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There are recognised knowledge gaps in understanding pharmaceutical tablet manufacturing processes, particularly those relating to the relationship between components in the formulation, processing conditions and the resultant final product characteristics. Being able to quantitatively visualise the microstructure of a tablet matrix is essential for better product and process understanding. Vibrational spectroscopic chemical imaging is an established tool in the pharmaceutical industry to characterise the size and distribution of components within tablets. Current methods, however, do not go beyond examining a sample's single exposed surface area. There are limitations associated with estimating domain size and shape statistics from a two-dimensional (2D) chemical image as the values obtained will depend on where the domain is sectioned, often over- or underestimating its true value. Three-dimensional (3D) imaging has been reported in the literature by using confocal Raman measurements to obtain a depth profile of a sample. However, this depth is typically less than 50 microns, which is smaller than the particle size of many commonly used excipients and active ingredients. This approach is therefore unsuitable for visualising the spatial arrangement of components within pharmaceutical products. For enhanced drug product and tablet manufacturing understanding, new 3D imaging techniques that can provide full visualisation of a tablet's microstructure must be developed. This thesis presents an alternative method of obtaining 3D chemical images of pharmaceutical tablets by combining Raman mapping and serial sectioning. 3D quantitative image processing methods were developed to provide a means of objectively assessing differences in the spatial distribution of components. A novel domain adjacency method was devised to calculate the contact surface area between each component to provide superior knowledge on the relative position of a material within the tablet matrix and quantify the association between components. To demonstrate the advanced understanding provided by the proposed 3D chemical imaging approach, the method was used to explore differences in the microstructure of tablets with known good and atypical stability behaviour. The physical structure of the tablets could be used to explain the difference in the stability and provided some insights into the possible types of manufacturing techniques used during production. By providing a means to quantify the 3D microstructure of a tablet matrix, these new capabilities provide a method to link product performance with 3D physical characteristics and thereby enhance manufacturing process understanding. This fundamental knowledge could be used to optimise formulation development through designing manufacturing processes that deliver effective medicines with desired performance characteristics, to ultimately reduce development timelines.

Malaria and tuberculosis (TB) are diseases which have both plagued humanity for thousands of years and remain a significant cause of global death to this day. In 2019 there were a predicted 229 million cases of malaria worldwide causing 409,000 deaths. In the same period there were 10 million cases of active TB and a corresponding 1.4 million deaths. Though treatment for malaria and TB can be effective and curative, treatment failure rates owing to resistance to front-line antimalarial and anti-TB drugs threatens to undermine currently available therapies – therefore the development of novel drugs is essential to the continuing treatment and eradication of these diseases. This work describes three projects focussing on the development of novel peroxide and non-peroxide containing drugs for the treatment of malaria and TB. Synthetic endoperoxide antimalarials, such as 1,2,4-trioxolanes and 1,2,4,5-tetraoxanes, are promising successors for current front-line antimalarials, semisynthetic artemisinin derivatives. However, limited solubility of second-generation analogues in biologicallyrelevant media represents a barrier to clinical development. We present methodology for the synthesis of nonlinear analogues of second-generation tetraoxane antimalarials E209 and N205 to investigate reduced molecular symmetry on in vitro antimalarial activity and physicochemical properties. While maintaining good antimalarial activity and metabolic stability, head-to-head comparison of linear and nonlinear counterparts showed up to 10-fold improvement in FaSSIF solubility for three of the four analogues studied. Pharmacokinetic studies in rats comparing a selected nonlinear analogue 2.9b and its parent N205 showed improvement in oral absorption and exposure in vivo with more than double the AUC and a significant increase in oral bioavailability (76% versus 41%). These findings provide support for further in vivo efficacy studies in preclinical animal species. Lengthy treatment duration is an issue in current anti-TB therapy. This is caused in part by the ability of Mycobacterium tuberculosis (Mtb) to enter a non-replicative persistent (NRP) state characterised by reduced metabolism and a phenotypic tolerance to most anti-TB drugs. A recent high-throughput screen highlighted the peroxide-containing antimalarial drug artemisinin as an inhibitor of DosRST signalling – a signalling pathway implicated in the ability of mycobacteria to enter NRP state. We used molecular docking studies to design and synthesise a series of peroxide-containing hybrids of the emerging indole-2-carboxamide (I2C) and benzothiazinone (BTZ) classes of MMPL3 and DprE1 inhibitors respectively. We hypothesised that the peroxide moiety would inhibit DosRST signalling, sensitising Mtb to the anti-TB pharmacophore. Of the I2C scaffold only 4.11 maintained anti-TB activity with a modest IC50 of 2.634 µM. All synthesised BTZ hybrids maintained single-digit nanomolar potency with the exception of 4.17 which displayed near maximum inhibition at the lowest concentration tested (0.457 nM). Compounds 4.11 and 4.16, and parent compounds NITD304 and BTZ043 were submitted for DosRST inhibition assays. At this stage, DosRST signalling inhibition data has only been obtained for 4.11 which indicated a dual mode of action of DosRST inhibition and MMPL3 inhibition. This result provides a case for further characterisation of interaction with components of the DosRST regulon. DprE1 is a promiscuous target for drug discovery with several classes of covalent and noncovalent inhibitors reported in literature. Covalent interaction is mediated by a nucleophilic cysteine residue Cys387 which is implicated in the mechanism of action of the BTZs and other nitro-aryl DprE1 inhibitors. The non-covalent inhibitor OPC-167832 possesses potent activity against Mtb comparable to the covalent inhibitor BTZ043. Molecular docking studies of OPC167832 showed that Cys387 was placed only 3.7 Å away from C3 of the coredihydrocarbostyril ring. We hypothesised that incorporation of an electrophilic warhead at this position would enable covalent attachment of the dihydrocarbostryril scaffold to generate covalent inhibitors. These inhibitors could be used in head-to-head studies with non-covalent counterparts to investigate resistance acquisition of covalent versus non-covalent inhibitors. A range of ring-closed and ring-opened covalent analogues were designed and synthesised, guided by molecular docking studies. All synthesised ring-opened analogues were found to be inactive in vitro against Mtb while dihydrocarbostryril containing control compounds 5.10a,b were found to maintain activity. This suggests that the interactions of the dihydrocarbostyril ring system are important to the potency of this class of inhibitors. Ring-closed analogues may better preserve this interaction and are the subject of synthesis by another group member at the time of writing.

Part I: Synthesis of a Stable, Fluorinated Thromboxane A2 Analogue and its Application as a Hapten for Monoclonal Antibody Generation. Cardiovascular disease (CVD) is the leading cause of death worldwide, accounting for more deaths per annum than cancer, diabetes, and respiratory diseases combined. One contributing factor to CVD is the overexpression of thromboxane A2 (TxA2)-a member of the prostanoid family of natural products that assists with blood clot formation. Pharmacological inhibition of TxA2 has so far failed to meet expectations due to issues related to selectivity, toxicity, or efficacy. To overcome these problems, a TxA2-specific monoclonal antibody has been proposed; however, the short half-life of TxA2 (t½ = 32 s, pH 7.4) renders this impossible. In this respect, 10-fluorothromboxane A2 (F-TxA2) has been developed as a stable TxA2 mimic using a 15 step process from a key building block that the Aggarwal group has used routinely in the synthesis of several prostaglandins. Unfortunately, this route to F-TxA2, and other thromboxanes of interest, is particularly long and involves significant synthetic challenges. Alongside this, F-TxA2 itself is not immunogenic and must be attached to a large carrier protein such as keyhole limpet hemocyanin (KLH) in a structural design known as an antigen, which is required to elicit an immune response. This thesis details the development of both an improved strategy towards the total synthesis of thromboxanes, with a focus on thromboxane B2 (TxB2) a natural metabolite of TxA2, as well as the synthesis of a tetra-valent F-TxA2-containing antigen. In the former, a key oxidative ring-expansion provided rapid access to the core of the thromboxanes in the form of a hemi-acylal intermediate. However, the sensitive nature of this species and its downstream intermediates towards epimerisation ultimately required various route re-designs. This was able to be overcome with a diastereoselective dynamic kinetic resolution process to provide stereocontrolled access to a unique acylal that is currently being evaluated for its potential in the improved total synthesis of TxB2 and eventually F-TxA2. In the latter work, F-TxA2 isolated from the initial total synthesis was rendered immunogenic by coupling to a tri-lysine peptide and conjugating to KLH. This strategy allowed for the preparation of a tetra-valent F-TxA2-containing antigen that has since been administered to murine hosts for the potential generation of TxA2-specific monoclonal antibody that could find use as a novel therapeutic for the treatment of CVD. Part II: Difunctionalisation of C-C -Bonds Enabled by Strained Boronate Complexes. Cyclobutanes are becoming increasingly prevalent within drug discovery programmes; however, access to this type of scaffold is generally limited by the lack of synthetic methods that permit its construction. In this respect, the synthesis of cyclobutanes, particularly those of defined geometry, is often the limiting step in medicinal chemistry. This section discloses the development of a modular approach to the diastereoselective synthesis of 1,1,3-trisubstituted borylcyclobutanes using bicyclobutyl boronate complexes. These strained intermediates were prepared through the reaction of bicyclobutyl lithium with boronic esters and found to be highly reactive with a broad range of electrophiles, facilitating the introduction of both a migrating group and an electrophile across the central C-C -bond in a formal difunctionalisation process. The unique reactivity of these highly strained boronate complexes was studied using in situ IR spectroscopy and showcased in a reaction with benzaldehyde, which was found to be complete after only 5 min at −78 °C. Furthermore, unique electrophiles such as carbon dioxide, which has not previously been shown to react with other boronate complexes, was amenable to the process. Density functional theory (DFT) calculations were also undertaken to provide a model for the stereochemical outcome of the process, and finally, the boronic ester handle was further derivatised into a range of useful functional groups to provide a third point of diversification. This methodology may find use within drug discovery programmes to rapidly build-up functionalised cyclobutanes for use in screening libraries.

Three mesoporous silica excipients (Syloid®silicas AL-1 FP, XDP 3050 and XDP 3150) were formulated with three model drugs known for their poor aqueous solubility, namely phenylbutazone, indomethacin and imipramine in an attempt to enhance the extent and rate of their dissolution. Although other forms of mesoporous silica have been investigated in previous studies, the effect of inclusion with these specific Syloid® silica-based excipients with phenylbutazone, indomethacin and imipramineare unknown. This work reports a significant enhancement for both the extent and rate of drugs release for all three forms of Syloid® silica at 1:1, 2:1 and 1:3drug:silica ratios over a period of 45minutes. An explanation for this increase was determined to be conversion from crystalline to the amorphous form and an enhanced drug loading ability within the pores. Differences between the release profiles of the three silicas was concluded to be a consequence of the physicochemical differences between the three forms. Overall, this study confirms that Syloid® silica-based excipients can be used to enhance dissolution, and potentially therefore bioavailability, for compounds with poor aqueous solubility, such as phenylbutazone, indomethacin and imipramine. In addition, it has been confirmed that drug release can be carefully tailored based on the choice of Syloid® silica and desired release profile. The second part of this study investigated the effect of microwave heating through the application of microwave differential thermal analysis to eight model pharmaceutical compounds and a set of four model excipients. Benzocaine, haloperidol, ibuprofen, indomethacin, ketoprofen, naproxen, imipramine and phenylbutazone were analysed, along with four excipients, namely β-cyclodextrin, D-mannitol, stearic acid and Syloid® silica (XDP 3050) using microwave differential thermal analysis. Samples were heated by microwave irradiation at 5 °C/min to a minimum of 160 °C, held isothermally and then slowly cooled to room temperature. Thermal profiles were analysed and compared with data obtained using differential scanning calorimetry (DSC), x-ray powder diffraction (XRD)and hot stage microscopy (HSM). Overall, it was found that the process of microwave heating produced different thermal profiles to those seen using traditional, conductive heating. Investigating differences in thermal profiles can be a useful way to consider the effect of microwave induced heating on formulations which can, in turn, help guide formulation choices. The latter part of this study describes the analysis and characterisation of polyvinylalcohol (PVA)-based hydrogel polymer beads, developed for the embolisation of vessels, specifically focussing on the quantitative and qualitative aspects of water within such beads in the absence and presence of a model drug, namely imipramine. The utilisation of Microwave differential thermal analysis (MWDTA) for the characterisation of bound water within the polymer beads was unsuccessful because there was no cooling system and as a result, most of the water evaporated during the measurement. Following successful incorporation within the beads, thermogravimetric analysis (TGA) permitted determination of the total water content within the beads (96.8 %) and differential scanning calorimetry (DSC) indicated the water within the beads was apportioned as 15.8 % non-freezing, 25.1 % loosely bound and the remaining 55.9 % unbound. In the presence of drug, the size of the beads decreased with an average diameter reduction from 121.4 μm to 78.5 μm, coupled with a reduced total water content of 95.4 %, coupled with a reduced percentage of loosely bound water. This study confirms the ability of TGA and DSC to characterise the differing types of water within the beads and indicates the relative changes in water content in the presence of model drugs.

