丙型病毒性肝炎联合治疗药物相互作用及肝靶向利巴韦林的实验研究

发布时间：2018-03-05 07:57

本文选题：丙型病毒性肝炎　切入点：利巴韦林　出处：《中国人民解放军医学院》2014年博士论文　论文类型：学位论文

【摘要】：第一部分：丙型病毒性肝炎联合治疗药物相互作用的实验研究 1.目的本研究通过考察抗炎保肝药物水飞蓟素、甘草酸二铵、苦参素或双环醇与利巴韦林（RBV）同时给药，对大鼠口服RBV毒副作用及药代动力学的影响，为临床HCV联合治疗提供依据。 2．方法 2.1用MTT法测定RBV对HepG2的毒性以及不同保肝药物对其细胞毒性的影响。 2.2将四种抗炎保肝药物分别与高剂量（180mg/kg）或低剂量（60mg/kg）的RBV同时给药，连续10天或14天，测定其对大鼠体重增长、血常规及生化学指标的影响。 2.3以同位素标记的RBV作为内标的液相色谱串联质谱(LC-MS/MS)法测定RBV单独给药及其与保肝药物联合给药时大鼠血浆中RBV及其代谢产物的浓度，考察抗炎保肝药物对RBV药代动力学的影响。 3.结果 3.1水飞蓟素在30μM浓度下即显示出对RBV细胞毒性的保护作用，保护率为101.14%，100μM的保护率为215.05%。 3.2高剂量利巴韦林重复给药导致大鼠死亡，体重增长减慢，WBC、RBC、HGB下降和转氨酶升高等血常规及生化指标异常。四种抗炎保肝药物均能显著降低高剂量RBV所致的动物死亡数，拮抗RBV所致的血小板升高;水飞蓟素和双环醇能够显著升高RBV所致的WBC降低，苦参素能够显著升高RBV所致的HGB降低，水飞蓟素能够显著升高RBV所致的HCT降低；在生化指标方面,甘草酸二铵对RBV所致的ALT的升高具有显著的抑制作用,双环醇和苦参素对RBV所致的AST的升高具有显著的抑制作用;四种保肝药物对其它生化指标没有显著影响。低剂量利巴韦林重复给药导致大鼠体重增长减慢，但是对血常规和生化指标均无显著影响。甘草酸二铵和苦参素与低剂量RBV联合用药，能够显著加重RBV对大鼠体重增长的抑制；水飞蓟素和双环醇能够显著拮抗RBV对雌性大鼠体重增长的抑制作用，对雄性大鼠作用不显著。甘草酸二铵与RBV合用,血小板计数和PCT均显著升高;对于雌性大鼠，苦参素与RBV合用，能够显著升高RBV所致的TP降低;水飞蓟素和苦参素与RBV合用，能够显著降低雄性大鼠的肌酐水平。 3.3建立了以同位素标记的RBV作为内标的LC-MS/MS检测大鼠血液中RBV及其代谢产物浓度的方法，有效克服了内源性物质的干扰，灵敏度可达到10ng/ml;药代动力学研究结果表明水飞蓟素和双环醇与RBV合用后，RBV的药-时曲线下面积（AUC）和最大血药浓度（Cmax）均显著低于RBV单独给药。甘草酸二铵和水飞蓟素与RBV合用，大鼠血浆中RBV代谢产物的AUC和Cmax显著低于RBV单独给药。 4.结论水飞蓟素能够拮抗RBV的细胞毒性，四种抗炎保肝药物均能降低高剂量RBV所致的大鼠死亡率，并能部分改善血常规和生化指标；可见抗炎保肝药物能够降低高剂量RBV重复给药所致的毒副作用。低剂量RBV重复给药，对大鼠体重增长也有显著影响，但是与正常组相比，RBV单用或与保肝药物合用，对血常规及生化指标均无显著影响，表明其没有系统毒副作用；推测对体重的影响可能与药物的胃肠道副作用影响了大鼠对食物的摄取、吸收和消化。在药代动力学方面，水飞蓟素和双环醇能够显著降低RBV的AUC和Cmax，可能与抑制RBV的吸收或促进RBV在胃肠道的代谢有关；二者与RBV合用，有可能降低RBV的疗效。因此，该类药物与RBV联合用药的合理性，特别是其对利巴韦林抗HCV疗效的影响需要进一步的研究确认。第二部分：以胆酸为载体的肝靶向利巴韦林的实验研究 1.目的本研究通过考察以胆酸为载体的肝靶向RBV偶合物大鼠灌胃给药的生物利用度，以发现口服生物利用度较高的靶向偶合物。 2.方法 2.1靶向偶合物和RBV经口给药，于给药后不同时间采血，以LC-MS/MS法测定RBV血药浓度，计算主要的药代动力学参数。 2.2靶向偶合物和胆酸同时经口给药，于给药后不同时间采血，以LC-MS/MS法测定RBV血药浓度，，计算主要的药代动力学参数。 3.