Oral drug delivery is considered the most popular route of delivery because of the ease of administration, availability of a wide range of dosage forms and the large surface area for drug absorption via the intestinal membrane. However, besides the unfavourable biopharmaceutical properties of the therapeutic agents, efflux transporters such as Pglycoprotein (P-gp) and multiple resistance proteins (MRP) decrease the overall drug uptake by extruding the drug from the cells. Although, prodrugs have been investigated to improve drug partitioning by masking the polar groups covalently with pre-moieties promoting increased uptake, they present significant challenges including reduced solubility and increased toxicity. The current work investigates the use of amino acids as ion-pairs for three model drugs: indomethacin (weak acid), trimethoprim (weak base) and ciprofloxacin (zwitter ion) in an attempt to improve both solubility and uptake. Solubility was studied by salt formation while creating new routes for uptake across the membranes via amino acids transporter proteins or dipeptidyl transporters was the rationale to enhance absorption. New salts were prepared for the model drugs and the oppositely charged amino acids by freeze drying and they were characterised using FTIR, 1HNMR, DSC, SEM, pH solubility profile, solubility and dissolution. Permeability profiles were assessed using an in vitro cell based method; Caco-2 cells and the genetic changes occurring across the transporter genes and various pathways involved in the cellular activities were studied using DNA microarrays. Solubility data showed a significant increase in drug solubility upon preparing the new salts with the oppositely charged counter ions (ciprofloxacin glutamate salt exhibiting 2.9x103 fold enhancement when compared to the free drug). Moreover, permeability studies showed a 3 fold increase in trimethoprim and indomethacin permeabilities upon ion-pairing with amino acids and more than 10 fold when the zwitter ionic drug was paired with glutamic acid. Microarray data revealed that trimethoprim was absorbed actively via OCTN1 transporters while MRP7 is the main transporter gene that mediates its efflux. The absorption of trimethoprim from trimethoprim glutamic acid ion-paired formulations was affected by the ratio of glutamic acid in the formulation which was inversely proportional to the degree of expression of OCTN1. Interestingly, ciprofloxacin glutamic acid ion-pairs were found to decrease the up-regulation of ciprofloxacin efflux proteins (P-gp and MRP4) and over-express two solute carrier transporters; (PEPT2 and SLCO1A2) suggesting that a high aqueous binding constant (K11aq) enables the ion-paired formulations to be absorbed as one entity. In conclusion, formation of ion-pairs with amino acids can influence in a positive way solubility, transfer and gene expression effects of drugs.

Tuberculosis (TB), an infection caused by human pathogen Mycobacterium tuberculosis, continues to kill millions each year and is as prevalent as it was in the pre-antimicrobial era. With the emergence of continuously-evolving multi-drug resistant strains (MDR) and the implications of the HIV epidemic, it is crucial that new drugs with better efficacy and affordable cost are developed to treat TB. With this in mind, the first part of this thesis discusses the synthesis of libraries of derivatives of pyridine carboxamidrazones, along with cyclised (1,2,4-triazole and 1,2,4-oxadiazole) and fluorinated analogues. Microbiological screening against M. tuberculosis was carried out at the TAACF, NIAID and IDRI (USA). This confirmed the earlier findings that 2-pyridyl-substituted carboxamidrazones were more active than the 4-pyridyl-substituted carboxamidrazones. Another important observation was that upon cyclisation of these carboxamidrazones, a small number of the triazoles retained their activity while in most of the remaining compounds the activity was diminished. This might be attributed to the significant increase in logP value caused by cyclisation of these linear carboxamidrazones, resulting in high lipophilicity and decreased permeability. Another reason might be that the rigidity conferred upon the compound due to cyclisation, results in failure of the compound to fit into the active site of the putative target enzyme. In order to investigate the potential change to the compounds’ metabolism in the organism and/or host, the most active compounds were selected and a fluorine atom was introduced in the pyridine ring. The microbiological results shows a drastic improvement in the activity of the fluorinated carboxamidrazone amides as compared to their non fluorinated counterpart. This improvement in the activity could possibly be the result of the increased cell permeability caused by the fluorine. In a subsidiary strand, a selection of long-chain , -unsaturated carboxylic esters, -keto, -hydroxy carboxylic esters and -keto, -hydroxy carboxylic esters, structurally similar to mycolic acids, were synthesised. The microbiological data revealed that one of the open chain compound was active against the Mycobacterium tuberculosis H37Rv strain and some resistant isolates. The possible compound activity could be its potential to disrupt mycobacterial cell wall synthesis by interfering with the FAS-II pathway.

