重楼克感胶囊的药代动力学研究

【作者】 尹兴斌；

【导师】 倪健； 孙建宁；

【作者基本信息】 北京中医药大学 ， 中药制药学， 2013， 博士

【摘要】 目的建立生物样品中重楼皂苷Ⅰ、重楼皂苷Ⅱ、重楼皂苷Ⅵ、重楼皂苷Ⅶ、重楼皂苷H、重楼皂苷V及金丝桃苷的含量测定方法；进行重楼、贯叶金丝桃和重楼克感胶囊的药代动力学研究,明确两药同时给药的药代动力学相互作用。方法1采用LC-ESI-MS/MS法测定重楼皂苷Ⅰ、重楼皂苷Ⅱ、重楼皂苷Ⅵ、重楼皂苷Ⅶ及金丝桃苷在beagle大血浆中的含量。2采用LC-ESI-MS/MS法测定重楼皂苷H、重楼皂苷V在beagle犬血浆中的含量。3选用6只健康Beagle犬作为试验动物,给药前12小时禁食不禁水。重楼皂苷Ⅰ重楼皂苷Ⅱ、重楼皂苷Ⅶ、重楼皂苷Ⅶ四种化合物标准品按剂量与25mL0.5%CMC-Na制备为混悬液,灌胃给药。分别于给药前和给药后0.5、1、2、3、4、6、8、10、12、24小时取血,测定各时间点的浓度,Excel软件绘制药时曲线,Kinetica4.4分析软件计算药代动力学参数。4选用6只健康Beagle犬作为试验动物,给药前12小时禁食不禁水。重楼提取物、贯叶金丝桃提取物和两者提取混合物按剂量与25mL0.5%CMC-Na制备为混悬液,灌胃给药。分别于给药前和给药后0.5、1、2、3、4、6、8、10、12、24小时取血,测定各时间点的浓度,Excel软件绘制药时曲线,Kinetica4.4分析软件计算药代动力学参数。5采用SPSS17.0软件进行T检验分析重楼、贯叶金丝桃口服给药后的药代动力学相互作用。6采用HPLC去测定建立重楼皂苷Ⅰ、重楼皂苷Ⅱ、重楼皂苷Ⅵ、重楼皂苷Ⅶ及金丝桃苷在beagle犬血浆中的含量。7选用6只健康Beagle犬作为试验动物,给药前12小时禁食不禁水。重楼克感胶囊按剂量灌胃给药。分别于给药前和给药后0.5、1、2、3、4、5、6、8、12、24小时取血,测定各时间点的浓度,Excel软件绘制药时曲线,Kinetica4.4分析软件计算药代动力学参数。结果1建立了同时测定beagle犬血浆中重楼皂苷Ⅰ、重楼皂苷Ⅱ、重楼皂苷Ⅵ、重楼皂苷Ⅶ及金丝桃苷的LC-ESI-MS/MS方法,五种成分保留时间分别为6.20min、6.82min、2.76min、2.08min、1.32min,检测范围为10-5000ng/mL;低浓度批内及批间精密度均<20%,中、高两个浓度批内及批间精密度均<15%,低、中、高三个浓度的准确度均在80～120%的范围内;五种成分的回收率均大于90%,并且回收率测定的RSD值均小于15%。2建立了同时测定重楼生物样品中重楼皂苷H、重楼皂苷V的LC-ESI-MS/MS方法,两种成分的专属性好,保留时间分别为2.44min、2.93min,检测范围为1-50ng/mL;低浓度批内及批间精密度均<20%,中、高两个浓度批内及批间精密度均<15%,低、中、高三个浓度的准确度均在80-120%的范围内；回收率均大于90%,并且回收率测定的RSD值均小于15%。3重楼皂苷Ⅰ、重楼皂苷Ⅱ、重楼皂苷Ⅵ、重楼皂苷Ⅶ标准品口服给药后Tmax分别为3.17±0.41、3.33±0.52、3.00±0.00、3.17±0.41h；Cmax分别为272.30±27.66、192.40±29.11、148.28±16.91、208.33±37.92ng/mL; AUC0-24分别为2482.95±361.031696.88±50.14、1420.86±230.19、913.06±261.35ng-h/mL; AUC0-∞分别为3025.06±439.58、1955.21±94.40、1689.75±272.60、2243.20±352.15ng·h/mL; MRT分别为13.95±2.15、11.77±1.83、12.98±0.45、12.24±1.54h,T1,2分别为9.41±1.67、7.82±1.78、8.14±0.37、7.47±1.03h。4重楼提取物、贯叶金丝桃提取物口服给药后重楼皂苷Ⅰ、重楼皂苷Ⅱ、重楼皂苷Ⅵ、重楼皂苷Ⅶ、重楼皂苷H、重楼皂苷V及金丝桃苷的Tmax分别为3.83±0.41、3.00±0.00、2.83±0.75、3.67±0.52、3.00±0.00、3.00±0.00、2.17±0.41小时：Cmax分别为276.01±66.28、173.01±37.55、143.90±29.60、184.43±18.38、21.28±2.19、23.06±3.10、4063.87±360.91ng/mL; AUC0-24分别为2049.21±663.23、1417.37±242.56、1324.69±300.89、1652.71±223.28、170.66±41.45、207.63±30.09、27030.28±1064.07ng-h/mL; AUC0-∞分别为2610.08±1023.73、1657.53±312.23、1598.51±381.91、2077.97±463.66、196.44±53.30、239.70±37.06、29215.12±1355.75ng·h/mL; MRT分别为14.36±3.77、12.29±3.37、13.29±3.63、14.68±4.39、10.03±3.98、11.87±1.78、8.80±1.33h,T1/2：分别为9.31±2.74、7.77±2.43、8.36±2.66、9.70±3.22、6.22±3.55、7.20±1.10、5.72±1.11h。