EMA (European Medicines Agency), require that biological products should be extensively tested for their similarity to the original drug (in the case of a biosimilar) as well as to products from different batches (batch-to-batch comparison). The issued guidelines focus, among other tests, on physicochemical characterisation of these molecules. The suggested analytical techniques, however, are only vaguely named in the specifications, leaving the final decision to the manufacturers. The present work focuses on the use of different combinations of analytical techniques with an aim to demonstrate similarity or dissimilarity between two or more samples. The selected instrumental techniques are characterised by their simplicity and are able to detect structural differences and microheterogeneity of the active ingredient in different samples, aggregation, degradation and post-translational modifications (PTMs). Seven studies were completed in total, each one to a different extent, and these included protein therapeutics such as insulin and monoclonal antibodies. The applied techniques served for primary (MS),* secondary (far-UV CD, FT-IR) and tertiary structure (near-UV CD, fluorescence) comparison of the examined samples. Particle size comparability and detection of aggregation was achieved with DLS, and higher-order structure comparison with 1D 1 H-NMR. Coupling of the techniques with temperature-dependent measurements enabled further comparison on the thermal stability of the samples and provided confidence in the observed (at room temperature) results. The acquired empirical experience pointed out the advantages and disadvantages of each technique compared to the rest of the techniques, possible solutions to the encountered challenges, and the cases that one technique can be used instead of another or as complementary to it. Finally, a potential SOP is suggested, advising on which biophysical techniques should be used depending on the structure of the protein that is examined and its formulation.

Protein kinases are major drug targets for many diseases. Among these are the STE20/SPS1-related proline/alanine-rich kinase (SPAK) and the oxidative- stress-responsive kinase 1 (OSR1), which are two related serine/threonine protein kinases. Both kinases are key components of the WNK-SPAK/OSR1 signalling pathway that has emerged as a key regulator of electrolyte homeostasis, body fluid and blood pressure. Various knock-in and knock-out SPAK and OSR1 mouse models exhibited reduced blood pressure. This highlighted SPAK and OSR1 kinases as promising targets in the treatment of hypertension. Encouraged by this, this project was initiated to discover specific WNK-signalling inhibitors by targeting SPAK and OSR1 kinases as potential novel antihypertensive agents. My work led to the identification of an allosteric pocket located in the highly conserved C-terminal domains of SPAK and OSR1, which influences their kinase activities. Using in silico screening, Rafoxanide, an anti-parasitic agent, was identified as a novel allosteric inhibitor of SPAK and OSR1. Additionally, high throughput screening led to the discovery of the clinically used agent, Verteporfin, as a novel and potent WNK-signalling inhibitor. Moreover, several fragment-binders to the C-terminal domain of OSR1 kinase were identified using NMRfragment based screening. In addition, the NMR backbone assignments of the C-terminal domain of OSR1 kinase were determined and used to map the previously unknown binding site of different OSR1 and SPAK inhibitors. Collectively, these findings have significantly advanced the field of SPAK and OSR1 kinase inhibition and provided key tools that will facilitate the future discovery of other SPAK and OSR1 kinase inhibitors.

The use of proteins as therapeutics is one of the fastest growing sectors of the pharmaceutical industry, particularly monoclonal antibodies. However, a significant challenge in the development of such protein-based medicines is to counter aggregation of the proteins in solution (as these drugs are typically administered by injection). In solution form, aggregation of the normally monomeric protein ingredient affects therapeutic efficiency and reduces shelf life. Moreover, the rapid formation of aggregates in patients during the administration of therapeutic proteins can lead to immunological reactions which could be fatal. Hence, the long term storage of proteins in solution is discouraged. Lyophilization (vacuum drying) is considered to be an effective route for ensuring longer shelf life and better stability of protein therapeutics. However, aggregation can still occur, because the driving forces for aggregation (covalent as well as non-covalent interactions such as hydrogen bonds, van der Waals forces and hydrophobic interactions) are influenced by lyophilization induced changes in the pH, temperature, exposure to interfaces, and dehydration stress. Lyoprotectants such as sugars can counter the undesirable consequences of lyophilization depending upon their nature and potential. Several mechanisms have been proposed for the role of the lyoprotectants. The comprehensive investigation into the inherent nature and influence of lyoprotectants on a protein therapeutic during lyophilization is hence important. In this project an attempt has been made to develop a novel nanoscale screening methodology for the detection, quantification, characterization and prevention of protein aggregation. Initially, ferritin and then a polyclonal IgG (antiglucose-6-phospate dehydrogenase antibody) antibody have been used as model proteins for these studies. The effect of lyophilization on the level of aggregation of IgG was studied and compared to reports in the literature. IgG was exposed to seven cycles of lyophilization, where each cycle of lyophilization was followed by reconstitution and characterization. IgG was also lyophilized with different excipients (sucrose and mannitol, alone and in combination) in different molar ratios. In the liquid state, the formulations were characterized on the basis of particle size and antigen binding activity, whereas in the dry powdered form, the formulations were characterized by studying morphology, thermal stability, and secondary structural alterations in order to establish a relationship amongst the indicated properties. Atomic force microscopy (AFM), dynamic light scattering (DLS) and single particle tracking (Nanosight) were used to study particle size. The identification of different components at the nanoscale and general morphology were screened by AFM and scanning electron microscopy (SEM). Subsequently, the thermal properties and structural alterations respectively were analysed by differential scanning calorimetry (DSC) and infra read spectroscopy (ATR-FTIR) spectroscopy. The antigen binding activity was investigated by performing an indirect ELISA assay on the lyophilized formulations. Lyophilization of ferritin and IgG caused a significant decrease in the proportion of monomeric species was confirmed by AFM, DLS and Nanosight. Dimeric, lower-multimeric and larger aggregates existed in variable proportions for both ferritin and IgG. Powdered lyophilized ferritin formulations showed aggregation, increased crystallinity (concomitant decrease in amorphicity), porosity and flakiness which in case of IgG increased with repeated lyophilization. A consistent increase in the extent of aggregation (unfolding of Fabs and Fc) was detected by DSC and an increase in the beta-sheet structure coupled with structural re-arrangement within the components by ATR-FfIR. The presence of sucrose in IgG formulations resulted in reduced aggregation and enhanced porosity. The inclusion of Mannitol promoted crystallinity, decreased porosity when used alone, however, improved the efficiency of sucrose in combined formulations. The nature of crystals formed by mannitol during lyophilization was shown by SEM and confirmed by AFM. The data obtained from DLS, NTA, AFM, DSC,ATR, and SEM was consistent with by ELISA results which indicated a significant fall in IgG activity upon repeated lyophilization, and improvement in the activity when IgG was formulated with sucrose, which significantly enhanced in combination with mannitol. The benchmark provided by this work would serve as a precursor for developing a novel screening standard for optimizing and improving the therapeutic efficiency of other proteins besides furnishing a detailed account of the disparity in correlating the data from multiple novel techniques. The findings of our work can be directly translated to biotech and biopharm industries for the enhancement of protein based therapeutics.

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.

Homoeoprophylaxis (HP) is the use of homoeopathically prepared substances to prevent targeted infectious diseases in recipients. Its first use in an epidemic of Scarlet Fever was documented in 1801. It has been used throughout the world since then for both short-term and long-term preventative purposes. The effectiveness and safety of Golden�s long-term HP program using homoeopathically prepared substances to prevent targeted infectious diseases in recipients was tested through two research projects. The effectiveness of the program could not be established with statistical certainty given the limited sample size and the low probability of acquiring an infectious disease. However, a possible level of effectiveness of 90.3% was identified subject to specified limitations. Further research to confirm the effectiveness of the program is justified. Statistically significant results were obtained that confirmed the safety of the program both in absolute terms as well as compared to all other methods of disease prevention studied. It also appeared possible that a national immunisation system where both vaccination and HP were available to parents would increase the national coverage against targeted infectious diseases, and reduce the incidence of some chronic health conditions, especially asthma.

An analysis of over 34,000 free-text messages assigned by pharmacists to prescription orders over a 12-month period showed a sub-optimal exchange of information with the physician. Focus groups and observational research were conducted to provide a more in-depth understanding of the factors involved. The use of CPOE did not reduce opportunities for personal interaction. The capability to communicate electronically facilitated a non-interruptive workflow, beneficial for staff time and for limiting distractions. It also improved clinical documentation, which helped coordinate care of patients between members of the pharmacy team. However, the research identified several barriers to the effectiveness of communication via the CPOE system, including: the increased frequency of messages sent; poor display characteristics of the message; poor access to information to inform decision-making; one-way communication; and no assigned responsibility to respond. These factors need to be considered in the design of systems and supported by interprofessional training to optimise communication between the professionals.

Clays and clay minerals (phyllosilicates) have been used for millennia to treat a range of human maladies, such as infected wounds and diseases of the skin. The unique chemistry of phyllosilicates allows them to support the wound environment and encourage healing. Their physicochemical properties can also be utilised to develop modified drug-release formulations and also enables their incorporation into polymer matrices for the development of advanced wound care materials. By developing novel antibacterial phyllosilicate-polymer composite materials it should be possible to support wound healing, whilst simultaneously treating infections locally to avoid systemic adverse effects and prevent the development of antimicrobial resistance. In this research project the clay minerals kaolin (KN), refined montmorillonite (rMMT), montmorilonite K10 (MMTK10), Laponite® RD (LRD), and Laponite® XL21 (LXL21) were investigated for their differing structure and physicochemical properties. Their ability to adsorb and desorb the antibacterial agents tetracycline (TC), doxycycline (DC) and ciprofloxacin (CIP) was determined through a series of adsorption kinetics and isotherm studies. LRD and LXL21 were shown to have the highest drug-carrying capacity and were also able to relinquish this drug-load to inhibit the proliferation of key wound pathogens; Staphylococcus epidermidis, Propionibacterium acnes, and Pseudomonas aeruginosa. XRD and FTIR analyses demonstrated that these drug molecules could be adsorbed into the interlayer space and edge groups of the Laponite® particles. LXL21-CIP composites were successfully incorporated into alginate polymer matrices through interaction of the exposed edge-groups on LXL21 and the hydroxyl groups of the alginate to produce novel nanocomposite film and foam materials. Selection of candidate materials was initially undertaken qualitatively with the support of a tissue viability nurse at the Royal Liverpool and Broadgreen University Hospitals NHS Trust. Important properties for wound-dressings such as adsorptive capacity, water vapour transmission rate, and keratinocyte compatibility were measured quantitatively and compared to materials already used for wound care in the UK. Both the film and foam materials were shown to have properties that would be beneficial for wound healing and were also able to release CIP in a controlled manner with notable activity against S. epidermidis, P. acnes, and P. aeruginosa. The nanocomposite film formulation developed in this research project showed promise for future clinical applications and future work should be undertaken to further optimise their manufacture and fully characterise their ability to support the healing of infected wounds. Although the nanocomposite foams require further research, the work presented in this thesis suggests they could also be promising materials for wound care applications.

Intravenous lipid emulsions are used ubiquitously through the medical field as a source of parenteral nutrition. Development of new formulations requires an understanding of the metabolism of the product. Current methods of rate determination and of metabolism analysis have several drawbacks. Radioactive labelled assays have lower biological relevance, are time consuming due to separation steps and require long substrate preparation. Existing fluorescence assays based around triglyceride hydrolysis are impractical in emulsion systems due to high signal-noise ratio as well as the use of non-specific fluorescent dyes. Colorimetric methods such as the non-esterified fatty acids (NEFA) assay is expensive, requires multiple steps and specialised machinery. Due to the limitations of these techniques we developed a novel fluorescent assay using a lipoprotein lipase specific substrate incorporated into lipid emulsions. The lipoprotein lipase substrate, EnzChek® Lipase Substrate, green fluorescent, 505/515, is based on a triglyceride structure with a fluorescent dye at the Sn1 position and a dark quencher at the Sn2 position. LPL cleaves preferentially at the Sn1 position of triglycerides, which separates the dye from the quencher creating a fluorescent signal. The signal can then be detected using a fluorescent plate reader. Both lipid emulsion particles and EnzChek are substrates for LPL, so the hydrolysis of EnzChek is analogous for native emulsions particles. Over time, in the presence of LPL, fluorescent signal increases as more EnzChek is hydrolysed in tandem with emulsion particles. We have designed a range of emulsions with varied oil and surfactant composition. Using the EnzChek emulsion assay detailed above we are able to follow the rate of metabolism in real-time. This assay has been tested and found to be robust and reproducible. It is able to investigate differences in metabolism between Soybean oil, medium-chain triglyceride and fish oil emulsions. As well as changes in surfactant type and concentration.

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.

