【作者】 颜苗；

【导师】 李焕德；

【作者基本信息】 中南大学 ， 药理学， 2010， 博士

【摘要】 甘草酸(glycyrrhizic acid, GL)是甘草中最主要的活性成分,水解后产生二分子葡萄糖醛酸(glcuronic acid)和一分子甘草次酸(glycyrrhetinic acid, GA)。由于三萜皂苷母核18位手性碳原子(C-18)构型的不同,甘草酸存在一对差向异构体,即18α-甘草酸(α-GL)和18β-甘草酸(β-GL),两者水解后可生成相应的18α-甘草次酸(α-GA)和18β-甘草次酸(β-GA)。早期对甘草酸、甘草次酸的研究大多没有将两个异构体明确区分,但随着具有我国自主知识产权的“手性肝药”异甘草酸镁在2006年上市,甘草酸18位差向异构体的差异日益受到国内外学者的关注。从药物转运的角度,本文研究了甘草酸18位差向异构体及其水解产物对P-糖蛋白的影响。目的研究甘草酸18位差向异构体及其水解产物对P-gp功能和表达的影响,探讨其对P-gp的作用是否具有立体选择性；通过研究甘草酸18位差向异构体水解产物在Caco-2细胞上对P-gp底物转运的影响,进一步确证功能和表达实验结果；通过研究甘草酸制剂对他林洛尔药动学的影响,初步探讨甘草酸制剂与P-gp底物在临床上可能发生的相互作用。方法1. RP-HPLC法分离并测定甘草酸制剂中的18α-、18β-甘草酸采用Phenyl-Hexyl (4.6 mm×250 mm,5μm)色谱柱,流动相为10mmol·L-1醋酸铵溶液(pH=7.75)-乙腈(83:17),流速1.0 mL/min,柱温38℃,检测波长250 nm。2.细胞培养及单层模型的建立采用MEM培养基常规培养Caco-2细胞、脐静脉内皮细胞(ECV304),用于研究甘草酸18位差向异构体及其水解产物对P-gp功能和表达的影响。采用苯基溴化四氮唑蓝法(MTT),判断各试验药物的体外最大非细胞毒性剂量,保证试验过程中细胞的活性,并为甘草酸18位差向异构体及其水解产物对P-gp功能和表达以及P-gp底物转运影响的研究提供与细胞作用的最大浓度。3.罗丹明123摄取实验研究药物对P-gp功能的影响以ECV304细胞作阴性对照细胞,维拉帕米作为抑制P-gp功能的阳性对照,使用流式细胞术测定罗丹明123(Rho-123)荧光值,分析甘草酸18位差向异构体及其水解产物对P-gp底物—Rho-123被Caco-2细胞摄取的影响,以此反映P-gp功能的变化。4.实时荧光定量PCR检测MDR1 mRNA表达以环孢素A作为上调MDR1 mRNA表达的阳性对照,不加药处理的Caco-2细胞作为阴性对照,Real-time PCR检测MDR1 mRNA,研究甘草酸18位差向异构体及其水解产物对MDR1 mRNA表达的影响。5.流式细胞术和Western blot检测P-gp蛋白表达以环孢素A作为诱导P-gp蛋白表达的阳性对照,不加药处理的Caco-2细胞作为阴性对照,采用流式细胞术(FCM)和Western blot分别研究α-GL和β-GL、α-GA和β-GA对P-gp蛋白表达的影响。6. Caco-2细胞单层模型上P-gp转运实验在TranswellTM12孔板上建立Caco-2细胞单层模型,使用酶标仪测定转运液中Rho-123的浓度,分别考察药物瞬时作用下对Rho-123双向转运的影响以及药物干预72h后Rho-123双向转运的变化。HPLC测定转运液中他林洛尔的浓度,进一步在Caco-2细胞单层模型上考察药物干预72h后他林洛尔外向转运的变化。7.甘草酸制剂对他林洛尔药动学的影响采用2×2双交叉试验设计。14名受试者随机等分为2组,一组先服用复方甘草酸苷片6天,每天三次,每次三片,第7天服用他林洛尔片1片,即试验组；另一组先服用安慰剂6天,每天三次,每次三片,第7天服用他林洛尔片1片,即对照组。受试者服药前及服药后0.25、0.5、0.75、1、1.5、2、2.5、3、4、6、8、12、16和24h取静脉血5mL置肝素化离心管中,分离出血浆,置-70℃冰箱中保存待测。采用HPLC法测定血浆中他林洛尔的浓度。使用DAS2.1.1软件计算药动学参数,SPSS 13.0软件进行统计分析。结果与结论1.各制剂中α体和β体甘草酸的组成比各不相同,亟待制定甘草酸制剂中异构体含量的相关质量标准。2.α-GL、β-GL对P-gp的影响作用方向呈现相反的趋势。中、高浓度(10μmol·L-1、60μmol·L-1)α-GL对P-gp功能表现出抑制作用,但没有呈现出剂量依赖性。β-GL各浓度对P-gp功能表现出诱导作用,也没有表现出剂量依赖性；中、高浓度(10μmol·L-1、60μmol·L-1)α-GL下调MDR1 mRNA表达,β-GL只在高浓度60μmol·L-1上调了MDR1mRNA表达；高浓度(60μmol·L-1)β-GL在蛋白水平对P-gp有诱导作用,α-GL各浓度没有表现出对P-gp表达的影响。转录水平上α-GL、β-GL对P-gp的影响与α-GL、β-GL对CYP3A的影响呈现一定的同向性,甘草酸18位差向异构体对CYP3A与P-gp的影响有相似的立体选择性,其机制是否与孕烷X受体(PXR)有关,有待进一步研究。3.α-GA、β-GA对P-gp的作用可能存在程度上的差别,而且18α-、18β-甘草次酸对P-gp的影响与其母体甘草酸的作用趋势并不一致。中、高浓度(1μmol·L-1、10μmol·L-1)α-GA对P-gp功能表现出诱导作用,并呈现出剂量依赖性。高浓度(10μmol·L-1)β-GA对P-gp功能表现出诱导作用；高浓度(10μmol·L-1)α-GA上调MDR1 mRNA表达,实验浓度β-GA未影响MDR1 mRNA的表达；高浓度(10μmol·L-1)α-GA在蛋白水平对P-gp有诱导作用,实验浓度β-GA没有表现出对P-gp表达的改变。甘草酸苷水解后与其苷元甘草次酸对P-gp的影响存在区别,其机制有待进一步研究。4.P-gp底物转运试验中,高浓度(10μmol·L-1)α-GA瞬时作用于Caco-2细胞单层模型,或者干预单层模型72h后,均能诱导P-gp底物Rho-123外向转运；高浓度(10μmol·L-1)β-GA瞬时作用于Caco-2细胞单层模型没有表现出诱导作用,但在干预Caco-2单层模型72h后,表现出对Rho-123外排的诱导；他林洛尔的外向转运受α-GA、β-GA干预72h后的影响与Rho-123受α-GA、β-GA干预72h的结果相似。α-GA诱导P-gp底物外排的能力比β-GA强。鉴于α-GA、β-GA对Caco-2细胞干预72h后均能不同程度的促进P-gp底物Rho-123和他林洛尔的外向转运,甘草酸上调MDR1,诱导外源性物质的外排,可能是甘草解毒的机制之一。5.他林洛尔药动学试验表明,复方甘草酸苷没有增加他林洛尔的不良反应,总体上也没有对其药动学产生影响。但有可能是因为样本数量不够多、群体的个体差异而使药动学参数改变没有表现出统计学意义。也有可能是试验设计服用复方甘草酸苷的时间不够长,未能产生对肠道P-gp的诱导。虽然国内外多数研究采用了他林洛尔作为P-gp相关人体试验的探针药物,其是否能准确反映P-gp的变化情况,有待进一步确证。鉴于本研究体外实验发现α-GA可能比β-GA有更强的诱导P-gp的能力,α体甘草酸制剂对P-gp底物药动学的影响有待进一步研究,并且由于纯α体甘草酸制剂临床应用时间还不长,临床上与其他药物同时使用时,应注意观察是否有药物相互作用的发生。根据本研究的提示和相关文献报道,差向异构体之间虽然只是立体构型的不同,但对药物转运体的影响可能会有不同的表现,在药物研究与开发中应当引起一定的重视。更多还原