重楼、贯叶金丝桃提取混合物口服给药后重楼皂苷Ⅰ、重楼皂苷Ⅱ、重楼皂苷Ⅵ、重楼皂苷Ⅶ、重楼皂苷H、重楼皂苷V及金丝桃苷的Tmax分别为4.00±0.00、3.00±0.00、3.17±0.75、3.67±0.52、3.33±0.52、3.17±0.41、2.00±0.00小时；Cmax分别为250.40±46.29、174.58±30.67、154.29±21.49、181.86±13.34、20.72±2.22、19.94±2.38、3542.58±317.45ng/mL; AUC0-24分别为2152.78±415.34、1330.68±205.22、1288.47±191.00、1604.96±134.07、171.41±53.52,167.99±42.00、25336.45±3142.54ng-h/mL; AUC0-∞分别为2649.38±458.35、1548.87±175.72、1557.49±292.46、1999.47±273.62、194.17±63.73、198.56±60.95、27530.00±3546.90ng·h/mL; MRT分别为14.25±1.50、12.39±2.91、13.31±1.90、14.53±2.87、10.37±2.50、12.34±3.37、9.04±0.65h,T1/2分别为9.05±1.84、8.81±2.93、9.62±3.15、10.18±2.36、7.18±1.80、8.15±3.06h、6.03±1.10h。5通过对六种药代动力学参数进行T检验比较,发现混合给药时七种成分的药代动力学参数只有Cmax差异有统计学意义(P=0.013),其余参数均无显著性差异。6建立了同时测定重楼生物样品中重楼皂苷Ⅰ、重楼皂苷Ⅱ、重楼皂苷Ⅵ、重楼皂苷Ⅶ的HPLC方法,四种成分的专属性好,检测范围为0.5-100μg/mL;低浓度批内及批间精密度均<20%,中、高两个浓度批内及批间精密度均<15%,低、中、高三个浓度的准确度均在80～120%的范围内;回收率均大于90%,并且回收率测定的RSD值均小于15%。7重楼克感胶囊口服给药后重楼皂苷Ⅰ、重楼皂苷Ⅱ、重楼皂苷Ⅵ、重楼皂苷Ⅶ、金丝桃苷的Tmax分别为4.00±0.00.3.83±0.41、3.83±0.41、3.67±1.21、2.17±0.41h, Cmax分别为7.58±0.83、7.42±1.57、5.70±1.49、6.80±1.37、11.42±1.15ng/mL,AUC0-24分别为65.75±4.68、54.66±5.62、53.26±13.88、61.97±13.53、51.61±12.74ng·h/mL, AUC0-∞分别为85.63±7.17、82.03±15.41、70.31±12.01、76.79±14.32、68.89±16.30ng·h/mL, MRT分别为15.77±1.95、13.72±4.71、15.53±2.54、13.80±3.29、6.85±1.43h,T1/2分别为10.12±2.31、8.71±4.01、9.45±1.73、8.65±2.77、5.37±1.32h。结论1建立的重楼皂苷Ⅰ、重楼皂苷Ⅱ、重楼皂苷Ⅵ、重楼皂苷Ⅶ、重楼皂苷H、重楼皂苷V及金丝桃苷的含量测定方法中样品回收率高,能够满足给药后各个时间点血浆药物浓度的测定。在本测定条件下的稳定性考察中,七种成分含药基质的低浓度偏差均在±20%以内,中、高浓度点的偏差均在±15%以内,能够保证检测结果的准确性和重现性。2对beagle犬口服重楼皂苷Ⅰ、重楼皂苷Ⅱ、重楼皂苷Ⅵ、重楼皂苷Ⅶ四种标准品后的药代动力学进行了研究,通过药时曲线可以看出,四种重楼皂苷的吸收、消除趋势相似,在0.5h均可在血液中发现,4小时内迅速吸收达到最大血药浓度,4小时后开始逐渐消除。分析药时曲线相似的原因为四种化合物同为皂苷类成分,母核类似,分子量接近,口服给药后在被胃肠道吸收的趋势类似。对beagle犬口服重楼提取物后重楼皂苷H、重楼皂苷V的药代动力学进行了研究,两种成分的吸收、消除趋势与前四种重楼皂苷类似。对beagle犬口服贯叶金丝桃提取物后金丝桃苷的药代动力学进行了研究,与四种重楼皂苷的吸收消除趋势相比,金丝桃苷在动物体内的吸收、消除更为迅速。2小时内迅速吸收达到最大血药浓度,2小时后开始逐渐消除。分析其原因为金丝桃苷较重楼皂苷极性小,脂溶性更强,易于透过胃肠道上皮细胞,更容易被胃肠道吸收。对beagle犬口服重楼提取物、贯叶金丝桃提取物和两者提取混合物后的药代动力学进行了研究,并对两药的相互作用进行了统计分析,结果表明重楼皂苷单体化合物给药与重楼提取物给药比较结果表明四种重楼皂苷对彼此的吸收消除没有影响、重楼药材中的其他成分对四种重楼皂苷的吸收消除没有影响。重楼提取物、贯叶金丝桃提取物及两药合用比较结果表明重楼克感胶囊的处方配伍对重楼、贯叶金丝桃的吸收消除影响较小,两药合用时能够发挥相加作用,按照各自的吸收特性被机体吸收,两药的作用能够形成互补,提高单独给药的治疗作用。3建立的重楼皂苷Ⅰ、重楼皂苷Ⅱ、重楼皂苷Ⅵ、重楼皂苷Ⅶ的HPLC方法中样品回收率高,能够满足给药后各个时间点血浆药物浓度的测定。在本测定条件下的稳定性考察中,四种成分的低浓度偏差均在±20%以内,中、高浓度点的偏差均在±15%以内,能够保证检测结果的准确性和重现性。4对beagle犬口服重楼克感胶囊后的药代动力学进行了研究,分析了beagle犬口服重楼克感胶囊后重楼皂苷和金丝桃苷在体内的吸收情况。结果表明重楼克感胶囊和提取混合物口服给药后五种成分的MRT、T1/2无显著性差异,但重楼克感胶囊给药后重楼皂苷Ⅱ、重楼皂苷Ⅵ的Tmax明显增大,表明胶囊给药能延迟重楼皂苷Ⅱ、重楼皂苷Ⅵ的达峰,减缓吸收速率,有一定的缓释效果,但对重楼皂苷Ⅱ、重楼皂苷Ⅵ的消除无影响。Tmax分别增加10、25min,推测胶囊壳的破裂过程延缓了两种化合物的分散吸收,影响了其药代动力学性质。通过对T1/2实验结果分析,为保证重楼克感胶囊的药效,应考虑将给药间隔改为每日三次,即8小时给药一次,有利于保证药物疗效,降低血药浓度波动。更多还原

