Doctor of Philosophy(PhD) / The present study deals with the synthesis, characterization, determination of anticancer activity of three mononuclear trans-planaraminepalladium(II) complexes code named TH5, TH6 and TH7 and three trinuclear complexes code named TH1, TH8 and TH14. The activity of the compounds against human cancer cell lines: A2780, A2780cisR and A2780ZD0473R, cell uptake, DNA-binding and nature of interaction with pBR322 plasmid DNA have been determined. Whereas cisplatin binds with DNA forming mainly intrastrand GG adduct that causes local bending of a DNA strand, TH5, TH6, TH7, TH1 and TH8 bind with DNA forming mainly interstrand GG adducts that causes more of a global change in DNA conformation. Although TH5, TH6 and TH7 each have two substituted pyridine ligands in a trans-geometry (3-hydroxypyridine in TH5, 2-hydroxypyridine in TH6 and 4-hydroxypyridine in TH7), the compounds differ in their activity against ovarian cancer cell lines, indicating that non-covalent interactions involving the hydroxyl group may be playing a significant role in activity of the compounds. Among trinuclear complexes TH1 is found to be significantly more active than cisplatin. It is actually twice as active as cisplatin against the parent cell line A2780, thirteen times as active as cisplatin against the cisplatin-resistant cell line A2780cisR and 11.5 times as active as cisplatin against the cell line A2780ZD0473R. Whereas the resistance factor for cisplatin as applied to the cell lines A2780 and A2780cisR cell lines is 12.9 that for TH1 is 1.98. The results suggest that TH1 has been able to significantly overcome resistance operating in A2780cisR cell line. The compound is soluble in water so that it may be taken orally. Provided it has favourable toxicity profile, TH1 has the potential to be developed into a highly active anticancer drug with a wider spectrum of activity than cisplatin. Although platinum drugs use a shot-gun approach to kill cancerous cells, widespread use in the clinic and increasing volume of their sale indicate that even in the genomic age, there is still need for shot-gun drugs in the clinic.

This thesis seeks to examine how funding arrangements for cancer drugs and other treatments, which are not available to everyone within the NHS, are made available to some, on the basis of exceptionality. The escalating costs of cancer treatment and the life threatening nature of cancer make resource allocation decisions for cancer drugs particularly acute, and the recent changes to funding arrangements for cancer drugs within the NHS receive particular scrutiny. In the three papers at the core of this thesis, the concept of exceptionality is explored from legal, ethical and empirical perspectives respectively. The first paper reviews the legal origin of exceptionality as the basis for the allocation of resources for expensive treatments, and explores how the concept has been interpreted by successive judicial reviews concerning access to cancer drugs. Particular attention is paid to the role of social factors in determining exceptionality. Choosing to fund treatment for one patient, and not another, involves a moral choice. In recognition of this, the Department of Health advocates that decision makers use an ethical framework to support decision making regarding exceptionality. The second paper examines the strengths and weakness of Daniels and Sabin’s Accountability for Reasonableness Framework, which is widely used to support resource allocation, focussing on the Relevance Condition, and its applicability to resource allocation within the NHS. The final paper reports the findings of an empirical study examining how PCTs interpret the concept of exceptionality in practice, providing the first comprehensive insight into the factors which are considered in determining whether a patient is exceptional, and exposing some of the external influences on the decision making process. In conclusion, it is argued that whilst the need for discretionary health funding decisions arises in rare circumstances, where this is necessary such decisions should be made on a national, or at least supra-regional basis, to ensure consistency and fairness. If we cannot afford to fund all effective cancer drugs, and other treatments, we should not hide behind the concept of exceptionality, but should have a national debate about how we reach a consensus on which drugs to fund, and about how we pay for those treatments. Whilst acknowledging that cancer is a dreadful disease, it is also argued that, in the absence of any convincing evidence that the management of cancer deserves preferential treatment, the special status of cancer funding within the NHS, which has become increasingly apparent in recent years, should come to an end.

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Thesis (Ph. D. in Interdisciplinary Studies: Engineering Management)--Vanderbilt University, Dec. 2008. / Title from title screen. Includes bibliographical references.

