The rational design of novel chemotherapeutics is based upon knowledge of the intermolecular interactions between ligands and their targets, and the physical, topological and dynamic properties of those ligands within the biological environment. The pyrrolo(2,1-c)(1,4)benzodiazepine-5-ones (PBDs) are a family of sequence-selective, DNA minor groove-binding antitumour agents that inhibit transcriptional events and DNA replication. The effect of pH and salt concentrations on the conjugation rates of two PBDs with short DNA sequences were quantified by HPLC analysis. To further understand how the binding interactions of PBDs with DNA are affected by varying electrolyte conditions, computational techniques including conformational searching and molecular dynamics simulations were applied. The effect of the environment on the conformation of ligands and their target interactions was further evaluated and utilised in the design of Keap1-Nrf2 protein-protein interaction (PPI) inhibitors. Inhibition of this PPI increases the free concentration of Nrf2, a redox transcription factor which plays important roles in controlling xenobiotic and oxidative stress. Inhibitors could therefore act as cytoprotective agents in a range of diseases. A series of peptides with minor sequence modifications that interact with Keap1 were evaluated using computational approaches including: conformational searching (MacroModel), simulated annealing and molecular dynamics (Desmond). Binding poses of peptides in complex with Keap1 based on the structural information from the crystal structure of the Keap1-Nrf2 complex (PDB entry 1X2R) were predicted using several molecular docking programs (Hex, GLUE, Glide, GOLD). Finally, a series of reference crystal structures of a small molecule and peptides in complex with Keap1 were used as templates for the rational design of potential inhibitors of the Keap1-Nrf2 PPI. ‘Scaffold hopping’ (Spark) and free energy perturbation (FEP) (MCPRO) calculations were applied during the inhibitor design to provide possible replacements for key structural elements. This resulted in a virtual library of potential non-peptide inhibitors of the Keap1-Nrf2 PPI for future synthesis and evaluation.

The prenatal use of medicines and recreational substances is of significant importance because there is insufficient information on the effects of medicines and recreational substances on pregnancy outcomes. In addition, literatures on health beliefs of pregnant women about medicine and recreational substance use are lacking. The aim of this thesis was to investigate medicine and recreational substance use during pregnancy in an antenatal population of London. The study was approved by the ethics committee. The first part of the thesis was a prospective cohort study of medicine and substance use across all trimesters (using survey methods), and the pregnancy outcomes (using the medical records); the second part was a qualitative study of the health beliefs of pregnant women which employed semi-structured telephone interviews and the Health Belief Model as a framework for data collection and analysis. The results of the prospective study demonstrated that the prevalence of use of prescription, over-the-counter and complementary and alternative medicines during at least one trimester were 32.5%, 50.2% and 57.1% respectively. The prevalence of exposure to alcohol, cigarette and illicit substances were 16.0%, 3.5% and 0.9% respectively. However, due to limited sample size, the study could not demonstrate an association between the medicines and substances used and increased risk of congenital anomalies in the baby. The qualitative study indicated that pregnant women’s adherence to medicines could be explained by women’s perception of the severity of a medical condition, risks of non-adherence to the medicine as well as anxiety about the risks of the medicine on the foetus. In the case of substance use, a low risk perception could be used to explain women’s behaviour. Healthcare professionals have a responsibility to counsel pregnant women about the benefits or risks of medicines and substances, informed by the best evidence, and guided by the women’s perceptions.

Chimeric proteins (e.g. fusion proteins, bispecific antibodies) often have more than one therapeutic function. All of the clinically validated examples (e.g. Enbrel®) are produced using recombinant strategies. Considering the recombinant fusion of two different proteins, there is a need to consider the design of the construct, specifically the polypeptide linker. Challenges remain to make small families of chimeric proteins for preclinical studies as well as to produce these proteins at manufacturing scale. A possible alternative strategy is a recombinant-chemical conjugation approach using separate proteins and heterobifunctional reagents. The concept of conjugating two proteins together has been known for several decades, but there have been limitations because many protein conjugation reagents were not site-specific. To explore strategies to prepare chimeric proteins more efficiently, site-specific heterobifunctional reagents were developed. These heterobifunctional reagents were derived from PEG and were designed to undergo site-specific protein conjugation at one PEG terminus by either mono- or bis-alkylation Michael reaction. The other reagent terminus comprised a functional moiety that was capable of undergoing a reaction with a reactive group not found on a protein (e.g. aldehyde and hydrazine). These bio-orthogonal moieties would then be matched to undergo reaction to give Protein1-(PEG)2×5k-Protein2 conjugates with PEG being used as a spacer. To prepare Protein1-(PEG)2×5k-Protein2 it was first necessary to generate protein-PEG5k-X(Y) intermediates. This was accomplished using only 1-2 molar equivalents of either mono- or bis-alkylating-PEG5k-X/Y reagents with a conversion achieved up to 75%. The X and Y functional groups were hydrazine and aldehyde. Two heterodimers (IFN-(PEG)2×5k-HSA and amylase-(PEG)2×5k-lipase) were then prepared by coupling two of the protein intermediates together. Purified amylase-(PEG)2×5k-lipase heterodimer was active when tested for both lipase and amylase activity. IFN-(PEG)2×5k-HSA heterodimer was shown to have comparable specific activity to Pegasys®.