【Abstract】 1Simultaneous determination and pharmacokinetic study of polyphyllin I, polyphyllin Ⅱ, polyphyllin Ⅵ and polyphyllin Ⅶ in beagle dog plasma after oral administration of four compounds and Rhizoma Paridis Extracts by LC-MS-MSFor the first time, a rapid and specific LC-MS-MS method was developed for the analysis of polyphyllin Ⅰ, polyphyllin Ⅱ, polyphyllin VI and polyphyllin Ⅶ in beagle dog plasma. The analysis was carried out on an Agilent Zorbax XDB-C18reversed-phase column (100×2.1mm,1.8μm) by isocratic elution with acetonitrile and water (50:50, v/v). The flow rate was0.25mL/min. All analytes including internal standards were monitored by selected reaction monitoring with an electrospray ionization source. Linear responses were obtained for polyphyllin Ⅰ, polyphyllin Ⅱ, polyphyllin VI and polyphyllin Ⅶ ranging from10to5000ng/mL. The intra-and inter-day precisions (RSDs) were less than6.66and9.15%. The extraction recovery ranged from95.53to104.21%with RSD less than8.69%. Stability studies showed that polyphyllin Ⅰ, polyphyllin Ⅲ, polyphyllin Ⅵ and polyphyllin Ⅶ were stable in preparation and analytical process. The results indicated that the validated method was successfully used to determine the concentration-time profiles of polyphyllin I, polyphyllin Ⅱ, polyphyllin VI and polyphyllin Ⅶ.The method was applied to study the pharmacokinetics of four compounds. Six male beagle dogs were fast for12h, and then four compounds were given orally as solutions respectively. Blood samples (0.5mL) were collected from the femoral vein of dogs in their front legs before administration and after that at0.5,1,2,3,4,6,8,10,12and24h.The blood samples were immediately centrifuged at12,000rpm for10min to separate out plasma and stored at-20℃until analysis. All the values were expressed as means standard deviation (SD). The pharmacokinetic parameters were analyzed using Kinetica4.4software (Thermo Scientific, USA).Polyphyllin Ⅰ plasma concentration reached a maximum at3.17±0.41h after administration with an average Cmax of272.30±27.66ng/mL. The area under the curve (AUC0-∞) was3025.06±439.58h ng/mL and MRT was13.95±2.15h.Polyphyllin Ⅱ plasma concentration reached a maximum at3.33±0.52h after administration with an average Cmax of192.40±29.11ng/mL. The area under the curve (AUC0-∞) was1955.21±94.40h ng/mL and MRT was11.77±1.83h.Polyphyllin Ⅵ plasma concentration reached a maximum at3.00±0.00h after administration with an average Cmax of148.28±16.91ng/mL. The area under the curve (AUC0.