Breast cancer is the most common female malignancy in the western world. Currently, tamoxifen remains the gold standard anti-hormone for ER+ breast cancer. However de novo resistance to tamoxifen is a huge clinical problem. What is more, as many as 50% of initial responders develop acquired resistance to tamoxifen and relapse. Aberrant growth factor signalling has been linked to the resistant phenotype, in particular EGFR and IGF-IR signalling and often gives a more aggressive disease type and poorer patient outlook. There is a substantial clinical need for new anti-breast cancer therapeutics that target the resistant phenotype or prevent this from occurring. One hypothesis is to combine both anti-hormonal therapies with anti-EGFR therapies, in hope of preventing resistant growth. Many downstream pathways of EGFR have been targeted to develop novel therapeutics against however, these compounds are almost certain to give severe adverse reactions, furthermore a complex tablet regime or IV injections have a negative effect on patient compliance. A novel transcutaneous delivery system would give a more patient friendly alternative, with patients re-applying a patch or cream once a day, with rapid termination of dosing in the event of adverse event This would also go some way to prevent endometrial complications associated with tamoxifen use. The in vitro simultaneous delivery of 4-hydroxytamoxifen and signal transduction inhibitors LY294002 and PD98059 along with EPA determined that all compounds were capable of permeating porcine skin and nipple. Masses able to permeate were 5.14 0.93,6.97 0.99, 5.06 0.93 and 1121.6 143 &mu;gcm2 across skin respectively and 21.14 2.75,18.2 2.55,25.1 6.89 and 3081.2 252.7 &mu;gcm2 across porcine nipple. These compounds were formulated with 2.5% v/v DMSO and ethanol and 4% w/v Cab-o-sil. Fish oil was shown to be a skin friendly vehicle. Ki-67 assays confirmed that incubation with fish oil maintained skin viability and H &amp; E staining confirmed no obvious histological effects. Growth Studies were performed on MCF-7 and TamR cells and showed that the combination of these compounds was able to reduce cell growth to 4.04 0.15 and 2.47 1.4 % of control growth when incubated at 25 &mu;M PD98059 &mu;M LY294002 x 10<super>7</super>M 4-hydroytamoxifen and 1 &mu;LmL<super>-1</super> fish oil. This combination of compounds, without 4-hydroxytamoxifen, was able to reduce the migratory capacity of TamR cells (P = < 0.005). Results confirmed that the simultaneous transcutaneous delivery of multiple anti-breast cancer agents is possible and that these show promising anti- tumorogenic actions post skin.

In this study, the topical delivery and anti-psoriatic properies of the major anti-inflammatory constituent of fish (eicosapentaenoic acid, EPA) were studied, in combination with other anti-psoriatic agents with a view to the development of a new therapeutic regimen. In in vitro models of skin permeation EPA was delivered successfully and presence of fish oil also enhanced the delivery of betamethasone dipropionate (BD) to the lower basal layers of the epidermis. The same enhancement was not seen with salicylic acid and aspirin. Investigations on the anti-inflammatory action properties of fish oil were also successfully carried out. Fish oil alone inhibited the expression of key inflammatory enzymes in the skin, cyclooxygenase-2 (COX-2) and 5-lipooxygenase (5-LOX) along with a major eicosanoid, prostaglandin E2, comparable to that of BD. In HaCaT keratinocyte cell culture, the anti-psoriatic properties of fish oil were further demonstrated by both growth inhibitory effects and the induction of pro- apoptotic markers. The final part of the study investigated in vivo a potential new model of psoriasis: the defolliculated mouse. The model responded well to formulations containing BD, denoted by a reduction in the epidermal thickness associated with the mutation. This suggests a role in screening of new therapeutic compound. Treatment with fish oil, however, caused a thickening of the epidermis, contradictory to the initial hypothesis. This was further confirmed when expression of growth markers Ki67 and K17 were found to be increased. Concurrently, Optical Coherence Tomography was utilized successfully in the in vivo studies, providing a rapid, non-invasive technique of live measurement of skin modulation without the need of sacrificing the animals for individual observations. In summary, despite unexpected outcome with the in vivo studies, the study provided ample evidence to support the incorporation of fish oil in the treatment of inflammatory skin diseases such as psoriasis.