【Abstract】 Glycyrrhizic acid (GL) is the main active component of licorice (Radix Glycyrrhiza), one molecule of GL can be transformed to two molecule of glucuronic acid and one molecule of glycyrrhetinic acid (GA) after hydrolyzation. Base on the defferent configuration of C18g-H bond of triterpene saponin mother nucleus, GL has two different epimers:α-GL andβ-GL and they can be hydrolyzed to correspondingα-GA andβ-GA. They were not distinguished at most previous researches. But the difference of C-18 epimers of glycyrrhizic acid has attracted more and more attention after magnesium isoglycyrrhetate was marketed in 2006. The effects of C-18 epimers of glycyrrhizic acid and their hydrolysis products on P-glycoprotein (P-gp) were introduced in this paper.OBJECTIVEStudy the effects of C-18 epimers of glycyrrhizic acid and their hydrolysis products on the function and expression of P-gp and investigate that whether there are stereoselectivity on them. Confirm the experimental result of function and expression by studing the changes of transmembrane transport of P-gp substrates on Caco-2 cell monolayers. Study the effects of glycyrrhizin preparations on the pharmacokinetics of talinolol, initially investigate possible drug interaction between glycyrrhizin preparations and P-gp substrates.METHODS1. Separation and determination of 18a-GL and 18β-GL in glycyrrhizin preparations by RP-HPLCThe chromatographic analysis was carrid out on a Phenyl-Hexyl Column (4.6 mm×250 mm,5μm) with the mobile phase consisting of 10 mmol·L-1 ammonium acetate (pH 7.75)-acetonitrile (83:17 v/v) at a flow-tate of 1.0 mL·min-1. The detection wavelength was 250 nm. 2. Cell culture and establishment of Caco-2 cell monolayer modelCaco-2 cell and ECV304 cell were cultured with MEM (Modified Eagle’s Medium) and were applied to study the effects of GL and GA epimers on the function and expression of P-gp. MTT (Methyl thiazolyl tetrazolium) assay was applied to determine the maximum non-cytotoxic dose of each test drugs to ensure the activity of cells during the test and to consult the maximum test concentration of each drugs.3. Rho-123 uptake experiments assay the the function of P-gpECV304 cell was used as negative cell control. Verapamil was used as positive control of the inhibitory effect on the function of P-glycoprotein. Flowcytometry were used to study the effects of C-18 epimers of GL and GA on the uptake of rhodamine-123 which is a substrate of P-gp in Caco-2 cell.4. Real-time PCR assay MDR1 mRNACyclosporin A was used as positive control of up regulation