This study describes an investigation into the biological roles of selected glycine receptor (GlyR) subtypes in startle disease/hyperekplexia using zebrafish and mouse models. Firstly, I investigated the molecular basis of a novel zebrafish mutant dhx37nig1 with defective glycinergic transmission and designed morpholinos to knockdown DEAH box RNA helicase-37 and GlyR α and β subunits to recapitulate the phenotype. Secondly, using site-directed mutagenesis and molecular genetics techniques, I introduced a dominant- negative mutation (p.R271Q) common in human startle disease into vector- containing zebrafish GlyR α2 and GlyR α4 subunit cDNAs as an alternative method for targeted gene knockdown in zebrafish. Using CRISPR/Cas9 technology, I also generated a zebrafish glra2 knockout and developed a diagnostic PCR to genotype mutants with a large 26 base pair deletion. The glra2 knockout line was used to explore the role of the GlyR α2 subtype in locomotion and brain and spinal motor axon morphology. Thirdly, using bioinformatic tools, I investigated the GlyR α4 subunit in vertebrate species, looking for molecular evidence to explain why this gene is a pseudogene in humans. This revealed that, in addition to an in-frame stop codon, the human GlyR α4 subunit gene contains at least two other damaging mutations (p.E59K and p.Y204C) that were also present in ancient humans such as Denisovans. Lastly, using molecular genetic techniques, I identified candidate mutations in the shaky mouse, a mutant with an exaggerated startle response and tremors. This revealed a missense mutation causing the amino acid change p.Q177K in extracellular loop F of the GlyR α1 subunit. This shaky mutant showed defective integration into synaptic GlyRs as well as decreased current amplitudes with significantly faster decay times. My study highlights how the use of modern genetics techniques and model organisms can provide new insights into the biology of glycinergic transmission that underlies neurological disease.

Oral delivery of therapeutic peptides poses serious challenges in the field of drug delivery. The main concerns in developing an oral formulation of therapeutic peptide is that the peptide can be susceptible to enzymes in the gastrointestinal tract, poor solubility, short circulation time in plasma and fast renal clearance. This study focuses on improving the oral availability of therapeutic peptide by its encapsulation in a biodegradable polymer called GCPQ. GCPQ has been reported previously to solubilize small molecular weight hydrophobic drugs and peptides. GCPQ also demonstrated to improve their oral bioavailability. In the present study, two therapeutic peptides, Methionine enkephalin (anticancer peptide) and Labyrinthopeptin (anti-noiciceptive peptide) was characterized and formulated with GCPQ nanoparticles. Labyrinthopeptin, a hydrophobic peptide having poor solubility was solubilized using GCPQ nanoparticles and their oral uptake was evaluated in vivo. Anti-noiciceptive activity of Laby GCPQ B2 formulation was evaluated in rat CFA model and compared against Laby glycofurol formulation. Therapeutic effect of Laby GCPQ B2 oral and nasal formulation was found to be significantly higher to control at time points 240 min and 40 min respectively as demonstrated in rat CFA model. Laby glycofurol formulation also showed significant therapeutic effect (40 and 60 min) after nasal administration as a result of permeation enhancing property of glycofurol itself. Concentrations of Laby in plasma were observed to be higher for Laby GCPQ B3 oral and nasal formulation compared to Laby glycofural formulation, suggesting that GCPQ improves oral uptake of Laby peptide. However, concentrations of Laby in plasma for Laby GCPQ B1 formulation were lower than that it was observed for Laby GCPQ B3 and Laby glycofural formulation, suggesting that the characteristics of GCPQ can be modified to control the uptake of Laby peptide. Similarly, Methionine enkephalin, a peptide having poor stability in the gut was formulated with GCPQ B1 & B2 and characterized in vitro and in vivo. Their therapeutic effect was evaluated in nude mice tumor xenograft model. Significant reduction in tumor volume for mice treated with MENK-GCPQ B2 oral formulation was observed compared to MENK formulation and control. The concentration of MENK following oral administration of MENK GCPQ B2 formulation (100 mg/kg) were variable and at nanogram levels. However, this concentration could be sufficient to cause therapeutic effect in mice xenograft model. The concentrations of MENK in plasma were observed to be at nanogram levels (298 ng/mL), 180 min after administration of MENK-GCPQ B1 IV formulation. Following administration of MENK IV formulation, the concentrations of MENK in mice plasma were not detectable after 5 min. This result also suggests that only a fraction of MENK was encapsulated in MENK GCPQ B1 formulation. Although, there were significant differences in pharmacokinetic effect between MENK and MENK GCPQ B1 IV formulations, the therapeutic effect of MENKGCPQ B1 IV formulation in tumor xenograft model was only significant to that of control but insignificant to that of MENK IV formulation. Excess MENK concentration resulting from MENK-GCPQ B1 IV formulation may not have improved the therapeutic effect due to saturation of OGFr receptor and clearance from the system. An alternative mode of MENK-GCPQ administration such as subcutaneous or intra-peritoneal could delay MENK release in the systemic circulation and thereby enhancing the therapeutic effect compared to MENK only formulation.

