双环醇对利福平、异烟肼在大鼠体内药代动力学的影响
摘要
目的:分别建立鼠血浆中利福平和异烟肼浓度的反相高效液相色谱检测方法。研究双环醇与利福平和异烟肼联合应用后,对利福平和异烟肼药代动力学特征的影响。
方法:1.利福平血药浓度的测定:血浆样品经甲醇沉淀蛋白,0.22μm滤头过滤后,直接进样分析;分析柱:Shim-pack VP-ODS(5μm,250mm×4.6mm):流动相:甲醇:水=70:30(pH=6):流速:1mL.min~(-1):检测波长:336nm。异烟肼血药浓度的测定:血浆样品经10%三氯醋酸沉淀蛋白后,上清液加桂皮醛衍生化后直接进样;分析柱:Shim-pack VP-ODS(5μm,250mm×4.6mm);流动相:0.02mol.ml~(-1)磷酸二氢钾:乙腈=48:52;流速:1.0mL.min~(-1);检测波长:340nm。2.36只Wistar大鼠随机分为6组:即利福平组(Ⅰ)、异烟肼组(Ⅱ)、双环醇+利福平组(Ⅲ)、双环醇+异烟肼组(Ⅳ)、利福平+异烟肼组(Ⅴ)、双环醇+利福平+异烟肼组(Ⅵ),药物剂量:双环醇为15.85mg.kg~(-1)、利福平为63.4 mg.kg~(-1)、异烟肼为31.7 mg.kg~(-1),每天一次,连续给药4天。第4天在给药前和给药后0.25、0.5、1、1.5、2、4、6、8、12、24h取血,进行血药浓度的测定。用DAS2.0药动学程序计算药动学参数,SPSS(11.5)软件对数据进行统计学处理。
结果:利福平在1.25-80.0μg.ml~(-1)范围内具有良好的线性关系(R~2=0.9995),平均回收率为101.54%,日内和日间差均＜5%。异烟肼在0.25-32.0μg.ml~(-1)范围内具有良好的线性关系(R~2=0.9996),回收率为98.64%,日内和日间差均＜5%。双环醇与利福平合用,双环醇与异烟肼合用,双环醇与利福平和异烟肼合用,各时间点中除了三药合用组6小时时间点的血药浓度与对照组有显著性差异(P＜0.05)之外,其余时间点的血药浓度值在各组间均无显著性差异(P＞0.05);药动学参数AUC_(0-t)、AUC_(0-∞)、MRT_(0-t)、MRT_(0-∞)、t_(1/2z)、T_(max)、CL_(z/F)、V_(z/F)、C_(max)各组间均无显著性差异(P＞0.05)。
结论:1.本研究建立了良好的异烟肼和利福平药物浓度测定方法,两种方法均简便准确快速,重现性好,适合于生物样品的测定。2.双环醇与利福平合用,双环醇与异烟肼合用,以及双环醇与利福平和异烟肼两药合用,均未见对利福平和异烟肼药动参数有明显影响,提示临床上三药可以联合使用,不需要对利福平或异烟肼的给药剂量或给药方案进行调整。
Objectives:To establish reverse phase high-performance liquid chromatography methods to determine the plasma concentration of Rifampin and Isoniazid in rat respectively,and observe the effects of Bicyclol on the pharmacokinetics parameters of Rifampin and Isoniazid in rats.
Methods:1.The concentration of Rifampin in plasma was detected by reverse phase high performance liquid chromatography.After Serum protein was precipitated with methanol and filtrated with 0.22μm membrane,the sample were injected and analysized.Chromatographic column:Shim-pack VP-ODS(5μm,250mm×4.6mm); Mobile phase:methanol and water(70:30,Ⅴ:Ⅴ);Flow rate:1.0ml.min~(-1);Ultraviolet wave lengh:336nm.The concentration of isoniazid in plasma was detected by reverse phase high performance liquid chromatography.The plasma sample was injected directly for determination after being deproteinized with 10%trichloroacetic acid and reacted with cinnamaldehyde.Chromatographic column:Shim-packVP-ODS (5μm,250mm×4.6mm);Mobile phase:KH_2PO_4 and acetonitrile(48:52,Ⅴ:Ⅴ);Flow rate:1.0ml.min-1;Ultraviolet wave lengh:340nm.2.Thirty-six rats were divided randomly into six groups:rifampin group(Ⅰ)、isoniazid group(Ⅱ)、bicyclol+ rifampin group(Ⅲ)、bicyclol+isoniazid group(Ⅳ)、rifampin + isoniazid group(Ⅴ)、bicyclol +rifampin + isoniazid group(Ⅵ).Drug dosage:bicyclol was 15.85 mg.kg~(-1)、rifampin was 63.4 mg.kg~(-1)、isoniazid was 31.7 mg.kg~(-1),once every day for 4 days. Plasma samples were obtained on day 4 at 0、0.25、0.5、1、1.5、2、4、6、8、12、24h after administration.Concentrations of rifampin and isoniazid were determined by reverse phase high-performance liquid chromatography.Pharmacokinefics parameters were evaluated by DAS2.0 pharmacokinetics pragramme.Parameters were analyzed by SPSS(11.5)software.
