Cardiovascular disease (CVD) is currently a global epidemic and is now the leading cause of mortality worldwide. The two major approaches for the prevention of CVD in the developed world are public health based Policies and clinical based Strategies focusing on high-risk individuals. Pharmaceutical companies have developed a range of treatments to tackle the causes of CVD and these include the development of anti-inflammatory, anti-hypertensive and anti-cholesterol drugs. However, unpleasant side-effects often exist with prescription drug-taking. Sourcing of natural food components from foods to provide protection against the development of CVDis a useful strategy to help combat illness. This thesis aims to utilise Irish macroalgae as a source of bioactive compounds which can be delivered in the food vehicle Bread to confer a Heart health effect to the consumer. Hypertension, one of the major risk factors associated with CVD may be controlled by inhibiting enzymes of the reninangiotensin aldosterone System (RAAS). Inhibition of the enzyme renin is an important strategy for the control of hypertension as renin is the initial and rate limiting enzyme of the RAAS. Inhibition of the circulating enzyme platelet activating factor acetylhydrolase (PAF-AH) is also important in the control of atherosclerosis development. PAF-AH generates two pro-inflammatory mediators lysophosphatidylcholine (LPC) and oxidized non-esterified fatty acids (oxNEFAs). Both of these mediators are involved in promotion of atherosclerotic plaque which may be lead to high blood pressure development. Macroalgae are part of the regular diet of many Asian cultures and have a tradition of being consumed in many coastal regions of the Western World. Regular consumption of macroalgae is associated with a decline in the prevalence of breast cancer and diabetes mellitus development. Species of macroalgae belonging to the group known as Rhodophyta or the red macroalgae are known to contain levels as high as 47 % protein. Several bioactive peptides, including ACE-Iinhibitory peptides were isolated previously from macroalgae protein extracts and hydrolysates. In this thesis the extraction and isolation of renin and PAF-AH inhibitory peptides from the macroalgae Palmaria palmata was carried out. The effectiveness of the isolated renin inhibitory tridecapeptide IRLIIVLMPILMA was further explored in terms of its capacity to survive gastrointestinal (GI) digestion and its ability to lower blood pressure in vivo in spontaneously hypertensive rats (SHRs). The Palmaria palmata protein hydrolysate from which these peptides were identified was subsequently incorporated in to bread and the effects of its addition were observed in terms of volume, colour, texture profile, moisture, crumb structure, sensory attributes and renin inhibitory activity.

The efficacy of protein-based medicines is frequently compromised by rapid clearance from circulation. Achieving prolonged and optimal pharmacokinetics (PK) is necessary to avoid dose dumping while ensuring that a therapeutic concentration can be maintained between each time the the dose is administered. Optimised PK properties are important for therapeutic proteins, such as interferon alpha 2a (IFN) which is used to treat infections to ensure complete pathogen kill. The conjugation of poly(ethylene glycol) (PEG) to a protein, which is known as protein PEGylation, is a clinically proven strategy to increase the circulation half-life of proteins. However, most clinically used PEGylated proteins, including PEGylated IFN α-2, are heterogeneous mixtures comprised of positional conjugation isomers. There is a genuine need to develop efficient strategies for site-specific and site-selective PEGylation to obtain more homogeneous PEGylated products. The work described in this thesis examines the use of bis-alkylation PEGylation reagents (i) to site-specifically conjugate PEG to IFN consensus (IFN-con) at the two cysteine thiols in either of its two native disulfides and (ii) to site-selectively conjugate PEG at two histidines which have been added or substituted into the IFN structure. Initial work focused on PEGylation at the existing disulfides of IFN-con which is approved for the treatment of HCV patients who have failed first line treatment of PEG-IFN-α/ribovarin. IFN-con is more potent than IFN α-2 and PEGylated IFN-con is not clinically available. Disulfide PEGylation of IFN-con was then conducted using just 1 molar equivalent of the bis-alkylation PEG reagent (20 kDa PEG or 2×20 kDa PEG), which yielded 28–57% conversion to the mono-PEGylated products. Purified PEG-IFN-con displayed 3-fold higher activity than the clinically used PEG-IFN α-2a (Pegasys®). These results support continued investigation of disulfide PEGylated IFN-con as a potential long-acting treatment for HCV non-responders. While utilising the native disulfides for site specific conjugation is an improvement over the existing methods for PEGylating IFN, it was hypothesised that bis-alkylation PEGylation could be accomplished by conjugation to two histidines at a slightly acidic pH. This second strategy would therefore provide a means to engineer the site of conjugation within IFN (i.e. siteselective PEGylation). Bis-alkylation PEGylation was investigated with IFN alpha-2a that had been herein focused on confirmation by either trypsin or CNBr digestion that the site of PEGylation on IFN alpha-2a was achieved at the His8-tag. To achieve true site-selectivity it was hypothesised that two histidines as a conjugation site could be rationally added to a region of IFN alpha-2a distant from the receptor binding site. This method could potentially be applied to any protein and avoids challenges associated with engineering a free cysteine or incorporation of a non-native amino acid to achieve site-selectivePEG conjugation. Genes encoding for IFN alpha-2a analogs were constructed by site-directed mutagenesis and IFN alpha-2a variants were successfully expressed as soluble and active protein in identical yields as the production of native IFN alpha-2a. Site-selective PEGylation at the engineered histidines was as efficient as PEGylation at the N-terminal His8-tag. The use of the bis-alkylation PEG conjugation reagent proceeded with a similar efficiency that is generally observed for other amine conjugation methods. A good level of biological activity was retained for all conjugates produced (ranging from 10-75%). A IFN -2a variant that had been PEGylated at position 106 was produced in the highest yields (33%) and displayed the highest level of bioactivity (75%),which is greater than any reported PEGylated conjugate of IFN. In contrast, Pegasys® which is the most widely used PEG-IFN conjugate in the clinic has an activity of ~7%.

