【作者】 刘艺平；

【导师】 李焕德；

【作者基本信息】 中南大学 ， 药理学， 2012， 博士

【摘要】 目的：考察地塞米松与维拉帕米对P-糖蛋白(P-gp)功能及表达影响的时效与量效关系及其对阿米替林与去甲替林外排速率及中枢分布的影响；评估P-gp介导的体外相互作用与脑内相互作用的相关性,探讨利用P-gp介导的相互作用促进药物疗效与减少不良反应的可行性。方法：1.采用HaloC18(2.1mm×100mm,2.7μm)色谱柱,纯净水-乙腈(v/v,60：40)为流动相,采用Waters Micromass Quattro PremierXE二极质谱仪为检测器,建立UPLC-MS/MS同时测定阿米替林与去甲替林含量的方法。2. Caco-2细胞采用MEM培养基进行常规培养,P-糖蛋白表达通过荧光免疫法验证；采用MTT法考察维拉帕米和地塞米松对Caco-2细胞活性的影响；使用流式细胞术考察地塞米松、维拉帕米对罗丹明123摄取的影响。使用免疫荧光与流式细胞术研究地塞米松、维拉帕米对P-糖蛋白表达的影响。3.将Caco-2细胞接种于transwell板进行培养,构建单层细胞模型,用细胞电位仪、萤光黄及普奈洛尔转运实验对模型进行验证。建模成功后考察地塞米松、维拉帕米对阿米替林与去甲替林的双向跨膜转运的影响。4.396只昆明种雄性小鼠被随机分成3组,经单次或多次给药后,研究地塞米松与维拉帕米对阿米替林与去甲替林中枢转运的影响。结果：1.在实验条件下,地塞米松、维拉帕米及内标物质分离良好,色谱峰尖锐且半峰宽小于0.25s。一次进样的运行时间小于4min。最低检测限为10ng·mL-1。阿米替林与去甲替林在10～2500ng·mL-1浓度范围内线性良好,方法准确、灵敏、简单。2. Caco-2细胞经复苏后培养成功,荧光免疫法证实了Caco-2细胞中P-糖蛋白强表达。MTT法证实地塞米松、维拉帕米浓度小于100μmol·L-1时,对细胞活性无影响。Rh-123的摄取实验发现在0.01～25μmol·L-1范围内,与空白对照组相比,地塞米松组降低了细胞内荧光强度,降低的幅度为10～60%,荧光强度与浓度呈负相关；而维拉帕米增强了细胞内荧光强度(浓度>0.1μmol-L-1),荧光强度的增加与剂量正相关。免疫荧光实验发现,在1-50μmol·L-1浓度范围内,地塞米松组荧光强度显著强于空白对照组(P<0.05)；维拉帕米组在0.1-50μmol·L-1范围内荧光强度均显著强于空白对照组(P<0.05)。在较低浓度(0.01-0.1μmol·L-1)时,地塞米松对P-gp的表达无影响；维拉帕米浓度为0.01μmol·L-1时,对P-gp表达无影响。3.显微镜下显示Caco-2单层细胞排列紧密且完整,荧光黄及普萘洛尔跨膜实验证实细胞旁路转运通透性低,而跨细胞转运通路通透性良好。跨膜转运实验证实了阿米替林与去甲替林是P-gp的弱型底物,地塞米松增加了阿米替林和去甲替林的外排速率(增加幅度为10%-60%),而维拉帕米减少了阿米替林和去甲替林的外排速率(减少幅度为10%～20%)。4.在体动物实验表明地塞米松能减少阿米替林与去甲替林脑组织浓度-时间曲线下面积(AUC)值,减少程度大约为40～50%,而维拉帕米能增加阿米替林与去甲替林AUC值,增加程度大约为10%～50%。结论：地塞米松能增强P-糖蛋白的功能,同时诱导其表达,增加了阿米替林与去甲替林的外排速率,减少中枢分布,而维拉帕米能抑制P-糖蛋白功能,诱导其表达,总体表现为抑制了P-gp的外排速率,增加阿米替林与去甲替林的中枢分布。体外与体内研究结果基本一致,说明P-gp介导的中枢相互作用可以通过体外研究实现,利用P-gp介导的相互作用能改变中枢的药动学,达到增强药物疗效,减少药物的不良反应的目的,亦可用于中枢药物过量时的解毒。更多还原

