Inhibition of DT-diaphorase (NAD(P)H:quinone oxidoreductase, EC 1.6.99.2) by 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and flavone-8-acetic acid (FAA): implications for bioreductive drug development

The tumour blood flow inhibitors 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and flavone-8-acetic acid (FAA) have been shown to potentiate the antitumour activity of several bioreductive drugs in vivo. Whilst the induction of hypoxia as a result of blood flow inhibition is presumed to be responsible for enhancing the activity of bioreductive drugs, no studies have examined potential interactions between DMXAA or FAA and enzymes involved in bioreductive drug activation. Both FAA and DMXAA are competitive inhibitors of the enzyme DT-diaphorase (NAD(P)H:Quinone oxidoreductase EC 1.6.99.2) with respect to NADH, with Ki values of 75 and 20 microM, respectively. Cytochromes P450 reductase and b5 reductase activities are not significantly inhibited by FAA, whereas DMXAA partially inhibits cytochrome b5 reductase activity. The cytotoxicity of the indoloquinone EO9 (3-hydroxymethyl-5-aziridinyl-1-methyl-2-[1H-indole-4,7-dione] prop-beta-en-alpha-ol) against DLD-1 (IC50 = 0.32+/-0.08 microM) was significantly reduced when combinations of EO9 and FAA (IC50 = 12.26+/-5.43 microM) or DMXAA (IC50 > 40 microM) were used. In the case of menadione (which is detoxified by DT-diaphorase), combinations of menadione with FAA or DMXAA were more toxic (IC50 = 7.46+/-2.22 and 9.46+/-1.70 microM, respectively) than menadione alone (IC50 = 22.02+/-1.59 microM). Neither DMXAA nor FAA potentiated the activity of tirapazamine in vitro. These results suggest that the use of DMXAA and FAA to potentiate the activity of bioreductive drugs where DT-diaphorase plays a central role in either activation or detoxification may be inappropriate. The fact that FAA in particular does not inhibit other key enzymes involved in bioreductive activation suggests that it may be useful in terms of identifying DT-diaphorase-activated prodrugs.