The Hypoxia-Activated ProDrug AQ4N Penetrates Deeply in Tumor Tissues and Complements the Limited Distribution of Mitoxantrone

O. Tredan, A. B. Garbens, A. S. Lalani, I. F. Tannock
2009 Cancer Research  
Hypoxic tumor cells are likely to be resistant to conventional chemotherapy, in large part because many anticancer drugs are unable to penetrate into poorly oxygenated tumor tissue. Here, we used quantitative immunofluorescence to study the distribution of mitoxantrone and AQ4N in tumor tissue. AQ4N is a prodrug activated under hypoxic conditions to AQ4, which is structurally similar to mitoxantrone and inhibits topoisomerase II. We characterized the penetration of mitoxantrone and AQ4N/AQ4
more » ... ne and AQ4N/AQ4 through multilayered cell cultures (MCC) and in relation to blood vessels and hypoxic regions in human tumor xenografts. We also studied tumor growth delay after treatment with each agent alone and with the combination. In both MCC and xenografts, mitoxantrone is taken up by proximal cells and penetrates slowly to distant regions. In contrast, AQ4N rapidly penetrates MCC and tumor tissue in vivo, and AQ4N (or its reduced form AQ4) is detected at high concentration within hypoxic regions. The targeting of mitoxantrone to oxygenated regions and AQ4N/AQ4 to hypoxic tumor regions results in effective drug exposure over the entire tumor after combined treatment and increases tumor growth delay compared with either drug alone. The combination of a clinically used anticancer drug with limited tissue penetration and a structurally related drug activated in regions of tumor hypoxia is an effective strategy to overcome chemoresistance due to the tumor microenvironment. This study supports clinical evaluation of AQ4N in combination with conventional anticancer agents, such as mitoxantrone. [Cancer Res 2009;69(3):940-7] Materials and Methods Drugs and reagents. AQ4N was supplied by Novacea, Inc., as a solution diluted in 0.9% sodium chloride (30 mg/mL, stored at 4jC). Mitoxantrone (Mayne Pharma) was obtained from the hospital pharmacy as a solution diluted in sodium chloride at a concentration of 2 mg/mL. Both drugs were injected i.v. into mice via the tail vein. Purified rat anti-mouse CD31 monoclonal antibody was purchased from BD Pharmigen, and the Cy3-conjugated goat anti-rat IgG secondary antibody was purchased from Jackson Immuno Research Laboratories, Inc. The hypoxia-selective agent EF5 and Cy5-conjugated anti-EF5 antibody were provided by Dr. C.J. Koch (University of Pennsylvania). Cell cultures and tumors. EMT-6 mouse mammary sarcoma cells were obtained originally from Dr. R. Sutherland (University of Rochester). The EMT6 cell line was selected because it readily allows the growth of MCCs, and we have previously studied the penetration of multiple drugs, including mitoxantrone, using this tissue culture model (13). MDA-MB-231 human Requests for reprints: Ian F. Tannock, Princess Margaret Hospital, Suite 5-208,
doi:10.1158/0008-5472.can-08-0676 pmid:19176397 fatcat:hhquwf5krbb33juwn6h7ykwqbi