In vivo activation of the hypoxia-targeted cytotoxin AQ4N in human tumor xenografts.

AQ4N (banoxantrone) is a prodrug that, under hypoxic conditions, is enzymatically converted to a cytotoxic DNA-binding agent, AQ4. Incorporation of AQ4N into conventional chemoradiation protocols therefore targets both oxygenated and hypoxic regions of tumors, and potentially will increase the effectiveness of therapy. This current pharmacodynamic and efficacy study was designed to quantify tumor exposure to AQ4 following treatment with AQ4N, and to relate exposure to outcome of treatment. A single dose of 60 mg/kg AQ4N enhanced the response of RT112 (bladder) and Calu-6 (lung) xenografts to treatment with cisplatin and radiation therapy. AQ4N was also given to separate cohorts of tumor-bearing mice 24 hours before tumor excision for subsequent analysis of metabolite levels. AQ4 was detected by high performance liquid chromatography/mass spectrometry in all treated samples of RT112 and Calu-6 tumors at mean concentrations of 0.23 and 1.07 microg/g, respectively. These concentrations are comparable with those shown to be cytotoxic in vitro. AQ4-related nuclear fluorescence was observed in all treated tumors by confocal microscopy, which correlated with the high performance liquid chromatography/mass spectrometry data. The presence of the hypoxic marker Glut-1 was shown by immunohistochemistry in both Calu-6 tumors and RT112 tumors, and colocalization of AQ4 fluorescence and Glut-1 staining strongly suggested that AQ4N was activated in these putatively hypoxic areas. This is the first demonstration that AQ4N will increase the efficacy of chemoradiotherapy in preclinical models; the intratumoral levels of AQ4 found in this study are comparable with tumor AQ4 levels found in a recent phase I clinical study, which suggests that these levels could be potentially therapeutic.

Hypoxia-selective targeting by the bioreductive prodrug AQ4N in patients with solid tumors: results of a phase I study.

PURPOSE: AQ4N is a novel bioreductive prodrug under clinical investigation. Preclinical evidence shows that AQ4N penetrates deeply within tumors and undergoes selective activation to form AQ4, a potent topoisomerase II inhibitor, in hypoxic regions of solid tumors. This proof-of-principle, phase I study evaluated the activation, hypoxic selectivity, and safety of AQ4N in patients with advanced solid tumors. EXPERIMENTAL DESIGN: Thirty-two patients with cancer (8 glioblastoma, 9 bladder, 8 head and neck, 6 breast, and 1 cervix) received a single 200 mg/m(2) dose of AQ4N before elective surgery. AQ4 and AQ4N levels in 95 tissues (tumor, healthy tissue) were assessed by liquid chromatography-tandem mass spectrometry. Tissue sections were also analyzed for AQ4 fluorescence using confocal microscopy, and for expression of the hypoxia-regulated glucose transporter, Glut-1. RESULTS: Activated AQ4 was detected in all tumor samples with highest levels present in glioblastoma (mean 1.2 microg/g) and head and neck (mean 0.65 microg/g) tumors; 22 of 32 patients had tumor AQ4 concentrations > or = 0.2 microg/g, levels previously shown to be active in preclinical studies. In 24 of 30 tumor samples, AQ4 was detected at higher concentrations than in adjacent normal tissue (tumor to normal ratio range 1.1-63.6); distant skin samples contained very low concentrations of AQ4 (mean 0.037 microg/g). Microscopic evaluation of tumor sections revealed that AQ4 colocalized within regions of Glut-1+ hypoxic cells. CONCLUSIONS: AQ4N was activated selectively in hypoxic regions in human solid tumors. Intratumoral concentrations of AQ4 exceeded those required for activity in animal models and support the evaluation of AQ4N as a novel tumor-targeting agent in future clinical studies.