Summary Background Elevated blood lipids (particularly cholesterol and sub-fractions) contribute to the risk of developing cerebral, peripheral and cardiovascular disease and associated complications which are leading causes of morbidity and death. Statins reduce the risk of suffering vascular events, with or without decreasing cholesterol levels. Statin prescribing continues to increase but there is scope to improve prescribing and dosing, particularly in primary care. However, there is insufficient empirical evidence to inform approaches to quality improvement. Methods Following pilot work, we designed a new model of primary care based pharmacist-led intervention for General Practitioners (GPs) and nurses. The aim of the intervention (called Statin Outreach Support, SOS) was to improve statin prescribing by GPs, in line with recent evidence, targeting patients at highest risk of suffering a vascular event. Eleven trained pharmacists worked in SOS allocated practices one day per week for a year. During this period, the pharmacist met three times with all GPs, all nurses and other practice staff. Between meetings, pharmacists used patient level clinical and prescribing data to identify eligible patients and help practices initiate, up-titrate the dose or switch to simvastatin 40mg where indicated. The effectiveness of SOS was tested in a prospective single blind cluster randomised controlled trial. Usual care (UC) practices received no pharmacist support during the study. With a mean of 1.7 years follow up, the study had over 90% power (at 5% significance) to detect a difference of 12% in the proportion of patients with controlled cholesterol after practices had received the SOS intervention. Results Thirty one practices were recruited from the UK’s largest Health Board area. At randomisation, 16 practices were allocated to the SOS intervention and 15 to UC with 4,040 patients included at baseline. Recruited practices showed few differences compared with invited, non participating practices. Practices and patients randomised to each arm of the study had similar distributions with respect to age, complications, cholesterol levels and statin prescribing. The mean age was 68 years; 53% male, 45% ischaemic aetiology. Fifty nine percent had no statin prescribed at baseline; only 51% had cholesterol controlled. Follow up included 7586 patients in 29 practices (one practice had disbanded between recruitment and randomisation and another practice dropped out). Compared with UC, the SOS intervention achieved the primary endpoint of increasing the proportion of patients prescribed Simvastatin 40mg with controlled cholesterol (SOS 44.9% vs. UC 27.9%; odds ratio 1.79 (95% CI: 1.61, 1.98), p< 0.001). Secondary endpoints were also improved in the SOS arm practices. The intervention effect was strong and consistent across most subgroups including a positive impact on patients from practices in areas of greater socioeconomic deprivation. Conclusion A pragmatic, new, complex intervention was developed, tested and shown to be effective in a cluster randomised controlled trial with good internal and external validity. If implemented on a wider scale, in practices with comparable characteristics and baseline prescribing, the SOS intervention has the potential to reduce the burden of vascular events for patients with vascular disease. This work provides a convincing evidence base for the role of pharmacists collaborating with primary care practices, to improve statin prescribing and drug based cholesterol management, for patients at highest risk of suffering vascular events.

This thesis describes work towards the biomimetic synthesis and understanding the biosynthesis of two families of natural products: prodiginines and quartromicins. Prodiginines are a large family of red pigmented tripyrrole antibiotics. Although they have not been used clinically, the promising anti-cancer, immunosuppressive and antimalarial activity they display at non-toxic doses has generated renewed interest in their utilisation. The synthesis of an analogue of the proposed pyrrole-2-carboxyl-RedO intermediate in prodiginine biosynthesis has been achieved. The resulting NAC thioester and analogues of it have been used to investigate the prodiginine biosynthetic pathway in Streptomyces coelicolor, and to examine the production of prodiginine analogues by mutasynthesis. Quartromicins, novel anti-viral antibiotics, are a structurally unique group of spirotetronate natural products produced by Amycolatopsis species. They are unusual symmetric macro cyclic compounds which possess a 32-membered carbocyclic structure with four spirotetronic acid units connected by enone or dienone linkers in a head-to-tail fashion. These macrocyclic compounds are intriguing because they have alternating endo- and exo- spirotetronic acid units, with the opposite "comers" being identical. Although the quartromicins have therapeutic potential, very little is known about their biosynthesis. In this research a biosynthetic pathway to the quartromicins has been proposed based on hypothetical pathways to related natural products. The synthesis of the two putative key intermediates in quartromicin biosynthesis has been achieved. An improved method for the synthesis of exomethylene tetronates has been developed, and novel rearrangements have been discovered. The two putative key intermediates have been used to investigate the biomimetic synthesis of the carbon skeleton of the quartromicin algycone, and mass spectrometric evidence for formation of homo- and heterodimers, and a heterotetramer of the key intermediates has been obtained.

