The nasal route is highly promising for the delivery of drugs exerting local effects in the nose or for therapeutic molecules having systemic or CNS effect. This is attributed to the fact that the nasal epithelium is highly vascularized and permeable, which ensures rapid absorption of the drug. The limitation of short residence time of the formulations in the nose and poor bioavailability of hydrophilic drugs could be overcome by the inclusion of bioadhesive agents into formulation. The main objective of this study was to develop novel bioadhesive microspheres and liposomes entrapping the anti-Parkinson drugs ropinirole hydrochloride (RH). The microspheres were prepared via spray drying in combination with chitosan or sodium alginate and the liposomes were prepared using the ethanol-based proliposome method. This study has investigated the potential of powdered mucoadhesive microparticles and liquid liposomes for nasal delivery via Miat® nasal insufflator and nasal spray devices respectively. Optimum mucoadhesive chitosan microparticles were prepared by co-spray drying of chitosan glutamate and ropinirole hydrochloride (90:10 w/w). Characterization studies have revealed that the drug following spray drying was amorphous and the microparticles were spherical and offered drug entrapment efficiency values in the range of 93 - 99%. The optimum formulation provided maximum swelling capacity and slowest drug release. Ex vivo toxicity study using isolated sheep nasal mucosa proved the safety of the optimized formulations for intranasal delivery. Investigation of powder delivery demonstrated that the Miat® nasal insufflator could deliver 90% of the dose with the first puff regardless of the loading weight used to fill the capsule fitted into the nasal device. The spray cloud had elongated shape and was homogenous; this is expected to enhance the impaction of the formulation in the nose following delivery from the nasal device. The properties of sodium alginate microparticles prepared via spray drying were highly dependent on inlet temperature of the spray drier, affecting particle morphology and product yield percent. The best performing particles were obtained when the inlet temperature was 140oC. Alginate to RH ratio had marked effect on particle size (2.60 - 4.37µm), entrapment efficiency (101 – 109%), physical state of the encapsulated RH, and morphology and surface smoothness of the particles as shown by scanning electron microscopy (SEM). In vitro drug release profile showed the amount of sodium alginate in formulations has controlled the rate of drug release. Results revealed that RH-alginate microparticles in 90:10 w/w polymer to drug ratio was the best performing spray dried formulation. Toxicity study proved safety of RH loaded sodium alginate for intranasal delivery. In contrast to RH-chitosan microparticles, particle trajectories was found from the cloud generated from emitted powder and laser diffraction demonstrated that powder was less likely to deposit in the lower respiratory tract owing to particle agglomeration. Ethanol-based proliposome technology produced oligolamellar liposomes from lipid ethanolic solutions as revealed by transmission electron microscopy (TEM). The resultant liposomes entrapped approximately 23.30% of the drug. Using five different bioadhesive agents, inclusion of any of these agents (0.2% w/v) caused a decrease in drug entrapment except for carboxymethyl chitosan which had no effect on the drug entrapment (25.97%). Investigation of aerosolized liposome dispersion using a range of nasal spray devices demonstrated integrity of liposomes were not changed (i.e particle size, Span, and drug entrapment efficiency were unaffected) and RH-loaded liposomes were efficiently delivered from the devices. In conclusion, the finding of this study explored mucoadhesive microspheres entrapped the anti-Parkinson drug, RH, and can potentially be applicable for nasal delivery to enhance nose to brain transport using nasal insufflator for improvement of the symptoms of Parkinson disease and Restless legs syndrome. Similar findings using nasal sprays were found for liposomes. In vivo studies are required in the future to determine the amount of the drug that may reach the blood circulation and brain.

