Identification of CYP isozymes involved in benzbromarone metabolism in human liver microsomes

Benzbromarone (BBR) is metabolized to 1'-hydroxy BBR and 6-hydroxy BBR in the liver. 6-Hydroxy BBR is further metabolized to 5,6-dihydroxy BBR. The aim of this study was to identify the CYP isozymes involved in the metabolism of BBR to 1'-hydroxy BBR and 6-hydroxy BBR and in the metabolism of 6-hydroxy BBR to 5,6-dihydroxy BBR in human liver microsomes. Among 11 recombinant P450 isozymes examined, CYP3A4 showed the highest formation rate of 1'-hydroxy BBR. The formation rate of 1'-hydroxy BBR significantly correlated with testosterone 6β-hydroxylation activity in a panel of 12 human liver microsomes. The formation of 1'-hydroxy BBR was completely inhibited by ketoconazole in pooled human liver microsomes. On the other hand, the highest formation rate of 6-hydroxy BBR was found in recombinant CYP2C9. The highest correlation was observed between the formation rate of 6-hydroxy BBR and diclofenac 4'-hydroxylation activity in 12 human liver microsomes. The formation of 6-hydroxy BBR was inhibited by tienilic acid in pooled human liver microsomes. The formation of 5,6-dihydroxy BBR from 6-hydroxy BBR was catalysed by recombinant CYP2C9 and CYP1A2. The formation rate of 5,6-dihydroxy BBR was significantly correlated with diclofenac 4'-hydroxylation activity and phenacetin O-deethylation activity in 12 human liver microsomes. The formation of 5,6-dihydroxy BBR was inhibited with either tienilic acid or α-naphthoflavone in human liver microsomes. These results suggest that (i) the formation of 1'-hydroxy BBR and 6-hydroxy BBR is mainly catalysed by CYP3A4 and CYP2C9, respectively, and (ii) the formation of 5,6-dihydroxy BBR is catalysed by CYP2C9 and CYP1A2 in human liver microsomes.