Cancer is the most common cause of the human death in the UK. Every year 3.2 million Europeans are diagnosed with cancer, and the figure is projected to rise due to the aging population. Although significant advances are being made in the fight against the disease, cancer remains a key public health concern and a tremendous burden on UK society in financial and social terms. This thesis evaluated two classes of compounds that can be potentially developed as anti-cancer agents. 4-(4-Methyl-2-(methylamino)thiazol-5-yl)-2-(4-methyl-3-(morpholinosulfonyl)phenylamino)pyrimidine-5-carbonitrile (S-134, 5g) is a novel cyclin-dependent kinase 9 (CDK9) inhibitor showing nano-molar growth inhibitory potentials in established human cell lines. Biochemical assays have confirmed that S-134 primarily inhibits CDK9 at the protein and cellular levels, respectively. The detailed mechanistic investigation demonstrated that the compound caused wild-type p53 stabilisation and cell cycle arrest at the G2/M transition. Cancer cell death induced by S-134 was proven by Annexin-V/PI staining, caspase-3 assay and PARP cleavage. Transcription and expression of anti-apoptotic proteins Mcl-1, Bcl-2 and XIAP were reduced by the inhibitor, as proved by RT-PCR and Western blots respectively. Compound 2-methoxy-5-(3,4,5-trimethoxyphenethyl)phenyl 2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole-3-carboxylate-N-oxide (ZJU-6, 6g) a semi-synthetic derivative of the natural medicinal compound Erianin (6a) was designed to introduce anti-oxidant property and enhance anti-angiogenic activity of Erianin. The study of ZJU-6 revealed that while the effect of cellular growth inhibition and the transcription of Bcl-2 were reduced by the structural modification, the suppression of tubulin polymerisation and anti-angiogenic properties were enhanced compared to Erianin. Compound BI 2536, 4-(8-cyclopentyl-7-ethyl-5-methyl-6-oxo-5,6,7,8-tetrahydropteridin-2-ylamino)-3-methoxy-N-(1-methylpiperidin-4-yl)benzamide (10), is one of the most potent and selective inhibitors of Polo-like kinase 1 (Plk1) and is a current experimental candidate for the treatment of cancer. A racemic BI 2536 was synthesised using multiple synthesis routes, which aimed to use as a lead compound for the discovery of novel Plk1 inhibitors.

The pulmonary delivery of drugs is an attractive route of administration because of the large surface area and high permeability of the airway epithelium. The large majority of inhaled drugs are used to manage asthma and Chronic Obstructive Pulmonary Disorder (COPD), such as inhaled corticosteroids and β2-adrenergic receptor agonists. Local delivery of small molecules often results in sub-optimal pharmacokinetics characterised by short absorption times (tmax) and high systemic concentrations (Cmax). Numerous drug delivery strategies have been attempted to increase lung retention time, including drug encapsulation in microspheres, the use of polymeric excipients, or the formation of low solubility drugs. So far, drug conjugation strategies have been limited to decreasing the prodrug solubility. The non-permanent conjugation of small molecules to a large hydrophilic polymer has not been studied for pulmonary delivery. The rationale behind such a strategy is that small molecules are mainly absorbed through the epithelium by passive diffusion, the absorption rates being positively correlated to the drug lipophilicity and molecular weight. This project has therefore been looking at the production, characterisation, in vitro and ex vivo evaluation of polyethylene glycol (PEG)-ester conjugates for the sustained delivery of drugs to the lung. This thesis presents the successful oxidation and subsequent esterification of PEG of various molecular weights with prednisolone (a corticosteroid) and salbutamol (a β2-adrenergic receptor agonist). This study illustrated the feasibility of a polymeric drug conjugate strategy for sustained release of drugs to the lung. The conjugates exhibited good in vitro stability which was translated into improved pharmacokinetics and longer residence time ex vivo in the isolated and perfused rat lung. Further studies must be conducted to fully assess the role of esterases in the pulmonary hydrolysis of the conjugates and in vivo experiments would be necessary to verify the safety of the conjugates and efficacy of the drug.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a widely used surface chemical analysis technique that is traditionally employed to characterise the first few molecular layers of a material interface. The ability of this technique to accurately reflect the surface chemistry of polymers, biomaterials and many other solid materials is well documented. However, the majority of research that utilises this technique is based upon a qualitative rather than quantitative assessment of the material under investigation. The qualitative analysis of a range of traditional tablet and bead formulations containing drug and multiple excipients was performed in order to identify key diagnostic ions for all the different components. The lateral distributions of the ions across the surfaces of these formulations were imaged. Two different methods were then used to perform a qualitative analysis of the surfaces and results from these experiments were compared to the bulk composition. The effect of surface roughness on the ability to produce reproducible quantitative analyses from ToF SIMS ion yield data was