This thesis presents a novel investigation into the use of two advanced imaging techniques in the field of pharmaceutics. The first is surface dissolution imaging(SDI).This technique utilises ultra-violet (UV)light and a Complementary Metal Oxide Semiconductor(CMOS)detector chip to image and film a wide range of dissolution phenomena. The second technique is focus variation microscopy(FVM).This powerful light microscope combines conventional microscopy with vertical scanning allowing for both high resolution images and surface topography data to be obtained. The introduction of this work thus provides a brief history and review into the design and development of dissolution imaging and the wide range of research that has currently been conducted. An introduction in to surface texture measurement and focus variation microscopy are also given to give the reader context as to how these are used in the detailed research. This thesis focuses on the second generation of the surface dissolution instrument (SDi2) which has an additional dosage cell, compared to its predecessor the SDI300,and the ability to record images in both the UV and visible ranges simultaneously. Chapter 3 therefore details and describes the development of a methodology that allows the utilisation of dissolution imaging for the purposes of monitoring accurate swelling of hydrophilic matrices. The successful results showed a set of specific parameters needed to be followed in relation to both absorbance threshold and the width of the measurement zone. This developed method was also successfully used in the swelling determination of other hydrophilic polymeric matrices in chapter 4. The results also showed this methodology to be a material sparing technique as compared to the traditional methods of investigating swelling processes. Chapters 5-7 uses gemfibrozil (GEM),indomethacin (INDO) and propranolol hydrochloride as model drugs in salt formation, solid dispersions and liquisolid compact formulations. Focus variation is used in understanding the surfaces that are prepared prior to intrinsic dissolution rate determination (IDR). Techniques such as X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC)and scanning electron microscopy (SEM) were used for salt and solid dispersion confirmation as well as morphological characterisation. The results from the focus variation for both the salts and solid dispersions indicated that compacts prepared for IDR determination picked up the tooling "imprint" on the surfaces which can impact on IDR values obtained. A web-like phenomenon was also observed for the solid dispersions during IDR imaging which seemed to increase with increasing polymer content. This process explained the lower IDR values obtained for the solid dispersion relative to that of the "pure drug (INDO)". The use of the whole dose cell showed the dissolution of the solid dispersion to increase with increasing polymer content. Chapter 7 also recorded the first simultaneous drug release and swelling of liquisolid compacts. The information from this thesis therefore demonstrates the versatility and use of both imaging techniques in providing vital information for the formulator in the early stages.

Introduction: Lateral flow immunoassays (LFA) are routinely used to detect illicit drugs in biological matrices. Most recently, latent fingerprints (LFPs) have been analysed using this method. There is a lack of screening tests that identify MDMA(ecstasy) despite it being used by 2.6 million people in Europe and 524,000 in England and Wales. MDMA (pKa 9.9) has been shown to be excreted in sweat. This thesis explored the utility of LFPs as a biological matrix for screening for MDMA in the field via a LFA as a Point-of-Care test (POCT). Method: The Ridgeway (Intelligent Fingerprinting Ltd., UK) was employed to quantify LFPs and they were further characterised using ATR-FTIR to measure LFP mass and composition. The Ridgeway was used to investigate fingermarks sampling methods and variables known to influence sweat production including deposition pressure,environmental conditions and certain drugs. An optimised plate immunoassay was used to establish a suitable binding pair (Anti-MDMA and BSA-MDMA). Further characterisation included establishing an ideal Alexa Fluor 488 TFP:Anti-MDMA ratio and confirming cross-reactants. Results were validated with Surface Plasmon Resonance (SPR). A LFA suitable for MDMA detection using LFPs was achieved by testing different antibody binding concentrations against different extraction buffer compositions. Optimisation of the LFA identified: cross-reactants, cartridge run-time and cut-off level. Fluorescent LFA signals were measured using Intelligent Fingerprinting Reader 1000 (Intelligent Fingerprinting Ltd., UK). Results: A significant positive correlation was calculated between LFP mass and Ridgeway score [p=0.02] advising a relationship between the two measurements. Higher Ridgeway scores corresponded with sebaceous LFPs after ATR-FTIR analysis. An optimal sampling method was determined as: cumulative deposition of10 LFPs [p<0.1] and either hand can be used. To collect eccrine only LFPs, Carex soap with lint free tissue were most suitable [p<0.01] compared to antibacterial handgel. Different environmental climates did not change the sample quantity deposited for both natural and eccrine LFPs. Drug use, including methadone, significantly increased Ridgeway scores in the addict population versus the control population[p<0.01]. Comparable LFPs were collected via consistent deposition pressure (300 -400 g) and glass deposition surface. Both plate assay and SPR results confirmed the binding pairs' suitability for the MDMA LFA. Monoclonal Anti-MDMA detected MDMA at 0.15 ng/mL and KD values of 0.3 and 0.5 nM were calculated, respectively. A ratio of 7.3:1 was calculated as the ideal labelling ratio. A sensitive LFA, which produced a sharp-drop in signal, was designed using an extraction buffer coupled with 20 ng Anti-MDMA and 120 µg/mLBSA-MDMA. This established a cut-off value of 60 pg in 10 µL which generated excellent accuracy (0.98), sensitivity (1.0) and specificity (0.95). Immunoassay methods recognised Methylenedioxy-N-ethylamphetamine and para-Methoxy-Nmethylamphetamine as key cross-reactants at 55 pg in 10 µL. Conclusion: A sensitive MDMA LFA was successfully designed to identify MDMA inLFPs at a cut-off value of 60 pg in 10 µL for use in drug screening. For this purpose,an optimal LFP collection method was also established.

The naked mole-rat (NMR) is a fascinating animal which has unique biological features including eusociality, strict subterranean inhabitation and poikilothermy. It is the longest-living rodent, showing negligible senescence over the majority of its lifespan and high resistance to diseases such as cancer and neurodegeneration. This animal is therefore a compelling system for understanding ageing and age-related diseases. Recent evidence suggests that protein homeostasis (proteostasis) mechanisms may play a vital role in mediating the resistance to multiple forms of stress and diseases and, subsequently, contribute to the exceptional longevity of the NMR. However, this view has been predominately based on protein-level and/or cell viability analyses, and our knowledge is still limited about the modulation of proteotoxic stress responses at the transcriptional level due to a lack of reliable and validated molecular tools. This thesis sets out to develop, optimise and apply new methods to investigate two important and complex proteostatic mechanisms, namely the unfolded protein response (UPR) in the endoplasmic reticulum (ER) and macroautophagy (autophagy), in the NMR. Using these methodologies, the effects of pharmacologically induced in vitro stress and the effects of disease-related neurotoxic protein species on the UPR and autophagy in NMR fibroblasts were investigated. In Chapter 3, RNA-based methods, including an Xbp1 splicing assay and quantitative PCR (RT-qPCR) assays were successfully established to probe the activation and outputs of the UPR in a NMR kidney fibroblast cell line in response to tunicamycin (TU) and thapsigargin (TG)-induced ER stress. In Chapter 4, differences between the UPR of NMR kidney fibroblasts and mouse homologues were identified, where a notably higher threshold of pharmacologically induced UPR activation was observed in the NMR under conditions of mild ER stress. In Chapter 5, LC3B turnover and transcriptional changes of autophagy markers under rapamycin (RA) and chloroquine (CQ)-treated conditions were monitored in an NMR skin fibroblast cell line, where the sensitivity of the NMR skin fibroblasts to CQ, when compared to NMR kidney fibroblasts and mouse NIH3T3 embryonic cells, seemed to be partly attributed to the downregulation of TFEB, a master transcription factor of autophagy. In Chapter 6, the effects of amyloid-beta (Aβ) and α-synuclein oligomers, which are believed to be the major pathogenic species of Alzheimer's and Parkinson's diseases, respectively, on the UPR and/or autophagy were investigated in NMR and mouse cells using a combination of molecular and cellular tools. Although no significant changes of UPR markers were observed under Aβ oligomer-treated conditions, the chronic toxicity of wild-type α-synuclein oligomers seemed to be associated with downregulation of genes encoding ER chaperones and autophagy proteins. In Chapter 7, we demonstrate the utility of rational design to create a protein-specific binding probe for the NMR LC3B protein by introducing a peptide derived from a LC3-interacting region (LIR) motif into the inter-repeat loop of a consensus-designed tetratricopeptide repeat protein (CTPR). The results provide proof-of-concept validation of using CTPR-based probes to detect proteins in emerging animal models. Having established a set of reliable methods to investigate the molecular details of the UPR and autophagy in the NMR, we have demonstrated unique features of the NMR, at the transcriptional level, when different forms of in vitro stress are employed. Exploiting these assays to measure the UPR and autophagy, as well as other proteostastic mechanisms, in the NMR under more disease-relevant conditions, may ultimately shed light on therapeutic developments to combat age-related neurodegenerative diseases.

Despite advancements in the drug discovery process, drug attrition rate in the late stages of clinical trials still remains high. Better understanding of drug efficacy in the pre-clinical stage would potentially translate into increased clinical trial success rate and reduce the economic burden of failed trials. However, visualising intracellular drug uptake and distribution at high resolution to improve the pre-clinical drug discovery process is still a challenge for scientists. Stimulated Raman scattering (SRS) microscopy represents a powerful imaging tool for visualising drug molecules inside cells with high resolution, without the need for additional labels, or nanoparticle sensors as used in many other optical imaging technologies. It provides Raman imaging with minimal spectral distortion and a quantitative output, allowing accurate determination of intracellular drug concentrations. Ponatinib is a clinically approved tyrosine kinase inhibitor that targets BCR-ABL and is used to treat chronic myeloid leukaemia (CML). Drug resistance is a widespread problem in CML treatment, where ponatinib resistant patients have very limited treatment options. Ponatinib has an alkyne moiety in its structure that makes it inherently Raman active in the cellular silent region of the Raman spectrum. In this thesis, SRS microscopy was used to image intracellular ponatinib label-free with high sensitivity and specificity in live human CML cell lines, in the context of ponatinib resistance. SRS imaging of ponatinib was optimised in Chapter 3, enabling ponatinib imaging at nanomolar treatment concentrations as well as determination of absolute ponatinib concentrations in both ponatinib sensitive and resistant CML cells. In Chapter 4, it was determined that ponatinib is sequestered into the lysosomes, with a higher lysosomal concentration found in drug resistant cells. This was associated with increased lysosome biogenesis. Target engagement studies showed that treatment with chloroquine reduced ponatinib accumulation in lysosomes, but did not resensitise cells to ponatinib, confirming a BCR-ABL independent resistance mechanism in this CML cell model. To demonstrate further utility of SRS microscopy, it was applied to spheroid imaging in Chapter 5. CML cell lines formed three-dimensional (3D) cell 'aggregates' rather than spheroids with tight cell-cell contacts, and could not be used for SRS imaging. However, spheroid growth was successfully optimised in a breast cancer cell line T47D; and live T47D spheroids were imaged using SRS microscopy.

