【作者】 赵瑞柯；

【导师】 彭文兴；

【作者基本信息】 中南大学 ， 药剂学， 2009， 硕士

【摘要】 一、目的通过考察盐酸度洛西汀对Caco-2细胞上P-糖蛋白功能和表达的影响以及对P-糖蛋白底物他林洛尔人体药动学的影响,探讨盐酸度洛西汀对P-糖蛋白的作用。二、方法（一）盐酸度洛西汀对Caco-2细胞上P-糖蛋白功能和表达的影响1.细胞培养以及形态学验证用MEM培养基对Caco-2细胞进行常规培养,建立Caco-2细胞模型,流式细胞术进行P-糖蛋白表达验证。2.细胞毒性试验用MTT法考察盐酸度洛西汀对Caco-2细胞的毒性,选择细胞存活率大于90%的药物浓度作为最大无毒浓度进行下一步细胞试验。3.罗丹明-123外排试验用维拉帕米作为P-糖蛋白功能抑制的阳性对照,用流式细胞仪测定细胞内罗丹明-123的荧光强度,考察度洛西汀对P-糖蛋白介导的罗丹明-123外排作用的影响,从而判断药物对P-糖蛋白功能的影响。4.P-糖蛋白表达的测定药物与细胞作用72h后采用流式细胞术分析度洛西汀对Caco-2细胞上P-糖蛋白表达的影响。（二）盐酸度洛西汀对他林洛尔人体药动学的影响1.给药方法采用随机开放,两周期自身前后对照试验设计,12名健康受试者第一周期单剂量口服他林洛尔1片（50mg）,第二周期受试者服用度洛西汀（30mg/次,Bid）,连续服用6天,于第七天晨加服他林洛尔1片（50mg）。洗脱期:1周。2.血样采集方法在他林洛尔服药前及服药后0.5,1,1.5,2,3,4,5,6,7,9,12,24,36,48和60h由肘静脉取血5mL置肝素化离心管中,分离出血浆,置-70℃冰箱中保存待测。3.测定方法采用HPLC-ESI-MS/MS法测定血浆中他林洛尔的浓度。三、结果（一）盐酸度洛西汀对P-糖蛋白作用的研究结果1.细胞培养以及形态学验证Caco-2形态正常;流式细胞仪测定显示Caco-2细胞高度表达P-糖蛋白,适合用于盐酸度洛西汀对P-糖蛋白功能和表达影响的研究。2.细胞毒性试验盐酸度洛西汀在≦10μmol/L时,与细胞培养72h后细胞的存活率大于90%,因此,盐酸度洛西汀的最大无毒浓度为10μmol/L。3.盐酸度洛西汀对罗丹明-123外排的影响与阴性对照组相比,低、中、高浓度（0.2、5、10μmol/L）的盐酸度洛西汀显著降低了罗丹明-123从Caco-2细胞的外排（p＜0.05）,但不同药物处理组间差别不明显。4.盐酸度洛西汀对P-糖蛋白表达的影响Caco-2细胞与低、中、高浓度（0.2、5、10μmol/L）的盐酸度洛西汀培养72h后,其荧光强度与阴性对照组相比,没有显著性差异。说明试验浓度的盐酸度洛西汀对P-糖蛋白的表达无明显作用。（二）盐酸度洛西汀对他林洛尔人体药动学的影响1.他林洛尔的测定他林洛尔在2.0～240.0ng/mL浓度范围内线性关系良好。萃取回收率均大于86.6%,方法回收率为94.3%～105.6%,日内、日间RSD均小于13%,稳定性试验RSD均小于10.5%。2.盐酸度洛西汀对他林洛尔药动学的影响受试者多剂量服用盐酸度洛西汀后,他林洛尔的AUC_0-60和C_max由单用时的1348.54ng·h/mL,91.90ng/mL分别增加至1498.30ng·h/mL和125.21ng/mL。AUC和C_max分别增加了11%和36%。等效性分析显示两周期AUC和C_max比值的90%置信区间分别为77%—106%和68%—112%,均落于等效范围之外。他林洛尔的其它药动学参数T_max,t_1/2,CL/F,V/F在两周期间并无显著性差异。四、结论本试验首次从体内、体外两方面考察了盐酸度洛西汀对P-糖蛋白功能的影响。体外试验显示:低、中、高浓度（0.2、5、10μmol/L）的盐酸度洛西汀均抑制了P-糖蛋白的功能,降低了罗丹明-123从Caco-2细胞中的外排,但是共同培养72h并不影响P-糖蛋白的表达。这说明盐酸度洛西汀对P-糖蛋白的影响仅为功能上的抑制,其抑制的可能原因为竞争性抑制,或者抑制ATP酶等,其具体机制还需进一步研究。体内试验显示:多剂量服用度洛西汀使得他林洛尔的AUC和C_max增加,提高其生物利用度,但并不影响其他的药动学参数。我们推测可能的原因是盐酸度洛西汀抑制了肠道P-糖蛋白的功能,增加了其吸收程度。试验结果提示我们盐酸度洛西汀和P-糖蛋白底物合用时可能通过抑制P-糖蛋白功能减少药物外排,增加其血药浓度,使得疗效增加,但同时应注意因药物浓度增加导致的药物不良反应。更多还原

