Polymer conjugation is a strategy able to enhance the tumour selectivity of anticancer drugs and to prolong their half life. Seventeen polymer-drug conjugates containing traditional chemotherapy (e.g. paclitaxel) have entered clinical trials and novel conjugates containing experimental drugs (e.g. the anti-angiogenic agent TNP-470) are emerging. Dopamine (DA) is an endogenous regulator of angiogenesis (the process of new blood vessels formation) and is able to retard the development of angiogenesis-dependent tumours when administered at a low non-toxic dose. However, the clinical application of DA in cancer treatment is hindered by its short half life (2 min) and severe side effects. DA is also a natural substrate of tyrosinase, a physiological enzyme present in the skin and over-expressed in malignant melanoma. Derivatives of DA have been designed as a new class of melanoma-specific anticancer prodrugs but their development is still limited to the preclinical stage. This study focuses on the development of novel polymer conjugates of DA and DA derivatives with the aim of enhancing their therapeutic potential as anti- angiogenic drugs and melanoma-specific agents, respectively. Polyglutamic acid (PGA) was selected as the polymeric carrier and the conjugation of DA was optimised using a carbodiimide and N-hydroxysuccinimide mediated coupling. The synthesis of the PGA-DA conjugate was confirmed by IH-NMR and 13C_NMR spectroscopic analysis. The total DA content was quantified by IH-NMR spectroscopic analysis and found to be in the range 10.8-14.9% w/w. The free DA content was determined by HPLC analysis and always found to be below 1 % of the total DA. PGA-DA as an anti-angiogenic drug. DA exerts its anti-angiogenic effect by binding to its D2 receptor and inhibiting the pro-angiogenic action of the vascular endothelial growth factor (VEGF). Thus, the anti-angiogenic activity of PGA-DA was assessed in two VEGF- dependent systems: an in vitro scratch assay, concerning the migration of human umbilical vein endothelial cells (HUVEC) and an in vivo Miles assay, evaluating vascular permeability. After a short incubation (1 h), both DA and PGA-DA reduced HUVEC migration to a similar extent (-50% VEGF inhibition). In contrast, after a longer incubation time (24 h), DA proved inactive while PGA-DA was even more effective (complete inhibition of VEGF). Similarly, a single injection of PGA-DA reduced the VEGF-induced vascular permeability for longer timeframes (24 h, 60% reduced dye extravasation) than DA (inactive at 24 h). Binding and degradation studies were performed to investigate the mechanism of action of PGA-DA. The inability of PGA-DA to bind to the D2 receptor and its degradation in the presence of cathepsin B suggested that the anti- angiogenic activity of the conjugate was mediated by the release of DA. PGA-(DA derivative) as melanoma-specific conjugates. The ability of DA to act as a substrate of tyrosinase was exploited in the development of an enzymatically activated conjugate. DA was chemically modified to act as a linker between the polymer and the drug. A model drug (p-nitroaniline (pNA)) or a cytotoxic drug (nitrogen-mustard) were coupled to DA. The ability of these DA derivatives to undergo enzymatic activation was assessed by a UV -Vis assay and oximetry. There were two main findings: (i) the linker- mustard was a substrate of tyrosinase; (ii) the linker-pNA was not recognised by tyrosinase as a substrate. Computational studies were used to probe this difference and showed that pNA prevented the activation of the DA derivative by altering its conformation in the active site of tyrosinase. An attempt to conjugate PGA to a DA derivative was also carried out using the method optimised for DA. Further characterisation is required to confirm the identity of the product. To conclude, the relevance of this work is two-fold: (i) polymer conjugation extended the anti-angiogenic activity of DA, making its translation to the clinic a plausible target; (ii) the development of the first melanoma-specific polymer-drug conjugate was undertaken.

