【作者】 徐文；

【导师】 陈大为； 何仲贵；

【作者基本信息】 沈阳药科大学 ， 药剂学， 2006， 博士

【摘要】 冬凌草甲素为抗癌中药冬凌草中的主要抗癌活性成分，具有资源丰富、毒性低、药理活性强等优势，近年来吸引了广大药学工作者的注意。但目前对于该药的研究主要集中在药理活性方面，对于其制剂研究较少，并且不系统，目前也没有合适的制剂投放到市场。本文从制剂学角度对冬凌草甲素进行了系统的研究。通过对冬凌草甲素的理化性质和初步的药物动力学研究，解析了冬凌草甲素的体内外性质，并据此对冬凌草甲素的制剂进行了筛选。通过对羟丙基-β-环糊精复合物、注射用亚微乳、固体脂质纳米颗粒和脂质体这四种微粒制剂的考察和比较，研制出冬凌草甲素羟丙基-β-环糊精复合物和冬凌草甲素脂质体，并对这两种制剂进行体内外评价。首先，本文对冬凌草甲素的理化性质进行了考察，侧重于平衡溶解度、油水分配系数和稳定性。冬凌草甲素在水、常用油中的溶解度较差；具有中等的疏水性；在溶液状态时对光、热的稳定性较差，在碱性条件下稳定性更差，不能长期放置。通过测定该药以及降解产物的红外光谱推测该药的活性基团α-亚甲基环戊酮遭到破坏。根据冬凌草甲素的理化性质，对文献关于冬凌草甲素的提取纯化工艺进行改进，采用浸渍法提取虽提取率稍低，但出膏率明显降低，有利于纯化；用硅胶柱提取时，缩短柱长，提高上样量，以利于药物晶体的析出。改进后的工艺可以提高冬凌草甲素的产率和纯度。其次，测定了冬凌草甲素溶液在大鼠体内的药动学和在小鼠体内的分布。大鼠静脉注射冬凌草甲素溶液后，血药浓度迅速下降，然后呈现4～6h的平台，接着是缓慢消除，消除半衰期约为10h。在所测定的给药剂量范围内(5～15mg／kg)冬凌草甲素的药物动力学特征呈非剂量依赖性。大鼠静脉注射冬凌草甲素溶液后(给药剂量为10mg／kg)有大约相当于给药剂量16.4％的冬凌草甲素以原型从胆汁中排泄，并且主要发生在前6个小时，提示存在肝肠循环，药时曲线的平台可能是肝肠循环造成的。大鼠灌胃给予冬凌草甲素溶液后血浆中药物浓度迅速升高，很快达到峰值，但绝对生物利用度很低，并且在实验所测定的范围内呈现出剂量依赖性，高剂量是低剂量和中剂量的两倍多。大鼠口服冬凌草甲素溶液后，48 h内以粪便形式排泄量和肠内容物的量只占给药剂量的6.52％，表明造成大鼠口服冬凌草甲素溶液绝对生物利用度低的原因可能是首过效应，而剂量依赖现象则是由于首过代谢饱和所造成的。大鼠腹腔注射冬凌草甲素溶液后(给药剂量为10 mg／kg)的绝对生物利用度为12.64％，表明冬凌草甲素有强的肝首过代谢。灌胃给予冬凌草提取液后(给药剂量相当于冬凌草甲素40 mg／kg)冬凌草甲素的绝对生物利用度仍很低，只有11.4％，但较给予相同剂量的冬凌草甲素溶液提高了1.5倍，表明冬凌草甲素中的其它成分可能影响冬凌草甲素的吸收。小鼠静脉注射冬凌草甲素溶液后分布广泛，在心、肺和肾中分布较高，但在心脏中消除很快，在脑中分布很低。最后，对冬凌草甲素的制剂进行了研究。考虑到该药口服生物利用度低，所以主要考察静脉注射制剂。因为冬凌草甲素的水溶性差，而使用有机溶剂增溶会增强毒副作用并降低患者的顺应性，所以我们研究了不含有机溶剂的微粒制剂，包括羟丙基-β-环糊精(HP-β-CD)复合物、注射用亚微乳、脂质纳米颗粒和脂质体。HP-β-CD可以增加冬凌草甲素在水中的溶解度，可以满足临床给药的需要，对于该药在溶液中的稳定性差无改善，但将复合物制成粉针后解决了冬凌草甲素稳定性差的问题。大鼠静脉注射冬凌草甲素-HP-β-CD后该药的分布稍快于普通溶液，消除速度无差别；在小鼠体内分布实验表明注射复合物增加了肺中的药物浓度并稍降低了在心脏中的浓度。初步安全性实验表明该复合物适于静脉注射给药。对冬凌草甲素制成注射用亚微乳、固体脂质纳米颗粒和脂质体的可行性进行了分析，采用单相溶液冻干法将冬凌草甲素制成前体脂质体可获得较高的包封率并且解决了药物稳定性差的问题。通过处方优化确定单相溶液冻干法制备冬凌草甲素的处方、工艺。得到的脂质体平均粒径为119nm，分布均匀，包封率约为63％，放置12 h内包封率没有明显下降，无药物晶体析出。初步的制剂安全性实验表明冬凌草甲素脂质体静脉注射不会导致溶血，无刺激性，适合静脉注射给药。大鼠药动学实验表明冬凌草甲素脂质体药动学行为不同于注射冬凌草甲素溶液，分布变快但是消除变慢，消除半衰期延长了约1倍，药时曲线下面积增加了66.5％。小鼠静脉注射冬凌草甲素脂质体后的组织分布研究表明药物在心脏中的分布下降，在5 min时为注射溶液的1半；在肝中的分布上升，在5 min时是注射溶液的4倍，在1h是注射溶液的4.3倍；在脾和肺中的分布稍有降低，但在脑中的分布提高了约1倍。结果表明冬凌草甲素脂质体可能降低药物的毒性并利于肝癌的治疗。根据理化性质推测该脂质体可能会取得较好的肿瘤靶向性。更多还原

