This thesis details the design, synthesis and evaluation of novel small molecule inhibitors of the histone methyltransferase DOT1L and the ubiquitination facilitator Keap1. The thesis is in two parts as outlined below. Part 1: The first part of this thesis details efforts towards identification of novel small molecule inhibitors of DOT1L, a histone methyltransferase which has been implicated in the development and proliferation of mixed lineage leukaemias (MLL). This work aims to optimise the drug-like properties of published DOT1L inhibitors while retaining potency, through further exploration of the nucleobase template. Structure-activity relationships (SARs) identified polar substituents and small heterocycles as favourable replacements for the halogen in 5-ITC, a small molecule inhibitor of DOT1L. Alternative nucleobase templates also demonstrated comparable DOT1L inhibition. To demonstrate proof of concept, a polar nitrile substituent was translated into the inhibitor Br-SAH as a direct replacement of the bromide. Activity was retained and a crystal structure obtained which demonstrated the nitrile occupied the same hydrophobic pocket. This work also demonstrated the use of a nitrile as a non-traditional replacement for heavy halogen atoms. Part 2: The second part of this thesis details identification of novel inhibitors of the Keap1-Nrf2 protein-protein interactions (PPI) using an approach based on kinetic target-guided synthesis (kTGS). Keap1 is a dimeric cytoplasmic protein that mediates the ubiquitination of Nrf2, a transcription factor that acts as a regulator of cellular antioxidant responses. Disruption of the PPI between Keap1 and Nrf2 has been shown to have a therapeutic benefit in diseases associated with oxidative stress and inflammation as well as providing a potential route to chemopreventative agents for cancer. Biased kTGS was applied as proof of concept. A biased ligand was designed and screened against a focused library of azides. The 1,3-dipolar cycloaddition products formed in the presence of the Keap1 Kelch domain were evaluated and validated through chemical synthesis and screening. A novel triazole structure was identified with improved activity over the initial biased fragment thus demonstrating kTGS as a valid approach for identifying novel inhibitors of the Keap1-Nrf2 PPI interaction.

Tuberculosis is a major global health problem, which ranks alongside HIV as a leading cause of death worldwide. Adherence to tuberculosis treatment regimens is quite low, particularly in paediatric patients, and the aversive taste of these medicines is often cited as a major reason for this. In the first part of this thesis, the taste of these four drugs was assessed using both a human taste panel and the rodent Brief Access Taste Aversion (BATA) model. Human EC50 (i.e. the concentration of drug which elicits 50% of the maximum taste response) values were determined for each drug and the BATA model was identified as being useful for the assessment of formulations containing isoniazid, rifampicin and ethambutol. The ability of an in vitro technique, the Insent TS-5000Z electronic tongue, to detect and assess the taste of isoniazid, rifampicin, pyrazinamide and ethambutol dihydrochloride was investigated. The best correlation between human responses and electronic tongue responses was observed for ethambutol dihydrochloride. The latter half of this thesis focused on the use of hot melt extrusion (HME) as a processing technique to develop taste masked polymeric formulations of isoniazid and rifampicin and a fixed dose combination containing both isoniazid and rifampicin. A fixed dose combination (FDC) formulation containing 20% w/w isoniazid and 30% w/w rifampicin was produced using Eudragit E-PO as a carrier. The extrudate was milled and incorporated into a dispersible tablet. The weight uniformity, thickness, hardness, disintigration time and content uniformity of the tablets were investigated and found to conform to the specifications for solid dosage forms. The dispersible tablet was found to effectively mask the taste of the drugs when dispersed in water with the drug release remaining below the human EC50 value for each drug.