Substituted indoles (2-arylindoles) and related structures are known to exhibit potent anti-cancer activity against human breast cancer cell lines, and a range of other therapeutic targets. This activity, and other factors such as their biological activity, the fact that they are privileged structures, and the presence of the indole nucleus in various commercial anti-cancer drugs led to the choosing of indoles for the current study as a starting point for the development of new treatments against glioma. Investigation began on determining the anti-cancer activity of a variety of indoles against glioma cell lines (1321N1 and U87MG) using a number of different cell-based assays and also to compare them with conventional anti-cancer drugs. The aim was to find potent anti-cancer compound(s), amongst the compounds tested, and by studying its preliminary structure-activity-relationships (SAR), try to determine how the active compound(s) may be exerting their effects. The SAR screening was divided into two main groups: indoles without a 2-aryl group and indoles with a 2-aryl group. The most potent compound identified, and its analogues, were further tested on the non-cancerous SVGp12 cell line to check for specificity of these indoles towards cancer cells, wherein it was found that these compounds were not specific to any particular cell type. Furthermore, activity was also observed for the best lead compound in the glioblastoma short-term culture, IN859, in which it gave a relatively low micromolar IC50 value (400 μM). The results indicated that the anti-cancer activity of these compounds started within 2 h and therefore it was speculated that the mechanism of action of these compounds might work through the generation of reactive oxygen species (ROS). A ROS-detection kit was used to demonstrate this hypothesis, a result which was later corroborated using flow cytometry, and also provided quantitative analysis of the amount of ROS generated. It was further hypothesised that in the cells studied, autophagy was mediated due to excessive ROS generation. This was also confirmed over a similar time course by quantifying the amount of fluorescence generated in the 1321N1 and U87MG cell lines when labelled with acridine orange (a dye used to detect the formation of autophagosomes during autophagy) using flow cytometry. Moreover, the use of an autophagy inhibitor, 3-methyladenine, was shown to inhibit autophagy in these cell lines, again validating this hypothesis. In conclusion, it has been demonstrated that the ability of certain substituted privileged indoles possessing a 2-aryl group and having an attached –OH group to it may have a rapid, deleterious effect on the viability of a primary short term culture (IN859) and glioma cell lines (1321N1 and U87MG). The mechanism of action of these indoles to cause cell death may be via the generation of ROS, leading to cell death initiated by autophagy. Another short separate study was also performed in order to investigate the anti-cancer activity of an anionic host defence peptide, Cn-AMP2, on the above mentioned cell lines. This peptide was found to exhibit a modest cytostatic effect on both the cell lines but at higher concentrations (> 1 mM) and only when the serum concentrations were weaned down from 10 % to 2.5 %.

In the past two decades only modest advancements in glioma treatment have been made, with patient prognosis and median survival time following diagnosis only increasing from 3 to 7 months. A substantial body of clinical and preclinical evidence has suggested a role for aspirin in the treatment of cancer with multiple mechanisms of action. Aspirin is one of the most widely used drugs, successfully taken as an analgesic, antipyretic, anti-inflammatory agent and for prevention of strokes and ischemic diseases. The effects on cell viability, proliferation, apoptosis and migration of aspirin and aspirin derivatives were tested on primary glioblastoma cell cultures, BTNW911 and BTNW 914, and the well-established cell lines, SVG-p12, 1321N1, GOS-3, U87 MG, using the PrestoBlue assay, CFDA-SE, PI/annexin V, and live imaging receptively. The effects on cell viability following 24 and 48 hour incubation of four aspirin derivatives (PN508, PN517, PN526 and PN529) were compared to cisplatin, aspirin and di-aspirin, establishing IC50 values, showing PN517 to be the most potent analogue, and in some cases greater efficacy than cisplatin. Aspirin analogues showed greatest efficacy in the first 24 hours, while cisplatin increased in efficacy with time showing a lower IC50 value in all cell lines at 48 hours. Cell proliferation was assessed over 3 to 10 days, with each treatment decreasing proliferation and the largest effect of PN517 found in BTNW914 cells. PN517 treatment decreased the population of G0/G1 phase cells in cell cycle analysis, decreased cyclin D1 and EGFR activation, and total EGFR expression. Apoptosis was induced by PN517 in a concentration and time dependent manner in both the cell lines and short term cultures, with activation of both intrinsic and extrinsic pathways. Finally, PN517 reduced migration in both the Boyden chamber and scratch assays, but did not inhibit invasion. In conclusion, these data support the further development of PN517 as a novel therapeutic drug for the treatment of glioma.

