【作者】 方海明；

【导师】 许建明；

【作者基本信息】 安徽医科大学 ， 老年医学， 2010， 博士

【摘要】 研究背景:肠道代谢能力在新药筛选、研发及口服制剂生物利用度中的作用日益受到重视。药物在肠道可以产生I相和II相代谢作用,其中CYP3A是肠道主要I相代谢酶,在人体肠道中占总CYP酶的70%。肠道上皮细胞同样表达如P-糖蛋白(P-gp)多种转运蛋白,CYP3A和P-gp底物具有广泛交叉,能够产生协同作用,共同形成药物肠道吸收的生物屏障。CYP3A4和P-gp常常能够被同一种物质诱导或者抑制,产生药物间交互作用,药物或者其代谢产物浓度呈数量级式的增加或者减少,从而影响疗效,甚至产生不良反应。奥美拉唑和雷贝拉唑口服生物利用度低且存在个体差异性,肠道代谢作用对二者生物利用度的影响研究报道很少,很有必要进行探讨。目的;评价奥美拉唑和雷贝拉唑肠道首过代谢及探讨部分机制;初步探讨连续服用奥美拉唑和雷贝拉唑对肠道P-gp功能及蛋白表达的影响。方法:经门静脉及十二指肠置管建立肠道-血管通路IVAP大鼠模型。分别经十二指肠(id, 3–9 mg·kg-1)、门静脉(ipv,1.5–6 mg·kg-1)或者外周静脉(iv,1.5–6 mg·kg-1)给予奥美拉唑或者雷贝拉唑,HPLC法测定血药浓度并进行药动学分析,肝脏摄取率(EH)和肠道摄取率(EI)分别由浓度时间曲线下面积(AUC)计算;采取平行对照设计,经十二指肠给予酮康唑(60mg·kg-1)或者维拉帕米(9mg·kg-1),30min后分别经十二指肠(6mg·kg-1)或者外周静脉(3mg·kg-1)给予奥美拉唑或者雷贝拉唑,比较酮康唑或者维拉帕米对两种药物肠道与静脉药动学过程的影响。另外一独立实验,30只大鼠随机分成5组,6只每组,分别为奥美拉唑组、雷贝拉唑组、地塞米松组、维拉帕米组及正常对照组,分别灌胃给予奥美拉唑、雷贝拉唑、地塞米松、维拉帕米,对照组给予等剂量生理盐水,连续7d,d8处死动物,分别取空肠、回肠、结肠制成外翻肠囊,并置于含95%O2和5%CO2混合气体的Krebs-Ringer’S缓冲液中,在囊内(即浆膜侧)注入1mL罗丹明123(5μg·mL),在囊外(即粘膜侧)每隔10min取样500μL,至90min,荧光分光光度计测定罗丹明123浓度。同时取空肠、回肠、结肠组织制备成蜡块,采用免疫组化方法的测定不同肠段P-gp蛋白表达情况并用软件进行平均光密度分析。结果:IVAP模型中,奥美拉唑各给药途径药动学参数AUC、Cmax值随剂量成比例增加。id给予不同剂量奥美拉唑,均在15min以内达到峰浓度,提示奥美拉唑在大鼠肠道内能快速吸收。全肠道内奥美拉唑残留量占给药剂量百分比为0.01～0.05%,提示奥美拉唑在大鼠肠道接近完全吸收。id给予3.0和6.0 mg·kg-1后,其绝对生物利用度分别为20.73±2.49%和22.07±2.92%;EH分别为50.00±4.16%, 60.19±1.27%,EI分别为58.59±1.54%, 44.41±9.13%。雷贝拉唑各给药途径药动学参数AUC、Cmax值随剂量成比例增加。id给予不同剂量雷贝拉唑,均在9min以内达到峰浓度,提示雷贝拉唑在大鼠肠道内能快速吸收。全肠道内雷贝拉唑残留量量占给药剂量百分比为0.15～0.25%,提示雷贝拉唑在大鼠肠道几乎完全吸收。id给予3.0和6.0 mg·kg-1后,其绝对生物利用度分别为15.28±2.05%和27.16±2.47%;EH分别为16.77±2.85%, 27.79±3.43%,EI分别为81.65±2.28%, 62.26±4.69%。id预先给予酮康唑或者维拉帕米后,显著增加id途径下奥美拉唑和雷贝拉唑的AUC和Cmax值,但对奥美拉唑和雷贝拉唑静脉药动学参数无显著性影响。连续给予奥美拉唑后,肠道P-gp分泌罗丹明123的能力显著下降,按受抑制程度分,空场>回肠>结肠;肠道P-gp蛋白表达显著降低,按受抑制程度分,空场<回肠<结肠。连续给予雷贝拉唑后,肠道P-gp功能和蛋白表达水平虽然有所降低,但是与对照组比较,不具有统计学差异性。结论:奥美拉唑和雷贝拉唑均经历显著的肝脏和肠道代谢作用,奥美拉唑肝脏和肠道首过代谢程度相当,但雷贝拉唑肠道首过代谢程度显著高于肝脏。增加药物剂量可以明显增加雷贝拉唑生物利用度,但是奥美拉唑生物利用度增加不明显。肠道CYP3A介导代谢和P-gp介导的转运过程均参与奥美拉唑和雷贝拉唑肠道首过代谢过程,抑制肠道CYP3A和P-gp功能可以显著增加二者口服生物利用度。连续口服奥美啦可以显著抑制大鼠肠道P-gp活性和蛋白表达水平,但连续口服雷贝拉唑对大鼠肠道P-gp活性和蛋白表达水平不产生显著性影响。更多还原