Parkinson's disease (PD) is a progressive neurodegenerative disorder affecting millions of people globally. Like other age-related conditions, inheritance of genetic variations contributes to PD pathogenesis. Mutations in leucine-rich repeat kinase 2 (LRRK2) are linked with familial forms of late-onset PD. Importantly, the LRRK2 locus has been identified by genome-wide association studies to contribute to risk of sporadic disease. These observations suggest that the study of LRRK2 cell biological function and dysfunction might shed light on the pathogenesis of PD. LRRK2 is a large multidomain cytosolic protein reported to play a role in a variety of cellular functions such as cytoskeletal dynamics, vesicular trafficking and autophagy. Mouse models deficient of LRRK2 or harbouring the pathogenic human G2019S mutation do not show typical PD brain pathology. However, reported phenotypic kidney pathology in LRRK2 knockout mice provides a rationale to investigate LRRK2 knockout and G2019S knockin kidneys to further elucidate the biological role of LRRK2. Using an unbiased quantitative proteomic approach, significant alterations in protein levels associated with cytoskeletal, lysosomal, vesicular trafficking and control of protein translation were observed in Lrrk2 knockout but not G2019S knockin tissue. Lysosomal protein accumulation and changes in expression of a subset of cytoskeletal proteins were validated using orthogonal techniques in independent cohorts of mice across several age time points. Very few protein changes were observed in brain or varied in opposite directions in knockout versus knockin mice. A role for LRRK2 in the endo-lysosomal pathway was further confirmed in primary kidney cells from LRRK2 knockout mice. Overall, these results imply LRRK2 co-ordinated responses in protein trafficking and cytoskeletal dynamics, and argue against a simple dominant negative role for the G2019S mutation.

Superparamagnetic iron oxide nanoparticles (SPIONs) have gained signif- icant interest over the past decades because of their wide range of appli- cations. In biomedicine, SPIONs had been used extensively in the past as MRI contrast agents but they are currently being investigated for hyperther- mia therapies, magnetic manipulation and as part of diagnostic devices. The main aim of this study is to develop a method to synthesise positive MRI enhancing iron oxide nanoparticles (T1) and use these as the diagnos- tic component to produce a theranostic (therapeutic and diagnostic) agent. An aqueous flow-based synthesis method was assembled and tested producing 25nm iron oxide nanoparticles with T1 enhancement, CMDxUS- PIONs. The devised synthetic method enabled the fabrication of the desired nanoparticles without the use of organic solvents and at higher outputs than previously reported with flow-based methods, with 12 clinical doses being produced per hour using a lab-scale system. Surface functionalisation of CMDxUSPIONs with aptamers for active targeting was accomplished using a recently discovered anti-annexin 2A aptamer (ACE4). Particle uptake results show that ACE4-CMDxUSPIONs presented at least a two-fold increase in cell uptake when compared to un- modified CMDxUSPIONs. Further modifications of CMDxUSPIONs included drug-loading with cisplatin. Cisplatin loaded CMDxUSPIONs (CPt-CMDxUSPIONs) were achieved following pre-optimisation using a Design of Experiments ap- proach. The drug-loaded CPt-CMDxUSPIONs containing 0.64mg of CPt/mg of iron were able to retain both the activity of free cisplatin (in vitro) and the imaging capabilities of CMDxUSPIONs. In vivo experiments using a 4T1 mammary carcinoma cell line in Balb/C mice showed that CPt-CMDxUSPIONs were trackable in a 1T preclinical MRI and therapeutically, CPt-CMDxUSPIONs reduced tumour size and minimised cisplatin’s nephro- toxicity. Moreover, preclinical MRI scans show that it is possible to use CMDxUSPIONs as reporters for nanoparticle uptake detected by a T1-T2 signal switch in MRI. Iron oxide based theranostic systems reported in this thesis could play an important role in future cancer treatments by enabling a personalised therapy approach and modifying current chemotherapeutic drug biodistribu- tion as a carrier. The facile synthesis together with the addition of therapeu- tic agents to iron oxide nanoparticles demonstrates that the production of an experimental theranostic is possible.

