Following nutrient ingestion, glucagon-like peptide 1 (GLP-1) secreted from intestinal L-cells mediates anti-diabetic effects, most notably stimulating glucose-dependent insulin release from pancreatic β-cells but also inhibiting glucagon release, promoting satiety and weight reduction and potentially enhancing or preserving β-cell mass. These effects are through the GLP-1 receptor (GLP-1R) which is a therapeutic target in type 2 diabetes. The present study focused on desensitisation and re-sensitisation of GLP-1R-mediated signalling and interactions of orthosteric and allosteric ligands. Data demonstrate GLP-1R desensitisation and subsequent re-sensitisation following removal of extracellular ligand with ligand-specific features. Following GLP-1-mediated desensitisation, re-sensitisation is dependent on receptor internalisation, endosomal acidification and receptor recycling. Re-sensitisation is also dependent on endothelin converting enzyme-1 (ECE-1) activity, possibly through proteolysis of GLP-1 in endosomes, facilitating disassociation of receptor-β-arrestin complexes leading to GLP-1R recycling and re-sensitisation. ECE-1 activity also regulates GLP-1-induced activation of extracellular signal regulated kinase (ERK) and generation of cAMP possibly through a G protein independent/β-arrestin dependent mechanism. By contrast, following GLP-1R activation by the orthosteric agonist, exendin-4, or allosteric agonist, compound 2, re-sensitisation was slow and independent of ECE-1 activity. Thus, different ligands depend on different events during GLP-1R trafficking which could be important for re-sensitisation and signalling, particularly that mediated by scaffolding around β-arrestin. As the GLP-1R is targeted therapeutically at orthosteric and allosteric sites, this study examined activation of the GLP-1R by orthosteric and allosteric agonists and in particular interactions between ligands of these sites. Challenging the GLP-1R with the allosteric ligand, compound 2, along with GLP-1 9-36 amide, a low affinity, low efficacy metabolite of GLP-1 7-36 amide, results in synergistic receptor activation. This may be important for therapeutic approaches with allosteric ligands, as metabolites of GLP-1 may be present in vivo at concentrations higher than the classic endogenous ligand. Indeed this could present a novel therapeutic approach.

No description available.

No description available.

Organosilica nanoparticles, due to their versatile properties, attract considerable attention for pharmaceutical applications. The first chapter of this thesis gives an introduction to organosilica materials and traditional methods of their synthesis, provides a literature review on functionalisaUon of silica nanoparticles, in particular PEGylation, and describes recently investigated routes of their application in pharmaceutical science. This is followed by a brief description of methods and techniques used in the current study for synthesis and characterisation of organosilica nanoparticles. Chapter 3 represents a novel size-controlled method of synthesis of thiolated and PEGylated organosilica nanoparticles from 3-mercaptopropyltrimethoxysilane (MPTS) using various organic solvents as media. The mechanism of the nanoparticle formation is proposed, the dependence of nanoparticles size on solvent's dielectric constant is determined, and other factors affecting the nanoparticle size are investigated. Thiol-groups on the surface of organosilica nanoparticles are available for a direct conjugation. Therefore, their surfaces were funcUonalised with two types of fluorescent dyes and PEG maleimide of two molecular weights (750and 5000 Oa). Additionally, polymer- and surfactant-templated mechanisms of MPTS nanoparticle formaUon are reported. The synthesis chapter is followed by studying the properties of thiolated and PEGylated organosilica nanoparticles. Mucoadhesive properties of silica nanoparticles were investigated using a newlyintroduced in vitro method assessing their retention ability on the urinary bladder by the means of fluorescence microscopy. The main factors affecting mucoadhesive properties of MPTS nanoparticles were revealed and thiolated nanoparticles were demonstrated to be promising mucoadhesive materials for intravesical drug delivery. PEGylated nanoparticles were found to be less mucoadhesive which led to the hypothesis of providing better permeation by PEGylation. This was considered in the subsequent chapter (chapter 5), studying the barrier functions of the cornea using thiolated and PEGylated nanoparticles. The "whole eye" in vitro method combined with the fluorescence microscopy demonstrated a good permeation of the latter through de-epithelialised ocular tissue into the stroma. This revealed the interaction of nanoparticles with the corneal surface to be a more impcrtant factor than the particle size, determining their permeation ability. As most organs of the human body are covered with biological hydrogels, the ability of silica nanoparticles to permeate through biological tissues has risen an interest in studying their capability to diffuse through such gels. Therefore, the diffusion of thiolated and PEGylated nanoparticles in different polymer solutions, which were selected as prototypes of biological hydrogels, was studied using NanoSight tracking analysis with fluorescent detection. The main factors affecting the diffusion of organosilica nanoparticles were revealed, and it was demonstrated that the presence of strong attractive interactions between the nanoparticles and macromolecules present in solutions can hamper the diffusion. This was followed by studying biocompatible properties of organosilica nanoparticles using an alternative to traditional methods, a slug mucosal irritation test, revealing their non-irritant nature. Finally, the general results of the current study are summarised and discussed in the last chapter of the thesis, proposing plans for the future work and expansion of the investigations on organosilica nanoparticles presented so far.

