A fragment-based approach to drug design has recently emerged in which small chemical groups that bind to adjacent sites on a receptor are identified, optimised, and linked together to generate a high-affinity lead compound. Due to its sensitivity, nuclear magnetic resonance (NMR) is a particularly useful technique for the detection of low-affinity fragment binding. An aim of this project was to explore further the utility of NMR with regards to fragment ligand binding using a well- understood model system - the Src Homology 2 (SH2) domain from v-Src kinase. This choice was supported by a large body of structural, functional, and thermodynamic data, coupled with a decade of pharmaceutical investigation into several SH2 domains. Our ongoing biophysical studies of v-Src SH2 required determination of the apo solution structure of this domain. A high quality structural ensemble was obtained using standard NMR techniques and a combination of manual and automated assignment methods. The internal hydrogen bonding, ionisation states, and backbone dynamics of the apo v-Src SH2 domain was also explored using NMR. The perturbation of v-Src SH2 backbone chemical shifts and dynamics by interaction with fragment ligands yielded insights into SH2 domain binding behaviour and specificity. Computational approaches were used to identify potential fragment ligands for v-Src SH2. A small library of these molecules were screened in vitro using a recently-proposed 19F-NMR competition screening approach, which was optimised for the detection of low-affinity fragments. Follow-up NMR and calorimetry experiments confirmed the screening results and provided further characterisation of the novel fragment ligands. Such compounds may be useful as phosphotyrosine mimetics in SH2-related drug design. A novel 31P-based screening experiment was also proposed. These studies have furthered our understanding of the SH2 domain, in terms of its binding specificity and drug design, and of the NMR screening approaches useful for fragment-based lead discovery.

The hypothesised association between leukotriene receptor antagonists (LTRA) and suicide is not clear. In this thesis, causality assessments of Individual Case Safety Reports (ICSRs) and a novel pharmaco-vigilance method using electronic healthcare records (EHRs) are used to examine the strength of this hypothesis. This thesis is also the first to describe the application of the self-controlled case series (SCCS) methodology to the study of suicide and LTRA using UK primary care data. There are 5 components: 1) an initial screening of VigiBase, a global database of ICSRs for drugs associated with suicide; 2) a review of the literature for evidence of an association of suicide with montelukast; 3) an assessment of the strength of this association using causality assessments of ICSRs, and an overall causality assessment using multiple data sources in accordance to the Bradford Hill guidelines of causality; 4) Temporal-Pattern Discovery (TPD), a novel data-mining method, was applied to UK EHRs to complement signal detection; 5) an observational study using an SCCS methodology was used to examine whether LTRAs are associated with an increased risk of suicide attempts. We found montelukast to be amongst medication with the most number of global ICSRs for suicide-related adverse events in young people aged 2-17. However, causality assessment of ICSRs revealed that reports were often incomplete and did not strengthen this hypothesis. Also, complementary signal detection using the TPD method in EHRs did not detect a signal. Finally, the SCCS study using EHRs from a UK primary care database did not provide evidence to support the hypothesis; on the contrary we found the risk of suicide attempts decreased after cessation of a course of LTRAs in females (IRR=0.46 95%CI 0.22, 0.96). In conclusion, there is little evidence to support an association between suicide and montelukast, however this link cannot be excluded.

