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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.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]