Studies have shown an increasing use of herbal medicines alongside conventional drugs by patients in their disease management especially for chronic diseases, with the attendant risks of herb-drug interactions. In order to forestall this, adequate information about the pharmacological and toxicological profile of herbal medicines and how these would in turn affect the bioavailability of the co-administered drug is required. To evaluate potential herb-drug interactions that could occur in diabetes management in Nigeria- (a) An assessment of available data on the pharmacological and toxicological effects of plants used in diabetes management was conducted as a means of mapping those with identified potential risks for herb-drug interactions; (b) A field work study was carried out in different localities in Nigeria to identify potential pharmacokinetic interactions based on the prescription drugs and herbal medicines co-administered by diabetic patients; and (c) Experimental analysis of plant samples collected during the field work was done to assess their effects on known cell detoxification mechanisms and pharmacokinetic parameters. The results of the research have confirmed the continued use of a wide range of medicinal plants in diabetes management, many of which have not been thoroughly investigated. In addition, 50% of diabetic patients visiting healthcare facilities in Nigeria routinely manage their diabetes or existing co-morbidities with herbal medicines alongside prescription drugs. Even more worrying is the frequent use of unlabeled herbal preparations which would constitute a huge challenge in the proper identification of herb-drug interactions when they occur. Based on previously available data and the experimental results of this research, a number of these herbal medicines have been identified as having overlapping interactions with prescription drugs. There is therefore a need for better regulation of herbal medicine use alongside pharmacovigilance monitoring in Nigeria in order to forestall the occurrence of clinically relevant untoward herb-drug interactions.

The primary objective of this research was to investigate the possible effects of selected liquid crystal (LC) forming surfactants, namely ArlacelTM 2121, CrodafosTM CES and BrijTM system (BrijTM S721/ BrijTM S2) and selected oils, namely, Arlamol™ PS15E, Crodamol™ OP, Arlamol™ HD on formulation properties. The effects of the different excipients were monitored using the formulations thermal properties, water holding ability and ability to promote the permeation of ibuprofen (IBU and caffeine (CAF)) across silicone membranes. The melting endotherms of the ternary formulations containing 10% w/w Arlamol™ PS15E or Crodamol™ OP, 10% w/w surfactant and 80% w/w water resulted in high melting endotherm (>55˚C). However, the inclusion of 10% w/w Arlamol™ HD in equivalent ternary formulations lowered the melting endotherm to 45-50 ˚C, suggesting a destabilising effect of this oil. In addition, increasing the surfactant content of ternary formulations from 5% w/w to 10% w/w reduced the evaporation time of free water by 10-20 min. However, this change in the water holding ability was not the same for all surfactants. The results ranked the water holding ability of the surfactants as the Brij™ system > CrodafosTM CES > Arlacel™ 2121. The permeation profiles of IBU and CAF across model membranes showed significant enhancement (p <0.05) for both drugs from saturated formulations containing Crodamol™ OP with the Brij™ surfactant system. This was attributed to the uptake of Crodamol™ OP into silicone membrane (22.88%) and the good solubility of both model actives in this oil. In addition, the permeation results suggest that the Brij™ system was interacting with membranes at a greater extent compared with other surfactants. The thesis also investigated the hydration characteristics of the human nail using the Confocal Raman spectroscopy (CRS). The results showed the nail to absorb significant amounts of water (~15% w/w) into the top 5 µm of the nail plate after 10 min of hydration. The absorbed water was lost from the nail in a quick manner of 30 min which agreed with previous reports. The CRS was also used to monitor in vivo deposition of IBU from saturated propylene glycol (PG) solutions under infinite (occluded) conditions. The results were consistent with previous work, showing IBU signal inside the skin to increase consequently with PG content in the applied formulation. These results indicated that CRS can be used as a valid in vivo technique to monitor drug delivery.