No / Telomerase-targeted strategies have aroused recent interest in anti-cancer chemotherapy, because DNA-binding drugs can interact with high-order tetraplex rather than double-stranded (duplex) DNA targets in tumour cells. However, the protracted cell-drug exposure times necessary for clinical application require that telomerase inhibitory efficacy must be accompanied by both low inherent cytotoxicity and the absence of mutagenicity/genotoxicity. For the first time, the genotoxicity of a number of structurally diverse DNA-interactive telomerase inhibitors is examined in the Ames test using six Salmonella typhimurium bacterial strains (TA1535, TA1537, TA1538, TA98, TA100, and TA102). DNA damage induced by each agent was also assessed using the Comet assay with human lymphocytes. The two assay procedures revealed markedly different genotoxicity profiles that are likely to reflect differences in metabolism and/or DNA repair between bacterial and mammalian cells. The mutational spectrum for a biologically active fluorenone derivative, shown to be mutagenic in the TA100 strain, was characterised using a novel and rapid assay method based upon PCR amplification of a fragment of the hisG46 allele, followed by RFLP analysis. Preliminary analysis indicates that the majority (84%) of mutations induced by this compound are C→A transversions at position 2 of the missense proline codon of the hisG46 allele. However, despite its genotoxic bacterial profile, this fluorenone agent gave a negative response in the Comet assay, and demonstrates how unwanted systemic effects (e.g., cytotoxicity and genotoxicity) can be prevented or ameliorated through suitable molecular fine-tuning of a candidate drug in targeted human tumour cells. / CAEB, Balearic Islands and Yorkshire Cancer Research

Owing to their non-selective nature, anti-cancer drugs affect both cancerous and non-cancerous cells and present a major health risk to healthcare staff working with them. This project was conducted at Derriford Hospital, Plymouth, to investigate the extent of contamination with anti-cancer drugs on work surfaces and the environmental emissions of these drugs. In the Isolator study, surface contamination arising from the preparation of five anticancer drug infusions (epirubicin, fluorouracil, cisplatin, oxaliplatin and carboplatin) in a pharmaceutical isolator and external surfaces of infusion bags and syringes using a conventional syringe and needle technique was investigated and compared with that obtained using a closed system drug transfer device (Tevadaptor). Wipe samples were taken for a period of one week from pre-defined areas in a pharmaceutical isolator and from the surface of prepared Intra-Venous (IV) infusion bags and pre-filled syringes to obtain baseline data. Gloves and preparation mats used during this period were also collected. Following a one-week operator familiarisation period, the Tevadaptor device was then introduced for cytotoxic preparation and wipe-sampling of surfaces and collection of consumables was continued for a further week (intervention period). The samples obtained were then analysed by HPLC and ICP-MS. The baseline contamination data from Tevadaptor isolator study was undetected to 0.9 ng cm-2 (epirubicin), undetected to 3.58 ng cm-2 (5-FU) and 0.05-0.92 ng cm-2 (Pt) in the wipe samples from the pharmaceutical isolator surfaces; amounts on glove samples were 1100-6100 ng/glove (epirubicin), 300-8100 ng/glove (5-FU) and 1-6 ng/glove (platinum). During the intervention phase isolator surface contamination was not detected in all samples for 5-FU and epirubicin and platinum was detected on the isolator surfaces in the range of 0.002-0.09 ng cm-2. The use of Tevadaptor resulted in a reduction of contamination on external surfaces by a factor of 10 or more for all marker drugs. A ward study investigated the surface contamination in the oncology out-patient department caused by cisplatin, oxaliplatin, carboplatin and gemcitabine. The study compared the effect of using the Tevadaptor to prepare and administer anticancer drugs infusions on ward surface contamination to the current UK standard practice. A questionnaire was also distributed to participating staff members to assess the user-friendliness of Tevadaptor. Wipe samples were taken from pre-defined areas from the oncology out-patients department and gloves used by nursing staff for assembly and administration of the above drugs were also collected. Sample collection followed a similar schedule to the Tevadaptor isolator study. The baseline ward surface contamination ranged from undetected to 4.97 ng cm-2 (gemcitabine) and 3.1 ng cm-2 (platinum). In the case of gloves used by nursing staff the levels of contamination ranged from undetected to 1251 ng/glove (gemcitabine) and 405.4 ng/glove (platinum). The contamination on ward surfaces during the intervention phase ranged from undetected to 3.21 ng cm-2 (gemcitabine) and 2.69 ng cm-2 (platinum) and contamination levels on gloves ranged from undetected to 9252 ng/glove (gemcitabine) and 1319 ng/glove (platinum). During the intervention phase there was a reduction in frequency of contamination, even though the total amount of surface contamination by anticancer drugs did not always decrease in comparison to baseline data, presumably due to unaccounted spillages. A drain study investigated the presence of platinum in hospital wastewater as a measure of contamination caused by the excretion of platinum-based anticancer drugs by patients. Platinum was measured over a three week period in one of the main drains and in the effluent of the oncology ward. The study showed the presence of measurable quantity of platinum which ranged from 0.02 to 140 μg L-1 in the oncology effluent and 0.03 to 100 μg L-1 in the main drain. Data from this study was coupled with published measurements on the removal of the drugs by conventional sewage treatment and then concentration of platinum arising from each drug was predicted in recipient surface waters as a function of water flow rate. Although predicted concentrations were below EMEA guidelines warranting further risk assessment, the presence of potentially carcinogenic, mutagenic and teratogenic substances in surface waters is cause for concern. The results showed that a closed system drug transfer device (CSTD) used in conjunction with an isolator is highly efficient in reducing surface contamination with anti-cancer drugs. However, despite current best practice contamination on ward surfaces remained even after the use of a CSTD. Nursing as well as healthcare staff should be educated of these results and the risks of occupational exposure to low levels of anti-cancer drugs and the use of PPE should be emphasised. Results of the drain study form the basis of preliminary estimates of the likely concentrations of platinum-based drugs in surface waters and their potential environmental impacts.