Liposomes offer an ideal platform for the delivery of subunit vaccines, due to their versatility and flexibility, which allows for antigen as well as immunostimulatory lipids and TLR agonists to become associated with these bilayered vesicles. Liposomes have the ability to protect vaccine antigen, as well as enhance delivery to antigen presenting cells, whilst the importance of cationic surface charge for delivery of TB subunit vaccines and formation of an ‘antigen depot’ may play a key role in boosting cell-mediated immunity and Th1 immune responses. The rational design of vaccine adjuvants requires the thorough investigation into the physicochemical characteristics that dictate the function of a liposomal adjuvant. Within this thesis, physicochemical characteristics were investigated in order to show any effects on the biodistribution profiles and the ensuing immune responses of these formulations. Initially the role of liposome charge within the formulation was investigated and subsequently their efficacy as vaccine adjuvants in combination with their biodistribution was measured to allow the role of formulation in vaccine function to be considered. These results showed that cationic surface charge, in combination with high loading of H56 vaccine antigen through electrostatic binding, was crucial in the promotion of the ‘depot-effect’ at the injection site which increases the initiation of Th1 cell-mediated immune responses that are required to offer protection against tuberculosis. To further investigate this, different methods of liposome production were also investigated where antigen incorporation within the vesicles as well as surface adsorption were adopted. Using the dehydration-rehydration (DRV) method (where liposomes are freeze-dried in the presence of antigen to promote antigen encapsulation) and the double emulsion (DE) method, a range of liposomes entrapping antigen were formulated. Variation in the liposome preparation method can lead to antigen entrapment within the delivery system which has been shown to be greater for DRV-formulated liposomes compared to their DE-counterparts. This resulted in no significant effect on the vaccine biodistribution profile, as well as not significantly altering the efficacy of cationic liposomal adjuvants. To further enhance the efficacy of these systems, the addition of TLR agonists either at the vesicle surface as well as within the delivery system has been displayed through variation in the preparation method. Anionic liposomal adjuvants have been formulated, which displayed rapid drainage from the injection site to the draining lymph nodes and displayed a reduction in measured Th1 immune responses. However, variation in the preparation method can alter the immune response profile for anionic liposomal adjuvants with a bias in immune response to Th2 responses being noted. Through the use of high shear mixing and stepwise incorporation, the efficient loading of TLR agonist within liposomes has been shown. However, interestingly the conjugation between lipid and non-electrostatically bound TLR agonist, followed by insertion into the bilayer of DDA/TDB resulted in localised agonist retention at the injection site and further stimulation of the Th1 immune response at the SOI, spleen and draining lymphatics as well as enhanced antibody titres.

