【作者】 周燕；

【导师】 武新安；

【作者基本信息】 兰州大学 ， 药物分析， 2010， 硕士

【摘要】 目的：1.建立内/外源性胆汁淤积大鼠模型,获得表达不同水平Mrp2的大鼠模型；2.研究头孢哌酮(Cefoperazone, CPZ)在内/外源性胆汁淤积大鼠模型体内的药动学行为；3.初步阐明分布于肝脏的多药耐药相关蛋白2(Multidrug resistance associated protein 2, Mrp2)对CPZ药动学的影响。方法：1.内源性胆汁淤积大鼠模型的建立：Wistar大鼠颈部皮下注射苯甲酸雌二醇(Estradiol Benzoate, EB,11 mg/kg/d),连续注射4 d可得；外源性胆汁淤积大鼠模型的建立：实验前Wistar大鼠禁食12 h,经乙醚麻醉后,迅速固定,上腹正中切口(约3 cm)开腹,暴露肝门,游离胆总管,用眼科剪于胆总管中上1/3处剪“V”字形口,插入引流管(腰麻管),0号手术线双重结扎固定引流管,钝性分离大鼠腹部皮下隧道至颈部,开口,引流管过皮下隧道至颈外固定,远端结扎5d,于第6 d再通(即剪开引流管远端结扎处),再通3 d后获得外源性胆汁淤积大鼠模型。2. CPZ的药动学研究：将Wistar大鼠分为正常对照组,内源性胆汁淤积模型组；溶媒对照组(皮下注射等体积的橄榄油)；外源性胆汁淤积模型组；假手术组(大鼠被开腹,游离胆管后关腹)；灌胃茵栀黄颗粒3 d/7 d内源性胆汁淤积组；灌胃生理盐水3 d/7 d内源性胆汁淤积模型组；灌胃茵栀黄颗粒7 d外源性胆汁淤积组；灌胃生理盐水7 d外源性胆汁淤积模型组。各组大鼠在给予CPZ前进行胆管插管术(外源性胆汁淤积模型组除外)和股动脉插管手术,待大鼠恢复清醒状态后,通过股静脉给大鼠瞬间注射CPZ生理盐水溶液(20 mg/Kg),采集不同时间点的血样、收集不同时间段的胆汁,用含有内标的葛根素甲醇溶液(20μg/mL)沉淀蛋白。采用Cosmosil C18色谱柱(5μm,4.6×150 mm),以甲醇-0.03 mol/L醋酸铵缓冲液(20：80 v/v)为流动相,流速1 mL/min,检测波长为270nm,柱温40℃,进样量10μL的色谱条件测定CPZ在大鼠胆汁和血液中的浓度,并用DAS1.0软件估算相应的药动学参数。结果：1.内／外源性胆汁淤积大鼠模型的进食量,体重及活动量均明显减少,且其生化指标谷草转氨酶(Glutamic oxaloacetic transaminase, AST)、谷丙转氨酶(Glutamic pyruvic transaminase, ALT)、总胆红素(Total bilirubin, TBIL)、直胆红素(Direct bilirubin, DBIL)、总胆汁酸(Total bile acids, TBA)及碱性磷酸酶(Alkaline phosphatase, ALP)显著升高,而白蛋白(Albumin, ALB)和胆固醇(Cholesterol, CHOL)显著降低。外源性胆汁淤积大鼠模型的双耳、眼球及尾巴均明显黄染,尿液变黄,其肝脏肿胀,包膜张力变大,边缘钝厚,色泽暗红,其组织病理切片可见毛细胆管扩张,肝血窦变窄,汇管区毛细胆管大量增加等现象,再通手术后,其肝脏组织渐渐恢复正常,但有个别大鼠的肝组织出现了局部硬化的现象。内源性胆汁淤积大鼠模型未见双耳、眼球和尾巴的黄染,但尿液变黄,其肝脏肿胀并黄染,有淤血现象,且组织病理切片可见肝内胆管有所增生,肝细胞排列紊乱,小叶间质炎症等现象。2. CPZ的血浆样品和胆汁样品在0.16～160μg／mL浓度范围内的标准线性方程分别为Y=0.1703X+0.0019和Y’=0.1404X’+0.0011,其相关系数R2分别为0.9993和0.9996；血浆样品的日内、日间回收率均大于92.42%,精密度RSD均小于9.55%；胆汁样品的日内、日间回收率均大于91.22%,精密度RSD均小于8.06%。3. CPZ在内／外源性胆汁淤积大鼠模型体内的药动学行为较正常大鼠组发生了显著变化,其在内／外源性胆汁淤积大鼠模型体内的血药浓度显著增加而胆药浓度显著降低(P<0.01),且CPZ在内／外源性胆汁淤积大鼠体内经胆汁排泄的量较正常大鼠分别降低28.58%和59.23%,而其半衰期t1/2和平均滞留时间(Mean residence time, MRT)显著增加(P<0.01),而系统清除率(Systemic clearance, CLsys)和胆汁清除率(Bile clearance, CLbile)显著降低(P<0.01)。4. CPZ在茵栀黄颗粒治疗7 d的内源性胆汁淤积大鼠模型体内的动力学行为与灌胃7 d生理盐水内源性胆汁淤积大鼠模型的药动学学行为存在显著性差异,其t1/2分别为0.385±0.016 h和0.526±0.17 h(P<0.01)；其MRT分别为0.472±0.098 h和0.653±0.131 h(P<0.01)。但CPZ在茵栀黄颗粒治疗7 d的外源性胆汁淤积大鼠模型体内的药动学行为与灌胃7 d生理盐水外源性胆汁淤积大鼠模型的药动学行为间并无统计学意义(P>0.05)。结论：1.本实验通过大鼠颈部皮下注射EB和胆道梗阻-再通手术分别获得了典型的内／外源性胆汁淤积大鼠模型。2.肝细胞膜转运蛋白Mrp2在胆汁淤积大鼠模型肝脏上的表达显著降低,使CPZ不能被顺利排入胆汁,导致了CPZ在胆汁淤积大鼠体内经胆汁排泄的量显著降低,在血液循环中的浓度显著升高,使系统清除率也随之降低。3.茵栀黄颗粒作为临床上常用的治疗黄疸的辅助用药,灌胃给予内／外源性胆汁淤积大鼠模型7d后,根据CPZ的药动学行为变化初步推测茵栀黄颗粒的退黄作用机制之一可能是改变了Mrp2的表达或增强了Mrp2的转运能力。创新性：1.考察了CPZ在内／外源性胆汁淤积大鼠体内的药动学变化；2.从转运蛋白的角度初步阐明CPZ药动学行为改变的机理。更多还原