Evolving regulatory reforms have strengthened the global focus towards developing age- appropriate and acceptable formulations for paediatric patients. The European Medicines Agency (EMA) also mandates that acceptability of paediatric preparations should be evaluated with children as an integral part of pharmaceutical and clinical development. A review of literature highlights the current paucity of evidence to support the development of dosage forms that are suitable for children of different ages and health backgrounds. Important knowledge gaps also exist in the methodological considerations for conducting acceptability studies. The overall aim of this research was to determine the attitudes, preferences and behaviours of children, caregivers and healthcare professionals towards solid oral dosage forms. Age- adapted questionnaires were systematically developed and pre-tested in collaboration with young people, in keeping with the principles of patient and public involvement in research. A diverse sample of 590 school children and adolescents and over 400 caregivers were surveyed in hospitals, community pharmacies and schools. Attitudes towards dosage forms differed primarily based on age and prior use. Precedence for chewable dosage forms and tablets was identified, while capsules and multiparticulates administered with food were less acceptable. Another aspect of the research identified the prevalence of swallowing difficulties in healthy young adults. These participants showed a preference for taking two smaller dosage forms over a larger size, and for capsules over tablets. In an acceptability trial with placebo capsules, over 95% of young adults were able to swallow size #1 capsules. The inclusion of flavour had variable effects on acceptability . Healthcare professionals showed reservations towards solid oral dosage forms and many favoured liquid medicines for paediatrics. This thesis highlights the gap between regulatory expectations, industrial feasibility and clinical ideals. This should be addressed, to ensure that legislative reforms and global initiatives will have a positive impact on paediatric therapeutics in practice.

A first synthesis of the acyl side chain of azinothricin and a second-generation route to the A83586C side chain is described in this thesis. The successful strategy was based on an asymmetric aldol reaction, a stereoselective Roush-type crotylboration and a Trost asymmetric allylic alkylation reaction to set the stereocentres of the key intermediates 3.95 and 3.5, which were subsequently coupled together to give the side chain fragment 3.105 after 10 further steps. The total synthesis of several A83586C/azinothricin analogues is also described. These hybrid structures have been formed via a chemoselective coupling between the N-hydroxybenzotriazole activated ester of the acyl side chain and the appropriate fully deprotected cyclodepsipeptide hydrochloride salt. The first total syntheses of the natural products azinothricin and kettapeptin have also been achieved by this strategy.

Adverse Drug Reactions (ADRs) are a global health problem and a leading cause of death, illness and injury in economically developed countries. Therapeutic response, as well the occurrence of undesired effects, can differ significantly between children and adults and many drugs have not been sufficiently studied in the paediatric population. Existing studies on ADRs in children differ widely in study design and outcome reporting, and many are methodically problematic. The incidence and characteristics of ADRs in hospitalised children and factors associated with an increased risk of experiencing and ADR, were assessed in a large, prospective, observational study. 17.7% of all children experienced at least one ADR. Opiate analgesia and drugs used in general anaesthesia (GA) accounted for more than 50% of all drugs implicated in ADRs. Less than 1% of ADRs caused permanent harm or required admission to a higher level of care. Children post GA were more than six times more likely to experience an ADR than children who had not received a GA (HR 6.38; 95%CI 5.3-7.7). Other risk factors identified were increasing age (HR 1.05 for each year; 95%CI 1.04-1.07), increasing number of medicines (HR 1.25 for each additional medicine; 95%CI 1.22-1.28) and being an oncology patient (HR 1.89; 95%CI 1.36-2.63). The proportion of ADRs caused by GA agents and opiate analgesia has previously been underestimated. The cost of excess bed days due to ADRs, has been estimated to be £2 Million per year for a 400-bed adult hospital. The cost of excess bed days in our study was only £35,000 per year for a 300-bed hospital. Other parameters and methods might need to be considered when assessing the financial impact of ADRs in children. Cisplatin is used in cancer treatment and causes irreversible hearing loss in 42-88% of children. Cathechol-O-Methyltransferase (COMT) and Thiopurine-S-Methyltransferase (TPMT) genetic variants have been associated with hearing loss in paediatric patients, but findings from subsequent studies are contradictory. The occurrence of COMT and TPMT genetic variants in a UK population was examined in a retrospective, multicentre cohort study. Known risk factors for ototoxicity were confirmed; increasing cumulative dose of cisplatin younger age (p< 0.01), cranial radiotherapy (p <0.028) and exposure to vincristine p< 0.091). The association with COMT and TPMT genetic polymorphisms could not be replicated. ADRs in children are common and improving medicines safety in children remains a vital aspect of clinical care. New assessment tools are aiming to address some of the challenges faced by clinicians and researchers. Our knowledge of pharmacokinetics and pharmacodynamics in children is still limited, but advances in the field of paediatric pharmacogenomics are beginning to translate into improved medicines safety and efficacy for children. Further work about non-Oncology ADRs would benefit from a focus on high impact events that are described in this thesis.