Oral liquid formulations are ideal dosage forms for paediatric, geriatric and patient with dysphagia. Dysphagia is prominent among patients suffering from stroke, motor neurone disease, advanced Alzheimer’s and Parkinson’s disease. However oral liquid preparations are particularly difficult to formulate for hydrophobic and unstable drugs. Therefore current methods employed in solving this issue include the use of ‘specials’ or extemporaneous preparations. In order to challenge this, the government has encouraged research into the field of oral liquid formulations, with the EMEA and MHRA publishing list of drugs of interest. The current work investigates strategic formulation development and characterisation of select API’s (captopril, gliclazide, melatonin, L-arginine and lansoprazole), each with unique obstacles to overcome during solubilisation, stabilisation and when developing a palatable dosage from. By preparing a validated calibration protocol for each of the drug candidates, the oral liquid formulations were assessed for stability, according to the ICH guidelines along with thorough physiochemical characterisation. The results showed that pH and polarity of the solvent had the greatest influence on the extent of drug solubilisation, with inclusion of antioxidants and molecular steric hindrance influencing the extent of drug stability. Captopril, a hydrophilic ACE inhibitor (160 mg.mL-1), undergoes dimerisation with another captopril molecule. It was found that with the addition of EDTA and HP-β-CD, the drug molecule was stabilised and prevented from initiating a thiol induced first order free radical oxidation. The cyclodextrin provided further steric hindrance (1:1 molar ratio) resulting in complete reduction of the intensity of sulphur like smell associated with captopril. Palatability is a crucial factor in patient compliance, particularly when developing a dosage form targeted towards paediatrics. L-arginine is extremely bitter in solution (148.7 g.L-1). The addition of tartaric acid into the 100 mg.mL-1 formulation was sufficient to mask the bitterness associated with its guanidium ions. The hydrophobicity of gliclazide (55 mg.L-1) was strategically challenged using a binary system of a co-solvent and surfactant to reduce the polarity of the medium and ultimately increase the solubility of the drug. A second simpler method was developed using pH modification with L-arginine. Melatonin has two major obstacles in formulation: solubility (100 μg.mL-1) and photosensitivity, which were both overcome by lowering the dielectric constant of the medium and by reversibly binding the drug within the cyclodextrin cup (1:1 ratio). The cyclodextrin acts by preventing UV rays from reaching the drug molecule and initiated the degradation pathway. Lansoprazole is an acid labile drug that could only be delivered orally via a delivery vehicle. In oral liquid preparations this involved nanoparticulate vesicles. The extent of drug loading was found to be influenced by the type of polymer, concentration of polymer, and the molecular weight. All of the formulations achieved relatively long shelf-lives with good preservative efficacy.

Background: The role of the community pharmacist has evolved from compounding and dispensing to providing patient focused services which require more patient interaction. Previous research has described pharmacist consultation skills as not optimal or patient centred. The aim of the thesis was to add an in depth understanding about the possible reasons behind this. Method: The thesis comprises three studies; the first study used focus groups to investigate community pharmacists’ experiences and perceptions of their consultations with patients. The second study was the first nationwide questionnaire based study to investigate consultation skills training received by community pharmacists. The final study was a feasibility study to investigate the use of an innovative interactional-analysis methodology known as the Roter Interactional Analysis to audio recorded community pharmacy consultations. Results: The results showed while community pharmacists enjoy speaking to patients, a number of factors limit the quality of these interactions. The nationwide questionnaire results indicates that a large number of community pharmacists have not had any formal consultation skills training and seek more advanced consultation skills training. Analysis showed consultation skills training could influence confidence and had a positive impact on the delivery of more patient facing services. The use of an interactional analysis system is a useful tool to develop future consultation skills training in community pharmacy. Conclusion: The thesis has provided a more in depth understanding of the consultation based challenges facing community pharmacists, community pharmacy as a profession and researchers investigating pharmacist-patient interaction. It has also identified many areas which require further development if community pharmacists are going to undertake high quality consultations. It will be important for these to be fully considered if any future proposed changes to community pharmacy roles are to be successful.

I would like to thank my primary supervisor Dr. Sheng Qi for the invaluable support and encouragement received during my PhD time at UEA. Thanks also to my supervisors Prof. Peter Wilde and Prof. Duncan Craig for their help. I would also like to express my gratitude to Prof. Pete Belton, Dr. Francesca Baldelli Bombelli from the UEA School of Pharmacy for their precious scientific collaboration. Thanks to Dr. Patrick Gunning, Dr. Nicola Woodwards and Mr. Andrew Kirby, Dr. Paul Kroon and Mr. Mark Winterbone from the Institute of Food Research for their help with Nanosight, pendant drop and biological experiments. I would like to thank all the friends that have been part of my life in Norwich, for the happy times together and for being supportive when I mostly needed. A special thanks goes to Yohan for being a good friend and taking care of me as a brother. Thanks to my housemates Desirè and Hanae, for their friendship and their constant support. Thanks to Francesca and Alberto for all the laughes and their invaluable help and care. Thanks to Lorina, Elisabetta, Marcello, Antonella and Tiziana for being good friends during these years. Thanks to all the other friends from the schools of Pharmacy and Chemistry. I would also like to thank my friends from Italy, in particular Ilaria for being a constant presence and support in my life. Finally I would like to thank my family, my outstanding parents for their love, their encouragement and support. Thanks to my sister Flavia, for being the special sister she is.

