Enzymology of the reduction of the novel fused pyrazine mono-N-oxide bioreductive drug, RB90740 roles for P450 reductase and cytochrome b(5) reductase

RB90740 is the lead compound in a series of fused pyrazine mono-N-oxide bioreductive drugs. These agents have potential application in cancer therapy, since they are more toxic to hypoxic than to aerobic cells as a consequence of their bioactivation by cellular reductase enzymes within the hypoxic regions of a tumour. In this study, mouse liver microsomes have been used to characterise the enzymology of the reductive activation of RB90740. Under hypoxic conditions, the reduction of RB90740 to its stable 2-electron reduced product RB92815 was supported by both NADH and NADPH, the former supporting a rate approximately 80% of the latter. Combining the two cofactors had no additive effect. Neither carbon monoxide nor metyrapone inhibited reduction of RB90740, indicating that P450 isozymes were not involved in the reduction of this compound. 2'AMP, an inhibitor of P450 reductase, did not inhibit formation of RB92815, whereas DPIC, another inhibitor but with a different mode of action, inhibited both the NADH and NADPH-dependent reduction of RB90740. Similarly, two selective inhibitors of NADH:cytochrome b(5) reductase, pHMB and PTU, completely inhibited both the NADH and NADPH-dependent reduction of RB90740. Our findings implicate P450 reductase, cytochrome b(5) reductase, and cytochrome b(5) in the activation of this compound. However, there is no clear relationship between the intracellular levels of P450 reductase and cytochrome b(5) reductase and the hypoxic toxicity of RB90740, which implies that other factors, in addition to drug activation, play a major role in controlling the toxicity of this particular bioreductive drug.