Introduction- Lacquers are ideal as topical drug carriers for the treatment of nail fungal infections. In this work, the Hansen Solubility Parameter (HSP) concept, which reflects the notion that ‘like’ seeks ‘like’, was investigated as a potentially useful tool in the rational formulation of an antifungal nail lacquer. This was done by evaluating the influence of HSPs, if any, on various steps of the lacquer development programme, including: (i) maximum drug loading in the lacquers, (ii) lacquer water resistivity and adhesion, (iii) drug release and ungual permeation from the films. Methods- HSPs of the antifungal drugs (i.e. amorolfine, terbinafine and ciclopirox), twelve polymers (as lacquer film-formers) the human nail and its models (bovine hoof and high density polyethylene(HDPE) sheets) were determined. Polymers were classified as ‘good’ or ‘bad’ depending on their HSPs and lacquers consisting of these polymers were then selected to test the potential usefulness of the HSP concept. Subsequently, the drugs’ saturation solubility in the lacquer films, adhesive strengths and water resistivity were determined. Finally, drug release and ungual drug permeation from the lacquer films were determined. Results- HSPs indicated that both the nail and hoof have a high affinity for polar and hydrogen bonding molecules, and that the antifungals have a high affinity for the nail. Moreover, the closer the HSPs of the drug and the polymer (i.e. ‘good’ polymers), the higher the drugs’ saturation concentrations achieved in the lacquer films. Smaller affinities between the drugs and the polymers (i.e. ‘bad’ polymers) allowed higher ungual drug permeation. However, HSPs could not predict the lacquers’ adhesive strengths or drug release Conclusion – HSP is a useful tool in the rational design of an antifungal nail lacquer.