investigated. A range of samples with different topographies were studied including polytetrafluoroethylene (PTFE), glass microscope slides, gold coated abrasive papers and gold coated precision measurement samples. The surface roughness was assessed by Atomic Force Microscopy and Laser Profilometry. Samples were analysed in imaging mode and the variance in ionization across the total image was measured for each sample. Evidence is presented that there is a relationship between ion yield and surface roughness, and that the surface roughness of the analysed surfaces will effect on any quantification approach in the processing of ToF-SIMS data. In addition, the presence of any orientation/directionality in surface features also needs to be evaluated when considering use of a quantitative approach. To investigate the effect of chemical environment on the ability to derive quantitative data from ToF SIMS analysis of pharmaceutical materials, drug loaded spun cast polymer films with low surface roughness were studied. ToF SIMS data were obtained for two chemically similar drugs in two different polymer matrices. In the majority of the samples there was no quantitative relationship between drug ion intensity and nominal bulk composition. Due to the large sample set, the multivariate technique, Principal Component Analysis (PCA) was employed to look at variance in secondary ion yields from the different samples. PCA is becoming more prevalent in ToF-SIMS data interrogation as it allows for a mathematically un-biased analysis of sample variables through the identification of the ions that account for the majority of the variance in the sample set. PCA successfully highlighted the impact of the chemical environment, showing secondary ion yields of drugs can be dependent on the surrounding matrix. PCA was also used to look at variance in two of the tablet samples and was successfully able to differentiate between the tablet samples with the lowest and highest concentrations of paracetamol. This thesis has demonstrated that surface topography and surface chemical environment or matrix will have a significant impact on ion yields in the ToF-SIMS experiments. These findings suggest caution in the use of ToF-SIMS for the quantitative analysis of complex chemically heterogeneous and topographically diverse pharmaceutical formulations.

Informal learning is a major factor influencing the professional development and practices of health professionals (Eraut, 1994). This thesis is an in-depth exploration of how this process occurs in pharmacy and involves a detailed study of practising pharmacists. The learning approach of these individuals is explored in relation to the variety of working situations in which they practise and the prevailing climate in relation to professional learning and development within the National Health Service (NHS). This study therefore adds to an understanding of the way in which informal learning shapes the practice of pharmacists and as such it has considerable implications for both future policy and practice. In the past there has been very little detailed research investigation into informal learning in pharmacy, although studies by Wilson, Schlapp & Davidson (2003) and Swallow et al (2006) have demonstrated the importance of this aspect of professional development. This study addresses that deficit, utilising semi structured interviews and focus groups to explore in some depth the nature of pharmacists’ informal learning and their perceptions of the effectiveness of current CPD practices in supporting such learning. The study reveals that pharmacists use a range of informal learning methods to develop in their careers post-qualification, including experiential learning and reflective practice. Many also continue to take further formal courses and qualifications. Practitioners perceive knowledge to be of particularly high value, and place less emphasis and value on the learning of skills, attitudes and behaviours, despite their comprising a vital part of practice. The role of helpful others (Eraut et al., 2004) plays a critical part in the professional learning and development of many pharmacists. They appear to value this support highly and in some cases rely on it due to the isolated nature of their practice situation. Paradoxically, whilst pharmacists acknowledge the need to provide evidence of their ongoing professional development, they often do not complete CPD records in practice. One of the main criticisms offered, in relation to the CPD system, was the perceived limitations of the RPSGB Plan & Record forms, and this was also used as a justification for not completing their records. Greater flexibility in the system was seen as vital for the full benefits and strengths of the CPD system to be realised. The change management process through which the RPSGB introduced CPD is critically examined and the literature on educational change processes utilised, to suggest ways in which the implementation process of CPD may have created the resistance evident in the pharmacists’ narratives. This thesis raises questions about the value that pharmacists and the pharmacy profession place on various types of learning. The importance of informal learning in the development of pharmacists is emphasised. The thesis also explores the apparent need for pharmacists to have access to appropriate helpful others and the need to ensure that the method used to record CPD is flexible and fit for purpose.

