【作者】 卢新建；

【导师】 张鉴； 初建设；

【作者基本信息】 山东大学 ， 制药工程， 2008， 硕士

【摘要】 目的:药物代谢酶CYP1A2、CYP2A6、NAT2和XO在药物代谢和前致癌物激活的过程中均起着重要的作用。CYP1A2、CYP2A6、NAT2和XO的活性与许多药物的疗效或毒性以及一些肿瘤的易感性密切相关;它们在人群中的分布存在种族差异,其活性也有明显的个体差异,四种酶不同活性状态的存在对化学物质的体内转化必定产生一定的影响。本课题以健康受试者为研究对象,从代谢表型探讨银杏叶片与药物代谢酶CYP1A2、CYP2A6、NAT2和XO的关系,预测银杏叶片与常用药物的相互作用,以便指导临床医师合理用药,预防和减轻药物间的相互作用,最大限度的提高药物的有效性和安全性。方法:以咖啡因作为药物代谢酶CYP1A2、CYP2A6、NAT2和XO的探针药物,为保证应用咖啡因代谢探针的正确性,采用岛津Shim-pack VP-ODS柱（4.6mm×150mm,5μm）,岛津Shim-pack C18预柱,流动相为乙腈（A相）,0.05%醋酸（B相）,0-24min流动相B的比例从2.5%线性增加至10%,流速为1.0mL.min^-1,柱温25℃,检测波长为280nm,建立了反相高效液相色谱（RP-HPLC）测定尿液中咖啡因代谢物的方法,采用该法测定30名受试者分别服用银杏叶片前后尿液内咖啡因5种主要代谢产物5-乙酰氨基-6-甲酰氨基-3-甲基尿嘧啶（AFMU）、1-甲基尿酸（1U）、1-甲基黄嘌呤（1X）、1,7-二甲基尿酸（17U）和1,7-二甲基黄嘌呤（17X）的相对含量,采用（AFMU+1X+1U）/17U比值反映CYP1A2的活性,17U/（AFMU+1U+1X+17X+17U）反映CYP2A6在体内的活性,AFMU/（AFMU+1X+1U）反映NAT2的活性,1U/（1X+1U）反映XO的活性,对连续28天服用治疗剂量的银杏叶片对人肝脏药物代谢酶CYP1A2、CYP2A6、NAT2和XO活性的影响进行系统研究。结果:RP-HPLC梯度洗脱直接进样法测定咖啡因5种主要代谢产物,具有样品前处理快捷有效,样品分析时间短,分离度和可靠性良好的特点,且可克服提取及分析过程对测定的干扰。服用银杏叶片前、服用银杏叶片28天,CYP1A2的平均活性分别为2.976±1.428,3.021±1.318,服药28天后比服药前升高1.51%,虽CYP1A2活性在服药后有所提高,但并无统计学差异;服用银杏叶片前、服用银杏叶片28天后,CYP2A6的平均活性分别为0.227±0.076,0.227±0.072,服药28天后与服药前相比基本没有变化,而且无统计学差异;服用银杏叶片前、服用银杏叶片28天后,NAT2的平均活性分别为0.447±0.172,0.391±0.147,服药28天后比服药前降低12.5%,,而且有明显统计学差异（P＜0.05）;服用银杏叶片前、服用银杏叶片28天后,XO的平均活性分别为0.723±0.060,0.728±0.074,服药28天后比服药前升高0.69%,虽XO活性在服药后有所提高,但并无统计学差异。结论:以咖啡因探针法测定正常人肝脏药物代谢酶CYP1A2、CYP2A6、NAT2和XO活性的RP-HPLC梯度洗脱直接进样法,为国内深入开展CYP1A2、CYP2A6、NAT2和XO四种酶的研究开拓了一种新方法;服用治疗剂量的银杏叶片对CYP1A2、CYP2A6和XO活性无明显影响,但是可以明显减低代谢酶NAT2的活性,说明银杏叶片不能通过药物代谢酶系统影响其他经CYP1A2、CYP2A6和XO酶代谢的药物临床疗效,但是可以影响经NAT2代谢的药物临床疗效。长期或者大剂量使用银杏叶片对CYP1A2、CYP2A6、NAT2和XO活性的影响值得进一步研究。更多还原

