Background: Administering medication to patients with dysphagia (PWD) is a challenging process for patients and healthcare professionals (HCPs). This study aimed to improve those administrations by focusing on the development of the elements of a pharmacy service providing individualised guidance on the administration of medication to older PWD in care homes. The objectives were to:- assess the feasibility of a pharmacy service promoting guidance on the administration of medication to PWD, - identify and develop theory on the elements that affect the administration of medication to PWD in care homes, - identify outcomes for the modelling process previous to a large scale intervention. Methods A questionnaire was designed to evaluate the acceptability by HCPs of a pharmacy service for PWD in hospital wards. Qualitative interviewing was used in care homes to explore the perceptions of nurses on the administration of medication to PWD and nurse’s acceptability of a pharmacy service providing individualised medication administration guides (I-MAGs). Observational drug rounds were carried out in care homes to describe the quality, type and frequency of errors in the administration of medication to PWD. Results I-MAGs were well received on the hospital wards and nurses felt more confident and time efficient in their practice when the I-MAGs were present on the ward. Interviews identified the isolating environment of the care home, the importance of formulation choice, the lack of awareness of dysphagia and gaps in nurse’s pharmaceutical knowledge as barriers in the medicines management of PWD. Observational drug rounds revealed that medicine administration errors (MAEs) in care homes (excluding time errors) are three times more frequent in PWD than in those without. Conclusion: Medicines management for PWD requires a multidisciplinary approach from several HCPs and consequently PWD could benefit from interventions that overcome the practice barriers between those HCPs.

The aim of my PhD project was the development of novel hydrogels for wound care applications using autoclave-mediated cross-linking in the polymer mixtures. Transparent, bubble-free and flat hydrogels were produced. The two polymers that gave the best results and therefore were used to continue the work, were poly(vinyl alcohol) and poly(methyl ether-alt-maleic anhydride), which is also known as Gantrez® AN. These dressings were then fully characterised. We took advantage of the fact that the autoclaving step allowed the gelation in a controlled way, to synthesise another class of hydrogel materials known as ‘superporous’ hydrogels (SPH), whose production usually involves the use of complicated set up and the addition of initiators and cross-linkers. We developed a straightforward method composed of few simple steps to synthesise SPHs of different shapes and thicknesses from aqueous mixtures of PVA and Gantrez® AN. These materials and their physicochemical properties were then investigated. Using the same combination of polymers again it was also possible to produce physically cross-linked hydrogels applying a slightly modified version of the well-known freeze-thawing technique. The cryogels produced were studied and their properties were compared with those of the autoclaved (chemically cross-linked) samples. We developed three new wound dressing prototypes and two novel methods for the synthesis of hydrogels and SPHs that could make a difference, not just in the wound management sector. New products, but above all an innovative and environmentally-friendly approach to biomaterials manufacturing can derive from our work at Reading School of Pharmacy. From the method of synthesis of the materials we present in this thesis, to their extensive characterisation, our work aimed to give a valid and useful contribution to the wound management field.

Down syndrome (DS) is the most common human aneuploidy. It results from the presence of three copies, or trisomy, of human chromosome 21 (Hsa21). DS is associated with a plethora of characteristic clinical features, most notably learning disability, altered body morphology, congenital heart disease and early-onset Alzheimer’s disease (AD-DS). Despite knowledge of its primary cause, pathological mechanisms underlying DS are poorly understood, including potential deficits in essential signalling processes. This thesis investigates Wnt/β-catenin signalling dysfunction in DS. The Wnt signalling pathway is a fundamental transduction cascade with key roles in development, cancer and neurodegeneration. Particularly, mounting evidence suggests that AD neuropathology may be underscored by critical dysfunction of canonical Wnt signalling. Given the close relationship between AD and DS, it is proposed in this thesis that Wnt abnormalities may also be present in the DS brain. This hypothesis is thus investigated, combining bioinformatics with RNA and protein analysis in DS mouse models and humans. The evidence gathered here suggests canonical Wnt signalling is dysfunctional in the DS brain. Most importantly, Wnt signalling activity is suppressed in the adult DS hippocampus. Furthermore, this thesis identifies the Hsa21-encoded kinase DYRK1A, an essential contributor to DS, as a novel, bimodal Wnt signalling regulator. DYRK1A may both suppress and enhance Wnt activity, depending on the activation state of the pathway. It is proposed that, in DS, dosage imbalance of DYRK1A may substantially affects Wnt signalling, with a complex array of resulting transcriptional changes to Wnt target genes. This mechanism may contribute to several developmental and adult features of DS, particularly learning disability and AD-DS. Overall, these findings may provide key evidence for the global understanding of this condition, and targeting Wnt signalling may open unexplored avenues for therapeutic development.

