【作者】 孙亮；

【导师】 柴逸峰； 陈万生；

【作者基本信息】 第二军医大学 ， 药物分析学， 2013， 博士

【摘要】 中药注射液是中医药在长期发展的基础上形成的一种新剂型，由于其自身特点，中药注射液成分复杂，临床适应症较多，在发展过程中也出现过一些影响重大的不良反应事件。一些中药注射液的质量标准落后，缺乏控制其质量稳定性的评价指标，物质研究基础不足；药物进入体内的入血成分不明确，药动学特征不够清晰，药物之间相互作用研究并不多见。这些不足大大影响了中药注射液在临床的安全有效应用，难以适应中药现代化要求，因此提高中药注射液的质量标准与用药安全性势在必行。痰热清注射液由黄芩、熊胆粉、山羊角、金银花和连翘组成，主要用于风温肺热病痰热阻肺证，是治疗急性支气管炎和急性肺炎等疾病的中药制剂，被广泛应用于呼吸科、儿科、外科、肿瘤科等。目前痰热清注射液缺少权威的质量控制体系，其体内过程也并不清楚，对其它药物的疑似影响也有报道。因此本文以痰热清注射液为研究对象，以HPLC、LC-MS/MS、LC-Q-TOF/MS联用技术为主要监测手段，在对痰热清注射液质量控制研究和痰热清注射液主要成分及西罗莫司体内定性定量分析方法建立的基础上，深入探讨痰热清注射液体内代谢动力学特征和及与西罗莫司的相互作用机制，具体内容包括：1、采用HPLC-DAD-ELSD技术对11批不同批次的痰热清注射液建立了双谱指纹图谱，并进行了相似度分析。在此基础上以LC-Q-TOF/MS技术对痰热清注射液的化学成分进行了鉴别，共鉴别出53种化合物，并根据二级质谱特征离子区分了部分同分异构体，同时对指纹图谱的部分峰进行了指认，使其代表性增强。对痰热清注射液中的主要成分绿原酸、咖啡酸、黄芩苷、熊去氧胆酸与鹅去氧胆酸建立了HPLC-DAD-ELSD含量测定方法，并进行了方法学考察。结果表明各成分线性关系良好（r>0.99），日内、日间精密度均小于5%，重现性好，精密度高，成功用于不同批次痰热清注射液的测定，为更好地控制痰热清注射液质量提供了科学依据，并为体内药代动力学研究所需的质量稳定可靠的供试样品提供了保证。2、采用LC-Q-TOF/MS技术对痰热清注射液的入血成分进行了鉴定，共鉴别出20余种入血成分。再采用LC-MS/MS技术建立了对痰热清注射液中5种主要活性成分，即绿原酸、咖啡酸、黄芩苷、熊去氧胆酸与鹅去氧胆酸，在大鼠血浆中的定量分析方法。采用蛋白沉淀为血浆样品前处理条件，沉淀剂为甲醇：乙腈＝3：1，以芦丁、葛根素为内标，色谱分析采用Zorbax SB-C18柱(3.5μm，100mm×2.1mm)，流动相为乙腈与水（含0.1%甲酸），采用梯度洗脱，流速为0.3mL·min(-1)，柱温为35℃。运用Agilent G6410A型串联质谱的多反应监测(MRM)模式负离子检测，并进行了系统的方法学评价。结果显示，在各自的生物样品浓度范围内，均呈良好线性，r>0.99；各成分的线性范围分别为：30～14933ng·mL(-1)，27～13333ng·mL(-1)，50～50333ng·mL(-1)，550～55000ng·mL(-1)，480～48000ng·mL(-1)，日内、日间精密度均小于14%，提取回收率在70%～105%之间。该方法操作简单，选择性好，灵敏度高，成功用于痰热清注射液大鼠血浆的药代动力学研究，为临床合理用药提供参考依据。最后以LC-Q-TOF/MS技术对痰热清注射液主要入血成分的代谢产物进行了初步鉴别，初步明确了痰热清注射液在体内的代谢特征。3、本章建立了一种简单、快速、灵敏的LC-MS/MS方法来测定大鼠全血中西罗莫司的浓度。蛋白沉淀法可有效提取全血中的西罗莫司，内源性杂质不干扰测定，标准曲线线性关系良好（r>0.99），最低定量限为2.5ng·mL(-1)，高、中、低3个浓度下测得各成分的日内和日间精密度RSD均低于15%，相对回收率、绝对回收率均符合要求。本方法各项指标符合生物样品分析测定要求，且操作简单，选择性好，灵敏度高，适用于痰热清注射液与西罗莫司合并用药后的药代动力学研究。从整体药代动力学角度上考察了在不同时间注射等量痰热清注射液及在相同时间注射不同剂量痰热清注射液对西罗莫司在大鼠体内过程的影响，结果显示痰热清注射液在体内对西罗莫司都表现为Cmax升高，AUC0→t增大，对其消除相并没有明显影响。4、采用人肝微粒体体外孵育实验，系统考察了痰热清注射液及5个主要活性成分对CYP1A2、CYP2C9、CYP2C19、CYP2D6、CYP3A4和CYP2E1这6种酶活性是否产生抑制作用，从而影响西罗莫司在肝微粒体的代谢。并以正己烷、二氯甲烷、乙酸乙酯及水对痰热清注射液进行了液液萃取，再以指纹图谱的液相条件为基础，以不同时间间隔对痰热清注射液进行了分段收集。结果显示，痰热清注射液对这6种酶均有不同程度的抑制作用，但其5个主要成分及除水提液外的其它萃取液无明显抑制，而痰热清注射液0-5min段和30min以后的两段收集液有不同程度的抑制作用。初步证实了痰热清注射液是通过抑制CYP450酶系从而抑制西罗莫司的代谢使其生物利用度升高，但具体抑制性成分仍有待考察。更多还原

