title: The 2-nitroimidazole EF5 is a biomarker for oxidoreductases that activate the bioreductive prodrug CEN-209 under hypoxia.
abstract: OBJECTIVE
  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 positron emission tomography imaging of hypoxia, can detect not only hypoxia but also the one-electron reductases required for activation of these hypoxia-targeted prodrugs.
  
  
  METHODS
  Aerobic and hypoxic covalent binding of [(14)C]-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 γH2AX formation. Bioreductive metabolism was modulated in HCT116 tumor xenografts by overexpression 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 γH2AX 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 (R(2) = 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.
authors.affiliation: School of Medical Sciences, The University of Auckland, Auckland, New Zealand.
authors.forename: Jingli
authors.initials: J
authors.lastname: Wang
authors.forename: Annika
authors.initials: A
authors.lastname: Foehrenbacher
authors.forename: Jiechuang
authors.initials: J
authors.lastname: Su
authors.forename: Rita
authors.initials: R
authors.lastname: Patel
authors.forename: Michael P
authors.initials: MP
authors.lastname: Hay
authors.forename: Kevin O
authors.initials: KO
authors.lastname: Hicks
authors.forename: William R
authors.initials: WR
authors.lastname: Wilson
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D003497:Cyclic N-Oxides / Q000378:metabolism; D017341:Etanidazole / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D005260:Female; D045325:HCT116 Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D006845:Hydrocarbons, Fluorinated / Q000032:analysis* / Q000378:metabolism; D007202:Indicators and Reagents; D051379:Mice; D010088:Oxidoreductases / Q000378:metabolism; D011355:Prodrugs / Q000494:pharmacology; D000077704:Tirapazamine; D014183:Transplantation, Heterologous; D014227:Triazines / Q000378:metabolism; D005466:Fluorocarbons
chemical_list: D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D003497:Cyclic N-Oxides; D017341:Etanidazole; D006657:Histones; D006845:Hydrocarbons, Fluorinated; D007202:Indicators and Reagents; D010088:Oxidoreductases; D011355:Prodrugs; D000077704:Tirapazamine; D014227:Triazines; C516729:5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide; C472752:CETN2 protein, human; C468783:H2AX protein, human; C096011:2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide; D005466:Fluorocarbons