BACKGROUND AND PURPOSE: Systemic delivery of hydrophobic therapeutics represents substantial formulation challenges impeding optimal benefits due to side effects and sub-therapeutic drug levels at the target site consequently leading to progression of multi-drug resistance. This thesis describes three distinct novel nanotechnology-based strategies with desirable aerosolization characteristics for delivery via the airways, aimed at enhancing the therapeutic efficacy of hydrophobic drugs for pulmonary and neurological disorders. METHODS: The first approach involved the development of dry powder microparticles for pulmonary delivery of antifungal amphotericin B nanocomplexes, prepared by co-grinding the drug with ascorbic acid. Nanocomplexes developed were characterized for molecular interactions by FT-IR, size, zeta potential, morphology, in vitro aerodynamic behavior and antifungal activity. The second strategy entailed design of liposomes co-encapsulating rifampicin and ibuprofen using Design of Experiment, targeted to the mannose and/or scavenger receptors on the alveolar macrophages where TB infection resides. Spray dried microparticles were characterized for in vitro aerodynamic behavior and macrophage uptake using the flow cytometer in RAW 264.7 cells. The third approach involved the development of Kolliphor® HS 15 micelles incorporating neuroprotective agents CNB001 or curcumin and Kolliphor® TPGS micelles encapsulating curcumin for the treatment of neurodegeneration and neuroblastoma respectively. Nasal delivery of these micellar systems was intended for brain targeting. Micelles were characterized for size, charge, aerosol droplet size distribution, drug release, morphology and in vitro cellular studies on SH-SY5Y cells. RESULTS: Successful development of nanocarrier-based systems with a high encapsulation efficiency greater than 80% for all the systems was achieved, with particle size desirable for the end-use. Spray dried microparticles of amphotericin B nanocomplexes with L-leucine showed a high fine particle fraction of around 58% signifying likely deposition in the peripheral airways, to the areas of fungal infection. There was no loss of antifungal activity against Candida spp on complexation of amphotericin B. Microparticles of liposomes encapsulating antitubercular drugs showed good aerosolization, and up to 65% fine particle fraction on addition of L-leucine could be achieved. An enhanced in vitro cellular uptake was evident for negative-charged liposomes targeted to the scavenger receptors and the mannosylated liposomes targeted to the mannose receptors on the macrophage cell line RAW 264.7. Finally, Kolliphor® micelles encapsulating CNB001 or curcumin showed desired aerosol droplet size for delivery to the posterior nasal olfactory epithelium with median size of 42.75-54.86 μm when aerosolized by the NasalTM Mucosal Atomization Device. The formulations intended for neuroprotection showed improved cellular viability, reduction in reactive oxygen species and nuclear morphology in the in vitro Parkinson’s model. CONCLUSION: The nanotechnology-based formulations combined with administration to or through the airways using commercially available delivery devices, represent a highly attractive formulation strategy for delivery of hydrophobic agents to the target site at a therapeutic level to combat issues of multi-drug resistance.