Results:The linear relation of rifampin was excellent within the range of 1.25-80.0 ug.ml~(-1)(R~2=0.9995),and the mean recovery was 101.54%Daily and daytime RSD were less than 5%.The linear relation of isoniazid was excellent within the range of 0.25-32.0 ug.ml~(-1)(R~2=0.9996),and the mean recovery was 98.64%.Daily and daytime RSD were less than 5%.There groups that respectively used bicyclol in combination with rifampin,or with isoniazid,or with both rifampin and isoniazid, were considered.In each time points,there was no significant difference(P＞0.05) among the drug concentrations in blood of the other groups.Only in time point of six hours,the drug concentration in blood of the three drug combination group differed from the control group(P＜0.05).Pharmacokinetic parameters,included AUC_((0-t))、AUC_((0-∞))、MRT_((0-t))、MRT_((0-∞))、t_(1/2z)、T_(max)、CL_(z/F)、V_(z/F)and C_(max),were found no significant difference(P＞0.05)among the groups.
Conclusion:1.The methods of determination of isoniazid and rifampicin concentration are established,and they are simple,rapid,accurate,reproducible and suitable for the determination of biological samples.2.Bicyclol has no effects on the pharmacokinetic parameters of rifampin or/and isoniazid,when it combinates with two major anti-tuberculosis drugs.That means bicyclol may combine with rifampin and isoniazid in clinical therapy.It is no need to adjust dose and regimen of rifampin or isoniazid.
方法:1.利福平血药浓度的测定:血浆样品经甲醇沉淀蛋白,0.22μm滤头过滤后,直接进样分析;分析柱:Shim-pack VP-ODS(5μm,250mm×4.6mm):流动相:甲醇:水=70:30(pH=6):流速:1mL.min~(-1):检测波长:336nm。异烟肼血药浓度的测定:血浆样品经10%三氯醋酸沉淀蛋白后,上清液加桂皮醛衍生化后直接进样;分析柱:Shim-pack VP-ODS(5μm,250mm×4.6mm);流动相:0.02mol.ml~(-1)磷酸二氢钾:乙腈=48:52;流速:1.0mL.min~(-1);检测波长:340nm。2.36只Wistar大鼠随机分为6组:即利福平组(Ⅰ)、异烟肼组(Ⅱ)、双环醇+利福平组(Ⅲ)、双环醇+异烟肼组(Ⅳ)、利福平+异烟肼组(Ⅴ)、双环醇+利福平+异烟肼组(Ⅵ),药物剂量:双环醇为15.85mg.kg~(-1)、利福平为63.4 mg.kg~(-1)、异烟肼为31.7 mg.kg~(-1),每天一次,连续给药4天。第4天在给药前和给药后0.25、0.5、1、1.5、2、4、6、8、12、24h取血,进行血药浓度的测定。用DAS2.0药动学程序计算药动学参数,SPSS(11.5)软件对数据进行统计学处理。
结果:利福平在1.25-80.0μg.ml~(-1)范围内具有良好的线性关系(R~2=0.9995),平均回收率为101.54%,日内和日间差均＜5%。异烟肼在0.25-32.0μg.ml~(-1)范围内具有良好的线性关系(R~2=0.9996),回收率为98.64%,日内和日间差均＜5%。双环醇与利福平合用,双环醇与异烟肼合用,双环醇与利福平和异烟肼合用,各时间点中除了三药合用组6小时时间点的血药浓度与对照组有显著性差异(P＜0.05)之外,其余时间点的血药浓度值在各组间均无显著性差异(P＞0.05);药动学参数AUC_(0-t)、AUC_(0-∞)、MRT_(0-t)、MRT_(0-∞)、t_(1/2z)、T_(max)、CL_(z/F)、V_(z/F)、C_(max)各组间均无显著性差异(P＞0.05)。
结论:1.本研究建立了良好的异烟肼和利福平药物浓度测定方法,两种方法均简便准确快速,重现性好,适合于生物样品的测定。2.双环醇与利福平合用,双环醇与异烟肼合用,以及双环醇与利福平和异烟肼两药合用,均未见对利福平和异烟肼药动参数有明显影响,提示临床上三药可以联合使用,不需要对利福平或异烟肼的给药剂量或给药方案进行调整。
Objectives:To establish reverse phase high-performance liquid chromatography methods to determine the plasma concentration of Rifampin and Isoniazid in rat respectively,and observe the effects of Bicyclol on the pharmacokinetics parameters of Rifampin and Isoniazid in rats.
