Approximately 50% of patients with adult acute lymphoblastic leukemia (ALL) relapse after initial chemotherapy. The introduction of novel chemotherapeutics that target leukemia or leukemia-harboring tissues could improve response rates. AQ4N (banoxantrone; 1,4 bis[[2-(dimethylamino)ethyl]amino]-5,8-dihydroxyanthracene-9,10-dione bis N-oxide) is designed to have anti-tumor activity following a bioreduction step to AQ4, a DNA topoisomerase II inhibitor. Studies in rats and monkeys revealed that multiple doses of 10–20 mg/kg AQ4N were well tolerated. However, lymphopenia and atrophy of lymph nodes and spleen suggested selective activity in lymphoid cells. We postulated that AQ4N might have therapeutic potential in ALL. In a proliferation assay, using 3 previously established ALL cell lines, we compared in vitro anti-leukemic activity of AQ4N and AQ4 with daunorubicin, a commonly used component of induction therapy. AQ4 and daunorubicin displayed similar in vitro activity with IC50 values of 2–9 nM. AQ4N displayed little activity with IC50 values of 600–2,500nM. Subsequently, we evaluated in vivo efficacy of AQ4N in a xenotransplant model of human ALL. Eighteen NOD/scid mice were inoculated with primary human ALL cells. Leukemic cell counts (LCC) in peripheral blood were determined weekly. Engraftment was observed 23 days after inoculation and LCC increased exponentially. After 55 days mean LCC reached 0.5x106 cells/mL (range 0.1–0.9) and treatment was started. The cohort was randomized into 6 groups of 3 animals. Each group received three daily i.v. injections. Three groups received 3x20, 3x60 or 3x120 mg/kg AQ4N, respectively. Two groups received 3x2.0 or 3x5.0 mg/kg daunorubicin, respectively. Control mice received saline. Six days after the first injection, AQ4N and daunorubicin appeared equally effective. In the AQ4N treated groups mean LCC had decreased from 0.41, 0.48 and 0.55x106/mL to 0.016, 0.013 and 0.005x106/mL in the 20, 60 or 120 mg/kg treated groups (94.2, 97.3 and 99.2% reduction, respectively). In daunorubicin treated animals, mean LCC decreased from 0.15 and 0.17x106/mL to 0.014 and 0.004 x106/mL in the 2.0 or 5.0 mg/kg treated groups (90.6 and 97.2% reduction, respectively). Eleven days after the first administration, mean LCC remained at 0.01, 0.02 and 0.03x106/mL in the 20, 60 or 120 mg/kg AQ4N treated groups and at 0.02x106/mL in the group that received 5.0 mg/kg daunorubicin. However, in the group that received 2.0 mg/kg daunorubicin LCC had returned to pre-treatment values and leukemia progression was resumed. LCC in control treated animals increased throughout the study. In AQ4N treated animals a strong (90–99%) decrease of murine leukocyte counts occurred 6 to 11 days after treatment, supporting the hypothesis that AQ4N exerts specific activity in the leukocytic compartment. In contrast, in daunorubicin treated animals, murine leukocyte counts remained unaffected. The dose regimens used in this study were above the MTD in NOD/scid mice as toxicity was observed in all AQ4N and the 5.0 mg/kg daunorubicin treated groups. However, this systemic toxicity was a specific artefact for the NOD/scid strain as administration of three doses of 120 mg/kg AQ4N to BALB/c mice did not result in any toxicity. In conclusion, AQ4N may be a promising agent for the treatment of human ALL.

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