Overcoming the excellent barrier properties of the human skin represents the major challenge of this route of delivery. The Metered dose transdermal spray (MDTS®) is a technology developed by Acrux Ltd (AUS). This passive delivery system has the potential to avoid skin irritation. An informed choice of solvents is one of the strategies to design efficient transdermal formulations. Therefore, it is the aim of this thesis to develop optimal volatile formulations and to investigate the enhancement effects of solvents on drug permeation through the skin. A secondary objective is to evaluate the influence of solvent thermodynamic activity on drug permeation and the transport of solvent through skin on drug delivery. Miscibility, solubility, solvent uptake and residence time studies were conducted as a basis for formulation development. The effects of selected solvents on drug permeation were studied using a flow through diffusion cell design, under clinical dosing conditions of use. The influence of formulation related parameters such as solvent dose, supersaturation, combination of solvents and solvent thermodynamic activity, on the amount of ibuprofen permeated through human epidermis was further studied in vitro. The permeation of octyl salicylate, Propylene glycol and polyethylene glycol 200 was monitored by HPLC/UV, GC/MS and LC/MS, respectively, from selected volatile formulations. From these studies, it was found that ibuprofen permeation from all the residual phases studied was solvent dependent. Superior enhancement was obtained using polyethylene glycol 200 followed by propylene glycol, dipropylene glycol and transcutol®. The excipients in which ibuprofen had higher solubility showed a promotion of drug transport. Furthermore, concentration-dependent effect on ibuprofen permeation from solvents was observed. Significant improvements in the permeation of ibuprofen through human skin were achieved using combinations of solvents. The residual phase composed of polyethylene glycol: octyl salicylate (5:1 and 5:10 % (w/v)) were the best solvent/vehicle for ibuprofen permeation. The findings indicate that drug transport appears to be a function not only of the residual phase excipients, but also of the ultimate fate of the excipients after application. Finally, the reported findings demonstrate the potential of volatile formulations to optimise the efficiency of drug delivery to the skin.

Infections by encapsulated bacteria are a major cause of neonatal mortality. Escherichia coli are isolated in a significant proportion of these infections with 80-85% of isolates expressing the K1 polysaccharide capsular antigen. Systemic E. coli K1 infection is age-dependent, the basis of which is likely the capacity of the pathogen to translocate from the gastrointestinal (GI) tract into the bloodstream. This step in pathogenesis is poorly characterized. Post-partum development of the GI microbial population (microbiota) and host tissue may modulate susceptibility to E. coli K1. Two-day old (P2) neonatal rats were susceptible to infection with E. coli K1 strain A192PP, whereas P9 neonates were refractive. This variation was not caused by the capacity of the pathogen to colonize the GI tract. The P2-P9 microbiota was significantly different to that of the adult, but very little variation occurred between the neonatal groups examined. Suppression of the P9 microbiota using antibiotic treatment did not increase susceptibility E. coli K1. A substantial degree of developmental GI tissue expression was observed over P2-P9, including the up-regulation of components of the small intestinal (α-defensin peptides Defa24 and Defa-rs1) and colonic (trefoil factor peptide Tff2) mucus barrier. Colonization with E. coli K1 was dysregulated Tff2 expression in P2 tissues, likely due to IL-1β and NFκB signalling, and was accompanied by a decrease in the mucin Muc2. Alpha-defensin expression was up-regulated in P9 tissues. These results indicated that the intestinal barrier function of the P9 GI tract is more developed than the P2 equivalent. E. coli K1 colonization may compromise the development of the colonic mucus barrier in P2 neonates. This supports the hypothesis that the developmental state of the GI tissue, but not the microbiota, modulates susceptibility to systemic E. coli K1 infection.

