【作者】 李必波；

【导师】 李少林；

【作者基本信息】 重庆医科大学 ， 肿瘤学， 2012， 博士

【摘要】 黄芩苷（Baicalin）是从唇形科植物黄芩的干燥根中提取的单一有效成分，能够抑制多种肿瘤细胞的增殖并对放化疗所致的肝损伤有防护作用。但由于其难溶性而导致制剂溶出度低、生物利用度差（仅2.2%±0.2%）,限制了黄芩苷达到抗肿瘤所需的药物浓度，临床上现用于治疗急性黄疸型、急性无黄疸型及慢性肝炎，目前市售口服制剂仅有常规的片剂和胶囊。固体分散体技术是一种被广泛用于提高难溶性药物溶出速率的新技术，能使难溶性药物以微晶、无定形、分子状态分散在载体中而呈固体分散体，分别被称着简单低共熔混合物、共沉淀物（又称玻璃态固熔体）、固态溶液。药物在载体中的分散状态直接影响药物的溶出速率，其中分子分散状态最快，其次是无定形态，微晶态最慢。不同的载体则直接影响药物在其中的分散形态。因此，本文利用固体分散体技术，以聚乙烯吡咯烷酮（PVP）为载体，用溶剂法将黄芩苷制备成芩苷－PVP共沉淀物，装入胃溶胶囊作为口服制剂，并对其制备工艺、溶出度、物相、处方工艺、稳定性和药代动力学进行了研究。拟解决黄芩苷在临床应用中存在的不足，提高药物服用后血药浓度，扩大其在临床上抗肿瘤方面的应用。本文完成的主要工作和结论如下：1.建立紫外光谱法测定黄芩苷－PVP共沉淀物胶囊的药物含量及四种溶出介质中的溶出度，为制剂处方筛选和质量控制奠定了基础。采用岛津UV-3150紫外可见分光光度计，在278nm单波长处，测定黄芩苷－PVP共沉淀物胶囊的药物含量及在0.1mol·L^-1HCl溶液、蒸馏水、pH4.5溶液、pH6.8溶液中的溶出度。结果显示：含量测定时，黄芩苷浓度在3.473～9.261μg·m L^-1范围内，线性关系良好（r=0.9998），平均加样回收率为98.85%。溶出度测定时，在0.1mol·L^-1HCl溶液中，黄芩苷浓度在1.158～15.049μg·m L^-1范围内，线性关系良好（r=0.9998），平均加样回收率为99.47%；在蒸馏水、pH4.5溶液、pH6.8溶液中，黄芩苷浓度在1.158～12.734μg·m L^-1范围内，线性关系良好（r=0.9999），平均加样回收率分别为99.63%、99.82%、98.75%。该测定方法简便、快速，结果准确可靠、重现性好，可作为该制剂的质量控制方法。2.改进黄芩苷－PVP共沉淀物的制备工艺，降低生产成本，进一步提高难溶性药物高黄芩苷的溶出度及药辅比。运用改进工艺的共沉淀法制备药辅比分别为1∶2、1∶3、1∶4的黄芩苷－PVP共沉淀物并制成胃溶胶囊。采用紫外光谱法测定其药物含量及在0.1mol·L^-1HCl溶液、蒸馏水、pH4.5溶液、pH6.8溶液中的溶出度并与原料药胶囊对比，采用X－射线粉末衍射法考察共沉淀物中黄芩苷晶体特征。结果显示：药辅比分别为1∶2、1∶3、1∶4的黄芩苷－PVP共沉淀物胶囊在0.1mol·L^-1HCl溶液中的溶出度为原料药胶囊的4.8倍、21.5倍、30.4倍；在蒸馏水中为原料药胶囊的1.7倍、3.3倍、3.4倍；在pH4.5溶液中为原料药胶囊的2.06倍、2.02倍、2.09倍，在pH6.8溶液中的溶出度与原料药胶囊相近。X－射线粉末衍射表明药辅比分别为1∶2、1∶3、1∶4的共沉淀物中黄芩苷的晶体峰均消失。通过改进工艺制备的黄芩苷－PVP共沉淀物可有效地降低生产成本，可显著提高黄芩苷的溶出度及药辅比，提高效果远高于文献报道的结果。3.确定了制备黄芩苷-PVP共沉淀物的处方及工艺。通过前期对各种药辅比的黄芩苷-PVP共沉淀物胶囊的制备工艺、处方配比、溶出度考察及X-衍射考察，作者最终确定黄芩苷-PVP共沉淀物的处方为药辅比1:3，并试图大量制备。作者通过对黄芩苷-PVP共沉淀物的制备工艺条件进行适当的调整，现单次可制备黄芩苷-PVP共沉淀物28.00g，溶出度试验表明，大批量生产的药辅比为1:3的黄芩苷-PVP共沉淀物胶囊溶出度无改变，可达到中国药典2010年版附录对胶囊溶出度所要求：在45min时间里，溶出度值大于70%。联合多方法对黄芩苷-PVP共沉淀物进行了物相鉴定。采用高精度原子力显微镜（AFM）、溶出度测定、红外光谱法（IR）、X-射线粉末衍射法（XRD）、差示扫描量热法（DSC）等方法进行考察。AFM结果显示：黄芩苷以分子状态存在于PVP高分子中，且与PVP形成了分子间氢键；溶出度结果显示：在以蒸馏水为溶出介质中，共沉淀物胶囊的溶出度为原料药胶囊的3.6倍，表明药物高度分散在辅料中；XRD结果显示：共沉淀物中黄芩苷的晶体峰消失，表明黄芩苷以无定形或分子态存在于PVP高分子中；IR结果显示：共沉淀物在3550cm^-1～3200cm^-1间出现两个宽的吸收峰，说明有游离及缔合的羟基存在，表明有分子间氢键的形成；DSC结果显示：共沉淀物中黄芩苷的熔点从223℃变为219℃，说明黄芩苷以高度分散状态存在于PVP中。溶出度实验、DSC、XRD和AFM分析证实黄芩苷以无定形或分子状态的高度分散载体材料中，而药物分子与载体之间的氢键形成有利于维持这种高度分散的稳定性。同时表明高精度的原子力显微镜能更直观地从分子水平上分析药物与辅料的空间构象及其相互作用，克服了传统方法的缺陷，为开发新型黄芩苷-PVP固体分散体制剂打下基础。4.考察了黄芩苷-PVP共沉淀物的稳定性。影响因素考察结果显示，高温（60℃或40℃）、高湿（25℃,RH90%士5%或25℃，R H75%士5%）、强光对黄芩苷-PVP共沉淀物胶囊含量、溶出度、性状、重量影响均较大，其中高湿影响最大。加速实验表明，黄芩苷-PVP共沉淀物胶囊按市售包装（置双铝箔密封包装内），在40℃士2℃, RH75%士5%条件下放置6个月，黄芩苷-PVP共沉淀物胶囊的含量及溶出度降低。长期试验表明，黄芩苷-PVP共沉淀物胶囊按市售包装（置双铝箔密封包装内），在25℃士2℃，R H60%士5%条件下放置1年，黄芩苷-PVP共沉淀物胶囊的含量、溶出度、性状、重量均无明显变化，提示本品有效期可暂定为1年。5.建立了高效液相色谱法测定beagle犬血浆中黄芩苷的含量。色谱条件为，色谱柱：Hypersil ODS250mm x4.6mm,5μm，保护柱：Hypersil ODS4.0mmx3.0mm,5μm，流动相：甲醇：0.1%H3PO445：55（v／v），内标：大豆苷元甲醇液，流速：0.8mL／min，UV检测波长：278nm，进样量：50μL，柱温：30℃。采用液液萃取及固相萃取法处理血浆样品，并通过氮气吹干浓缩，能够提取并初步分离ng级的黄芩苷样品。结果显示血浆中杂质不干扰主药和内标的测定，这表明该方法专属性强，3.648-364.8ng／mL浓度范围内线性关系良好，R=0.998（n=6），萃取回收率在80%-90%之间，精密度RSD均小于10%，黄芩苷血浆样品在室温条件下，冻融条件下，长期条件下稳定性均良好。符合生物样本的分析要求。表明该法准确、可靠，可用于beagle犬血浆中黄芩苷的定量检测。6.考察了黄芩苷-PVP共沉淀物在beagle犬的体内药代动力学。以黄芩苷原料药胶囊为参照制剂，采用自身对照交叉给药法，对6只beagle犬进行体内药代动力学实验。药-时曲线及药动学参数显示：与黄芩苷原料药胶囊相比，黄芩苷-PVP共沉淀物胶囊使药物在犬胃及结肠的吸收显著增加，相对生物利用度为338.2％±93.2%。证实了通过进一步降低黄芩苷在载体中的分散粒径，则可进一步提高黄芩苷的溶出速率和生物利用度的假设。更多还原