∞,) was1689.75±272.60(h ng/mL) and MRT was12.98±0.45h.Polyphyllin Ⅷ plasma concentration reached a maximum at3.17±0.41h after administration with an average Cmax of208.33±37.92ng/mL. The area under the curve (AUC0-∞) was2243.20±352.15(h ng/mL) and MRT was12.24±1.54h.The method was applied to study the pharmacokinetics of f Rhizoma Paridis Extracts. Polyphyllin I plasma concentration reached a maximum at3.83±0.41h after administration with an average Cmax of276.01±66.28ng/mL. The area under the curve (AUC0-∞) was2610.08±1023.73(h ng/mL) and MRT was14.36±3.77h.Polyphyllin Ⅱ plasma concentration reached a maximum at3.00±0.00h after administration with an average Cmax of173.01±37.55ng/mL. The area under the curve (AUC0-∞was1657.53±312.23(h ng/mL) and MRT was12.29±3.37h.Polyphyllin Ⅵ plasma concentration reached a maximum at2.83±0.75h after administration with an average Cmax of143.90±29.60ng/mL. The area under the curve (AU0-∞) was1598.51±381.91(h ng/mL) and MRT was13.29±3.63h.Polyphyllin Ⅶ plasma concentration reached a maximum at3.67±0.52h after administration with an average Cmax of184.43±18.38ng/mL. The area under the curve (AUC0-∞) was2077.97±463.66(h ng/mL) and MRT was14.68±4.39h.2Simultaneous determination and pharmacokinetic study of polyphyllin H, polyphyllin Vin beagle dog plasma after oral administration of Rhizoma Paridis Extracts by LC-MS-MSFor the first time, a rapid and specific LC-MS-MS method was developed for the analysis of polyphyllin H, polyphyllin V in beagle dog plasma. The analysis was carried out on an Agilent Zorbax XDB-C18reversed-phase column (100×2.1mm,1.8μm) by isocratic elution with acetonitrile and water (50:50, v/v). The flow rate was0.25mL/min. All analytes including internal standards were monitored by selected reaction monitoring with an electrospray ionization source. Linear responses were obtained for polyphyllin H, polyphyllin V ranging from1to50ng/mL. The intra-and inter-day precisions (RSDs) were less than6.66and9.15%. The extraction recovery ranged from95.53to104.21%with RSD less than8.69%. Stability studies showed that polyphyllin H, polyphyllin V were stable in preparation and analytical process. The results indicated that the validated method was successfully used to determine the concentration-time profiles of polyphyllin H, polyphyllin V.The method was applied to study the pharmacokinetics of f Rhizoma Paridis Extracts. Polyphyllin H plasma concentration reached a maximum at3.00±0.00h after administration with an average Cmax of21.28±2.19ng/mL. The area under the curve (AUC0-∞) was196.44±53.30(h ng/mL) and MRT was10.03±3.98h.Polyphyllin V plasma concentration reached a maximum at3.00±0.00h after administration with an