The work described within this thesis takes the form of several different but related projects. The radionuclide, 99mTc, is widely used in nuclear medicine departments and researched in many laboratories throughout the world today and it is the intention of this thesis to contribute to the growing library of knowledge in this field. In chapter three, the technetium complex of tiron, a disulphonato derivative of catechol, is assessed for its radiopharmaceutical efficacy using both animal and human models. The no carrier added [ 99mTc] Tctiron preparation appears to form a single radiochemical species under the reaction conditions described, thought to be [99mTc] [TcO (tiron) 2,]⁵⁻, based on the known reaction of technetium with catechol. A small highly charged ion could be expected to clear efficiently through the renal system, an expectation upheld by the animal studies and not observed in the human studies, where it is heavily protein bound. Chapters four and five are both concerned with thioether complexes of technetium, a donor group little studied with this particular metal. Firstly, a series of dithiadicarboxylic acids were synthesised or purchased and their subsequent reactions at both no carrier added [99mTc] and carrier added [99Tec] levels are discussed. Secondly, a macrocycle, potentially hexadentate through a S4 N2, donor set, was synthesised and derivatised and the reactions of these ligands with 99mTc and 99Tc are examined. In both cases, comprehensive animal studies are performed using two types of rodent, which show mixed hepatobiliary and renal clearance.

In the present study, we investigated the long term antiproliferative potential of iron chelators Salicylaldehyde Isonicotinoyl Hydrazone (SIH), Pyridoxal Isonicotinoyl Hydrazone (PIH) and their caged-derivatives 2-Nitrophenyl Ethyl-SIH and –PIH (2NPE-SIH and 2NPE-PIH) using human primary fibroblast cell line FEK4 and the spontaneously immortalised human keratinocyte cell line, HaCaT as models. We then extended the study to additional hyperproliferative skin keratinocyte cell lines notably MKPS (immortalised psoriatic cell line) as well as PM1 and MET2 that represent two cancerous skin cell lines isolated at different stages of malignant transformation of squameous cell carcinoma (SCC) from a single adult individual. Iron depletion with SIH and its UVA-activated caged-derivative (i.e. 2NPE-SIH) led to significant cell death in all cell models presumably as a result of inhibition of G1/S progression in cell cycle. PIH and 2NPE-PIH on the other hand only caused transient growth retardation in cells due to delayed S Phase but with no apparent toxicity. The growth inhibitory/retardation effects of SIH/PIH or UVA-activated caged-SIH/PIH were related to their iron-chelating properties, as their saturation with iron could reverse their antiproliferative activity in the analysed skin cells. Taken together the results suggested that 2NPE-PIH which possesses very high iron chelating potential, but low antiproliferative activity (i.e. upon uncaging by UVA) is more suitable for skin photoprotection. In contrast, 2NPE-SIH which remains inactive inside the cells until its strong iron binding activity and high antiproliferative properties are activated by UVA, may offer a highly selective and dose-controlled alternative for treatment of hyperproliferative skin disorders such as skin cancer.

No description available.

