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17 allylarnino, 17-demethoxygeldanamycin (17-AAG) is a heat shock protein inhibitor (HSP90) that is the first of its class to go into phase I clinical trials. This thesis describes pre-clinical and clinical studies with 17-AAG directed towards rational development of the drug. The first section describes the development and validation of pharmacodynamic (PD) markers. A molecular signature of inhibition of HSP90, i. e. depletion of client proteins c-RAF-1, CDK4 and induction of the co-chaperone heat shock protein 70 (HSP70), was validated in human cancer cell lines and human peripheral blood leukocytes (PBLs) in vitro. The pharmacokinetic (PK) - PID relationship was established in tumour and PBLs in two human ovarian tumour xenograft models. The second section of the thesis describes the phase I clinical trial of 17-AAG. The highest dose reached was 450 mg/m2/wk, and dose limiting toxicities of diarrhoea and hepatotoxicity were seen in 2/9 of patients at this dose. It was possible to demonstrate potentially therapeutic plasma levels and PID changes in PBLs and tumour tissue at the dose level of 450 mg/m2/wk, which was the recommendedd ose for phase 11tr ials. The third section of the thesis addresses the combination of 17-AAG and platinum compounds. The effects of the combination were studied in vitro and in vivo and will provide the pre-clinical validation for a combination phase I trial in the future. The fourth section discusses the study of 17-AAG in malignant melanoma prompted by the indication of clinical activity in the phase I clinical Mal. c-DNA microarrays were used to study gene expression changes in response to 17-AAG in two melanoma cell lines (SKMEL-2 and SKMEL-5). The changes in gene expression were used to try and identify new PID markers and understand factors influencing sensitivity of malignant melanoma cells to 17-AAG.

Microvesicles are shed constitutively, or upon activation from both normal and malignant cells. Although recent studies have reported various nonlytic virus release mechanisms, this mode of virus transmission to secondary sites of infection has remained unclear. This study identified that Coxsackie virus B1 (CVB1) entry into HeLa cells results in apoptosis and production of virus-induced apoptotic microvesicles (vaMVs) by infected cells. Flow cytometery and fluorescence microscopy data illustrated that these vaMVs carry and disseminate CVB1 virions to new host cells via a non lytic MV-to-cell viral mechanism. Inhibition of MV production by siRNA knockdown of CAPNS1 in HeLa cells suggested that these vesicles mediate the spread of apoptosis to secondary sites of infection and the vaMVs could mediate non lytic MV-to-cell transmission. This thesis also identified a new mechanism for multi-drug resistance involving the efflux of anticancer drugs from cancer cells mediated by release of microvesicles, removing the drug from treated cancer cells. Immunoblotting and flow cytometery data showed that transcriptional silencing of calpain by siRNA knockdown of CAPNS1 in PC3M cells prior to drug treatment inhibits MV release and results in induced apoptosis in cells. This mechanism contributes to understanding the reasons for insensitivity to drug-induced apoptosis and the induction of drug-detoxification by cancer cells. This study has yielded important information about how to circumvent drug resistance to improve cancer chemotherapy. Furthermore, fluorescence microscopy results postulate that induction of MV release with agonist agents and anticancer drugs, results in damage to the host plasma membrane, which must be resealed immediately using activated Iysosomes if the host cell is to survive and proliferate.

