CYP3A1/2 and 2C9/10 in rat liver microsomes mediate 22α-and 24-hydroxylation of 18α-glycyrrhetic acid
GAO Kai, WEI Yu, YANG Jing, DUAN Xu-hua
2007, 12(11):
1255-1260.
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AIM: To identify the cytochrome P450 isoforms involved in 22 α-hydroxylation and 24-hydroxylation of 18α-glycyrrhetic acid (GA) in rat livers. METHODS: Kinetic analysis of the rates of formation of monohydroxylated metabolites of GA, including 22 α-hydro-GA and 24-hydro-GA, was performed using rat liver microsomes at substrate concentrations ranging from 25 to 200 μmol/L. Seven selective inhibitors or substrate probes specific for different CYP isoforms were applied for screening the isoform (s) responsible for mono-hydroxylated metabolism of GA hydroxylate. RESULTS: The formation of metabolites of GA depended on incubation time(10-40 min),substrate concentration (25-200 μmol/L) and microsome protein concentration (0.25-1.0 g/L). The kinetic behaviors of 22α-hydroxylation and 24-hydroxylation of GA were described well by a Michaelis-Menten equation [Vmax were (7.89±1.43) and (3.38±0.95) μmol·min-1·g-1,Km was (33.5±8.6) and (67.8±17.9) μmol/L, respectively]. Inhibition experiments showed that troleandomycin (6.25-100 μmol/L and erythromycin (15.7-250 μmol/L) as potent CYP3A1/2 inhibitors, reduced 22α-hydroxylation in a dose-dependent manner (the maximum inhibitory rates were 82.4 % and 45.7 %, respectively),while sulfaphenazole, which was an inhibitor towards 2C9/10 did not display significant inhibition. 22α-hydroxylation of GA correlated well with erythromycin N-demethylase activities (r =0.864, P<0.01, n =10). Sulfaphenazole (6.25-100 μmol/L) as potent CYP2C9/10 inhibitors, reduced 24-hydroxylation of GA in a dose-dependent manner, the maximum inhibitory rate was 69.5 %, while troleandomycin, which were inhibitors as CYP3A1/2 did not display significant inhibition. The 24-hydroxylation of GA did not correlate with erythromycin N-demethylase activities (r = 0.310, P> 0.05, n = 10). CONCLUSION: CYP3A1/2 and CYP2C9/10 in rat liver microsomes mediate the 22α-hydroxylation and 24-hydroxylation of GA.
