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EF5 as a predictive biomarker for activation of the new hypoxia-targeting prodrug SN30000.

e13597 Background: SN30000 (CEN-209), a benzotriazine-N-oxide, is a second generation analogue of tirapazamine (TPZ) and is currently in late stage preclinical development as a hypoxia-targeted prodrug. Here we test the hypothesis that the 2-nitroimidazole imaging agent EF5, currently in clinical development for both immunohistochemical and PET imaging of hypoxia, can also detect the one-electron reductases required for activation of this class of bioreductive prodrug.


METHODS
Aerobic and hypoxic covalent binding of 14C-EF5, and reductive metabolism of TPZ and SN30000 by LC/MS/MS, was determined in 14 human tumor cell lines and in SiHa and HCT116 transfectants that over-express NADPH:CytP450 oxidoreductase (CYPOR). The DNA damage response to the bioreductive prodrugs was measured by γH2AX formation, and cell killing by clonogenic assay. Bioreductive metabolism was modulated in HCT116 tumor xenografts either by over-expression of CYPOR or breathing of hyperbaric oxygen, air, or 10% oxygen following SN30000 administration.


RESULTS
Over-expression of CYPOR induced similar increases in EF5 binding and reduction of TPZ and SN30000, and similar enhancement of γH2AX formation in response to the bioreductive prodrugs in isogenic paired cell lines. Hypoxic EF5 binding and reduction of TPZ and SN30000 were highly correlated in the panel of human tumor cell lines (r2 = 0.65, p = 0.0005) suggesting that EF5, TPZ and SN30000 share one-electron reductases. In HCT116 xenografts, CYPOR over-expression also significantly increased EF5 binding and SN30000 reduction, and modification of tumor hypoxia caused similar changes to the bioreductive activation of both agents. This resulted in a strong correlation between EF5 binding and SN30000-induced γH2AX formation (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 one-electron reductase activity as well as hypoxia in individual tumors.