Doxorubicin is one of the most effective anti-cancer agents; however its use is associated with adverse cardiac effects, including cardiomyopathy and progressive heart failure. Mitochondrial function and integrity are crucial for cellular processes in general and play an important role during diseased development. These characteristics of the mitochondria make them the prime target for treatments for majority of diseases and in particular of the cardiovascular system. The mitochondria are also considered to play an integral role in the manifestation of the cardiotoxic effects of compounds such as doxorubicin. The current project is designed to investigate the cardiotoxic effects of doxorubicin at tissue, cellular and protein level. In addition, it is investigated whether the inhibition of the mitochondrial permeability transition pore (mPTP) with cyclosporin A (CsA) or the inhibition of mitochondrial fission with the mitochondrial division inhibitor (mdivi-1) protects against the detrimental effects of doxorubicin on cardiac function. We also investigated whether co-treatment of doxorubicin with either CsA or mdivi-1 has any negative interaction with the cytotoxicity of doxorubicin against cancer cells. Langendorff results indicated that doxorubicin caused a time dependent reduction in the haemodynamic function of the heart as well as causing an increase in the infarct size to risk ratio in both naïve conditions and in conditions of ischaemia and reperfusion. Detrimental effects of doxorubicin on cardiac function were abrogated by co-treatment of doxorubicin with CsA or mdivi-1 in naïve conditions and in conditions of ischaemia and reperfusion. Cell viability data of isolated cardiac myocytes revealed that doxorubicin caused a concentration dependant decrease in the viability of neonatal cardiac myocytes as well as causing a reduction in the time taken to depolarisation and hypercontracture under sustained oxidative stress, all of which were prevented when co-treated with either CsA or mdivi-1. Doxorubicin significantly elevated the levels of p-Akt, p-Erk, p-Drp1 and p-p53. Co-treatment with CsA prevented the increase in the levels of p-Akt and p-Erk caused by doxorubicin in both naïve and IR condition whereas mdivi-1 prevented the increase in the levels of p-Erk, p-Drp1 and p-p53 and caused further increase in the levels of p-Akt. Using sinusoidal muscle length change during contraction and relaxation, it is demonstrated that doxorubicin caused a decrease in the power output, peak force and force during shorting. Detrimental effects of doxorubicin on work-loop contraction were abrogated when doxorubicin was co-administered with CsA. To conclude, results demonstrated that doxorubicin caused cardiotoxicity at tissue, cellular and protein level in both naïve conditions and in conditions of ischaemia and reperfusion injury. In addition, it is shown that the inhibition of mitochondrial permeability transition pore with CsA or the inhibition of the mitochondrial fission with mdivi-1 protect against doxorubicin-induced toxicity without affecting its anti-cancer properties.