Research into responsive polymeric insulin delivery systems for the management of diabetes mellitus is gaining increasing interest due to the rise in the incidence rate and the burden of daily multiple subcutaneous insulin injections that needs to be endured by the patient. The present study attempted to formulate a nanoparticulate glucose responsive insulin delivery system from a natural polymer chitosan, using a safe glucose sensor, phenyl boronic acid (PBA), which is known to interact with glucose. In the present project, a new method for the production of chitosan tripolyphosphate (TPP) nanoparticles via ultrasonication was developed and optimised. The electrostatic method of tagging PBA onto chitosan was unsuccessful, but the method of N-reductive alkylation of introducing the PBA was successful. Evidence of PBA bonding on to chitosan was assessed by FTIR, ToF-SIMS, DSC and glucose adsorption sensitivity measurements. Glucose adsorption sensitivity to PBA-bonded chitosan polymer was directly related to the amount of PBA functionality within the conjugates and the physical nature of the matrices (porous or crystalline) as revealed by scanning electron microscopy (SEM). The nanoparticles showed glucose concentration dependent swelling with swelling decrease at a glucose concentration above 2.5mg/ml. Encapsulation of insulin into the nanoparticulate matrix was achieved by both the ionotropic gelation and polyelectrolyte complexation methods. Smaller particles with z-average between 140 – 150nm, lower Pdi and zeta potential between 17.5-19.1mV were characteristic of particles produced by PEC, whilst slightly larger particles with z-averages between 170-200nm, higher Pdi and zeta potential between +17.6-21.6mV were noticed for the particles produced by ionotropic gelation. Higher encapsulation of insulin of about 90% was achieved using the PEC method as compared to 34% from the ionotropic gelation series. The amount of drug encapsulated in both methods was pH dependent. In vitro xxi glucose dependent insulin release studied on PEC formulations showed a glucose and fructose concentration dependent release which was affected by the buffer system used. Lower insulin release from higher concentration of the sugars was attributed to the formation of bidentate interaction between the diols in the sugar and PBA, which restricts further expansion of the nanoparticles and hence reduces insulin release. This was confirmed by the SEM images of the nanoparticles after exposure to buffer, glucose and fructose in buffers at pH 7.4. Nanoparticles exposed to fructose showed more spherical and intact matrices whilst the buffer samples showed fragmented particles. The samples exposed to glucose showed some degree of fragmentation but not high as compared to that of nanoparticles exposed to buffer. The release of insulin from this formulation was therefore dependent on a complex interplay between the components of the buffer and the amount of sugar present.

Canarium patentinervium Miq. belongs to the family of Burseraceae best known for producing resins of economic, medicinal, and cultural values such as frankincense, myrrh, and copal. This family consists of 18 genera and 700 species of trees. In the Asia-Pacific region, about 20 species of Burseraceae are used to treat haemorrhoids, heal wounds and to treat skin infections. This plant has been used to heal wounds amongst the indigenous people of Malaysia. Furthermore no pharmacological and phytochemical studies have been reported on this species. This study was undertaken to screen the phytochemical and pharmacological aspects of this plant. Qualitative phytochemical properties of the crude extract was determined for the presence of tannins, flavonoids, alkaloids, saponins or sterols. Phytochemical analysis of Canarium patentinervium Miq. revealed presence of tannins and flavonoids in the ethanol extract of leaves and barks. Bioassay guided fractionation led to isolation of eight secondary metabolites by chromatography which were identified by NMR techniques. Two coumarins (scopoletin and scoparone), five phenols (cynaroside, hyperin, (+)-catechin, lioxin and syringic acid) and a norsesquiterpene with cyclohexenone ring (vomifoliol). The latter three compounds were isolated for the first time from the genus Canarium. The plant and the isolated compounds were then subjected to six biological assays comprising of antibacterial, antioxidant, anticancer, anti-inflammatory, anti-acetylcholinesterase and anti-parasitic activities. Infectious diseases remain the leading cause of death worldwide and bacteria have become more resistant to conventional antibiotic and the search for novel antimicrobial agents from medicinal plants has become crucial. Antibacterial test was done using disc diffusion method, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and