999感冒灵对不同种属CYP2E1酶活性的影响

Effect of 999 Ganmaoling on CYP2E1 Enzyme Activity in Liver Microsomes from Different Species

目的:研究999感冒灵(感冒灵)对大鼠肝脏的影响,比较感冒灵对大鼠、小鼠和人不同种属CYP2E1酶活性的影响,为临床安全性再评价及合理使用提供实验依据。方法:大鼠随机分为空白组、感冒灵组(4.53 g·kg-1),单次灌胃给药,14 d后动物称重,处理;全自动生化仪检测血清丙氨酸氨基转移酶(ALT),天门冬氨酸氨基转移酶(AST),尿素氮(BUN),肌酐(SCr),肌酸激酶(CK),取肝脏、肾脏及心脏,计算脏器指数;对肝脏进行苏木素-伊红(HE)染色,光学显微镜下观察其组织形态学变化。将人、大鼠和小鼠肝微粒体分别与不同浓度的感冒灵或对乙酰氨基酚(APAP)在37℃预孵育15 min后,加入CYP2E1的探针底物氯唑沙宗和还原型辅酶Ⅱ(NADPH)继续孵育30 min,用LC-MS/MS方法测定6-羟基氯唑沙宗的生成量,计算半数抑制浓度(IC50)。结果:与空白组比较,感冒灵组大鼠肝脏、肾脏及心脏指数、血清生化指标、肝脏病理组织学无明显变化。感冒灵在人、大鼠和小鼠的肝微粒体中对CYP2E1的IC50分别为6.516,344.2,932.8 mg·L-1,APAP在人、大鼠和小鼠的肝微粒体中对CYP2E1的IC50分别为52.4,453.8,806.5 mg·L-1。各种属中感冒灵和APAP对CYP2E1抑制程度趋势一致。结论:不同种属对感冒灵抑制CYP2E1活性敏感程度依次为人、大鼠及小鼠,该差异可能与大剂量感冒灵对大鼠肝脏无明显毒性影响有关;提示药物在进行临床前安全性评价时宜采用不同种属动物尤其是考察对肝脏代谢的影响,在临床需关注含有APAP的复方感冒药与其他药物联合用药时对体内药酶代谢的互相作用的影响,为临床合理用药提供依据。

Objective:To study the effect of 999 Ganmaoling on rat liver and compare the effect of Ganmaoling on the enzyme activity of CYP2E1 in different species of rats,mice and humans,so as to provide theexperimental basis for the clinical safety reevaluation and rational use.Method:Rats were divided into two groups: blank control and Ganmaoling group,which were administrated respectively with 0.5% CMC-Na or equal volume of Ganmaoling at the dose of 4.53g·kg^-1 for oral administration.The rats were weighed after 14d,and then treated the next day.Activity of serum alanine aminotransferase (ALT),aspartate aminotransferase (AST),creatine kinase (CK),urea nitrogen (BUN)and creatinine (SCr)were detected by automatic biochemical analyzer.The liver,kidney and heart were collected.The ratio of tissue weight to body weight was counted.HE staining was used in the slices of liver sample,and the histopathological change in liver was observed by light microscopy.Human, rat and mouse liver microsomes were incubated respectively with different concentrations of Ganmaoling or APAP at 37℃ for 15minutes,then chlorzoxazone,as CYP2E1probe substrate,and nicotinamide adenosine denucleotide hydro-phosphoric acid (NADPH)were added and incubated for another 30minutes.The content of 6-hydroxychlorzoxazone was detected by LC-MS/MS method.The inhibition rate concentration (IC50)was calculated.Result:Compared with blank control group,the liver,kidney and heart weight index,serum biochemical indicators,and liver histopathology in Ganmaoling group showed no significant change.The IC50 values of Ganmaoling for CYP2E1in liver microsomes of human,rat and mouse were 6.516,344.2,932.8mg·L^-1 respectively.The IC50values of APAP for CYP2E1in human,rat,and mouse liver microsomes were 52.4,453.8, 806.5mg'L^-1,respectively.Ganmaoling and APAP showed a consistent trend of inhibition of CYP2E1in various genera.Conclusion:The order of the inhibitory activity of Ganmaoling against CYP2E1of different species from high to low was human,mouse and rat.The difference may explain the reason that large-dose Ganmaohng has no obvious toxicity to rat liver.The results suggest that different species of animals shall be adopted in::drug pre- clinical safety assessments,especially for the study of effect on liver metabolism.In clinical practice,more attention shall be paid to the interaction between compound cold medicine containing APAP and other drugs,in order to provide the basis for rational clinical drug use.