Bis-alkylation for disulfide-bridging PEGylation has emerged as a valid strategy for protein conjugation. Proteins can be efficiently modified to add a three-carbon methylene bridge between the two sulfurs in a disulfide bond. The so-called C3 bis-sulfone reagent is a linear poly (ethylene glycol) (PEG) that has been functionalised at one terminus with a latently reactive bis-alkylation moiety capable of undergoing sequential Michael reactions. Latency is achieved by utilising leaving groups that must undergo elimination to unmask an ,-unsaturated double bond needed for Michael addition. Structural modifications of these reagents are thought to alter the solvent availability or electrophilic character of the Michael acceptor to modulate conjugation reactivity with a protein. It was therefore hypothesised that by modifying the structure of C3 bis-sulfone reagent, it would be possible to obtain reagents with different reactivity. This variable reactivity can then be exploited with bifunctional reagents to allow the preparation of protein-protein conjugates in an efficient manner. Several synthetic targets and strategies were examined to prepare different types of di-PEG and multifunctional reagents for protein conjugates. A small family of di-PEG bis-alkylating reagents with different molecular weights was prepared and the conjugation efficiency was compared to linear C3 PEG reagents of the same overall molecular weight. Molecular dynamic studies were used to understand how the PEG chain affected the linker reactivity. Results showed that PEG-linker interactions were found to be less pronounced for reagents that contained two 10 kDa PEG chains (di-PEG2×10) when compared to a linear C3 reagent with a single PEG of 20 kDa (PEG20) with the same overall molecular weight. While the presence of a second PEG chain was found to influence conjugation efficiency, the modification of the bis-alkylating Michael acceptor in C3 reagent was also explored as a means to vary reactivity. Acetylenic ketones were examined as bis-Michael acceptors in the preparation of two C1 reagents with distinct structural features (aliphatic and aromatic). An aliphatic C1 reagent was prepared without leaving groups but was found to have less reactivity when compared to an aromatic C3 reagent. Semi-empirical studies suggested that this lower reactivity could be attributed to less electron-withdrawing aliphatic structure and to stereoelectronic effects. Aliphatic C1, while less reactive was found to undergo a double Michael addition and consequently allowed re-bridging of a reduced disulfide. In contrast, aromatic C1 required leaving groups to modulate the higher reactivity observed but was not found to re-bridge a reduced disulfide. The C3 bis-sulfone reagent is known to undergo elimination much more slowly at slightly acidic pH values. This is important because conjugation will not proceed until elimination has occurred. The need for elimination was used as a basis for the synthesis of hetero-bifunctional reagents that could be used for hetero-functional protein-protein conjugates, such as bispecific Fab-PEG-Fab conjugates. For potential scalability, effort was focused on preparing mono-sulfone-PEG-bis-sulfone hetero-bifunctional reagents (MpB reagents) that could be utilised in a one-pot reaction sequence to give hetero-functional protein conjugates. Reactions using the MpB reagent showed the potential to allow the sequential conjugation of two Fab molecules by altering the pH conditions of the reaction mixture in a single reaction vessel. This variable reactivity can provide a synthetic platform for controlled sequential conjugation that can allow the efficient preparation of protein-protein conjugates.

The pyrrolobenzodiazepines (PBDs) are members of a family of DNA minor-groove binding anticancer agents. Some PBD compounds have very potent in vitro cytotoxicity and in vivo antitumour activities. Topical dosage forms in some cases offer more advantages than oral or parenteral dosage forms as more localised delivery of drug is possible with comparatively lower side effects. However, there are only a few anticancer topical formulations currently available for the treatment of skin cancer. Therefore, the main objective of this project was to develop topical formulations using PBD compounds for the palliative treatment of late stage melanoma patients, and for the treatment of non-melanoma skin cancers and potentially actinic keratosis. In order to develop suitable topical formulations for PBD compounds, a number of formulations were initially evaluated using anthramycin, a model PBD compound. In addition, the permeation and retention of the penetration enhancers (solvents) in human skin were also evaluated. Single solvent analysis results showed that Transcutol® and propylene glycol (PG) were the most highly permeable solvents. For all formulations, anthramycin absorption was comparatively higher in PG:PGML (propylene glycol monolaurate) (90:10). A focussed library of PBD C8-conjugates and a PBD-dimer (SJG-136) were also evaluated using dimethyl sulphoxide as a solvent. Potential topical efficacy was confirmed for a number of the compounds based on skin flux values and in vitro potency data. KMR-04-175 and KMR-28-24 were ranked as the most topically effective PBD conjugates. SJG-136 which has reached Phase II clinical trials, also showed higher skin absorption values compared with the other PBD compounds (except for KMR-28-24). This is the first report describing the preparation and evaluation of topical formulations of PBD compounds. The results are encouraging and suggest that topical formulations of PBD compounds could be efficacious for the topical treatment of skin cancers and potentially actinic keratosis.