death kinetic assay with ampicillin as the positive control. The ethanol extract of leaves and the hexane extract of bark displayed remarkable antibacterial activity against both Gram-positive bacteria and Gram-negative bacteria. All isolated compounds tested against S.aureus ATCC 11632 showed bacterial growth inhibition. Scopoletin, scoparone, hyperin, cynaroside and syringic acid had bactericidal effect <100 µg/ml. Only scopoletin had bactericidal effect and complete kill at MBC 50.00 µg/ml. Bacterial infections have been known to generate extensive formation of free radicals which is becoming increasingly recognized in the pathogenesis of the many human diseases. The role of free radicals and active oxygen is becoming increasingly recognized in the pathogenesis of the many human diseases, including cancer, neurodegenerative diseases, ageing, and atherosclerosis. Five various antioxidant assays with different mode of action [2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), β-carotene bleaching assay and superoxide dismutase (SOD) assay] were used to test the antioxidant scavenging abilities of this plant. Vitamin C (L-ascorbic acid), quercetin and trolox were used as positive controls. The ethanol extract of leaves and barks displayed superior antioxidant capacities. The EC50 values of the samples were consistently low in SET methods (ABTS, DPPH and FRAP) superior to standard as opposed to HAT method (β-carotene bleaching assay). Hyperin and (+)-catechin exhibited the most consistent free radical scavenging capability across the five antioxidant assay. Hyperin and (+)-catechin have significantly lower IC50 (0.75±0.03 µg/ml and 0.94±0.27 µg/ml respectively) compared to SOD enzyme (IC50 1.59±04 µg/ml). Scopoletin exhibited potent antioxidant activity compared to scoparone with significantly lower EC50 values in ABTS (IC50 1.08±0.03 µg/ml) compared to ascorbic acid (EC50 1.54±0.03 µg/ml) and lower values in FRAP assay (FRAP value 49.00±0.64 µg/ml) than quercetin (FRAP value 86.00±0.24 µg/ml) and ascorbic acid (FRAP value 347.00±0.23 µg/ml). Vomifoliol had potent β-carotene bleaching activity with IC50 6.85±0.37 µg/ml. Infections and free radical generation are recognized in the pathogenesis of cancer. The ethanol and chloroform extract of barks showed significant anticancer activities with GI50 values of 34.40µg/ml and 23.44µg/ml. The most susceptible cell lines were found to be the breast cancer cell line, MDA 468. Scopoletin displayed potent anticancer effect against breast cancer cell line MDA 468 (GI50 0.09±0.25 µg/ml) and colorectal cancer cell line HT-29 (GI50 0.17±0.05 µg/ml), the latter being more significant that positive control doxorubicin (GI50 0.66±0.60 µg/ml). In recent years, roles have been identified for several inflammatory cells and for a large number of inflammatory mediators in important pathologies not previously linked to inflammation, such as Alzheimer’s disease and cardiovascular disorders including atherosclerosis, as well as cancer. Recently, reports have appeared regarding so-called “dual inhibitors,” agents that inhibit not only cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), but also 5-lipoxygenase (5-LOX). Chloroform extract of the barks had the lowest 5-LOX inhibition (IC50=29.53±0.0 3μg/ml) when compared to NDGA (IC50=29.19±0.02 μg/ml). Ethanol extract of leaves had superior COX-1 inhibition (IC50 = 0.60±0.01µg/ml) compared to COX-2 inhibition (IC50 = 1.07±0.01 µg/ml), whereas the barks had superior COX-2 inhibition (IC50 = 9.39±0.03 µg/ml) as opposed to COX-1 (IC50 = 11.41±0.03 µg/ml). All isolated compounds exhibited significantly lower 5-LOX inhibition than NDGA. Scopoletin and scoparone were potent inhibitors of 5-LOX recording lowest IC50 values (IC50 0.34±0.01 µg/ml and 0.20±0.01 µg/ml respectively). However (+)-catechin had a more comprehensive anti-inflammatory activity with dual inhibition of 5-LOX (IC50 16.10±0.03 µg/ml) and COX (COX-1; IC50 12.08±0.02 µg/ml, COX-2; IC50 83.89±0.03 µg/ml). Syringic acid exhibited potent 5-LOX inhibition (IC50 1.38±0.03 µg/ml) and moderate COX-1 inhibition (IC50 34.89±0.02 µg/ml). Furthermore, oxidative and inflammatory processes are among the pathological features associated with the central nervous system in Alzheimer’s disease. There is evidence that acetyl cholinesterase (AChE) inhibitors have an anti-inflammatory role through action against free radicals and amyloid toxicity, as well as through decreasing release of cytokines from activated microglia in the brain and blood. Chloroform extract of the barks displayed the best activity (IC50 = 88.59±0.14 μg/ml) as opposed to positive control, galanthamine (IC50 = 0.74±0.06 μg/ml). The ethanol extract of barks and leaves follow through with IC50 = 186.00±0.15 μg/ml and IC50 = 201.24±0.15 μg/ml