BACKGROUND: Clinical research using herbal medicines requires specific considerations such as production method, which leads to chemical and pharmacological variability. Asthma is a leading chronic respiratory disease, remains sub-optimally controlled despite conventional costly treatments. Nigella sativa L. (NS) is a traditional herbal treatment for asthma, but lacking well-established scientific evidence. Objectives To develop an integrated research strategy incorporating preclinical and clinical research focusing on NS use in asthma by employing a chemically and pharmacologically well-characterised NS preparation in a well-designed clinical trial. METHODS: Distinct NS preparations were chemically characterised for thymoquinone (main active compound) concentration by High Performance Liquid Chromatography. Human T-lymphocyte, monocyte and A549 epithelial cells were utilised to assess the in-vitro anti-inflammation/immunomodulatory activity of NS preparations. The most potent and suitable NS preparation was clinically evaluated for efficacy as add-on treatment for asthmatics in a phase-II randomised double-blind placebo-controlled clinical trial (RDBPCT). Asthma Control Test (ACT) was the primary outcome. Pulmonary function, blood eosinophils and serum inflammatory markers were secondary outcomes. RESULTS AND DISCUSSION: Ten different NS preparations were obtained, showing variability in thymoquinone concentration and in-vitro anti-inflammatory/immunomodulatory activities. Two thymoquinone-rich oily preparations (a super critical fluid extract and a commercial product registered in Saudi Arabia) showed the best in-vitro activity via inhibiting inflammatory cytokines in human cellular models. In the RDBPCT including 80 asthmatics, the commercial product capsules showed significant improvements in ACT, eosinophilia and some serum cytokines without serious side effects. CONCLUSION: This project addressed important requirements to optimise clinical research using herbal medicines particularly for NS in asthma. Preclinical research on the chemistry and activity of the investigational NS product were the basis for the clinical trial. The RDBPCT revealed a higher level of evidence for the add-on NS treatment in asthma within the Saudi healthcare. This strategy is suggested for future clinical phytotherapeutic research.

In this study, indomethacin-loaded self-nanoemulsifying drug delivery systems (SNEDDS) were developed in liquid, solid and carrier-mediated formulations in order to improve the solubility of this model poorly water soluble drug. Liquid SNEDDS based on CapryolTM 90 (oil phase), Cremophor® RH 40 (surfactant) and Transcutol® HP (co-surfactant) were thermodynamically stable and produced clear nanoemulsions upon dilution. Optimized liquid formulations were transformed into solid SNEDDS by adsorption onto the inert carriers Syloid® XDP 3150, Neusilin® US2 and Florite® PS-200. Ratios of adsorbent: liquid SNEDDS of 1:1.5 and 1:2 resulted in solid SNEDDS formulations that exhibited fair to passable powder flow properties. Carrier-based solid SNEDDS formulations were developed using the solid self-emulsifying carriers Gelucire® 44/14 and Gelucire® 48/16 and prepared by hot melt extrusion. The absorbent-based solid SNEDDS maintained the self-nanoemulsification properties of the original liquid SNEDDS formulations, with solid state analysis suggesting that the drug had remained in a dissolved state within these formulations. Similarly, physical characterization of the carrier-based solid SNEDDS formulations indicated that the drug was molecularly dispersed within the system and that the self-nanoemulsifying properties of the carrier were unchanged. The only exception was those formulations prepared at the highest drug: carrier ratio (3: 10). For both absorbent-based and carrier-based solid SNEDDS, the in vitro dissolution efficiency was significantly higher than that obtained for the pure drug. However, incorporation of adsorbents into Gelucire®-based solid SNEDDS formulations resulted in reduced dissolution of the drug. Gelucire®48/16-based solid SNEDDS prepared at 50oC were more physically stable to storage at 30oC/75% RH for 6 months than formulations processed at 40oC, suggesting that complete melting of the carrier during manufacture is essential for production of physically stable formulations. Overall, a range of liquid, solid and carrier-based SNEDDS formulations were successfully developed and offer useful alternatives to improving the solubility of poorly water-soluble drugs.

