Rational enzyme-directed prodrug development : exploiting tumour hypoxia to target the bioactivation of cytotoxic prodrugs

Conventional cancer chemotherapy often lacks specificity and is consequently associated with significant normal tissue toxicities. Molecular chemotherapy offers the potential to target the activation of inert prodrugs by utilising tumour-specific catalytic enzymes to restrict cytotoxicity to neoplastic tissues. Appropriate expression of therapeutic enzymes can be achieved by exploiting the genetic distinctions that exist between tumour and normal tissues through the use of tissueor disease-specific promoters. Alternatively, the unique physiological differences that arise in solid tumour masses as a consequence of the abnormal vascular architecture might also be exploited to achieve therapeutic selectivity. The most conspicuous of these differences is the presence of areas of low oxygen tension (hypoxia) arising through both diffusion and perfusion-limited oxygen availability. Hypoxia is an important cause of radioresistance and is a independent prognostic indicator for local recurrence, metastatic spread and overall survival. Evidence also implicates hypoxia in chemotherapeutic resistance and genetic instability, as well as the progression of and selection for an aggressive neoplastic phenotype. Attempts to eliminate tumour hypoxia have met with some success, but the opportunity to utilise thisphenomenonfor therapeutic gain, through the exploitation of the unique reductive tissue environment have lead to the development of hypoxic-specific cytotoxins. These bioreductive prodrugs rely. on the natural complement of tumour enzymes to catalyse their activation under low tissue oxygen tensions. Levels of these reductases are potentially heterogeneous and are often down-regulated in the neoplastic state. The artificial reintroduction of high levels of reductive enzyme expression may be of significant therapeutic value, particularly if expression is restricted to the hypoxic tissue enviroment in which the prodrugs will be activated. This might be achieved through the utilisation of the specific cisacting sequences that are responsive to hypoxia-regulated transcription factors. A diverse spectrum of genes are known to be induced as a consequence of oxygen deprivation, being involved in systemic oxygen supply, vascular tone, neovascularisation, iron homeostasis, glucose metabolism, drug detoxification and protein chaperoning. The details of the cis-acting sequences and transcription factors that mediate this oxygen-sensitive gene control are beginning to emerge. This provides the opportunity to exploit these defined sequences to regulate therapeutic genes in a hypoxia-responsive manner. This thesis describes the evaluation of three potential prodruglenzyme paradigms that may have application in this context. Further, the potential of hypoxia-response-elements to specifically regulate heterologous genes in response to low oxygen tension is described. The application of such an oxygen-regulated gene-directed enzyme/prodrug therapy to solid tumours may provide chemotherapeutic specificity aimed at a clinically important tumour subpopulation. 1 Table of