INITIAL CHARACTERIZATION OF THE MAJOR MOUSE CYTOCHROME-P450 ENZYMES INVOLVED IN THE REDUCTIVE METABOLISM OF THE HYPOXIC CYTOTOXIN 3-AMINO-1,2,4-BENZOTRIAZINE-1,4-DI-N-OXIDE (TIRAPAZAMINE, SR-4233, WIN-59075)

The benzotriazine di-N-oxide SR 4233 (tirapazamine, WIN 59075) is currently in phase I clinical trials as the lead compound in a series of novel and highly selective antitumour hypoxic cytotoxins. Reductive bioactivation is thought to proceed via a one-electron reduced, oxidizing nitroxide radical and also forms the inactive single N-oxide SR 4317 via radical disproportionation or a second one-electron reduction. In mouse fiver microsomes reductive metabolism is catalysed predominantly by cytochrome P450 (70%) and cytochrome P450 reductase (30%). The aim of the present study was to examine which cytochrome P450 isozymes may be involved. Reduction of SR 4233 to SR 4317 was monitored by HPLC analysis. Metabolism by microsomes from both control and dexamethasone-induced BALB/c male mice was 70% inhibited by carbon monoxide. The cytochrome P450 inhibitor SKF 525A, following aerobic preincubation, also inhibited SR 4233 reduction by 58%. Reduction was induced 2-3-fold by dexamethasone and was not accountable by increases in cytochrome P450 reductase or DT-diaphorase. The induction data and the greater degree of inhibition of SR 4233 reduction by metyrapone compared to alpha-naphthoflavone suggested a possible involvement of Cyp2b, Cyp2c and Cyp3a cytochrome P450 subfamilies. Both Cyp3a (7.4-fold) and Cyp2b (1.8-fold) type enzymes were shown by western immunoblot analysis to be induced by dexamethasone, the latter correlating more closely with increased SR 4233 reductase activity and also with the 2-fold induction of benzphetamine N-demethylase, a Cyp2b-type enzyme. No inhibition of SR 4233 reduction was seen with erythromycin or cyclosporin A which act as substrates/inhibitors for Cyp3a-type enzymes, but inhibition was seen with p-nitrophenol and tolbutamide which are substrates for Cyp2e1- and Cyp2c-type enzymes, respectively (11% and 25% inhibition in induced microsomes). SR 4233 itself inhibited benzphetamine N-demethylase, which is catalysed by Cyp2b-type enzymes but not erythromycin N-demethylase which is catalysed by Cyp3a-type isoforms. Immunoinhibition studies with epitope specific monoclonal antibodies were consistent with the major involvement of phenobarbitone- and steroid-inducible products of the Cyp2b and Cyp2c subfamilies. These forms contributed at least 53% and 26%, respectively, of the cytochrome P450-associated SR 4233 reductase activity in the induced microsomes. The findings support our earlier conclusion that cytochrome P450 is the major SR 4233 reductase in mouse liver and provides leads as to the possible involvement of specific isoforms in human tumours and normal tissues.