effect on the expression in mRNA level of MDR1 gene. Caco-2 cell without drug dealing was used as negative control. Real-time PCR was used to measure the expression of MDR1 mRNA in Caco-2 cell and to study the effects of C-18 epimers of GL and GA products on the expression of MDR1 mRNA.5. Flow cytometry and Western blot assay P-gp proteinCyclosporin A was used as positive control of up regulation effect on the expression of P-gp protein. Caco-2 cell without drug dealing was used as negative control. Flow cytometry (FCM) and Western blot were applied to study the effects of C-18 epimers of GL and GA on the expression of P-gp protein, respectively.6. P-gp transport experiments on Caco-2 monolayersAppropriate Caco-2 monolayers were established in TranswellTM plates. ELISA Reader was applied to detect the concentration of Rho-123 in transfer fluids. The bi-directional transports of Rho-123 after treated by instantaneous action and incubation for 72 h ofα-GA andβ-GA were studied. HPLC was applied to detect the concentration of talinolol in transfer fluids. The efflux of talinolol in Caco-2 monolayers after treated for 72 h byα-GA andβ-GA was also investigated.7. Effects of glycyrrhizin preparation on the pharmacokinetics of talinololA two-cycle double crossover design was used.14 subjects were randomized into 2 groups. One group administrated the glycyrrhizin preparation for 6 day (3 times one day,3 tablets once) and then administrated talinolol one tablet on the 7th day (Test Grop). Another group administrated the placebo for 6 day (3 times one day,3 tablets once) and then administrated talinolol one tablet on the 7th day (Control Grop). Before the administration of the preparation and at 0.25,0.5,0.75,1,1.5, 2,2.5,3,4,6,8,12,16 and 24 h after the administration of the preparation,5 mL of the venous blood was taken respectively. The blood samples were centrifuged in a centrifugal tube with heparin to separate the plasma, which was stored at-70℃until testing. HPLC was used to measure the concentration of talinolol in human plasma. DAS (Drug and Statistics 2.1.1) and SPSS 13.0 were used in the statistical analysis.RESULTS AND CONCLUSION1. The composition ratios ofα-GL andβ-GL in different glycyrrhizin preparations were different. Quality specifications about the composition ratios of epimers of GL should be established.2. The effects ofα-GL andβ-GL on P-gp show an opposite trend. At middle and high concentrations (10μmol·L-1、60μmol·L-1), a-GL inhibited the function of P-gp and with on dose dependent while P-GL induced the function of P-gp at three test concentrations with no dose dependent too; At middle and high concentrations (10μmol·L-1、60μmol·L-1),α-GL down-regulated the expression of MDR1 mRNA. At high concentrations (60μmol·L-1),β-GL up-regulated the expression of MDR1 mRNA; At high concentrations (60μmol·L-1),β-GL induced the expression of P-gp protein