【作者】 王进；

【导师】 郭瑞臣； 张岫美；

【作者基本信息】 山东大学 ， 药理学， 2010， 博士

【摘要】 目的：创建灵敏度高、特异性强的,适合岩白菜素体内药代动力学研究的液相色谱-二级质谱联用(HPLC-MS/MS)生物样本浓度测定方法。建立灵敏度高、专属性强、方便可行的高效液相色谱法(HPLC)并且首次测定昆明种(KM)小鼠血浆及组织中岩白菜素浓度,评价岩白菜素在健康小鼠体内的吸收、分布、代谢、排泄过程。采用建立的HPLC-MS/MS血药测定方法首次进行口服岩白菜素后人体药代动力学研究。进行中国健康志愿者单次单剂量口服岩白菜素(250mg)的药代动力学研究,了解其人体吸收、分布、消除体内过程规律。为制定科学合理的岩白菜素临床给药方案和深入的药效机制研究提供依据。建立高灵敏度和精确度、专属性强的液相色谱——级质谱联用(HPLC-MS)血药浓度测定方法。并对健康志愿者单次单剂量口服复方岩白菜素片(岩白菜素与马来酸氯苯那敏)中小剂量联用的氯苯那敏(4mg)进行人体药代动力学规律研究。应用膜片钳技术记录岩白菜素对大鼠海马中枢神经元和GABAA受体转染的HEK293细胞膜上Y-氨基丁酸(GABA)-激活电流(IGABA)的调制作用,同时对比岩白菜素对谷氨酸(Glu)-激活电流(IGLU)的影响,分析研究岩白菜素的中枢镇咳作用的可能分子作用靶点和机制。应用膜片钳记录分析岩白菜素对GABAA受体介导的IGABA的量效关系及电压-电流(I-V)曲线,分析其作用特点和规律。试采用液相色谱和质谱法及膜片钳等技术多方面分析研究岩白菜素的药代动力学规律和药效学主要作用机制,为新药开发和中药有效化合物研究提供较新的系统思路。方法：岩白菜素(1)色谱条件：采用Agilent SB-C18 (50mm×2.1mm,3.5μm)色谱柱；流动相为乙腈：水(10：90,V/V),在线脱气；流速0.2ml·min-1；柱温20℃；检测波长250nm；进样量20μl。(2)质谱条件：ESI离子源,负离子模式,雾化压力40 psi,干燥气(N2)流速9 L·min-1,干燥气温度为350℃,毛细管电压4000V,多级反应监测(MRM)方式,m/z：327.1→192(岩白菜素),m/z：354→185.1(内标),碰撞能量15V(岩白菜素)和30V(内标),碎片电压为100V。氯苯那敏(1)色谱条件：Venusil XBP-C18(5μm, ID4.6×150mm)色谱柱；流动相为甲醇：0.005M醋酸铵溶液(60：40,V/V),0.40μm微孔滤膜过滤,在线脱气；流速0.8ml·min-1；柱温25℃；进样量10μl。(2)质谱条件：API-ES离子源,正离子模式,喷雾气(N2)压力50psig,干燥气(N2)流速13.0L·min-1,干燥气温度350℃,毛细管电压4000V,选择性监测质荷比(m/z)为275.1(氯苯那敏)和256.1(苯海拉明)的分子离子峰,碎片电压分别为90V和70V。样本处理：(1)岩白菜素取血浆样品0.5ml,加入400ng·mL-1甲砜霉素内标溶液20μL,涡旋混匀,加4ml提取液(乙酸乙酯),涡旋混合1min,5000 r·min-1离心5min,取上层萃取液3.5ml,于40℃水浴N2吹干,残渣用100 u l流动相溶解,进样20μL。(2)氯苯那敏取血浆0.5ml,加入200ng·mL-1的苯海拉明内标溶液50u l,混匀,加入5ml氯仿,涡旋混匀2min,4000rpm离心5min,吸取下层有机相4ml,40℃水浴N2吹干,残渣用100μl流动相复溶。20名健康志愿者,于试验前一天晚禁食,试验当日晨抽取空白血样后,立即空腹口服复方岩白菜素试验制剂(250mg岩白菜素+4mg氯苯那敏)。不同时间取血,采用HPLC-MS/MS法和HPLC-MS法检测血浆中岩白菜素原型和氯苯那敏的浓度变化,采用DAS2.0实用药代动力学程序计算其药代动力学参数,分析其药代动力学特点。同期观察不良反应。昆明种小鼠104只,岩白菜素尾静脉注射给药3mg·kg-1后静脉取血及解剖取小鼠的心、肝、脾、肾、小肠、肺、脑,HPLC法测定其浓度或组织含量变化,评价小鼠体内的药代动力学及组织分布特征参数。应用全细胞式膜片钳技术和外膜外向式单通道技术,记录岩白菜素对大鼠海马中枢神经元和GABAA受体转染的HEK293细胞膜上ICABA的调制作用,并记录其剂量-效应变化和I-V曲线,分析作用特点。同时采用全细胞模式记录观察岩白菜素对IGLU的影响。分析岩白菜素的中枢镇咳作用的可能机制。结果：本实验创建的岩白菜素体内测定HPLC-MS/MS方法灵敏度高,专属性强,方便可行。岩白菜素在0.25～60ng·ml-1范围内线性关系良好,最低定量限可达0.25ng-mL-1,成功测定出岩白菜素主要人体药代动力学参数：t1/2为2.850±1.823h,Tmax为1.725±0.638h,AUC0～12为49.013±14.613ng·ml-1·h, Cmax为15.216±5.023ng·ml-1, AUC0～∞为51.352±15.194 ng·ml-1·h。HPLC-MS方法测定氯苯那敏的定量下限可达0.2ng·ml-1,氯苯那敏在0.2～20ng·ml-1范围内线性关系良好。联用的小剂量氯苯那敏在口服复方岩白菜素片后人体内主要药代动力学参数：血药浓度达峰值Tmax为2.650±0.763h,t1/2为11.480±3.605h, AUC0～36为98.636±22.863ng·ml-1·h, AUC0～∞为113.173±32.390ng·ml-1·h, Cmax为6.636±1.533ng·ml-1。小鼠静脉注射岩白菜素后体内药代动力学参数：t1/2为1.16±0.03 h, Cmax为2443.3±557.6 mg·L-1, AUC0-8为2482.6±281.4 mg·ml-1·h,AUC0-∞为2482.9±331.0mg·ml-1·h, Vd为1.07±0.08 L·kg-1。岩白菜素经静脉给药后,很快分布于全身,在体内组织分布广泛,代谢快。在动物体内主要分布于血液、肝脏和肾脏组织,其他组织中含量较少,但脑中含量相对肺组织较高。