Aim: To identify type(s) and possible cause(s) of medicine-related problems (MRPs) from the SA and ME patients' perspectives. Setting and Method: The study was a cross-sectional study. Patients were from SA and ME origins, aged over 18 and prescribed three or more regular medicines. Patients were identified through previous medicine use reports (MUR), patient medication records (PMR) or when presenting with a prescription. The data were collected in 80 face-to-face semi-structured interviews in seven pharmacies in London using MRPs tool, 8-item MMAS and EuroQol questionnaire (EQ-5D-3L). Interviews were audiotaped, transcribed verbatim and analysed thematically using Gordon’s coding frame and Nvivo 10 software. The SPSS 21.0 software was used for analysis of descriptive data obtained quantitatively. Results: Participants (61% male) had mean (SD) age 58 (13.4) years and on a mean (SD) of 8 (4) medicines. Final analysis showed the following types of MRPs: adverse drug reactions and drug interactions; intentional non-compliance; cognitive, physical and sensory problems and issues with concurrent use of herbal and alternative therapies. Problems with drug prescribing; lack of information; monitoring and review; repeat prescriptions; GP surgery and pharmacy service were also identified. Interviews revealed that several factors contribute to the development of MRPs; some appeared to be specific to SA and ME cultures and others were similar to the general population. Many of these factors could be expected to influence patient’s safety, adherence, and informed decision-making. Conclusion: This study demonstrated that SA and ME patients have their own problems and needs with both medicine use and service access. By uncovering particular problems experienced by these groups the study can inform healthcare professionals to support SA and ME patients in the use of their medicines.

No description available.

No description available.

The p53/MDM2 protein-protein interaction is the most widely characterised proteinprotein interaction to date. As of 2014, there are over 20 compounds that have been shown to the p53-MDM2 protein-protein interaction, however many compounds have not progressed into clinical trials due to their high hydrophobicity. Herein we describe the synthesis, molecular modelling, physical characterisation and biological testing of novel inhibitors of the p53/MDM2 protein-protein interaction based on the natural product chlorofusin. The first focus is a combinatorial library generated in the Searcey laboratory of known p53/MDM2 protein-protein interaction inhibitors with the desire to generate novel analogues and study their interactions with the protein through NMR spectroscopy and molecular modelling. These compounds were tested by in a fluorescence polarisation assay and also in cell lines overexpressing MDM2 as well as p53-null cells as a comparator. This generated two novel compounds shown to have activity selectively for the p53/MDM2 protein-protein interaction. The second chapter focuses on simplified substitutions of the azaphilone (the chromophore portion of chlorofusin, a natural product inhibitor of the p53-MDM2 proteinprotein interaction): initially with simple fused bicyclic carboxylic acids and later using click chemistry substitutions. Interestingly, in vitro studies showed that the click analogues retained activity or activity improved when the peptide portion was removed and hence further studies of the click amino acid analogues were generated. This library generated one analogue that was active in vitro as well as selectively in MDM2-overexpressing cell lines. The third chapter focusses on the azaphilone chromophore present in the natural product chlorofusin. The Sonogashira precursor used to generate azaphilone analogues was synthesised using a methodology adopted by Porco et al and subsequent analogues were generated using a novel double-Sonogashira approach followed by functionalisation published by Boger et al. Once the azaphilone was synthesised, metholodogies were trialled in order to condense the azaphilone with the chlorofusin peptide in order to create analogues containing both the peptide and small molecule portions of chlorofusin. In addition, molecular modelling was attempted to generate novel binding analogues.