As the proteins responsible for the initiation and propagation of the action potential in excitable cells, voltage-gated sodium channels (NaVs) are crucial drug targets. Antiarrhythmics, anaesthetics, and antiepileptic drugs all target NaVs. Yet despite the difference in NaV subtypes affecting these diseases, the drugs used to treat them are able to act while showing little subtype specificity. This is explained by drugs displaying state-dependant binding and kinetics. Hence an understanding of the structural changes in different gating states of NaVs is vital for structure-based drug design, of more effective and selective compounds. To date there is no atomic-resolution structure of a functional eukaryotic NaV, although the structures of several prokaryotic NaVs which share pharmacological characteristics with their eukaryotic counterparts have been determined. However these structures are in variety of conformations and the relationships between them remain unclear. This study presents biophysical techniques complementary to structural ones to investigate the gating of NaVs, using the channel from Magnetococcus Marinus (NaVMs) as a case study. Oriented circular dichroism (oCD) spectroscopy is used to report on the orientation of the pore-lining α-helices within the membrane, based on quantitative analysis of oCD data. Single molecule FRET spectroscopy is able to measure interatomic distances across biological molecules, and here is used to investigate the changes in pore diameter at the gate of NaVMs. Also presented is a new web-based application which uses protein structure files to define the orientation of α-helices that comprise the channel.

Many Picornaviridae family members infect humans. Enterovirus (EV), the largest genus, causes diseases ranging from the common cold to fatal heart disease and paralysis. Human parechoviruses (HPeV) are at least as prevalent as EVs. Currently, no antipicornavirus drugs are used clinically and the large number of viruses precludes vaccination. New drugs are therefore required. Picornavirus infection exploits viral and host proteins, lipids and cellular systems, all potentially targetable by inhibitors. In addition to compounds with a known target, natural products are potential sources of new drugs. Plant material such as berries are cheap, and some extracts have proven antiviral activity. The aim of this project was to repurpose known drugs, approved and used for several years for various medical indications, as well as identify new compounds from natural sources. Coxsackievirus A9 (CAV9) was used as a typical enterovirus and a plaque reduction assay was used to assess antivirus activity. Both approaches gave promising agents. Fluoxetine and dibucaine caused a complete CAV9 inhibition at low concentrations. All drug resistant mutants (DRM) against these compounds had a protein 2C I227V mutation, suggesting this protein is targeted, in accord with previous work on coxsackievirus B3. The identification of DRMs with a single mutation suggests that drug resistance could be problematical if dibucaine and fluoxetine are used clinically. A previously observed interaction between lipid droplets (LDs) and CAV9 and HPeV1 led to studies of LD targeting agents. DGAT inhibitors A922500 and betulinic acid were the most active compounds, in addition to promising results from aspirin and metformin. Redcurrant extract showed antiviral activity, in addition to antioxidant and photodynamic activity. Some microalgae extracts also showed antiviral effects. Although much works remains to be done to fully develop these novel approaches, they have great potential to combat the serious effect of picornavirus infections.

Background: The thesis explores the role of pharmacy in the lived experience of patients with severe mental illness (SMI) who experience excess morbidity and mortality due to cardiometabolic risk (CMR), metabolic syndrome (MetS) and related diseases. Aims and objectives: To understand and explain the role of pharmacy in providing care for CMR, MetS and related diseases in patients with SMI; achieved by 1. systematically reviewing the published evidence 2. exploring and document the views, perceptions, and experiences of patients with SMI, their informal carers, caring dyads (caring dyad defined here as a patient and their informal carer), care and pharmacy professionals 3. providing strategic recommendations for pharmacy for clinical practice, policy, and research to improve care. Methods, results, and key findings: To address research objective 1. a systematic review of 33 articles found that face-to-face interactions of pharmacists with others consistently and significantly improved process outcomes such as the rate of blood tests 2. framework analysis of semi-structured interviews conducted with 16 patients, 8 informal carers, 21 care professionals and 11 pharmacists identified three themes: (i) CMR, MetS and related diseases, psychotropic medication, psychotropic medication side-effects and SMI (ii) barriers and (iii) facilitators to the role of pharmacy. Framework analysis contrasting and comparing data from each individual within 6 caring dyads identified three themes: (i) enhanced closeness (ii) dissonance, and (iii) balance within the caring dyad. In-depth meaningful impactful interactions between pharmacists and patients, informal carers, and caring dyads were infrequent. 3. Changes to pharmacy practice and policy could facilitate face-to-face interactions between pharmacists and other care professionals and more importantly patients, informal carers, and caring dyads. Conclusion: Patients', informal carers', and caring dyads' desires and needs were not met. Changes to pharmacy practice could facilitate increased face-to-face interactions and encourage person-centred care with the goal of building long lasting trusting relationships which is key in this vulnerable population.

The purpose of this study is to improve the solubility, dissolution rate and permeability of poorly water-soluble drugs by understanding the mechanism of dissolution at molecular level of Flufenamic acid and Carbamazepine co-crystals in the presence of polymers. This study has been separated into four sections: (1) Formation of pharmaceutical co-crystals: Three pharmaceutical co-crystals of poorly water soluble active pharmaceutical ingredient (API) of Flufenamic acid (FFA) and Carbamazepine (CBZ) were synthesized, including 1:1 Flufenamic acid-theophylline co-crystal (FFATP CO), 1:1 Flufenamic acid-nicotinamide co-crystal (FFA-NIC CO) and 1:1 Carbamazepine-nicotinamide co-crystal (CBZ-NIC CO). The results of Fourier Transform Infrared spectroscopy (FTIR), Differential scanning calorimetry (DSC) and X-ray Powder Diffraction (XRPD) confirmed the formation of co-crystals. (2) The effect of polymers on the surface dissolution of co-crystals: The influence of three polymers (polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), and a copolymer of N-vinly-2- pyrrolidone (60%) and vinyl acetate (40%) (PVP-VA)) on the surfaces of FFA-TP CO, FFA-NIC CO and CBZ-NIC CO was studied using Atomic force Microscopy (AFM), Scanning electron microscopy (SEM) and Raman spectroscopy. It was found that the co-crystals have different dissolution mechanisms, and that addition of polymers can alter the dissolution properties of co-crystals by interacting with the crystal faces. (3) The molecular interactions between the drugs, co-formers and polymers were investigated using Nuclear Magnetic Resonance (NMR) and Diffusion Ordered Spectroscopy (DOSY). It was found that the type of a polymer, its concentration, and the interaction of the polymer with a co-former in solution will significantly affect the FFA and CBZ co-crystals (4). Molecular modelling of free drug molecules with coformers and polymers in the presence of water molecules: Results indicate bulk precipitation could be occurring for FFA molecules in solution and that PVP-VA was an effective precipitation inhibitor for all three co-crystals studied in solution. Overall, PVP was an effective polymer for surface precipitation inhibitor and PVP-VA was the most effective inhibitor for precipitation in solution.

Conformity to the FDA, it is of importance for pharmaceutical industries to maintain good manufacturing practice with precision. Developing the best quality tablets that meet changing demands of patients remains a key purpose for this particular study. In this project, three main stages of tablet production including granulation, drying and tableting are investigated for identifying optimum parameters in achievement of final granule and tablet quality attributes. Particular attention is given to drying process in fluidized bed using conventional batch lab-scale and continuous sixsegmented industrial-scale. The Twin Screw Granulator (TSG) and Fluidized Bed (FB) dryer are the focus of this research project as they are strongly interrelated and are the key units located upstream in the process. Given the strong association between the twin-screw parameters and its effect on granules formation and consequent drying, it is timely to investigate the downstream impact of twin screw. Therefore, the uniqueness of this project is integrating twin screw granulation with fluidized bed drying process to set the parameters of processing and formulation on two scale production. This unique involvement of twin screw has further assisted in determining the subsequent transformation during fluidised bed drying process. Moreover, this project aimed at visualizing the spatial distribution of moisture content within a single granule as a function of fluidized bed drying time through a novel quantitative non-destructive NIR chemical imaging technique. This technique has the potential to determine or predict the drying stages that the granules exhibit in drying process at any point of time. The effect of formulation parameters including liquid solid (L/S) ratio, binder viscosity and Lactose primary particle size, as well as process parameter including screw configuration were examined in both scale productions. For the work conducted using small laboratory scale equipment, FB process parameters such as air temperature and air velocity were investigated with all other formulation and process variables of TSG kept constant (i.e., 150M lactose as the primary powder, water as the granulation liquid binder, L/S of 0.1, screw speed of 250rpm and 16 kneading elements (KE) in each screw). Furthermore, when TSG both formulations (i.e., varying the L/S ratio; L/S of 0.089, 0.104 and HPC binder viscosity of different concentrations; 2%, 5%, 10%, 15%) and process parameters (varying number of kneading elements, conveying element only; 0KE, 4KE, 8KE,16KE in each screw) were investigated, all other FB process parameters (i.e., air velocity of 2m/s, air temperature of 50ºC) kept constant. In industrial scale (consgima-25 powder to tablet line), one TSG formulation parameter (varying lactose powder primary particle size) was investigated with keeping all other TSG and FB parameters fixed (i.e., water as granulation liquid binder, L/S of 0.1, screw speed of 250 rpm, 2 kneading element zones in each screw, FB drying air temperature of 50ºC, air flow rate of 400 m3 /hr. and FB drying time of 800 seconds). And one TSG process parameter (varying screw configurations, conveying element only, 1 zone of kneading element and 2 zones of kneading element) was investigated with keeping all other TSG and FB parameters constant (i.e., 150M lactose as the granulation primary powder, water as the granulation liquid binder, L/S of 0.1, screw speed of 250 rpm, FB drying air temperature of 50ºC, air flow rate of 400 m3 /hr and FB drying time of 800 seconds). Granule breakage during FB drying in laboratory scale was evident; however, minimal breakage was achieved when higher L/S ratio, more kneading elements and lower binding viscosity are used. In addition, lower L/S ratio, a smaller number of kneading elements, lower binder viscosity and smaller lactose primary particle size was shown to follow a fast-drying regime. Furthermore, tablet attributes including tensile strength and dissolution were examined. Robust quantitative NIR-CI analytical method was developed and successfully obtained a spatial moisture content distribution across the radial distance of a single granule with MATLAB image processing software.