Methods:1.The concentration of Rifampin in plasma was detected by reverse phase high performance liquid chromatography.After Serum protein was precipitated with methanol and filtrated with 0.22μm membrane,the sample were injected and analysized.Chromatographic column:Shim-pack VP-ODS(5μm,250mm×4.6mm); Mobile phase:methanol and water(70:30,Ⅴ:Ⅴ);Flow rate:1.0ml.min~(-1);Ultraviolet wave lengh:336nm.The concentration of isoniazid in plasma was detected by reverse phase high performance liquid chromatography.The plasma sample was injected directly for determination after being deproteinized with 10%trichloroacetic acid and reacted with cinnamaldehyde.Chromatographic column:Shim-packVP-ODS (5μm,250mm×4.6mm);Mobile phase:KH_2PO_4 and acetonitrile(48:52,Ⅴ:Ⅴ);Flow rate:1.0ml.min-1;Ultraviolet wave lengh:340nm.2.Thirty-six rats were divided randomly into six groups:rifampin group(Ⅰ)、isoniazid group(Ⅱ)、bicyclol+ rifampin group(Ⅲ)、bicyclol+isoniazid group(Ⅳ)、rifampin + isoniazid group(Ⅴ)、bicyclol +rifampin + isoniazid group(Ⅵ).Drug dosage:bicyclol was 15.85 mg.kg~(-1)、rifampin was 63.4 mg.kg~(-1)、isoniazid was 31.7 mg.kg~(-1),once every day for 4 days. Plasma samples were obtained on day 4 at 0、0.25、0.5、1、1.5、2、4、6、8、12、24h after administration.Concentrations of rifampin and isoniazid were determined by reverse phase high-performance liquid chromatography.Pharmacokinefics parameters were evaluated by DAS2.0 pharmacokinetics pragramme.Parameters were analyzed by SPSS(11.5)software.
Results:The linear relation of rifampin was excellent within the range of 1.25-80.0 ug.ml~(-1)(R~2=0.9995),and the mean recovery was 101.54%Daily and daytime RSD were less than 5%.The linear relation of isoniazid was excellent within the range of 0.25-32.0 ug.ml~(-1)(R~2=0.9996),and the mean recovery was 98.64%.Daily and daytime RSD were less than 5%.There groups that respectively used bicyclol in combination with rifampin,or with isoniazid,or with both rifampin and isoniazid, were considered.In each time points,there was no significant difference(P＞0.05) among the drug concentrations in blood of the other groups.Only in time point of six hours,the drug concentration in blood of the three drug combination group differed from the control group(P＜0.05).Pharmacokinetic parameters,included AUC_((0-t))、AUC_((0-∞))、MRT_((0-t))、MRT_((0-∞))、t_(1/2z)、T_(max)、CL_(z/F)、V_(z/F)and C_(max),were found no significant difference(P＞0.05)among the groups.
Conclusion:1.The methods of determination of isoniazid and rifampicin concentration are established,and they are simple,rapid,accurate,reproducible and suitable for the determination of biological samples.2.Bicyclol has no effects on the pharmacokinetic parameters of rifampin or/and isoniazid,when it combinates with two major anti-tuberculosis drugs.That means bicyclol may combine with rifampin and isoniazid in clinical therapy.It is no need to adjust dose and regimen of rifampin or isoniazid.
引文
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[1]Toit L C,Pillay V,Danckwerts M P.Tuberculosis chemotherapy:current drug delivery approaches[J].Respiratory Research 2006,7:118
[2]Kaplwitz N.Mechanisms of liver injury.[J].Hepato,2000;32:39-47
[3]王新华.抗结核病药物对肝功能的损害及其防治[J].中国全科医学 2004,7(10):736.
[4]Zhang JY.New drugs derived from medicinal plants[J].Therapie,2002,57(2):137-150
[5]陈燕琴,高同军,迟俭,等.双环醇片治疗抗结核药物性肝损害临床疗效和安全性分析[J].传染病信息,2005,18(4):188-189
[6]刘耕陶.双环醇的抗病毒与肝细胞保护作用及其作用机制[J].中国新药杂志,2001,10(5):325-327.
[7]赵冬梅,刘耕陶.双环醇对刀豆蛋白所致小鼠肝细胞核DNA损伤的保护作用[J].中华医学杂志,2001,81:844-848.
[8]Wang H.Li Y.Protective effect of bicyclol on acute hepatic failure induced by lipopolysaccharide and D—galactosamine in mice[J].Eur J Pharmacol,2006,534(3):194-201
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[9]王建军,成军,刘妍,等.基因表达谱芯片筛选双环醇作用HepG2细胞后的差异表达基因[J].世界华人消化杂志,2004,12(7):1711-1714
[10]周建凤,陈书长,白春梅,等.双环醇片防治化疗药物性肝损害的研究[J].肝脏,2007,12(4):286-287
[11]孙建民,朱增红,胡庆军.双环醇联合利巴韦林治疗丙型肝炎肝硬化的疗效观察[J].透析与人工器官,2007,18(3):16-17
[12]姚光弼,徐道振.兰培,等.双环醇治疗2200例病毒性肝炎的安全性和疗效分析[J].中国新药与临床杂志,2005,24(6):421.426.
[13]计焱焱,程能能,姚光弼.30例健康志愿者口服双环醇片剂的药代动力学研究[J].中国临床药理学与治疗学,2001,6(3):218-221
[14]鞠美华,李燕.双环醇在大鼠和人肝微粒体的代谢[J].药学学报,2005,40(2):111-116
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