【Abstract】 ObjectiveTo evaluate the effect of duloxetine on P- glycoprotein function by investigating the effects of duloxetine on the function and expression of P-glycoprotein in Caco-2 cells and quantitating the effect of orally administered duloxetine on the pharmacokinetics of talinolol.Method1.The effects of duloxetine on the function and expression of P-glycoprotein in Caco-2 cells1.1 Cell culture and morphology validated of cellsCaco-2 cells were cultured with MEM（Minimal Essential Medium） which contains 10%FBS（Fetal Bovine Serum） in a humidified atmosphere of 95%air and 5%CO₂ at 37℃.The expression of p-glycoprotein in the cells was identified by flow cytometer.1.2 CytotoxicityMTT（diphenyltetrazolium bromide） assay was used to determine the non-cytotoxicity dosage of duloxetine in Caco-2 cells.The dosage at which the survival rate of cells is above 90%was considered as the highest non-cytotoxic dosage.1.3 Effects of duloxetine on the efflux of rhodamine-123 in Caco-2 cellsThe effect of duloxetine on P-glycoprotein function was analyzed using rhodamine-123 assay.Flow cytometry was used to determine the intracellular rhodamine -123 concentration and verapamil was used as the positive control.1.4 Effect of duloxetine on P-glycoprotein expressionThe expression of P-glycoprotein in Caco-2 cells was measured by flow cytometry after incubation with duloxetine for 72h.2.The effect of orally administered duloxetine on the pharmacokinetics of talinolol2.1 Dose AdministrationThe study was conducted in a self-controlled two-period experiment in a randomized,open-labeled design in 12 healthy Chinese volunteers.In the 1^st period,the volunteers received a single oral dose of 50 mg talinolol. After 1 week washout,the volunteers received 30 mg duloxetine twice daily for 6 consecutive days.On the 7^th day,they received 30 mg duloxetine and 50 mg talinlolol concomitantly.2.2 Blood samples collectionSerial blood samples were collected from an in-dwelling venous catheter（anti-coagulated with sodium heparin） at 0、0.5、1、1.5、2、3、4、5、6、7、9、12、24、36、48、60 h after talinolol administration.The plasma was separated immediately and frozen at-70℃until analysis. 2.3 Analytic MethodConcentration of talinolol in plasma was determined by HPLC-ESI-MS/MS.Results1.Effects of duloxetine on the function and expression of P-glycoprotein in Caco-2 cells.1.1 Cell Culture and Morphology ValidatedThe expression of P-glycoprotein is plentiful in Caco-2 cell and Caco-2 cell is suitable for evaluating the effect of duloxetine on P-glycoprotein function and expression.1.2 CytotoxicityThe 90%cells survived after incubation with duloxetine when the concentration of duloxetine was no more than 10μmol/L.The max concentration without cytotoxicity for duloxetine is 10μmol/L.1.3 Effects of duloxetine on the efflux of rhodamine-123 in Caco-2 cellsVarious concentrations of duloxetine decreased the efflux of rhodamine-123 significantly compared with negative control,but the differences between dose groups were not significant.1.4 The effects of duloxetine on the expression of P-glycoproteinThe P-glycoprotein expression in Caco-2 cells after incubation with various concentrations of duloxetine（0.2、5、10μmol/L） was not significant different with the negative control.So,duloxetine in these concentrations doesn’t modulate the expression of P-glycoprotein in Caco-2 cells.2.The effect of duloxetine on the pharmacokinetics of talinolol2.1 Determination of talinololThe calibration curve was linear in the range of 2.0～240.0 ng/mL for talinolol.The average extraction recoveries for talinolol were above 86.6%.The methodology recoveries were between 94.3%and 105.6%. The intra-day and inter-day RSD were less than 13%and the RSD of stability test were no more than 10.5%.2.2 The effect of duloxetine on the pharmacokinetics of talinololAfter subjects treated with duloxetine,the AUC_0-60 and C_max of talinolol increased from 1348.54 ng·h/mL to 1498.30 ng·h/mL and 91.90 ng/mL to 125.21 ng/mL,respectively.The AUC_0-60 and C_max of talinolol were increased by 11%and 36%,respectively.The 90%CIs for the ratios of AUC（0.77-1.06） and C_max（0.68-1.12） were not fell within the accepted range for lack of interaction（0.80-1.25）.No significant difference in any other pharmacokinetic parameters was observed between the two periods(T_max,t_1/2,CL/F,V/F).DiscussionThe study for the first time evaluated the effect of duloxetine on the P-glycoprotein function in vitro and in vivo.In vitro study showed that various concentrations of duloxetine inhibit the function of P-glycoprotein and decrease the effiux of rhodamine-123,but doesn’t modulate the expression of P-glycoprotein in Caco-2 cell.This suggested that duloxetine just influence the function of P-glycoprotein.The possible reason is competitive inhibition or inhibition of ATP enzyme.In vivo study showed that a 6 -day treatment period with duloxetine increased the oral bioavailability of talinolol,but other pharmacokinetic parameters were not changed.Because talinolol is not metabolized to a relevant extent,we speculate that the increase in oral bioavailability of talinolol by co-administration of duloxetine can be attributed with high certainty to an inhibition of P-glycoprotein in the small intestine.Our results suggest that duloxetine could inhibit the function of P-gp in vitro and in vivo,and caution should be exercised when duloxetine is to be co- administered with drugs that are P-gp substrate.更多还原