Four carboxylate terminated half generation polyamdioamine (PAMAM) dendrimers (G3.5- G6.5) were conjugated with the active components of cisplatin, {Pt(NH3)2}2+, or oxaliplatin, {Pt(R,R-dach)}2+, to form dendrimer platinate complexes. The platinum content of the cisplatin containing complexes was measured using inductively coupled plasma atomic emission spectroscopy. The number of cisplatin molecules was found to increase with increasing dendrimer size, 22 – 94, however the assumed bidentate binding mode allows for a total of 32 – 256 cisplatin moieties per dendrimer. Whilst the number of cisplatin moieties increases with dendrimer size, a decrease in the binding efficiency is also observed with dendrimer size, 68 – 37% (G3.5- 6.5). This decrease is probably due to the close proximity of surface groups of larger dendrimers, i.e. more branched, increasing the difficulty in deprotonating the carboxylic acids to obtain the carboxylate required for coupling. The release of the platinum from the dendrimers, in the active form, was evaluated using 1H NMR or inductively coupled plasma mass spectroscopy (ICP-MS). An increase in the number of active cisplatin molecules released is observed with larger dendrimers, four from the G3.5 dendrimer to 59 from the G6.5 dendrimer, with the release efficiency increasing from 18 – 63%. Diffusion ordered spectroscopy NMR was also used to determine the size of the dendrimer platinate complexes. The free dendrimers increased from 2.72 to 5.93 nm, cisplatin dendrimer platinates from 1.66 to 4.01 nm and oxaliplatin dendrimer platinates from 4.03 to 14.51. The dendrimer platinates and free dendrimers were tested in vitro against the A2780 and A2780cis cancer cell lines and their activity compared to cisplatin. In all cases the dendrimer platinates showed reduced activity when compared to cisplatin whilst the free dendrimer showed no activity in vitro, IC50 > 100 mM. The in vivo activity of the G6.5 dendrimer platinate was tested in nude mice bearing A2780 xenografts and compared to cisplatin using a single dose of equivalent concentration and the G6.5 dendrimer platinate was shown to have a similar activity in vivo as cisplatin after 6 days. Similar dendrimer-platinate complexes were formed with the active component of oxaliplatin, {Pt(R,R-dach)}2+. The dendrimer-platinates were found to contain 12-63 platinum molecules per dendrimer, a reduction on the number observed for the complexes formed with the active component of cisplatin. The size of the oxaliplatin dendrimer-platinates was also determined to be greater than the cisplatin equivalent, most likely as a result of aggregation due to the hydrophobic nature of the dach ligand. The number of platinum groups released from the dendrimer is also lower than the equivalent cisplatin complexes with only 2-43 platinum moieties being released. The platinum was shown to be released from the dendrimer in an initial burst release within the first 2 hours, accounting for 85% of the releasable platinum. The {Pt(R,R-dach)}2+ dendrimer-platinates were shown to be more cytotoxic in vitro than the free {Pt(R,Rdach)} 2+ complex. In a further study a folic acid targeting group was also attached to the dendrimers to allow active targeting of cancer cells through the folate receptor. Folic acid was conjugated to 1,6-diamminohexane via an amide coupling reaction. The linker was then attached to the dendrimer through a further amide coupling reaction prior to the addition of the platinum complexes. The resulting complexes were found to contain 3-8 folate targeting groups depending on dendrimer size. The targeted cisplatin dendrimerplatinates were shown to be more cytotoxic than the non-targeted complexes however the opposite was observed for the {Pt(R,R-dach)}2+ dendrimer-platinates. The uptake of platinum by the cells increased for the folate targeted cisplatin dendrimer-platinates but decreased for the {Pt(R,R-dach)}2+ dendrimer-platinates.

Cisplatin is a leading anti-cancer drug used in the treatment of various cancers. However, its clinical use is limited by its undesirable toxic side effect profile and the potential of certain tumours to be or to develop resistance to cisplatin. Encapsulation of cisplatin within a vesicular structure such as non-ionic surfactant vesicles (NIVs) was developed to overcome these limitations. Characterisation studies showed that the size and negative surface charge of cisplatin NIVs could be exploited in enhancing their uptake by the mononuclear phagocytic system present in the lungs, liver and spleen. Physicochemical stability of the systems over a 15 month time period was demonstrated in relation to vesicle size and surface charge; the absence of colloidal aggregation and chemical stability of the lipid components. Vesicle entrapment efficiency of cisplatin was improved with increasing cisplatin concentrations used for lipid hydration but subsequent precipitation of drug limited the usefulness of such an approach. Removal of unentrapped cisplatin by the use of diafiltration and resuspension in lower concentrations of cisplatin solution overcame this problem but resulted in drug leakage from the vesicles over time. Preliminary in vitro and in vivo studies were used to evaluate NIVs. In vitro studies confirmed the potential of NIVs in enhancing the anti-cancer effect of cisplatin in comparison to free drug in a murine B16-F0 murine melanoma cell line. In vivo rodent studies compared cisplatin NIVs with free drug solution administered as single doses by intravenous or pulmonary routes of delivery. Intravenous delivery demonstrated more representable results with greater accumulation of cisplatin in the lungs when administered as a NIVs formulation in comparison to free drug solution. In conclusion, NIVs have great potential to be a viable delivery platform for the administration of cisplatin.