Temporal lobe epilepsy (TLE) is the most common type of epilepsy and exists with memory loss as a comorbidity. The conventional therapy available to treat these disorders achieves only modest therapeutic efficacy at best. This study investigates two potential treatments: phytocannabinoids to alleviate seizures, and a novel cognitive enhancer to restore/halt memory deficits. The anti-convulsant properties of cannabidiol (CBD) were first examined with regards to the neuropathology of two major types of hippocampal interneurons expressing parvalbumin (PV) and cholecystokinin (CCK) which are thought to dysfunction during epilepsy. Immunohistochemistry experiments using an in vivo kainic-acid induced epileptic rat model, revealed that PV- and CCK-immunopositive interneurons were significantly affected during epilepsy. This effect was greatly reduced following CBD treatment, suggesting that CBD exerts a neuroprotective function. The effects of CBD on the intrinsic membrane properties of these interneurons, together with hippocampal pyramidal cells, were further investigated in acute brain slices of rat seizure models of TLE (in vivo kainic acid-induced and in vitro Mg2+ free-induced). Whole-cell recordings revealed that bath application of CBD (10 μM) normalised the firing frequency of epileptic adapting pyramidal cells to healthy control levels. A similar effect was seen in hippocampal CCK-immunopositive Schaffer collateral associated (SCA) interneurons. In contrast, CBD resulted in an increased firing of PV-immunopositive interneurons, thus increasing their excitability and restoring the impaired membrane properties of the cells apparent in the epileptic models. The effects of cannabidivarin (CBDV), a similar cannabinoid compound, on the intrinsic membrane properties of these cell types were also evaluated. Additionally, CBDV affected excitatory postsynaptic currents by reducing excitation. In an attempt to address the memory impairment aspect associated with TLE, I investigated the neuronal effects of a5AM21, a novel potential memory enhancer. Electrophysiological experiments revealed that a5AM21 preferentially acts on 5-containing gamma (γ)-aminobutyric acid (GABA) type A (GABAA) receptors, reducing their inhibitory effects. Furthermore, data obtained using behavioural experiment paradigm, the eight-arm radial maze, suggest a significant improvement in short- and long-term memory retrieval in rats treated with a5AM21. In conclusion, the results reveal the potential mechanisms of action of two therapies to alleviate seizures and memory impairment, and the future goals would be to combine CBD/CBDV and a5AM21 as a promising novel targeted therapy for TLE.

Layer II (L II) Medial Entorhinal Cortex (MEC) stellate cell (SC) intrinsic membrane properties vary along the MEC dorsal-ventral axis. This has been attributed partly to altered HCN and K+ conductances (Garden et al. 2008; Giocomo and Hasselmo 2008). The subthreshold active T-type CaV3.2 Ca2+ channels, though, are also expressed in the MEC (Huang et al. 2011). CaV3.2 channels are known to influence neuronal excitability but their effects on dorsal and ventral LII MEC SC properties remain unknown. To investigate this, I obtained acute brain slices from CaV3.2 wild type (CaV3.2+/+) and null (CaV3.2-/-) 5-8 week old mice and made electrophysiological recordings from dorsal and ventral L II MEC SC. CaV3.2-/- ventral neurons displayed significantly reduced input resistance but little difference in resting membrane potential (RMP) compared with CaV3.2+/+ ventral neurons. Consequently, depolarizing steps resulted in fewer action potentials in CaV3.2-/- ventral SC than in wild type neurons. In contrast, dorsal CaV3.2-/- and CaV3.2+/+ SC properties were similar. Furthermore, CaV3.2+/+ ventral cells had a significantly higher α excitatory post synaptic potentials (αEPSP) summation ratio (at 50 Hz) in comparison to CaV3.2-/- ventral neurons. The Cav3 inhibitors, NiCl2 and TTA-P2, also significantly reduced input resistance and action potential firing in CaV3.2+/+ ventral neurons, whilst having little effect on CaV3.2+/+ dorsal or CaV3.2-/- neurons. Furthermore, voltage-clamp experiments revealed a significantly greater T-type Cav3.2 Ca2+ current in ventral than dorsal neurons. Our results suggest that Cav3.2 channels selectively affect L II MEC ventral SC properties, thereby contributing to the intrinsic membrane gradient across the MEC dorsal-ventral axis.