In chapter two, the development of new a nanoparticulate carrier system for gene delivery was described. The new nanocarrier consists of a blend matrix formed by a poly (lactic-eo-glycolic acid) (PLGA) and Poly(ethylene glycol) bis (3-aminopropyl) terminated (also known as JeffamineTM). Nanopartic1es were formulated based on a 50:50 weight ratio of PLGA:Jeffamine using a modified emulsification-solvent diffusion technique. The potential of these blended matrix nanoparticles for encapsulation efficiency of Calf Thymus DNA and release profile were also studied. The achieved encapsulation efficiency of Calf Thymus DNA was approximately 84% for 0.4% theoretical loading with regard to total amount of PLGA. The PLGA: Jeffamine blended nanoparticles provided continuous and controlled release of Calf Thymus DNA. The PLGA:Jeffamine nanopartic1es were also coated with PLGA-PEGMA&75and PDMAEMA-PEGMA block copolymers using a simple physical adsorption method. After surface coating of the nanoparticles, zeta potential value showed significant reduction of surface charges from -38 mV to near zero value, while TEM micrographs showed a well defined core-shell nanoparticle. In chapter three, A facile route to biocompatible poly (lactic acid-coglycolic acid)-co-poly (ethyleneglycol methacrylate) (PLGA-PEGMA) block co-polymers was described utilising a combination of ring-opening polymerisation (ROP) and Radical Addition Fragmentation Transfer (RAFT) methods. A series of PLGA-PEGMA polymers varying in comonomer content and block length were synthesised with low polydispersities. All the block co-polymers formed micelles in aqueous solution as shown by dynamic light scattering, while critical micelle concentrations were found to be in the micromolar range. The polymer micelles were able to encapsulate model drugs(carboxyfluorescein and fluorescein isothiocyanate) and selected copolymer micelles incubated with 3T3 fibroblasts as a model cell line were rapidly taken up as indicated by fluorescence microscopy assays. The combination of the polymer chemistries opens the way to highly flexible syntheses of micellar drug carrier systems. In chapter four, multifunctional and modular block co-polymers prepared from biocompatible monomers and linked by a bioreducible disulphide linkage have been prepared using a combination of ring-opening and atom-transfer radical polymerizations (ATRP). The presence of terminal functionality via ATRP allowed cell-targeting folic acid groups to be attached in a controllable manner, while the block co-polymer architecture enabled well-defined nanopartic1es to be prepared by a water-oil-water double emulsion procedure to encapsulate DNA with high efficiency. Gene delivery assays in a Calu-3 cell line indicated specific folatereceptor-mediated uptake of the nanoparticles, and triggered release of the DNA payload via cleavage of the disulfide link resulted in enhanced transgene expression compared to non-bioreducible analogues. These materials offer a promising and generic means to deliver a wide variety of therapeutic payloads to cells in a selective and tuneable way.