A medicine will not elicit its desired therapeutic effect if the patient is not able or willing to take it. The specific needs of the target population must be taken into account in the design of medicines. Evaluation of the effect of formulation factors on patient’s acceptability could guide the development of better medicines for children. Flexible solid oral dosage forms, including multiparticulates and (oro)dispersible formulations, offer advantages over conventional solid and liquid dosage forms to meet the needs of paediatric patients. These advantages include favourable stability profile, suitability for taste masking, flexibility of dose titration and convenient administration. The overall aims of this research were to identify barriers for the development of acceptable medicines for children, to explore methodology for palatability and patient’s acceptability testing and to generate evidence of acceptability of flexible solid oral dosage forms. Methodological tools for the assessment of palatability and acceptability were developed and the use of such tools was explored through a series of investigations in healthy volunteers using model placebo formulations. Pharmaceutical formulation work was performed to optimise formulation design and choice of excipients, integrating manufacturability and patient’s acceptability criteria. A direct comparison between palatability and acceptability outcomes in children and adults was performed, which highlighted the value of conducting studies in adults to provide initial guidance on formulation design. Some of the key formulation factors that affect acceptability of flexible solid oral dosage forms were identified, which can be used to guide the development of more palatable and acceptable medicines. This research also evidenced methodological barriers in the assessment of palatability and patient’s acceptability which are thoroughly discussed in this thesis and will need to be overcome in the future. The knowledge generated by this research is applicable not only to the development of medicines for children, but also for other subsets of the population.

Niemann-Pick Type C (NP-C) is a lysosomal storage disorder with neurological and visceral pathology, for which there is currently no major disease modifying treatment. Loss of NPC1 function, a late endosomal transmembrane protein, leads to systemic intracellular lipid accumulation. The subsequent premature death is usually associated with neurological manifestations, such as neurodegeneration and neuroinflammation. This project focuses on the development and preclinical evaluation of gene therapy for NP-C in a mouse model using an adeno-associated viral (AAV) vector. The vector would be capable of delivering and expressing human NPC1 in the mouse brain and providing therapeutic benefit. AAV vectors exhibit efficient and widespread gene delivery throughout the brain, however their limited packaging capacity can be a constraint for larger genes. In this project extensive construct modifications were carried out to incorporate the relatively large NPC1 cDNA into a functional AAV serotype 9 vector, where NPC1 is controlled by a constitutively active neuronal promoter. Initial in vivo testing demonstrated successful NPC1 over-expression in administered mouse brains, compared to endogenous NPC1 levels in unadministered controls. No indications of toxicity were observed as a result of exogenous NPC1 overexpression in vivo. A series of preclinical proof of concept survival studies were subsequently carried out on the Npc1-/- model, where newborn Npc1-/- mice were administered with 4.6 x 109 vector genomes of AAV9-NPC1 via intracerebroventricular injections. Treated Npc1-/- mice exhibited an increased lifespan (median survival - 116.5 days) compared to untreated Npc1-/- mice (median survival - 75.5 days). Low dose treated mice exhibited permanent normalisation of locomotor function and significant neuronal rescue in all brain regions analysed. A subsequent study with a 65-fold dose increase resulted in an additional significant extension of lifespan, along with improved weight maintenance. Combined, these results demonstrate the potential beneficial use of gene therapy for NP-C and support the further development of this approach.