大鼠海马中枢神经元和转染GABAA受体的HEK细胞对外加GABA敏感。神经元上此GABA激活电流可被特异性GABAA受体的选择性拮抗剂荷包牡丹碱(bicuculline)所完全阻断。岩白菜素可明显增强GABA诱导的突触后抑制性电流IGABA。0.02-10mmoL·L-1岩白菜素对IGABA增强有浓度依赖性变化,量效曲线呈“s”型且EC50为0.81mmol·L-1。加入岩白菜素后GABA量效曲线可明显上移,但IGABA的最大值在加入岩白菜素前后基本不变。单通道记录发现岩白菜素对GABAA受体的影响主要是受体门控性通道的开放概率增加。此外,海马神经元同时给予50μmol·L-1 Glu和1mmol·L-1岩白菜素,IGlu在加入岩白菜素前后无明显改变(P>0.05)。结论：创建报道的岩白菜素体内测定HPLC-MS/MS方法灵敏度高,专属性强,方便可行。岩白菜素在0.25-60ng·ml-1范围内线性关系良好,最低定量限可达0.25ng.mL-1。健康志愿者口服岩白菜素250mg后达峰时间短,吸收迅速,半衰期不长显示人体内消除速度较快。药代动力学特点符合二室模型。建立的HPLC-MS方法测定氯苯那敏定量下限可达0.2ng·mL-1,氯苯那敏在0.2-20ng·ml-1范围内线性关系良好。可很好的检测复方给药后血药浓度很低的氯苯那敏。也可用于单方及其它剂型的检测。口服该药后吸收较快且完全,比岩白菜素半衰期长可在体内停留较长时间。小鼠静脉给药后岩白菜素的半衰期短,分布广泛,其中肝脏肾脏分布高,说明岩白菜素消除代谢快,发现脑组织相对肺组织含量高,可能与中枢性镇咳有关。岩白菜素能够增强突触后抑制性电流IGABA；且岩白菜素自身无法激活IGABA,与GABA合用可使GABAA受体开放的概率增加,从而增强IGABA。说明岩白菜素可能提高GABA和GABAA受体的亲和力来增强IGABA。岩白菜素对IGLU无影响。提示岩白菜素中枢镇咳作用的分子作用机制可能与GABA密切相关。岩白菜素和氯苯那敏安全性高,实验期间未见不良反应发生。启示：液相色谱-质谱法和膜片钳技术综合应用联合运用现代先进检测技术,对新药或中药有效化合物的药代动力学和药效学机制综合评价有重要意义。岩白菜素的镇咳及相关作用机制研究进一步研究岩白菜素对GABA等各类受体的作用和体内外实验效应,可探究岩白菜素类镇咳等主要作用的分子作用机制,为指导临床应用和开发新药提供明确的理论依据。进行岩白菜素体内代谢过程及代谢产物相关研究进一步对岩白菜素生物体内转化过程,代谢产物及其活性进行更详尽的研究,有利于明确岩白菜素类的全面作用规律和机制。开发岩白菜素靶向制剂开发在呼吸中枢部位高分布的岩白菜素靶向制剂,减少在肝脏肾脏组织的分布有助于提高其治疗作用。更多还原

【Abstract】 Objectives:To firstly establish highly sensitive, simple and selective high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) methods for the determination of bergenin in plasma after oral administration of bergenin tablets.To study the pharmacokinetics and tissue distribution. of bergenin in healthy mice after 3mg-kg"’bergenin intravenous injection by HPLC, to evaluate its pharmacokinetic parameters and rule.To apply HPLC-MS/MS method to the determination of bergenin concentration in human plasma after single dose (250mg) oral administration.To establish the HPLC-MS method for determine the low concentration of chlorpheniramine in human plasma after single dose(4mg) oral administration in complex tablets consists of bergenin and chlorpheniramine.To investigate the modulatory effect of bergenin onγ-aminobutyric acid (GABA)activated currents(IGABA)-patch clamp technique was performed on rat hippocampal pyramidal neurons and HEK293T cell (transfected of GABAA receptor)Methods:.Bergenin (1) Chromatographic conditions:A C18 column (50mm×2.1mm,3.5μm) (Agilent, U.S.A) was used for chromatographic separations. Bergenin and I.S. were eluted with mobile phase of acetonitrile-water(10:90, V/V)at a flow-rate of 0.2 mL/min. The total run time for a sample was about 6.5 min. The detection occurred at 25℃. (2) Mass spectrometric conditions:The ion source was set at the negative ionization mode (ESI+) and ion collection was occurred