The long term objective of this grant is to understand at the cellular and molecular level the mechanism for both the specific toxicity of tirapazamine (TPZ) for hypoxic cells and for its effects on aerobic cells that may be associated with some of its clinical side effects. These side effects include muscle cramping and a general fatigue after repeated administration of TPZ. The basis for the specific antitumor effects of this drug is its ability to be metabolized to a highly toxic radical within hypoxic cells in solid tumors. These cells are considered to be a problem in cancer treatment of solid tumors, both by radiotherapy and chemotherapy. Killing these cells at doses that are not toxic to well-oxygenated cells provides the basis for increasing the efficacy of standard anticancer therapy. Phase II and III trials of this drug are currently in progress, but the frequency of administration of the drug with radiotherapy is limited by its side effects. This laboratory proposes to investigate two new hypotheses relating to the mechanism to hypoxic cytotoxicity of TPZ and muscle cramps. First, they propose that the enzyme responsible for the intracellular metabolism of TPZ is located close to the nuclear matrix and preferentially damages DNA close to these regions. The second hypothesis is that the effects on muscles are a consequence of preferential damage under aerobic conditions to mitochondria, which destroys their normal transmembrane potential, thereby inhibiting ATP production. They will investigate these two hypotheses by determining the enzyme(s) responsible for the intranuclear TPZ metabolism using an in vitro assay of TPZ metabolism, and will determine whether DNA protein crosslinks formed by TPZ under hypoxia are made preferentially to the nuclear matrix. Loss of the transmembrane mitochondrial potential will be investigated using Rh123 accumulation in cells in vitro and in tissues in mice. Since it is likely that the mitochondrial damage is produced by OH radicals produced by the Fenton reaction, the researchers will inhibit this reaction using clinically used iron chelators. These investigations should lead to a better ability to select patients for TPZ administration and to allow more frequent administration of drug by reducing its side effects. In this way the researchers expect to increase the clinical efficacy of this new anticancer agent.
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