【Abstract】 Objective:Cytochrome P450 enzyme 1A2（CYP1A2）,Cytochrome P450 enzyme 2A6 （CYP2A6）,N-acetyltransferase-2（NAT2）,and xanthine oxidase（XO） play an important role while the drugs are metabolized and carcinogens are activated,their activation is closely related to curative effect or toxicity of a lot of drugs and susceptibility of some tumors,different active state of this enzymes must has certain influence on transformation of chemical material in vivo.To investigate the effect of ginkgo biloba tablets on CYP1A2,CYP2A6,NAT2 and XO activities in human and to forecast the drug-drug interaction of ginkgo biloba tablets,the activities of CYP1A2, CYP2A6,NAT2 and XO are assayed by metabolic probe which are contributed to predict medicine curative effect,prevent and mitigate medicine adverse effect, improve drug safety and instruct clinician to prescribe rationally.Methods:Caffeine was used as a metabolic probe for CYP1A2,CYP2A6,NAT2 and XO,a HPLC method of metabolites of caffeine in the urine with direct injection was established for assuring the assay accuracy.The Shim-pack VP-COD column（4.6mm×150mm,5μm） and Shim-pack C18 pre-column were used.The mobile phase was acetonitrile and the gradient program was acetonitrile 2.5%to 10% in 0 to 24 min.The flow rate was 1.0mL.mim^-1,and the column temperature was 25℃.The detective wavelength was at 280nm.In thirty volunteers,before and after ginkgo biloba tablets administration,urine samples were collected.The contents of five major metabolites of caffeine,5-acetylamino-6-formylamino-3-methyluracil（AFMU）,1-methylxanthine（1X）,1-methyluricacid（1U）,1,7-dimethylurica cid（17U） and 1,7-dimethylxanthine（17X） in the urine were determined by RP-HPLC with direct injection method,and then the activity of CYP1A2,CYP2A6,NAT2 and XO were evaluated by respectively calculating the（AFMU+1X+1U）/17U, 17U/（AFMU+1U+1X+17X+17U）,AFMU/（AFMU+1X+1U）and 1U/（1X+1U）.The effect of therapeutic dose ginkgo biloba tablets administrated for 28 days were investigated simultaneously on CYP1A2,CYP2A6,NAT2 and XO respectivelyResults:A new HPLC method of five metabolites of caffeine in the urine with direct injection was developed.The method is simple,rapid and accurate.It was found that the average activities of CYP1A2 were 2.976±1.428,3.021±1.318 before treatment,after taking medicine 28days respectively,There were no statistical significancet between before treatment and after taking medicine 28 days.It was found that the average activities of CYP2A6 were 0.227±0.076,0.227±0.072 before treatment,after taking medicine 28days respectively,There were no statistical significance between before treatment and after taking medicine 28 days.It was found that the average activities of NAT2 were0.447±0.172,0.391±0.147 before treatment,after taking medicine 28days respectively,There were statistical significance between before treatment and after taking medicine 28 days.It was found that the average activities of XO were 0.723±0.060,0.728±0.074 before treatment,after taking medicine 28days respectively,There were no statistical significance between before treatment and after taking medicine 28 days.Conclusions:The method to measure simultaneously the activity of CYP1A2, CYP2A6,NAT2 and XO should contribute to simplifying and optimizing the caffeine drug metabolizing enzyme assays.Ginkgo biloba tablets have no influence on CYP1A2,CYP2A6and XO,so ginkgo biloba tablets do not modify the efficacy of drugs taken simultaneously which were metabolized by CYP1A2,CYP2A6 and XO.But ginkgo biloba tablets may modify the efficacy of drugs which metabolized by NAT2 taken simultaneously in slow acetylator.It is necessary to further study the effect of ginkgo biloba tablets administrated for long-term or in large dose on CYP1A2,CYP2A6,NAT2 and XO in human.更多还原