Supplementary Tables S1-S5 from Hypoxia-Selective Targeting by the Bioreductive Prodrug AQ4N in Patients with Solid Tumors: Results of a Phase I Study

Abstract 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/m2 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 μg/g) and head and neck (mean 0.65 μg/g) tumors; 22 of 32 patients had tumor AQ4 concentrations ≥0.2 μg/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 μg/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.

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 μg/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. [Mol Cancer Ther 2009;8(12):3266–75]

525 AQ4N (banoxantrone; 1,4-Bis{[2-(dimethylamino)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione bis-N-oxide) is a novel therapeutic agent that targets the treatment-resistant hypoxic fraction of tumors. AQ4N is a highly soluble and penetrating prodrug that is enzymatically reduced to a cytotoxic DNA-binding agent, AQ4, preferentially in conditions of low oxygen tension. Combination therapy using AQ4N in addition to radiation and/or chemotherapy targets both the oxygenated and hypoxic regions of tumors, increasing effectiveness of treatment in preclinical models. AQ4N is well-tolerated in man and is currently in Phase I clinical trial. A major contributor to acquired drug resistance is the multi-drug resistance phenotype (MDR) caused by overexpression of ABC family transmembrane protein pumps. Substrates for MDR include anthracyclines (eg daunorubicin) and anthracenediones (eg mitoxantrone). We have investigated whether AQ4N and AQ4, which are structurally related to mitoxantrone, are substrates for the prototypical MDR protein PGP/MDR1. The intrinsic fluorescence of these molecules was used to directly evaluate transport by PGP using flow cytometry. The measurement of steady state levels in MDR1-positive cell lines revealed no sensitivity to the PGP inhibitor verapamil for AQ4, in contrast to a 3-5-fold increase for mitoxantrone (indicative of cellular accumulation). Direct measurements of efflux from isogenically matched PGP-overexpressing cells confirmed no verapamil-dependent efflux of AQ4, contrasting sharply with a dramatic efflux of mitoxantrone and daunorubicin. Survival assays using the MDR1-positive cells revealed no differential toxicity of AQ4 or AQ4N compared to the control parental cell lines, whereas mitoxantrone showed reduced sensitivity in the PGP-overexpressing line, demonstrating a functional relevance of the efflux observations. The potential roles of other transmembrane pumps in transport of AQ4, and the significance of these findings in in vivo models are also under further investigation. These data suggest that AQ4N is not a substrate for MDR, which is one of the major barriers to the effectiveness of anthraquinones and other agents. AQ4N is also effective under conditions of hypoxia, another well-known characteristic of treatment resistant tumours. Taken together, these features indicate significant clinical potential, and the utility of AQ4N, both as monotherapy and in combination with established regimes, is under evaluation in a range of Phase I trials.