Dysregulation of mitochondrial dynamics is an emerging theme in neurodegenerative disorders, including Parkinson disease (PD). Autosomal recessive PD (ARPD) can be caused by mutations in the mitochondrial kinase PINK1 or the cytosolic E3 ubiquitin ligase Parkin. PINK1 and Parkin act in a common genetic pathway to control mitochondrial dynamics. To gain insights into the regulation of mitochondrial dynamics, binding partners of PINK1 were identified using different approaches: yeast two-hybrid screening of cytosolic and membrane bound forms of PINK1 and affinity purification of epitope-tagged PINK1 from a stable cell line under basal conditions and during mitochondrial stress. Many potential substrates of PINK1 were identified, including proteins with roles in mitochondrial dynamics. CluH was persistently identified in the PINK1 co-precipitation studies. CluH is a large tetratricopeptide protein that is highly conserved. CluH has previously been implicated in the regulation of mitochondrial dynamics in yeast, slime mold, plants and the fruit fly, but remains uncharacterized in mammals. I therefore investigated the expression profile of CluH in mouse tissues, and the subcellular localization in human cells using confocal microscopy. I also examined whether CluH could regulate mitochondrial morphology and function. I demonstrated that siRNA knock-down of CluH led to hyperfused mitochondria while over-expression of CluH led to fragmented mitochondria. Knock-down of CluH also resulted in decreased complex I activity, suggesting that CluH is required for mitochondrial integrity. The domains of CluH required for altering mitochondrial dynamics were also determined. Finally, I examined the interplay of CluH with the fission/fusion machinery and found that CluH can interact with fission proteins MiD49 and Drp1, consistent with a role for CluH in promoting mitochondrial fission. Taken together, these studies have identified CluH as a new player in mitochondrial dynamics, which interacts with the ARPD protein PINK1.

In the light of problematic challenges in assessing the antimicrobial properties of medical materials and devices it is necessary and important to explore novel applications of existing analytical techniques. Isothermal microcalorimetry, whether as a static or modified flow-through system, was shown to have great potential with this area. The growth and metabolism of microbes, as with any other process, involves a change in enthalpy (heat). This is measurable using isothermal microcalorimetry, allowing real-time detection of growth and changes in metabolic performance of microorganisms. While there are a number of standard methods to assess microbial growth, for example, turbidometric measurements, calorimetry offers the benefits of being non-invasive, not dependent on sample transparency and being capable of measuring whole sections of medical devices. This work demonstrates the application of calorimetry in testing the efficacy of antimicrobial wound dressings both in freely suspended cultures (planktonic cells) and in sessile, matrix enclosed communities (biofilms). A flow-through system was developed by modifying existing ampoules to allow placement of ‘test’ tubing within, as well as to allow the flow of bacteria cultured in an external bioreactor. The flow-through system was designed as a testing system for putative products and materials aimed at reducing or eliminating device related biofilms. The calorimetric work described in this thesis was corroborated using classical microbiological methods with the aim of bridging the gap between these disciplines and to demonstrate the validity of calorimetry as a useful analytical method in microbiological studies.

A major bottleneck in drug discovery is the production of soluble human recombinant protein for functional, biochemical and structural analyses. The level of recombinant protein expression is controlled by a complex relationship between both biological and engineering variables. Due to the inter-play between these variables and standard experimental methods, the identification of the key variables which lead to improved protein expression can sometimes be missed. This thesis presents a framework which underpins the generation of large quantities of soluble recombinant protein in E. coli in a rapid and cost-effective manner. To achieve this goal, Design of Experiments (DoE) was first employed in combination with microwell plate (MWP) fermentations to investigate the wide array of protein expression variables. These tools are well suited to high-throughput expression requirements as they afford large savings in time, cost and resource requirements. The information generated from these MWP experiments was then exploited to devise a strategy for reproducing the process within stirred- tank reactors (STRs). The DoE methodology was first used to identify relevant protein expression variables including fermentation variables (media type and fermentation time), protein induction variables (inducer concentration and induction time) and environmental variables such as oxygen transfer rate, temperature and pH. Ten factors were screened overall at the microwell scale and three were investigated further through optimisation designs. The application of DoE led to a robust understanding of the process and resulted in protein yields five-fold greater than those obtained under standard shake-flask conditions. The most significant factors were post-induction period and shaking speed, the latter of which is strongly related to the mass transfer coefficient, faa. In order to translate this stable and optimised small-scale expression system to a production-scale stirred-tank reactor (STR), an understanding of the engineering parameters at both scales of operation was crucial. This need was complicated by significant differences between the MWPs and STRs such as geometry, mode of aeration and agitation, and the effects of surface tension. In this work, the MWP fermentation results led to the hypothesis that operation at a constant kia value would facilitate predictable scale translation. However, there currently exists very little published work on the characterisation of kia within MWPs. Miniature oxygen probes were, therefore, used to characterise MWP kia values directly via the static gassing-out method over a range of square-well MWP formats and shaking speeds. This information was then used to translate the performance of a 3ml MWP E. coli fermentation, on the basis of matched faa, to STR working volumes of 51 and 451. The efficacy of scale-up was confirmed by performing F tests on pairs of profiles for cell growth and expression levels of recombinant firefly luciferase. This rapid, accurate and direct method of kia characterisation within MWPs enabled a 15,000-fold direct scale-up of fermentation performance in terms of cell growth and protein expression from MWP to STR.