Mucosal delivery of biotherapeutics as a non-invasive means of delivery could potentially be enhanced using nanoscale therapeutic carriers. However, nanoparticles do not readily cross the mucosal barriers, with the epithelium severely restricting their translocation into the systemic circulation. Translocation of nanocarriers across the mucosae may be improved by employing ligands capable of exploiting receptor-mediated cell uptake processes. This work explores the potential of vitamin B12 transport pathway for mucosal delivery of B12-decorated model nanoparticles and investigates the cell trafficking pathways involved in these processes. Cyanocobalamin (vitamin B12) was chemically modified to produce the α-ω-aminohexylcarbamate B12 derivative – as a suitable bioconjugate – which was then conjugated to fluorescent, carboxy-functional nanoparticles (<200 nm). These systems were applied to intestinal Caco-2 monolayers, expressing the relevant proteins involved in B12 trafficking and endocytic processes. Vitamin B12-conjugated nanoparticles demonstrated notably increased cell uptake and transport capacities in Caco-2 monolayers, compared to their unconjugated counterparts. Importantly, the cell uptake of B12-conjugated nanoparticles occurred via a pathway that was different to that used by both soluble B12 and unmodified nanoparticles. B12-conjugated nanoparticles circumnavigated the lysosomal compartment and were transported by a route perturbed by caveolae-specific inhibitors, unlike the clathrin-mediated trafficking of soluble vitamin B12. These previously unreported observations are important and have potential implications in the field of bioconjugate and nanocarrier-mediated drug delivery. Epithelial cell uptake and transport of B12-conjugated nanoparticles was also investigated in airway-derived Calu-3 cells, shown to express the B12-intrinsic factor receptor, cubilin. B12-nanoparticles showed markedly larger cell uptake and transport capacities in Calu-3 layers, with B12-conjugation dramatically influencing the intracellular trafficking of the particles in a similar way to Caco-2 cells. The B12 endocytotic machinery therefore shows potential for delivery of nanocarrier-associated therapeutics across the airways. Present work also aimed to establish methods for the production of stable nano-sized protein crystals displaying a slow drug release profile, based on evidence that protein therapeutics which are formulated in this manner, offer beneficial drug-delivery properties and can be targeted using biological ligands. Nano- and micron-sized insulin crystals were prepared by an adaptation of the batch crystallisation approach. The crystals were stabilised using a chemical crosslinker, namely β-[Tris(hydroxymethyl) phosphino] propionic acid (THPP). The resulting insulin crystals were generally stable in the absence of crystalisation buffer, displayed a slow-release profile, with the released insulin retaining its biological activity. This study therefore shows that formulating protein bioactives in this form is possible and may provide a promising strategy to develop biotherapeutics with improved drug delivery properties.

Guanylyl cyclase - cyclic guanosine monophosphate (cGMP) signalling has been demonstrated to play an important role in the endogenous cardioprotective signalling of the myocardium during early reperfusion. It is proposed that infarct limitation is afforded by elevating cGMP and activating protein kinase G and its distal targets. It was hypothesised that increasing the activity of soluble guanylyl cyclase (sGC) would limit myocardial ischaemia-reperfusion injury. Primarily using the rat isolated perfused heart method, the experiments reported in this thesis investigate the role of exogenous targeting of sGC during early reperfusion, specifically exploring targeting different redox states of the enzyme and their effects on myocardial infarct size. The novel sGC stimulator BAY 41-2272 and activator BAY 60-2770 were selected to investigate this hypothesis. Both administration of BAY 41-2272 and BAY 60-2770 during early reperfusion significantly limited infarct size compared to controls. This was associated with elevated total tissue cGMP levels. Inhibition of nitric oxide could not completely abrogate this protection, but exogenous perfusion of nitric oxide along with BAY 41-2272 showed synergistic action. Oxidation of the prosthetic haem group by ODQ abrogated the protection afforded by BAY 41-2272 but potentiated the protection afforded by BAY 60-2770. Targeting both the reduced and oxidised forms of sGC together did not afford additive protection, in fact it reduced the protection afforded compared to the individual treatments. Preliminary data also suggest that targeting the particulate form of guanylyl cyclase increases activity of Akt signalling during early reperfusion suggesting common signalling between soluble and particulate guanylyl cyclase. These data suggest that targeting sGC during early reperfusion can afford cardioprotection by limiting infarct size. The relationship between cGMP elevation and infarct size needs to be investigated further. Nevertheless, these studies suggest that sGC may be a tractable target for the therapeutic management of acute myocardial infarction.

The work described in this thesis is concerned with the design and evaluation of microsphere-based systems for drug delivery into the colon. In initial experiments, techniques were devised for the preparation of microspheres from two sustained-release acrylic polymers, Eudragits RL and RS, using emulsification-solvent evaporation techniques. For Eudragit RS microspheres containing the drug 5-aminosalicylic acid, the rate of drug release could be controlled by the type and concentration of surfactant used for preparation. Consequently, formulations could be produced which released encapsulated drug instantaneously or over many hours. The surfactants may have been altering the structure of the microsphere drug-polymer matrix. Two novel analytical techniques were employed to characterise Eudragit RS microspheres containing sulphasalazine. Fourier transform-Raman spectroscopy was successfully used as a non-destructive method for qualitative and quantitative microsphere characterisation. The technique provided good agreement with a UVspectrophotometric method in quantifying the amount of drug in microsphere samples. X-ray photoelectron spectroscopy was used to estimate the concentration of sulphasalazine at the microsphere surface for samples produced with or without the use of surfactant. Across a wide range of microsphere drug loadings, the surface drug content remained remarkably constant, but was consistently lower in the samples produced using surfactant. In a parallel programme of work, using gamma scintigraphy, the transit rate of different sizes of radiolabelled materials through the human colon was investigated to determine whether there was an optimal size to maximise colon residence. There was no clear evidence of any difference in the colon residence time of 0.2 mm particles, 5 mm tablets, or 8.4 mm tablets, under normal conditions and during accelerated transit. In healthy subjects, 50% of a dose of 0.2 mm particles resided in the ascending colon for an average of 11 hours. Finally, an in vivo biopharmaceutical evaluation of sulphapyridine-containing Eudragit RS microspheres in the human colon was undertaken. For this study, a neutron activation technique was developed for microsphere radiolabelling. Microspheres could be successfully radiolabelled by incorporation of samarium oxide followed by neutron irradiation. However, it was necessary to minimise the period of irradiation and the amount of incorporated samarium oxide, since high levels of both were found to adversely affect microsphere performance. The in vivo investigation revealed that the colonic bioavailability of sustained-release microencapsulated sulphapyridine was less than 50% of unencapsulated sulphapyridine powder. This shortfall was possibly due to an interaction of the drug with colonic bacteria or in vitro/in vivo differences in microsphere drug release characteristics.

Despite intensive research, cancer remains one of the major causes of worldwide morbidity. It is widely believed, however, that if currently available anti-cancer drugs could be delivered specifically to tumours then the disease would have been mastered. The delivery of prodrug converting enzymes by clostridial spores specifically to the anoxic centres of tumours is one potential delivery mechanism. This is due to the extreme selectivity of spores to germinate solely in the hypoxic regions of tumours. Once germinated, the expression of a prodrug converting enzyme converts a systemical1y administered prodrug to a highly toxic drug only in the tumour. Previous studies using Clostridium acetobutylicum and Clostridium beijerinckii as the delivery vehicle highlighted that prodrug converting enzyme expression is only found in tumours. However, no significant anti-tumour affect was observed. Two possible reasons were evolved. Firstly, expression of the prodrug converting enzyme may be low, and/or, secondly, the tumours may not be colonised sufficiently to promote an antitumour effect. Preliminary studies identified that Clostridium sporogenes NCIMB 10696 may represent a more suitable host. Higher spore titres could be prepared and, once administered, higher cell counts are found in the colonised tumours. Prodrug converting enzymes with improved kinetics over pre-existing enzymes have also been identified. Once effective gene transfer systems and expression systems had been developed, suitably high levels of several different prodrug converting enzymes, in particular nitroreductases, were obtained. Initial in vivo studies on one of the early recombinant strains identified a definite anti-tumour effect. Since those initial studies, further improvements to expression have been made. It is hoped that a more significant anti-tumour affect would result from using these improved strains. It is the ultimate aim of CDEPT to have the prodrug converting enzymes integrated into the host genome so as to negate the use of antibiotics. Towards this, studies on the use of both classical and novel integrative technologies have been investigated.

Hepatitis C virus (HCV) is a major cause of chronic liver disease, leading to hepatic steatosis, fibrosis, cirrhosis and hepatocellular carcinoma. A vaccine is currently not available, while the standard of care is effective in only 50% of treated patients. The first specific anti-HCV drugs have been recently approved, and new classes of targeted agents are under clinical trials/investigation. Nevertheless, improved treatment strategies are needed, in order to bypass the rapid emergence of resistance. All the viral non-structural proteins are a possible target for the identification of novel and selective antivirals. Among them, the NS3 helicase is still underexploited, with no known inhibitor under pre-clinical or clinical development. This enzyme plays a crucial role in the virus life cycle: it catalyses the separation of double-stranded RNA strands, which is necessary for genome amplification and translation. Due to its essential function, the NS3 helicase was chosen as a target for the identification of new, specific anti-HCV compounds. Different computer-aided techniques were employed to identify potential smallmolecule inhibitors of the enzyme. Two structure-based virtual screenings of commercially available compounds were performed on the main nucleic acid binding site. A series of candidate inhibitors was evaluated in the HCV replicon assay, yielding two primary hits with low μM activity. Secondly, the model of the one known inhibitor co-crystallised with the enzyme was used as a starting point for a shape-comparison screening of small molecule libraries. A new series of compounds was selected and evaluated for anti-HCV activity, and one of them was found to inhibit the viral replication at a low μM concentration. Several new derivatives of the initial hits were synthesised, belonging to four main structural families: bis-aromatic piperazine derivatives, symmetrical phenylendiamine compounds, differently substituted thieno-pyrimidines, and triphenyl-pyrrolone analogues. Inhibition of HCV replication in the replicon assay was evaluated for the new compounds prepared and several structures showed a range of activity from low-μM to nM.

Natural products are an important source for drug discovery. At present there is a resurgent interest in pharmacognosy as a platform for new combinations of active principles to provide highly potent and low-cost medications to treat a growing population with an increasing longevity. This product studied phytochemical interactions in Artemisia annua plant extracts using anti-plasmodium and anti-proliferation assays to identify interactions with potential therapeutic implications. To enable the study a rapid tandem quadrupole mass spectrometry (TQD) method was developed for metabolites in the plant and the validation indices showed the method to be robust, quick, sensitive and adequate for a range of applications.

Since their inception the B-Iactam antibiotics have become one of the most important classes of phannaceutical agents, both therapeutically and economically, in modern day usage for the treatment of a wide spectrum of bacterial infections. However, due to the versatility of bacteria many previously treatable species are developing resistance to the antibiotics currently available and so there is ever a need to develop more ~-lactam antibiotics, which are effective and yet safe. A major drawback to the ~-lactams is the degree of immunologically adverse reactions they induce. It was the aim of this study to develop both mechanistic and immunological methods to enable the prediction of a B-lactam's potential to induce an allergic response and to determine if a relationship between these responses and the molecular properties of the ~-lactams was present. In this study a database pertaining to frequency by which 70 p-lactams induce adverse reactions has been compiled and used to produce 27 QSAR models. A highly sensitive assay for the quantitation of cross-reactivity between B-lactams and serum anti-benzylpenicillin antibodies has been developed and used to determine the cross-reactivity potential of 31 ~-lactams and to develop 18 QSAR models. All of the QSARs developed suggest that the shape and electron separation of the ~-lactams are crucial to the development and extent of adverse response or crossreactivity induced by a specific p-lactam antibiotic, new or old. The QSARs developed will enable the design and development of new ~-lactam antibiotics which present a significantly lower risk of inducing immunologically mediated adverse responses when used therapeutically. Two sensitive assays for the quantitative detennination of the cytokines IL2 and IL4 in lymphocyte culture supernatants have been developed, and have been shown to have a potential use in the prediction of the type of immunological response initiated following p-Iactam stimulation of a sensitised individual.

Sulphathiazole is a highly polymorphic model system exhibiting at least five polymorphic forms: I, II, III, IV, and V. Polymorph stability is known to be susceptible to solvent environment, and it is established that 1-propanol stabilizes the most metastable form I. This study examines the effect of a range of alcohols on polymorph selection and attempts to elucidate the mechanism. The role of the alcohol functional group in the polymorph selection process is thus investigated and evaluated. Crystals were characterized using optical microscopy, SEM, PXRD, DSC, IR, and single-crystal X-ray diffraction for their polymorphic identity. The role of solvent in the stabilization of polymorphs was investigated by visualizing and calculating energy requirements for the interaction of each solvent molecule with α- and β-dimers of sulphathiazole, using Cerius2 modeling software and GRID based systematic search simulation. These studies showed that solvent had a significant impact on polymorph selection. In common with 1-propanol, 1-butanol was found to stabilize form I by inhibiting the formation of the β-dimer, which is necessary for nucleation of and transformation to forms II-IV. Shorter chain alcohols and branched chain alcohols such as methanol, 2-propanol, and ethanol did not stabilize form I but stabilized forms II, III, and IV, respectively, showing that it is not only the alcohol functionality but also the steric effects of the alkyl chain that contributed to the effect. Sulphathiazole form I normally has a needlelike morphology. Form I with a modified rodlike morphology was produced by crystallization from 1-propanol with the addition of methanol in low concentration, showing that it is possible to control the morphology and selectively isolate polymorphs. Indomethacin is known to exhibit at least five polymorphs but only the stable γ Form and metastable α Form are reported to be reliably produced by standard methods. The metastable α Form has an undesirable fibrous needle-like morphology. The current study focused on producing crystals of α Indomethacin with a well-defined morphology using additives. Adipic acid, myristic acid, oleic acid and structurally related 3-indoleacetic acid were selected as additives and their impact on the morphology and polymorphism of indomethacin were investigated in this study. Additives did not change the needle-like morphology of α-indomethacin but less fibrous and less aggregated well defined needles were observed in presence of adipic acid, oleic acid and 3-indole-3-acetic acid.

