Abstract 2263: NADPH-cytochrome P450 reductase reduction of TH-302, a hypoxia-activated prodrug

Abstract

Abstract TH-302 is a 2-nitroimidazole containing inactive prodrug and undergoes a hypoxia specific activation and the release of a DNA crosslinking toxin. This process has been believed to be mediated by the initial reduction by cytochrome P450 reductase (P450R) to the radical anion (RA). In the presence of oxygen (air), the RA is oxidized back to original prodrug, producing superoxide (SO) and the consequent lack of cytotoxicity. In the absence of oxygen (hypoxia), the RA has a longer half life and is presumed to undergo further reduction to an electron rich hydroxyl amine which then eliminates to release the toxin. We now have data which suggest that further reduction may not be necessary for elimination and that TH-302 can release the toxin at the RA stage. We have monitored individually the formation of the RA and the subsequent reactions in an in vitro system using P450R. The rate of initial reduction to the RA was monitored by the production of SO under air. The subsequent reaction was monitored by the disappearance of parental compound under both air and hypoxia by LC/MS. To assess the SO production, we used proxyl fluorescamine as the detecting reagent. TH-302 produced a fluorescent signal rapidly in the presence of NADPH and P450R. This signal was abolished by the addition of superoxide dismutase, confirming the production of SO. TH-302 alone, TH-302 with NADPH or TH-302 with P450R did not generate detectable SO. Pimonidazole (pimo), the classic 2 nitroimidazole hypoxia labeling reagent, is structurally very related to TH-302 but is incapable of an elimination reaction. Pimo produced SO under air at a similar rate to that of TH-302. These results suggest that both TH-302 and pimo are reduced to the RA by P450R at similar rates. To evaluate the 2nd step, we measured the disappearance of parental TH-302 in the presence or absence of NADPH and P450R under either air or hypoxia. TH-302 was very stable under air in the presence of 50 µM NADPH and P450R. Under hypoxia, TH-302 rapidly reacted. Curiously, TH-302 reacted specifically under hypoxia without enzyme with high levels of NADPH (500 µM). This raises the possibility that in cellular systems, non enzymatic activation under hypoxia could be at least part of the mechanism of TH-302 activation. Pimo can't eliminate and has been shown by others to undergo subsequent reductions to the hydroxyl amine. Pimo was similarly stable in the presence of NADPH and P450R under air but was metabolized under hypoxia at much lower rate as compared to TH-302. This dramatic difference between TH-302 and pimo in the rate of subsequent reaction after formation of the RA, suggests two hypotheses. One possibility is that the radical anion of TH-302 is a better substrate for further reduction relative to the structurally related pimo. The other is that TH −302 is eliminating at the radical anion stage rather than requiring further reduction to a hydroxyl amine species. Further efforts to discern between these hypotheses are in progress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2263.