Background and Purpose: Optimal benefit in the treatment of lung cancer is impeded due to systemic side effects, sub-therapeutic drug levels at the tumour site and the development of multidrug resistance. This thesis describes three related but distinct strategies aimed at enhancing drug treatment of lung cancer. Methods: The first approach entails design of micelles using three amphiphilic derivatives of chitosan (TPGS-chitosan, retinoic acid-chitosan-PEG and lipoic acid-chitosan-PEG (LACPEG)) for the pulmonary delivery of siRNA. Polymers where characterized using FT-IR and 1H NMR. Micelles prepared from these polymers were characterised for their size, zeta potential, morphology and toxicity in A549 and PC-9 cells. Micelle complexation with siRNA was assessed using agarose gel electrophoresis and the PicoGreen assay. Gene knockdown was assessed by MTS assay and western blotting. LACPEG and LACPEG/DSPE-PEG nanocarriers were loaded with gefitinib and re-characterised for their physicochemical properties, entrapment efficiency and activity (cell death) in vitro. In vivo biodistribution of these nanocarriers was assessed in CT26 and LLC-tumour bearing BALB/c and C57BL6 mice, respectively. The third approach to drug delivery employed mixed nanocarriers of DSPE-PEG or TPGS and PLGA-PEG, for co-delivery of gefitinib and genistein. Results: Successful synthesis of amphiphilic derivatives of chitosan. Further optimisation needed for micelles prepared using the first strategy with respect to siRNA complexation, as although 100% complexation was observed using agarose gel electrophoresis and the PicoGreen assay, at an NP ratio of 1:160, this was not reflected in cell culture. LACPEG micelles, showing the smallest size, best stability and lowest toxicity, were further studied and mixed with micelles of DSPE-PEG, for improved loading of gefitinib (45% compared to 80%). These showed improved antitumour activity in vitro and whole body prolonged circulation with tumour accumulation and uptake in vivo. Similar results were obtained with gefitinib and genistein mono- and co-loaded PLGA-PEG/TPGS nanocarriers. Conclusion: LACPEG/DSPE-PEG and PLGA-PEG/TPGS nanocarriers showed promise for the delivery of anticancer drugs, demonstrating a synergistic activity from the carrier itself, as well as the co-delivery of both therapeutic molecules. Further studies are warranted for siRNA complexation.

Flucloxacillin and co-amoxiclav are both associated with drug-induced liver injury (DILI). HLA genotype is an important predictor of DILI susceptibility but it is likely that non-HLA risk factors also contribute. This study aimed to characterise non-HLA risk factors in larger cohorts (155 flucloxacillin and 165 co-amoxiclav adjudicated cases) than previously. Drug causality of the cases was assessed using the RUCAM method which showed 88.1% of the cases were either highly probable or probable but 11.9% of them indicated a possible causality for the drug. Variants showing associations in previous candidate gene, genome-wide association and exome sequencing studies were genotyped to extend these findings. A SNP (rs2476601) in PTPN22, which encodes a protein involved in T-cell-receptor signalling had already been shown to be a risk factor for co-amoxiclav DILI. This was confirmed by genotyping co-amoxiclav DILI cases (n=99) (OR=2.74, 95% CI=1.58–4.77; P=4.1x10-4). There was also a significant effect for flucloxacillin DILI (OR=1.9, 95% CI=1.1–3.1; P=0.02). Exome sequencing performed previously on 66 UK co-amoxiclav DILI cases reported significant associations for several variants, including rs117511121 in IL12RB1 and rs145855109 in TPH1. Additional cases (n=99) were genotyped for rs117511121, confirming the association (OR 6.5, 95% CI=1.5-27.8; P=0.012). No association with IL12RB1 genotype was seen for flucloxacillin DILI. Functional analysis of IL12RB1 using reporter gene constructs revealed significantly lower luciferase activity for the variant constructs. The TPH1 variant was confirmed to be associated with co-amoxiclav DILI (n=99) (OR=14.73, 95% CI=2.94–73.92; P=0.013). Polymorphisms in the following genes showed no significant association with DILI due to either drug: FMO5, GPX1, GSTM1, GSTT1, HFE, KCNJ1, SHMT1, SLCO1B1, SOD2, ST6GAL1 and UGT1A1. The findings for PTPN22 and IL12RB1 confirm the relevance of T cell responses to co-amoxiclav DILI. Odds ratios of 17 for DILI risk can be calculated for individuals with the at risk HLA alleles (A*02:01 and DRB1*15:01) and the PTPN22 and IL12RB1 variants, assuming an additive model. PTPN22 is also relevant to flucloxacillin DILI but, though biologically plausible as a risk factor, appears minor compared with HLA-B*57:01.