The most successful antimicrobial agents in clinical use are of microbial origin and of these the greatest variety has been found in the genus Streptomyces. However, the resistance of the pathogenic microbes to the commonly used antibiotics is increasing as a result of the wide-spread and long-term use of these antibiotics. Therefore, understanding the strategies that bacteria use to become resistant is of crucial need. Streptomycetes are Gram positive bacteria, commonly found in soil and are known antibiotic-producers. The focus of this thesis was to underpin the mechanism of resistance to penicillin G in isolated strains of streptomycetes that exhibit elevated resistance to penicillin G and to characterise these organisms. Moreover, to investigate the interaction between penicillin G and PBPs in Streptomyces strains and investigate the relationship between growth rate and penicillin G resistance in Streptomyces in vitro. Ninety six Streptomycetes were isolated and characterized. Morphological examination and the16s rRNA sequences of these strains indicated that strains belong to the species Streptomyces. The MICs and MBCs for penicillin G for the isolated Streptomyces strains were measured by plate culture. Some strains showed growth up to 400 μg/ml with penicillin G, which indicate that the strains were highly resistant against penicillin G. Some strains were unable to grow at penicillin concentrations above 200μg/ml. Also, The MICs of penicillin G for isolated Streptomyces strains were measured using a novel OxoPlates® system in 96-well culture format employing Mueller-Hinton broth culture. The MICs of all strains ranged from 1-100 μg /ml. Results indicate that the sensitivity of Streptomyces strains of penicillin G is not directly related to β-lactamase production in the panel of isolates examined. There was no correlation between the MICs of penicillin G and the growth rate in these isolates. Likewise, there was no association between the position of beta-lactamase producing and non-beta-lactamase producing strains on the phylogenetic tree and their beta-lactamase xii activity. Beta-lactamase producing and non-producing strains refers to the same ancestral origin clade. Additionally, the comparative analysis of 16S rRNA gene sequence and phylogenetic relationship of strain (W43) revealed that the isolate clustered with (W76) Streptomyces lividans strain YLA0. Bocillin (a penicillin binding protein stain) staining in β-lactamase producing strains showed staining throughout the mycelia whereas in non β-lactamase producing strains staining only occurred in certain parts of the mycelia. Bocillin also revealed that in spores PBPs were located on both poles of the spores. Streptomyces coelicolor has the ability to grow at high concentrations of penicillin G up to 640 μg/ml in continuous culture. It also has the capacity to grow at very low amounts of dissolved oxygen in continuous culture. Significantly, there was a correlation between the growth rate of S. coelicolor and the resistant to penicillin G. S. Coelicolor was more sensitive to penicillin G at a high dilution rate. Furthermore, our strategy of using the Bug-Lab for monitoring the progress of S. Coelicolor 1147 in continuous culture, even at low concentrations of cells in real time was successful.

Hydrogen sulfide (H2S) is the simplest endogenously produced thiol and has an indispensable role in cardiovascular homeostasis. It has been shown that exogenous H2S supplementation protected the heart against myocardial ischaemia/reperfusion injury through a mechanism which is yet to be defined. In this thesis, it was hypothesised that controlled application of thiol/H2S donors at reperfusion would mitigate acute myocardial infarction. We sought to characterise the cardioprotection and molecular targets of three H2S donors (mesna, GYY4137 (a slow-releasing, non-mitochondrial targeted H2S donor) and AP39 (a mitochondria-targeting H2S donor)). This characterisation was conducted using a broad range of experimental models and techniques including anaesthetised rat model of ischaemia/reperfusion injury, Western blotting and mitochondrial studies using isolated cardiomyocyte mitochondria, namely subsarcolemmal and interfibrillar mitochondria. Mesna did not limit infarct size when it was given pre-ischaemia or at reperfusion. GYY4137 and AP39 significantly limited infarct size when given specifically at the time of reperfusion through different mechanisms. Cardioprotection established by GYY4137 was mediated mainly by triggering of PI3K/Akt/GSK-3β signalling at reperfusion with partial dependency on eNOS activity. Selective mitochondrial delivery of H2S at reperfusion using AP39 had no effect on Akt, eNOS, GSK-3β and ERK1/2. In isolated mitochondria, AP39 inhibited Ca2+-sensitive opening of PTP in subsarcolemmal and interfibrillar mitochondria through attenuation of mitochondrial reactive oxygen species generation. The studies presented in this thesis provided novel mechanistic insights into cardioprotection by H2S. These studies suggest that targeted delivery of H2S represents a novel and effective adjunctive therapy to ameliorate the injurious effects of reperfusion which contribute to acute myocardial infarction.

Adverse drug reactions remain a major health issue with delayed type hypersensitivity reactions developing in a high number of individuals. The cellular immunological processes that underlie drug-specific responses in hypersensitive patients have been previously described; however the involvement of the humoral immune system has not been studied in great detail. Consequently, this thesis explores the nature of the piperacillin-specific B cell response in hypersensitive patients and compares the cellular and humoral immune response that develops in patients with cystic fibrosis (CF) exposed to repeated courses of the drug. Initial studies involved characterization of B cell proliferation, B cell phenotype and the nature of total and drug-specific IgG antibody secretions using peripheral blood mononuclear cells (PBMC) from hypersensitive patients. For comparison PBMCs from 2 groups of individuals were assessed: piperacillin naïve healthy volunteers and piperacillin tolerant patients with CF. ELISA, and ELISpot were used to detect piperacillin-specific B cells responses and IgG secretion. T lymphocyte proliferation was assessed with the lymphocyte transformation test (LTT). T lymphocytes from hypersensitive patients, but not tolerant patients or naïve donors were stimulated to proliferate in the presence of the drug. The peak concentration for T cell activation was 1 mM. Phenotypic assessment of hypersensitive patients B-cells revealed an increase in CD19+CD27+ expression in response to piperacillin treatment in vitro. IgG secreting immortalized B-cell lines also expressed a pure CD19+CD27+ phenotype. Piperacillin stimulation of hypersensitive patient PBMC also led to an increase in the secretion of IgG. In contrast, IgG secretion was not detectable following piperacillin stimulation of PBMC from tolerant patients and healthy controls. Western blotting and mass spectrometric methods were applied to characterize -lactam-protein covalent binding. Bovine serum albumin (BSA) binding was time- and concentration-dependent with hapten densities (i.e., the extent of selective lysine residue modification) and anti-piperacillin antibody binding affinity increasing with increasing molar ratios. Lysine residues in BSA at positions 4, 12, 131, 132, 136, 211, 431, 524, and 537 were modified by piperacillin. Epitope profiles also showed similar lysine residues were modified with amoxicillin, benzylpenicillin and flucloxacillin though the extent of ionisation at each site of modification was drug-dependent. A hapten inhibition ELISA used to assess the specificity of the antidrug antibodies revealed the total antibody binding to aztreonam, amoxycillin, benzylpenicillin and penicillin V BSA adducts. This indicates a lack of cross-reactivity with piperacillin-specific IgG antibodies. Subsequently, LTT and ELISA were employed to screen the piperacillin-specific T cell response and IgG antibodies during piperacillin therapy. It was established that piperacillin-specific T cells were detectable on and following clinical diagnosis of hypersensitivity. Moreover, piperacillin-specific T cell responses were detected in a small number of patients currently classified as drug tolerant. A significant difference in piperacillin-specific IgG was observed when plasma form LTT positive and negative blood samples were compared. LTT positivity was associated with higher levels of piperacillin-specific IgG. Furthermore, a significant decrease in piperacillin-specific IgG was seen 24 h post-desensitisation (graded drug challenge). Piperacillin-specific T cell clones isolated from hypersensitive patients were used to explore the effect of plasma bearing anti-piperacillin IgG on the T cell response. Eleven piperacillin-specific CD4+ and CD8+ T-cell clones were generated from 2 hypersensitive patients. All clones were stimulated to proliferate with piperacillin in a concentration-dependent manner. IFN-γ and IL-5 secretion was seen to predominate following piperacillin stimulation. There were no differences in piperacillin-specific T-cell proliferation when piperacillin-specific antibody bearing plasma and plasma from naive volunteers were compared. However, attenuation in IFN-γ secretion was observed with plasma bearing anti-piperacillin antibodies alone. Collectively, the data presented in this thesis begins to describe the different components of the drug-specific humoral and cellular immune response that develops in piperacillin hypersensitive patients with CF.

Background: Cardiovascular disease (CVD) is one of the major causes of morbidity and mortality worldwide. Clinical guidelines, based on the results of randomised controlled trials, state that effective secondary prevention therapies should be prescribed following a diagnosis of particular CVD unless there are contraindications. Although evidence shows that use of evidence based pharmacotherapies after diagnosis of CVD reduces mortality and disease progression, many inequalities exist in prescribing practice. Many studies have documented that women and the elderly are less likely to receive evidence based therapies than men and the young, respectively. Greater socioeconomic deprivation has also been shown to be associated with lower rates of prescribing of therapies. However, prior studies have all focussed on one particular CVD or failed to adjust for confounders. Also, few studies have examined trends in the prescribing of evidence based pharmacotherapies over time and documented whether prescribing inequalities are static, narrowing or widening. This project aims to describe the pharmacotherapy received by patients with CVD in Scotland, and to describe the factors associated with prescribing of evidence based pharmacotherapy. Methods: In this retrospective cohort study I examined a linked database of primary care records (Continuous Morbidity Records) and secondary care records (Scottish Morbidity Records) covering 238064 individuals in Scotland (approximately 6% of the total population) from 1997 to 2005. Patients with a first diagnosis (defined as a first hospitalisation or first recording of the diagnosis in primary or secondary care) of myocardial infarction (MI), angina, and peripheral arterial disease (PAD) were identified. Patients who died within the first 30 days of diagnosis/first hospitalisation were excluded from further analysis. Data on prescribing of evidence based therapies (angiotensin converting enzyme inhibitors (ACEI)/angiotensin receptor blockers (ARBs), β-blockers, statins and antiplatelet agents [aspirin or clopidogrel]) within 30 days of diagnosis was obtained from primary care database records. Multivariable logistic regression was conducted to examine the association between prescribing of evidence based pharmacotherapies and age, sex, socioeconomic status, comorbidities and year of diagnosis. Results: Between 1997 and 2005, 4305 (83.4%) patients with a first diagnosis of MI, 7210 (98.6%) with angina, and 3385 (95.8%) with PAD had survived to 30 days after their first diagnosis. Increasing age was associated with lower odds of being prescribed evidence based therapies. This association persisted after adjustment for sex, socioeconomic status, year of diagnosis, and comorbidities. In general, older patients ≥ 85 were significantly less commonly prescribed evidence based therapy (EBTs), however they were significantly prescribed nitrates (OR 1.29; 95% CI 1.05-1.59, P < 0.01) for angina. Generally men were more likely to be prescribed evidence based therapies than women. After adjustment, prescribing of evidence based therapies was significantly higher in men with a MI for β-blockers (OR 1.18; 95% CI1.04-1.33, P < 0.01), ACEI/ARBs (OR1.26; 95% CI1.05-1.47, P < 0.01) in angina, and statins in men (OR 1.39; 95% CI1.01-1.93, P < 0.04) with PAD and coronary heart disease (CHD). In contrast, men diagnosed with isolated PAD were significantly less commonly prescribed statins than women (OR 0.73; 95% CI0.59-0.91, P < 0.004). Prescribing of evidence based therapies varied negligibly between the most deprived and least deprived patients. These minor differences disappeared after adjustment except for β-blockers which were significantly less likely to be prescribed for patients who had been diagnosed with angina and were residing in quintile 9 compared to the least deprived area (OR 0.76, 95% CI 0.58-1.00, p= 0.05). Prescribing of evidence based therapies increased between 1997 and 2005, particularly for ACEIs/ARBs, β-blockers, statins and antiplatelet agents. Generally the presence of comorbidities was associated with lower odds of being prescribed evidence based therapies. When comparing prescribing rates between the different diagnoses, patients with a first MI were more likely to be prescribed ACEI/ARBs, β-blockers, statins, aspirin and clopidogrel compared to angina. All evidence based therapies were less likely to be prescribed for those with PAD compared to patients with a MI or angina. Conclusion: In conclusion, I have shown that prescribing of evidence based therapies has improved over time, though rates remain low. Prescribing evidence based therapies is inequitable, though not always significant, for age, sex, and socioeconomic status. Concomitant disease decreased the odds of being prescribed evidence based therapies. More studies are needed to identify the reasons for the prescribing inequalities and low rates observed. Further studies are needed to examine the existence of other inequalities in using evidence based therapies such as dosing and to find strategies to improve prescribing rates.