Background: Plant UDP-dependent glycosyltransferases (UGTs) play important roles in biology via the glycosylation of secondary metabolites. The future prospects of UGTs look promising, for example, it may serve as promising path for progress in expanding drug targets and synthesising glycan-based drug with enhanced bioactivity. Nevertheless, the current poor understanding of UGTs at molecular level (e.g. kinetic and structure) has led to a limited understanding of their biological roles and has also hampered their potential applications. Aims: This project aims to 1) build up a mass spectrometry (MS) based approach to study families of UGTs including their substrate specificities, kinetic parameters and mechanisms of action (Chapter 3); 2) identify catalytic key amino acids (ckAAs) in the various UGTs (Chapter 4); and finally, 3) apply the methods above in the study of selected Rhamnosyltransferases (RhaTs) 78D1 and 89C1 (Chapter 5). Methodology: A triple quadrupole MS (QQQ-MS) was used as this instrument required limited modification of substrates and provided direct monitoring of the glycosylated product. ‘Full scan mode’ gave the initial screening of any potential glycosylated product, and the ‘product ion’ mode provided additional confirmation of the formation of the glycosylated product. The ‘multiple reaction monitoring (MRM)’ mode quantified the products formation as a function of reaction time and provided kinetic data of the UGTs (Chapter 3). The study of catalytic key amino acids (ckAAs) was based on the multiple sequence alignment (MSA) method via the AA sequence comparison with template UGTs that have known crystal structures. Subsequent site-directed mutagenesis (SDM) was used to substantiate/disprove the functional role of potential ckAAs: mutants (with potential ckAAs mutated) were checked by MS to find out whether the original activities were maintained and/or new activities were gained. A further activity comparison (kcat/KM) between the active mutant and the wild type (WT) could indicate the influence from a particular AA (Chapter 4). Results and conclusions: 29 recombinant UGTs from groups B, D, F, H and L were examined. New donor activities (e.g. UDP-GlcNAc towards 73B4 and 78D2) were reported. Full kinetic studies of these WT UGTs indicated that they followed the Bi-Bi sequential mechanism. Whilst most followed the Bi-Bi random sequential mechanism, exceptions could be found such as that for 73B4 facilitating the UDP-GlcNAc reaction (Chapter 3). Based on the interactions between the donor and template UGT (e.g. VvGT1, PDB 2C1Z), mutations of the potential ckAAs oriented towards the sugar (positions C2, C3, C4 and C6), phosphate and uridine were designed with alanine and an AA with a similar structural and chemical character. An activity comparison (kcat/KM) between the WT and active mutants indicated that most of the potential ckAAs within the PSPG motif inferred from MSA, were conserved and possibly followed a similar interaction pattern. However, exceptions could be found (e.g. 78D2 D380). Taking the results of both MSA and SDM together, the ckAAs in the active sites in each target UGT towards a specific donor were identified (Chapters 4 and 5). Additionally, the study of Rhamnosyltransferases (RhaT) 78D1 and 89C1 showed that new activities were acquired by point mutagenesis: 78D1 N375Q acquired UDP-Glc and UDP-GlcNAc activities; and 89C1 H357Q acquired UDP-Glc activity (Chapter 5).