【Abstract】 Objective:1. To establish rat model of intrahepatic/extrahepatic cholestasis and obtain rat models with different expression of Mrp2;2. To investigate the pharmacokinetics behavior of cefoperazone (CPZ) in rat model of intrahepatic/extrahepatic cholestasis;3. To clarify the influence of hepatocyte membrane transporters Mrp2 on the pharmacokinetics of CPZ.Methods:1. Estradiol benzoate (EB,11 mg/kg/d) was used to replicate rat model of intrahepatic cholestasis. Rats were under ether anesthetized, a 3-cm incision was made just below the xiphoid process. A sterile PE-10 cannula was inserted into the proximal portion of the common bile duct and held in position with two silk sutures. The distal portion of the cannula was then occluded and brought out through the lower end of the midline incision and secured in the cervical part for 5 days. Then, cut the ligation of tube on the 6th day for 3 days, extrahepatic cholestasis rats were replicated.2. Wistar rats were divided into normal control group, intrahepatic cholestasis group, dissolvant control group (subcutaneous injection of olive oil), extrahepatic cholestasis group, sham operation group (rats opened abdomen, freed bile duct and closed abdomen), intrahepatic cholestatic group interfered with Yinzhihuang granules (gave Yin-zhi-huang granules to rat model of intrahepatic cholestatic for 3 d or 7 d), extrahepatic cholestatic group interfered with Yin-zhi-huang granules (gave Yin-zhi-huang granules to rat model of extrahepatic cholestatic for 7 d). Rats fasted for 12 h before experiment. Under ether anesthesia, the femoral artery was cannulated for the collection of blood samples (except for the extrahepatic cholestasis group). The bile duct was cannulated for the collection of bile samples. After the animals had recovered from anesthesia, CPZ (dissolved in saline) was intravenously injected at a dose of 20 mg/kg. Blood samples (0.25 mL) were collected from the femoral artery at different time points. Bile specimens were collected in fractions at different time period. The samples were determined by HPLC. Chromatographic conditions to determine of CPZ in plasma and bile (Reverse phase column:Cosmosil-ODS C18,5μm, 4.6×150mm, Kyoto, Japan) was been used. The mobile phase consisted of methanol-water containing 30 mM/L ammonium acetate buffer (20:80, v/v). Prior to use, the mobile phase was filtered through a 0.45μm hydrophilic membrane filter. The mobile phase was delivered at a flow rate of 1.0 mL/min. Detection was performed at a wavelength of 270 nm at 40℃. The sample injection volume was 10μL. Internal standard was puerarin). Plasma concentration-time data and bile concentration-time data for CPZ were analyzed by the DAS 1.0 statistical software for pharmacology.Results:1. The food intake, body weight and activity were decreased significantly in intrahepatic/extrahepatic cholestasis rats (P<0.01), and their biochemical indicators of AST, ALT, TBIL, DBIL, TBA and ALP significantly increased, while ALB and CHOL significantly reduced (P<0.01). In rat models of extrahepatic, the ears, eyes, tail and urine were yellowing, the liver was swelling, capsular tension of large, blunt the edge of thick, dull red color, and the histology of extrahepatic cholestasis rat was capillary duct