Natural products play a pivotal role in the treatment of cancer; identification of compounds such as taxanes and the vinca alkaloids were seminal landmarks in natural product drug discovery. Jerantinine A (JA), a novel Aspidosperma alkaloid isolated from plant species Tabernaemontana corymbosa, was previously reported to possess cytotoxic activity against vincristine-resistant nasopharyngeal carcinoma cells and is therefore an ideal candidate for biological investigation. Furthermore, Tabernaemontana corymbosa has been placed in the endangered list of threatened species by the International Union for Conservation of Nature (IUCN) thus making it a priority to elucidate the biological activity of this alkaloid. Herein, we report detailed biological evaluation of JA on various human-derived carcinoma cell lines. Our preliminary screens showed that significant inhibition of cell growth and colony formation accompanied time- and dose-dependent induction of apoptosis in human cancer cell lines after treatment with JA. Dose-dependent accumulations of cleaved PARP and caspase 3 further confirmed apoptosis. Profound G2/M cell cycle arrest was observed 24 h after treatment in all cell lines. Characteristics of mitotic arrest including inhibition of tubulin polymerisation, microtubule disruption, and aneuploidy were clearly observed. DNA fragmentation was also evident in cells treated with JA. Indeed, significant increases in phosphorylated-γH2AX were indicative of DNA damage caused by double strand breaks and were relatively similar to levels caused by vincristine. Investigations into JA’s ability to overcome vincristine resistance demonstrated that it is not a substrate of Pgp. The role of reactive oxygen species (ROS) in acquired resistance and cell death have also been widely studied. JA induced significant levels of ROS in treated cells, possibly contributing to their apoptotic destiny. Proteomic analyses also corroborated the phenotype of JA-treated cells with increased expression of ROS-neutralising enzymes, aberrant expression of proteins involved in the spindle assembly checkpoint critical to mitosis, and decreased expression in all tubulin proteins detected by LC-MS/MS. A genome-wide RNAi screen revealed several candidate genes involved in mediating sensitivity to JA. The genes corresponding to c-Jun-N-terminal kinases, JNK1/2, were selected for subsequent investigation based on their involvement in multiple pathways that were identified using bioinformatic tools. JNK1/2 were knocked down in MCF-7 and MDA-468 cells and then treated with JA. MTT assays revealed some loss of sensitivity, suggesting that these proteins were indeed involved in mediating cell sensitivity to JA.

Graphene oxide (GO) and reduced GO (r-GO) nanomaterials exhibit great potential for several biomedical applications. Of foremost importance is to determine any potential health hazards related in their exposure. In this research, we hypothesised that the different material properties evidenced by GO and r-GO would elicit different biological responses. The first objective of this work was to synthesize Go and r-GO and characterize their physiochemical properties. The second aim was to investigate whether the two-distinct surface chemistries of GO and r-GO influenced their biological effect. The potential toxicity of these nanomaterials was investigated using the normal lung fibroplast cell line MRC-5 and cancerous epithelial lung cell line A549. The cytotoxicity of graphene derivatives was concentration-, time- and cell-dependent and varied according to the material used. Thus, the surface chemistry of graphene plays a critical role in its biocompatibility. Non-cancerous cells had a higher sensitivity to GO cytotoxicity than cancer cells. R-GO was highly biocompatible to MRC-5 cells and for A549 cells had a minimal effect of cell viability. At 37C˚, GO and r-GO were moderately hemolyric at concentration of 125 µg/ml and highly haemolytic at concentration of 300 µg/ml. Exposure of cells to both graphene derivatives led to reactive oxygen species (RO5) generation without genotoxicity. GO, but not r-GO, led to autophagy in both cell lines, possibly inhibiting the PIP3-Akt/mTOR pathway. For both cell lines and at non-lethal concentrations, GO downregulated the expression of glycogen synthase kinase-3 (GSK-3ß). GO was also found to dysregulate both Wnt/b-catenin and Akt cell signalling pathways which are vital for cellular function. The finding relating to cell signalling provide an insight to the safety of GO which is important to its use in cancer therapy.

Shape-based approaches have many potential areas for development in the future for application to in silico pharmacology. Further exploration of the role of molecular shape may lead to better understanding of the substrate specificity of enzymes and the possibility to reduce toxic effects that may be caused by ligands binding to undesired target proteins. Methods exploiting molecular shape for activity and toxicity prediction might have a great influence on the drug discovery process. There are different approaches that might be used for this purpose, e.g. shape fingerprints and shape multipoles. Both methods describe the shape of molecules, discarding any chemical information, using numerical values. Focusing only on shape can lead to identifying novel core structures of molecules, with improved properties. Molecular fingerprints are binary bit strings that encode the structure or shape of compounds; shape is measured indirectly by alignment to a database of standard molecular shapes – the reference shapes. The Shape Database should represent a wide range of possible molecular shapes to produce accurate results. Therefore, this was the main focus of the investigation. The shape multipoles method is a fast computational method to describe the shape of molecules by using only numbers and therefore it requires low storage needs and comparison is performed by simple mathematical operations. To describe the shape, it uses only 13 values (3 quadrupole components and 10 octupole components). The performances of both methods in grouping compounds based on shared biological activity were evaluated using several test sets with slightly better results in case of shape fingerprints. However, the shape multipole approach showed potential in finding differences in shape between enantiomers. Among the possible applications of the shape fingerprints method are solubility prediction (on comparable level as well-established methods) and virtual screening.

Inhalation represents a potentially attractive delivery route for biologics, especially those designed to treat pulmonary diseases such as asthma, cystic fibrosis or lung cancer. Delivery directly to the site of action should increase local concentrations of drug, whilst reducing systemic side effects. However, there is limited knowledge regarding the mechanisms of pulmonary clearance, with gaps in understanding; where molecules are absorbed, the mechanisms involved, regional variability throughout the lung, and how to control pulmonary retention and/or facilitate cellular uptake. The work presented in this thesis details the development of a SPECT/CT imaging protocol to determine the pulmonary retention and tissue redistribution of technetium labelled antibodies and their fragments in vivo, in mice, to begin to address these knowledge gaps. The SPECT/CT imaging method was applied to a whole monoclonal murine immunoglobulin G1 (mlgG1), as well as its Fab and scFv fragments and a small protein (FN3) in order to determine whether diffusion controlled pathways were important in pulmonary antibody clearance. Additionally, the pulmonary retention of mutant mlgG1 with differing binding affinities to the murine neonatal Fc receptor (mFcRn) were assessed in order to determine whether antibody transport across the epithelium occurred via active transcytosis. It was determined that 54.4 ± 0.63 % of the total instilled dose of a whole monoclonal antibody remains in the lung over 24 hrs, with Fab and scFv fragments cleared significantly quicker with 28.7 ± 0.73 % and 34.9 ± 0.85 % respectively of the total instilled dose remaining in the lung at 24hrs. The pulmonary retention of the 11 kDa FN3 protein was also assessed with 21.0 ± 0.65 % remaining in the lungs after 24 hrs. No evidence of build up of any protein was detected in the oesophagus/stomach, suggesting little contribution by mucociliary clearance. Very little build up of whole antibody, Fab or scFv was observed in the liver or kidneys. However, very clear evidence of renal filtration of the 11 kDa fragment was observed. There was no difference in the pulmonary retention of wild type IgG and any of the mFcRn binding mutants. Additional investigation of antibody retention rates in the murine house dustmite (HOM) model of asthma, although not making use of the SPECT/CT method, showed that antibody is cleared more rapidly from the diseased lung than the normal lung. It was also shown that the expression pattern of the mFcRn receptor is the same in the normal and HDM exposed lung and so this increase in clearance rate occurs via passive diffusion controlled processes. This is most likely a result of increased paracellular transport due to disrupted mucosal barrier function. The SPECT/CT imaging method developed has proven to be a simple and reliable method to assess, non-invasively, pulmonary antibody retention in vivo. Overall it appears that antibody transport across the pulmonary epithelium occurs predominantly via diffusion controlled mechanisms, which include both paracellular transport and nonspecific transcytosis by pinocytotic routes. Additionally, in the mouse, neither receptor mediated transport by mFcRn nor mucocillary clearance is important in the pulmonary clearance of antibodies.

Abstract During the course of the studies outlined in this thesis, a new approach for the synthesis of the tropane alkaloid, (±)-physoperuvine has been developed using a highly efficient one-pot tandem process which involved the Overman rearrangement and a ring closing metathesis reaction. An asymmetric one-pot tandem process has also been employed for the synthesis of the natural product, (+)-physoperuvine. This methodology was also applied to the generation of a late-stage intermediate that could be used in the synthesis of carbocyclic nucleosides, such as noraristeromycin. In the second part of this thesis, an ether-directed Pd(II)-catalysed Overman rearrangement which had previously been developed by the Sutherland group was applied in conjunction with a cross-metathesis reaction for the stereoselective synthesis of the guanidine alkaloid, (+)-monanchorin in a fourteen-step synthesis. Further employment of this process provided the first synthesis of clavaminol A, C and H from (R)-glycidol in a rapid and efficient manner. In a similar fashion, (2S,3R)-enantiomers were also synthesised from (S)-glycidol. In addition to this, using similar chemistry, an intermediate protected enone was prepared using a cross-metathesis reaction as the second key step in an approach towards the synthesis of an NO-inhibitor.

The prevalence of obesity worldwide has more than doubled since 1980. The World Health Organisation (WHO) estimates that more than one in ten adults in the global population is obese. Cardiovascular and metabolic health can be improved with moderate weight loss; losses of 5%–10% have been found to improve conditions such as diabetes, hypertension and cholesterol and low-density lipoprotein (LDL) levels. Within the UK, a number of weight management programmes that depend on lifestyle intervention (tier 2) and others that supplement this with drug therapy (tier 3) and surgery (tier 4) are available. The guidelines produced by the Scottish Intercollegiate Guideline Network (SIGN) advocate that weight management programmes address changes to diet, physical activity and behaviour. For patients with a body mass index (BMI) ≥30 kg/m2 or ≥28 kg/m2 in patients with comorbidities, orlistat can be considered as a drug intervention on a case-by-case basis following a full risk and benefit assessment. The objective of the Glasgow and Clyde Weight Management Service (GCWMS), a specialist weight-loss programme, is for patients to lose at least 5 kg. There are a number of metabolic disorders that are associated with obesity. One such disorder is type 2 diabetes mellitus, where weight loss is a standard recommendation to improve blood glucose control. Randomised controlled trials (RCTs) of orlistat indicate that the drug is effective in promoting weight loss and improving metabolic control for those patients with the comorbidity of type 2 diabetes and obesity. There are several different groups of anti-diabetic drugs that can be used to manage diabetes. The effects of the different medications on body weight are considerable. Some, such as biguanides (metformin), dipeptidyl peptidase-4 inhibitors (DPP-IV), Glucagon-like peptide-1 agonist (GLP-1) and sodium-glucose co-transporter-2 inhibitors (SGLT2), either have no effect on weight or can cause weight loss. Others, such as sulfonylureas (SUs) and thiazolidinediones (TZDs) can lead to weight gain. This thesis explores the impact of lifestyle interventions in weight management services, and the impact of drug interventions, on weight loss and glycaemic control. It is supported by the results of five complementary studies that reviewed the effect of orlistat on type 2 diabetes and assessed the impact of the prescription patterns of anti-diabetic drugs in addition to the effects of these pharmacological interventions on weight change in comorbid patients. The first aim of this thesis is to review the evidence of the effects of orlistat on diabetic outcomes. The second aim is to evaluate the lifestyle interventions, and phase 2 of the GCWMS. Finally, the third aim is to determine the prescribing patterns of anti-diabetic drugs, and to observe the association between anti-diabetic medications and weight change. This thesis addresses the following objectives: 1. To undertake a systematic review and meta-analysis of published studies in order to review the evidence of the effects of orlistat on weight loss, specifically concerning glycosylated haemoglobin (HbA1c) and fasting plasma glucose (FPG), using the Cochrane review methodology; 2. To investigate the proportion of patients losing 5 kg of weight, commencing from their entry into the GCWMS programme, until the end of the lifestyle phase of treatment, for individuals of different ages, genders, and socioeconomic groups; 3. To study the proportion of patients losing 5 kg of weight, commencing from their entry into the GCWMS programme, until the end of phase 2, with the three different interventions of orlistat, low-calorie diet (LDL), and further weight loss (FWL); 4. To investigate the proportion of patients referred to the GCWMS on weight-neutral, mixed, and weight-gaining anti-diabetic medications; 5. To investigate the effect of baseline anti-diabetic medications on weight change for patients within a weight management programme. Chapter 2 presents the first study, which was a systemic review that considered the evidence collected in RCTs on the efficacy of orlistat for type 2 diabetes and weight loss. The effects were considered at the biochemical level and included the levels of glycosylated haemoglobin (HbA1c) and fasting plasma glucose (FPG) in people with overweight and obesity. The results, collected from 2,802 participants in 12 trials, were combined into a meta-analysis. The overall finding was that a combination of orlistat and lifestyle intervention yielded superior results. When the results were compared, it was evident that patients who are overweight or obese who were subjected to combined lifestyle and drug intervention lost more weight and had better glycaemic control than patients who were subjected to lifestyle interventions only. Chapter 3 presents the second study which appraised the effectiveness of a real-life NHS lifestyle weight management intervention in reducing body weight by ≥5 kg. The study followed 23,650 patients referred to the GCWMS, of whom 7,329 attended at least two lifestyle intervention sessions. Those individuals had either a BMI of ≥30 kg/m2, with obesity-related comorbidities, or a BMI of ≥35 kg/m2 and were aged ≥18 years. The lifestyle interventions included a combination of a 600 kcal deficit diet, exercise, and behavioural changes. 30% of the overall group succeeded in losing ≥5 kg. Out of those who completed the programme, however, a considerably higher number (46%) lost ≥5 kg. The greatest losers were men, those aged ≥40 years, those with a BMI ≥50 kg/m2, and those from areas that are more affluent. Chapter 4 presents the third study which focused on patients who lost ≥5 kg in phase 2 of the treatment provided by GCWMS which comprised a low-calorie diet (LCD), orlistat 120 mg, three times a day, or further weight loss (FWL). Participants on LCD were prescribed a 1,200 or 1,500 calorie plan; however, those on FWL repeated the lifestyle phase. There were 3,262 participants who attended at least two sessions in phase 2; these were divided into three categories: 536 who took orlistat, 1,043 who followed a LCD and 1,683 who were selected FWL. By the end of phase 2, the levels of success in terms of weight loss across the groups varied from 31% of participants in the orlistat group to 22% of participants in the LCD group and 83% of participants in the FWL group who lost ≥5 kg. Chapter 5 presents the fourth study, which evaluated the pattern of anti-diabetic drug prescriptions for comorbid patients referred to the GCWMS. The study also looked at the proportion of patients who were referred prior to and after the publication of updated SIGN guidelines for the prescription of anti-diabetic medication. In total, the study enrolled 3,063 participants who received anti-diabetic medications, of whom 47.8% received weight-neutral medications, 39.4% had mixed-effect medications and 12.7% took weight-gaining drugs. Prior to the publication of the SIGN guidelines, 11.6% of participants were on weight-gaining drugs, a proportion that did not change significantly one year after the release of the guidelines. Weight-neutral drugs were more commonly prescribed to women, those with a higher BMI and young people. No relationship was observed between the Scottish Index of Multiple Deprivation (SIMD) and anti-diabetic drug prescriptions. Weight-gaining drugs such as SUs and TZDs were more commonly prescribed to older patients and those with lower BMIs. Chapter 6 presents the fifth and final study, which investigated the effect on body weight of anti-diabetic medications in 998 participants following the lifestyle phase of the GCWMS. By the end of the programme, patients who were on weight-neutral anti-diabetic drugs achieved a mean weight change of -3.3 kg (95% confidence interval [CI]: -3.8 to -2.9 kg) and those on weight-gaining drugs achieved a mean weight change of -2.5 kg (95% CI: -3.2 to -1.8 kg), p =0.05.
Among those who completed the programme, the difference was statistically significant (p =0.005). The association between weight change and anti-diabetic drug type was not explained by differences in sex, initial BMI or age. To conclude, there was a clinically and statistically significant change in weight, HbA1c and FPG in patients with obesity and type 2 diabetes who used orlistat. Of the patients following the GCWMS lifestyle phase, less than 50% succeeded in losing at least 5 kg, with patients who completed the programme being more successful. Participants who lost weight in the lifestyle phase were selected for FWL and experienced the greatest weight loss by the end of phase 2. Those who were unsuccessful in losing 5 kg through the lifestyle programme, were offered orlistat and LCD. The large sample size increased the precision of the results, while the stratification for potential confounding factors increased the study’s validity. A higher proportion of patients were prescribed weight-neutral medications, compared with mixed and weight-gaining anti-diabetic medications. The proportion of patients on weight-gaining diabetes drugs referred to the GCWMS did not alter appreciably following the release of the SIGN guidelines. By the end of the lifestyle treatment phase, patients receiving weight-neutral drugs (metformin, DPP-IV, GLP-1, and SGLT2) were more successful in losing weight than those receiving weight-gaining drugs (SUs, TZDs, and any combination including insulin). The main recommendation from this research are, that further studies are carried out to better establish the best timing of use of orlistat within a weight management programme, that the intensity of phase 2 of the GCWMS is increased, and that prescribers take account of a patient’s current BMI prior when prescribing anti-diabetic medication, especially when recommending weight loss and referring to a weight management programme.