【Abstract】 Baicalin, a single active ingredient extracted from the dried roots ofScutellaria baicalensis of the Labiate family, is a flavonoid used to treatacute icteric, acute non-icteric and chronic hepatitis. Current studies haveshown that baicalin is able to inhibit multiple cancer cells from growing ormultiplying and protect the liver from drug-induced injuries. Poorsolubility of the currently marketed oral preparations including tablets andcapsules has resulted in poor dissolution and bioavailability. The absolutebioavailability of baicalin has been found to be only2.2±0.2%in rats afteroral administration.Solid dispersion technology is a new technology used to improvedissolution rates of insoluble drugs within solid dispersions into carriers inmicrocrystallite, amorphous and molecular phases that are called simpleeutectic mixtures, coprecipitates （also called vitreous state solid solutions）and solid solutions, respectively. Drug dispersity in a given carrier directlyaffects its dissolution rate and places molecular, amorphous and microcrystalline phases in a descending order of sequence in terms ofdissolution rate. In addition, different carriers also have a direct impact ondrug dispersion morphology.In this study, baicalin-PVP coprecipitate were prepared using solventmethod of solid dispersion technology to increase the dissolution rate andstability of baicalin in its carrier, and its preparation process, dissolution,prescription process, stability and pharmacokinetics were studied. The aimof this study was solving the shortcomings of baicalin, improvig the plasmaconcentration of baicalin after oral administration, and expanding itsapplication of anticancer action in clinical practice.The whole work of this paper have been completed, therefore, we candraw the conclusions as follows:1. The UV method was established to determine of content ofBaicalin-Polyvinylpyrrolidone （PVP）coprecipitate capsule and itsdissolution rate in four different solutions, to provide the basis forscreening prescription and quality control. In278nm wavelength, thecontent of Baicalin-PVP coprecipitate capsule and its dissolution rate in0.1mol·L^-1HCl solution, distilled water, pH4.5solution and pH6.8solution were determinated by SHIMADZU UV-3150. During thedetermination of the content, the calibration curves was linear in theconcentration range of3.473～9.261μg·m L^-1, r=0.9998（n=6）, therecovery rate was99.06%～99.48%, RSD＜1%（n=3）. During determining the dissolution rate in0.1mol·L^-1HCl solution, linearity wasgood within the concentration range of1.158～15.049μg·m L^-1, r=0.9998（n=7）, the recovery rate was99.15%～99.58%, RSD≤0.70%（n＝3）; in distilled water, linearity was good within the concentration rangeof1.158～12.734μg·m L^-1, r=0.9999（n=6）, the recovery rate was98.25%～101.56%, RSD≤0.71%（n＝3）; in pH4.5solution, linearity wasgood within the concentration range of1.158～12.734μg·m L^-1, r=0.9999（n=6）, the recovery rate was98.25%～101.56%, RSD≤0.61%（n＝3）; in pH6.8solution, linearity was good within the concentration rangeof1.158～12.734μg·m L^-1, r=0.9999（n=6）, the recovery rate was98.37%～99.14%, RSD≤0.58%（n＝3）. The