【Abstract】 Background: It has been widely hypothesized that intestinal apical secretion and/or CYP3A-mediated metabolism may profoundly affect a drug’s bioavailability (BA); CYP3A is a major phase I drug metabolizing enzyme that potentially mediates the biotransformation of more than 50% of known drugs with its gene expression levels being 30% and 70% of total cytochromes P450 in human liver and intestine, respectively. P-glycoprotein (P-gp) is thought not only to affect the secretion of a variety of drugs in the small intestine of the body, but it can also affect the metabolism and disposition of drugs. CYP3A and P-gp have wide overlapping substrate specificity and may have synergisitic effects on form the barrier to oral drug absorption. However, the ability to directly assess the potential importance of these two intestinal processes in vivo has represented a major technological challenge with little success being demonstrated to date.Objective: The aim of this study was to evaluate the effects of intestinal and hepatic extraction on the bioavailability of omeprazole and rabeprazole and the possible involvement of cytochromeCYP3Aand P-gp in this process in rats.The second aim of this study was to investigate the effects of consecutive administration of omeprazole or rabeprazole on the intestinal P-gp activity and protein expression in rats.Methods: The current study was performed in intestinal and vascular access ported (IVAP) rats to quantify and differentiate the components of intestinal and hepatic first pass extraction of omeprazole or rabepzole mediated by CYP3A and P-gp. Omeprazole or rabeprazole was administered by intravenous injection (i.v., 1.5–6 mg·kg-1), inrtraportal infusion (ipv, 1.5–6 mg·kg-1) or intraduodenal instillation (id, 3–9 mg·kg-1). The blood concentrations of omeprazole or rabeprazole were measured by the high-performance liquid chromatography. Extraction ratios in the liver and intestinal tract were determined from the area under the plasma concentration-time curve (AUC).The roles of intestinal efflux by means of P-gp and/or metabolism by CYP3A on the first pass intestinal extraction of omeprazole or rabepzole were differentiated by using ketconazole (an inhibitor of CYP3A) or verapamil(a P-gp inhibitor). Omeprazole or rabepzole was administered by id, ipv or iv to rats alone or 30 min after the id administration of ketoconazole(60mg·kg-1) or verapamil(9mg·kg-1), respectively.In another separated experiment, 30 rats were divided into five groups randomly as omeprazle, rabeprazole, verapamil, dexamethasone and control group, respectively. Drugs (omeprazle, rabeprazole, verapamil and dexamethasone) were given to rats by intragastric administration once a day for 7 days. The control group was given the same dose of saline. On d8, rats were sacrificed, everted sac of jejunum, ileum and colon were prepared quicly and put into Krebs-Ringers solution given with 95%O2 and 5%CO2, 1mL rhodamine 123(5μg·mL) was injected in the serosa side and 500μL samples was obtained from the mucosa side very 10min for 90mins. The activity of P-gp of segments of rat intestine was determined by the secretion of rhodamine 123 using everted sac of jejunum, ileum and colon respectively. Expression of P-gp in jejunum, ileum and colon of rat were immunohistochemistry assays.Results: In IVAP rat model, pharmacokinetic parameters of AUC and Cmax of omeprazole were increased propotionally after id, ipv, and iv administration at various doses. After id administration of various doses, all reached peak concentration within 15mins, and unchanged omeprazole in the whole gastrointestinal were 0.01~0.05% of the given dose, indicating rhat omeprazole can be absorbed rapidly and completely in rats. The bioavailability after id administration of 3 and 6 mg·kg-1 of omeprazole was 20.73±2.49% and 22.07±2.92%, respectively. And the EH was 50.00±4.16% and 60.19±1.27%, EI was 58.59±1.54%, 44.41±9.13%, respectively. Pharmacokinetic parameters of AUC and Cmax of rabeprazole were increased propotionally after id, ipv, and iv administration at various doses. After id administration of various doses, all reached peak concentration within 9mins, and unchanged rabeprazole in the whole gastrointestinal were 0.15～0.25% of the given dose, indicating rhat rabeprazol can be absorbed rapidly and completely in rats. The bioavailability after id administration of 3 and 6 mg·kg-1 of rabeprazol was 15.28±2.05% and 27.16±2.47%, respectively. And the EH was16.77±2.85% and 27.79±3.43%, EI was 81.65±2.28%, 62.26±4.69%, respectively. Compared with the control group, the presence of ketoconazole (60 mg·kg-1) or verapamil (9 mg·kg-1) significantly increased AUCand Cmax of id-administered omeprazole or rabeprazle, while it had no significant effect on those of omeprazole or rabeprazle administered by iv ports. Consecutive administration of omeprazole for 7ds significantly inhibited intestinal P-gp activity and protein expression of rat respectively, while consecutive administration of rabeprzole have no siginificant effects on them.Conclusion: Oral omeprazole or rabeprazole undergoes marked intestinal first pass metabolism in rats. Omeprazole has a comparable hepatic and intestinal first pass metabolism in rat, while intestinal metabolism of rabeprazole significant higer than that of in liver. The BA of rabeprazole may increase dose-dependently, while that of omeprazole may not increase with oral dose. CYP3A-mediated metabolism and P-gp mediated effluex are both involve in the intestinal metabolism of omeprazole or rabeprazole.Inhibition of CYP3A and P-gp may marked increase the BA of omeprazole or rabeprazle. Consecutive administration of omeprazole significantly inhibited intestinal P-gp activity and protein expression of rat, while rabeprzole have no siginificant effects on them.更多还原