The aim of this thesis was to highlight the use of hot melt extrusion (HME) as a manufacturing process in the development of single and multi-dose, multi-layered solid oral dosage forms using celluloses to control drug release. Furthermore the ability of HME to be used as a technique to manufacture multi-layered tablets was analysed becoming cognisant of the factors that influence drug release from such systems. The impact of HME, polymer type, drug type, drug loading and tablet dimensions on the amount of drug released and the drug release mechanisms were defined and utilised to produce a clinically relevant fixed dose combination (FOG) that contained aspirin and simvastatin. Cellulose based formulations containing HPG, HPMG and MG were successfully manufactured using hot melt extrusion as a rapid continuous process for the production of solid oral dosage forms that offered sustained and controlled drug release. Furthermore, HME was successfully used to manufacture cellulose based single dose and fixed dose combination (FOC) multi-layer tablets. The application of layers reduced the surface area to volume (SA V) ratio of the drug layers, significantly affecting the amount of drug released, while polymer swelling characteristics were also identified as contributing factors that modulated drug release. The promising performance of the HME multi-layered cellulose based formulations at controlling drug release led to the design of a clinically relevant FOC that contained simvastatin and aspirin. The FOG had to sustain the release aspirin while delaying the release of simvastatin to when it is optimally effective, with less than 10% of maximum simvastatin release occurring before 2 hours. The combination of dissolution, mechanical and process analysis proved successful in the selection of candidate formulations and tablet designs. Results confirmed that 32.75% aspirin and 32.3% simvastatin were suitable formulations, and therefore progressed as candidate formulations for future testing beyond this study

Azithromycin, a macrolide antibiotic, has a poor stability in solution . The hydrolysis of the a-glycosidic bond in acidic condition yields the major degradation product of azithromycin that is desosaminylazithromycin. One answer to this problem is the application of the freeze-drying process. However, freeze-drying is known as an expensive and time consuming process thus the optimum freeze-drying condition is absolutely important for cost-effective manufacturing. In this thesis, a new, more efficient freeze-drying process for an azithromycin formulation has been successfully developed. Integrated pharmaceutical freeze-dried product development beginning from characterization of the formulation solution, freeze-drying cycle optimization, stability study, and scale-up process was performed. Various thermoanalytical techniques including Differential Scanning Calorimetry, Freeze Drying Microscopy, Thermogravimetric Analysis, and Dynamic Vapor Sorption were used. Scanning Electron Microscopy, X-Ray Powder Diffraction, Raman Microscopy, and HPLC were used as complementary methods to fully characterize the formulation. A significant reduction in freeze-drying time compared to the established process from 98 to 46 hours was achieved without compromising product quality. This time saving could not only increase the production efficiency but also reduce the production cost due to lower power consumption by the freeze-dryer. The stability of the developed formulation was demonstrated for 6 months in 40 °C/75 % RH. The design space of primary drying process of azithromycin formulation was successfully developed based on the heat and mass transfer equation. This model was proven useful to predict the outcome of the product temperature which is the most important parameter that determines the product quality outcome in the freeze-drying process and furthermore makes the scale-up process straightforward. The last part of this thesis discussed the promising possibility of a new drug delivery system based on azithromycin encapsulation in liposomes. Freeze-drying and spray-drying were proven useful in the drying process of this liposomal azithromycin formulation.