Disruption of the interaction between the ubiquitination facilitator protein Keap1 and the transcription factor Nrf2 is a potential strategy for enhancing the expression of antioxidant and detoxification gene products that are regulated by this cap ‘n’ collar basic-region leucine zipper transcription factor. Agents that disrupt this protein-protein interaction may be useful pharmacological probes and future cancer chemopreventive agents. The activity of Nrf2 is thought to be regulated by at least two other proteins; the nuclear protein prothymosin α and the proteasome associated sequestosome-1 that compete for the Keap1 binding site. The aim of this project is to develop compounds that directly inhibit the Keap1-Nrf2 protein-protein interaction and thereby enhance Nrf2 activity by a mechanism different to that of existing chemopreventive agents. Linear and cyclic peptides based on the binding motifs of Nrf2, prothymosin α and sequestosome-1 were designed and synthesised. An optimised series of peptides was then developed with further changes to the sequence to improve the binding profile. The peptides were shown to inhibit the Keap1-Nrf2 protein-protein interaction, determined using a fluorescence polarisation assay. A small series of stearoyl capped peptides were also developed for use in cell based assays. The peptides can be ranked in order of affinity and used to determine a structure activity relationship for interaction with the Keap1 protein. Cyclic inhibitors were generally more potent than the linear peptides, however, the most potent peptide was a linear hybrid sequence based upon the Nrf2 and sequestosome-1 binding motifs, and with an IC50 of 115 nmol/L was more active than either native sequence alone and equivalent in activity to the Nrf2 Neh2 domain protein. Finally, a small library of peptidomimetics designed using the peptide template and molecular modelling studies were also synthesised, resulting in five small molecules with IC50 values in the micromolar range.

Cancer of the mammary gland is the most common type of cancer in women worldwide, and the vast majority of breast cancers originate from a cluster of malignant cells in the epithelial tissue of the breast, which initially confines the ductal carcinoma in situ. Research has shown that the signalling pathways that increase differentiation and maintain proliferation in normal epithelial cells are of utmost importance for sustaining this barrier against malignant cells. As a model for normal mammary epithelial cells, the MCF-12A cell line was used to determine factors that are required for cell cycle progression of these cells. A discontinuous treatment assay was developed in which the MCF-12A cells were treated with epidermal growth factor (EGF) and insulin at two distinct times to induce cell cycle re-entry. The use of these chemically defined growth factors enabled us to determine that continuous stimulation with mitogenic factors is not required for these cells to re-enter the cell cycle. An initial activation of the MAP kinase pathway and an up-regulation of the transcription factor c-Myc, followed by activation of the PI3K pathway, resulted in full competence to progress into S phase. The order in which the growth factors were applied, and thus the sequence in which the subsequent proteins were triggered, was of great importance for successful S phase entry. We found that estradiol (E2) was unable to induce the factors necessary for cell cycle progression. Furthermore, we report for the first time that E2 did not affect estrogen-regulated genes which normally are under the control of a ligand-bound estrogen receptor (ER). We suggest that the mechanism by which the ligand-activated ER usually interferes with the estrogen responsive element in the promoter region of the target genes is defective in the MCF-12A cell line. The results presented here may contribute to new approaches in chemotherapy, taking advantage of the diverse molecular mechanism in place for cell cycle progression and proliferation in malignant cells compared to normal mammary epithelial cells.

In the 21st century, cancer is becoming the curse of the ageing population in developed countries. No satisfactory therapies are available for many tumour types, and application of existing therapies is limited by severe side effects. Thus, there is a great need of new approaches in cancer treatment. DAB-Am16 is a dendritic polymer with a globular structure, consisting of poly(propylene imine) branches that emerge from a diaminobutane core. Its intrinsic tumoricidal activity in mouse models was published in 2005, but no information on the mechanism of action was available. This thesis presents novel findings on the pathways underlying the anti-cancer activity of this dendrimer in vitro and in vivo. Extensive chemical characterisation of DAB-Am16 confirmed its stability and purity. Severe time- and concentration-dependent cytotoxicity was observed for a panel of human tumour cell lines, while a small population of persisters was identified. Toxicity was accompanied by a delayed or abrogated cell cycle. There was an increased number of S phase cells, while the ability to synthesise DNA or to undergo mitosis was progressively lost with increasing DAB-Am16 concentration. The following cell death was found to be apoptotic and was biased in a cell cycle phase dependent manner. The order of apoptotic events upon DAB-Am16 exposure was determined. Finally, an in vivo experiment confirmed that DAB-Am16 has a pronounced effect against human pancreatic cancer xenografts in mice, while being well tolerated by the animals. Post mortem examination of tumour tissue revealed cell cycle blockage of tumour cells from DAB-Am16-treated mice. However, disposition for further proliferation was not diminished, and no significant difference in tumour vascularisation was observed.