Cancer chemoprevention aims to prevent, delay, or reverse carcinogenesis. Thai Traditional Medicine (TTM) could be a source for cancer chemopreventive agents and – more broadly –could play a role in cancer prevention. Using an ethnopharmacology approach this thesis aims to understand the pharmacological basis of some of these botanical drugs and to discover new extracts which could be useful in cancer prevention. Interviews with 33 TTM practitioners revealed the five characteristics of cancer in TTM (mareng), which is described as an accumulation of waste, chronic inflammation, chronic illnesses (krasai), bad condition of body fluids (‘luead’ and ‘namlueang’), and imbalance of dhātu si. Further analysis of preventive methods led to the five strategies for preventing mareng. To link TTM actions to pharmacology, we proposed that three strategies, removal of waste, liver protection, and prevention from krasai, can be linked to the antioxidant system. After screening of fifty-two extracts, fifteen exhibited protective effect in a liver cancer cell line. Among them, ethanol extracts of Thunbergia laurifolia leaves (TLe) and Senegalia rugata leaves (SR1e) exhibited the most potent activities in the induction of NQO1 enzyme and glutathione. Upregulation of antioxidant genes and radical scavenging were among their protective mechanisms. While TLe induced NQO1 expression, SR1e upregulated the expression of Nrf2. Both extracts did not induce CYP1A1 expression nor reduce cell viability of primary rat hepatocytes which provided preliminary safety profile. Using HPLC-HRMS-SPE-ttNMR, we could identify some active constituents in the extracts. This is the first report analysing how cancer is perceived in TTM, what prevention strategies are used, linking this to pharmacological models, and on chemopreventive properties of TLe and SR1e and some of their constituents. The evidence supports the potential use of these medicinal plants in cancer prevention. Future work should be performed with more TTM practitioners and use in vivo models.

Introduction: A “flipped classroom” uses technology to shift the traditional lecture outside the scheduled class time and uses the face-to-face time to engage students in interactive activities. / Aim of the study: Assess the feasibility, acceptability and effectiveness of using the “flipped classroom” teaching format with MPharm students in two pharmacy schools in the UK: UCL School of Pharmacy and the School of Pharmacy and Biomedical Sciences at University of Portsmouth. / Methods: An experimental mixed methods design was employed, with final year MPharm students in two phases; 1) a qualitative study using focus groups 2) a quasi-experiment measuring knowledge acquisition and satisfaction by delivering a session on rheumatoid arthritis, in two teaching formats: the flipped classroom and the traditional lecture. / Results: The flipped classroom approach was preferred over the traditional lecture for delivering a pharmacy practice topic, and it was comparable or better than the traditional lecture with respect to knowledge acquisition. In addition, this teaching approach was found to overcome the perceived challenges of the traditional lecture method such as fast pace instructions, student disengagement and boredom due to lack of activities and/ or social anxiety. However, high workload and difficult or new concepts could be barriers to pre-class preparation, and therefore successful flipped classroom. The flipped classroom encouraged learning scaffolding where students could benefit from application of knowledge, and interaction with peers and the lecturer, which might in turn facilitate learning consolidation and deep understanding. This research indicated that the flipped classroom was beneficial for all learning styles. / Conclusion: Implementing the flipped classroom at both pharmacy institutions was successful and well received by final year MPharm students. Given the attention now being put on the Teaching Excellence Framework (TEF), understanding effective methods of teaching to enhance student achievement and satisfaction is now more valuable than ever.