Research in the field of titanium-based complexes as potential anticancer drugs has led to impressive results in vitro, but there is still scope for further improvements. Significant effort has been put into synthetic research and biological evaluation of titanocene derivatives, as well as the identification of their main biological targets. Surprisingly, the area of computer-aided drug design (CADD) has not been utilized up-to-date for the design of novel titanium-based compounds. The work within the thesis describes the computational approaches employed to study the mode of action of titanium-based anticancer agents, highlighting the structural features required for biological activity. Consequently, novel titanium-based derivatives were designed and synthesized. Furthermore, the synthetic attempts for amidosilyl-substituted titanocene and ferrocene derivatives with potentially enhanced activity are also reported. The first chapter provides a thorough introduction into the developments in the field of metal-based antitumour agents with a particular emphasis on titanium-based agents and assessment of their cytotoxic activity. Chapter 2 describes the first systematic receptor- based docking approach utilized to understand the binding mode of titanium-based agents against human serum albumin (RSA). The first 3D-QSAR study, which is reported in Chapter 3, was performed on a series of titanocene complexes to gain insight into the key structural features vital for their biological activity and to advance the design of potent titanocene anticancer agents. The successful synthesis of six novel benzyl-substituted titanocene derivatives, which have been designed based on the 3D-QSAR analysis is described in Chapter 4. Chapter 5 focuses on the attempts made for the synthesis of a range of amidosilyl-substituted titanium and iron-based metallocenes. Although the amidosilyl- substituted derivatives could not be obtained as pure compounds, six titanium and iron- based metallocenes were successfully synthesized. Chapter 6 summarizes the results obtained and provides suggestions for areas of future research.

Improving the water solubility of hydrophobic drugs still remains one of the foremost important issues in the formulation of a drug. This thesis describes a novel complexation method based on biodegradable poly(y- glutamic acid) (PGA) for the effective solubilisation and delivery of the hydrophobic antifungal drug amphotericin B. Poly(y-glutamic acid) is a biosynthetic polymer derived from Bacillus anthracis and is known to be biocompatible and non-toxic towards humans and the environment; the sodium salt of which is highly water soluble. The preparation of an amphotericin B complex comprises of three steps: (i) initially the molecular weight reduction of PGA from 1500 kDa to 30-110 kDa by alkaline hydrolysis followed by (ii) hydrophobisation of PGA by activation with N-hydroxysuccinimide and then (iii) the complexation of amphotericin B with PGA to produce a highly water soluble PGA/amphotericin B complex. The complex shows attenuated toxicity compared with traditional amphotericin B deoxycholate (Fungizone®) and comparable to the liposomal amphotericin B formulation (Ambisome®). The complex retains its broad-spectrum antifungal activity against Candida sp. In comparison to amphotericin B deoxycholate, the AmB complexes show a marked reduction in the pro-inflammatory response which is thought to be responsible for infusion-related adverse effects. The complex shows enhanced uptake by macrophage which may further contribute towards the lower toxicity of the complex. In vivo studies demonstrate that AmB complexes to be more effective compared with Fungizone® with the complex constructed from the high molecular weight PGA the most efficacious. Analyses by DSC and XRPD show that the complex is amorphous in nature although it proves to be stable for more than 6 months at 4°C and more than two months at 37°C. Finally, water solubility of chemotherapeutic agents such as SN-38, etoposide and resveratrol could also be improved using the same concept.

The enzyme α-methylacyl coenzyme A racemase (AMACR) is overexpressed in a variety of cancers, including prostate cancer. siRNA knockdown of the AMACR gene has identified it as being a potential therapeutic target. The racemase is involved in B-oxidation of branched-chain fatty acids and in bile acid biosynthesis of cholesterol derivatives, in both cases providing the required (S)-stereoisomer for the branched-chain acyl-CoA oxidase. This is achieved by equilibration of the naturally produced (R) and (S) stereoisomers of pristanoyl-CoA and (R)-THC-CoA. We have synthesised a range of potential inhibitors of the enzyme that are based on the natural substrates of the enzyme. These designed inhibitors incorporate a range of functional groups that aim to probe the mechanism of action of the enzyme, as well as the ability of AMACR to tolerate alterations to the a-methyl moiety. We have also synthesised a number of truncated analogues of CoA in order to discover how well such modifications of this moiety are accepted by the enzyme. In addition to this, modelling studies on the enzyme have revealed the existence of an alternative binding pocket close to the active site. A range of compounds designed to exploit this novel pocket have also been synthesised, in order to provide inhibitors that are specific for AMACR. A number of these compounds have been assayed against the racemase from Mycobacterium tuberculosis (MCR). These compounds include the first designed inhibitor of AMACR that does not include a CoA moiety. The initial results obtained suggest that these substrates act as reversible inhibitors of the enzyme.

