The characteristic reduction and binding of nitroimidazoles to cellular macromolecules in the absence of oxygen allows their use for detection and characterization of hypoxia. The biodistribution of a new nitroimidazole, EF5 (2-[2-nitro-1 H-imidazol-1-yI]-N-(2, 2, 3, 3, 3-pentafluoropropyl) acetamide), in mice bearing EMT6 tumors is described. Detection methods based on radioactivity and monoclonal antibody techniques are com-pared for liver and tumor. All nonexcretory tissues demonstrated similar levels of radioactivity at 0.5 hr postinjection of drug, demonstrating equivalent access of EF5 to all tissues. At 24 hr, when unbound drug has been cleared, the tissues with the highest binding are the liver, esophagus, bladder and tumor. Typically, liver tissue contains the highest level of radioactivity at this time. Examination of tumor and liver tissue by use of fluorescence microscopy and Cy3-bound monoclonal antibodies specific for EF5 adducts showed that the patterns of binding in tumor are considerably more heterogeneous than those of liver. Histograms of fluorescence intensity, with use of these antibodies, demonstrate average and maximal binding higher in tumors than in the liver. This divergence from the radioactivity data was determined to be unrelated to sampling error, differential antibody access or staining efficiency of liver vs. tumor tissue. A possible cause is the scavenging of radioactive drug metabolites by liver. The data presented herein suggest that EF5 is useful as a hypoxia detector and that monoclonal antibody detection methods can give detailed information on the distribution of EF5 binding. This technology may allow an accurate estimation of the oxygenation and/or nitroreductase levels in both tumor and normal tissues.

Cells derive much of their energy from oxygen consumption via oxidative phosphorylation. When consumption of oxygen exceeds supply, hypoxia results. Tumors are often characterized by hypoxia because of their heterogeneous blood supply. As early as 1953, Gray et al suggested that the curability of some human cancers by radiation might be limited by oxygen depletion in the tumor cells (Gray et al., 1953). It has been confirmed that hypoxia does limit radiocurability in cellular and rodent systems(for review, see Moulder and Rockwell, 1987). The proof of the effects of hypoxia on the radiocurability of human tumors is more controversial, but it is generally accepted that hypoxia is treatment-limiting in certain tumors, for example, oral squamous cell cancer and cervical cancer (Gatenby et al., 1988; Hockel et al., 1991; Overgaard, 1992; Hockel et al., 1993). It is also possible that many other tumor types develop some