respectively. Only scopoletin, scoparone, vomifoliol and syringic acid showed AChE inhibition at IC50 <100 µg/ml. Syringic acid exhibited good AChE inhibition (IC50 29.53±0.19 µg/ml), lowest of all compounds tested. Choline is the precursor of phosphatidylcholine (PC), a main component of Leishmania promastigote membranes. Therefore, inhibition of choline formation may de¬crease Leishmania survival. This hypothesis can be tested by using inhibitors of the acetylcholinesterase enzyme (AChE), which catalyzes the hydrolysis of acetylcholine to choline and acetic acid, as leishmanicidal compounds. The hexane extract of leaves showed moderate antileshmanial activity with IC50 values of 257.40±0.30 μg/ml. Only ethanol extracts showed activity against Giardia intestinalis and Entamoeba histolytica at concentration of 500 μg/ml. Scopoletin was tested against all three parasite amd it was more potent against Leishmania donovani (IC50 163.30±0.32 µg/ml) and MIC of >200 µg/ml for both Giardia intestinalis and Entamoeba histolytica. Six kinds of major biological effects were evident in the crude and compounds namely, antioxidant, antibacterial, anti-inflammatory, anti-AChE, anti-parasitic, and anticancer all of which were reported for the first time from this plant. Given the aforementioned evidence it is tempting to speculate that Canarium patentinervium Miq. represents an exciting scaffold from which to develop leads for treatment of inflammatory and oxidative stress related diseases.

Malaysia is ranked the 12th richest country for its biological diversity of plant species by the Convention on Biological Diversity and this project aims to contribute to existing knowledge of Malaysian rainforest plants, Acalypha wilkesiana, Duabanga grandiflora, Archidendron ellipticum and Pseuduvaria macrophylla, for the treatment of cancer. A. wilkesiana (Euphorbiaceae) whole plant EtOH and EtOAc extracts inhibited growth of breast cancer MDA-MB-468 cells (GI50: 22.7 and 15.9 μg/ml) and revealed preference over non-transformed MRC5 fibroblasts (GI50: 46.6 and 53.3 μg/ml, respectively). EtOH and HEX extracts were able to impair cell survival and colony-forming abilities in MDA-MB-468 cells after 24 h. Detection of increased MDA-MB-468 sub-G1 cell populations after 48 h treatment to EtOH and HEX extracts, suggest that cells may be undergoing apoptosis. A. ellipticum (Leguminosae) crude polar bark and leaf extracts inhibited MDA-MB-468 cell growth (GI50 of bark EtOH, EtOAc extracts: 1.7 and 40.4 μg/ml, respectively and leaf EtOH and EtOAc extracts: 9.3 and 9.3 μg/ml, respectively). However, MDA-MB-468 cell growth was unaffected by HEX extracts (> 200 μg/ml). Separation of crude extracts revealed sub-fractions of greater activity, in particular a 50-fold enhanced potency in sub-fractions of HEX extract thus overcoming masking or antagonistic activity in the crude mixture. Following 24 h, bark and leaf extracts impaired MDA-MB-468 cells’ proliferative and colony-forming abilities at 1X and 2X GI50 values suggesting significant damage was induced leading to observed cellular senescence and inhibition of cell proliferation. After 48 h exposure to EtOH and HEX extracts, MDA-MB-468 cells accumulated cellular damage, possibly affecting microtubule functions resulted in activation of apoptosis as shown by increased of sub-G1 and G2/M MDA-MB-468 cell populations and presence of phosphatidyl serine on the outer membrane of cells. Modest levels of flavonoid and phenolic compounds were found in bark and leaf extracts, which correlated to moderate level of free radical scavenging activity observed. D. grandiflora (Lythraceae) bark and leaf extracts revealed growth inhibitory effects against colorectal cancer HCT116 cells (GI50 of bark Water, EtOH, EtOAc and HEX extracts: 42.4, 37.5, 21.69 and 28.9 μg/ml, respectively; leaf Water, EtOH, EtOAc and HEX extracts GI50: 38.0, 40.9, 24.7 and > 200 μg/ml, respectively). Separation of crude bark extracts resulted in fractions of greater activity, whereas separation of leaf extracts revealed reduced activity suggesting synergistic activity in the mixture. Following 24 h, bark and leaf extracts impaired HCT116 cells’ proliferative and colony-forming abilities at 1X and 2X GI50 values indicating significant damage incurred leading to observed cellular senescence and cell proliferation inhibition. After 48 h exposure to D. grandiflora extracts, increased sub-G1 HCT116 cell population and a G1/0 cell cycle block accompanied by decreased S and G2/M cell populations were measured. Detection of phosphatidyl serine on cells’ outer membrane and activated apoptotic caspase 3 protein confirmed D. grandiflora extracts induced apoptosis. Highest levels of flavonoid and phenolic compounds were found in polar bark and leaf D. grandiflora extracts, which may correlate to the highest free