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Title: A fluorogenic hypoxia-activated prodrug strategy for targeted compound delivery and imaging of tumours
Author: O'Connor, Liam John
ISNI:       0000 0004 6497 8542
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2016
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Hypoxia, a common feature in solid tumours, is associated with aggressive phenotypes, decreased genomic stability, and with increased resistance to all forms of cancer therapy. The significant differences between the hypoxic microenvironment, and normal tissues, present a therapeutic opportunity through the design of bioreductive hypoxia-activated prodrugs (HAPs). This strategy aids the selective delivery of chemotherapeutics to hypoxic regions, reducing their effects in non-hypoxic tissue. A variety of chemical functionalities that undergo oxygen dependent enzyme mediated reduction have been utilised in the development of HAPs. Despite some clinical successes of HAPs, compound delivery to hypoxia remains a key challenge, and the biodistribution of any HAP is currently unclear. Therefore, we sought to develop HAPs, which become fluorescent upon reduction, and subsequently fragment to release a biologically active cargo molecule. A proof of concept fluorogenic bioreductive group was initially designed and synthesised, based on a 6 nitroquinoline scaffold. A BET bromodomain inhibitor was installed as the cargo molecule of therapeutic interest for delivery to hypoxic conditions, affording a novel fluorogenic HAP for biological evaluation. We demonstrated that following reduction of the nitro group to the corresponding amine, fragmentation is induced to release the cargo BET bromodomain inhibitor. When treated with reductase enzymes under hypoxic conditions, reduction and fragmentation occurred as desired. Under normoxic conditions, however, the novel fluorogenic HAP remained hydrolytically stable. Furthermore, following reduction to the corresponding amine and fragmentation products, a significant increase in fluorescence intensity was observed. This work represents the first example of a fluorogenic bioreductive group, and is the subject of a patent application. Following the initial demonstration of fluorogenic bioreductive groups, we sought to improve the group's spectrophotometric properties, by focusing on achieving fluorescence in the near infrared (NIR) region of the electromagnetic spectrum. This window represents a favourable range of wavelengths for surgical fluorescence imaging and in vivo interrogation. By exploiting an aminostyryl chromenone based NIR fluorophore, introduction of oxygen sensitive functionalities was considered, including the nitro group. This work led to the investigation of azides as hypoxia responsive moieties, which undergo bioreduction. This dissertation demonstrates, for the first time, the use of azides as fluorogenic molecules for the detection of hypoxia in a range of in vitro assays and in 3D tumour models. The oxygen dependent enzymatic reduction of azides, a functionality traditionally considered as biologically inert, has implications for their use in other areas of chemical biology, such as click chemistry. Further studies focused on exploiting NIR fluorophore scaffolds already employed in surgical fluorescence imaging to develop fluorogenic HAPs. We were able to show for the first time, in a cancer cell line that a single bioreductive core is capable of fragmenting to release two distinct molecules, following intracellular reduction. Recommended studies to follow this work are discussed, which may improve the efficiency of the fragmentation process of HAPs, and also considers alternative technologies that might be used in the design of bioreductive groups for imaging. An efficient protocol for the design, synthesis and evaluation of molecularly targeted HAPs, comprising experiments of increasing biological evaluation, is also disclosed. In addition, an improved synthesis of the widely used 2 nitroimidazole bioreductive group is also reported.
Supervisor: Conway, Stuart ; Hammond, Ester Sponsor: Medical Research Council ; Cancer Research UK
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available