In biological systems, many proteins have specific binding sites for small-molecules or other proteins critical for their activity and function. Discovery of small-molecules that inhibit such protein interactions is useful in understanding and controlling protein function in disease. Hypoxia inducible factor (HIF1) is a heterodimeric transcription factor and its C-terminal activation domain (CTAD) interacts with the CH1 domain of p300 forming a complex known to regulate many genes. Spectral variants of green fluorescent protein were fused to the CTAD and CH1 to monitor the interaction between these proteins. Fluorescence resonance energy transfer (FRET) between these two chromophores occurred when the complex formed. A homogeneous screening assay was then developed for small-molecules with potential to inhibit the formation of the CTAD-CH1 complex. As part of the assay validation, some new small-molecule inhibitors previously tested by an alternative heterogeneous assay were found to inhibit within the same 100-500 μM concentration range. The new homogenous assay has promising potential for high-throughput screening of large chemical libraries. Novobiocin, a member of the aminocoumarin family can act as an antibacterial or anticancer agent. The clinical use of this class of antibiotics has been limited due to their low water solubility, low activity against gram-negative bacteria, and toxicity against cancer. Glycosyltransferases have been established as important tools in new drug development and are used here to improve water solubility and cell uptake. Glycosylation can be achieved enzymatically or chemically. A mass spectrometry based high-throughput screening (HTS) method was developed and used to find novel glycosylated aminocoumarins generated using a panel of glycosyltransferases and native/non-native sugar donors. The novobiocin derivatives were also re-synthesized chemically. The MIC for novobiocin in a DNA gyrase assay was 1 μM, and the derivatives showed similar MICs. However against a panel of human cancer cell lines these derivatives showed more than twice the activity.

E.coli is one of the most widely utilised hosts for protein expression due to its rapid growth, low production costs and high product yields. Often proteins are deposited as insoluble inclusion bodies that later require refolding to achieve biological activity. As a result of misfolding and aggregation for many proteins refolding is the yield limiting step in their production. Relevant therapeutic proteins obtained from E.coli include the α-helical barrel proteins (e.g. interferon-α2). Many proteins derived from E.coli are further modified after refolding by the covalent conjugation of poly(ethylene glycol) (PEG). This is known as PEGylation and several PEGylated α-helical barrel proteins are now routinely used in the clinic. PEGylation is used to address the short circulation half-life, immunogenicity and poor stability associated with many protein-based therapeutics. Our method of PEGylation is site specific. Conjugation occurs by bis-alkylation and takes advantage of the presence of the two free thiols from native disulfide bonds that have been reduced. The conjugated product has PEG linked to the protein through a three-carbon bridge spanning the two thiols derived from the native disulfide. Currently proteins are first purified and then a PEG reagent is used to covalently conjugate PEG to the protein. The PEG-protein conjugate is then purified. This means the protein has to be purified twice which can reduce yields. PEGylating the protein during its initial refolding would avoid the need of two downstream purification processes resulting in a more efficient process with an improved product yield. Therefore the aim of this project is to integrate the process of protein folding and PEGylation to make the production of PEGylated proteins more economically viable allowing their widespread use in the clinic. In this project the following hypotheses will be tested i) Reducing the number of purification steps that need to be performed to improve the overall yield of recovered protein, ii) The ability of PEG to impart the properties of a glycosyl group or a chaperone and protect the protein against aggregation during the folding process, iii) our method of PEGylation in particular should promote the formation of the protein’s disulfides bond and therefore the protein’s thermodynamically stable native state and iv) that the specificity of the conjugation can still be maintained despite the exposure of more sites of conjugation. Here we examine this process with different model alpha helical barrel proteins namely leptin, IFN-β and EPO. In each case the protein was denatured and fully reduced then refolded in the presence of a thiol specific, bis-alkylation PEG reagent allowing us to effectively capture the cysteine thiols during the refolding process. For IFN-β which is highly prone to aggregation, refolding yields in the presence of the PEG reagent were much improved suggesting that our method of PEGylation had a stabilising effect on the protein structure during refolding. This improved stability was also found to benefit the protein after PEGylation. Isolation of the purified PEGylated IFN- conjugate could be achieved in a single purification step in good yield. A similar activity to that generated on PEGylation of the fully folded protein was observed suggesting that for a protein with an accessible disulfide PEGylation did not significantly affect its folding. Some work was also carried out on RNase A and T1 which contain multiple inaccessible disulfides. In this instance PEGylation appeared to hinder the refolding process either by sterically hindering the formation of the protein’s native structure or by incorrect disulfide bond formation. The work described herein therefore suggests that it is possible to refold and PEGylate proteins within a single step but the effectiveness of this approach is likely to be protein dependant.