An ideal treatment for lymphoma and leukemia is the use of highly selective compounds to eliminate diseased cells with minimal systemic toxicity to normal tissues (cf. imatinib mesylate; Gleevec). AQ4N (1,4 bis[[2-(dimethylamino)ethylamino}-5,8-hydroxyanthracene-9,10-dione bis N-oxide) is designed to have little or no toxicity until selectively activated by bioreduction in hypoxic cells to AQ4 (reduced AQ4N), a highly potent DNA topoisomerase II inhibitor. In a series of studies, AQ4 has been shown to have potent cytotoxicity on lymphoma and leukemia cell lines in vitro and AQ4N has selective activity in lymphatic tissues in vivo. The IC50 of AQ4, was 0.63, 12.0, 90.5 and 150 nM in Namalwa, Daudi, Ramos, and Raji human lymphoma cell lines and 1.0, 6.0, and 20 nM in HL-60, KG1a and K562 human leukemia cell lines. On several of the tumor lines the activity of AQ4 was more potent than doxorubicin (i.e. IC50 for Dox was 20.3 nM on Namalwa). AQ4N also had anti-proliferative activity at μM levels indicating a potential mechanism for activation by these cell lines. In repeat dose toxicology studies of AQ4N in pigmented rats and cynomolgus monkeys, the maximum tolerated doses (MTD; rats: 20 mg/kg/wk x 6; monkeys 6 mg/kg/wk x 6) resulted in lymphoid tissue atrophy. A decrease in lymphocyte levels and atrophy of the spleen, thymus, and mandibular and mesenteric lymph nodes were observed at terminal sacrifice of the animals. In contrast, there was an absence of myelosuppression and only mild neutropenia and minor bone marrow atrophy at the MTD. Administration of radiolabeled AQ4N (14C-benzene) to pigmented rats and cynomolgus monkeys indicated persistence of AQ4N radioactivity in lymphoid tissues for several weeks after a single dose (rats: 20 mg/kg (130–140 μCi/kg); monkeys: 10 mg/kg (135 μCi/kg)). For example, in rats the half-life of radioactive AQ4N in the spleen was 538 hrs with 0.9 μg AQ4N/g tissue (spleen) remaining one week after dosing. Monkeys demonstrated a similar effect with 76.5–86.8 μg AQ4N/g tissue observed in the spleen one week after treatment. Other tissues contained significantly less radioactive AQ4N with the exception of the liver (67.9–78.6 μg AQ4N/g tissue) and adrenal cortex (78.7–86.6 μg AQ4N/g tissue). While some hypertrophy and eosinophila was observed in the adrenal glands, liver toxicity was not observed at the MTD in the repeat dose cynomolgus monkey toxicology study. Overall, these initial findings indicate that AQ4N is active in vitro against human lymphoma and leukemia cell lines and selectively targets lymphoid tissues in vivo suggesting the potential benefit of AQ4N in the treatment of lymphoproliferative diseases.

4134 Based on promising pre-clinical data the bioreductive topoisomerase II inhibitor AQ4N (1,4-bis-{[2-(dimethylamino-N-oxide)ethyl]amino}5,8-dihydroxyanthracene-9,10-dione) is currently in Phase I clinical trial in combination with radiotherapy. Increasingly, curative-intent interventions utilise chemo-radiation protocols. The aim of this study was to evaluate AQ4N-potentiated chemo (cisplatin)-radiotherapy response in human lung tumour xenografts. The Calu-6 cell line was selected for study from a panel of lung lines. This was on the basis of IC50 values for: (1) the toxic metabolite of AQ4N (AQ4); 0.2μM (vs. average for panel of 0.09 μM), and (2) cisplatin; 0.4 μM (vs. average 1.17μM). Calu-6 xenografts were established from the ID implant of 2x106 cells in 0.1ml of a 1:1 matrigel: serum free RPMI mix. Tumours were treated when a size of 240-280mm3 was attained. Fractionated radiotherapy was administered over 3 or 5 days (2 Gy per fraction) and cisplatin was given at a dose of 2mg/kg 6 h after the first fraction. To evaluate the effect of AQ4N on this treatment response, a single 60mg/kg dose of AQ4N was given 30 minutes prior to the first radiation dose. The growth delay for tumours to achieve a volume four times that at the time of treatment was 9 ± 2 days (mean ± se) for tumours treated with 3 x 2Gy + cisplatin compared with untreated controls. Additional treatment with AQ4N increased this to 14 ± 2 days. This growth delay was identical to that achieved with 5 x 2 Gy + cisplatin, suggesting that the benefit of AQ4N treatment equates to two 2 Gy fractions. AQ4N metabolism was evaluated in S9 fractions derived from Calu-6 cells grown in vitro, or as xenografts in nude mice, using HPLC. The rates of formation of AQ4 and the intermediate AQ4M were 15 and 132 μmol/min/mg protein for cells in culture, respectively. Using lysates of Calu-6 xenografts, AQ4 and AQ4M were generated at rates of 1± 0.2 and 16± 5 μmol/min/mg. Addition of the haem precursor hemin enhanced the rates of AQ4 and AQ4M accumulation by 11 and 2 fold respectively in the xenograft lysates, perhaps suggesting a greater reliance on cytochrome p450 proteins for the conversion of AQ4M to AQ4 than in the initial conversion of AQ4N to AQ4M in vivo. These data support the future clinical evaluation of AQ4N in chemo-radiation regimes including cisplatin-based chemo-radiation in carcinoma of the lung.