Abstract:
Purpose. Benzotriazine-N-oxide bioreductive prodrugs such as tirapazamine and its improved analogue CEN-209 (SN30000) have potential for exploiting hypoxia in tumors. Here, we test the hypothesis that the 2-nitroimidazole EF5, in clinical development for both immunohistochemical and PET imaging of hypoxia, can detect not only hypoxia but also the one-electron reductases required for activation of these hypoxia-targeted prodrugs. Experimental design. Aerobic and hypoxic covalent binding of [14C]-EF5 was determined in human tumor cell lines, including lines with overexpression of NADPH:cytochrome P450 oxidoreductase (CYPOR), and reductive metabolism of tirapazamine and CEN-209 by mass spectrometry. DNA damage response was measured by gammaH2AX formation. Bioreductive metabolism was modulated in HCT116 tumor xenografts by over-expression of CYPOR and breathing of hyperbaric oxygen or 10% oxygen. Results. Overexpression of CYPOR induced similar 2- to 4-fold increases in EF5 binding and metabolic reduction of tirapazamine and CEN-209 in SiHa and HCT116 cell lines, and similar enhancement of gammaH2AX formation. EF5 binding and metabolic reduction of the prodrugs were highly correlated in a panel of 14 hypoxic tumor cell lines. In HCT116 xenografts, CYPOR overexpression also significantly increased EF5 binding and CEN-209 reduction, and modification of tumor hypoxia caused similar changes to the bioreductive activation of both agents resulting in a strong correlation between EF5 binding and CEN209-induced DNA damage (r2 0.68, p < 0.0001) at the individual tumor level. Conclusions. EF5 binding is a promising stratification biomarker for benzotriazine-N-oxide bioreductive prodrugs because of its potential for interrogating reductase activity as well as hypoxia in individual tumors.
