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Summary Preclinical studies demonstrate that in vivo AQ4N enhances the anti-tumour effects of radiation and chemotherapeutic agents with a dose-modifying factor of approximately 2.0. With careful scheduling no, or very little, additional normal tissue toxicity should be observed. AQ4N is a bioreductive prodrug of a potent, stable, reduction product which binds non-covalently to DNA, facilitating antitumour activity in both hypoxic and proximate oxic tumour cells. AQ4N is clearly different in both its mechanism of action and potential bystander effect compared to previously identified bioreductive drugs. In particular AQ4N is the only bioreductive prodrug topoisomerase II inhibitor to enter clinical trials. Targeting this enzyme, which is crucial to cell division, may help sensitize tumours to repeated (fractionated) courses of radiotherapy. This is because in principle, the bioreduction product of AQ4N can inhibit the topoisomerase activity of hypoxic cells as they attempt to re-enter the cell cycle.

S-y AQ4N (l,4-bLs-q25ddimthyai oo y}amin5dydroxy-anthrane-9,10-dione) is a novel alkylaminoanthraquinone N-oxide which, on reducin, forms a stable DNA affinic cytotoxi compound AQ4. The The failure to cure tumours with radiotherapy and chemotherapy has been attributed in part to the presence of treatment-resistant subpopulations of hypoxic tumour cells (Bush et at., 1978). Bioreductive agents provide a novel approach to this problem: reduction of the prodrug within a hypoxic cell to produce a cytotoxic metabolite should selectively target this sub population within tumours. Since normal tissus contain few, if any, poorly oxygenated cells, systemic reductive activation and its attendant toxicity should be minimal. Several classe of bioreductive agents have been described, icluding the nitroimidazoles, benzotriazene di-N-oXides and mitosenes (Workman, 1992). Using a different class of compounds, i.e. the anthraquinones, we have developed a novel alkylamnoanthraquinone N-oxide, AQ4N (1,4-bis-{[2-(di-methylamino-N-oxide)ethylarmino}5,8-dihydroxyanthracene-9,10-ione), which is susceptible to reduction under hypoxic conditions (Patterson, 1993). AQ4N is a weak DNA-binding agent and weak topoisomerase H inhibitor, critically, the electrically neutral N-oxide function prevents stable binding to the DNA helix (Patterson, 1993). In contrast, the reduction product of AQ4N, i.e. AQ4 (1,4-bis-{[2-(dimethylamino) ethylnamino}5,8-dihydroxyanthracene-9,10-dione) (Figure 1), is a cationic compound with high affinity for DNA. Interaction of AQ4 with DNA is facilitated by the planar, eletrondeficient antraquinone chromophore intercalating between adjcent DNA bases. Tlis complex is further stabiised by electrostatic interactions and hydrogen bonding with the deoxyribose phosphate backbone, as has been observed for similar anthraquinones (Denny and Wakelin, 1990 al., 1992). This will allow metabolism of the drug in cells which will contribute, when the hypoxic stimulus is removed, to the main growth frction within the tumour. Secondly, the drug can be combined with an agent which is selectively toxic to well-oxygenated cells to evahuate the effect of targeting the two subpopulations of cells (Brown and Lemmon, 1991;Grau and Overgaard, 1991). We have used both these approaches to study the effect of AQ4N on tumour growth in vivo. Firstly, we tested the anti-tumour effect of AQ4N when combined with hypobaric hypoxia. Following this we combined AQ4N with radiation as both single and fractionated doses. Materiak an mth Tumour systemThe T50/80 tumour is a poorly differentiated mammary carcinoma which arose in a B6D2FI mouse. Tumour and breeding colonies for mice were obtained from Dr JV Moore, Paterson Laboratory, Christie Hospital, Manchster, UK.Male B6D2FI mice aged 8-12 weeks were used for all studies, which were carried out in accordance with the UK. Animals (Scientific Procedures) Act 1986. The tumour has been maintained by intadermal passage for up to ten passages and then re-establshed from frozm stock. Tumour brei (0.05 ml) w...

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SummaryThe ability of the bioreductive drugs AQ4N and tirapazamine to enhance the anti-tumour effect of cyclophosphamide was assessed in three murine tumour models. In male BDF mice implanted with the T50/80 mammary carcinoma, AQ4N (50-150 mg kg -1 ) in combination with cyclophosphamide (100 mg kg -1 ) produced an effect equivalent to a single 200 mg kg -1 dose of cyclophosphamide. Tirapazamine (25 mg kg -1 ) in combination with cyclophosphamide (100 mg kg -1 ) produced an effect equivalent to a single 150 mg kg -1 dose of cyclophosphamide. In C3H mice implanted with the SCCVII or RIF-1 tumours, enhancement of tumour cell killing was found with both drugs in combination with cyclophosphamide (50-200 mg kg -1 ); AQ4N (50-200 mg kg -1 ) produced a more effective combination than tirapazamine (12.5-50 mg kg -1 ). Unlike tirapazamine, which showed a significant increase in toxicity to bone marrow cells, the combination of AQ4N (100 mg kg -1 ) 6 h prior to cyclophosphamide (100 mg kg -1 ) resulted in no additional toxicity towards bone marrow cells compared to that caused by cyclophosphamide alone. In conclusion, AQ4N gave a superior anti-tumour effect compared to tirapazamine when administered with a single dose of cyclophosphamide (100 mg kg -1 ).