dilatation, hepatic sinusoids become narrow and so on. After reconstruction surgery, liver tissue began to return to normal gradually, but some of the organizations had cirrhosis phenomenon. In rat models of intrahepatic, the liver was swelling, and yellow dye, a large number of intrahepatic bile duct hyperplasia, congestion phenomena, disordered arrangement of liver cells, lobular interstitial inflammation.2. The assay of CPZ in plasma samples and bile samples showed good linearity (0.9993 and 0.9996 respectivel) over a relatively wide concentration range from 0.16 to 160μg/mL. The standard linear equations were Y= 0.1703X+0.0019and Y’= 0.1404 X’+0.0011, respectively. The precision of CPZ at low to high concentrations were better than 9.55%and 8.06%for plasma samples and bile samples, respectively. The recovery of the method exceeded 88.3%and 92.42%for CPZ in plasma samples and bile samples.3. The pharmacokinetics behavior of CPZ in rat models of intrahepatic/ extrahepatic cholestasis were significant different from normal control rats. The concentrations of CPZ in plasma were significantly increased (P<0.01), but concentrations of CPZ in biliary were significantly decreased (P<0.01). The amounts of CPZ excreted by bile were reduced 28.58%and 59.23%, respectively. The t1/2, MRT and the CLsys were increased. However, CLbile were significant decreased (P<0.01).4. The pharmacokinetics behavior of CPZ in rat model of intrahepetic cholestasis rats treated by Yin-zhi-huang granules was significant different from intrahepatic cholestasis rats treated by physiologic saline. The t1/2 of them was 0.385±0.016 h and 0.526±0.17 h, respectively. The MRT of them was 0.472±0.098 h and 0.653±0.131 h, respectively. However, the pharmacokinetics behavior of CPZ was no significant difference between extrahepatic cholestasis rats treated by Yin-zhi-huang granules and extrahepatic cholestasis rats treated by physiologic saline (P>0.05). Conclusion:1. The rat models of intrahepatic cholestasis were replicated by injecting EB. Based on the bile duct obstruction-reconstruction, the rat models of extrahepatic cholestasis were replicated.2. The expression of efflux transporter Mrp2 are markedly reduced in cholestatic rat, which means that a large number of CPZ can not be smoothly excreted into the bile and cause concentration of CPZ in the blood increased significatly, the account of bile excretion was significantly reduced.3. Yin-zhi-huang granules commonly used in clinic as an adjuvant treatment of jaundice. Based on the pharmacokinetics of CPZ, the pharmcological effects of Yin-zhi-huang granules may be caused by adjusting the expression of Mrp2 or enhancing the transport function of Mrp2.New Ideas:1.To investigate the pharmacokinetics behavior in rats model of intrahepatic/extrahepatic Cholestasis;2. To interpret the pharmacokinetics behavior in cholestasis rats by Mrp2.更多还原