發(fā)布時(shí)間：2018-03-05 07:57

  本文選題：丙型病毒性肝炎　切入點(diǎn)：利巴韋林　出處：《中國(guó)人民解放軍醫(yī)學(xué)院》2014年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：第一部分：丙型病毒性肝炎聯(lián)合治療藥物相互作用的實(shí)驗(yàn)研究 1.目的 本研究通過(guò)考察抗炎保肝藥物水飛薊素、甘草酸二銨、苦參素或雙環(huán)醇與利巴韋林（RBV）同時(shí)給藥，對(duì)大鼠口服RBV毒副作用及藥代動(dòng)力學(xué)的影響，為臨床HCV聯(lián)合治療提供依據(jù)。 2．方法 2.1用MTT法測(cè)定RBV對(duì)HepG2的毒性以及不同保肝藥物對(duì)其細(xì)胞毒性的影響。 2.2將四種抗炎保肝藥物分別與高劑量（180mg/kg）或低劑量（60mg/kg）的RBV同時(shí)給藥，連續(xù)10天或14天，測(cè)定其對(duì)大鼠體重增長(zhǎng)、血常規(guī)及生化學(xué)指標(biāo)的影響。 2.3以同位素標(biāo)記的RBV作為內(nèi)標(biāo)的液相色譜串聯(lián)質(zhì)譜(LC-MS/MS)法測(cè)定RBV單獨(dú)給藥及其與保肝藥物聯(lián)合給藥時(shí)大鼠血漿中RBV及其代謝產(chǎn)物的濃度，考察抗炎保肝藥物對(duì)RBV藥代動(dòng)力學(xué)的影響。 3.結(jié)果 3.1水飛薊素在30μM濃度下即顯示出對(duì)RBV細(xì)胞毒性的保護(hù)作用，保護(hù)率為101.14%，100μM的保護(hù)率為215.05%。 3.2高劑量利巴韋林重復(fù)給藥導(dǎo)致大鼠死亡，體重增長(zhǎng)減慢，WBC、RBC、HGB下降和轉(zhuǎn)氨酶升高等血常規(guī)及生化指標(biāo)異常。四種抗炎保肝藥物均能顯著降低高劑量RBV所致的動(dòng)物死亡數(shù)，拮抗RBV所致的血小板升高;水飛薊素和雙環(huán)醇能夠顯著升高RBV所致的WBC降低，苦參素能夠顯著升高RBV所致的HGB降低，水飛薊素能夠顯著升高RBV所致的HCT降低；在生化指標(biāo)方面,甘草酸二銨對(duì)RBV所致的ALT的升高具有顯著的抑制作用,雙環(huán)醇和苦參素對(duì)RBV所致的AST的升高具有顯著的抑制作用;四種保肝藥物對(duì)其它生化指標(biāo)沒(méi)有顯著影響。低劑量利巴韋林重復(fù)給藥導(dǎo)致大鼠體重增長(zhǎng)減慢，但是對(duì)血常規(guī)和生化指標(biāo)均無(wú)顯著影響。甘草酸二銨和苦參素與低劑量RBV聯(lián)合用藥，能夠顯著加重RBV對(duì)大鼠體重增長(zhǎng)的抑制；水飛薊素和雙環(huán)醇能夠顯著拮抗RBV對(duì)雌性大鼠體重增長(zhǎng)的抑制作用，對(duì)雄性大鼠作用不顯著。甘草酸二銨與RBV合用,血小板計(jì)數(shù)和PCT均顯著升高;對(duì)于雌性大鼠，苦參素與RBV合用，能夠顯著升高RBV所致的TP降低;水飛薊素和苦參素與RBV合用，能夠顯著降低雄性大鼠的肌酐水平。 3.3建立了以同位素標(biāo)記的RBV作為內(nèi)標(biāo)的LC-MS/MS檢測(cè)大鼠血液中RBV及其代謝產(chǎn)物濃度的方法，有效克服了內(nèi)源性物質(zhì)的干擾，靈敏度可達(dá)到10ng/ml;藥代動(dòng)力學(xué)研究結(jié)果表明水飛薊素和雙環(huán)醇與RBV合用后，RBV的藥-時(shí)曲線(xiàn)下面積（AUC）和最大血藥濃度（Cmax）均顯著低于RBV單獨(dú)給藥。