The process of developing pharmaceuticals requires expertise from numerous different scientific areas. Four separate studies have been undertaken on Pharmaceuticals Testing, Process Development, Business Strategy and Process Validation within this industry. New pharmaceuticals generally require multi-step reactions, which increasingly feature the involvement of biological synthesis to improve the optical purity and thus efficacy and safety of the drug. Two of the problems with employing biological synthesis are the high level of inhibition observed and the potential difficulty with which these batch based reactions are combined with semi-continuous chemical synthesis. The first study characterises the inhibition of CHMO, a promising oxygenase enzyme, with the aid of flow cytometry using different systems: o A range of substrates, based around the natural substrate cyclohexanone but with differing ring size and increasing chain length. o Different CHMO catalysed reactions - isolated enzyme, free cell and immobilised whole cell. As expected, reactions with CHMO expressed in E. coli TOP 10 [pQR239] in their immobilised form reduced the observed reaction rate. Unexpectedly, for the more rapidly converted substrates (generally those closest to cyclohexanone), immobilisation was found to increase the inhibition observed. It has been postulated that this is due to an oxygen shortage for maintaining cell metabolism and a time based inhibitory effect. Advantages of immobilised cells are that they can be rapidly removed from the reaction broth allowing greater integration with other processes and can be recycled for multiple re-use. To facilitate their industrial use, the large scale production of immobilised whole cells is required. Whilst immobilised cell reactions are industrially employed, how such large quantities of immobilised cells are produced is yet to be reported. The feasibility of immobilisation of oxygenase expressing cells has been assessed in this first study, using the flow cytometry as a tool for assessing cell damage in the key step of cell separation. Within the pharmaceutical development process drug molecules are rigorously tested in clinical trials. However the metabolites likely to be produced in the body, which may be active (and preferable drug candidates to the parent molecule) or toxic (and thus responsible for the failure of the drug in the final stages of clinical trials) are often ignored. Within the human body the oxygenase enzymes Cytochrome P450s (CYPs) are responsible for the primary metabolism of more than 90% of drugs. The second study assesses different methods of identifying the CYP responsible for metabolism and discusses the importance of being able to produce gram scale quantities of metabolites. This study indicated that the best currently feasible option of CYP identification is the employment of Bactosome (individual CYP enzymes expressed in bacteria) with a selective inhibitor pre-screen. The scientific complexity of the pharmaceutical development process makes effective strategic planning and decision making difficult. Whilst the necessity of business plans to enable companies to secure finance has helped scientists to gain an understanding of their market and associated business risks, business decisions such as when to invest and how much, often rely solely on the company's tolerance of risk, collective intuition and experience. The third study investigates the business strategy of the pharmaceutical development process. StrategyDynamics modelling has been employed to create a living model of a start-up contract research organisation. The model demonstrates the advantages of being able to predict key resource bottlenecks, contrast different business decisions such as growth strategy and plan for future events and changes in technology and markets. This modelling can potentially save companies from expensive trial and error approaches and help to manage risk. Regulatory pressure within the pharmaceutical development industry and the importance of validation is increasing. In the fourth study the application of Process Validation to the areas of pharmaceutical development process in the first three studies are investigated. For CHMO biocatalysis the reproducibility of immobilised experiments was assessed, for drug metabolite production the importance of change validation, i.e. assay robustness, was determined and for the Strategy Dynamics modelling an approach to validating the model has been detailed.