Polarity is dependent on the asymmetric distribution of proteins, which can be especially challenging in neurons as the nucleus, where transcription takes place, can be far away from the final location of the protein. One essential mechanism by which protein compartmentalization can be achieved is through the targeting of specific mRNA transcripts to specific cellular regions where they are locally translated. Neurotrophin 3 (NT3) is secreted by blood vessels and is required for the initial growth of proximal sympathetic neuron axons along the vasculature. NGF on the other hand, is secreted by the sympathetic target tissue and is required for sympathetic neuron survival and the final innervation of target organs. Although both neurotrophins act through the common receptor TrkA, NGF supports TrkA internalisation and retrograde signalling from distal axons to cell bodies to regulate gene expression, whereas NT3 can only induce a local signalling response in axons and is unable to support retrograde TrkA signalling. I performed a Digital Gene Expression Tag Profiling of axonal and cell body mRNA obtained from rat superior cervical ganglia cells maintained with NT3 with the aim of identifying mRNAs localized in subcellular compartments and to understand the control of mRNA stability and local protein translation in axons. I found that several hundred transcripts are significantly enriched in axons of sympathetic neurons when compared to cell bodies. Rapid Amplification of cDNA Ends (RACE) analysis of untranslated regions (UTRs) of candidate genes showed that at least some of the transcripts enriched in axons possess different UTRs. The most abundant transcript in axons was SCG10 (or Stmn2), a regulator of microtubule dynamics. Furthermore, I identified a member of the Hu protein family as an RNA binding protein that interacts with the UTR of SCG10 and will discuss its potential role in neurotrophin-dependent regulation of SCG10 mRNA stability and translation.

Nitric oxide (NO) is a highly reactive, ubiquitous respiratory tract product with numerous biological activities. After examining the methodology of the measurement of NO in the exhaled breath, we found that chronic cigarette smoking (n=74, 40 female) lowered exhaled NO, while acute smoking (n=24, 13 female) increased exhaled NO. Since these effects may have related to the chemical reactivity of NO, we studied the effect of oxidant (nitrogen dioxide), antioxidant (ascorbic acid), and D-arginine exposure on exhaled NO. While nitrogen dioxide exposure (n=10, 7 female, 1.5 ppm for 20 minutes) lowered exhaled NO between 1 and 3 hours after exposure, ascorbic acid exposure (n=20, 14 female, 1g daily for 2 weeks) had no effect. D-arginine exposure (n=8, 7 female, 2.5g) increased exhaled NO, suggesting that NO may be formed non-enzymatically from arginine. Finally, the nasal exchange dynamics of NO were studied. NO release was a positive function of luminal gas flow due to a change in mucosal equilibrium. A mathematical model allowed the determination of the nasal mucosal NO concentration and diffusing capacity. In summary, the chemical reactivity of NO may affect biological activity. The human nose provides a convenient source of NO producing mucosa for further in vivo study.

In recent years, the chemical and pharmaceutical industries have faced increased development times and costs with fewer novel chemicals being discovered. This has resulted in many companies focusing on innovative research and development as they consider this key to business success. In particular, a number of leading industrial organisations have adopted the principles of Whole Process Design (WPD). WPD considers the optimisation of the entire product development process, from raw materials to end product, rather than focusing on each individual unit operation. The complexity involved in the implementation of WPD requires rationalised decision-making, often with limited or uncertain information. This thesis assesses the most widely applied methods in Multi-Criteria Decision Analysis (MCDA) in conjunction with the results of two interviews and two questionnaires that identified the industrial requirements for decision-making during WPD. From the findings of this work, a novel decision-making methodology was proposed, the outcome of which allows a decision-maker to visually interpret their decision results with associated levels of uncertainty. To validate the proposed methodology, a software framework was developed that incorporates two other decision-making approaches, the Analytical Hierarchy Process (AHP) and ELimination Et Choix Traduisant la REalité trois (ELECTRE III). The framework was then applied to a number of industrial case studies to validate the application of the proposed methodology.