average Cmax of23.06±3.10ng/mL. The area under the curve (AU0-∞) was239.70±37.06(h ng/mL) and MRT was11.87±1.78h.3Development and validation of a highly sensitive LC-ESI-MS/MS method for the determination of hyperoside in beagle dog plasma:application to a pharmacokinetic study.A highly sensitive, rapid assay method has been developed and validated for the analysis of hyperoside in beagle dog plasma with liquid chromatography coupled to tandemmass spectrometry with electrospray ionization in the positive-ion mode. The assay procedure involves extraction of hyperoside and ginsenoside Re (IS) from beagle dog plasma. Chromatographic separation was carried out on an Agilent Zorbax XDB-C18(100×2.1mm,18μm) column by isocratic elution with acetonitrile and water (50:50, v/v) at a flow rate of0.25mL/min with a total run time of2.0min. The MS/MS ion transitions monitored were464.4±463.4for hyperoside and947.12→-969.60for IS. Linear responses were obtained for hyperoside ranging from10to5000/7g/mL. The intra-and inter-day precisions (RSDs) were less than5.38and3.39%and the extraction recovery ranged from94.39to100.78%with RSD less than3.82%. Stability studies showed that hyperoside was stable in preparation and analytical process.The sensitivity and specificity of the assay were found to be sufficient for accurately characterizing the plasma pharmacokinetics of hyperoside in dogs following oral administration. Hyperoside plasma concentration reached a maximum at2.17±0.41h after administration with an average maximum concentration (Cmax)of4063.87±360.91ng/mL. The area under the plasma concentration-time curve from time zero to infinity (AUC0-∞) was29215.12±1355.75(h ng/mL) and MRT was8.8±1.3h.4Simultaneous determination of polyphyllin Ⅰ, polyphyllin Ⅱ, polyphyllin Ⅳ and polyphyllin Ⅶ from Rhyzoma Paridis dog plasma by a rapid HPLC and its application to pharmacokinetic study after oral administrationFor the first time, a rapid and specific RP-HPLC-UV method was developed for the analysis of polyphyllin Ⅰ, polyphyllin Ⅱ, polyphyllin Ⅳ and polyphyllin Ⅶ in beagle dog plasma. The analysis was carried out on a Diamonsil C18(2) reversed-phase column (250×4.60mm,5μm) by isocratic elution with acetonitrile and0.1%phosphoric acid (50:50, v/v). The flow rate was1.00mL/min and the detection wavelength was set at203nm. Linear responses were obtained for hyperoside ranging from0.5to50μg/mL. The intra-and inter-day precisions (RSDs) were less than9.23and9.24%with the accuracy (%) ranging from87.65to107.25%. The