大鼠肝微粒体CYP3A1/2和CYP2C9/10参与甘草次酸羟化代谢  

CYP3A1/2 and 2C9/10 in rat liver microsomes mediate 22α-and 24-hydroxylation of 18α-glycyrrhetic acid

作　　者：高凯[1] 余伟[2] 杨静[2] 段徐华 

机构地区：[1]湖北省妇幼保健院药剂科 [2]武汉大学医学院药理学系

出　　处：《中国临床药理学与治疗学》2007年第11期1255-1260,共6页Chinese Journal of Clinical Pharmacology and Therapeutics

摘　　要：目的:对参与18α-甘草次酸(GA)羟化代谢的细胞色素P450(cytochromeP450,CYP)亚型进行研究。方法:采用大鼠肝微粒体体外代谢GA的孵育方法和高效液相色谱(HPLC)技术,通过分析甘草次酸在肝微粒体中形成的单羟化代谢物的酶促动力学,分析其酶学模型,然后用不同的CYP同工酶选择性抑制剂和底物进行抑制实验,初步选出介导甘草次酸单羟化代谢所涉及的CYP同工酶。结果:大鼠肝微粒体羟化代谢GA呈反应时间(10～40min),底物浓度(25～200μmol/L)和蛋白浓度(0.25～1.0g/L)依赖性。GA代谢为22α-羟-GA和24-羟-GA的Vmax分别为(7.9±1.4)μmol.min-1.g-1和(3.4±1.0)μmo1.min-1.g-1,Km分别为(33±9)μmol/L和(68±18)μmol/L。抑制性研究可见:TAO和Ery剂量依赖性抑制22α-羟-GA形成,最大抑制率分别为82.4%和45.7%,而Sul无显著抑制作用;Sul剂量依赖性抑制24-羟-GA形成,抑制率依次为26.8%、45.3%和69.5%,而TAO和Ery的抑制作用不显著。红霉素N-脱甲基酶活性与22α-羟化代谢速率高度相关(r=0.864,P<0.01,n=10),与24-羟化代谢速率无明显相关(r=0.310,P>0.05,n=10)。结论:大鼠肝微粒体CYP3A1/2和CYP2C9/10分别参与了GA的C-22α和C-24羟化代谢。AIM： To identify the cytochrome P450 isoforms involved in 22α-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 22α-hydro-GA and 24-hydm-GA, was performed using rat liver microsomes at substrate concentrations ranging from 25 to 200μmol/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 hydmxylate. REULTS： The formation of metabolites of GA depended on incubation time （ 10 - 40 min）, substrate concentration （ 25 - 200μmol/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 - 100mol/L and erythmmycin （ 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-hydmxylation 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 troleandomy- cin, 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.

关 键 词：18α-甘草次酸 高效液相色谱 体外代谢 肝微粒体 

分 类 号：R969.1[医药卫生—药理学]