Human inflammatory/innate immune responses lie at the core of resistance to infectious disease and determine the nature of pathophysiological reactions of hematopoietic cells like leukaemia and allergy. The crucial step in these events is the ability of immune cells to function properly, which depends on their adaptation to stress caused by pro-inflammatory stimulation. The mechanisms underlying this crucial biochemical dogma, and its role in normal and pathological cross-links between immune cells, are not well understood. This PhD programme was devoted to investigation of these important problems. We found that the inflammatory mediator interleukin 1 beta (IL-1β), derived from human innate immune cells, triggers production of the major hematopoietic growth factor, the stem cell factor (SCF) in MCF-7 human epithelial cells. This process is controlled by the hypoxia-inducible factor 1 (HIF-1) transcription complex, which regulates cellular adaptation to inflammatory/hypoxic stress by promoting angiogenesis and glycolytic degradation of the glucose. Translational mechanism, which is majorly dependent on the mammalian target of rapamycin (mTOR) kinase pathway underlies IL-1β-induced HIF-1 accumulation and also contributes to SCF biosynthesis. The effect is applicable in both in vitro and in vivo systems. Further experiments demonstrated the involvement of this biosynthetic mechanism in the differential control of normal and pathological functions of inflammatory cells including monocytes, basophils and mast cells. Our results also demonstrated possible biochemical mechanisms regarding the cross-talk between inflammation and SCF-dependent blood cancer (leukaemia), which remains a serious medical burden worldwide. Finally, the pathways investigated could be further considered as potential therapeutic targets for pharmacological correction of human inflammatory reactions and treating cancer/leukaemia by classic and principally novel approaches, such as utilisation of gold nanoparticles.

Hyperprolactinemia (hyperPRL) is a highly prevalent adverse side effect in antipsychotic therapy as most antipsychotic drugs are dopamine D2 receptor antagonists. Peony-Glycyrrhiza Decoction (PGD, 芍藥甘草湯) is a classic Chinese herbal formula initially used to treat muscle pain and spasm. Our pilot clinical studies have confirmed the effectiveness of PGD in alleviating antipsychotic-induced hyperPRL in patients with schizophrenia. In the present study, we further examined the effects of PGD, its individual herbal preparations and major compounds in suppressing prolactin (PRL) hyperactivity in in vitro and in vivo models and underlying mechanisms. PGD treatment significantly suppressed PRL secretion in MMQ cells, an exemplary model of hyperPRL that is derived from pituitary adenoma cells. PGD also suppressed PRL synthesis of MMQ cells in a dose-dependent manner; however, these suppressive effects were completely abolished by pretreatment with 10 μM haloperidol, a dopamine D2 receptor antagonist. PGD did not affect hyperactive PRL in GH3 cells that lack the D2 receptor expression, but PGD significantly increased the expressions of the D2 receptor and dopamine transporters (DAT) in PC12 cells. In the rat model of hyperPRL produced by repeated injection with the dopamine blocker metoclopramide (MCP), PGD (5 - 10 g/kg daily) treatment for 14 days significantly reduced elevated serum PRL and the reduced magnitude was similar to that of 0.6 mg/kg bromocriptine (BMT), a dopamine D2 receptor agonist used for treating hyperPRL. Both PGD and BMT did not alter serum estradiol, but PGD reversed MCP-induced decreased serum progesterone to control level, whereas BMT did not. Similar to BMT, PGD treatment displayed a great effect in reversing the MCP-induced reduction of the expressions of D2 receptor, DAT and tyrosine hydroxylase in both the pituitary and the hypothalamus, in particular the arcuate nucleus, but both had least effects on the expressions of PRL in the pituitary and hypothalamus. The anti-hyperPRL effects of individual herbal preparation and major compounds of PGD were further examined in cultured cells. The three herbal preparations, Paeoniae Radix (PR) alone, Glycyrrhiza Radix (GR) alone and the pooled PR and GR individual preparation (PR+GR), and the three major constituents 18β-glycyrrhetinic acid (GA), liquiritigenin (LQ) and paeoniflorin (PF), were tested. All these preparations and constituents displayed significant effects in suppressing PRL hyperactivity and enhancing the expressions of dopamine mediators. However, PR had the most robust anti-hyperPRL effects compared to PGD and other preparations and constituents. The present study provides experimental evidence confirming the clinical effects of PGD in suppressing antipsychotic-associated hyperPRL. Not only D2 receptor is involved in the anti-hyperPRL effect of PGD, it is also associated with the modulation of other dopamine mediators and sex hormones. The finding that the magnitudes of the anti-hyperPRL effects of PGD and of combined PR and GR are similar suggests that boiling the herbs together or or separately shows the same effects. Additionally, PR preparation appears to be more efficacious in reducing hyperPRL compared to GR preparation which deserves to be further investigated.
published_or_final_version
Chinese Medicine
Doctoral
Doctor of Philosophy

Inhaled administration is the mainstay of asthma and chronic obstructive pulmonary disease (COPD) management using either a pressurised metered dose inhaler (pMDI) or a dry powder inhaler (DPI). Poor disease control and increased hospitalisations is linked to poor inhaler technique. Previous studies to assess inhaler technique have used subjective measures and there is very limited data about the inhalation characteristics used by patients when they use their inhalers. Inhalation flow profiles when patients use their pMDI and inhalation pressure profiles when they use DPIs have been measured using 659 subjects (106 children with asthma [CHILD], 361 adults with asthma [ADULT], 142 COPD [COPD] and 50 healthy volunteers [HEALTHY]) in 5 separate studies. All patient studies used their real life inhaler technique. One of the studies also evaluated the value of using a pMDI co-ordination aid and training these patients to prolong their inhalation whilst a different one investigated the impact of using enhanced training when using a DPI. The first study, 20 CHILD, 57 ADULT and 32 COPD subjects, revealed that the mean (SD) inhalation flows through a pMDI were 108.9 (40.4), 146.0 (58.8) and 107 (50.6) L/min, respectively and only 7, 10 and 10 used a slow flow. In the second pMDI study involving, 20 CHILD, 130 ADULTS, 31 COPD patients, their flows were 70.5 (36.4), 131.4 (60.8) and 70.9 (28.1) L/min and 5, 53 and 10 used their pMDI with good co-ordination. However only 3, 6 and 9 patients had good co-ordination and slow flow. In the third study, 71 ADULT patients, the mean (SD) pMDI inhalation flow was 155.6 (61.5) L/min which decreased (p<0.001) to 112.3 (48.4) when the pMDI was fitted with a co-ordination aid. This was due to the increased resistance to airflow from the aid. Inhalation flow further reduced (p<0.001) to 73.9 (34.9) L/min when patients were trained to prolong their inhalations. Their inhaled volumes did not change whereas mean (SD) inhalation times were 1.60 (0.21), 1.92 (0.80) and 2.66 (1.03) seconds (p< 0.001) respectively. There was a good correlation between their inhaled volume and forced vital capacity with a ratio of 0.7 suggesting that the patient used a full inhalation. A DPI study, involving 16 CHILD, 53 ADULT and 29 COPD patients, measured inhalation characteristics through different DPIs (low to high resistance) when patients used their real life DPI inhalation manoeuvres. The inhalation characteristics were lower in CHILD and highest in ADULT. Overall flows were higher when using low resistance DPIs but the pressure changes and the acceleration of the inhalation flow were significantly higher with high resistance DPIs which suggest more efficient de-aggregation of the formulation. There was a tendency for more problems with low resistance DPIs than high resistance DPIs. The last study involved CHILD, ADULT, COPD and HEALTHY subjects (50 of each) when they inhaled through a Spiromax and a Turbuhaler (similar resistance) after standard verbal inhalation technique training and when using enhanced training with an IN-Check Dial. The order of inhalation characteristics was HEALTHY > ADULT > COPD > CHILD. Significant (p<0.001) improvements in the inhalation flows, pressure changes and acceleration of the flow were achieved in all groups after the enhanced training. The studies provide an insight into the inhalation characteristics of patients when they use different inhalers. The main problem with pMDI use was short inhalation times and when patients were trained to prolong their inhalation then flows reduced. Enhanced training when using a DPI significantly improved the technique of all patients.