The aim of the project was to investigate the potential of ionic polysaccharide/drug complexes as controlled release drug delivery systems. Two highly purified alginates from Laminaria hyperborea and Ascophyllum nodosum were characterised in terms of molecular weight, polydispersity and M:G ratio using gel permeation chromatography (GPC), low-speed sedimentation in the analytical ultracentrifuge and GPC combined with multi-angle laser light-scattering. Viscometric and nephelometric studies provided evidence that, above certain concentrations of propranolol, there was an interaction between propranolol and alginate in deionised water resulting in the formation of an insoluble complex, which dissociated in the presence of counter-ions, for example, sodium chloride. Binding studies were undertaken using equilibrium dialysis in order to quantify this interaction in the presence and absence of sodium chloride. These indicated that there was a one-to-one stoichiometric relationship between propranolol and the carboxyl group on each uronic acid reSidue of the alginate and that negative co-operativity was occurring, such that the binding of one propranolol molecule to the alginate made it more difficult for subsequent propranolol molecules to bind. The possible in-vacuo three-dimensional structure of the molecular complex was modelled using computational molecular modelling techniques. A freeze-dried complex of propranolol and alginate was prepared and characterised. In vitro investigations indicated that drug release from the complex (formulated as a suspension in deionised water or in isotonic glycerol solution) was delayed compared with release from a propranolol solution. The release of propranolol from the propranolol/ alginate complex was assessed in vivo using the anaesthetised rat as an animal model for nasal delivery. It was found that the rate of absorption of propranolol from the complex was much slower and was sustained over a greater period of time, compared with absorption from a propranolol solution. In addition, the bioavailability of the drug from the complex was comparable to that of the solution and to that of an intravenous dose carried out in rats by other workers (Hussain et al 1980b).

The effect of nanoparticle charge and conjugation to the cell penetrating peptide Tat on the uptake of nanoparticles into MRC-5 fibroblasts was investigated using a range of advanced imaging techniques including atomic force microscopy (AFM), scanning ion conductance microscopy (SICM) and fluorescence microscopy. New technologies are required to investigate the uptake of nanoparticles since although many studies have examined their destination within cells, little work has been done looking at the interaction between the nanoparticles and cell which also plays an important role. Polyacrylamide nanoparticles showed a clear correlation between increased positive charge and cellular uptake. These results obtained using fluorescence microscopy correlated well with those observed using AFM. Nanoparticles with low levels of positive charge caused minor effects on bilayer structure such as fusion of holes in the bilayer, whilst negatively charged nanoparticles had no effect on the bilayer. Polyacrylamide nanoparticles conjugated to the cell penetrating peptide Tat displayed greater cell uptake and increased effects on the bilayer depth and coverage than the similarly charged nanoparticles alone. This indicated that the peptide leads to enhanced uptake by additional mechanisms other than just the presence of a positive charge. In contrast, silica nanoparticles with high levels of positive and negative charge entered cells to a similar extent. However, when the interaction with the cell membrane was imaged using Scanning Surface Confocal Microscopy (SSCM), there were differences. Nanoparticles possessing a negative charge were often found to be associated with extensions of the membrane. Positively charged particles were also found associated with membrane extensions in some cases but were also observed isolated on the membrane. The results highlight the importance of using multiple imaging techniques to investigate cellular interaction and uptake in order to provide a complete picture of all the processes involved.

Randomised controlled trials (RCTs) play a fundamental role in the development and marketing activities of pharmaceutical companies. They are the primary means of evaluating the tolerability, safety and efficacy of a drug, and for providing information relevant for pricing and reimbursement decisions and clinical decision-making. RCTs require a substantial investment by pharmaceutical companies and the financial consequences of poorly or sub-optimally designed trials are potentially substantial. Revenue does not materialise unless a licence to market a product is granted and sales may be restricted if a trial fails to provide evidence of sufficient strength or relevance for those involved in product adoption decisions. From a pharmaceutical company's perspective, the value of RCTs can therefore be judged on the contribution they make to the performance of a drug in the market and hence on their contribution to the performance of the firm. Consequently the design choices made in the planning of RCTs are effectively investment appraisal decisions. However, the application of investment appraisal techniques to RCT design has not previously been proposed. The purpose of this thesis is to consider how private sector investment appraisal methods might be applied to RCT design decision-making and to explore aspects of the practicalities of application. A general investment appraisal model is presented and its application to determine profit maximising RCT designs is illustrated. Considering the cost side of the investment appraisal equation, it is shown how decision-makers' requirements for cost-effectiveness evidence derived from trials could have a significant impact on the major determinants of cost (sample size and study duration) depending on their specific preferences for evidence defined over key components of RCT design. Considering the revenue side of the investment appraisal equation, it is shown how discrete choice analysis could be used to incorporate decision-makers' preferences for RCT designs into the planning of studies. Specifically, it is shown how the predicted probabilities derived from the application of this technique could be used within an investment appraisal framework. Directions for future research into the application of investment appraisal to RCT design are proposed.