radical scavenging activity measured. P. macrophylla (Annonaceae) extracts collectively displayed greatest HCT116 cell growth inhibition (EtOH, EtOAc and HEX extracts GI50: 5.2, 1.6 and 5.4 μg/ml) and separation of EtOH and EtOAc extracts revealed even greater activity in sub-fractions. After 24 h, polar extracts at 2X GI50 completely impaired HCT116 cells’ proliferative and colony-forming abilities suggesting extract-induced significant damage led to inability of cells to proliferate. Analysis of cell cycle distribution revealed increased sub-G1 HCT116 cell population and high levels of early apoptotic HCT116 cells following 48 h exposure to P. macrophylla extracts coupled with detection of caspase 3 activation confirming execution of apoptosis. Modest levels of flavonoid and phenolic compounds were detected in EtOH, EtOAc and HEX extracts, which correlated to modest free radical scavenging activity. The findings in this project should justify further separation and in vitro investigations.

A tropical shrub from Euphorbiaceae (Juss.) family, namely Acalypha wilkesiana var. macafeana hort. was investigated for its antioxidant and antibacterial properties. The antioxidant properties were assayed by Ferric Reducing Antioxidant Power (FRAP) assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and β-carotene bleaching assay. Assessment of its antibacterial properties was conducted with pour plate disc diffusion assay and dilution methods. The plant was collected, dried, grinded and soaked continuously in four different solvent starting from non-polar to polar solvent: hexane, ethyl acetate, ethanol and water. The crude extracts were concentrated under pressure and kept in -20 °C prior to investigation. The ethanol extract of A. wilkesiana var. macafeana hort. exhibited good antioxidant and antibacterial activities with results more potent than the standards used. To further locate the bioactive constituents of the plant, we fractionated the ethanol extract leading to five fractions, namely F1, F2, F3, F4 and F5. Both antioxidant and antibacterial assays were conducted upon all the five fractions. Results showed profound activity from F5 of both antioxidant and antibacterial properties which warrants further investigation. To shed light on the active constituents of F5, identification was done with high performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LCMS) and nuclear magnetic resonance (NMR). Separation was obtained with reversed phase HPLC which showed one major compound and 6 minor compounds. The major compound was collected with a fraction collector and identified as geraniin via interpretation and comparison of its NMR shifts, while the other 3 compounds were identified by fragmentations of LC-MS. The compounds identified are β-glucogallin, potentillin and sanguiin H-6. All identified compounds are ellagitannins, except for β-glucogallin which is a gallotannin. The in vitro cell-based assay was performed to HepG2 cells to assess the ability of antioxidants like ellagitannins to protect cells against oxidative insults, and F5 was observed to be able to protect cells against cell death induced by t-BHP insults in a dose-dependent manner. F5 was also found non-toxic in the concentration needed to protect the cells, which is 100 µg/mL. We then explored the synergistic property of the tannin fraction, F5 with commercial antibiotics and observed F5 and ampicillin inhibit the growth of Staphylococcus aureus synergistically. Field Emission Scanning Electron Microscopy (FESEM) analysis was able to demonstrate that the bactericidal mechanism of F5 involves cell wall lysis as the result illustrates indentation of the cell surface and some showed total collapse of the cells. To explore its ability to be formulated and used as a topical agent for treating bacterial infections, a preliminary formulation was made incorporating F5, and formulated with 3 different bases. The formulation made with the paraffin base was observed able to exert the antibacterial property of F5 against Staphylococcus aureus in the in-vitro assay. In vivo animal study on guinea pigs with an incised cut infected with Staphylococcus aureus and treated with the formulation showed that the closure and healing of the wound was faster than Burnol®. Our results indicate possible use of ellagitannins from A. wilkesiana var. macafeana hort. with ampicillin to treat Staphylococcus aureus infections as it is bactericidal via a mechanism involving cell lysis. It also illustrates the possibility to be used as a topical wound healing agent with respect to its antibacterial and antioxidant properties. Ellagitannins from A. wilkesiana var. macafeana hort. can be viewed as a possible bactericidal agent that can contribute to the development of topical antibacterial drug or cosmetics in tropical countries.

