鹅去氧胆酸在肝微粒体体外代谢的种属差异研究

Species difference in chenodeoxycholic acid glucuronidation in liver microsomes

目的:比较鹅去氧胆酸(chenodeoxycholic acid,CDCA)在人及小鼠、大鼠、犬、家兔、猪、豚鼠、牛、羊等实验动物肝微粒体中葡萄糖醛酸化的代谢差异。方法:将CDCA分别与上述肝微粒体和尿苷二磷酸葡糖醛酸(uridine diphosphate glucuronic acid,UDPGA)进行孵育启动葡萄糖醛酸化反应。样品采用超高效液相色谱-质谱联用仪(ultra performance liquid chromatography coupled with mass spectrometer,UPLC-MS),色谱柱为ACQUITY UPLC BEH C18(100 mm×2. 1 mm,1. 7μm)分离,以分别含有5 mmol·L-1乙酸铵的20%乙腈(A)-80%乙腈(B)为流动相进行梯度洗脱,电喷雾离子源(ESI)负离子模式检测。CDCA的代谢轮廓用热图可视化及主成分分析进行差异研究。酶促动力学用于比较不同种属肝微粒体CDCA葡萄糖醛酸化代谢特征的相似性。结果:本实验在不同种属中共发现了6种CDCA的葡萄糖醛酸化代谢产物(M1~M6),其中M5是在所有种属中均产生的主要代谢产物。在相同的肝微粒体和底物浓度下,CDCA葡萄糖醛酸化主要代谢产物M5在犬肝微粒体中的代谢活性最强,而在猪肝微粒体中的代谢活性最弱。热图可视化和主成分分析发现羊和人最相似。根据酶促动力学参数,CDCA葡萄糖醛酸化主要代谢产物M5的内在清除率(CLint/μL·min-1·mg-1)依次为人(14. 34)<大鼠(58. 86)<牛(82. 17)<豚鼠(104. 77)<羊(159. 83),大鼠的内在清除率与人最为接近。结论:CDCA葡萄糖醛酸化途径的定量不能用作所有物种尿苷二磷酸葡萄糖醛酸转移酶1A3(UDP-glucuronosyltransferase,UGT1A3)活性评判的精准依据,就酶促动力学的相似轮廓而言,如果一个药物证明由UGT1A3催化代谢,则在临床前研究中推荐采用大鼠为模型,以保证药物在体内暴露与人体试验尽量接近。

Objective:To compare chenodeoxycholic acid(CDCA)glucuronidation in human and animal liver microsomes among different species at metabolites and kinetics levels.Methods:Glucuronidation was initiated by incubating CDCA with liver microsomes and uridine diphosphate glucuronic acid(UDPGA).UPLC-MS was used to analyze CDCA glucuronide.The chromatography separation was performed on ACQUITY UPLC BEH C18(100 mm×2.1 mm,1.7μm)column by gradient elution.Mobile phase A consisted of 5 mmol·L-1 ammonium acetate in 20%acetonitrile,mobile phase B consisted of 5 mmol·L-1 ammonium acetate in 80%acetonitrile,electrospray ionization(ESI)source was applied.Heatmap was used to visualize the metabolic profile of CDCA,while principle component analysis(PCA)was used to tell the differences of CDCA glucoronidation between human and animals.Metabolic kinetic sstudy was used to compare species with similar metabolic profile of CDCA glucuronidation.Results:Six UDPGA-dependent metabolites(M1~M6)were clearly observed in humans and other animals.M5 played a predominant role in all species.At the same liver micrsome concentration and substrate concentration,CDCA had the highest degree of glucuronidation in dog liver microsomes.Pig showed the weakest capacity of chenodeoxycholic acid metabolism.Based on the metabolic profile indicated by heatmap and PCA score plots,it was found that sheep was the closest to humans.Further,calculated from the kinetic parameters,the intrinsic clearance(CLint/μL·min-1·mg-1 protein)of CDCA glucronidation represented by M5 generation followed the order as below:humans(14.34)<rats(58.86)<cattle(82.17)<guinea pigs(104.77)<sheep(159.83).Rats showed the closest CLintvalue with human.Conclusion:CDCA glucuronidation is various in quantity in different species.In terms of similarity in pharmacokinetic profile,rats are recommended to be used in preclinical trials other than other species in the case that the drug candidate has been proved to be metabolized by UGT1 A3.