【作者】 曹亚杰；

【导师】 程泽能；

【作者基本信息】 中南大学 ， 药剂学， 2007， 硕士

【摘要】 一、目的研究氟非尼酮对细胞色素P450亚家族:CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP2E1和CYP3A活性的影响,为氟非尼酮的药理研究及临床药动学研究以及预测临床药物相互作用研究提供参考。二、方法1.体外实验方法获取大鼠及人肝脏标本后,以差速离心法制备肝微粒体,用卡马斯亮兰法进行蛋白定量。在37℃的水浴中建立肝微粒体孵育体系,加入药动学实验证明为有效剂量的一系列浓度的氟非尼酮和相应的CYP的探针药物进行孵育。分别以咖啡因、咪哒唑仑、甲苯磺丁脲、氯唑沙宗、美托洛尔、美芬妥因为探针药,探测加入氟非尼酮组和未加氟非尼酮组CYP1A2、CYP3A4、CYP2C9、CYP2E1、CYP2D6、CYP2C19的活性。2.体内实验方法按照随机对照的原则,32只大鼠雌雄各半,性别内各随机分成4对,然后性别间随机组合成4组,每组8只大鼠,一组做对照,另三组分别给予250,500,1000mg/kg/day（每天两次）氟非尼酮的混悬剂,连续灌胃给药5日,处死动物,制备肝微粒体。分别以咖啡因、咪哒唑仑、甲苯磺丁脲、氯唑沙宗、异喹胍、美芬妥因为探针药,测定大鼠CYP1A2、CYP3A4、CYP2C9、CYP2E1、CYP2D6、CYP2C19的活性。并采用Western-blot方法对活性有显著改变的酶进行蛋白表达半定量分析。三、结果1.体外实验部分在大鼠肝微粒体中加入氟非尼酮,发现对CYP2C19、CYP2C9和CYP2E1有抑制作用,而人肝微粒体中加入氟非尼酮后未发现明显的抑制作用。2.体内实验部分与安慰剂组相比,氟非尼酮250 mg·kg^-1·day^-1组导致氯唑沙宗代谢物产率增加了约723%,甲苯磺丁脲代谢物产率增加了约142%,咪哒唑仑代谢物产率增加了约129%;氟非尼酮500 mg·kg^-1·day^-1组导致氯唑沙宗代谢物产率增加了约14倍,甲苯磺丁脲代谢物产率增加了约52%,咪哒唑仑代谢物产率增加了约232%;氟非尼酮1000 mg·kg^-1·day^-1组导致氯唑沙宗代谢物产率增加了约12倍,甲苯磺丁脲代谢物产率增加了约74%,咪哒唑仑代谢物产率增加了约85%;经统计分析,CYP2E1活性（氯唑沙宗代谢物产率）显著增加（P＜0.05）,而CYP3A4活性（咪哒唑仑代谢物产率）和CYP2C9活性（甲苯磺丁脲代谢物产率）的也有所增加,但没有统计学意义。不同剂量组分别反映CYP1A2、CYP2C19和CYP2D6活性的咖啡因、美芬妥因和异喹胍代谢物产率均无明显变化。酶活性的改变与剂量没有相关性。CYP1A2、CYP2C19和CYP2D6的活性均无显著性改变。对给药组及对照组的微粒体中CYP2E1及CYP3A4的蛋白表达采用western-blotting进行了测试,结果表明给药后CYP2E1的蛋白表达较对照组有显著增加。四、结论1.氟非尼酮在大鼠肝微粒体中对CYP2C19、CYP2C9和CYP2E1有抑制作用,而在人肝微粒体中对CYP450无明显抑制作用,其IC₅₀值均大于788.2μmol·L^-1。2.氟非尼酮在体内诱导CYP2E1,CYP3A4和CYP2C9的活性,并使CYP2E1和CYP3A4蛋白表达增加。更多还原

【Abstract】 OBJECTIVESTo investigate the inhibit or induce effects of fluorofenidone on the activities of cytochrome P450.METHODS1.In vitroCaffeine,midazolam,tolbutamide,chlorzoxazone,debrisoquine,and mephenytoin were incubated as probes with or without fluorofenidone respectively to study the effect of fluorofenidone on cytochrome P450 activities including CYP1A2,CYP3A4,CYP2C9,CYP2E1,CYP2D6 and CYP2C 19 in rat and human liver microsomes.2.In vivoRats were administrated by gastrogavage with different doses(250 500,and 1000 mg·kg^-1·day^-1,respectively)of fluorofenidone twice daily for five consecutive days.Liver microsomes were extracted at the sixth day. Probe drugs including caffeine,tolbutamide,mephenytoin,debrisoquine, chlorzoxazone and midazolam were used to estimation CYP1A2,CYP2C9, CYP2C19,CYP2D6,CYP2E1 and CYP3A4 activities,respectively. Concentrations of the probe drugs and their metabolites in the incubated system were determined by HPLC and the cytochrome P450 activities were measured by the production of metabolites.The protein contents of CYP2E1 and CYP3A4 in the liver microsomes were determined by western-blotting method.RESULT 1.In vitroOur data indicated that fluorofenidone has not significant inhibition on the activities of CYP450 in human but has inhibition on CYP2C19、CYP2C9 and CYP2E1.2.In vivoIn 250 mg·kg^-1·day^-1dose group,fluorofenidone pretreatment cause 723%,142%and 129%increase of chlorzoxazone 6-hydroxylation, tolbutamide 4-hydroxylation and midazolam 1-hydroxylation respectively.In 500 mg·kg^-1·day^-1dose group fluorofenidone pretreatment cause 14 fold,52%,and 232%increase of chlorzoxazone 6-hydroxylation, tolbutamide 4-hydroxylation and midazolam 1-hydroxylation,respectively.In 1000 mg·kg^-1·day^-1dose group fluorofenidone pretreatment induce 14 fold,74%and 85%increase of chlorzoxazone 6-hydroxylation, tolbutamide 4-hydroxylation and midazolam 1-hydroxylation,respectively.Significant differences in ennyme activitiy between control group and treatment groups were observed for CYP2E1（P＜0.05）.The activity of CYP2C9 and CYP3A4 increased but not significantly.There are not differences in caffeine 3-demethylation,mephenytoin 4-hydroxylation and debrisoquine 4-hydroxylation in different doses and there is no correlation between the dose and the changes of activities.CONCLUSIONSFluorofenidone has not inhibition effect on CYP450 in human liver microsome but it has inhibition effect on CYP2C19,CYP2C9 and CYP2E1 in rat liver microsome.The value of IC₅₀of CYP2C19 was about 400μmol·L^-1and the values of IC₅₀of CYP2C9 and CYP2E1 were; higher than 788.2μmol·L^-1.Fluorofenidone is a potential CYP2E1,CYP3A4 and CYP2C9 inducer in vivo.The protein expression of CYP2E1 and CYP3A4 are increased after fluorofenidoneadministration.更多还原