Abstract

Methylation at the 2-position of the imidazole ring of IBN (I), a 1, 5-substituted imidazole-containing compound, was carried out to minimize its inhibition of rat cytochrome P450 (CYP)3A activity. The resulting analog 2-MIBN (II) exhibited an inhibitory potency 70-fold weaker (K_i = 25 μM) than that of I (K_i = 0.3 μM) toward CYP3A, the major rat liver microsomal P450 isoform(s) for the metabolism of I and II by rat liver microsomes in the presence of NADPH. The structural modification did not switch the major metabolic pathways for I and II, but significantly decreased the affinity of II to the metabolizing enzyme(s) as reflected by the difference in theirK_i values for CYP3A. Enzyme kinetic studies also demonstrated that I had a lower apparentK_m (0.3 μM) than II (18 μM), but an apparent V_max 14 times lower than II. This finding indicates that methylation at the imidazole ring reduced the affinity of the compound to CYP3A, but increased the catalytic capacity, turning I as a substrate of low K_mvalue but low capacity into a compound of highK_m but high capacity for the metabolism. Our results suggest the significance of substrate concentration in comparing the metabolic stability of compounds with different kinetic parameters. Although higher intrinsic clearance is implied for I when the substrate concentration is below or close to itsK_m value, higher metabolic rate was constantly seen with II over micromolar range. The different kinetic parameters of I and II may also explain the observation that no significant difference in pharmacokinetic behavior was seen after an i.v. administration of I and II to the rat.