Natural products have been the source and inspiration of numerous drugs. However, as they need to be isolated from natural sources, they are often obtained in minute quantities. Hence, total syntheses of natural products and their analogues give us a better chance to look into their biological activities and perform SAR studies for drug discovery. During my doctoral tenure I have synthesised a number of natural products and also some fragments. CPI-2081, a 2:1 mixture of two pentapeptides 1.8 and 1.9, were isolated from Streptomyces species NCIM 2081. These peptides are potent inhibitors (IC50 36.9 ± 1.8 nM) of the cysteine protease papain and inhibit cancer cell migration. However it was not clear which peptide is responsible for the observed biological activities. Both peptides have been prepared synthetically in our lab and they are being tested. Myriastramide C (2.23) is a modified cyclic octapeptide isolated from M. clavosa. The complete series of myriastramides A-C represent the first peptide metabolites isolated from M. clavosa. They were assumed to be cytotoxic, however myriastramide A was found inactive against 10 different human cancer cell lines. Myriastramides B and C were isolated in insufficient quantities so similar tests cannot be performed. Furthermore, the stereochemistry of the tryptophan residue in myriastramide C was not confirmed due to insufficient material isolated. Myriastramide C has been synthesised in our laboratory using L-tryptophan, however a mixture of products were obtained due to the two proline residues in the peptide. A pure product was purified by our collaborator and further NMR and biological studies is underway. Goadsporin (3.14) is a linear oligopeptide that was isolated in 2001. Its unique structure contains 19 amino acids, six of which are cyclised to form four oxazole and two thiazole rings. Goadsporin was found to be active specially in streptomycetes, with the ability to promote morphogenesis and secondary metabolism. It can also promote antibiotic production. The synthesis of this natural product was challenging, and I I unfortunately with the limited time of my PhD study, we did not complete the total synthesis. However, we have synthesised several fragments of it, and they are going to be tested in the future. Breitfussin B (4.35) was first isolated in 2007 from the sample of Thuiaria breitfussi collected from Bear Island. Its highly unsaturated nature prevented its structure elucidation until 2012: Hanssen et al. proposed a structure using a combined atomic force microscopy and computational approach. However no biological activity was reported. The total synthesis of breitfussin B was completed in our lab, and tested for its antimicrobial ability. Further biological studies are underway.

Background: Community pharmacists have not fulfilled expectations for an extended role and their education and training is recognised as contributing to this. Postgraduate diplomas may provide the additional development required. These courses are predominantly distance learning based despite evidence that multifaceted approaches are more effective. Furthermore, the role of learning theory in developing or assessing pharmacist education is unclear. UEA obtained funding to provide a workplace based diploma for community pharmacists based in eastern England. The aim of this PhD was to investigate the role of this diploma in community pharmacist development. Methods: Mixed methods were used. In-depth interviews were conducted with a purposive sample of 15 diploma students in October 2011 after one year of the course. Follow-up interviews were completed one year later. Interviews were conducted in summer 2012 with four community pharmacy employer representatives. A service provision, employment and CPD survey was conducted annually with diploma students (n=39) and a comparison group (n=18). A patient satisfaction survey was conducted in the main workplace of these pharmacists at the outset of the course and repeated as it concluded. Results: Students described positive effects on their development and practice including improved confidence and inter-professional relationships. The diploma scaffolded learning and a reduction in the potential barriers to CPD was demonstrated. The opportunities for interacting with peers and other healthcare professionals were important. Workload pressures were detrimental. Employers recognised pharmacists needed development but favoured training they controlled. No significant change was seen in the provision of services or patient satisfaction. Discussion: This work contributes to understanding community pharmacists’ needs from a learning theory perspective. Professional isolation impacts negatively on the development of their practice and the social learning facilitated by the diploma appears fundamental to the positive results obtained. Whether a workplace based diploma is the best way to achieve this is unclear.

The putative CACHD1 gene was identified following a systematic search for ligand-binding proteins as novel drug targets. The CACHD1 gene encodes CACHD1 and has a predicted protein structure similar to that of the [alpha]2[delta] family of voltage-gated Ca2+ channel (VGCC) auxiliary subunits, which modulates the biophysical properties and plasma membrane expression of VGCCs. On this basis, the hypothesis that CACHD1 represents a novel [alpha]2[delta]-like VGCC auxiliary subunit was tested.