Pulmonary delivery is increasingly seen as an attractive, non invasive route for the delivery of forthcoming protein therapeutics. However, the lung presents specific barriers for macromolecular/protein therapeutics. Susceptibility to enzymatic degradation, poor absorption across epithelial barrier, as well as lung induced immunogenicity are significant challenges to be addressed if this approach is to be adopted as a viable alternative for protein delivery. In this context, nanomedicine and, in particular, polymer therapeutics (PT) is emerging as a powerful alternative strategy to overcome these limitations. To this aim, in the present PhD project we utilised non-covalent polymer-protein nanocomplexes to allow reversible complexations of protein formulation and investigate their potential to protect and release them at the target site following pulmonary administration. It has been recognised that nanocarriers conjugated with specific ligands can increase the interaction with epithelial cells which makes ligand targeted delivery a very attractive area for in-depth research. In Chapter 3, we describe protein complexes with a new 'complexing excipient' - vitamin B12-targeted poly(ethylene glycol)-block poly(glutamic acid) (PEG-b-pGA) copolymer. Native B12 was first activated at the 5'-hydroxyl group of the ribose moiety using 1,1' carbonyldiimidazole (CDI) followed by the addition of α II dibenzocyclooctyne-amine giving α-dibenzocyclooctyne-vitamin B12. The product was precipitated, purified by preparative TLC and characterised by mass spectrometry ready to react with azide terminated linear PEG-b-poly(glutamic acid) via copper-free azide alkyne cycloaddition to afford the desired targeted B12-PEG-b-pGA copolymers. The synthesis of azide-terminated linear PEG-b-pGA started from a commercially available azide-PEG-amine 3500 precursor acting as a macroinitiator for NCA polymerisation. Following click reaction, a purification step by dialysis yielded the desired B12-PEG3k-b-pGA copolymer. These form complexes in sub 200 nm size with a model protein interacting via ionic interaction, suitable for cellular targeting and intracellular delivery. Initially we confirmed expression of vitamin B12-internalisation receptor (CD320) by Calu-3 cells of the in vitro lung epithelial model used and demonstrated enhanced B12 receptor-mediated cellular internalisation of B12-targeted complexes, relative to non-targeted counterparts or protein alone. The nanocomplexes were spray-dried into inhalable particles with aerodynamic diameter within the suitable range (<5 µm) for lower airway deposition. in vivo lung tolerance studies of copolymer-protein complexes demonstrated that repeated administration of formulated dry powders over a 3 week period in healthy BALB/c mice induced no significant toxicity or indications of lung inflammation, as assessed by cell population count and quantification of IL-1β, IL-6, and TNF-α pro-inflammatory III markers. Importantly, the in vivo data appear to suggest that B12 targeted polymer complexes administered as dry powder enhance lung retention of their protein payload, relative to protein alone and non-targeted counterparts. Next, to test the versatility of this formulation approach, we sought to investigate the inclusion of different targeting ligands detailed in Chapter 4 and 5. Studies have suggested a potential use of another essential vitamin - biotin - as an endocytosis ligand. Biotin receptor was found overexpressed in cancer cells, relatively more than folate or vitamin B12 receptors, and investigated as ligand for targeted delivery of nano-systems and prodrugs. Furthermore, this vitamin is a promoter of cell growth in many cell types which does not limit this delivery system only to target cancer cells. To this aim we synthesised biotin-terminal PEG2k-b-poly(glutamic acid) linear copolymers using commercially available biotin-PEG2k-amine suitable to act as a macroinitiator for N-carboxyanhydrides (NCA) ring opening polymerisation using the same strategy utilised previously for the B12 targeted B12-PEG-b-pGA polymer synthesis. These also form complexes in sub-200 nm size with a model protein lysozyme. Flow cytometry data demonstrated statistically significant increased cellular internalisation by biotin receptor positive A549 cells of biotin-PEG2k-b-GA10/30:lysozyme nanocomplexes relative to untargeted mPEG2k-b-GA30:lysozyme control complexes or 'free' Cy5-lysozyme. By fluorescently labelling lysozyme and tagging IV biotin targeted copolymers after cellular internalisation with Alexa Fluor™ 488 conjugated streptavidin, co-localisation analysis of the two fluorescently labelled polymer and protein using Pearson's correlation analysis was performed on several confocal images. An average r value of ~ 0.5 was obtained, suggesting that at 1 h after the complexes were applied to, and internalised by A549 cells, lysozyme and biotin-PEG2k-b-GA10 copolymers were co-localised. These promising preliminary results encouraged us for further in vitro investigation in understanding the cellular internalisation mechanism that might be responsible for targeted polymer-protein complex uptake. Therefore, work was carried out to probe the mechanisms of entry into the cells of targeted biotin-PEG2k-b pGA:lysozyme nanocomplex, and whether it significantly differs to free lysozyme or non-targeted mPEG2k-b-GA30:lysozyme nanocomplexes. In this respect, a panel of pharmacological inhibitors was used to investigate which endocytic mechanisms were responsible for cellular uptake of our targeted nanoparticles. Dynamin which plays a vital role in vesicular trafficking processes by facilitating vesicle pinching of membranes of cells and organelles was found to play a major role in the uptake of all 3 samples, with lysozyme predominantly taken up via the clathrin mediated pathway while mPEG2k-b-GA30:lysozyme and biotin-PEG2k-b GA10/30:lysozyme preferred the caveolae-mediated pathway; V inhibiting the caveolae pathway had higher impact on the uptake of biotin-PEG2k-b-GA10/30:lysozyme nanocomplexes. The use of targeted PEG-b-pGA copolymer delivery systems to successfully complex and deliver model protein lysozyme to epithelial cells in vitro and in vivo pave the way for further investigations, wherein the PEG-b-pGA copolymer can be used to deliver protein or peptide therapeutics and maintain or improve the therapeutic effect. Furthermore, it was envisaged that the PEG-b pGA copolymer delivery system can potentially be applied not only to proteins but also small molecule therapeutics formulated in nano sized entities, which became the focus of the final chapter of this thesis. Finally, in Chapter 6, antibiotic cationic peptide colistin was successfully formulated into nanocomplexes with mPEG2k-b-GA30 copolymer and complexation was confirmed by agarose electrophoresis and transmission electron microscopy. Colistin:mPEG2k-b-GA30 r = 1:2 nanocomplexes possess enhanced bacteriostatic effect in non-pathogenic E. coli XL1 and bactericidal activity in pathogenic E. coli E2348/69 compared to free colistin applied at the same concentration. It should be noted that colistin:mPEG2k-b-GA30 r = 1:2 formulation showed enhanced bactericidal effect in EPEC E2348/69 infected of HeLa cells, compared to free colistin by killing extra 2 logs of EPEC after 2 h VI treatment. This result strongly indicates that PEG-b-pGA delivery system can be utilised widely aiding the delivery of proteins and small molecule drugs.

The field of Computer Aided Drug Design (CADD) has experienced substantial developments over the last few decades thanks to a rapid growth incomputing power. In particular, Molecular Dynamics (MD) simulations and associated techniques have earned increased attention within the pharmaceutical sector thanks to their rising accuracy and diminishing cost. However, there are still limitations in the usage of these methods, due to thedifficulty of sampling the rugged energy landscapes of protein-ligand complexes. The main theme of this work is to address the sampling problem of MD methods for predicting the binding free energies of different biomolecular complexes. This work starts using MD simulations as a sampling technique for a relative free energy calculation protocol using the Sire Open Molecular Dynamics (SOMD) software. This protocol was then integrated in a ligand design workflow to optimize the binding selectivity of cyclophilin (Cyps) inhibitors. Cyps are proteins known to play a vital role in various diseases, such as cancer, Alzheimer and viral infections. Most Cyp inhibitors to date,however, are cyclic peptides that have potency in the nanomolar range but produce severe side effects, are complex to synthesize and display complex pharmacokinetic profiles. Thus, there is a need for new selective smallmolecules targeting specific Cyps isoforms, in order to gain new insightsfor the inhibition of these therapeutically vital proteins. The computational workflow was able to suggest auspicious designs that they will be synthesized and characterized using biophysical techniques from Alison Hulme's lab. Following, MD simulation methods were employed for the more challenging task of predicting the absolute free energies of binding of protein-ligand complexes. For this purpose, an Alchemical Free Energy (AFE) protocol was generated and its efficiency was evaluated in the Statistical Assessment of Modelling of Proteins and Ligands (SAMPL6) challenge. SAMPL challenges involve a series of blinded predictions of standard binding freeenergies for toy host-guest molecules. The results obtained from our protocol were ranked among the top submissions in terms of accuracy and correlation with experimental data. Encouraged by these results, we wanted to compare the efficiency of the AFE protocol versus a Markov State Modelling (MSM) protocol for the calculation of the standard binding free energy of a ligand to the intrinsically disordered protein c-Myc. The oncoprotein c-Myc is overexpressed in over 70% of human cancers and its inhibition has been considered the holygrail in cancer therapy. Due to its structural elasticity it is difficult to perform structure-based drug design methods for the discovery of novel compounds. The results showed that MSM can describe accurately the binding process of the ligand to the oncoprotein c-Myc, but the binding free energies were similar with the ones of the AFE protocol. Finally, an adaptive sampling protocol was established for the computation of the standard binding free energy and binding selectivity of lead-like ligands for the flexible protein MDM2. MDM2 is a vital protein that acts as an inhibitory mechanism of the transcription factor p53. p53 plays animportant role in the regulation of cellular processes and suppression of tumor development. For this reason, it is important to develop methods for the discovery of novel ligands that could inhibit the MDM2-p53 interaction through binding to the MDM2 protein. The results of the adaptive sampling study were encouraging as the protocol was able to predict binding selectivity trends for the MDM2-ligand complexes approximately six times faster than the original AFE protocol.

As life expectancy increases and fertility rates decrease, the growing ageing population poses a significant challenge to the healthcare systems of developed countries. Ageing as the major risk factor for chronic diseases constitutes the primary target to reduce the burden of diseases and improve human health. However, ageing is a complex process and predicting potential interventions into it requires system-level approaches. In this thesis, I present the development of two computational methods using biological data to predict novel genetic and pharmacological interventions to ameliorate ageing. My first study focused on identifying repurposable drugs to delay human ageing. Several computational drug-repurposing studies have been developed, but most of them focus on predicting geroprotectors using animal models data, even though certain aspects of ageing may be human-specific. Using drug-protein interaction information, I searched for drugs targeting a significant proportion of human ageing-related genes and pathways. The top-ranked drugs included a significant number of known geroprotectors, validating the capability of the method to discover drugs to modulate ageing. On the top of the list was tanespimycin, a heat shock protein inhibitor, whose geroprotective properties we validated experimentally. My second study centres on determining the molecular mechanisms associated with healthy lifespan, and how to use this information to find new genetic interventions to delay ageing. In recent years, the number of transcriptomic studies of mouse models of ageing has increased dramatically, providing the opportunity to compare gene expression changes of long- and short-lived strains. I showed that differences in healthy lifespan are associated with expression changes in genes regulating mitochondrial metabolism. Using these gene sets as biomarkers of lifespan, I compared the mouse models of ageing against 51 genetically engineered mice and predicted candidate genetic and pharmacological interventions with the potential to delay ageing. Through computational studies I predicted a narrowed down list of candidate genetic and pharmacological interventions to delay mouse and human ageing and validated several predictions made by other researchers using different methods, confirming the robustness of computational methods to identify new anti-ageing interventions. With the discovery of tanespimycin as a new geroprotector, I revealed that a little proteostatic stress is good for longevity and that we can trigger this hormetic response pharmacologically. I exposed the complexity of ageing as I found multiple mechanisms to delay ageing, most of which were tissue-specific, and found evidence for new candidate hallmarks of ageing and novel biomarkers of lifespan.

Antimicrobial resistance is a growing threat that will have profound effects on global health and the economy. In order to avoid a worst-case scenario, there is a need to develop structurally distinct classes of antibiotics. Herein, the synthesis of cyclic lipopeptides based on the tridecaptins is reported. In particular, analogues where residues D-Trp5 and Phe9 were substituted for D-Cys and Cys respectively were synthesized and cross-linked with various benzylic linkers of various sizes. Ortho-xylyl, meta-xylyl and para-xylyl cross-linked Oct-TriA1 maintained strong activity against Escherichia coli and other ESKAPE pathogens (including carbapenem-resistant Acinetobacter baumannii). Interestingly, these analogues were resistant to enzymatic degradation in the presence of recently reported D-stereoselective peptidases. Secondly, brevicidine represents a promising candidate for further research and development and this ultimately relies on a synthetic route to acquire sufficient material. The first reported synthesis of brevicidine is reported within this thesis. Briefly, an on-resin approach was used to pre-form the sensitive macrocyclic ring with a modified Yamaguchi esterification. Subsequent Fmoc-SPPS and global cleavage yielded the desired peptide in impressively high yields. With a synthetic route in hand, early structure-activity relationship studies were commenced. Subtle alterations did not ablate the antimicrobial activity while theoretically stabilizing the peptide towards in vivo hydrolysis. Finally, attempts were made to synthesize various lipid tails to install on the depsipeptide globomycin. The natural and non-allo versions of linear globomycin were synthesized and tested using a recently developed fluorescence resonance energy transfer (FRET) assay developed by the Caffrey group.

An extremely large volume of new chemical entities, as well as existing active pharmaceutical ingredients (APIs), on the market, possess sub-optimal physicochemical properties; particularly, their solubility. The synthesis of multi-component pharmaceutical systems, including the lesser-studied eutectic and liquid systems, pose as approaches with which to improve the physicochemical properties of these APIs. Currently, one of the biggest development hurdles associated with multi-component systems, and in particular liquid systems, is their intermediate handling. Consequently, this research aims to use a continuous manufacturing technique, hot-melt extrusion (HME), to manufacture multi-component systems while removing their intermediate handling.