method is simple and rapid,and the results is accurate, reliable, and reproducible. It can be used tocontrol the quality of Baicalin-PVP coprecipitate capsule with satisfactoryresults.2.The preparation technology of baicalin-PVP coprecipitate wasimproved, the production costs were reducd and the dissolution andproportion of drug and accessories of high-insoluble drug baicalin werefurther improved. The baicalin-PVP coprecipitate with drug and accessoriesproportion of1:2,1:3,1:4were prepared respectively by improvedtechnology of coprecipitation method and made stomach capsule. UVspectroscopy was used to determine its drug content and dissolution in0.1mol·L^-1HCl solution, distilled water, pH4.5solutions, pH6.8solution and compared with the bulk drug capsules. The characterization ofbaicalin-PVP coprecipitate was determined by X-ray powder diffraction.RESULTS The dissolution of baicalin-PVP coprecipitate capsules withdrug and accessories proportion of1:2,1:3,1:4was4.8times,21.5times,30.4times of bulk drug capsules respectively in0.1mol·L^-1HClsolution;was1.7times,3.3times,3.4times respectively in distilled water;was2.06times,2.02times and2.09times respectively in pH4.5solution;was similar with bulk drug capsules in pH6.8solution. X-ray powderdiffraction indicated that baicalin crystal peak disappeared in baicalin-PVPcoprecipitate with drug and accessories proportion of1:2,1:3,1:4.therefore, the improved preparation technology of baicalin-PVPcoprecipitate can effectively reduce the production cost; can significantlyimprove the dissolution and proportion of drug and accessories. Theincreased times are much higher than that reported in literature.3.Baicalin-polyvinylpyrrolidone （PVP） coprecipitate with drug andaccessories proportion of1:3was prepared by the improved solvent methodof solid dispersion technology and attempt to prepare more each time. Now28.00g baicalin-PVP coprecipitate could be prepared each time through theappropriate adjustments of preparation conditions. dissolution tests showedthat the dissolution of baicalin-PVP precipitate capsule could be matchedthe capsule dissolution requirements of Chinese Pharmacopoeia2010, hedissolution up to70percent in45min.The coprecipitate was characterized using X-ray powder diffraction （XRD）, differential scanning calorimetry（DSC）, infrared spectrometry （IR） and dissolution testing. Furthermore,AFM·IPC-208B high-resolution atomic force microscopy （AFM） wasutilized to characterize the molecular morphology of baicalin within itscarrier and the interaction between baicalin and its carrier. The results ofXRD and DSC indicated that baicalin resided in PVP polymers in anamorphous or molecular phase, dissolution test results demonstrated thatthe dissolution rate of the coprecipitate was3.6times that of the activepharmaceutical ingredient （API）. The results of IR indicated the possibilityof the formation of intermolecular hydrogen bonds. The AFM·IPC-208Bfindings revealed that baicalin was dispersed in PVP polymers with amolecular size of2nm and either wrapped or surrounded by approximately0.4nm of a five-membered ring of PVP arranged along the carbon