Pharmaceutical care and medicines management are terms which are often used interchangeably to describe the patient-centred services provided by pharmacists. The programme of research presented within this thesis evaluated a range of topics within this area of endeavour. The extent of pharmaceutical care provision within the community pharmacy setting was examined in a cross-sectional survey in N. Ireland and across 15 European countries. The findings indicated that community pharmacists' provision of comprehensive pharmaceutical care can be deemed as limited in Northern Ireland (mean score 41.4% of the total achievable score) and across Europe (scores ranged from 56.6% in England to 29.4% in Moldova). To inform the development of a definitive RCT for novel post-discharge medicines management clinic (MMC), a feasibility study was carried out, and identified a number of methodological challenges, in particular, challenges around patient recruitment, attrition rates and clinic scheduling. A series of risk assessment models was developed to help with the targeting of patients to be recruited for the RCT referred to above. These models were designed to forecast the likelihood of patient early readmission to hospital (within 30 and 8 to 30 days) and high-cost readmissions. Important risk factors for readmission identified included the Charlson age-adjusted co-morbidity score, respiratory- and genitourinary-related primary diagnoses and number of medications prescribed on discharge, and new patient targeting criteria for the RCT to explore the impact of the MMC was developed. A systematic content analysis revealed that the print media coverage of medicine-related adverse effects requires significant improvement in order to properly inform the public on this matter. Specifically, the reporting of adverse effects, in terms of medicines implicated and organ systems affected, did not correspond with those commonly cited in scientific journal articles. Findings from the present research help contextualise and provide evidence based advice on a number of issues which need to be addressed in pharmaceutical care / medicines management practice and research.

Variable drug response and lack of information on drug disposition and action in rare diseases are challenges that need to be addressed for safe and effective provision of medicines. The main aim of work presented in this thesis was to address these challenges in selected areas of liver disease in children. Population pharmacokinetic and pharmacogenetic analyses in children with liver transplant revealed that tacrolimus apparent clearance decreased over time, and was higher in carriers of the CYP3A5*1 allele in both donors and recipients, however, the recipient genotype showed a more profound impact. A sub-analysis in CYP3A5 non-expresser recipients, revealed that tacrolimus clearance in this subpopulation was higher in carriers of the POR*28 allele. When compared to liver transplant patients, children with intestinal transplant exhibited higher tacrolimus clearance. Several genetic variations were investigated for correlation with phannacodynamic outcomes; of these, the IL-4 -590 C allele showed a trend towards association with acute rejection in intestinal transplant patients, while selected ABCB1 SNPs at 6 months post-transplant were associated with tacrolimus associated nephrotoxicity only in liver transplant patients. Data on the use of nitisinone for treatment of tyrosinemia type I in children, revealed that, compared to dietary treatment alone, the use of this agent is associated with enhanced patient survival and decreased need for transplant, especially if the therapy is initiated early after birth. A distinct α-fetoprotein profile was noted in children who developed hepatic cancers. The population pharmacokinetics of nitisinone was evaluated for the first time in the UK population and a gradual time dependent decrease in nitisinone clearance was observed over a period of five years. A secondary aim of the present thesis was to analyse the content, quality and trends of reporting of issues relating to liver/intestinal transplantation in UK and USA newspapers. The newspaper coverage of this topic was generally poor in quantity and in quality. The general slant regarding transplantation and donation was positive, but few articles provided information regarding the means through which an individual could become a donor. In general the current research demonstrated the usefulness of population approaches in analysing the pharmacokinetics of drugs prescribed to children and the role of genetics in variable pharmacokinetic and pharmacodynamic outcomes of narrow therapeutic index drugs such as tacrolimus.