extraction recovery ranged from85.07to91.82%with RSD less than5.55%. Stability studies showed that polyphyllin Ⅰ, polyphyllin Ⅱ, polyphyllin Ⅵ and polyphyllin Ⅶ were stable in preparation and analytical process. The results indicated that the validated method was successfully used to determine the concentration-time profiles of polyphyllin Ⅰ, polyphyllin Ⅱ, polyphyllin Ⅵ and polyphyllin Ⅶ.A rapid and specific RP-HPLC-UV method was developed for the analysis of hyperoside in beagle dog plasma. The analysis was carried out on a Diamonsil C18(2) reversed-phase column (250×4.6mm,5mm) by isocratic elution with acetonitrile and0.1%phosphoric acid (42:58, v/v). The flow rate was1.0mL/min and the detection wavelength was set at203nm. Linear response was obtained for hyperoside ranging from2to60μg/mL. The intra-and inter-day precisions (RSDs) were less than4.80and2.61%with the accuracy (%) ranging from94.81to98.49%. The extraction recovery ranged from89.85to91.32%with RSD less than7.381%. Stability studies showed that hyperoside was stable in preparation and analytical process. The results indicated that the validated method was successfully used to determine the concentration-time profiles of hyperoside.The method was applied to study the pharmacokinetics of Chonglou Kegan Capsule containing olyphyllin Ⅰ, polyphyllin Ⅱ, polyphyllin Ⅵ, polyphyllin Ⅶ and hyperoside. Polyphyllin Ⅰ plasma concentration reached a maximum at2.83±0.41h after administration with an average Cmax of49.94±4.82ng/mL The area under the curve (AUC0-∞) was545.68±14.51(h ng/mL). The MRT was15.41±2.25h.Polyphyllin Ⅱ plasma concentration reached a maximum at3.67±0.52h after administration with an average Cmax of19.47±2.65ng/mL. The area under the curve (AUC0-∞) was151.38±46.57(h ng/mL).The MRT was8.10±3.47h. Polyphyllin VI plasma concentration reached a maximum at3.83v0.41h after administration with an average Cmax of5.70±1.49ng/mL. The T1/2was calculated to be10.45±2.07h. The area under the curve (AUC0-∞) was70.31±12.01(h ng/mL). The MRT was15.53±2.54h.Polyphyllin VII plasma concentration reached a maximum at3.67±1.21h after administration with an average Cmax of6.80±1.37ng/mL. The area under the curve (AU0-∞) was76.79±14.32(h ng/mL).The MRT was13.80±3.29h. Hyperoside plasma concentration reached a maximum at1.67±1.67h after administration with an average Cmax of14.06±1.53ng/mL. The T1/2was calculated to be4.23±1.79h. The area under the curve (AUC0.∞was81.67±16.38(h ng/mL). The mean values of Ke was0.19±0.06h-1and MRT was6.45±1.83更多还原