Over the past few years, natural products have already become increasingly important in antiinfectious drug research. This is especially the case as innovations in omics' technologies, chemical biology and genetics have facilitated the discovery of new drugs and their targets. Natural products have a privileged role in the drug discovery due to their intrinsic cell permeability, structural diversity, rich functionality and stereochemistry. They also provide unique scaffolds for further drug optimization towards increased potency and selectivity. China has been using herbs in medicine for centuries and these are now attracting global interest. In response to the challenge of increased anti-microbial resistance our group established a ChinaUK collaborative team to explore the potential of Traditional Chinese Medicines (TCM) to reveal new drug leads. This project focuses on 19 traditional Chinese herbs which were screened for a bioactives using a well-established analysis pipeline. All the plants were provided from China and underwent authentication using DNA barcoding and bioactivity screening of the extracts, mainly focusing on anti-microbial activities. Based on rbcl based DNA barcoding data some samples were misidentified in China whilst some samples of the same species exhibited differences in minimum inhibitory concentrations (MIC). The initial focus was on an assay-based evaluation of isolated compounds from Artemisia annua against mycobacteria. Artemisia annua is native to temperate Asia, known have antimalarial, anti-inflammatory, antitumor and allelopathic activities. Natural products with relatively weak anti-mycobacterial activity with MIC of 250 µg/mL from Artemisia annua were found with. Several fractions, isolated using different chromatography techniques, showed very significant anti-tubercular activity against M. smegmatis. Purification, isolation and identification of two compounds (artemisinic acid and deoxyartemisinin) using various chromatography techniques and identification using high-resolution mass spectrometry and nuclear magnetic resonance (NMR) with the best activity have been performed along with toxicity assays and Schistosoma mansoni assays. The mode of action using computerpredicting docking with other known targets of M. tuberculosis. Based on the MIC pattern and docking algorithms it was predicted that artemisinic acid could probably target KasA protein of Mycobacterium. Next the antimicrobial properties of herbs, Dryopteris crassirhizoma Nakai were identified as potential drug lead against Methicillin-resistant Staphylococcus aureus (MRSA). Bioactive phloroglucinols from D. crassirhizoma were isolated, purified, and identified using a range of bioactivity screens coupled with chromatographic and metabolomics approaches. The antimicrobial properties of the extracts and fractions yielded MIC ranging from 3.25-50 µg/mL. The potential compounds responsible for MIC were tentatively identified through metabolite fingerprinting using flow infusion electrospray high-resolution mass spectrometry (FIE-HRMS). Additionally, the mode of action of the selected fractions was suggested using non-targeted metabolomics analysis with other known antibiotics and docking with known targets. Metabolomics data shows that the anti-MRSA activity was linked to the altered carbohydrate metabolism that could reflect the inhibition of growth of the cells. This study requires further validation for the compounds from D. crassirhizoma using genomics, proteomic, and lipidomic analyses and in silico studies. Overall, our study can help find different naturally available compound and help in the identification of targets to fight different diseases including the drug resistant disease.

Background: There is growing evidence that excipients incorporated into beclomethasone propionate metered-dose inhalers, for the purpose of modifying aerosol particle size, may have effects on drug pharmacokinetics. This body of work is focused on using in vitro and in vivo systems to investigate the effect of non-volatile excipients present in inhaled formulations of beclomethasone dipropionate (BDP) on inhaler biopharmaceutics. Methods: A cellular model (Calu-3) and an artificial model, the Parallel artificial membrane permeability assay (PAMPA), were employed to determine glycerol effect on drug membrane permeability. Additionally, the interaction between glycerol, BDP and a lipid monolayer model was studied using X-ray diffraction. The effect of non-volatile excipient on drug dissolution rate was also assessed by testing prototype MDI formulations with varying excipient amounts (glycerol and isopropyl myristate - IPM) and evaluating in vitro dissolution profiles. Finally, different formulations with and without excipient were applied to a rodent model to assess the effect of excipient on in vivo drug pharmacokinetics.

Routinely collected healthcare data are increasingly being used as a source for research into the effectiveness and safety of drugs. Although these data have great potential, they require preparation before they can be used in research, a process which, amongst a number of other tasks, typically involves quantifying patients' exposure to the drug of interest. The aim of this thesis was to develop and validate a set of flexible, reusable functions for generating common drug exposure variables based on routinely collected prescribing data. Six main classes of method for quantifying drug exposure were identified through a review of pharmacoepidemiological research; ever use vs. never use, use at a specified time point, daily dose or duration, persistence and discontinuation, adherence, and population level measures. The information obtained on these methods, their applications and the potential variations within each class formed the basis for developing an R package, prescribeR, which contains a range of functions designed to simplify and standardise the generation of drug exposure variables, and to provide a structure for reporting how these variables were produced. The utility of the package was then demonstrated by applying it to two exemplar clinical studies, using a cohort of 5,571 patients with epilepsy constructed using linked data within the NHS Greater Glasgow and Clyde Safe Haven environment. In the first, prescribeR was used to quantify persistence to anti-epileptic drugs over the first 365 days of follow-up for cohort patients in order to assess differences in persistence across different drugs, as well as to compare persistence in new and existing users and patients prescribed monotherapy and combination therapy. All of the required persistence measurements for this study were generated using the prescribeR package, highlighting the relative ease of generating exposure data for a large cohort of patients and a number of different drugs. In the second, the package was used to examine the effects of adjusting drug exposure definition on the estimated number of patients exposed to various drugs, the estimated exposure durations. The association between levetiracetam exposure and all-cause mortality was estimated using a range of time-fixed and time-varying exposure definitions, and a wide range of hazard ratios and significance levels were observed across the resulting models, highlighting that the selected definition of drug exposure can potentially have a large impact on the results observed in clinical research.

Background: Cardiovascular diseases are responsible for a large proportion of deaths globally and disproportionately affects developing countries. Diabetes and hypertension are major contributors to the burden of cardiovascular diseases worldwide (WHO, 2017; IDF, 2017; WHO, 2013). In Jamaica, the prevalence of diabetes and hypertension was estimated at 12% and 22% respectively, and has been attributed to an ageing population, increased prevalence of obesity, sedentary living and unhealthy diet (MOHW, 2018; WHO, 2018; WHO Global Data Repository). Access and the appropriate use of essential chronic disease medicines is an effective public health strategy against the morbidity and mortality associated with diabetes and hypertension. The consequences of inappropriate drug therapy are poor health outcomes and increased health care costs to individuals and society. The National Health Fund (NHF), which was implemented in 2003, provides financial assistance for medicines to Jamaicans with one or more of 16 specific chronic diseases, including diabetes and hypertension. This benefit is available to all Jamaicans regardless of age, sex or socio-economic status. To be eligible, individuals are required to have their physician sign an enrollment application confirming their diagnosis (es). Although medicines can be accessed with no fee at point of service at public pharmacies, over 80% of the pharmacies in Jamaica are privately run and may be a more convenient and accessible option for many patients (PAHO, 2012). NHF enrollees have the option of accessing their medicines at a subsidized cost through this network of private pharmacies. Affordability of medicines was highlighted as a barrier to accessing chronic disease medicines, with approximately 25% of Jamaicans reporting not taking medicines due to unaffordability (PAHO, 2012). This study included a literature review, which highlighted multiple barriers to accessing medicines in developing countries. The extent, to which these barriers affected access and use of the NHF, was explored in this study. Objective: This study set out to answer three research questions related to access and use of the NHF in Jamaica among adults with diabetes or hypertension: 1) What are the factors predicting enrollment in the National Health Fund? 2) What is the drug utilization patterns and the factors associated with adherence to medicines among NHF enrollees with diabetes or hypertension? 3) What was the effect of a major health policy (the removal of user fees from primary health facilities in 2008) on access and use of the NHF? Design and methods: The study population was community dwelling adults between the ages of 18 and 59 years with diabetes or hypertension. Data were derived from two disparate sources, the Jamaica Health and Lifestyle Survey (2008) and the NHF pharmacy claims data. Multiple quantitative methods were used to analyze the data. Multivariate logistic regression models were used to identify predisposing, enabling, need and contextual factors associated with enrollment in NHF (n=626), and to identify factors predicting adherence among the enrollees (2008, n=20, 264; 2017, n=77, 454). Interrupted Time Series (ITS) models were used to examine the impact of the removal of user fees from public health facilities on access and use of the NHF (n=49,599; n=74,520). Results: Five years following the implementation of the NHF individualized drug benefits, only 25% of adults (18-59 years) with diabetes and/or hypertension were enrolled. Low enrollment was mainly seen among younger adults. While higher enrollment was observed among those with comorbid hypertension and diabetes. Adherence levels among this population of enrollees was also low at just over 50% in 2017, which represents a decline from 2008 (p < 0.001). Multiple factors were found to be predictive of adherence in both years examined. Howvever, out-of-pocket (OOP)/unsubsidized expense had the strongest effect on adherence when compared to the other predictors included in the model, with lower adherence consistenly observed among individuals with the lowest monthly out-of-pocket (OOP)/unsubsidized expense on medicines. Individuals who obtained multiple drug therapies; those with comorbid conditions; those who lived in the Southeast Health Region; females and those less than 45 years old were also significant predictors of medication adherence but with relatively small effects. Using independent sub-group analysis, the study found that the removal of user fees from public health facilities in 2008, increased NHF enrollment among specific sub-groups (females, urban residents, residents within the Southeast health region and adults ages 18 to 39 years and 45 to 54 years). However, the policy was not effective at increasing the rate of NHFCard users each month. Conclusion: The results of this study suggests that adults between the ages of 18 and 59 years, with diabetes or hypertension had suboptimal access to essential chronic disease medicines through the NHF. Additionally, continued access measured by adherence to drug therapy was low and showed indications of geographic and socio-economic differences. The study also found that different factors are associated with different levels of access, for example need and predisposing factors were the primary drivers of NHF enrollment, while enabling factors were primary drivers of medication adherence among those already enrolled. These findings highlight the need for interventions in Jamaica to increase access and use of the NHF. Considering the multiple factors predicting access and the limited effectiveness of the policy to remove user-fees from public health facilities, interventions must take a multidimensional approach and target those most in need.

Deep learning methods have experienced a revolution, driven by their successful application in fields such as computer vision and natural language processing. In this thesis, we describe several novel methodologies leveraging deep learning for applications to pre-clinical drug discovery. First, we propose a generative approach to the design of molecular linkers which incorporates basic 3D information. In large-scale tests, we find that our method substantially outperforms a database-based approach, the previous de facto approach for this problem. Through a series of case studies, we demonstrate the application of our approach to scaffold hopping, fragment linking and PROTAC design. We then extend this framework to incorporate physically meaningful 3D structural information, providing a richer prior for the generative process, and also apply our method to molecular elaboration tasks, such as R-group design. We then turn our attention to predictive modelling, in particular structure-based virtual screening. We find that the advances in convolutional neural networks (CNNs) for general computer vision tasks are applicable to structure based virtual screening. In addition, we propose two techniques to incorporate domain-specific knowledge into this framework. First, we show that limitations in docking necessitate the use of multi-pose scoring and demonstrate the benefits of an average scoring policy. Second, we propose a transfer learning approach to construct protein family specific models, utilising knowledge of the differences between protein families. Finally, we investigate how a generative approach can be used to improve the training and benchmarks sets employed in structure-based virtual screening. We propose a deep learning method that generates decoys to a user's preferred specification in order to control decoy bias or construct sets with a defined bias. We show that our approach significantly reduces the bias contained in such sets. We validate that our generated molecules are more challenging for docking-based approaches to separate from bioactive compounds than previous decoys. In addition, we show that CNN-based structure-based virtual screening methods can be trained on such compounds.

Objectives: In the present project, I attempted to uncover novel and clinically important drug-gene interactions (DGIs) and drug-drug-gene interactions (DDGIs) among 50 commonly used chronic drugs and 50 commonly used chronic drug combinations in the UK. Methods: Using the UK Biobank (cross-sectional) cohort and 3 other Scottish cohorts (longitudinal), I have studied the association of 162 genetic variants in important genes with three drug response phenotypes for the 50 selected drugs/combinations. This has generated a total of 48,600 findings divided equally between the two studies (DGIs and DDGIs), which I have made accessible via two online databases. I then undertook further replication for our top findings utilizing the UK Biobank primary care data. Results: We identify 8 novel associations after Bonferroni correction, 3 of which are replicated or validated in the UK biobank or have other supporting results: The C-allele at rs4918758 in CYP2C9 was associated with a 25% (15-44%) lower odds of dose reduction of quinine, p=1.6×10-5; the A-allele at rs9895420 in ABCC3 was associated with a 46% (24-62%) reduction in odds of dose reduction with doxazosin, p=1.2×10-4, and altered blood pressure response in the UK Biobank; the CYP2D6*2 variant was associated with a 30% (18 %- 40%) reduction in odds of stopping ramipril treatment, p=1.01×10-5, with similar results seen for enalapril and lisinopril and with other CYP2D6 variants. I have also detected two other novel findings with directionally consistent results in the replication cohort with p-values close to significance levels (amlodipine- rs868853 (ABCC4)-lower odds for daily dose reduction and clopidogrel-rs12353214 (PTGS1)-decreased drug stopping risk)). In addition, out of 3 novel DDGIs, one association was validated using an alternative phenotype in UK Biobank. In the discovery cohort, carrying the G allele at rs9516519 (T > G) variant in ABCC4 transporter was linked with a 4.72 (2.44-9.13) times increased risk of stopping bisoprolol or atorvastatin treatments when they were used concomitantly (p=1.48 × 10-5). In the replication cohort, this drug combination was associated with a great SBP reduction (~ 8 mmHg drop in mean SBP (p < 2 × 10-16)) and the presence of the rs9516519 (T > G) variant increased this effect. Finally, 19 DG associations were identified that replicated previous study findings including but not limited to the association of CYP2C9*3 with increased gliclazide side effects and the association of CYP2C8*3 with reduced pioglitazone efficacy. We also report some other novel and potentially important associations from both the DG and DDG interaction studies. Conclusion: The work in this thesis highlights the value of using large population datasets for pharmacogenomic discovery and has identified novel findings that may impact on clinical care.