Over the last half a century, hot-melt extrusion (HME) technology has emerging as an extensively studied new strategy of solid dispersion manufacture to achieve enhanced aqueous solubility and hence improved bioavailability for BCS Class II drug. During HME processing, .a number of parameters, such as barrel temperature, screw rotation speed and, screw element configuration, may significantly influence of the properties of the extrudates. However, the determination of those parameters has been often experience oriented rather than rationality directed. In this thesis, Flory-Huggins theory was adapted in combination with melting point depression measurement to address the mixing between the model drug and the chosen excipient as a function of temperature. For each studied binary system, a temperature-composition phase diagram was constructed depicting the direct relationship between interspecies mixing and temperature. Upon construction of such a phase diagram, the mixing between drug and excipient may be split into three zones, namely the soluble, intermediate and, the insoluble zone, by two boundary curves. It was understood that these two boundary curves could be critical in determining HME processing temperatures for binary systems when morphology alterations were concerned. Moreover, a free energy of mixing-composition phase diagram might be generated to reveal spontaneity and extent of mixing as temperature changes. Such information might be used to determine whether increase in mechanical input (kinetics) could significantly compensate the lack of thermodynamic forces during morphology alterations. In a study of amorphous drug dispersions (ADD), results indicated that ADD was only achievable when the HME processing temperature exceeded the solubility curve (boundary between the soluble region and the intermediate region). At low temperatures when spontaneous. interspecies mixing was prevented, increased screw speed would significantly improve amorphization of the model drug. Conversely, when processed at high temperatures and the mixing is thermodynamically favoured, changes in screw speed would not bring significant effects to the solid-state properties of the extrudates. The subsequent drug dissolution studies, however, revealed significantly different ranking of formulations for solubility enhancement due to dissolution mechanism change with test medium pH variations. It was discovered that extreme HME processing parameters, such as particularly high processing temperature or screw speed, could result in uncontrollable extrudate physical properties such as undesired cavity increase, and thus, exhibit unsatisfying dissolution profiles. The adaptation of Flory-Huggins theory and the use of those phase diagrams were also expanded into small molecular material processes. In the latter chapters, an ibuprofen-isonicotinamide cocrystal suspension in a noncomplementary carrier excipient was formulated using single step HME processing for the first time. Upon construction of temperature-composition and free energy-composition phase diagrams, it was established the relationship between solubility of cocrystal components in the chosen carrier excipient and the HME processing temperature. By doing so, appropriate HME processing temperature was selected for the chosen ternary system to achieve increased co crystal yield in the carrier. It was also proved in the last experiment chapter the drastic influence of the incorporation of mixing/kneading elements to the screw configuration on the cocrystal yield within the carrier matrix. Moreover, it was suggested that detailed arrangement of screw geometry ought to vary from case to case based on the cocrystallization mechanism along HME processing. It was emphasized that elements showing distinctively dispersive mixing capacities should be used in the melting zone, whereas those showing distinctively distributed mixing capacities should be employed in a later 'flushing' stage in the cooling zone.

The primary function of airway smooth muscle is to contract, regulating the airway tone and bronchial narrowing. Research extending over a number of decades has identified many of the major factors contributing to the degree of bronchial contraction at a cellular level including receptor mediated activation of airway smooth muscle by agonists, electrical and other membrane events, intracellular cascades and assembly of contractile and cytoskeletal proteins. Furthermore, airways function is also regulated by a number of key airway, lung and teleological events that collectively determine the bronchial tone. Therefore, the aim of this thesis was to investigate a number of factors influencing directly and indirectly the responsiveness of human bronchi. The tone of human bronchi was studied in isolated organ bath systems in both normal tissues and passively sensitized bronchi, a model that mimics some aspects of the airway wall of patients with allergic airways disease, since it is very difficult to obtain bronchi from such people. Furthermore, immunohistochemistry and PCR were carried out in bronchial tissues whilst immunoenzymatic assays were performed in cultured bronchial smooth muscle cells. A range of studies were carried out in order to investigate factors influencing bronchial responsiveness, including the pharmacological characterization of adenosine receptors, the effects of brain natriuretic peptide, the influence of lipopolysaccharide, the role of hyperglycemia, the activity of vasoactive intestinal peptide receptors and the influence of phosphodiesterase 3/4 enzymes and their interaction with both sympathetic (β2 mediated) and parasympathetic pathways. This study provides evidence for the role of adenosine AI receptor in causing human airway smooth muscle contraction, both directly, and indirectly following the activation of capsaicin sensitive sensory nerves, and the release of leukotrienes. Evidence is also provided for the relaxant effect of brain natriuretic peptide mainly regulated by the autocrine activity of epithelial cells. Furthermore, the contribution of neutral endopeptidases, capsaicin-sensitive sensory nerves, the mevalonate pathway and the protective influence of statins against lipopolysaccharide induced airway hyperresponsiveness has been demonstrated. Other results have shown that high glucose concentrations leads to an enhanced responsiveness of human airway smooth muscle, and that the Rho/ROCK pathway may play a crucial role for reducing lung function in hyperglycemic conditions. Finally, findings concerning the bronchial relaxation mediated by the selective activation of vasoactive intestinal peptide receptor 2 and the synergism of phosphodiesterase 3/4 inhibitors in association with antimuscarinics and β2-agonist drugs suggest new paradigms for treating obstructive lung diseases. Results presented in this thesis provide strong evidence of several novel pharmacological approaches to influence human airway smooth muscle tone.