甘草酸二銨和水飛薊素與RBV合用，大鼠血漿中RBV代謝產(chǎn)物的AUC和Cmax顯著低于RBV單獨(dú)給藥。 4.結(jié)論 水飛薊素能夠拮抗RBV的細(xì)胞毒性，四種抗炎保肝藥物均能降低高劑量RBV所致的大鼠死亡率，并能部分改善血常規(guī)和生化指標(biāo)；可見(jiàn)抗炎保肝藥物能夠降低高劑量RBV重復(fù)給藥所致的毒副作用。低劑量RBV重復(fù)給藥，對(duì)大鼠體重增長(zhǎng)也有顯著影響，但是與正常組相比，RBV單用或與保肝藥物合用，對(duì)血常規(guī)及生化指標(biāo)均無(wú)顯著影響，表明其沒(méi)有系統(tǒng)毒副作用；推測(cè)對(duì)體重的影響可能與藥物的胃腸道副作用影響了大鼠對(duì)食物的攝取、吸收和消化。 在藥代動(dòng)力學(xué)方面，水飛薊素和雙環(huán)醇能夠顯著降低RBV的AUC和Cmax，可能與抑制RBV的吸收或促進(jìn)RBV在胃腸道的代謝有關(guān)；二者與RBV合用，有可能降低RBV的療效。因此，該類(lèi)藥物與RBV聯(lián)合用藥的合理性，特別是其對(duì)利巴韋林抗HCV療效的影響需要進(jìn)一步的研究確認(rèn)。 第二部分：以膽酸為載體的肝靶向利巴韋林的實(shí)驗(yàn)研究 1.目的 本研究通過(guò)考察以膽酸為載體的肝靶向RBV偶合物大鼠灌胃給藥的生物利用度，以發(fā)現(xiàn)口服生物利用度較高的靶向偶合物。 2.方法 2.1靶向偶合物和RBV經(jīng)口給藥，于給藥后不同時(shí)間采血，以L(fǎng)C-MS/MS法測(cè)定RBV血藥濃度，計(jì)算主要的藥代動(dòng)力學(xué)參數(shù)。 2.2靶向偶合物和膽酸同時(shí)經(jīng)口給藥，于給藥后不同時(shí)間采血，以L(fǎng)C-MS/MS法測(cè)定RBV血藥濃度，，計(jì)算主要的藥代動(dòng)力學(xué)參數(shù)。 3.結(jié)果 3.1靶向偶合物RBV-Leu-CA和RBV-Ile-CA口服給藥后血漿中RBV的Cmax顯著低于RBV，但是Tmax及T1/2顯著大于RBV，因此其AUC與RBV相當(dāng)；靶向偶合物RBV-Leu-CA和RBV-Ile-CA的藥-時(shí)曲線(xiàn)表現(xiàn)為雙峰。 3.2膽酸與RBV-Leu-CA同時(shí)給藥，導(dǎo)致血漿中RBV的Cmax及AUC均顯著降低，分別只有RBV-Leu-CA單獨(dú)給藥的24%和26%。 4.結(jié)論 靶向偶合物RBV-Leu-CA大鼠經(jīng)口給藥，生物利用度略高于RBV，藥-時(shí)曲線(xiàn)表現(xiàn)為肝腸循環(huán)的特征吸收；膽酸對(duì)RBV-Leu-CA的吸收有顯著的拮抗作用，說(shuō)明RBV-Leu-CA是通過(guò)膽酸介導(dǎo)的特異性途徑吸收的。
[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.

【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R512.63

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