Gaucher disease is a lysosomal storage disorder caused by mutations in the GBA1 gene encoding the enzyme glucocerebrosidase (GCase). Deficiency of GCase causes the accumulation of its substrate glucosylceramide in both visceral organs and the brain. Enzyme replacement therapy is successfully used to ameliorate the visceral pathology, however there is no treatment available for the lethal neurodegeneration. This research focuses on Gaucher disease type II, the most acute neuronopathic form, in which the neuropathology results in death during early infancy. The aim of this project is to intravenously administer adeno-associated viral vector (AAV) based gene therapy to a GCase-deficient mouse model of acute neuronopathic Gaucher disease and assess improvement in lifespan, behaviour, brain and visceral pathology. The untreated Gba1 knock-out mice die 12-14 days after birth following severe neurodegeneration. The AAV vector carrying the functional human GBA1 gene under control of a ubiquitous promoter was intravenously administered to neonatal knock-out mice, with treated animals showing a significant increase in their lifespan (p=0.0081). Since the animals did not develop any evident pathological symptoms, they were sacrificed at 55 days of age for a short-term study. The neuropathology was ameliorated and several of the most affected areas of the brain were partially rescued. The analysis of liver, spleen, lung and heart tissues revealed promising improvements in the visceral pathology. A consequent long-term study was performed on 180-day-old treated mice, with the aim to compare intravenous and intracranial administration of the viral vector. In order to enhance the therapeutic effects of the treatment and improve gene expression in the central nervous system, a novel construct where the GBA1 gene is controlled by a neuron-specific promoter was administered to neonatal knock-out mice. The severe neurodegeneration was further rescued and the life span of treated animals increased. Together, these encouraging results demonstrate that gene therapy could provide an effective treatment for the neuronopathic form of Gaucher disease, for which therapeutic needs are currently unmet.

The biopharmaceutics classification system (BCS) is a framework for classifying drug substances based on their aqueous solubility and permeability across a biological membrane. It is possible for individuals to request a waiver of in vivo bioequivalence (BE) studies for immediate-release solid oral dosage forms. Nowadays, the Food and Drug Administration allows the biowaiver for BCS Class I and Class III drugs. However, the findings in this thesis call attention to the unearthed loopholes in this regulation, given that formerly considered “inert” pharmaceutical excipients have shown unexpected impacts on the absorption of co-formulated drugs. By virtue of our studies, it has been exposed that a number of excipients used to aid tablet manufacturing or formulation development are able to enhance therapeutic efficacy of the drug by the modulation of transporter activity and expression. This, therefore, could characterize the excipient to have two applications; in manufacturing and drug formulation. Furthermore, the thesis outlines the modifications of the overall function of transporters, simply due to the sex of the organism. As a result, the work reported here emphasizes that the selection of excipients during formulation development is integral in preventing differences in therapeutic efficacy and side effect profiles between males and females.

Gaucher’s Disease (GD) is a rare recessive disorder produced by the dysfunction of the lysosomal enzyme Glucocerebrosidase (GCase). GCase catalyses the cleavage of the glycolipid Glucosylceramide. The lack of functional GCase leads to the accumulation of its lipid substrate in lysosomes causing GD. GD presents a great phenotypic variation, symptoms ranging from asymptomatic adults to early childhood death due to neurological damage. More than 250 mutations in the protein GCase have been discovered that result in GD. Being able to link structural modifications of each mutation to the phenotypic variation of GD would enhance the understanding of the disease. The aim of this work is to understand the structural dynamics of wild type and mutant GCase. A model of the complex of the enzyme GCase with its facilitator protein, Saposin-C (Sap-C) was generated using Protein-Protein docking (PPD). In this work, a knowledge-based docking protocol that considers experimental data of protein- protein binding has been carried out. Here, a reliable model of the enzyme GCase with its facilitator protein is presented and is consistent with the experimental data. To understand the structural mechanism of function of the enzyme GCase, it was imperative to study its structural dynamics and conformational changes influenced by its interaction with other components including lipid bilayer, facilitator protein or substrate. Coarse-Grained MD (CG-MD) was employed to study lipid self-assembly and membrane insertion of the complex. Classical Atomistic MD (AT-MD) was used to study the dynamics of the interactions between different components of the simulation. Furthermore, the results of ten different AT-MD simulations sampling 9 s have been analysed. An activation method of GCase by Sap-C has been proposed, the change in conformation of GCase when its facilitator protein is present has been highlighted, through the stabilization of the loops at the entrance of the binding site. The differences in protein-protein binding when GCase is mutated have also been emphasised. Finally, Anharmonic Conformational Analysis and Markov State Models have been used to build a kinetic model of the system. This model supports our activation mechanism hyphothesis.