The assessment of the benefits of pharmaceutical products through health technology assessments (HTAs) has become a feature of health care decision-making in numerous OECD countries, including England and Germany. Assessment outcomes vary between countries but, to date, there is a lack of research on the factors that affect those assessments. This thesis addresses this shortcoming by examining what determines the outcome of pharmaceutical benefit assessments in two countries that employ formalised HTA procedures. It takes a novel theoretical approach by employing a framework of policy paradigms to explain an empirical phenomenon other than policy change. The study presents a qualitative analysis that compares the reasoning processes that led to assessment outcomes in ten of the same cases of pharmaceuticals in England and Germany. It finds that benefit assessment outcomes are determined by how a similar set of themes around evidence gets interpreted and framed by a HTA body, e.g. the National Institute for Health and Care Excellence (NICE) in England and the Federal Joint Committee (FJC) in Germany. The study explains the differences in addressing a similar set of themes around evidence by reference to different HTA paradigms that are applied, namely a cost effectiveness paradigm in England and a patient relevance paradigm in Germany. The thesis makes a significant theoretical contribution because it demonstrates that policy paradigms can be used to explain empirical phenomena other than policy change. This requires an analysis of how paradigms are articulated in ‘normal decision-making’, much akin to Kuhn’s analysis on the connection between ‘normal science’ and paradigms. The study calls for an expansion of the current use of policy paradigms to include how they are operationalised in practice as this leads to a better understanding of the crucial elements of a paradigm.

Background and purpose: Biodegradable polymers, such as PLGA, are delivery vehicles used to treat posterior segment eye disease, but suffer from poor drug loading and initial burst release. This thesis describes a ‘system-within-system’, PLGA microparticles incorporating chitosan-based nanoparticles, for ranibizumab delivery, and chitosan-containing micelles for transscleral rapamycin delivery. Methods: Synthesis of chitosan-N-acetyl-L-cysteine (CNAC) and octanoyl-chitosan-poly (ethylene glycol) (OChiPEG) were confirmed using spectroscopy. Chitosan/TPP, chitosan/TPP-HA, CNAC and CNAC/TPP nanoparticles containing ranibizumab were prepared then incorporated in PLGA microparticles and characterised for their size, zeta potential, morphologies, solid-state properties, morphology, protein loading, stability, release, in vivo antiangiogenic activity and effects on cell viability. Rapamycin-loaded micelles comprised of OChiPEG and chitosan-incorporated TPGS, CPEG and TPGS/CPEG were also prepared. Micelles were analysed for their size, zeta potential, morphology, stability, solid-state properties, rapamycin entrapment efficiency and ex vivo scleral retention and permeation. Results: Chitosan-based ranibizumab-loaded nanoparticles measured 17 – 350 nm with a zeta potential of -1.4 to + 12 mV; microparticles measured 3.0 – 6.6 µm (-12 to + 9.7 mV). PLGA microparticles appeared mostly spherical; those prepared with chit/TPP, CNAC and CNAC/TPP had spherical nanoparticles on their surface. Microparticle protein content ranged from 13 – 69%. All preparations showed burst release except for the CNAC containing microparticles, which had the slowest release. Ranibizumab released from PLGA microparticles maintained structural integrity, and activity in cell culture. Chit/TPP-HA nanoparticles containing ranibizumab showed enhanced antiangiogenic activity relative to native ranibizumab. Microparticles showed no effect on cell viability up to a concentration of 12.5 mg/mL. Rapamycin loaded micelles measured 11 – 41 nm, with a zeta potential of -1.2 to + 6.8 mV, entrapment efficiency of 75 - 97% and a scleral retention of 7.5 – 44 µg/g. Conclusion: Micelles showed enhanced scleral retention with potential for topical ocular delivery of poorly soluble drugs. The CNAC-containing preparation has potential for the intraocular delivery of protein-based drugs.