The formulation of drugs in the amorphous state offers potential advantages, including enhanced apparent solubility and hence bioavailability, although this comes at the cost of instability for recrystallisation. Recently, the importance of surface-enhanced crystallisation (SEC) mechanism on five amorphous drugs has been identified, in which crystal growth at an exposed surface is several orders of magnitude faster than growth in the bulk, and this occasionally affects the crystallisation polymorph and morphology at the surface. The aim of this thesis is to investigate the effect of the surface on the stability of other amorphous APIs against recrystallisation. If a different stability is seen, then these studies are to be extended to examine the influence of the SEC mechanism on the physical stability in the presence or absence of polymer additives. Hot-stage polarised light microscopy (HS-PLM) was the primary technique used in this thesis to measure the recrystallisation behaviour. Confocal Raman spectroscopy was used to identify the polymorph of the crystals. Differential scanning calorimetry was employed to explore the miscibility between the drug and the polymer. In Chapter 3, paracetamol (PCM) was selected as a model drug. Amorphous PCM exhibits low stability against recrystallisation, with different crystal polymorphs found on exposed surfaces compared to the bulk, and it was assumed that this might be due to SEC mechanism. Here, the crystallisation rates at the surface and in the bulk of amorphous PCM as a function of temperature were measured and compared for the first time. The crystallisation rate at the surface was found to be faster by an order of magnitude than that of the bulk at temperatures above the glass transition temperature (Tg), and faster by two orders of magnitude at temperatures slightly higher than Tg. Moreover, the crystallisation at the surface was discovered to be of Form II, while the bulk crystals existed in Form III. The results offer a solution to the long-standing puzzle of why slightly different crystallisation conditions can lead to different crystallisation outcomes, and also indicate that SEC may be more widespread and important in formulation than recognised to date. In Chapter 4, the effect of the surface was studied on amorphous nifedipine (NIF) in the presence and absence of HPMC polymer to gain more understanding of the SEC mechanism. Amorphous NIF has no variance in the polymorphic form at the surface and in the bulk when it is recrystallised. The results in Chapter 4 reveal that mixing amorphous NIF with 1% HPMC inhibits the crystallisation rate at the surface by two orders of magnitude, but it has a significantly weak effect on crystal growth in the bulk at below Tg. In Chapter 5, a high throughput (HT) method for measuring the recrystallisation rates of amorphous drugs was established to obtain a crystallisation growth rate at various temperatures in one experiment using hot-stage microscopy. The recrystallisation rates of the HT method were generally comparable with those measured by the standard method. Hence, the HT method could be useful to screen how widespread the SEC mechanism is among other amorphous drugs, which could allow an understanding of the rationale behind the existence of the SEC phenomenon.

Under Finkelstein-type reaction conditions, 2ʹ-deoxy-5ʹ-O-tosylnucleosides reacted cleanly with potassium selenocyanate in refluxing acetonitrile. Following quenching of excess selenocyanate with benzyl bromide and column chromatography, pure materials were isolated. Three novel 2ʹ,5ʹ-dideoxynucleoside-5ʹ-selenocyanate analogues (of dA, dC and dG) and a previously reported compound (5ʹ-deoxythymidine-5ʹ-selenocyanate) were thus prepared on multi-gram scales. These seleno nucleosides were immobilised on controlled pore glass (CPG) which underwent Michaelis - Arbuzov (M-A) reactions with nucleoside-3ʹ-H-phosphonates. All sixteen possible 5ʹ-phosphoroselenolate-linked canonical dimer motifs were synthesised. The support bound thymidine analogue was also used in the synthesis of a tetranucleotide sequence with a single phosphoroselenolate-linkage at the 3ʹ-terminus ((dCp)3SedT). In a further demonstration of the utility of M-A chemistry, protected phosphoroselenolate-triester linked dimers were constructed in solution through the high yielding M-A reaction. Subsequent phosphitylation fashioned the corresponding dimer phosphoramidites. Coupling of these phosphoramidites to solid-supported substrates was optimised under both manual and automated conditions and required only minor modifications to the standard DNA synthesis cycle. Following initial unmasking of methyl-protected phosphoroselenolate triesters, pure oligodeoxynucleotides were isolated using standard deprotection and purification procedures and subsequently characterised by mass spectrometry and circular dichroism. The CD spectra of both modified and native duplexes derived from self-complementary sequences with A-form, B-form or mixed conformational preferences were essentially superimposable. These sequences were also used to study the effect of the modification upon duplex stability which showed context-dependent destabilisation (-0.4 → -6.3 °C per phosphoroselenolate) when introduced at either the 5'-termini or at juxtaposed central locations within A- or B-form duplexes. As found with other nucleic acids incorporating selenium, expeditious crystallisation of both a modified A-form decanucleotide and B-form dodecamer duplex was found and the structure of the former solved to a resolution of 1.45 Å using SAD X-ray crystallography. The DNA structure adjacent to the modification was not significantly perturbed. The phosphoroselenolate linkage was found to impart resistance to nuclease activity.

Polypeptides are an important class of drugs used in the clinic for a number of disease states. However, the development of subcutaneous peptide formulations still poses challenges such as aggregation (fibrillation) and short half-life. By utilising formulation approaches, this study aimed to circumvent both aforementioned challenges in a Glucagon-like peptide-1 agonist (GLP-1) termed G48. Kinetic assays revealed that the fibrillation of G48 is pH dependent. At acidic pH (pH 3.7) the peptide displayed off-pathway fibrillation, forming largely amorphous aggregates, whereas above neutral pH (7.4 and 8.5), the peptide showed typical nucleation-dependent fibrillation, leading to highly ordered structures. In all cases the β-sheet rich fibrillar structures were completely suppressed by judicious use of non-ionic surfactants capable of occupying the air-water interfaces. Exploiting the inherent overall negative charge of G48, a hyperbranched polycation (pKH) was synthesised and found to have moderate affinity for G48 (KD = 10 ⁻⁵ M). Following this, two affinity-based formulations were designed: (i) a binary complex composed of pKH and G48 and (ii) a ternary complex composed of pKH, G48 and a polyanion (OG). In vitro release studies revealed that in comparison to the control, which showed complete G48 release within 7 h, the binary and ternary systems slowed release markedly, resulting in ~20 and 40 % G48 release after 168 h. In vitro cytotoxicity studies carried out with human dermal fibroblasts revealed the formulations were non-toxic (PrestoBlue, 100 µg/mL, 32 h). No visual signs of toxicity or weight loss were observed during in vivo studies in which CD-1 mice were subcutaneously administered with the binary or ternary complex formulations containing a G48 dose of 1mg/mL/kg.

The advent of sustained drug delivery has resulted in many patient benefits including reduced frequency of drug administration and reduced side effects. Implantable drug delivery benefits from these advantages, but importantly, can be removed if adverse effects necessitate it. In this thesis, reservoir and matrix-type subcutaneous implants were produced and demonstrated adjustable release rates for several compounds. Reservoir-type implants were produced using 3D printing and showed promising and adjustable in vitro release rates for model compounds: methylene blue, ibuprofen sodium and ibuprofen free acid. These implants could be promising for localised delivery of chemotherapeutics, antibiotics, or anaesthetics. However, they were not prolonged enough for the treatment of chronic diseases (for example human immunodeficiency virus). Polymeric coatings were developed which prolonged the release from these implants and increased their potential as a drug delivery device for chronic conditions. Sterilisation is an important factor which must be considered when designing a new implantable device. Gamma radiation, microwave radiation and ultraviolet light were investigated as potential sterilisation methods for polymeric implants. Microwave radiation proved to be an ineffective sterilisation method. However, gamma radiation and ultraviolet light showed promise. Matrix-type implants containing levothyroxine sodium were fabricated using solvent casting and demonstrated promising in vitro release rates for the treatment of hypothyroidism (a common condition which can have debilitating side effects and result in high costs to the health service or patient if not managed effectively). The implants produced in this work have the potential to improve the treatment outcomes and quality of life of patients with hypothyroidism. The simple implantable devices produced in this thesis are easily prepared and have the potential to be applied to a wide range of drug compounds and clinical conditions. This simplicity also gives them potential to be used in the on demand production of personalised drug delivery devices.

Recent advances in computational resources have allowed Molecular Dynamics (MD) to be run in real-time and displayed using Virtual Reality (VR), creating a fully interactive and immersive experience. Narupa is an open source program for performing Interactive Molecular Dynamics in Virtual Reality (iMD-VR), which is shown to have benefits for performing complex molecular manipulation tasks (in comparison to more conventional interfaces). Here, it is applied to the study of protein-ligand systems, starting with an initial exploration of three protein targets of varying complexity, and later expanded towards a target of high pharmacological relevance (SARS-CoV-2 Mpro). iMD-VR is demonstrated to be a useful tool for complexing proteins to both small, drug-like molecules and larger, more rotationally complex oligopeptides. Sound is also explored as a mechanism for providing feedback during iMD-VR about information relevant to ligand docking, where audio cues indicate the formation of key hydrogen bonding interactions to a user. Furthermore, the utility of iMD-VR is explored as a tool for enhancing the rate of sampling in a chemical system: the free energies of a series of distinct unbinding pathways,which were quickly generated using iMD-VR, are estimated using Umbrella Sampling. Chiefly, an iMD-VR unbinding pathway can be used to quickly describe a route in reduced dimensional space to bias sampling along. Overall, this thesis explores the utility of using iMD-VR for understanding protein-ligand systems, from recreating experimental (crystallographic) protein-ligand structures, to novel ways of conveying key simulation data, to quickly generating unbinding pathways which can be used to guide biased sampling methods. iMD-VR shows great promise as a tool for studying chemical systems of pharmacological interest.

Many RHD allele molecular bases studies on serological D negative phenotype have been conducted in Caucasian and African populations. Study of RHD alleles was lacking in Arabs from Gulf (Omanis) which could be very helpful to come up with a molecular screening protocol for managing donors and patients carrying an RHD molecular variant. In the present study, we analyse the molecular background of serological D negative Omani population. A total of 203 dry blood samples on Whatman’s FTA card were collected from Omani cohort from different regions of the country. The samples were first analysed serologically followed by RHD genotyping using allele specific real-time PCR with melting curve analysis, a commercial allele specific PCR (BAGene kits), digital PCR and Sanger sequencing. Among the 203 serological D negative samples, 189 (93.1%) were phenotyped as true D negative. The true serological D negative samples indicated two patterns that were presence of 37 bp insertion in exon 4 that is unique for RHDѰ (n = 8) and presence of RHDѰ in cis to partial D without RHCE hybrid (n = 2). Total of 9 samples (4.43%) were reclassified as D positive (serological false D negative) indicated patterns that are; RHD-CE-Ds/RHD-CE(4)-D (n = 1), RHD-CE(5)-D in cis to weak D DAR2.00 (n = 1), partial D without RHCE hybrid (DIIIb) in cis to weak D type 45 (n = 1), RHD-CE(4)-D in cis to weak D DAR2.00 (n = 1), weak D type 4.2 in cis to weak D type 41 (n = 1), RHD-CE(4)-D in cis to RHD-CE(8-9)-D (n = 1), RHD-CE(4)-D in cis to weak D type 4.2 (n = 2,) and DVI.2 (RHD-CE(4-6)-D in cis or trans to DEL(IVS8-31T > C) (n = 1). Total of 5 samples were unclassified. Altogether, these studies showed serological limitation existence in 4.4% of the samples that reinforce RHD molecular typing and lay the foundation for the development of PCR based RHD genotyping screens in Oman.