Hypertension (HTN) is a major risk factor for cardiovascular diseases including stroke, coronary heart disease (CHD), chronic renal failure, peripheral vascular disease, myocardial infarction, congestive heart failure and premature death. The prevalence of HTN in Scotland is very high and although a high proportion of the patients receive antihypertensive medications, blood pressure (BP) control is very low. Recommendations for starting a specific antihypertensive class have been debated between various guidelines over the years. Some guidelines and HTN studies have preferred to start with a combination of an antihypertensive class instead of using a single therapy, and they have found greater BP reductions with combination therapies than with monotherapy. However, it has been shown in several clinical trials that 20% to 35% of hypertensive patients could not achieve the target BP, even though they received more than three antihypertensive medications. Several factors were found to affect BP control. Adherence and persistence were considered as the factors contributing the most to uncontrolled hypertension. Other factors such as age, sex, body mass index (BMI), alcohol intake, baseline systolic BP (SBP), and the communication between physicians and patients have been shown to be associated with uncontrolled BP and resistant hypertension. Persistence, adherence and compliance are interchangeable terms and have been used in the literature to describe a patient’s behaviour with their antihypertensive drugs and prescriptions. The methods used to determine persistence and adherence, as well as the inclusion and exclusion criteria, vary between persistence and adherence studies. The prevalence of persistence and adherence have varied between these studies, and were determined to be high in some studies and low in others. The initiation of a specific antihypertensive class has frequently been associated with an increase or decrease in adherence and persistence. The tolerability and efficacy of the initial antihypertensive class have been the most common methods of explaining this association. There are also many factors that suggest a relationship with adherence and persistence. Some factors in previous studies, such as age, were frequently associated with adherence and persistence. On the other hand, relationships with certain factors have varied between the studies. The associations of age, sex, alcohol use, smoking, baseline systolic blood pressure (SBP) and diastolic BP (DBP), the presence of comorbidities, an increase in the number of pills and the relationship between patients and physicians with adherence and persistence have been the most commonly investigated factors. Most studies have defined persistence in terms of a patient still taking medication after a period of time. A medication possession ratio (MPR) ≥ 80 has been used to define compliance. Either of these terminologies, or both, have been used to estimate adherence. In this study, I used the same definition for persistence to identify patients who have continued with their initial treatment, and used persistence and MPR to define patients who adhered to their initial treatment. The aim of this study was to estimate the prevalence of persistence and adherence in Scotland. Also, factors that could have had an effect on persistence and adherence were studied. The number of antihypertensive drugs taken by patients during the study and factors that led to an increase in patients being on a combination therapy were also evaluated. The prevalence of resistance and BP control were determined by taking the BP after the last drug had been taken by persistent patients during five follow-up studies. The relationship of factors such as age, sex, BMI, alcohol use, smoking, estimated glomerular filtration rate (eGFR), and albumin levels with BP reductions for each antihypertensive class were determined. Information Services Division (ISD) data, which includes all antihypertensive drugs, were collected from pharmacies in Scotland and linked to the Glasgow Blood Pressure Clinic (GBPC) database. This database also includes demographic characteristics, BP readings and clinical results for all patients attending the GBPC. The case notes for patients who attended the GBPC were reviewed and all new antihypertensive drugs that were prescribed between visits, BP before and after taking drugs, and any changes in the hypertensive drugs were recorded. A total of 4,232 hypertensive patients were included in the first study. The first study showed that angiotensin converting enzyme inhibitor (ACEI) and beta-blockers (BB) were the most prescribed antihypertensive classes between 2004 and 2013. Calcium channel blockers (CCB), thiazide diuretics and angiotensin receptor blockers (ARB) followed ACEI and BB as the most prescribed drugs during the same period. The prescription trend of the antihypertensive class has changed over the years with an increase in prescriptions for ACEI and ARB and a decrease in prescriptions for BB and diuretics. I observed a difference in antihypertensive class prescriptions by age, sex, SBP and BMI. For example, CCB, thiazide diuretics and alpha-blockers were more likely to be prescribed to older patients, while ACEI, ARB or BB were more commonly prescribed for younger patients. In a second study, 4,232 and 3,149 hypertensive patients were included to investigate the prevalence of persistence in the Scottish population in 1- and 5-year studies, respectively. The prevalence of persistence in the 1-year study was 72.9%, while it was only 62.8% in the 5-year study. Those patients taking ARB and ACEI showed high rates of persistence and those taking diuretics and alpha blockers had low rates of persistence. The association of persistence with clinical characteristics was also investigated. Younger patients were more likely to totally stop their treatment before restarting their treatment with other antihypertensive drugs. Furthermore, patients who had high SBP tended to be non-persistent. In a third study, 3,085 and 1,979 patients who persisted with their treatment were included. In the first part of the study, MPR was calculated, and patients with an MPR ≥ 80 were considered as adherent. Adherence rates were 29.9% and 23.4% in the 1- and 5-year studies, respectively. Patients who initiated the study with ACEI were more likely to adhere to their treatments. However, patients who initiated the study with thiazide diuretics were less likely to adhere to their treatments. Sex, age and BMI were different between the adherence and non-adherence groups. Age was an independent factor affecting adherence rates during both the 1- and 5-year studies with older patients being more likely to be adherent. In the second part of the study, pharmacy databases were checked with patients' case notes to compare antihypertensive drugs that were collected from the pharmacy with the antihypertensive prescription given during the patient’s clinical visit. While 78.6% of the antihypertensive drugs were collected between clinical visits, 21.4% were not collected. Patients who had more days to see the doctor in the subsequent visit were more likely to not collect their prescriptions. In a fourth study, 3,085 and 1,979 persistent patients were included to calculate the number of antihypertensive classes that were added to the initial drug during the 1-year and 5-year studies, respectively. Patients who continued with treatment as a monotherapy and who needed a combination therapy were investigated during the 1- and 5-year studies. In all, 55.8% used antihypertensive drugs as a monotherapy and 44.2% used them as a combination therapy during the 1-year study. While 28.2% of patients continued with treatment without the required additional therapy, 71.8% of the patients needed additional therapy. In all, 20.8% and 46.5% of patients required three different antihypertensive classes or more during the 1-year and 5-year studies, respectively. Patients who started with ACEI, ARB and BB were more likely to continue as monotherapy and less likely to need two more antihypertensive drugs compared with those who started with alpha-blockers, non-thiazide diuretics and CCB. Older ages, high BMI levels, high SBP and high alcohol intake were independent factors that led to an increase in the probability of patients taking combination therapies.

Submitted in partial compliance with the requirements of the Master’s Degree in Technology: Homoeopathy, Department of Homeopathy, Durban University of Technology, Durban, South Africa, 2015.
Homoeopathy has advanced as an empirical art and has become a modern age science backed by the kind of massive research data and critical reviews that helps scientific knowledge become acceptable within the global knowledge space. Two centuries ago, the practice of homoeopathy was possible with a few hundred proven remedies of that time but as this science developed through both research and collection of empirical data the number of remedies available increased exponentially. As both the study of remedies and the remedy selection process became more challenging, group analysis by famous homoeopaths such as Farrington (1992), Sankaran (2003), Scholten (1993), Mangliavori (in Vidal 2005) emerged. Currently, with a database of over 3000 remedies available for prescription, group analysis, though a contested approach has proven to be an adequate tool that helps consolidate mass homoeopathic data into meaningful groupings that makes both the study of remedies and their application in practice easier. Classifying homoeopathic remedies into groups by means of group analysis and allowing such methods and results thereof to go through rigorous critiquing refines homoeopathic knowledge and improves its ability to sustain itself as a competent science. Homoeopathy has emerged as a technologically inclined science, utilising various software programs enabling more thorough correlation of symptoms and remedies and so improving the prescription process. Software programs have proven to be very useful tools for the development of group analysis. The aim of this study was to extract the common characteristic symptomatology of five selected homoeopathic remedies belonging to the acidum family as represented in the known materia medica and repertory. The acidum remedies appearing in Radar® 10 repertory (Archibel 2008) were analysed in terms of rubric representation (frequency) and the top five were selected for inclusion in the study. The top five remedies were: Nitricum acidum, Phosphoricum acidum, Muriatic acidum, Sulphuricum acidum and Fluoricum acidum. All rubrics in which the selected remedies appeared were extracted using the homoeopathic software package Radar® 10 (Archibel, 2008) and analysed for sensations and active, passive and compensation reactions of the selected remedies, as per Sankaran (2002). The common primary sensations identified were, burning, sore, swelling, pressing, cramping, dryness, weakness, tearing, and coldness. There were also sensations of anxiety, restlessness, delirium, delusions, dullness, sadness and cheerfulness. Active reactions identified were: hot, heat, inflammation and sensitivity. Passive reactions identified were: coldness, numbness and weakness. Compensation reactions identified were: restlessness, hurriedness, intense reaction and passion. Miasmatic keywords as per Sankaran (2002) were used to determine the miasmatic tendencies of the selected top five acidum remedies. In general all acidum remedies appeared to have a dominant syphilitic miasmatic tendency but acidums were present through all the miasms. Their miasmatic tendencies are easily recognised when viewed in the light of their pathophysiological processes. The pathological tendencies of acidum remedies examined in this study include: haemorrhoids, general haemorrhage, syphilis, warts, neoplastic and non-plastic tumours, irritable bowel syndrome (reflux, colic, constipation), rheumatism, muco-cutaneous ulcerations (STIs, Xerostomia, diabetic ulcers) and bone diseases (osteoporosis, osteitis, and peri-osteitis). The results of this research were compared to the findings of Scholten (1993) and Mangialavori and Marotta (2005). It is the researcher’s view that Sankaran’s methodology of group analysis which he used to explore biological groups of remedies (plants and animals) is adequate for use with non-biological groups of remedies such as mineral acidums. The researcher found group analysis methodology worthy as a tool for classifying the mass data of acidum remedies into orderly sets of meaningful data. Group analysis is consistent with the laws and principles of homoeopathy and encourages the use of materia medica and repertory which are fundamental to the study and application of homoeopathic knowledge.
M

Submitted in partial compliance with the requirements of the Master’s Degree in Technology: Homoeopathy, Department of Homepathy, Durban University of Technology, Durban, South Africa, 2015.
Introduction This study was a double-blind, placebo controlled proving of Panthera leo 30CH (claw of a African lioness); the aim being to determine the effect of this substance induces on healthy persons and in doing do explore the homoeopathic therapeutic potential thereof, in addition a subsequent comparison with the provings of Lac leoninum (milk of a lioness) (Sankaran,1998) (Herrick,1998) was performed. Objectives The primary objective of this proving was to determine the effect that Panthera leo 30 CH on healthy provers so that the therapeutic potential of the substance could be determined. The secondary objective was to compare the Materia medica of Panthera leo with that of Lac leoninum (Herrick, 1998) and Sankaran (1998). Lastly, the third objective was to further develop the homoeopathic Materia medica by adding the remedy picture of Panthera leo. Methodology The drug proving of Panthera leo was conducted as a randomized, exploratory, double blind placebo controlled study. Thirty participants (healthy volunteers) who met the inclusion criteria (Appendix B) participated in the proving, twenty – four were given the verum and the remaining six placebo. Upon obtaining written informed consent the researcher performed a full case history and comprehensive physical examination on each prover before commencement of the proving. A subsequent follow up case history and physical examination at the end of the proving was also performed to establish whether provers had returned to their respective healthy states. The provers took their first dose of the proving remedy at a dosage of 1 dose three times daily for 2 days and recorded all symptoms they experienced in their respective journals according to the guidelines in Appendix C. On completion of the proving, all provers handed in their journals which were then transcribed, assessed and analyzed. An extensive comparison between the proving of Panthera leo and the two provings of Lac leoninum (Herrick, 1998) (Sankaran, 1998) followed. The respective remedies were compared and contrasted with respect to Materia medica and repertory and similarities and differences highlighted. Results A total of 1255 rubrics were obtained of which the mind section of the Materia medica weighed heavily comprising 35 % (435 rubrics) of the proving symptoms, the generals section (11% = 135 rubrics) and head section (9% = 109 rubrics) respectively. Therefore it can be suggested that this remedy’s sphere of action is limited largely to the mind (emotional state) however unique physical indications specifically regarding headaches are also within its sphere of action. Of the total, 1255 rubrics that were obtained only 11% (138 rubrics) where shared with the two other provings of the African lion (Herrick 1998 & Sankaran 1998); as suggested by Naude (2011) one can speculate that these shared symptoms may represent the unique mammalian component of the animal the differences however considerably outweigh the similarities between these provings and for this reason one can conclude that factors in addition to the species of animal from which the remedy is sourced further influence the resultant proving symptoms. Conclusion It can be concluded that Panthera leo 30CH produced clearly observable symptoms in healthy provers who participated in this proving, furthermore there was clear congruency between certain aspects of this proving and that of Herrick and Sankaran however such similarities were limited.