【Abstract】 TCM injection is a totally new dosage formation based on its long development,due to its specific characters like complicated constituents, wide therapeutic index, wehave met some serious adverse occasions. The present research background for the TCMis the quality control standards can’t match up with the current requirements, we alsodon’t have a target index to make sure the reproducibility of quality control, the basicelements of TCM hadn’t drawn great attention from the researchers. What’s more, theeffective ingredients and pharmacokinetic characters of drugs administrated in humanplasma are still unclear, we even can’t get much report about TCM’ drug-drug interactionwith other chemotherapy, all those drawbacks affect badly on the successful clinicalapplication of the TCM injection, which can’t keep up with the pace of the Modernizationof TCM, so it’s essential to improve the quality control of TCM injection and make suresafe drug administration.TRQ injection, consisting of scutellaria, pulvis ellis urs, cornu gorais, and lonicerajaponica, forsythia suspense, is targeting for lung fever syndrome and widely used astreatment for acute bronchitis, acute pneumonia in respiratory department, pediatricdepartment, surgical department, oncology department. To date there is no official qualitycontrol system for TRQ injection, we get few knowledge about its metabolism in vivo,and several repots had talked about its interaction with other chemotherapy. In this article,we took TRQ injection as the research target, via the monitor equipment such as HPLC,LC-MS/MS and LC-Q-TOF/MS, we have done some quality study of TRQ andsuccessfully determined5major components of TRQ injections, sirolimus respectively.Meanwhile, we processed further pharmacokinetic investigation of TRQ injection in vivoand its interaction with sirolimus, the detailed contents are as follow:1. We got fingerprints of11different batches of TRQ injection viaHPLC-DAD-ELSD, and finished the relative analysis, and also successfully identified53components by LC-Q-TOF/MS, and according to the specific product ion fragments, wehad distinguished some isomers, which made the fingerprints more representative. Forthe5major constituents(chlorogenic acid, caffeic acid, baicalin, ursodesoxycholic acid)of TRQ injections, a sensitive HPLC-DAD-ELSD determination method was proposed, we also validated the method, the result was satisfactory, with a calibration coefficientr2>0.99, both intra-day precision and inter-day precision below5%. We have successfullyapplied this method for determination of other TRQ injection batches. The work beforegave support for the quality control of TRQ injection and we could make sure the qualityof TRQ injection involved in the following pharmacokinetic study was stable andacceptable.2. We have identified more than20ingredients of TRQ injection admistrated intoplasma via LC-Q-TOF/MS technique. Then a LC-MS/MS method for simultaneousdetermination of5major constituents(chlorogenic acid, caffeic acid, baicalin,ursodesoxycholic acid, chenodeoxycholic acid) in mouss plasma was provided, we choseprotein precipitation as pretreatment condition, and the reagent was methanol:acetonitrile(3:1, v/v), rutin and puerarin were used as internal standard, we performed thechromatographic separation through a Zorbax SB-C18column(3.5μm,100mm×2.1mm)with gradient elution of acetonitrile and0.1%formic acid in water, the flow rate was0.3mL·min(-1), and column temperature was set at35oC. We chose MRM as the monitoringmode. The whole method was successfully validated, the calibration cure range was30(-1)4933ng·mL(-1),27(-1)3333ng·mL(-1),50-5033ng·mL(-1),550-55000ng·mL(-1),480-48000ng·mL(-1), for chlorogenic acid, caffeic acid, baicalin, ursodesoxycholic acid,chenodeoxycholic acid respectively. Both intra-day and inter-day precision were below14%, the recovery percentage ranged from70%(-1)05%. Altogether, this simple, specificand sensitive method successfully applied to pharmacokinetic study of TRQ injection,provided support for clinical therapy. Finally we primarily identified the constituents ofTRQ injection administrated via LC-Q-TOF/MS, gained some knowledge of itsmetabolism character in vivo.3. In this article, we proposed a simple, rapid, sensitive method for determinationsirolimus in mouse plasma. Protein precipitation can extract the sirolimus in whole bloodeffectively and eliminated the matrix effect. The results showed that coefficient r2wasmore than0.99of calibration curve, LOQ proved to be2.5ng·mL(-1), other parameterssuch as intra-day and inter-day precision were below15%, recovery test was alsosatisfactory. This method matched all the needs for quantitative analysis of biologicalsample, and was simple, specific, and sensitive, which can be suited for thepharmacokinetic study of TRQ injection combined with sirolimous administration. Forthe whole part of pharmacokinetic study, we had studied that the same amount of TRQ injection could affect sirolimus metabolism at different time, and the different amounts ofTRQ injection could affect sirolimus metabolism at the same time, the result suggestedthat TRQ injection promoted the Cmax, followed by the increase of AUC0-t, however itdidn’t seem to change sirolimus’s elimination in vivo.4. Via the liver microsomal incubation experiments in vitro, we had systematicallystudied whether TRQ injection and its5major constituents inhibited the activity ofCYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4and CYP2E1, thus affecting themetabolism of sirolimous in liver microsomal. The results indicated TRQ injectionaffected those6enzymes at some extent, but the5definited constituents didn’t work.Subsequently we selected liquid-liquid extraction as the pretreatment procedure, andreagents such as n-hexane, dichloromethane, acetate and water had been tested. We foundthat only water extracts inhibited the activity of enzymes, which indicated that inhibitionconstituents are strongly polar. The experiments validated that TRQ injection canimprove the bioavailability of sirolimus by inhibiting the activity of the CYP450enzymefamily, but the exact worked constituents still needed further investigation.更多还原