In the last 20 years biotherapeutic macromolecules have become the fastest growing sector within pharmaceutical industry. Their development, facilitated by the introduction of advanced molecular engineering techniques, has led to improved treatment options for patients with autoimmune conditions, various cancer types, and infectious disease. The complex molecular structure of these drugs render them susceptible to degradation and, as a result, many commercially available products are suitable for parenteral drug delivery only. Subsequently, the hypodermicneedle and syringe has remained the device of choice for biotherapeutic delivery, despite the many drawbacks associated with this method. Transdermal delivery has been an attractive alternative for many pharmaceutical formulators. However, few drugs possess the appropriate physicochemical properties required for crossing the human skin barrier. Microneedle (MN) technology combines micro-engineering and material sciences to fabricate micron scale projections manufactured onto a platform. That, when pressed against the skin, create aqueous apertures allowing drug delivery directly into the dermal tissue. This thesis explores the development of polymeric MN-based drug delivery systems, capable of facilitating intradermal and transdermal passage of biotherapeutic macromolecules. The model protein ovalbumin was incorporated into polymeric MN systems using commonly employed industrial manufacturing techniques and sterility was successfully demonstrated. Further these MN platforms were evaluated for intradermal delivery in vivo, highlighting the potential adjuvant effects of Gantrez®S-97. Subsequently, through industrial collaboration with market leading transdermal manufacturing company, Lohmann Therapie Systeme AG, the commercially available vaccine Pentavac® was successfully incorporated into dissolving MN arrays. This study has provided significant learnings for both academia and industrial partners, in relation to industrial manufacture of MN and biotherapeutic macromolecules. Finally, polymeric MN platforms were evaluated in vitro and in vivo for transdermal delivery of a therapeutically relevant monoclonal antibody, bevacizumab. This thesis provides significant evidence to support polymeric MN arrays, as minimally invasive intradermal and transdermal delivery platforms of biotherapeutic macromolecules. Focused input from key stakeholders, including: academia, industry, regulators, healthcare professionals and patients will be needed to ensure successful MN commercialisation.

Over 80% of therapeutic monoclonal antibody (mAb) products are expressed in Chinese Hamster Ovary (CHO) cells. While cell-based expression platforms are traditionally used for the exploration of novel mAb therapeutics in the discovery phase, cell-based technologies remain particularly time- and resource intensive for mAb screening. To circumvent these shortcomings, cell-free protein synthesis (CFPS) platforms have emerged as versatile alternative allowing for rapid and flexible production of therapeutic proteins. In contrast to in vivo systems, CFPS platforms do not require intact host cells, which make them completely independent of host cell metabolism in determining product yield and quality. Although the open-production system bears several advantages over the traditional cell-based platforms, CFPS platforms still face limitations such as low product yields, challenges with post-translational modifications (PTMs), and poor cost efficiency of CFPS. It is therefore of great interest, to develop a simple, more cost-efficient and active CHO CFPS platform for accelerated screening of therapeutic mAbs. This work addresses the current limitations by developing a cell-free protein synthesis platform using CHO extracts for the rapid production and evaluation of industrially relevant mAbs. This study is divided into three main parts: 1) Development of a CHO CFPS system, 2) Yield optimization of CHO CFPS, 3) Production and screening of therapeutic mAbs. Our platform was able to express and characterise four functional mAbs in the supernatant fraction of 25 μl coupled batch reactions including two low in vivo expressers and the blockbuster drug Trastuzumab. Total synthesis yields were increased up to 50-fold by supplementing the system with two accessory proteins, GADD34 and K3L. Using our optimized platform, mAb yields of up to 31.06 μg/ml could be achieved. The trend in cell-free expressed functional mAbs replicated previously reported results from cell-based expression. Based on the success of our platform, we suggest its use for rapid, low-scale antibody expression screening to accelerate molecule selection and development for the biopharmaceutical industry.

Development of acceptable medicines is central for adherence and effectiveness of treatment. However, the assessment of medicines' acceptability has not yet been standardised. Correlating in vitro and in vivo acceptability data would facilitate the development of acceptable medicines, but research in this area is limited. This thesis aims to evaluate and correlate in vivo and in vitro acceptability data of conventional tablets with a range of coatings. In two randomised studies participants, ranging from pre-school children to older adults, evaluated acceptability of tablets in two aspects: ease-of-swallowing and palatability. Sensory attributes which were statistically related to acceptability were established with high selectivity and specificity, i.e. bitterness, aftertaste, stickiness, slipperiness, and smoothness. Discrepancies found between children and adults responses indicated differences in their perception of tablets and acceptability. Also developed was an array of instrumental methods and results were investigated for links to sensory perception. A linear relationship between tribology and oral sensory perception, specifically for ease-of-swallowing, slipperiness and stickiness, of tablets was found and is described for the first time. Lastly, a vocabulary describing tablets has been collected and organized into a lexicon, which provides the first step towards the standardisation of sensory testing, as a part of acceptability evaluation.

Crystallisation is a fundamental unit operation in purification and separation process. It has been amply adopted in the pharmaceutical and fine chemical industry as a product of high purity can be obtained in a particulate solid form. This unit operation allows for tailoring product characteristics, providing extensive benefits to industries that operate in highly regulated environments. In particular, the pharmaceutical industry must abide with a complex set of regulations and laws imposed on drug products intended to protect the health of the public. Thus, in order to gain a competitive edge, pharmaceutical industries invest significant time and resources to optimize the manufacturing process. In this context, increasing research is focused on the optimization of crystallisation. One of the major issues that may occur during the purification process is the concomitant crystallisation of other substances together with the Active Pharmaceutical Ingredient (API), which may reduce the purity of the product below the compliance level. This thesis explores the effects of an externally applied electric field on suspended crystals and on crystallisation, aiming at the understanding of the fundamental principles of the interaction of the electric field with suspensions of particles and solutions of small organic molecules such as pharmaceutical compounds. Then, electric fields can be exploited as a process intensification tool to aid purification processes. For instance, rather than alleviating a concomitant crystallisation problem, the use of an externally applied field could make use of a concomitantly crystallising system to purify and separate two crystalline products in a single process step. The manipulation of suspended particles driven by electric field requires elevated electric fields which might result in the generation of electric current into the fluid system, inducing undesired chemical reactions that may affect the final product. To minimize electric current, non-polar solvents constitute of small molecules such as dioxane can be used. In Chapter 3, the solution behaviour of a number of APIs in non-polar solvents are studied in order to define suitable systems that resist electrolysis under strong electric fields, and therefore allow for the design of crystallisation processes under such conditions. From a number of thermodynamic models, the van 't Hoff equations presented good correlation values to the experimental data, and therefore it can be used to extrapolate and interpolate solubility data at any given temperature. In addition, solution behaviour was studied from determined activity coefficients, which showed that all the studied systems positively or negative ly deviated from ideality. The Wilson activity coefficient model was used to predict these deviations. From well-defined systems, particle and solution properties can then be related to the electric field phenomena, leading to the identification of the scientific principles behind the interactions between the particles and the electric field. Suspensions of APIs of small organic molecules in different apolar solvent were studied under the presence of non-uniform and uniform electric fields in Chapter 4, which enabled to assess the electrokinetic phenomena associated to the motion of the suspended crystalline particles. Under an electric field, suspended particles travelled to and accumulated on a particular electrode. The greater dielectric properties of the organic compounds compared to that of the apolar solvents induce s a motion of particles towards an electrode by positive dielectrophoresis. The collection of particles on an electrode of a particular charge is due to electrophoretic forces acting on the surface charge of the particle. The collection on a specific electrode was anticipated by the sign of the zeta-potential of the solid phase. Other phenomena such as electro-osmosis and electrorheological fluids were observed in the studied systems in the presence of a strong electric field. The investigation of equilibrated suspensions in an electric field raises the question of the effects of the energy input in metastable systems. Chapter 5 explores the effect of strong electric fields in crystallisation processes of solutions of a small organic compound (isonicotinamide) in a non-polar solvent (dioxane). An electric field directly applied to a supersaturated solution of isonicotinamide in 1,4-dioxane enhances the nucleation kinetics of the small organic molecules. Thus, in the presence of an electric field, nucleation occurs at higher temperatures and shorter induction times for a given solution composition compared to crystallisation processes in the absence of an externally applied field. A plausible explanation is the accumulation of monomers of the crystalline compound in the vicinity of the electrodes due to electrokinetic forces. This phenomenon could increase the local supersaturation, resulting in an effective increase of the frequency of successful attachment of building units. Then, the combined action of the electric field effects on suspended particles and crystallisation from solution can be applied to developing means to exploit the electric field phenomena for the separation of heterogeneous suspensions. In Chapter 6, a number of multicomponent mixtures have been successfully separated in-situ by the use of inhomogeneous electric fields. The separation of two crystallising compounds that collect on opposite electrodes under the electric field was achieved with high purity values by cooling crystallisation in the presence of the field. In addition, the separation of compounds that collect on counter electrodes was carried out by a two-step seeding/cooling crystallisation method driven by electric fields. Thus, the separation method can potential be implemented as a tool to aid separation and purification processes. This thesis investigated the scientific principles behind the interaction of the electric field with both particle suspensions and crystallising solutions. By relating particle and solution properties to the observed phenomena, we were able to anticipate the behaviour of particles and solutions in an electric field, and then exploit the phenomena to separate complex mixtures of two crystallising compounds in situ. The gained knowledge can be applied as an intensification tool to aid purification and separation processes.

Pharmaceutical care describes a range of patient-focused activities delivered by pharmacists. The activities aim to optimise medicines use for patients and to reduce harm from adverse events with medicines. This study was conducted in an NHS Scotland organisation, where the clinical pharmacy service has an established quality management system. It was evident that some gaps existed in the quality assurance parameters for clinical pharmacy services and pharmaceutical care, with there being no clearly defined route to report adverse events or near misses that arose from within the service. In quality management terms this meant it was difficult to determine whether optimal pharmaceutical care was being delivered, or to establish how accurate clinical pharmacists were in their pharmaceutical care activities. Additionally, this meant it was difficult to evidence areas for quality improvement. This study aimed to explore the perceptions, experiences and behavioural determinants of the hospital clinical pharmacists in relation to optimal and suboptimal pharmaceutical care within an NHS organisation in Scotland using a theoretical framework. The research used the concept of suboptimal pharmaceutical care to describe the gap between pharmaceutical care as intended and pharmaceutical care as delivered. This research used qualitative study design and a phenomenological approach, and was conducted in two phases with the first phase influencing the design of the second phase. In Phase 1, focus group methodology was used to determine perceptions of hospital clinical pharmacists to optimal and suboptimal pharmaceutical care. Study participants (n=20) were hospital clinical pharmacists recruited from hospitals across the NHS Scotland health board. A topic guide focused the discussions on the activities related to medicines reconciliation and Kardex/medicines review. Data generated from focus groups was in the form of written statements and audio-recorded narrative to describe participants' perceptions of barriers and enablers to providing optimal pharmaceutical care. The Theoretical Domains Framework (TDF), an integrative theoretical framework that describes behavioural determinants, was used to analyse the findings. Phase 2 used in-depth interviews to explore participants' (n=10) experiences of optimal and suboptimal pharmaceutical care. A semi-structured interview schedule was developed using TDF, to facilitate identification of behavioural determinants to the provision of optimal and suboptimal pharmaceutical care. Within Phase 1, participants perceived that there were barriers to the delivery of optimal pharmaceutical care, citing as contributory elements time factors, lack of policy and procedure, conflicting priorities (including uncertainty over efficiency versus thoroughness), poor underpinning knowledge of medicines by doctors, and inadequate skills in completing and documenting activities. In Phase 2, key determinants were elicited, including knowledge (of trainees), time, policy, procedure or guidance on suboptimal pharmaceutical care, and personal and professional barriers and enablers, including professional embarrassment and hierarchy. The study has allowed an exploration of an underacknowledged topic in clinical pharmacy practice and identified behaviours, including role uncertainty and embarrassment, that may contribute to lack of reporting on suboptimal pharmaceutical care. Recommendations have been made using behavioural change technique interventions and include educational interventions, skills training, modelling, enablement, persuasion, incentivisation, coercion, restriction and environmental restructuring. Implementation of these interventions and evaluation of their effectiveness will enable the organisation to have more robust quality assurance parameters within the clinical pharmacy service, and to ensure continued conformance with the quality management system. Across the wider clinical pharmacy community, lessons may be learned about perceptions and experiences relating to suboptimal pharmaceutical care, and consideration made to capturing the learning opportunities that can arise when considering suboptimal pharmaceutical care in practice.