Glioblastoma (GBM) is the most common primary brain tumour in adults and among the most aggressive of all tumours. For several decades, the standard care of GBM was surgical resection followed by radiotherapy alone. In 2005, a landmark phase III clinical trial coordinated by the European Organization for Research and Treatment of Cancer (EORTC) and the National Cancer Institute of Canada (NCIC) demonstrated the benefit of radiotherapy with concomitant and adjuvant temozolomide (TMZ) chemotherapy. With TMZ, the median life expectancy in optimally managed patients is still only 12-14 months, with only 25% surviving 24 months. There is an urgent need for new therapies in particular in those patients whose tumour has an unmethylated methyl- guanine methyltransferase gene (MGMT) promoter, which is a predictive factor of benefit from TMZ. In this dissertation, the nature of the interaction between TMZ and radiation is in- vestigated using both a mathematical model, based on in vivo population statistics of survival, and in vitro experimentation on a panel of human GBM cell lines. The results show that TMZ has an additive effect in vitro and that the population-based model may be insufficient in predicting TMZ response. The combination of TMZ with particle therapy is also investigated. Very little pre- clinical data exists on the effects of charged particles on GBM cell lines as well as on the concomitant application of chemotherapy. In this study, human GBM cells are exposed to 3 MeV protons and 6 MeV alpha particles in concomitance with TMZ. The results suggest that the radiation quality does not affect the nature of the interaction between TMZ and radiation, showing reproducible additive cytotoxicity. Since TMZ and radiation cause DNA damage in cancer cells, there has been in- creased attention to the use of poly(ADP-ribose) polymerase (PARP) inhibitors. PARP is a family of enzymes that play a key role in the repair of DNA breaks. In this study, a novel PARP inhibitor, ABT-888, is used in combination with both TMZ and radiation. The results show that ABT-888 significantly enhances TMZ and radiation cell killing, regardless of the MGMT status. In summary, the findings of this research demonstrate that the use of particle ther- apy and PARP inhibitors are particularly promising and might improve the treatment outcome in patients with GBM.

Bioactive compounds from plant origin have the potential to subside the biochemical imbalances induced by various toxins associated with mutagenesis and carcinogensis. Therefore new strategy are interested in finding a potent phytotherapeutic agent with non toxic properties. The goal of the present study to evaluate the potential of Doash- leaves water extracts Origanum majorana (2% w/v) as antimutagenic and anticarcinogenic effects against known• .. .mutagens classes (heterocyclic amines, polycyclic aromatic hydrocarbons, nitrosoamines and direct acting carcinogens) in vivo and in vitro. The antimutagenic activity was determined by Ames test using different strains of Salmonella Typhimurium. Results obtained showed that, Doash extract possesses powerful antimutagenic against different mutagene. In vivo study revealed that treatment of rats with Doash extracts for one day or 30 days caused a decrease in hepatic cytochrome P450 enzymes such as CYP2B, CYP2El and CYPIA, the latter P450 subfamily being closely associated with the inhibition of chemical carcinogens inactivation. Aqueous Doash tea extract diminishes the excretion of indirect acting mutagens in rats treated with ( 2-Amino-3-methylimidiazol-[4,50-f]quinoiine) IQ or (2- hydroxyamino-3-methyl-3H-imidazo[ 4,5,f] quinoline )PhIP; the antimutagenic effect associated with elevation of hepatic CYPIA2 expression which is involved in the metabolism of carcinogens (IQ and PhIP). HPLC/MS analysis of Doash extract showed that a highest peak related to high terpens contents. Based on these records, it appears that Doash tea extract can in vivo affect the mutagenicity of various structurally diverse promutagens including many food-borne carcinogen, by decreasing cytochrome P450-mediated activation. It was concluded that, daily intake of Doash tea may protect against the conversion of promutagene to mutagene and scavenging carcinogen from environmental contact. More study is needed to examine the mechanistic action of active ingredients of Doash extract fractions on cellular and molecular level.

