ANTIPYRINE AS A PROBE FOR HUMAN OXIDATIVE DRUG-METABOLISM - IDENTIFICATION OF THE CYTOCHROME-P450 ENZYMES CATALYZING 4-HYDROXYANTIPYRINE, 3-HYDROMETHYLANTIPYRINE, AND NORANTIPYRINE FORMATION

Background and objective: Antipyrine has been widely used as a probe d rug for human oxidative drug metabolism. To evaluate the role of antip yrine as a model drug, me have identified the cytochrome P450 enzymes involved in 4-hydroxyantipyrine, 3-hydroxymethylantipyrine, and norant ipyrine formation. Methods: We used the following methods for this stu dy: (1) determination of enzyme kinetics for antipyrine metabolite for mation in human liver microsomes, (2) inhibition studies with antibodi es and inhibitors, and (3) formation of metabolites by stable expresse d human P450 enzymes. Results: Antipyrine biotransformation could be d escribed by Michaelis-Menten kinetics: norantipyrine: maximum rate of metabolite formation (V-max), 0.91 +/- 0.04 nmol . mg(-1). min(-1); Mi chaelis-Menten constant (K-m), 19.0 +/- 0.8 mmol/L; 4-hydroxyantipyrin e: V-max, 1.54 +/- 0.08 nmol . mg(-1)min(-1); K-m, 21.2 +/- 0.4 mmol/L ; 3-hydroxymethylantipyrine: V-max, 0.83 +/- 0.04 nmol . mg(-1). min(- 1); K-m, 39.6 +/- 2.5 mmol/L. Antibodies against CYP3A4 inhibited the formation of 4-hydroxyantipyrine by 25% to 65%. LKM-2 antibodies (anti -CYP2C) caused a 75% to 100% inhibition of norantipyrine and a 58% to 80% inhibition of 3-hydroxymethylantipyrine formation. Sulfaphenazole inhibited the formation of 3-hydroxymethylantipyrine and norantipyrine by about 50%. Furafylline and fluvoxamine inhibited norantipyrine, 4- hydroxyantipyrine, and 3-hydroxymethylantipyrine formation by about 30 %, 30%, and 50%, respectively. Ketoconazole reduced formation of noran tipyrine, 3-hydroxymethylantipyrine, and 4-hydroxyantipyrine by up to 80%. Formation in stable expressed enzymes indicated involvement of CY P1A2, CYP2B6, CYP2C, and CYP3A4 in metabolite formation. Conclusion: A ntipyrine metabolites are formed by at least six hepatic cytochrome P4 50 enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C18, and CYP3A4). 4-Hy droxylation is mainly catalyzed by CYP3A4 and, to a lesser extent, by CYP1A2. The CYP2C subfamily contains the predominant enzymes for noran tipyrine formation, and CYP1A2 is also involved. Formation of 3-hydrox ymethylantipyrine is mediated by CYP1A2 and CYP2C9. Because several cy tochrome P450 enzymes are involved in the formation of each metabolite , antipyrine is not well suited as a probe for distinct human cytochro me P450 enzymes.