Biopharmaceutical forms a new class of medicine which is biologically active and produced by recombinant technology or obtained from living organisms. It can be diversified into a range of subtype of drugs such as therapeutic enzymes, monoclonal antibodies, subunit proteins, nucleic acid and genetic materials. They have been used extensively for clinical application involving disease treatments, prevention and diagnosis. Unlike small molecule compounds, their chemical structures are more complex and can crucially influence their activity. However, these highly ordered conformation are often transformed upon exposure to physical stress during manufacturing such as extreme temperature, pH and high shear. This posses a challenge for the development of biologics and highlights the need of a more friendly formulation technique for macromolecules. Electrohydrodynamic atomisation (EHDA) is a process where using electrical energy to break up a bulk liquid into fine jets. The process allows the fabrication of micro to nano-scaled structures including particles or fibres without using heat involved. This can avoid the thermally induced degradation emerged during the formulation of macromolecules. Additionally, numerous materials can be fabricated by EDHA such as polymers, hydrogels and ceramic, thus enabling the design of various drug delivery systems. The aim of this PhD project is to undertake a conceptual study using EHDA to formulate biopharmaceuticals. Four biologics, alkaline phosphatase (ALP), bevacizumab (Avastin®; a whole-length monoclonal antibody used for neovascularization treatment), poly(inosinic-cytidylic acid) (poly-IC; an immunopotentiator), and ovalbumin (a model vaccine antigen) were processed into composites with polymers including poly(vinylpyrollidone) (PVP), poly(ε- caprolactone) (PCL) and poly(lactide-co-glycolide) (PLGA). Micron-nano sized fibres and particles for implantable biologic formulations were produced. Material properties and the activity of the developed formulation were characterised to identify the best formulation for biopharmaceutics.

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that together with a-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and kainate receptors mediate the vast majority of the fast excitatory neurotransmission in the central nervous system. Given this role, any dysfunction in neurotransmission is likely to have a severe impact on brain physiology. Recent mutations have been reported in NMDAR subunits that cause patients to suffer with a variety of neurodevelopmental disorders. Here, we use multidisciplinary structural modelling, site-directed mutagenesis, electrophysiology and kinetic modelling techniques to investigate how de novo missense mutations in distinct regions of the GluN2B subunit, affect NMDAR function. We predicted that these mutations would have pathophysiological implications and we sought to examine their effects on the cellular and molecular function of NMDARs. We developed a virtually complete 3D model of the human GluN1-GluN2B receptor based on the recently solved crystal structures of the frog and rat NMDARs. The human NMDAR structure locates the positions of the residues of interest, allows deductions about their potential impact on the protein as well as provides insight into the binding sites for Mg2+ and memantine using molecular docking. The functional effects of the missense mutations were first analysed in recombinant NMDARs and revealed gain-of-function and loss-of-function phenotypes, with some lacking an overt phenotype. We selected four most profound phenotypes for study in hippocampal cultured neurons revealing how these mutations can compromise excitatory neurotransmission. In addition, we also explored the therapeutic potential of the FDA-approved channel blocker memantine both in heterologous system as well as on excitatory neurotransmission as a potential therapeutic. Overall, the results suggest strong correlations between the effects of the missense mutations with patient phenotypes. Moreover, the study indicates which pharmacotherapeutic interventions are most likely to be successful as targeted therapies.