Background Non adherence to medication in myocardial infarction patients ranges from 13-60% (Garavalia et al, 2009). Consistent use of secondary prevention medication after a coronary event is associated with lower adjusted mortality and higher survival rates compared with patients who are not compliant (Newby et al, 2006). Strategies to tackle the burden of non adherence could involve pharmacy care and services including Medication Use Review and the New Medicine Service with a motivational interview as part of the counselling session of a community pharmacist. Objective To investigate the feasibility and potential impact of a pharmacy care intervention involving motivational interviews and referral to the community pharmacy services, amongst patients with acute coronary syndrome, on adherence to medication and on health outcomes. Methods This thesis reports a prospective, intervention, controlled feasibility/pilot study. Seventy one patients discharged from a London Heart Attack Centre following acute treatment for a coronary event were enrolled and followed up for six months. Thirty two pharmacies in London were allocated into intervention or control sites. The intervention was delivered by community pharmacists face-to-face in the pharmacy, or by telephone as part of the New Medicine Service or a Medication Use Review. The consultation included a 15-20 minute motivational interviewing session aimed at improving protective cardiovascular medicine adherence. As this was a feasibility study, measures of uptake, workability and acceptability were gathered from all stakeholders. A measure of fidelity to the intervention was also performed. The primary outcome measure was adherence to secondary prevention medication using a self report adherence measure. Secondary clinical outcomes included blood pressure and LDL-C. Data collection of outcome measures took place at baseline, 3 months and 6 months. An intention-to-treat analysis was conducted for the outcome measures. Results Given a small sample size, the feasibility study was not powered to measure clinical outcomes. However, at 3 and 6 months there was a statistically significant difference in adherence between the intervention group and the control group (P= 0.026), (P=0.004) respectively. In addition, there was a statistically significant relationship between the level of adherence at 3 months and beliefs regarding medicines (P=0.028). Patients who reported better adherence expressed positive beliefs regarding the necessity of taking their medicines. However, given the small sample size, no statistically significant outcome difference in terms of recorded blood pressure and LDL-C was observed over the six months of the study. Conclusion The feasibility, acceptability and potentially positive clinical outcome of the intervention was demonstrated, along with a high level of patient acceptability. It had a significant impact on cardiovascular medicine taking adherence. But these findings must be interpreted with caution. The intervention should be tested in a larger trial to ascertain its full clinical utility.

This thesis describes a study into the formulation, manufacture and characterisation of mini-tablets and inserts for ocular use. Powder-based mini-tablets were formulated using the antibiotic chloramphenicol and a range of polymers. The effect of powder particle size on the quality of the products was investigated and was significant only for drug release from polyethylene oxide 8M mini-tablets. Transition temperature microscopy was used to assess drug distribution across the surface of the mini-tablets. Good contact of the nano-probe with the mini-tablet surface was the determining factor in the quality of the images created, with the residual particle shape after compaction playing a significant role. A novel approach to the manufacture of mini-tablets using 3-D printing was investigated. The fused deposition modelling approach was unsuccessful due to the difficulty in producing extrudates of the required polymers. Stereolithography was used to prepare a range of formulations, with polyethylene glycol (PEG) diacrylate as the base material and phenylbis (2,4,6-trimethylbenzoyl) phosphine oxide as photoinitiator. The quality of the 3-D printed tablets was variable and dependent on the relative content of these two ingredients and the equipment settings. The 3-D printed mini-tablets showed a slow first-order drug release profile, which was increased by the inclusion of pore formers such as low molecular weight PEG. The applicability of the stereolithography approach for 3-D printing of individualised ocular inserts was investigated. A cranial MRI scan of an adult male human was used, with permission, to generate a 3-D image of the eye, from which a personalised ocular insert was produced. A matching personalised flow-through dissolution chamber was constructed, in order to enable to assess the drug release profile from the inserts. Similarly to the 3-D printed mini-tablets, the drug release followed a slow first-order profile, and was increased by the presence of pore formers in the insert.