at the multi-reaction monitoring (MRM) mode, nitrogen was used as collision gas (with a collision energy of 15V for bergenin and 30V for thiamphenicol). Nebulizer gas was set at 40 psi, and spray gas at 9L-min-1. The temperature of the turbo ion spray source was 350℃. The capillary voltage was 4000V. The transitions of m/z 327.1^192 for bergenin and 354→185.1 for thiamphenicol (I.S.) were selected as quantification signals respectively. The scan time for bergenin was set at 0.4s.Chlorpheniramine (1)Chromatographic conditions:The separation was performed on a Venusil XBP-C18column (5μm, ID4.6×150mm), and eluted by the mobile phase consisted of a methanol-0.005M ammonia acetate solution (60:40, v/v) at a flow rate of 0.8mL/min. The column temperature was maintained at 25℃using a thermostated column compartment. (2)Mass spectrometric conditions:Mass spectrometric analysis was performed in the ESI positive ion mode, with spray gas pressure of 350 Pa, protective air of nitrogen gas at a flow rate of 13 L-min-1, capillary voltage of 4000 V, fragment electric voltage of 90 V for chlorpheniramine and 70 V for diphenhydramine. Full scan mode was performed for screening and library-assisted identification. The pseudomolecular ions [M-H]ˉ(m/z 275.1 for Chlorpheniramine and 256.1 for diphenhydramine) were used as the target ions for quantification in the selected ion monitoring (SIM) mode.Sample preparation:(1)Bergenin:0.5 mL plasma and 20μL I.S. solution (0.4μg·mL-1) were added to centrifuge tube containing 4 mL acetic ether, vortex-mixed for 2 min, then centrifuged at 5000 rpm (revolutions per minute) for 5 min.3.5 mL supernatant was transferred and evaporated to dryness under gentle stream of nitrogen in water-bath at 40℃. The residue was reconstituted with 100μL of acetonitrile-water (10:90, V/V) and 20μL supernatant was injected for analysis. Blank plasma samples, calibration series, QC and plasma samples were analyzed in one analytical sequence. (2)Chlorpheniramine:50μLof the internal standard (200 ng·mL-1 diphenhydramine) was mixed with 0.5 mL plasma sample, then 5mL methylene chloride were added, vortex-mixed for 2 min, and centrifuged at 4000 rpm for 5 min. The organic phase was transferred to a clean tube and evaporated to dryness under gentle nitrogen gas at 45℃. The residue was reconstituted with methanol-0.005M ammonia acetate solution (60:40, v/v), and 10μL was injected onto the LC/MS for analysis.Twenty healthy Chinese volunteers were orally given 254 mg complex tablets consists of 250 mg bergenin and 4mg chlorpheniramine with 200 mL water in the morning and 4 mL of