ON01910.Na (Rigosertib, Estybon®), a styryl benzylsulfone, is a Phase III stage anti-cancer agent. This non-ATP competitive kinase inhibitor has multi-targeted activity, promoting mitotic arrest and apoptosis. Extensive Phase I/II studies with ON01910.Na, conducted in patients with solid tumours and haematological cancers demonstrate excellent efficacy. However, issues remain affecting its development. These include incomplete understanding of anti-tumour mechanisms, low oral bioavailability and unpredictable pharmacokinetics. In an attempt to improve drug-likeness and ADME properties of ON01910.Na analogues, a novel series of (E)-styrylsulfonyl methylpyridine derivatives was designed and synthesised. The SAR of this novel series is discussed. The lead compounds TL-68, TL-77, and AH-123 are highly potent mitotic inhibitors. Their selective cytotoxicity to cancer cells was identified in the screening cascade. Impressively, TL-77 possesses excellent pharmaceutical properties, with improved oral bioavailability when compared to ON01910.Na. The detailed cellular mechanisms of TL-77 were further investigated in comparison with ON01910.Na. TL-77 exhibits potent anti-proliferative activity against a wide range of human tumour cell lines, and demonstrated > 2 fold greater potency in cancer cell lines over normal cells. Cell cycle analyses reveal that TL-77 evokes profound G2/M cell cycle arrest at ≥ 6 h in cancer cells, followed by the onset of apoptosis. In cell-free conditions, TL-77 as well as ON01910.Na potently inhibits tubulin polymerization. Mitotically arrested cells display multipolar spindles and misalignment of chromosomes, indicating TL-77 interfere mitotic spindle assembly in cancer cells. These effects are accompanied by induction of DNA damage, inhibition of Cdc25c (Ser198) phosphorylation [indicative polo-like kinase 1 (Plk1) inhibition], and downstream inhibition of cyclin B1. However, kinase assays failed to confirm the inhibition of Plk1. Non-significant effects on PI3K/AKT signal transduction are observed after TL-77 treatment. Analysis of apoptotic signalling pathways reveals that TL-77 down-regulates expression of B-cell lymphoma 2 (Bcl-2) family proteins [Bid (BH3 interacting-domain death agonist), Bcl-xl (B-cell lymphoma-extra large) and Mcl-1 (induced myeloid leukaemia cell differentiation protein)] and stimulates caspase activation. These effects are comparable to those elicited by ON01910.Na. Unlike ON01910.Na, however, TL-77 causes preferential toxicity in cancer cells when compared to normal cells and mediates rapid mitotic inhibitory effects. In summary, selective in vitro anti-tumour activity and multi-faceted mechanisms of action of a novel molecule TL-77 have been identified, presenting a strong rationale for further development of (E)-styrylsulfonyl methylpyridine derivatives as therapeutic agents for cancer.

