Identification of cytochrome P450 and arylamine N-acetyltransferase isoforms involved in sulfadiazine metabolism.

Sulfadiazine hydroxylamine has been postulated to be the mediator of the greatly increased rates of adverse reactions to sulfadiazine experienced by people with human immunodeficiency virus infection. Therefore, we investigated the in vitro human cytochrome P450 (P450) and N-arylamine acetyltransferase (detoxification) metabolism of sulfadiazine. Formation of both the hydroxylamine and 4-hydroxy sulfadiazine was NADPH-dependent in human liver microsomes (HLM). The average K(m) (+/-S.D.) and V(max) in HLM (n = 3) for hydroxylamine formation was 5.7 +/- 2.2 mM and 185 +/- 142 pmol/min/mg, respectively. Significant (p < 0.05) inhibition by selective P450 isoform inhibitor sulfaphenazole (2.1 microM; CYP2C9) indicated a role for CYP2C9 in the formation of the hydroxylamine. Hydroxylamine formation correlated strongly with tolbutamide 4-hydroxylation (CYP2C8/9) in HLM (r = 0.76, p < or = 0.004, n = 12). Fluconazole (CYP2C9/19 and CYP3A4 inhibitor at clinical concentrations) inhibited hydroxylamine formation, with one-enzyme model K(i) estimates ranging from 9 to 40 microM. Acetylation of sulfadiazine in human liver cytosol (HLC) correlated strongly with NAT2 activity as measured by sulfamethazine N-acetylation (r = 0.92, p < 0.001, n = 12). The average K(m) (+/-S.D.) and V(max) in HLC (n = 3) was 3.1 +/- 1.7 mM and 221.8 +/- 132.3 pmol/min/mg, respectively. The polymorphic acetylation of sulfadiazine may predispose slow acetylator patients to adverse reactions to sulfadiazine. On the basis of our K(i) estimates, clinical fluconazole concentrations of 25 microM would produce decreases of 40 to 70% in hepatic-mediated hydroxylamine production. Therefore, we predict that fluconazole may prove useful in the clinic as an in vivo inhibitor of sulfadiazine hydroxylamine formation to suppress adverse reactions to this drug.