In the field of drug development, knowing the chemical composition of a pharmaceutical product is important to develop a process that will avoid the formation of unwanted compounds. The isolation and the chemical synthesis of impurities provides useful information. Naloxone is an opioid derivative present in Suboxone® sublingual film. It has been demonstrated that naloxone-related impurities are formed during the storage of this pharmaceutical product. However, these compounds are unknown and therefore, synthesising them is the key to confirm their structure. In this instance, the semi-synthesis of these impurities was attempted. Analysis showed that the majority of the impurities are oxidation products: Alcohols, carboxylic acid or lactones, alpha-hydroxy ketones, and products of rearrangements. In total, 17 impurities were found and needed to be prepared. Various compounds were targeted. The benzylic oxidation of naloxone was first attempted. This compound is known as a naloxone-related impurity in Suboxone® sublingual film. Chapter II relates of the synthesis of the desired impurity following a literature procedure, and our attempts to obtain the desired product using other methods. The second chapter of this report relates of the oxidation of the cyclohexanone ring of naloxone, in order to prepare a di-carboxylic acid, alpha-hydroxy ketone and lactone. A synthetic route to the dicarboxylic acid was designed. The key oxidation step - the dihydroxylation of an alkene - required a deallylation followed by the Boc protection of the amine. The diol is also an intermediate to the synthesis of the alpha-hydroxy ketone. The preferred route towards this compound involved the mono-protection followed by the oxidation of the diol. However, some work still needs to be done to provide the desired compound. Another targeted impurity was a lactone, formed by Baeyer-Villiger oxidation of the parent ketone. However, the main reaction that seemed to happen was the oxidation of the basic nitrogen to form the corresponding N-oxide. Finally, Chapter IV contains the initial work towards the opening of the furan ring, in order to form two alpha-hydroxyketone products. To this end, the formation of an alkene followed by oxidation was the attempted strategy. The opening of the E ring using a stepwise Wolff-Kishner approach provided the desired alkene between positions 5 and 6.

Chronic neuroinflammation leads to excessive production of pro-inflammatory mediators and consequently neurodegeneration. The alleviation of neuroinflammation may mitigate neurodegenerative disorders, including AD which incidence is higher in women due to decrease of oestrogen during menopause. This hypothesis indicates oestrogens as important neuroprotective factors. However, they may cause detrimental side effects in periphery. Therefore, oestrogens can be substituted by natural compounds such as isoflavones which resemble oestrogen structure but lack detrimental side effects. Presented research investigated anti-inflammatory and neuroprotective properties of four novel isoflavone analogues: biochanin A derivatives with carbamate and dodecenoyl ester moiety (compounds 1 and 2) and daidzein derivatives with ethyl ester and chloropropyl triazole moiety (compounds 3 and 4). The research concluded that compounds 1, 2, 3 and 4 reduced LPS-upregulated levels of TNFα, IL-6, IL-1β, NO and iNOS in BV2 microglia. Biochanin A derivatives were less effective in inhibition of pro-inflammatory cytokines compared to daidzein derivatives. All tested compounds did not affect LPS-induced production of COX-2 and PGE2 and do not possess free radical scavenging properties. Only compound 4 decreased NF-κB activity. All tested compounds did not reduce phosphorylation of p38, JNK and ERK1/2 and upregulated ERE activity. Moreover, the anti-inflammatory properties of compounds 3 and 4 are Nrf2-, SIRT1- and ER-independent. Compounds 3 and 4 diminished H2O2-induced apoptosis in SH-SY5Y neuroblastoma and reduced caspase-3/-7 and -9 activity. Compound 3 upregulated Bcl-2 level. In summary, compounds 3 and 4 inhibited neuroinflammation and protected neurons against oxidative damage. Additionally, this study investigated the validity of HMC3 cells as a cellular model to study neuroinflammation. Amongst ODN 2006, IFNγ, LPS, and TNFα, TNFα induced the broadest inflammatory response in HMC3, upregulating Iba1, IL-6, and activating NF-κB and p38 signalling pathways. HMC3 microglia express ERα and ERβ therefore this cell line can be used to investigate properties of anti-inflammatory compounds acting via ERs.

This PhD project aims to develop a novel polymer-drug conjugate (PDC) via free radical polymerisation for extended release and decreased permeation of p-menthane 3,8-diol (PMD) when applied topically onto the skin. The rational behind this was the volatile nature of PMD (evaporates quickly) and reports of the side effects associated with the topical absorption of the PMD. For this purpose, firstly hyaluronic acid (HA) was chosen as a polymer to conjugate PMD but despite exploring various synthetic strategies, and changing reaction parameters including the molecular weight, drug, reaction time etc, a conjugate could not be produced. After these initial attempts, an alternative route was selected for the PDC synthesis based on synthesis of polymerisable PMD conjugate and its subsequent co- and homopolymerisation. PMD was conjugated with acryloyl chloride via an ester bond to form acryloyl-PMD (APMD), which was subsequently copolymerised with acrylic acid (AA) to form a series of copolymers poly(AA-co-APMD). The copolymers were characterised by 1 H NMR and FT-IR for their structural elucidation, which was then followed by molecular weight characterisation, thermal analysis by TGA and DSC, reactivity ratio studies, turbidimetric analysis and drug loading. The properties of these copolymers were affected by the molar ratios of AA and APMD, where the AA incorporation into the final copolymer was 3´ higher than the APMD. In order to assess the amount of drug (PMD) released from the copolymer, an in vitro experiment was performed by using porcine liver esterases (PLEs) to cleave the ester bond from the substrate (copolymer). It was found that ~45% of the drug was released over five days. To investigate the reason for the comparatively modest drug release, two experiments were performed to investigate the effect of copolymer molecular weight and enzyme activity on PMD release. It was found that molecular weight did impact on drug release whilst addition of fresh enzymes showed that ester bond cleavage was not limited by enzyme activity in our study. Penetration and permeation of the copolymer and free drug (PMD) through excised full thickness porcine ear skin was investigated. The Franz diffusion cell studies showed no permeation of the copolymer as compared to the PMD. Moreover, tape stripping revealed that almost ~90% of the copolymer was found on the outer surface of the skin as compared to PMD which was found in all the layers of the skin. A new model using planaria fluorescence assay was developed as a novel method to pre-screen potential irritants such as our copolymer or PMD. The model was developed using a range of known skin irritants to include non-, mild-, moderate- and strong-irritants. The results showed that this model was able to successfully differentiate strong irritants from the non-irritants, whilst the other irritant classes also showed good correlation between the fluorescence intensity (FI) of the vii planaria after irritant and then fluorescent dye exposure and the known literature primary irritation index (PII). This test demonstrated that that the copolymer is unlikely to be a significant irritant when applied topically. Overall, this project has demonstrated the feasibility of the copolymer approach as strategy to develop extended-release insect repellents whilst reducing transdermal permeation of the small molecular weight active ingredient and hence minimising any adverse systemic effects.

Detergents have historically been used to solubilise membrane proteins for structural studies and pharmacological research, however detergents can alter the lipid environment surrounding a membrane protein. The styrene-maleic acid (SMA) copolymer has been designed to solubilise membrane proteins from the cell membrane, with the lipid bilayer intact, thus forming styrene-maleic acid lipid particles (SMALP). This would retain the native conformation of the protein and therefore suitable for applications such as drug discovery. In this project, the adenosine 2A receptor (A2AR) and the calcitonin gene-related peptide receptor (CGRPR) were solubilised into SMALPs. Various techniques were used to characterise the SMA-solubilised receptors. Out of the SMA copolymers tested, SMA2000 was chosen as the copolymer to solubilise the GPCRs. The copolymer was also compared with the new diisobutylene-maleic acid (DIBMA) copolymer, which has better resistance to divalent cations than the SMA copolymer. Molecular techniques confirmed the expression of the GPCRs in membranes and after solubilisation into SMALPs. Radioligand binding assays demonstrated that the A2AR retained its binding capability when solubilised and purified. The binding assay showed that the A2AR was more stable in SMALPs than DIBMA lipid particles (DIBMALP). Various techniques were used to characterise the A2AR-SMALP, providing novel properties of GPCRs in SMALP. The x-ray radiolytic footprinting (XRF) was used to detect regions of the GPCR-SMALP which were exposed to hydroxyl modification. The transmembrane domain, and the intracellular surface of the SMA-solubilised A2AR were exposed to water, demonstrating the SMALPs can successfully be used in XRF. Fluorescence correlation spectroscopy (FCS) was implemented to characterise the pharmacology of a single ligand binding to a single receptor, where the pharmacological profile of the A2AR was successfully characterised when in a SMALP.SMALPs were also tested for their applicability in phage display in order to generate GPCR-specific nanobodies against receptors in their native conformation. The M13 phage used in this project were conjugated with a VHH nanobody. The A2AR-SMALP was immobilised onto ELISA plates for phage binding, where approximately 22% of the total phage were A2AR-SMALP specific, which was lower than the control Fab protein. Avi-tagged A2AR and CGRPR constructs were designed to improve the immobilisation of the SMALPs, to yield a higher enrichment of phage, specific to the GPCR of interest. Finally, the photoaffinity cross-linking assay was implemented in this project, which has potential implications in drug discovery as receptors can be locked into a particular conformation when cross-linked with a ligand. The technique can theoretically be applied to receptors in SMALPs. Residues of the extracellular loops 1 and 3 of the CGRPR were studied. The assay showed residues A199, N200 and N201 of the extracellular loop 1 forming crosslinks with the ligand, when substituted with azidophenylalanine. Overall, the project demonstrated techniques which are applicable to study SMA-solubilised receptors. Using the various techniques revealed novel properties of the GPCRs in SMALPs. SMALPs were also applied to the drug discovery technique, phage display, with limited success. Techniques were incorporated into this study to improve the applicability of SMALPs in phage display.

Drug-induced seizure is a major reason for compound attrition during drug development, hence testing the potential of novel agents to induce such neurotoxic events is a vital process. Currently, in vivo and in vitro animal assays are used for seizure-liability studies; yet controversy over the relevance, efficacy and cost of these methodologies has led to interest in the development of human based models, for increased translatability and data extrapolation. Human induced pluripotent stem cells (iPSCs) are a revolutionary platform for neurotoxicity testing. However, considerable variation in culturing protocols, growth media and analytical techniques exists, with no validated standard for drug-induced seizure-liability testing. In this thesis, this cutting-edge iPSC technology, in combination with concurrent morphological and functional analysis has considered several methods for generation of a robust, reproducible human seizure-liability model, capable of responding to ionic and pharmacological stimuli. Spontaneously differentiated neural cultures display electrical activity, but sporadic epileptiform activity, as observed with fluorescent calcium imaging. Moreover, weak functional activity and longevity and the absence of characteristic seizure-like activity was observed in isolated monocultures of neurons and astrocytes. Various co-culture protocols were developed and tested, displaying greater baseline activity, network interconnectivity and responses to pro-convulsant conditions than spontaneously differentiated cultures; highlighting the absolute requirement for both cell types to be present in culture. Final experiments introduced interneuronal populations to the established co-culture protocol; with preliminary results highly suggestive of providing a robust system which can be used for widespread seizure-liability assessment. This thesis provides the first comparison of iPSC-derived culture methods for seizure-liability testing, whilst factoring in several variables which currently exist in the literature; including growth medium, duration of differentiation and methods to control cell proliferation. In addition, a proposal for a validatory panel of pro-convulsant conditions for the inclusion of human iPSC-derived platforms in safety pharmacology studies is presented.