The assessment of medicines has moved from efficacy and safety to that of a benefit-risk balance and regulatory agencies and pharmaceutical companies are improving their processes in order to achieve greater consistency and transparency in decision-making. However, their efforts are largely independent and do not address the lack of consistency in decisions by different countries, albeit for the same medicine, resulting in the potential inaccessibility of important medicines. The aim of this study was the development and validation of a universal benefit-risk framework for use by regulatory authorities. A questionnaire, specifically developed for this study, was used to evaluate the current approaches to benefit-risk assessment of medicines by 14 regulatory agencies and 24 pharmaceutical companies. None of the 11 agencies (79%) and 20 companies (83%) that responded used a fully quantitative approach, but the majority used a qualitative system for benefit-risk assessment. The development of a universal benefit-risk framework for use by both regulators and industry, with the involvement of all stakeholders, was supported by the study participants. A comparison of the existing benefit-risk assessment frameworks used by agencies and companies identified the common elements. As no major differences were observed, an 8-step universal framework was developed which incorporated the other frameworks. To support the framework in the assessment of benefits and risks, a template for documenting the benefit-risk decision together with a user manual was also developed. Four regulatory agencies conducted a retrospective pilot study to investigate the feasibility of this framework, the benefit-risk template and user manual. Subsequently, a prospective study was conducted by TGA of Australia, Health Canada and HSA of Singapore. The agencies found the benefit-risk template was ‘fit for purpose’ in terms of the relevance of information supporting the benefit-risk decision, the documentation and communication and the relative importance and values of the benefits and risks. The results showed that the benefit-risk summary template was adequate to document benefits and risks, relevant summaries and ii conclusions for the emerging markets. The applicability and validity of the summary component of the benefit-risk template was evaluated by sixteen HSA clinical reviewers in a retrospective study. They found that the BR Summary Template was adequate to document benefits, risks, relevant summaries and conclusions. However, a revision of the BR Summary Template should include technical improvements and more details of safety information. The BR Summary Template was thought to be a useful tool for communicating benefit-risk decisions to a variety of stakeholders. The formats of publicly available reports from major regulatory agencies were compared and found to be generally similar. When compared to the BR Template, the listing of benefits and risks, assigning of weights and values, visualisation and a more detailed, systematic standardised structure were found to be absent. This research has demonstrated that the 8-step universal framework is of value for the assessment of benefits and risks of medicines by regulatory agencies and the template was found to be useful for documenting and communicating benefit-risk decisions.

Asthma is a chronic inflammatory disease of the airways characterised by early and late asthmatic responses (EAR & LAR) to allergen, airways hyperresponsiveness (AHR) to inhaled spasmogens, airway inflammation and airway oedema. Viral infections and lipopolysaccharide (LPS) from bacteria and environmental sources contribute to exacerbations of asthma and the development of insensitivity to corticosteroids. Complete insensitivity to oral corticosteroids is rare and most patients lie on a continuum of steroid responsiveness. This thesis aimed to examine the effect of viral infection and LPS in a guinea-pig model of asthma and determine the sensitivity to inhaled and systemic corticosteroids. Sensitised guinea-pigs challenged with ovalbumin displayed EAR, LAR, AHR to histamine, airways inflammation and airway oedema. Inoculation of guinea-pigs with parainfluenza-3 virus alone induced AHR to histamine and airway inflammation. However this response was not consistent. Inhaled LPS alone induced an immediate bronchoconstriction, AHR, airway inflammation and oedema and goblet cell hyperplasia. LPS co-administered with ovalbumin exacerbated the allergen response by lengthening the EAR, prolonging the bronchoconstrictor response to histamine, increasing airway inflammation and oedema and goblet cell hyperplasia. In guinea-pigs challenged with ovalbumin alone, treatment with inhaled fluticasone propionate (FP) and inhaled and systemic dexamethasone decreased the LAR, abolished AHR, airway inflammation and oedema. Responses to LPS alone were not reduced by inhaled dexamethasone or FP but partially reduced by systemic dexamethasone. Ovalbumin and LPS combined responses were insensitive to inhaled corticosteroids, except lavage fluid protein. These responses were partially sensitive to systemic dexamethasone, with the prolonged EAR, inflammation and airway oedema all reduced. The data in this thesis suggests that LPS inhalation exacerbates ovalbumin-induced functional and inflammatory responses rendering them insensitive to inhaled corticosteroids but partially sensitive to systemic corticosteroids. Thus, the experimental combination of ovalbumin with LPS might represent a useful preclinical model of corticosteroid-insensitive airway inflammation.