This thesis describes some of the physical, chemical and biopharmaceutical properties of a poorly soluble salicylate, salicylsalicylic acid (SSA). In the introduction influences on drug absorption from the gastro-intestinal tract are described. These influences are of physical, chemical, physiological and pharmaceutical origin and are discussed with respect to drugs that are known to exhibit a poor or variable absorption that is limited by their solubility and dissolution behaviour. Methods for improving gastro-intestinal absorption of such drugs are introduced and the advantages of their coprecipitate formation with polyvinylpyrrolidone (PVP) and various bile acids, cholic (CA) and deoxycholic (DOCA) are reviewed. Salicylsalicylic acid is presented as a drug suggested to show dissolution rate-limited absorption and was considered as a suitable model for investigating the influences of various pharmaceutical and formulatory manoeuvres on its absorption. The experimental sections are divided according to in vitro and in vivo disciplines. The in vitro studies describe the characterization of SSA with respect to purity, particle size distribution and surface area, crystallinity, melting point, solubility, pKa, partition coefficient, stability, dissolution rate and the influence of different media on its recrystallisation. These studies showed the drug to exhibit poor solubility and high stability in acidic media and established a relationship between particle size and dissolution rate. Parameters relevant to the pH-partition hypothesis indicated that, provided SSA was in solution, then its absorption would occur throughout the entire gastro-intestinal tract. The preparation of SSA coprecipitates and admixtures with PVP, CA and DOCA are described together with their subsequent characterisation for purity, crystallinity and, in the case of the SSA:CA systems, their in vitro dissolution. In vivo studies are preceded by an introduction to some basic pharmacokinetic concepts and the methods of pharmacokinetic analyses used in this thesis. Investigations were performed in man to establish a correlation between plasma and saliva salicylate levels following oral doses of SSA. This correlation was not shown and, following a report of the limited studies carried out in man, the majority of this section deals with studies in the rat. Various pharmacokinetic criteria were established between the dose of SSA given by intravenous and oral routes. The drug was formulated as a suspension and the influences of suspending agent and particle size were ascertained. A relationship between in vitro dissolution rate and in vivo absorption for material of varying particle sizes was established and, in an attempt to improve bioavailability, the absorption of SSA from coprecipitates and admixtures with PVP, CA and DOCA was determined. These formulatory manoeuvres met with varied success; indeed, only the SSA:CA 1:1 systems significantly improved drug availability. These studies are discussed with respect to the literature and suggestions for further work are presented.

Medication administration in dysphagic patients or those with an enteral feeding tube (EFT) is complicated with an increased risk of medication errors and complications than the general population. The range of issues that require an evidence base is broad and data are lacking, consequently much of the clinical guidance is consensus based. Following a literature review to identify the main evidence themes, an exploration of aspects of medication administration in this patient group was undertaken using survey, laboratory and literature review methods. Survey methodology was used to enable description of the reported clinical practice of relevant professionals and patients across the UK. Liquid medications subsequently identified as ‘problem’ were rheologically evaluated using a clinically derived method focussing on flow properties via EFTs. Crushing methods previously described were evaluated for dose recovery using three model drugs. Finally, a systematic review was undertaken to identify interventions that have been successfully targeted at improving medication related outcomes in this complex group of patients. Appropriate formulation choice for EFT administration exceeded 80% for both patient and nursing home cohorts. Reported medication administration practices were consistent with consensus guidance in the professional group but were not consistently applied by patient groups. The role of healthcare professionals in informing practice was inconsistent across care settings and warrants further evaluation. Laboratory assessment of liquid medication properties demonstrates a relationship between viscosity and administration difficulties. Tablet crushing methods which significantly reduce dosing accuracy were identified, and calls into question the continued use of equipment currently used for crushing tablets. A systematic review of the literature surrounding intervention strategies to improve medication related practice revealed an education and documentation based focus on preparation and administration steps with minimal evaluation of interventions targeting prescription quality. Retrospective mapping against the Theoretical Domains Framework highlighted a lack of focus on motivational factors which may negatively impact intervention sustainability. This exploration, from bedside to bench and back again, revealed that potentially suboptimal administration methods are common in clinical practice. New data and insights generated within this thesis should be translated into clinical practice to improve outcomes. However, further research is still required to understand motivations for changing practice, provide pharmaceutical data to support more specific guidance on formulation choice, and an evaluated intervention strategy to change and embed good practice, each of these aspects are a major work in their own right.