chainsequentially. An intermolecular hydrogen bond was formed between the4-OH of the glucuronide of baicalin and the O of the carbonyl group fromPVP in addition to the formation of intramolecular hydrogen bonds withinbaicalin. The AFM·IPC-208B technique was able to characterize the spatialconformation and interaction between the drug and carrier intuitively at themolecular level and confirmed the possible formation of hydrogen bondsbetween the drug and carrier speculated by IR; it can even distinguish thebinding-OH of the interaction, overcoming many of the disadvantages oftraditional methods. Therefore, AFM·IPC-208B can be used as a new characterization technique for coprecipitates in the future.4. The stability of baicalin-PVP precipitate was investigated. Theresults of influencing factor showed that high temperature （60°C or40°C）, high humidity （25°C, RH90%±5%, or25°C, the R H75%±5%）,strong light significantly influence the content, dissolution, traits andweight of baicalin-PVP precipitation capsule, and high humidity is thegreatest influencing factor. Long-term tests shows that, baicalin-PVPprecipitate capsule was stability in condition of25℃±2℃, R H60%±5%with commercial packaging （a pairs of aluminum foil sealed package）one year. The content, dissolution, traits and weight of baicalin-PVPprecipitate capsule did not change, which suggest that the validity of theproduct be tempertarily scheduled as1year.5. A high performance liquid chromatography method wasestablished to determine the content of baicalin in beagle dog plasma.Chromatographic conditions were, column: A Hypersil ODS250mm x4.6mm5μm guard column: Hypersil ODS4.0mm x3.0mm5μm, mobilephase: methanol:0.1%H3PO4,45:55（v/v） internal standard: daidzein,flow rate:0.8mL/min, UV detection wavelength:278nm, injectionvolume:50μL, column temperature:30℃. Liquid-liquid extraction、solidphase extraction and concentration were used to extract and separate thebaicalin from plasma samples in very low level. The results showed thatthe impurities in the plasma did not interfere with the determination of main drug and internal standard, a good linear relationship was obtained inrange of3.648-364.8ng/mL, R=0.998（n=6）. The extraction recoverieswere80%-90%, and RSD were less than10%. The stability of baicalin inplasma samples at room temperature, freeze-thaw conditions andlong-term conditions were good. It shows that the method is accurate,reliable, and can be used for the quantitative detection of baicalin in theplasma of beagle dogs.6The pharmacokinetics of baicalin-PVP coprecipitate capsules inbeagle dogs were investigated. Self-control cross-administration was usedto do pharmacokinetic experiment in six beagle dogs. As compared withbaicalin bulk drugs capsules, the results of concentration--time curve andpharmacokinetic parameters showed that baicalin-PVP precipitatecapsules could improve drug absorption in stomach and colon significantly,and relative bioavailability was338.2%±93.2%. The results confirmedthe assumption that the more particle size of baicalin in the carrier wasreduced, the more dissolution rate and bioavailability would be increased.更多还原