blood were collected before (0 h) and 0.5,1,1.5,2.0,3.0,4.0,5.0, 6.0,8.0,12.0 24.0and 36.0 h after administrations, serum were separated, collected and stored at-20℃for analysis. The main pharmacokinetic parameters were calculated by Drug and Statistic software (DAS, version 2.0.1, by Sun et al, China).Healthy mice plasma were collected from vein or decollated for dissection of liver, spleen, kidney, intestine, lung, heart and brain after introvenous injection of 3 mg·kg-1 bergenin. Use HPLC to study the pharmacokinetics and tissue distribution, of bergenin in healthy mice after 3mg·kg-1 bergenin intravenous injection.Whole-cell configuration of patch clamp technique and outside-out configuration for single channal recording were performed on rat hippocampal pyramidal neurons and HEK293T cell (transfectde of GABAA receptor)to investigate the modulatory effect of bergenin on y-aminobutyric acid(GABA)-activated currents (IGABA). I-V curve and dose-response of Bergenin were recorded in the experiment. Glutamate activated currents (IGlu) was also observed to study the potential antitussive mechanism of bergenin in central nervous system.Results:It is proved that the HPLC-MS/MS methods is highly sensitive, simple and selective. The linear range of the calibration curve for determination of bergenin in plasma by the method was 0.25-60 ng·mL-1, and the low limit of quantitation (LOQ) was 0.25 ng·mL-1. The main pharmacokinetic parameters of bergenin after single oral doses in human plasma were as follows:t1/2 2.850±1.823h, Tmax 1.725±0.638h, AUC0～12 49.013±14.13ng·ml-1·h, Cmax 15.216±5.023ng·ml-1, AUC0～∞51.352±15.194ng·ml-1·h.The linear range of the calibration curve for determination of chlorpheniramine in human plasma after oral administration of complex tablets consists of 250 mg bergenin and 4mg chlorpheniramine by the HPLC-MS method was 0.2-20 ng·mL-1, and the low limit of quantitation was 0.2ng·mL-1. The main pharmacokinetic parameters of bergenin after single oral doses in human plasma were as follows:Tmax 2.650±0.763h, t1/2 11.480±3.605h, AUC0～36 98.636±22.863ng·ml-1·h, AUC0～∞113.173±32.390ng·ml-1·h, Cmax 6.636±1.533ng·ml-1。The main pharmacokinetic parameters of bergenin after intravenous injection (3mg·kg-1) in mice plasma were as follows:t1/2 1.16±0.03 h, Cmax 2443.3±557.6 mg·L-1, AUC0-8 2482.6+281.4 mg·ml-1·h, AUC0-∞2482.9±331.0 mg·ml-1·h, Vd 1.07±0.08L·kg-1。