The role of cytochrome P450 and cytochrome P450 reductase in the reductive bioactivation of the novel benzotriazine di-N-oxide hypoxic cytotoxin 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233, WIN 59075) by mouse liver

Abstract

SR 4233 or WIN 59075 (3-amino-1,2,4-benzotriazine-1,4-dioxide) is a novel and highly selective hypoxic cell cytotoxin requiring reductive bioactivation for its impressive antitumour effects. Expression of appropriate reductases will contribute to therapeutic selectivity. Here we provide more detailed information on the role of cytochrome P450 and cytochrome P450 reductase in SR 4233 reduction by mouse liver microsomes. Reduction of SR 4233 to the mono-N-oxide SR 4317 (3-amino-1,2,4-benzotriazine-1-oxide) is NADPH, enzyme and hypoxia dependent. An inhibitory antibody to cytochrome P450 reductase decreased the microsomal SR 4233 reduction rate by around 20%. Moreover, studies with purified rat cytochrome P450 reductase showed unequivocally that this enzyme was able to catalyse SR 4233 reduction at a rate of 20-30% of that for microsomes with equivalent P450 reductase activity. Exposure to the specific cytochrome P450 inhibitor carbon monoxide (CO) inhibited microsomal reduction by around 70% and CO plus reductase antibody blocked essentially all activity. Additional confirmation of cytochrome P450 involvement was provided by the use of other P450 ligands: beta-diethylaminoethyl diphenylpropylacetate hydrochloride gave a slight stimulation while aminopyrine, n-octylamine and 2,4-dichloro-6-phenylphenoxyethylamine were inhibitory. Induction of SR 4233 reduction was seen with phenobarbitone, pregnenalone-16-alpha-carbonitrile and beta-napthoflavone, suggesting that cytochrome P450 subfamilies IIB, IIC and IIIA may be involved. Since cytochrome P450 and P450 reductase catalyse roughly 70 and 30%, of mouse liver microsomal SR 4233 reduction respectively, we propose that expression of these and other reductases in normal and tumour tissue is likely to be a major factor governing the toxicity and antitumour activity of the drug.