Submitted in partial compliance with the requirements of the Master’s Degree in Technology: Homoeopathy, Durban University of Technology, Durban, South Africa, 2016.
There are now several thousand remedies available to a homoeopath and this number is continually increasing with the increase in homoeopathic research and provings. This growing wealth of data does, however, make choosing a homoeopathic remedy difficult and some homoeopaths argue that the essence of homoeopathic remedies are being lost in this excess of data. In an attempt to more accurately see a remedy’s ‘picture’ and gain deeper insight into remedies, Scholten (1993), Sankaran (2005a) and Mangialavori (2010) developed different methods which can now be collectively referred to as “group analysis”. The aim of group analysis is to find symptoms, sensations and pathological tendencies that are common to all remedies within a group. This study involved applying Sankaran’s group analysis approach to the psychoactive plant drug remedies with the rationale of filtering and organizing the mass of data we now have available on this group. This will enable both students and professionals of homoeopathy to develop a deeper understanding, and hence greater utilization, of the psychoactive plant drug remedies. The following five homoeopathic remedies were chosen for this study on the grounds that they have all been extensively proved through both homoeopathic provings and cured clinical cases and there is a vast amount of literature available on these remedies in materia medica and repertories: • Anhalonium lewinii (Cactaceae family) • Cannabis indica (Hamamalidae family) • Coffea cruda (Rubiaceae family) • Nux moschata (Magnolianae family) • Opium (Papaveraceae family) A computer repertory search was conducted using RadarOpus (Archibel, 2014) to extract all rubrics containing the selected remedies. Parameters were set to retain only rubrics that have less than 50 remedies and at least two of the selected psychoactive plant remedies in them. This was to ensure that only well-defined, characteristic remedies were looked at. The rubrics were visually analyzed, compared and contrasted to determine the common sensations within them and mental, general and particular symptoms were analyzed in terms of Sankaran’s model of Vital Sensation (2005a). The vital sensation of the psychoactive plant drug remedies was found to be that of horror, fear or fright. All the remedies belonging to this group experience the sensation of horror either through their perception of pain or through dreams, visions, hallucinations or anxiety. This sensation pervades all these remedies which are constantly trying to escape this sensation by either increasing or decreasing their activity and sensitivity. The active reaction to the sensation of horror is to increase activity. This is expressed through increased sensitivity; mental clarity; sensations of contraction, fullness, heaviness, heat or moisture; delirium, hallucinations and instability. The passive reaction to the sensation of horror is to decrease activity. This is expressed through insensitivity; lack of mental clarity; sensations of expansion, emptiness, lightness, cold or dryness; sleep, stupor and unconsciousness. The compensation, or coping mechanism that psychoactive plant drug remedies develop, is a transcendence of their condition: they transcend, or escape, their condition by no longer feeling or doing anything, by becoming numb and insensitive. The researcher suggests that although the remedies of the psychoactive plant drug group can be classified according to different miasms, the over-riding miasm of this group is the sycotic miasm with its fundamental sense of having a ‘fixed weakness’ within themselves. The researcher also proposes that the psychoactive plant remedies have an affinity for the central nervous system and for ailments caused by strong emotions such as joy, anger, excitement, fear or fright. These remedies tend to produce pathologies of the central nervous system and sleep including increased reflexes, involuntary motions, trembling, jerking; weakness, atrophy, slowness, paralysis; unconsciousness; catalepsy; Autism Spectrum Disorders; hypersensitivity; insensitivity or absence of sensitivity; pain; formication; mental confusion, poor comprehension, nonsensical speech; memory disorders; delirium, hallucinations, schizophrenia; mood disorders; behavioural disorders; anxiety; insomnia, narcolepsy and nightmares. The researcher found group analysis to be a powerful methodology that, if employed correctly, can aid homoeopaths to learn and understand remedies in their ‘totality’.
M

The objective of the following study was threefold: to conduct a homoeopathic drug proving of Acacia xanthophloea 30CH on healthy individuals in order to elucidate the totality of signs and symptoms produced by the drug substance; to analyse the signs and symptoms gathered from the proving, collate the data, and convert the symptoms into materia medica symptoms and rubrics; to compare the symptoms of the proving to the doctrine of signatures. Methodology This proving study was a double-blinded, placebo controlled study. Thirty provers or participants were recruited for the study and the sample size was divided equally between two researchers (A. Gobind and G. Zondi). The sample was randomly divided into two groups, with one group receiving an active substance (verum group) and the other group receiving a placebo (control group). Amongst the 30 provers, 24 participants received verum and six received placebo. Each prover was given a journal to record their symptoms, a pen and nine powders. The duration of this proving study was six weeks including the first week of baseline during which time each prover was required to record their symptoms before taking the proving substance. This procedure was clearly explained to each prover and an information sheet with detail explanation of the proving procedure was provided and informed consent obtained. A thorough case history was taken in the initial consultation and physical examinations to ensure that individuals participating met the inclusion criteria and were healthy. Results Information derived both researchers was combined and the extraction of signs and symptoms then commenced. The symptoms were translated into materia medica and repertory format and graded accordingly. Thereafter the symptoms that emerged from the study were discussed and compared with the doctrine of signatures of Acacia xanthophloea. Provers experienced a wide range of symptoms mentally and emotionally e.g. anger, poor concentration and focus, disconnected feeling, depression, stress, anxiety, antisocial/ aversion to company, tranquillity. A large number of physical symptoms were noted by provers e. g. headaches, ailments of extremities (joint pain, weakness), female related symptoms (irregular menses, decrease/ increase libido etc.), eye symptoms, skin symptoms and general symptoms. The comparison of the symptoms of Acacia xanthophloea 30CH to the doctrine of signatures of the tree Acacia xanthophloea illustrated certain similarities, especially with regard to skin symptoms, eye symptoms, gastrointestinal symptoms and headaches. Conclusion The proving substance Acacia xanthophloea 30CH did produced signs and symptoms when given to healthy individuals as hypothesised. The symptoms when compared to the doctrine of signatures of the acacia xanthophloea tree did show correlation as hypothesised.
M

Submitted in partial compliance with the requirements for the Master’s Degree in Technology, Department of Homoeopathy, Durban University of Technology, Durban, South Africa, 2015.
INTRODUCTION Homoeopathy is based on the law of similars meaning the medicine that produces symptoms in a healthy individual will cure the same symptoms in a sick individual (Sankaran, 1991:5). AIM Conducting a proving on Ubiquinone 30CH will lead to an establishment of its therapeutic potential through the application of the law of similars thus adding to the Materia Medica and advancing Homoeopathy (Vithoulkas, 2002). It was hypothesised that the 30CH potency of Ubiquinone would clearly produce observable signs and symptoms in healthy prover’s. It was further hypothesised that a comparison of Ubiquinone to those remedies yielding the highest numerical value and total number of rubrics on repertorisation of the proving symptoms would elucidate differences and similarities between Ubiquinone and other Homoeopathic remedies to clarify its therapeutic indications. It was hypothesised that in this manner a better understanding of Ubiquinone and its relationship to other Homoeopathic remedies would be gained. Methodology The proving of Ubiquinone 30CH was a randomised, double blind placebo controlled study, using the 30th centesimal potency and a total of 26 participants who met the inclusion criteria. Each prover was provided with a journal to record their symptoms daily. The data extracted from the journals were added to the case histories and physical examinations to compile a proving profile. The identity of the substance was revealed and the information was correlated after completion of the proving. The symptoms found were translated into Materia Medica and repertory language. Once the proving was concluded, a comparison to the remedies yielding the highest numerical value and total number of rubrics on repertorisation - which is the technique of using a repertory to identify the Homoeopathic medicines whose Materia Medica corresponds most closely to the clinical picture of the patient and from amongst which a simillimum may be chosen (Swayne, 2000:183) - was compared to the proving symptoms. Results The remedy’s main influence was on the mental and physical state. The most prominent symptoms seen in the mental sphere were extreme irritability and exhaustion. There was a sense of emotional fragility with a desire to be alone. On the physical side, headaches were common and weakening pains of the extremities were experienced. It can be concluded that the 30CH potency of Ubiquinone, if used precisely according to Homoeopathic principles, can be applied to a clinical setting, as the extensive range of symptoms produced during the proving suggests an equally wide array of application of the remedy Ubiquinone. Conclusion One of the downfalls of Homoeopathy is the limited number of provings being done, (Vithoulkas, 2002). Vithoulkas (2002:143) maintains that in order for Homoeopathy to advance, it is necessary to perform provings on new substances to expand the Homoeopathic armamentarium. Increasing the number of remedies in the Materia Medica facilitates greater accuracy and individualisation when treating patients (Wright, 1999). According to Herrick (1998) numerous cases cannot be solved because many of the most important remedies have not yet been developed. The purpose of this study was to increase the knowledge of drug substances due to the limited amount of information in our current Materia Medicas, by investigating the therapeutic potential of Ubiquinone 30CH. The investigation supported the hypothesis that Ubiquinone would produce clearly observable signs and symptoms in healthy volunteers. It is essential that the proving symptoms be verified and expanded through clinical use and with further proving of Ubiquinone in various potencies so that it becomes a well utilised remedy in the future.

Submitted in fulfillment of the Master’s Degree in Homoeopathy, Durban University of Technology Durban, 2014.
The purpose of this research study was to determine any therapeutic significance of Malus domestica (domestic apple) in the potentised, homoeopathic form and to contribute this information to the body of the homoeopathic materia medica. It was further hypothesised that some proving symptoms experienced by the provers during the research study would show a resemblance to unique characteristics of the plant in terms of its natural appearance and cultural references. This involved a detailed doctrine of signatures analysis of Malus domestica based on an extensive literature review. Combining a proving research study with comparative analysis to the doctrine of signatures helped to clarify and verify the remedy’s potential therapeutic value. Methodology This homoeopathic proving was carried out in the form of a double blind placebo controlled study of Malus domestica 30CH with a total of 30 provers. The prover sample was randomly divided into two groups: 24 provers (80%) into the verum group and the remaining six provers (20%) into the placebo group. The provers were unaware of either the proving substance or the potency used. Participants were required to record their mental, emotional and physical status’ one week prior to administration of the proving powders as a form of control for comparison of symptoms post administration of the proving remedy. Thorough physical examinations and case histories of each prover were taken prior to and after the proving period. Provers ingested one powder three times a day for three days and recorded their symptoms daily in a journal. The duration of the proving period spanned five weeks. During this period researchers were in constant contact with all participants. Once the proving period was complete, all journals were gathered and the information therein translated into materia medica and repertory format so as to develop the remedy picture of Malus domestica 30CH. A comparison between the symptomatology produced in the provers and the doctrine of signatures was then performed. Results A wide range of symptoms were documented by the provers. The results revealed an affinity to the mental plane – the most striking being the theme of being disconnected and separated from others in the environment as well as within one’s self. Marked polarities were depicted as follows: Anger/irritability vs. calmness/tranquility; Depression/sadness vs. cheerfulness; Confusion vs. concentration/clarity of mind; Connection vs. disconnection. Physically, symptoms concerning the extremities were numerous, with provers describing symptoms of their limbs being disconnected/separated as well. Head symptoms were also numerous as provers described a wide range of headaches, as well as headaches that were associated with eye symptoms. Abdominal symptoms manifesting as cramping associated with diarrhoea and, in some cases, constipation were documented. Dizziness associated with the sensation of the head floating was common as well. Numerous themes arising from dreams were recorded by provers, with a clear affinity to family members as well as dreams of social gatherings and banquets. Provers dreamt of family members placed in precarious situations wherein the lives of their loved ones were at risk, whilst the emotion of guilt was expressed in certain dreams regarding parties and banquets. The comparison between the homoeopathic drug proving of Malus domestica 30CH and the doctrine of signatures brought many similarities to light, with most of these similarities relating to the mind, extremities and head. Conclusion As hypothesised Malus domestica 30CH produced clearly observable symptoms in healthy provers. On comparison, the proving remedy and the doctrine of signatures brought many parallels to light – the majority of which related to the mind, extremities and head. To gain a complete remedy picture of Malus domestica it is imperative that further research into the symptomatology of different homoeopathic potencies be conducted.

Submitted in fulfillment of the Master’s Degree in Homoeopathy, Durban University of Technology, Durban, South Africa, 2016.
Introduction The aim of this study was to determine the effect of Hoodia gordonii 30CH on healthy provers, and to record the clearly observable signs and symptoms produced and the subsequent comparison to its toxicology as a raw substance and to homoeopathic remedies of reportorial similarity. Methodology The investigation was a randomised, double-blind placebo controlled trial, using the substance in the 30th potency. This was prepared according to the German Homoeopathic Pharmacopoeia. A sample of 20 provers, in good health, was recruited. Sixteen received verum as the experimental group and four in the control group received a placebo. Each of the 20 provers received a journal in which they recorded symptoms on a daily basis for a period of six weeks, including a one week observation prior to taking the powders, and a period of five weeks after administration of the powders. The information from the journals and case histories was edited, collated and translated into materia medica and repertory language and used to compile a proving profile of the remedy by qualitative methods. The remedy was only revealed to the participants after completion of the proving study period. The researcher compared the similarities and new symptoms of the remedy with the existing knowledge of its toxicology to prove the first hypothesis. A detailed and extensive literature review of Hoodia gordonii’s unique characteristics was conducted. A further comparison with other similar remedies according to the highest numerical number of rubrics on repertorisation was conducted. Results The proving remedy produced a wide variety of symptoms on the mental, emotional and physical levels of which many had polarities. In broad terms the following were identified from the proving symptoms of Hoodia gordonii: • Increased confidence and feeling refreshed, renewed energy, cheerfulness, concentration, alertness, calm, forgetfulness, unhappy, sadness and depression with desire to be left alone. • Anxiety for unknown reason and for the future, about finances; restlessness and busyness. • Tiredness, fatigue and exhaustion. • Positive feeling regarding home and family. • Mental exhaustion and aversion to study. • Common sensations were throbbing, pulsating, sharp, aching, heat, pressing, heavy, pulling, splitting, cramps. • Perceived attack or danger in dreams. • Painless diarrhea and constipation, nausea, headaches, vertigo, sore red eyes, nasal congestion and sinuses, toothache, tonsillitis and dryness of throat; bronchitis, heart palpitations. • Menses with breast tenderness and increased sexual stimulation, joint pain. • Unquenchable thirst, polyuria, appetite increase and decrease, sleeplessness and sleepiness with deep sleep, cold, increase perspiration, influenza and depressed immune function. Conclusion The data obtained from this proving study of Hoodia gordonii when comparing the new symptoms of the remedy with similarities to the existing knowledge of its toxicology proved the first hypothesis to be true. Three hundred and twenty four rubrics were produced and 17 were new rubrics; of these a total of 20 rubrics that represented the essence of the remedy were selected and used in the repertorisation process. A comparison of the highest numerical value of rubrics with other similar remedies on repertorisation found Hoodia gordonii 30CH to be most similar to: Atropa belladonna, Phosphorus, Lachesis mutus, Sulphur and Veratrum album.
M