Bergenin was distributed widely in mice,the concentration in liver, and kidney is higher than others..The concentration of bergenin in brain is relatively higher than the concentration in lung.Bergenin was declined quickly in the body.The results showed that GABA-activated membrane currents were obviously increased by bergenin. The GABA-activated current (IGABA)was inhibited by bicuculline(10μmol·L-1) that showed the current was mediated by GABAA receptor. Dose-response relationship of bergenin(0.02,0.1,0.5,1,5,10mmol·L-1) on IGABA shows IGABA can be increased by bergenin within 0.02-10mmol/L.The dose response curve is "s" shape and EC50 is 0.81mmol·L-1. Bergenin shifted the GAB A dose-response curve upward,however, the maximum response keeps unchanged. The single channel activity was abolished by 10μmol·L-1 bicuculline confirming it was mediated by GABAA receptors. Bergenin did not induce any detectable single channel activity on its own. Results suggest that the effect of bergenin on GABAA receptors was achieved by affecting channel open probability, not channel conductance. IGLU keeped unchanged when bergenin was in or not.Conclusions: The firstly establishde HPLC-MS/MS methods is highly sensitive, simple and selective. The linear range of the calibration curve for determination of bergenin in plasma by the method is 0.25-60 ng·mL-1, and LOQ is 0.25 ng·mL-1. Bergenin is absorbed into the plasma rapidly not completely, and the short t1/2 showed that elimination of bergenin is quick.The HPLC-MS method developed in this study is suitable to meet the requirement of determination of low chlorpheniramine concentration in human plasma after its therapeutic oral doses in complex tablets consists of bergenin (125mg) and chlorpheniramine (2mg). The linear range of the calibration curve for determination of chlorpheniramine in plasma by the method is 0.2-20 ng·mL-1, and LOQ is 0.2 ng·mL-1. chlorpheniramine is absorbed into the plasma rapidly and completely. The t1/2 of chlorpheniramine is longer than bergenin shows that chlorpheniramin can be storaged in human body for a longer time.Pharmacokinetics and tissue distribution characteristics of bergenin in mice shows that bergenin is distributed widely and eliminated rapidly. Besides, the concentration of bergenin in brain is relatively higher than the concentration in lung shows that antitussive action of bergenin may be related with central nervous system.Bergenin can increase IGABA but it cannot activate current without GABA. The effect of bergenin on GABAA receptors was achieved by affecting channel open probability, not channel conductance.Bergenin has no effect on IGlu-It is the first report about possible central antitussive mechanism of bergenin.No adverse reactions of bergenin and chlorpheniramine were observed in the study.更多还原