罗通定在人、比格犬和大鼠肝微粒体代谢转化的体外比较研究

目的体外研究罗通定(rotundine,RTD)在大鼠、比格犬和人肝微粒体中酶代谢动力学及代谢产物差异。方法优化3个种属肝微粒体与RTD反应体系,应用LC-MS测定RTD在3种肝微粒体中的体外代谢消除,应用LC-MS/MS分析比较RTD在3种肝微粒体中代谢产物种类和生成量的差异,计算并比较相应的活性值。结果RTD在人肝微粒体中代谢转化最慢,其相应的动力学参数为Km=2.67μmol·L-1、Vmax=0.095μmol·L-1·min-1、T21=298±18.0min、CLint=14.6±0.91ml·min-1·kg-1;大鼠中相应的动力学参数为Km=3.24μmol·L-1、Vmax=0.122μmol·L-1·min-1、T12=71.0±2.30min、CLint=87.5±2.79ml·min-1·kg-1;比格犬中相应的动力学参数为Km=5.31μmol·L-1、Vmax=0.228μmol·L-1·min-1、T21=62.3±0.647min、CLint=139±1.43ml·min-1·kg-1。RTD在3个种属肝微粒体中均代谢产生4个O-去甲基后的羟基化同分异构体产物,但4个产物的相对生成百分比在不同种属肝微粒体中有一定差异。结论罗通定在体外人、大鼠和比格犬肝微粒体中主要的I相代谢途径相同,但是酶代谢动力学性质及代谢产物的生成量存在着一定的差异。

In vitro comparison of rotundine metabolism in liver microsomes of human,dog and rat

Aim To investigate the inter-species differences of rotundine (RTD) metabolism in liver microsomes of human,dog and rat by comparing enzyme kinetics of the parent drug and the formation of its major metabolites.Methods The incubation systems of RTD with liver microsome of the three species were optimized in terms of RTD concentration and incubation time.The concentration of RTD in incubates was determined by a validated LC-MS method.The metabolites of RTD in incubates were separated and identified by LC-MS/MS.Results The biotransformation of RTD by human liver microsome was the slowest among the three species.The Km,Vmax,CLint and T12 of RTD obtained from human liver microsome were 2.67 μmol·L-1,0.095 μmol·L-1·min-1,14.6±0.91 ml·min-1·kg-1 and 298±18.0 min,respectively. The corresponding kinetic parameters for rat and dog liver microsomes were 3.24 and 5.31 μmol·L-1,0.122 and 0.228 μmol·L-1·min-1,87.5±2.79 and 139±1.43 ml·min-1·kg-1 71.0±2.30 and 62.3±0.647 min.Four isomeric metabolites were all found in the liver microsomes of three species,which were formed in incubates by O-demethylation at four methyl groups of RTD. It was observed that the relative amounts of these four metabolites generated in liver microsomes of three species were different.Conclusions The major metabolic pathway of RTD is O-demethylation in the liver microsomes from all three species.However,the inter-species differences are observed in terms of relative amounts of the metabolites measured in incubates,as well as in metabolic characteristics of RTD.