Going blind is incomprehensible and with an aging population the number of people with blinding disease is increasing. Glaucoma and age related macular degeneration (AMD) are two major causes of blindness affecting people as they age. The only proven treatment for glaucoma is lowering of the intraocular pressure (IOP) which is best done by surgically placing a channel from the anterior chamber to allow aqueous outflow to drain into the subconjunctival space. The drainage channel can be formed by the use of a glaucoma drainage device (GDD) or by glaucoma filtration surgery (GFS). Both GFS and the use of a GDD often fail over time because local fibrosis (scarring) in the subconjunctival space blocks aqueous outflow resulting in the increase of IOP and disease progression. It was hypothesised that a more biocompatible GDD could be fabricated from a hydrogel, and that the hydrogel material could be used to restrict aqueous outflow to control the IOP. Hydrogels are widely used in ophthalmic applications including contact lens and intraocular lens. Since hydrogels are widely examined for use in drug delivery, it was also hypothesised that a hydrogel implant could be made for the subconjunctival space after GFS to stop tissue adhesion and to deliver locally an anti-fibrotic or anti-inflammatory drug to increase the chances for long-term surgical success. For AMD, the current treatment is intravitreal (IVT) injections of anti-VEGF antibodies approximately every 4-6 weeks. IVT injections are an invasive procedure and associated with some complications, but it is also becoming apparent that many healthcare systems around the world cannot cope with the increasing demands for IVT injections to treat AMD. To reduce the frequency for IVT injections, there is a need to develop formulations that allow a longer duration of action for therapeutic proteins in the back of the eye. Maintaining protein stability is a major challenge in formulation science and clinical use. It was further hypothesied that injectable hydrogels could also be used to formulate an antibody for IVT injection to display an extended residence time in the vitreous cavity. Free radical polymerisations of 2-hydroxyethyl methacrylate (HEMA) and 2-methacryloyloxyethyl phosphoryl choline (MPC) in the presence of a cross-linker, poly¬(ethylene glycol diacrylate) (PEGDA) were conducted to prepare HEMA-MPC co-polymer hydrogel films. Both HEMA and MPC are widely used in ophthalmic hydrogel products and MPC is known to be exceptionally biocompatible, although it must only be used as a co-polymer to ensure there are suitable processing and mechanical properties in the resulting hydrogel. Different HEMA-MPC hydrogels with increasing relative stoichiometries of MPC (0%-100% (w/w)) were prepared and characterised to determine if water flow through the gel was possible. Unfortunately the hydrogel films formed have low permeability (1.1×10-18 m2 s-1 pas-1) compared to the permeability required to control flow at a rate of 2 μL/min under 10-15 mmHg IOP, which is (6 ×10-14 m2 s-1 pas-1). Although the HEMA-MPC hydrogel films could not be used for flow control, they were further examined for use as potential implants for local tissue site drug delivery in subconjunctiva. HEMA-MPC hydrogels with 10% MPC were found to offer the best balance between water content, mechanical strength and drug loading and release that was required for the possible implantation drug loaded films derived from a range drugs (dexamethasone, pirfenidone and doxycycline). The process used for drug loading of dexamethasone was optimised by using, methanol and the in vitro half-life of DEX was increased from 1.8 to 9.1 days with release being sustained for more than 3 weeks. There are other causes of subconjunctival scarring, in particular trachoma, which is the main cause of blindness due to infection. Doxycycline is thought to be a good candidate drug for treating patients after trachoma surgery because it has both anti-bacterial and anti-fibrotic properties. As a water-soluble drug, doxycycline release could not be sustained for more than 3 days, so the 10% MPC films were modified with the incorporation of β-cyclodextrin (β-CD) in an effort exploit the possible affinity of doxycycline with β-CD to prolong doxycycline release. Several methods were examined to introduce β-CD into the HEMA-MPC films including the formation of HEMA-MPC films with pendant β-CD, the embedding of β-CD cross-linked particles within the hydrogel network and formation of an interpenetrating network (IPN) of β-CD and HEMA-MPC. Unfortunately, the release profile of doxycycline was similar in the modified and non-modified HEMA-MPC hydrogels. To evaluate hydrogels for use in IVT injections of antibodies, N-isopropylacylamide (NIPAAm) thermoresponsive hydrogels were evaluated. Three different macromolecular hydrophilic cross-linkers were evaluated; PEGDA, phosphorylcholine 3059 (PC 3059) and acrylated hyaluronic acid (Ac-HA). The prepared hydrogels were characterised regarding physical properties such as water content, water retention thermoresponsivness and protein release. The thermal responsiveness decreased with increasing cross-linker percentage. Modification in the type and percentage of cross-linker used allowed the preliminary screening of the different formulations. Hydrogel formulations made with 40 mg NIPAAm as monomer and 8 μL PEGDA, 20 mg PC3059 or 4 mg Ac-HA were able to sustain the release of antibodies for a month in a validated in vitro model of the eye.