Chronic ulceration of the lower limb represents a significant clinical challenge in today's aging society. Depletion of growth factors at the chronic wound site by proteinases and reactive oxygen species, can lead to a protracted non-healing wound. Growth factors are known to act in concert to promote wound repair, but their topical application rarely leads to a significant clinical improvement in chronic wounds, due to premature inactivation in the wound environment. The aim of this Study was to synthesise and characterise a polymer-growth factor conjugate and to investigate whether the novel concept, called Polymer-masking-UnMasking-Protein Therapy (PUMPT), may be used to generate bioresponsive, polymer therapeutics, promoting tissue repair. Dextrin and recombinant human epidermal growth factor (rhEGF) were chosen as a first model combination. Dextrin was first modified by succinoylation and conjugated to rhEGF. The synthesised dextrin-rhEGF conjugate exhibited increased stability towards proteolytic degradation by the clinically relevant enzyme, neutrophil elastase. The dextrin component was degraded on exposure to physiological levels of a-amylase, leading to sustained release of free rhEGF. Biological activity was assessed in proliferation assays in vitro, using cells involved in the normal healing response. Polymer conjugation reduced rhEGF bioactivity, however, after exposure to physiological concentrations of a-amylase, dextrin degradation led to restoration of bioactivity of rhEGF, to the level observed with unmodified rhEGF. Dextrin-rhEGF was demonstrated to induce phosphorylation of the epidermal growth factor receptor (EGFR), and confirmed a mechanism of action by stimulation of classical signal transduction pathways. Analysis of ex vivo acute and chronic wound fluids, confirmed the presence of a-amylase, at levels similar to those used in vitro. Chronic wound fluid also contained active neutrophil elastase. In an ex vivo organotypic model of acute corneal wound healing, a-amylase-exposed dextrin-rhEGF was demonstrated to enhance wound healing, above that of free rhEGF. In an in vivo model of impaired wound healing in the genetically diabetic (db/db) mouse, dextrin-rhEGF was observed to significantly enhance dermal wound healing, on topical application, at delayed intervals. This is the first application of the PUMPT hypothesis in vivo, and supports the further development of polymer-protein conjugates as bioresponsive nanomedicines for tissue repair.

Methods have been successfully developed and evaluated to aerosolise surfactant-coated pDNA particles and maintain pDNA viability. Furthermore, investigations have demonstrated that the pDNA particles and a transfection agent (DOTAP) can be incorporated into a generic HFA134a formulation and successfully aerosolised using a standard valve and actuator. Further studies demonstrated the performance of these formulations after four week accelerated stability conditions.

The skin presents an attractive target for the delivery and expression of plasmid DNA pDNA. Potential therapeutic benefits from cutaneous gene therapy approaches include the correction or alleviation of inherited skin disorders genodermatoses and genetic vaccination. The skin is a particularly suitable portal for genetic vaccination due to its innate immunogenic capabilities. However, delivery of pDNA to the epidermis is severely constrained by the stratum corneum SC, low transfection efficiency and rapid loss of pDNA associated with epidermal cells. Microfabricated microneedles are employed as a means of penetrating the SC for macromolecular delivery. Solid silicon microneedles with different heights and tip morphologies were made by careful manipulation of the etching process, along with hollow silicon microneedles and solid polymer microneedles. To address the low transfection efficiency and rapid loss of pDNA in skin, hydrogels formed from smart polymers were investigated to provide sustained release reservoirs of pDNA. Gene delivery studies were performed in freshly maintained ex vivo human skin delivery formulations of reporter plasmid pCMVp and pEGFP-Nl and a therapeutic plasmid pCMV.M were applied to skin prior to microneedle application and maintenance in an optimized organ culture system. The results indicate that it is possible to deliver and express genes in the epidermis using microneedles. However, morphology of microneedles, their application protocol, and pDNA formulation all contribute to the efficiency of trans-gene expression.

There is a substantial clinical need for improved therapeutic systems for the treatment of arthritis. This thesis concerns the development of a novel medication that is applied to the skin directly overlying the areas affected. The system comprises the co-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), plus a non-steroidal anti-inflammatory (NSAID) drug. The source of EPA and DHA is fish oil which contains high proportions of these compounds and, although still largely considered a 'neutraceutical', its therapeutic value is supported by an ever increasing body of evidence, both scientific and anecdotal. The NSAID examined was ketoprofen, a widely used drug, found in a number of topical preparations. Synergistic action involving the two substances would be expected to provide a multi-faceted attack on the aetiology of arthritis. Ketoprofen and EPA/DHA were successfully delivered across full-thickness porcine ear skin in-vitro, although the presence of thickening agent retarded permeation of the latter. The successful delivery of these compounds into the joint capsule of a porcine forelimb was also demonstrated in-vitro. A novel transcutaneous delivery model was developed and used to provide preliminary data for the uptake of EPA into an ex-vivo cartilage ex-plant post transcutaneous permeation. The last three chapters can be considered collectively as an investigation into the unexpected phenomenon of enhancement of EPA/DHA by ketoprofen and two main hypotheses were tested firstly, the formation of a Jt-jc ketoprofen / EPA complex - the existence of which was strongly supported by the NMR/molecular modelling work of Chapter 8 secondly, the ketoprofen inhibition of epidermal enzymes active upon EPA, discussed in Chapters 7 and 9. In summary, the development of a novel dual-action, transcutaneous anti-arthritic formulation is possible and has been supported by this work. Furthermore, a hitherto unknown topical delivery mechanism has been elucidated.