whileα-GL has no effect on the expression of P-gp protein at three test concentrations. The effects ofα-GL andβ-GL on the expression of MDR1 mRNA and CYP3A mRNA showed the same trend. The character that epimers of GL act on CYP3A and P-gp show similar stereoselectivity whether relate to PXR need further study.3. The actions on P-gp betweenα-GA andβ-GA may with different degree. The effects ofα-GA andβ-GA on P-gp were different from their corresponding parent bodies (GL). At middle and high concentrations (1μmol·L-1、10μmol·L-1),α-GA induced the function of P-gp and with dose dependent whileβ-GA also induced the function of P-gp at high concentration (10μmol·L-1); At high concentrations (10μmol·L-1), a-GA up-regulated the expression of MDR1 mRNA while P-GA has no effect on the expression of MDR1 mRNA at three test concentrations;α-GA up-regulated the expression of P-gp protein at high concentrations (10μmol·L-1) whileβ-GA has no effect on the expression of P-gp protein at three test concentrations. The difference between the effects of glycyrrhizin and glycyrrhetinic acid need further study.4. In the transport experiments of P-gp substrates on Caco-2 monolayers, high concentration (10μmol·L-1)α-GA induced the efflux of Rho-123 both in the instantaneous action test and the incubation for 72 h test. High concentration (10μmol·L-1)β-GA induced the efflux of Rho-123 only in the incubation for 72 h test. The transports from AP to BL were not affected by any test drugs. The efflux test of talinolol from BL to AP got the similar results with those of Rho-123. The inducibility ofα-GA was stronger than that ofβ-GA. Glycyrrhizin can up-regulate MDR1 mRNA, induce the excretion of xenobiotics, it may be one of the detoxification mechanism of licorice.5. The pharmacokinetics experiments of talinolol suggested that no adverse reaction was observed and glycyrrhizin did not affect the pharmacokinetics of talinolol on the whole. But maybe the small number of samples or individual difference leaded to the results with no significance. The administration time of glycyrrhizin maybe too short or the dose of glycyrrhizin maybe too small, then P-gp in intestinal tract was not induced. Although talinolol was used as a probe drugs in many researches, the accuracy of the assay need further confirmation. In the light of result of this research,α-GA may be stronger thanβ-GA to induce P-gp. Effects of α-GL preparations on pharmacokinetics of P-gp substrates need further study. Because the clinical application of a-GL preparations are not long enough, when a-GL preparations used in conjunction with other durgs, drug interaction should be observed.According to the results of our research and other references, effects of epimers on P-gp may be different even though only configurations are different between the two compounds. This information deserves attention in drug research and development.更多还原