Background: As people living with dementia (PLWD) gradually lose their ability to manage their medicines appropriately, family members or friends step in to assist them. A variety of medication and person-related factors affect the medication use process. Aim: To identify and examine the factors contributing to the challenges to medication use from the perspective of PLWD and from their caregivers in the community and care home setting, in order to help maintain optimal therapy for this patient population, with a focus on the use of appropriate drug formulations, along dementia progression. In addition, to make recommendations for assistance for PLWD and caregivers to help alleviate caregiver burden and optimise the medication use process. Methods: To enable a comprehensive examination of issues from perspectives of PLWD and their caregivers, in relation to care setting and dementia severity, participants were recruited from the community and care homes in London. Semi-structured interviews were conducted, and medicines administration was observed in 4 care homes providing different types of care (nursing, residential, and mixed). Interviews were audio-recorded and transcribed verbatim. A conceptual model was developed based on a review of the literature; the domains provided the framework for thematic analysis of the data to achieve the research aim. Analysis was an iterative process and constant comparison was employed across interview transcripts. Results: Community results identified 6 areas that affect medication use; these are caregiver burden, the PLWD’s autonomy, scheduling and administering of medications, choice of formulations, interactions with formal care, and lack of medication information. Care home results also identified 6 areas; organisational aspects of the medication round, interactions between staff and residents, the residents’ autonomy, choice of formulation, staff knowledge, and interactions within the care home. Findings have also identified how key changes along dementia progression affect medication use. These include the development of swallowing difficulties, increase in the number and variety of medications, appropriateness of formulations, decline in cognition and communication, behaviour changes, caregiver expectations for the future, the PLWD’s autonomy, transition from self to caregiver-led care, and changes in support needs. Conclusion: The study has identified challenges to medication use in PLWD and their caregivers along dementia progression and informed recommendations to optimise medication use and alleviate caregiver burden. Recommendations include a proposed medicines optimisation model for PLWD and their caregivers, suggestions for tailored consultations and medication use reviews, improvements for care home organisation, and specific recommendations for the pharmaceutical industry for the development of dementia-friendly formulations. Furthermore, suggestions are proposed to adapt the Family Caregiver Medication Administration Hassles Scale for caregivers of PLWD based on the findings.

The applicability of deformable vesicles for topical drug delivery to skin and nails is an area of great interest. The aim of this project was to develop lipidic and polymeric vesicles as drug carriers for the topical treatment of onychomycosis and cutaneous leishmaniasis. Drug-free and drug-loaded vesicles (transfersomes and polymersomes) were prepared and characterised for size, deformability, encapsulation efficiency, drug release, permeation into and through nail and skin and antifungal and anti-leishmanial activities. Relationship between vesicle deformability and their ability to deliver drugs to nails and skin was explored. Transfersomes and PMPC-PDPA polymersomes showed higher deformability compared to liposomes and PEGMA-MCH polymersomes, and were further tested. Terbinafineloaded transfersomes and polymersomes showed drug encapsulation efficiencies of 100% and 65% respectively, and very different drug release profiles, with transfersomes showing slow and incomplete release, while the pH-responsive polymersomes showed fast drug release at the appropriate pH. Fluorescent vesicles were found to be able to partially penetrate the nail plate, with transfersomes permeating to a deeper extent than polymersomes. Drug encapsulation in the vesicles increased its anti fungal potency against C. albicans and T. rubrum. Interestingly, drug-free polymersomes showed intrinsic antifungal activity against dermatophytes. Polymersomes were also investigated for their potential in the topical treatment of cutaneous leishmaniasis. Topically-applied polymersomes permeated into both healthy and infected mouse skin, with greater permeation through the infected skin. Amphotericin B-loaded polymersomes had encapsulation efficiencies of 20% and were significantly more active against Leishmania major and Leishmania mexicana in in vitro experiments compared to miltefosine, the only oral treatment for cutaneous leishmaniasis. It was shown that the deformable transfersomes and polymersomes can be used to encapsulate antifungal and anti-leishmanial drugs, can permeate into the skin and the nailplate, and have anti-fungal and anti leishmanial activities. Thus, their application in the topical treatment of skin and nail infections is promising.