【Abstract】 ObjectiveTo study the effect of dexamethasone (Dex) and verapamil(Ver) on the function and expression of P-glycoprotein, and their effect on the efflux ratio and the distritribution into brain of amitriptyline (Ami) and nortriptyline(Nor). To investigate the correlation between in vitro and in vivo results of drug interactions. And to assess the feasibility of predicting outcomes of interactions in central nervous system based on the results in vitro and improving the efficacy and decreasing adverse reactions by utilizing P-glycoprotein mediated interactions.Methods1. Blood samples were prepared with protein precipitation, and Halo C18column was performed with the mobile phase consisting water and acetonitrile, and samples were detected on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM).2. Caco-2cells were cultured with MEM which contains10%FBS(Fetal Bovine Serum) in a humidified atmosphere of95%air and5%CO2at37℃. The expression of P-glycoprotein in the cells was identified by immunofluorescence assay. Activity of Caco-2cells was assessed by MTT (Methyl thiazolyl tetrazolium) assay. Fluorescence spectrophotometric method and flowcytometry were used to study the effects of Ver and Dex on the efflux of Rhodamine123. The expression of P-glycoprotein in Caco-2cells was measured by immunofluorescence method.3. The efflux ratios (RE) of Ami and Nor were investigated with Caco-2cells monolayer model in transwell plates.4.396Kunming male mice were divided into three groups, and the concentration of Ami and Nor in plasma and brain tissues were detected by UPLC-MS/MS after simultaneous intraperitoneal injection Ami or Nor and a single dose or multiple doses of Dex or Ver.Results1. UPLC-ESI-MS/MS in the MRM model methodology provides a highly sensitive, selective, reliable and rapid method for determination of Ami and Nor. The linear calibration curves of Ami and Nor were obtained in the concentration10ng mL-1～2500ng mL-1. The extraction recoveries were74.3%-78.6%, intra-and inter-day precision of the assay at three concentrations were2.8%-14.1%with accuracy of95%-105%.2. Caco-2cells grew very well and the expression of p-glycoprotein was plentiful in Caco-2cells. Dex and Ver did not affect the activity of Caco-2cells when the concentrations were less than100μmol-L-1. Dex can decrease the fluorescence intensity obout20-50%, and there was a significant negative correlation between fluorescence intensity and the dosage when the concentration of Dex was less than25μmol·L-1. Ver can increase the fluorescence intensity in a dose-dependent manner.The study of expression of P-gp showed that there was no difference between Dex group(within0.01～0.1μmol·L-1) and control group in the fluorescence (P>0.05). When the concentration of Dex was within1～50μmol·L-1, the fluorescence of Dex group was significantly higher than that of control group (P<0.05); When the concentration of Ver was within0.1～50μmol·L-1, the fluorescence of Ver group was significantly higher than that of control group (P<0.05).3. The study of monolayer model showed that Ami and Nor are the poor substrates of P-glycoprotein and the efflux ratio of Ami and Nor were increased about10to60percents in presence of Dex and decreased about10to20percents in presence of Ver.4. Dex can decrease the value of AUC of brain tissue in mice about40to50percent and Ver increased the AUC about10to50percents. There were no differences in Cmax between Dex group, Ver group and control group.ConclusionDex can induce the function and the expression of P-gp, and Ver can inhibit the function and induce the expression of P-glycoprotein. Ami and Nor both are the poor substrates of P-gp. The efflux ratio of Ami and Nor can be increased by Dex and decreased by Ver. Results in vivo showed that distribution of Ami and Nor into the brain was increased by Ver and decreased by Dex.The studies showed that the results in vitro can predict the outcomes of drug-drug interactions in central nervous system. Inducer and inhibitor can modulate the function and expression of P-glycoprotein, which indicates that rational use inducer and inhibitor can improve the curative effect and decrease adverse reactions.更多还原