Cocktail探针法评价感冒治疗药物对大鼠肝CYP450酶活性的作用  

Effects evaluation of cold treatment drugs on the enzymatic activities of rat hepatic CYP450 utilizing the Cocktail probe method

作　　者：林于茜 罗佳怡 王淑美 谢媛媛 LIN Yu-qian;LUO Jia-yi;WANG Shu-mei;XIE Yuan-yuan(School of Chinese Materia Medica,Guangdong Pharmaceutical University,Guangzhou 510006,China;Guangdong Provincial Traditional Chinese Medicine Quality Engineering and Technology Research Center,Guangzhou 510006,China;Key Laboratory of Digital Quality Evaluation Technology of Traditional Chinese Medicine,National Administration of Traditional Chinese Medicine,Guangzhou 510006,China)

机构地区：[1]广东药科大学中药学院,广东广州510006 [2]广东省中药质量工程技术研究中心,广东广州510006 [3]国家中医药管理局中药数字化质量评价技术重点研究室,广东广州510006

出　　处：《药学学报》2025年第4期1103-1114,共12页Acta Pharmaceutica Sinica

基　　金：广东省药品监督管理局科技创新项目(2023ZDZ03)。

摘　　要：采用Cocktail探针药物法评价葛根汤等感冒治疗药物对大鼠肝细胞色素P450 (cytochrome P450,CYP450)酶活性的影响。优化大鼠肝微粒体体外孵育体系,基于液相色谱串联质谱技术(LC-MS/MS)建立同时测定大鼠肝微粒体CYP450酶中各亚酶探针代谢产物含量的分析方法,Cocktail探针药物法表征治疗风寒感冒的经典方葛根汤和临床上常用于治疗感冒的西药对乙酰氨基酚、马来酸氯苯那敏、无水咖啡因及盐酸那可汀等对CYP2D6、CYP2C9、CYP3A4等亚酶活性的影响,为它们在临床联合用药安全提供参考。结果表明,所建立的分析方法稳定可靠,符合生物样品测定要求。经优化后,孵育体系中大鼠肝微粒体蛋白浓度为3.00 mg·mL^(-1),孵育时间为240 min,终止剂为乙腈,3种CYP亚酶特异性探针底物右美沙芬、睾酮、甲苯磺丁脲的K_(m)值分别为21.49、87.33、354.7μmol·L^(-1),通过阳性抑制剂验证了所建立孵育体系的有效性。与空白对照组相比,当各西药浓度和葛根汤提取物浓度分别在100.00μmol·L^(-1)和3.00 mg·mL^(-1)以内时,葛根汤提取物、马来酸氯苯那敏及盐酸那可汀对大鼠肝微粒体CYP2C9、CYP2D6有抑制作用;无水咖啡因对大鼠肝微粒体CYP2C9、CYP3A4有抑制作用;对乙酰氨基酚无明显抑制作用;盐酸那可汀对大鼠肝微粒体CYP3A4的诱导作用大于阳性诱导剂活性的40%,提示与盐酸那可汀联合用药时,可能存在一定的药物相互作用风险,应注意药物治疗剂量。本实验按照广东药科大学实验动物福利伦理委员会指导政策严格执行(批准号:gdpulacspf2022137)。Cocktail probe drug method was used to evaluate the effect of cold treatment drugs such as Gegen Decoction on cytochrome P450(CYP450) enzyme activity in rat liver.The in vitro incubation system of rat liver microsomes was optimized.Based on LC tandem mass spectrometry(LC-MS/MS),an analytical method for simultaneous determination of the content of metabolites of each subenzyme probe in rat liver microsome CYP450enzyme was established.Cocktail probe drug method was used to characterize the effects of Gegen Decoction,a classical prescription for the treatment of wind-cold cold,and acetaminophen,chlorpheniramine maleate,anhydrous caffeine and nacotine hydrochloride,which were commonly used in the treatment of cold,on the activity of CYP2D6,CYP2C9,CYP3A4 and other sub-enzymes in rat liver microsomes,so as to provide reference for the safety of clinical drug combination.The results showed that the established analysis method was stable and reliable,which met the requirements of biological sample determination.After optimization,the protein concentration of rat liver microsomes in the incubation system was 3.00 mg·mL^(-1),the incubation time was 240 min,and the terminator was acetonitrile.The K_(m) values of three CYP subenzyme specific probe substrates dextromethorphan,testosterone and tolbutamide were 21.49,87.33 and 354.7 μmol·L^(-1),respectively.The effectiveness of the established incubation system was verified by positive inhibitors.Compared with the blank control group,when the concentration of each Western medicine and the concentration of Gegen Decoction extract were within 100.00 μmol·L^(-1) and 3.00 mg·mL^(-1),respectively,Gegen Decoction extract,chlorpheniramine maleate and noscapine hydrochloride had inhibitory effects on CYP2C9 and CYP2D6 enzymes in rat liver microsomes;anhydrous caffeine had inhibitory effects on CYP2C9 and CYP3A4 enzymes in rat liver microsomes;no significant inhibitory effect on acetaminophen;the induction effect of narcotine hydrochloride on CYP3A4 enzyme in rat liver microsomes

关 键 词：Cocktail探针法 CYP450酶 肝微粒体 葛根汤 对乙酰氨基酚 马来酸氯苯那敏 无水咖啡因 盐酸那可汀 药物相互作用 

分 类 号：R283.6[医药卫生—中药学]