Tirapazamine (TPZ) is a hypoxia selective bioreductive drug currently in advanced clinical trials in combination with chemoradiation. However, high toxicity restricts the number of doses of TPZ that can be administered, and poor extravascular transport limits diffusion of TPZ to target hypoxic regions of tumors. We have developed a series of benzotriazine(BT)-1-oxides that selectively enhance hypoxic cell killing by TPZ. Screening of 96 BT-1-oxides identified 16 analogues that produced hypoxic TPZ potentiation ratios [TPR = (IC50 TPZ)/(IC50 TPZ + BT-1-oxide)] of >5, and hypoxic cytotoxicity ratios [HCR = aerobic(IC50 TPZ + BT-1-oxide)/hypoxic (IC50 TPZ + BT-1-oxide)] of up to 3000 (TPZ alone HCR = 66 ± 10). No aerobic potentiation was observed. Hypoxic potentiation was confirmed by clonogenic assay of HT29 cell suspensions. The BT-1-oxides SN29112 and SN29254 (300 μM) provided an additional 4 logs of cell killing, relative to TPZ alone (30 μM), and were not cytotoxic in the absence of TPZ. Induction of the phosphorylated histone γH2AX was used to confirm that potentiation of TPZ hypoxic cell killing by the BT-1-oxides results from increased DNA breakage. There are two mechanisms that could contribute: (i) enhanced metabolism of TPZ; (ii) BT-1-oxide oxidation of TPZ-induced DNA radicals. While the BT-1-oxides increased the rate of TPZ metabolism, this was insufficient to account for the increase in hypoxic cell killing. Radiosensitization of hypoxic cells was used as a measure of DNA radical oxidizing efficiency. Radiosensitizing efficiency of the BT-1-oxides varied - SN29254 was as efficient a radiosensitizer as misonidazole (on a molar basis). These results suggest that DNA radical oxidation is the main mechanism of TPZ potentiation by the BT-1-oxides. The maximum tolerated dose (MTD) of SN29112 (0.24 mmol/kg) was decreased to 0.18 mmol/kg when administered with TPZ (0.13 mmol/kg; 42% MTD). Plasma pharmacokinetics at these doses showed that concentrations of BT-1-oxide providing potentiation in cell cultures are achievable in vivo. Interestingly, co-administration of SN29112 with TPZ slowed the plasma clearance of both TPZ and its major metabolite, the BT-1-oxide SR4317, increasing the AUC for both. The net effect of this should be prolonged exposure of tumor cells to TPZ and potentiators (both SN29112 and SR4317). The results of tumor excision assays in progress will be presented.

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