Administration of erucin (3 and 15 mg/kg) and sulforaphane (15 mg/kg) to rats for 10 days enhanced hepatic and pulmonary quinone reductase activity; sulforaphane also stimulated hepatic glutathione S-transferase, when monitored using 4-chloro-7-nitrobenzofurazan. Immunologically, both compounds increased the expression of hepatic and pulmonary glutathione S-transferases class a and u, but not n, proteins.

The basic premise of cancer immunotherapy involves targeting the hosts' immune response against tumour cells. Immunoregulatory proteins known as cytokines and chemokines represent a group of compounds that fulfils the prerequisites for potential immunotherapeutic agents.

FLIP is a well-established regulator of the extrinsic apoptotic pathway which is mediated through death receptors. Following death receptor ligation a protein complex called the DISC is formed and this is where procaspase 8 is gets activated by homodimerisation and autoprocessing. By binding to the DISC and dimerizing with procaspase 8, FLIP can prevent caspase 8 processing and thus inhibit the extrinsic apoptotic signalling cascade. .. " FLIP overexpression is a mechanism by which cancer cells survive apoptotic pressure therefore FLIP is an attractive target for anti-cancer therapies. However, strategies for targeting FLIP are challenging due to its lack of enzymatic activity and a crystal structure. Based on the structure of viral FLIP, a homology model of FLIP was built. Using a combination of molecular modelling, mutagenesis and functional assays, we determined the preferential sites and mode of interaction for FLIP-FADD. Moreover an homology model of procaspase 8 was also developed, again based on the structure of MC159 vFLlP. Using similar molecular modelling, mutagenesis and functional assays, we determined the mode of interaction of all three key apoptotic players at the DISC and a novel model of DISC assembly was developed. In addition, small molecule inhibitors of FLIP were developed. Having identified the critical region of the FLIP-FADD interaction, virtual screening was performed to identify candidate small molecules. The panel of candidate compounds was tested in a high throughput assay and this led to identification and prioritisation of a leading hit series. The selected compounds were further tested in a panel both cell-free and cell-based assays for FLIP-FADD inhibition and apoptosis induction. In summary, these studies provide insight into the modes of interaction between FLIP, FADD and procaspase 8 at the DISC and identify small molecule starting points for the development of inhibitors for therapeutically targeting FLIP for the treatment of cancer.

Macrophages (M0s) which can have a profound impact on tumour growth and are broadly classified into two subsets: 1) Inflammatory, tumoricidal "classically activated" Ml M0s or anti-inflammatory, pro-tumour "alternatively activated" M2 M0s. High M2 M0 density within tumours is associated with poor prognosis in patients. sacs proteins attenuate cytokine signalling via the JAKJSTA T pathway, and therefore regulate inflammatory responses. Our findings indicate that deletion of Socs2 and Socs3 genes in mice alter M0 subset homeostasis and polarisation towards Ml or M2 subsets, respectively. Opposing M0 subsets observed in these mice resulted in divergent tumour growth rates in both in a chemically inducible model of squamous cell carcinoma (SCC) and a syngeneic murine colorectal adenocarcinoma model Adoptive transfer FACS sorted bone SOCS2/3-null monocytes into WT tumour bearing mice resulted in similar trends in tumour growth are transferrable. Myeloid restricted deletion of SaCS3 in mice results in accelerated tumour growth accompanied by enhanced angiogenesis, and macrophage infiltration. These findings suggest that SOCS proteins have a profound influence on macrophage polarization and consequently tumour growth. Targeting SOCS proteins may be a viable therapeutic target in treatment of cancer.