The studies described in this thesis are concerned with understanding and rationalising the inter-relationships between structural and other physical properties of organic solids encompassing hydrates, polymorphs and homologous series. This is achieved through assessment of the crystal packing arrangements and the nature of the intermolecular interactions, which may have a profound impact on the stability and physical properties of organic materials. Such an understanding is critically important when selecting or designing materials that demonstrate specific defined properties. The first part of the thesis describes the relationships between hydrate and anhydrate phases, exemplified by tetraphenyl phosphonium bromide as a model system. The relative inter-relationships between the two main phases of this material are rationalised by characterisation of both structural and dynamic properties. The second aspect is focused on the influence of short-range interactions on the long-range periodicity and subsequent properties of materials using 4-hydroxy benzoic acid esters as a homologous series to explore odd-even alternations, seen for other series containing long chain functionalities. In this study, the nature of short-range interactions is found to correlate with the observed odd-even alternation in material properties, with deviations from these observations shown to correspond to the presence of significant disorder in the structures. The third aspect concerns the properties of a polymorphic pharmaceutical material, AZD7140. The crystal structures for both polymorphs were solved and assessment of hydrogen bonding motifs and thermodynamic evaluation using phase diagrams allowed the selection of a robust polymorph as a suitable development material. The final aspect of the thesis concerns strategies that may be employed to solve structures of disordered systems directly from XRPD data using direct space methodology.

The behaviour of pharmaceutical excipients was studied in 2H, 3H-perfluoropentane (HPFP), a partially fluorinated liquid considered as a highly suitable model propellant for medicinal pressurized metered-dose inhalers (pMDIs) used in drug delivery to the lungs. The additives chosen belonged to three main categories: ethylene oxide oligomers and derivatives, chosen for their wide range of applications in pharmaceutical formulations, their harmlessness and low cost a hydrophobically modified cyclodextrin that shows potential for controlled release via host-guest interactions and some interesting solubility pattern in HPFP a family of tetrahydroxy diesters that act as unprecedented organogelators for mixtures of HPFP and a partially fluorinated alcohol co-solvent. By studying ethylene oxide oligomers and derivatives in HPFP and fully fluorinated analogue PFP, it was found that the solubility of the oligomers could be tuned by varying the nature of their end-group and the polarity of the fluorinated liquids employed. Methyl groups, either placed at the extremities, thus blocking the hydroxyl end-groups of dihydroxyl end-capped polyethylene glycols, or directly added on the polymer backbone, clearly enhanced their solubility. The Lower Critical Solution Temperature initially observed was either displaced or removed via the presence of the additional methyl groups. On the other hand, such phase separation could also be induced by the subsequent addition of PFP it is shown that such behaviours are dominated by end-group/solvent interactions. This study was published in two papers, Journal of Pharmacy and Pharmacology, 2008, 60: p. 593-599 and International Journal of Pharmaceutics, 2008, 362: p. 147-152. As an attractive formulation means, the hydrophobic triacetylated-P-cyclodextrin (TAjJCD) was chosen amongst others for its partial solubility in model propellant HPFP. The observation that various batches of the same chemical entity lead to highly different solubility profiles in HPFP resulted in the full characterisation of two existing polymorphs for this compound. The monitored conversion of one form into the other was achieved, and the amorphous form could be fully recrystallysed into the higher energy form via a limited seeding process. HPFP acted as a discriminator of the two crystalline forms. More details may be found in Journal of Physical Chemistry C, 2008,112: p. 14570-15578. The monitoring of the solubility via solvent properties, as seen in the first part, was also used in the formation of self-assembled supramolecular organogels. The addition of non-solvent HPFP to an initially stable sol composed of a small tetrahydroxy diesters in the fluoroalcohol 1H, 1H-heptafluorobutanol initiated their coming out of solution. However, instead of simply precipitating, the tetrahydroxy diester molecules self-assemble into long cylindrical fibres that form a 3-dimensional network capable of entrapping the fluorinated liquids, leading to a system that is both solid-like macroscopically and liquid-like microscopically. Such gels are thermoreversible and can be obtained at gelator concentrations as low as 0.1 wt%, thus, surface tension and capillary action are thought to be the principal forces behind these structures, along with hydrogen-bonding between the gelators molecules, rather than a specific gelator-solvent interaction. This work is the reference for both Chemical Communications, 2005,31: p. 3998-4000 and a manuscript to be submitted.