结果 3.1靶向偶合物RBV-Leu-CA和RBV-Ile-CA口服给药后血浆中RBV的Cmax显著低于RBV，但是Tmax及T1/2显著大于RBV，因此其AUC与RBV相当；靶向偶合物RBV-Leu-CA和RBV-Ile-CA的药-时曲线表现为双峰。 3.2胆酸与RBV-Leu-CA同时给药，导致血浆中RBV的Cmax及AUC均显著降低，分别只有RBV-Leu-CA单独给药的24%和26%。 4.结论靶向偶合物RBV-Leu-CA大鼠经口给药，生物利用度略高于RBV，药-时曲线表现为肝肠循环的特征吸收；胆酸对RBV-Leu-CA的吸收有显著的拮抗作用，说明RBV-Leu-CA是通过胆酸介导的特异性途径吸收的。
[Abstract]:Part 1: Experimental Study on the interaction of combination of hepatitis C and viral hepatitis
1. purposes
The aim of this study is to investigate the effects of silymarin, diammonium glycyrrhizinate, Oxymatrine or bicyclic alcohol on Leigh Bhave Lin's side effects and pharmacokinetics of RBV in rats by investigating the effects of anti-inflammatory and hepatoprotective drugs, silymarin, diammonium glycyrrhizinate, Oxymatrine or bicyclic alcohol on RBV, and provide evidence for clinical HCV combined therapy.
2. method
2.1 MTT was used to determine the toxicity of RBV to HepG2 and the effect of different liver preservation drugs on its cytotoxicity.
2.2, four kinds of anti-inflammatory and hepatoprotective agents were administrated with high dose (180mg/kg) or low dose (60mg/kg) RBV at the same time, and 10 days or 14 days later, the effects of the two kinds of anti-inflammatory and hepatoprotective agents on body weight gain, blood routine and biochemical indexes in rats were measured.
2.3 the content of RBV and its metabolites in plasma of rats was determined by RBV labeled with LC-MS/MS as internal standard by liquid chromatography tandem mass spectrometry (LC-MS/MS). The effects of anti-inflammatory and hepatoprotective agents on RBV pharmacokinetics were investigated.
3. results
3.1 silymarin showed a protective effect on RBV cell toxicity at the concentration of 30 M, the protection rate was 101.14%, and the protection rate of 100 mu M was 215.05%.