The overall aim of the research is to develop a computational platform based on HINT paradigm for manipulating, predicting and analyzing biomacromolecular-ligand structure. A second synergistic goal is to apply the above methodology to design novel and potent anti-cancer agents. The crucial role of the microtubule in cell division has identified tubulin as an interesting target for the development of therapeutics for cancer. Pyrrole-containing molecules derived from nature have proven to be particularly useful as lead compounds for drug development. We have designed and developed a series of substituted pyrroles that inhibit growth and promote death of breast tumor cells at nM and μM concentrations in human breast tumor cell lines. In another project, stilbene analogs were designed and developed as microtubule depolymerizing agents that showed anti-leukemic activity. A molecular modeling study was carried out to accurately represent the complex structure and the binding mode of a new class of tubulin inhibitors that bind at the αβ-tubulin colchicine site. These studies coupled with HINT interaction analyses were able to describe the complex structure and the binding modes of inhibitors. Qualitative analyses of the results showed general agreement with the experimental in vitro biological activity for these derivatives. Consequently, we have been designing new analogs that can be synthesized and tested; we believe that these molecules will be highly selective against cancer cells with minimal toxicity to the host tissue. Another goal of our research is to develop computational tools for drug design. The development and implementation of a novel cavity detection algorithm is also reported and discussed. The algorithm named VICE (Vectorial Identification of Cavity Extents) utilizes HINT toolkit functions to identify and delineate a binding pocket in a protein. The program is based on geometric criteria and applies simple integer grid maps to delineate binding sites. The algorithm was extensively tested on a diverse set of proteins and detects binding pockets of different shapes and sizes. The study also implemented the computational titration algorithm to understand the complexity of ligand binding and protonation state in the active site of HIV-1 protease. The Computational titration algorithm is a powerful tool for understanding ligand binding in a complex biochemical environment and allows generating hypothesis on the best model for binding.

Adriamycin is a first line cancer treatment drug for breast cancer and many soft tissue carcinomas. Adriamycin is a highly effective anti-cancer drug. It has a fused ring system that is planar, hydrophobic and electron rich. These features allow intercalation into DNA which, along with its topoisomerase inhibition, results in its anti-cancer effectiveness. Despite its success, its use is limited by a dose-dependent cardiotoxicity. Adriamycin forms tight complexes with iron in varying iron:drug ratios. The chelated complex [Fe+3-Adriamycin (1:2)] oxidatively cleaves DNA via the generation of reactive oxygen species (ROS). Enzymes associated specifically with heart mitochondria increase ROS formation explaining why adriamycin is selectively cardiotoxic. Pretreatment of mice with the neural hormone melatonin eliminates the cardiotoxic effectiveness of adriamycin therapy without reducing the drugÆs antitumor effect (Wahab et al. 2000, Tumori 86: 157-162). This has recently been verified in human cancer patients (Lissoni et al. 1999, European Journal of Cancer 35: (12) 1688-1692). Melatonin is soluble in both lipid and aqueous environments and has no known side-effects. This study analyzes the mechanistic features of DNA damage by Fe+3-Adriamycin and how melatonin ameliorates this damage. An Fe+3-Adriamycin (1:2) complex + glutathione cleaves DNA; this oxidative DNA cleavage does not occur with adriamycin +/- glutathione or with FeADR in the absence of glutathione. Melatonin reduces this oxidative DNA cleavage by 67%. Using a supercoiled-to-nicked-circular-conversion assay in conjunction with spectroscopic analyses give results revealing : 1) Fe+3-Adriamycin (1:2) complex + glutathione forms a reactive intermediate. 2) Melatonin protects the DNA from cleavage by this reactive intermediate. 3) Enzymatic assays show that H2O2 is the primary ROS formed with downstream production of the hydroxy radical via the Fenton reaction. 4) Fe+3-Adriamycin (1:2) intercalates into DNA to the same degree as uncomplexed adriamycin. 5) Melatonin also binds to DNA but not by intercalation. These experiments indicate that the cardioprotective effect of melatonin in adriamycin therapy may stem from melatoninÆs interaction with a reactive intermediate from Fe+3-Adriamycin(1:2) complex + glutathione and/or a direct interaction of melatonin with DNA.