Preliminary flow cytometry experiments studied the ability of the peptides, CLNGGTC (u7) and GVSNKYFSNIHWG (Gu11G), derived from the binding region of uPA to inhibit uPA-FITC binding to U937 cells. The optimised trimethylated chitosan oligomer (TMO) was functionalised with chloroacetic acid to give 6-O-carboxymethyl N,N,N-trimethyl chitosan (CMTMO). Two peptides derived from the binding region of uPA, u7 and Gu11G were conjugated to the 6-O-carboxymethyl functionality using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide and hydroxy-2,5-dioxopyrrolidine-3-sulfonicated. Fluorescent derivatives of u7-CMTMO and Gu11G-CMTMO were then produced by reaction with the succinimidyl ester of 5-(and-6)-carboxyfluorescein and Oregon Green RTM 488-X, succinimidyl ester. The cell-association of the fluorescent u7-CMTMO and Gu11G-CMTMO was assessed in U937 cells (a histocytic lymphoma known to over-express uPAR) and DU145 cells (a human prostate carcinoma) over time at 37&deg;C and 4&deg;C. The development of an improved gene delivery system using Gu11G-CMTMO was then investigated in COS-7, DU145 and MCF-7 cells. It was shown that chitosan could be trimethylated in a controllable and repeatable manner by increasing reaction time. Increasing degree of trimethylation decreased cell viability, an effect more pronounced in polymeric (TMC) over oligomeric (TMO) derivatives. The Gu11G-CMTMO-FAM showed an increase in cell-associated fluorescence (>5 fold) compared to CMTMO-FAM on U937 cells. u7-CMTMO-FAM showed decreased cell association compared to CMTMO. When Gu11G-CMTMO was mixed with TMO/pDNA or PEI/pDNA in the transfection mixture the transfection efficiency, as measured by the luciferase expression, was equal to that of the transfection achieved with uncoated polyplexes. Although Gu11G was shown to bind uPAR selectively, it was not possible to translate into an improved conjugate.

Polymer therapeutics include water-soluble polymers designed as carriers for drugs, proteins or DNA. Over the last two decades, they have found increasing clinical use against cancer and other diseases. A growing number is in clinical trials or on the market. However, the polymers used so far have limitations including heterogeneity in structure, molecular weight, polydispersity, drug carrying capacity, lack of control of architecture and biodegradability of polymer backbone. Therefore, the aim of this study was to synthesise and characterise a library of linear/star homo/copolymers with potential for further development as second-generation polymer therapeutics. Atom Transfer Radical Polymerisation (ATRP) and (chain transfer agent - CTA) Free Radical Polymerisation (FRP) techniques were used to synthesise water-soluble amine-based acrylamide and methacrylate homo/copolymers. Nuclear magnetic resonance, gel permeation chromatography, infrared and titration were used for characterisation. In vitro cytotoxicity studies of the polymers towards a murine melanoma cell line were performed using a cell viability evaluating colorimetric assay. Molecular weights (from 3,000 to 550,000 g.mol --1) were successfully adjusted by varying either the initiator or CTA to monomer ratios. Semitelechelic homo/copolymers with either carboxylic acid or hydroxyl termini were obtained using mercapto-based CTA. Either stable or degradable star-shaped poly(dimethylaminoethyl methacrylate) were obtained by copper-mediated ATRP using previously synthesised multifunctional initiators (4, 5 or 8 initiating moieties). A linear increase of predictable molecular weight with monomer conversion and narrow polydispersity (1.3) were observed. Amongst other molecular parameters systematically tested, the amount of cationic residues had the most striking effect on the cell viability. To conclude, conditions were optimised for the synthesis of a library of water- soluble amine-based homo/copolymers with different molecular weight, composition, charge density and architecture using several polymerisation techniques. Preliminary evaluation of polymer cytotoxicity associated to molecular parameters is vital for intelligently designing future, novel, and biocompatible polymeric carriers.