3.2 high dose repeated Leigh Bhave Lin administration resulted in the death of rat, slow increase of body weight, WBC, RBC, HGB and decreased transaminases blood routine and biochemical abnormalities. Four kinds of anti-inflammatory hepatoprotective drugs can significantly reduce the high dose of RBV caused by animal deaths, antagonize the RBV induced platelet increased; silymarin and bicyclol can remarkably increase the RBV induced decrease in WBC, matrine can significantly increase the RBV induced decrease in HGB, silymarin can significantly increase the RBV induced decrease in HCT; in biochemical indicators, diammonium glycyrrhizinate on RBV induced the increase of ALT has significant inhibitory effect, significantly inhibited the increase of bicyclol and matrine the RBV induced by AST; four kinds of hepatoprotective drugs had no significant effects on other biochemical indexes. Low dose of Leigh Bhave Lin repeated administration leads to the body weight of rats growth slows down, but the blood and biochemical indexes No significant effect of diammonium glycyrrhizinate and Oxymatrine combined with low dose of RBV, can significantly inhibit the growth of RBV increased the body weight of rats; silymarin and bicyclol can significantly antagonize the inhibitory effect of RBV on the growth of body weight in female rats, no significant effect on male rats. Diammonium glycyrrhizinate combined with RBV. The platelet count and PCT were significantly increased; the female rats of oxymatrine combined with RBV, can significantly increase the RBV induced decrease in TP; silybin and Oxymatrine combined with RBV, can significantly reduce the serum creatinine levels in male rats.
3.3 LC-MS/MS method was established for detection of RBV and its metabolites in blood of rats in the subject to the isotope labeled RBV as, effectively overcomes the interference of endogenous substances, the sensitivity can reach 10ng/ml; pharmacokinetic studies showed that silymarin and bicyclol and together with RBV, the area of RBV concentration time curve under (AUC) and maximum plasma concentration (Cmax) were significantly lower than those of RBV administered alone. Diammonium glycyrrhizinate and silymarin combined with RBV and RBV metabolites in rat plasma AUC and Cmax were significantly lower than those of RBV administered alone.
4. conclusion
Silymarin can inhibit RBV cell toxicity, four kinds of anti-inflammatory drugs can reduce the liver of high dose RBV induced rat mortality, and can improve the blood routine and biochemical index; anti-inflammatory drugs can reduce visible liver of high dose of RBV caused by repeated drug toxicity. Low dose of RBV administered, there significant effect on the growth of body weight in rats, but compared with the normal group, RBV alone or in combination with liver protecting drugs, had no significant effect on blood routine and biochemical indicators show that the system has no toxic side effects; that the impact on body weight may be associated with drug adverse gastrointestinal effects influence of food intake in rats, absorption and digestion.
In pharmacokinetics, silymarin and bicyclol can significantly reduce the RBV of AUC and Cmax, may be associated with inhibition of RBV or RBV in the absorption promoting gastrointestinal metabolism; two combined with RBV, may reduce the effect of RBV. Therefore, reasonable of the drugs combined with RBV, especially is the effect of Leigh Bhave Lin anti HCV efficacy need further research to confirm.
The second part: an experimental study of liver targeting Leigh Bhave Lin with cholic acid as the carrier
1. purposes
In this study, we investigated the bioavailability of bile targeting RBV conjugates in rats by bile acid as a carrier to detect oral bioconjugates with high bioavailability.
2. method
2.1 the drug was given to the conjugates and RBV through the mouth. The blood was collected at different time after the administration. The blood concentration of RBV was measured by LC-MS/MS, and the main pharmacokinetic parameters were calculated.
2.2 target conjugates and cholic acid were administered orally at the same time, and blood was collected at different times after administration. The blood concentration of RBV was determined by LC-MS/MS method, and the main pharmacokinetic parameters were calculated.
3. results
3.1 target conjugates RBV-Leu-CA and RBV-Ile-CA after oral administration, the Cmax of RBV in plasma was significantly lower than that of RBV, but Tmax and T1/2 were significantly greater than RBV, so AUC and RBV were equivalent. The drug time curve of target conjugates RBV-Leu-CA and AUC showed Shuangfeng.
3.2 cholic acid and RBV-Leu-CA were administered simultaneously, resulting in a significant decrease in the Cmax and AUC of RBV in plasma, and only 24% and 26%. of RBV-Leu-CA alone.
4. conclusion
The medicine orally targeting RBV-Leu-CA conjugates in rat, the bioavailability is slightly higher than that of RBV, drug time curve showed the absorption characteristics of enterohepatic circulation; RBV-Leu-CA absorption of cholic acid significant inhibitory effects, indicating that RBV-Leu-CA is absorbed by means of specific bile acid mediated.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R512.63

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