title: Molecular and cellular pharmacology of the hypoxia-activated prodrug TH-302.
abstract: TH-302 is a 2-nitroimidazole triggered hypoxia-activated prodrug (HAP) of bromo-isophosphoramide mustard currently undergoing clinical evaluation. Here, we describe broad-spectrum activity, hypoxia-selective activation, and mechanism of action of TH-302. The concentration and time dependence of TH-302 activation was examined as a function of oxygen concentration, with reference to the prototypic HAP tirapazamine, and showed superior oxygen inhibition of cytotoxicity and much improved dose potency relative to tirapazamine. Enhanced TH-302 cytotoxicity under hypoxia was observed across 32 human cancer cell lines. One-electron reductive enzyme dependence was confirmed using cells overexpressing human NADPH:cytochrome P450 oxidoreductase and radiolytic reduction established the single-electron stoichiometry of TH-302 fragmentation (activation). Examining downstream effects of TH-302 activity, we observed hypoxia-dependent induction of γH2AX phosphorylation, DNA cross-linking, and cell-cycle arrest. We used Chinese hamster ovary cell-based DNA repair mutant cell lines and established that lines deficient in homology-dependent repair, but not lines deficient in base excision, nucleotide excision, or nonhomologous end-joining repair, exhibited marked sensitivity to TH-302 under hypoxia. Consistent with this finding, enhanced sensitivity to TH-302 was also observed in lines deficient in BRCA1, BRCA2, and FANCA. Finally, we characterized TH-302 activity in the three-dimensional tumor spheroid and multicellular layer models. TH-302 showed much enhanced potency in H460 spheroids compared with H460 monolayer cells under normoxia. Multicellular layers composed of mixtures of parental HCT116 cells and HCT116 cells engineered to express an oxygen-insensitive bacterial nitroreductase showed that TH-302 exhibits a significant bystander effect.
authors.affiliation: Threshold Pharmaceuticals, 170 Harbor Way, Suite 300, South San Francisco, CA 94080, USA. fmeng@thresholdpharm.com
authors.forename: Fanying
authors.initials: F
authors.lastname: Meng
authors.forename: James W
authors.initials: JW
authors.lastname: Evans
authors.forename: Deepthi
authors.initials: D
authors.lastname: Bhupathi
authors.forename: Monica
authors.initials: M
authors.lastname: Banica
authors.forename: Leslie
authors.initials: L
authors.lastname: Lan
authors.forename: Gustavo
authors.initials: G
authors.lastname: Lorente
authors.forename: Jian-Xin
authors.initials: JX
authors.lastname: Duan
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Cai
authors.forename: Alexandra M
authors.initials: AM
authors.lastname: Mowday
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Guise
authors.forename: Andrej
authors.initials: A
authors.lastname: Maroz
authors.forename: Robert F
authors.initials: RF
authors.lastname: Anderson
authors.forename: Adam V
authors.initials: AV
authors.lastname: Patterson
authors.forename: Gregory C
authors.initials: GC
authors.lastname: Stachelek
authors.forename: Peter M
authors.initials: PM
authors.lastname: Glazer
authors.forename: Mark D
authors.initials: MD
authors.lastname: Matteucci
authors.forename: Charles P
authors.initials: CP
authors.lastname: Hart
mesh_terms: D000818:Animals; D016466:CHO Cells; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D020552:Comet Assay; D006224:Cricetinae; D003412:Cricetulus; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D045325:HCT116 Cells; D019073:HT29 Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D015394:Molecular Structure; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009593:Nitroimidazoles / Q000737:chemistry / Q000494:pharmacology*; D010084:Oxidation-Reduction / Q000528:radiation effects; D010100:Oxygen / Q000494:pharmacology; D010752:Phosphoramide Mustards / Q000737:chemistry / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011355:Prodrugs / Q000737:chemistry / Q000494:pharmacology*; D018874:Spheroids, Cellular / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D014410:Tumor Stem Cell Assay
chemical_list: C468783:H2AX protein, human; D006657:Histones; D009593:Nitroimidazoles; D010752:Phosphoramide Mustards; D011355:Prodrugs; C552526:TH 302; D010100:Oxygen