The Major Contribution Of Cytochrome P450（CYP）3A4 Towards The Metabolism Of Schisanhenol

Objective To explore the major contribution of cytochrome P450(CYP) 3A4 towards the metabolism of schisanhenol was demonstrated in the present study, which promotes the deeper understanding of pharmacokinetics of Fructus schisandrae.Methods The present study aims to identify the metabolic behavior of schisanhenol(the major component of Fructus schisandrae) using in vitro incubation methods. After incubation of schisanhenol in the microsome incubation system, one major oxidized metabolite was formed, and Incubation mixture for Schisanhenol metabolism study,Chemical inhibition study,Screening the recombinant CYP isoforms involved in the metabolism of schisanhenol,To calculate the kinetic parameters, various concentrations of schisanhenol were incubated with pooled HLMs or recombinant CYP3A4. Preliminary experiments were performed to make sure that the formation of metabolite was in the linear range of both reaction time and the concentrations of HLMs or CYP3A4. All the incubations were performed in duplicate. The apparent Km and Vmax values were calculated from non-linear regression analysis of experimental data in accordance with the Michaelis-Menten equation.Results(1)After schisanhenol was incubated with liver microsomes(LMs) fromdifferent species, one big peak was eluted at 7.1 min, and this peak was not detected in control incubation control system without adding substrate.(2)Under the experimental conditions utilized, the metabolism of schisanhebol obeyed the Michaelis-Menten kinetics, as evidenced by Eadie-Hofstee plots. The kinetic parameters(apparent Km and Vmax) were calculated to be 9.2 μmol/l and 4.1 nmol/min/mg pro.(3)CYP3A4 showed high catalytic activity towards the metabolism of schisanhebol, but other CYP isoforms showed no activity. Furthermore, the kinetic study indicated that CYP3A4-catalyzed schisanhebol obeyed the Michaelis-Menten kinetics, as evidenced by Eadie-Hofstee plots. The kinetic parameters(apparent Km and Vmax) were calculated to be 5.8 u M and 13.3 nmol/min/mg pro.(4)The nonspecific inhibitors of CYPs ABT inhibited almost 90% activity of schisanhebol metabolism, indicating the major involvement of CYP isoforms in the metabolism of schisanhebol. Among the specific inhibitors of CYP isoforms, the specific inhibitor of CYP3A4 ketoconazole completely prevented the formation of schisanhebol’s metabolite. However, the inhibitors of other CYP isoforms exhibited weak inhibition towards the formation of metabolite, indicating no or little involvement of other CYP isoforms in the metabolism of schisanhebol. The correlation analysis showed that the metabolism of schisanhebol has high correlation with the formation of midazolam 1’-OH metabolite, furtherly showing the major involvement of CYP3A4 in the metabolism of schisanhebol.(5)Molecular docking method was employed to understand the interaction between schisanhebol and CYP3A4. The major amino acids showing interaction with schisanhebol are Thr309 and Ser119. Two possible attacking sites were given in the Figure. One is the hydrogen located in the phenyl ring(4.73 A), and one is the hydrogen located in the methoxy group(2.97 A).Conclusions The current study characterizes the schisanhenol pathway in human, the liver becomes a major detoxifying tissue for schisanhenol and CYP3A4 is demonstrated to play an important role.