Mini-dissertation submitted in partial compliance with the requirements for the Master’s Degree in Technology: Homoeopathy in the Department of Homoeopathy, Durban University of Technology, 2011.
The aim of this study was to determine the effect that Curcuma longa 30CH would have on healthy individuals, and record the particular signs and symptoms produced. These signs and symptoms determine the therapeutic indications of this remedy, so that it may be prescribed according to the homoeopathic Law of Similars. The second aim of this study was to analyse the symptomatology of Curcuma longa 30CH in relation to a Doctrine of Signatures analysis of the Curcuma longa plant, in order to facilitate a more comprehensive understanding of the materia medica of this substance. Design The homoeopathic proving of Curcuma longa in 30CH potency took the form of a double blind, randomized, placebo controlled trial. Thirty healthy provers were selected on the basis of them meeting with the necessary inclusion criteria (Appendix A). The provers were randomly divided into 2 groups, of which 20% (6 of the 30 provers) formed the placebo group and received non-medicated powders, and the remaining 80% (24 of the 30 provers) received medicated powders (verum). The 2 groups were not aware of the nature of the substance that they were proving or the potency used. The provers recorded their mental, physical and emotional states over a period of a week prior to taking the remedy in order to establish a baseline for comparison after the administration of the remedy. Both verum and placebo were dispensed in the form of 6 powders. Each powder was taken sublingually 3 times daily for 2 days or until the prover experienced the onset of any symptoms. Each prover kept a journal and recorded their proving signs and symptoms daily after administration of the remedy or the placebo. The data was collected and extracted from these journals and then assessed by the researcher for suitability to be included in the materia medica of Curcuma longa. All data gathered from the case histories (Appendix C), physical examinations and group discussions were also considered for inclusion. Results A variety of mental, emotional and physical symptoms were produced and included in the materia medica of Curcuma longa. There were a total number of 202 symptoms that were produced as a result of the remedy, which resulted in the formulation of 141 rubrics. The main mental and emotional symptoms that surfaced during the proving were depression, a deep sadness, changeability of moods, courage/confidence, relaxed/ calm and less anger, agility, increased concentration, and vivid dreams. The physical symptoms noted were diarrhea, change in energy levels (too much or too little energy), burning sensations, headaches, heart palpitations and increased breathing rates. The symptoms that came about during the proving clearly showed correlation and association with the nature and description of the Turmeric plant, this is in keeping with findings of previous provings (Pistorius, 2006; Webster, 2002; Speckmeier, 2008 & Pather, 2009), furthermore as suggested by Richardson-Boedler (1999:173) the Doctrine of Signatures analysis of the Turmeric plant facilitated in the interpretation of the proving symptoms and thus the materia medica of the remedy.

Mini-dissertation submitted in partial compliance with the requirements for the Master’s Degree in Technology: Homoeopathy, Durban University of Technology, 2011.
Introduction The purpose of this study was to determine the therapeutic potential of Curcuma longa 30CH when administered to healthy individuals, thus revealing the materia medica of the substance. It was also the aim of this study to compare the existing therapeutic indications of the substance to the proving symptomatology. Methodology The proving took the form of a double-blind placebo controlled study and was conducted by two Master’s in Technology: Homoeopathy students using 30 healthy subjects. Twenty four provers were given the active medication and six provers were given the placebo. The remedy was manufactured according to the German Homoeopathic Pharmacopoeia in 30CH potency. The proving ran for a period of six weeks. Results The symptoms extracted from the proving were placed in different sections according to the repertory and was compared to the Ayurvedic and Phytotherapeutic indications of Curcuma longa. There were 202 symptoms produced as a result of the remedy, 141 rubrics were formulated using these symptoms. The largest number of rubrics i ii was allocated to the mind, head and dreams section of the repertory, other smaller sections of prominence included the eye, ear, nose and throat sections. A wealth of information was gained once the comparison was made between Curcuma longa 30CH and the Phytotherapeutic and Ayurvedic indications of use. Similarities between the materia medica of Curcuma longa and the Phytotherapeutic indications of use were found to exist with respect to sections such as eye, nose, face, stomach, stool, respiration, back, extremities, skin and generals. Conclusion The administration of Curcuma longa 30C to healthy provers according to the methodological protocol of this study resulted in the production of a variety of defined proving symptoms which comprise the materia medica thereof (first objective of the study). The subsequent comparison of the proving symptoms with the existing indications of Turmeric as an Ayurvedic and Phytotherapeutic medicine (second objective of the study) revealed clear correlations in a variety of defined areas.

Submitted in fulfillment of the Master’s Degree in Homoeopathy, Department of Homoeopathy, Durban University of Technology, Durban, South Africa, 2016.
Aim The aim of this homoeopathic proving study was to determine and document the arising symptomatology of Acacia xanthophloea (Fever tree) in the potentised homoeopathic form, 30CH, and to provide this data for inclusion to the homoeopathic materia medica. The results of this proving study and comparative analysis to African traditional medicinal uses of this substance confirms the potential therapeutic value of the remedy. Methodology The homoeopathic drug proving of Acacia xanthophloea 30CH was conducted in the form of a double blinded placebo controlled study. The investigation consisted of a total of 30 provers divided equally between two researchers ((A. Gobind and G. Zondi). The sample was randomly divided into two groups in which 24 provers(80%) were assigned into the verum group and the remaining six provers (20%) were allocated to the placebo group. All provers were requested to record their daily symptoms on the physical, mental and emotional planes in their journals for one week prior to administration of the proving substance. This formed as a mode of control for the comparison of symptomatology for the pre-proving and post proving period. A thorough case history was taken and physical examination performed on each prover before the commencement of the proving and after the duration of the proving period. Each prover received a total of nine powders. Starting on day 8 of the study the provers consumed one powder three times a day for three days and documented their daily symptoms in a journal. The duration of the proving term was six weeks in total. During this interval the researcher maintained consistent contact with the provers. Upon completion of the proving period all journals were collected and the information contained within these journals was translated into the materia medica and repertory format. This facilitated the establishment of the remedy portrait of Acacia xanthophloea 30CH. A subsequent comparison between the symptomatology that materialised in the provers and the African traditional medicinal uses was duly conducted. Results An extensive range of symptoms was reported by the provers. The outstanding themes that emanated from this proving on the mental plane include anger, anxiety, aversion to company, cheerful, depression, irritability, mood swings, restlessness, tranquillity and stress. A broad range of headaches were described with some headaches being associated with the eye. There were many eye symptoms displayed by the provers which include itching, redness, burning sensation and pain. The stomach symptoms revealed marked increased thirst, changeable appetite, bloating, constipation and diarrhoea with watery stools.The female genitalia / sex indicated several symptoms ranging from painful menstruation, bleeding and copious blood flow.The greatest number of symptoms in a system was associated with extremities, producing the greatest number of rubrics in the repertory section. Dream themes depicted by the provers were especially visionary, about family and friends in addition to other themes. The correlation process between the homoeopathic drug proving of Acacia xanthophloea 30CH and the African traditional medicinal use of the substance brought several resemblances to light.There were clear similarities with the eye symptoms, gastrointestinal symptoms and headaches. Conclusion As hypothesised Acacia xanthophloea 30CH did produce distinctly observable signs and symptoms when administered to healthy provers. The symptoms that emerged during the proving provide evidence that an overlay exists between the remedy Acacia xanthphloea 30CH and the traditional use of the crude substance Acacia xanthophloea. The researcher proposes that further research should be conducted to determine the symptomatology of various homoeopathic potencies so that a complete image of the remedy Acacia xanthophloea 30CH can be established and the clinical applications can be broadened.
M

Dissertation submitted in partial compliance with the requirements of the Masters Degree in Technology: Homoeopathy, Durban Institute of Technology, 2003.
The purpose of this randomised double-blind placebo-controlled study was to evaluate the efficacy of Oioscorea vil/osa cream in the treatment of menopausal syndrome in terms of subjective and objective data; and to compare the subjective data with that obtained from a concurrent study of ProgestoNat\xAE cream (McTeer, 2003).
M

Dissertation submitted in partial compliance with the requirements for the Master‟s Degree in Technology: Homoeopathy, Durban University of Technology, 2011.
Until recently, various attempts have been made to simplify the prescription process in homoeopathic prescribing. The doctrine of signatures, miasmatic theory, the homoeopathic repertory and more recently, kingdom analysis by authors such as Sankaran (1994) and Scholten (1993) are some of the attempts that have been made to understand the materia medica. With the materia medica constantly expanding and considering that plants make up a significant percentage of the materia medica (Kayne, 2006), it is evident that new systems of homoeopathic prescribing are continually needed to help practitioners both study the remedies and prescribe more accurately. Aim The Solanaceae plant family are an important and well utilised plant family in homoeopathy (Vermeulen, 2004). Considering this, it was felt that a study investigating the relationship of the natural history of the family to its general and mental symptoms be conducted in order to apply a previously unexplored research paradigm in order to create a greater understanding of Solanaceae remedies utilised in homoeopathy. The study conducted was a non empirical correlation study of the Solanaceae plant family‟s natural history and general and mental symptoms manifested in Solanaceae remedies utilised in homoeopathy. The aims of the study were to establish if commonalties existed between general and mental symptoms of individual remedies belonging the Solanaceae family and their natural histories, as well as to establish if collective commonalities and correlations existed between the general and mental symptoms and the natural history of the Solanaceae family as a whole. Methodology The homoeopathic remedies obtained from the Solanaceae family of plants for the study were analysed in terms of rubric representation (size) using homoeopathic software packages, Radar 10.4 (Archibel, 2009b) and v Encyclopedia Homoeopathica (Archibel, 2009a) a sample selection was chosen. This selection was analysed in terms of general and mental rubrics. Qualitative thematic analysis was used to establish commonalities in keyword concepts between the respective natural histories of the studied family and their respective general and mental symptoms. Keywords obtained from data tables which included criteria such as habitat and distribution, plant description, active principles (primary alkaloids), uses, physiological action if ingested, historical significance, mythology and toxicology were subjected to thesaurus consultation and tabulated in an attempt to identify synonyms relating to the general and mental symptoms of individual remedies of the sample group in the study. This facilitated in the grouping of similar themes. Once commonalities pertaining to each individual species and remedy was further tabulated and discussed in terms of keywords relating to their natural histories, a collective analysis of common correlations between the plant family as a whole was performed. Results Common themes related to general and mental symptoms and to the natural histories of species in the study included “aggression” found in Atropa belladonna, “depression” found in Solanum dulcamara, “anxiety” found in Datura stramonium, “confusion” found in Hyoscyamus niger, “burns” found in Capsicum annuum, “ convulsions” found in Nicotiana tabacum , “ hallucinations” found in Mandragora officinarum and “delirium” found in Solanum nigrum. Common themes relating to general and mental symptoms and the natural history of the Solanaceae plant family as a whole included convulsions, hallucinations, confusion and anxiety. These themes were further compared to themes exhibited in Solanaceae studies conducted by Mangialavori (2007) and Sankaran (2002).

Mini-dissertation submitted in partial compliance with the requirements for the Master's Degree in Technology: Homoeopathy, Durban University of Technology, 2010.
Introduction The aim of the study was to determine the effect of Acridotheres tristis 30CH on healthy volunteers (provers) and to record the clearly observable signs and symptoms produced, so that Acridotheres tristis 30CH may be prescribed according to the Law of Similars, as required by homoeopathy. Methodology The proving of Acridotheres tristis took the form of a randomised, placebocontrolled trial on 30 healthy volunteers who met inclusion criteria. The 30th centesimal potency of the tail feather was administered as lactose powders and fifty percent of provers were randomly administered an identical placebo-control substance. The collection of the data from the provers took the form of a journal in which each prover‟s symptoms were recorded for six weeks, including a one-week observation period prior to taking the powders, and a proving period of five weeks after administration of powders. On completion of the proving, each journal was assessed by the researcher to determine the suitability of the recorded symptoms for inclusion in the materia medica of Acridotheres tristis. These symptoms were then translated into the language of the materia medica and repertory and the remedy picture then formulated. Data from case histories, physical examinations and group discussions were also taken into account during the analysis of the proving symptoms. Results The homoeopathic drug proving of Acridotheres tristis, conducted as a doubleiii blind, randomised and placebo-controlled study produced a wide range of symptoms. In the collated edited data arising from the proving 396 journal entries were extracted and 595 rubrics in total were formulated, of which 56 rubrics were new. The main symptoms belonging to the mental and emotional spheres of this remedy include depression, anxiety, memory weakness and isolation accompanied by a need for solitude. The characteristic physical symptoms include headaches, dizziness, extreme fatigue, skin eruptions especially pimples and rashes, haemorrhoids, numbness and severe dysmenorrhoea. Other symptoms indicate a possible use in the treatment of gastro-intestinal complaints which include nausea, abdominal cramps, abdominal distention and heartburn. Conclusion The investigation supported the hypothesis that Acridotheres tristis would produce clearly observable signs and symptoms in healthy volunteers. It is essential that the proving symptoms be verified and expanded through clinical trials and further provings of Acridotheres tristis in various potencies, so that it becomes well-utilized remedy in the future.