This Project is one of three that together constitute a coordinated and comprehensive Program for the development of new bioreductive drugs for the exploitation of hypoxic cells in tumors. The approaches focuses on analogs of tirapazamine (TPZ), a drug with proven clinical ability but which does not fully exploit the potential of this new class of anti-cancer agents. The broad objective of the present Project is to develop a detailed understanding of the features of TPZ-like drugs that determine delivery to hypoxic tumor cells (and normal tissues) in vivo, and to use this information to develop analogs with superior utility. The hypothesis and approaches are: (1) The rate of overall bioreductive drug metabolism, and extent of entrapment in cells, determine the ability of TPZ analogs to penetrate hypoxic tumor tissue. Drug metabolism and uptake will be quantified in single cell suspensions, at a range of O2 concentrations. Tissue diffusion properties will be investigated using a novel tissue culture model, multi- cellular layers (MCL), developed in this laboratory. (2) Optimization of the ability of TPZ analogs to penetrate hypoxic tumor tissue will improve therapeutic efficacy of combining the analogs with radiation and cisplatin. Plasma pharmacokinetic parameters will be measured, and used along with in vitro cytotoxicity (Project 2) and penetration data (MCL studies) to predict activity against hypoxic cells. The model will be tested against measured cell killing in combination with radiation and cisplatin. (3) Physiology-based, high-throughput screens, designed to evaluate delivery to hypoxic cells, will facilitate optimization of bioreductive drug design. These studies will exploit the selectivity of liquid chromatography/mass spectroscopy (LC/MS) for simultaneous evaluation of MCL penetration of multiple compounds. (4) Alteration of physicochemical properties (especially log P) and/or reductase specificity can be used to reduce retinal toxicity of TPZ analogs relative to anti-tumor efficacy. We will measure retinal damage with compounds having a wide range of lipophilicities and enzyme substrate specificities. We will perform experiments to understand the mechanism of this toxicity.
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