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, hypoxiaselective 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 gH2AX 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.

Activity of the hypoxia‐activated pro‐drug TH‐302 in hypoxic and perivascular regions of solid tumors and its potential to enhance therapeutic effects of chemotherapy

“…27 The 2-nitroimidazole portion of TH-302 undergoes electron rearrangement forming a radical anion in regions with less than about 0.5% oxygen. 27 The radical anion can then fragment to the toxic bromoisophosphoramide mustard which can diffuse to surrounding tissues.…”

“…27 The 2-nitroimidazole portion of TH-302 undergoes electron rearrangement forming a radical anion in regions with less than about 0.5% oxygen. 27 The radical anion can then fragment to the toxic bromoisophosphoramide mustard which can diffuse to surrounding tissues. Therefore, the diffusible toxic bromo-isophosphoramide coupled with doxorubicin or docetaxel may have a heightened effect on the expression of biomarkers gH2aX, cleaved caspase-3 (or -6) and reduction in Ki-67.…”

“…Under aerobic conditions, TH-302 is fragmented to its radical anion and then back oxidized to its pro-drug form. 27 A byproduct of this reaction is the generation of superoxide via the reduction of oxygen. Superoxides can react with free iron generating hydroxyl radicals; these radicals in turn can act as oxidants and damage DNA.…”

Efficacy and safety of the hypoxia-activated prodrug TH-302 in combination with gemcitabine and nab-paclitaxel in human tumor xenograft models of pancreatic cancer

“…18 The 2-nitroimidazole moiety of TH-302 is a substrate for intracellular 1-electron reductases and, when TH-302 is reduced under hypoxic conditions, Br-IPM is released. TH-302 exhibits hypoxia-selective in vitro cytotoxicity cross a wide variety of human cancer cell lines 19 and in vivo anti-tumor efficacy in a panel of human tumor xenograft models. 20,21 Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumors in humans, with a 5-year survival rate of less than 5%.…”

Dual Targeting of Hypoxia and Homologous Recombination Repair Dysfunction in Triple-Negative Breast Cancer

“…Several classes of hypoxia-activated prodrugs (HAP) have been rationally developed to exploit tumor hypoxia (14). These include the clinical stage benzotriazine di-N-oxide HAP tirapazamine (25) and nitrogen mustard prodrugs TH-302 (26) and PR-104 (27), in addition to advanced preclinical compounds such as the tirapazamine analogue SN30000 (28) and a nitro-chloromethylbenzindoline (nitroCBI) that is a prodrug of a potent DNA minor groove alkylator (29). These agents are enzymatically reduced in hypoxic tumor tissue to DNA-damaging metabolites that are selectively toxic to hypoxic cells.…”