【Abstract】 Oridonin is the major antitumor components of Rabdosia rubescences which is traditionally used in China for the treatment of a variety of cancers. Due to the low toxicity of oridonin, abundant resource of Rabdosia rubescences and the obvious pharmacological activity of oridonin, this drug has attracted special attention and was regarded as a promising anticancer drug. But most research on oridonin is about its pharmacological activity and mechanism, and to our knowledge, no systemic research on the dosage form of this drug is reported and no successful product has been put onto the market.This paper systemically studied the drug as viewed from dosage from. We mastered the in vitro and in vivo behavior of oridonin through preformulation study, and designed the proper dosage forms for it. We studied the possibity of preparing oridonin-hydroxypropyl-β-cyclodextrin （HP-β-CD） complex, submicron emulsion, liposome and solid lipid nanoparticles, and found HP-β-CD complex and liposome are suitable drug delivery systems for oridonin. Oridonin-HP-β-CD complex and liposome were prepared and evaluated by us.Firstly, this paper determined the physical-chemical property of oridonin, especially for its equilibrium solubility, partition coefficients for the n-octanol—water/buffer solution systems and stability.The equilibrium solubility of oridonin in water is 0.725 mg·mL^-1 at 25℃and has lower values in basic buffer solutions. The equilibrium solubility of oridonin in organic media was also detected and oridonin has high solubility in high polarity organic media such as methanol and ethanol, while the solubility is poor in low polarity organic media such as petroleum ether and n-hexane. The partition coefficients for the n-octanol—water(lgP_app) of oridonin is 1.66, which showed oridonin has moderate hydrophobicity. The partition coefficients for the n-octanol—buffer solution systems varied little with the pH values of phosphorate buffer solution. The stability of oridonin in solution is poor. Light and high temperature can reduce the stability of oridonin in solution. Oridonin was less stable along with the increase of pH values at the interval of 4～10 and was most stable at pH4. According to the physico-chemical properties of oridonin, we improved the process of the extraction and purification of oridonin, and by the new process more oridonin was got and the purity of oridonin was also enhanced.Secondly, this paper studied the pharmaeokineties of oriodnin in rats and its distribution in mice.After intravenous administration, the plasma concentration of oridonin first decreased rapidly and then more slowly, that is to say, the plasma concentration of oridonin after intravenous administration decreased bi-exponentially and the terminal elimination half-life was relatively long （about 10 h）. After intravenous administration, the pharmacokinetic parameters of oridonin were dose-independent at three doses, 5, 10 and 15 mg/kg. These results show that oridonin exhibits linear kinetics following intravenous administration over the dose range studied. The enterohepatic circulation may pay contribution to the plateau in the plasma concentration-time curves.The oral absolute bioavailability of oridonin was rather low （4.32-10.85％） and appeared to be dose-dependent. Considering the amount of unchanged oridonin recovered from the gastrointestinal tract and feces 48 h after oral administration （the mean value was approximately 6.52％）, the low oral absolute bioavailability values are most likely due to hepatic, gastric, and/or intestinal first-pass effects. While the dose-dependent phenomenon may be resulted from the saturation of first-pass metabolism.After intraperitoneal administration of oridonin solution to rats at a single dose of 10 mg/kg, the bioavailability of oridonin was 12.64％. This result shows that hepatic first-pass effect may be the main reason for the low oral bioavailability of oridonin.The distribution of oridonin after intravenous administration of oridonin at a dose of 20 mg/kg to mice was detected. After oridonin was administrated, it distributed widely and quickly except brain. The concentration of oridonin in heart, lung and kidney was high, but the concentration of oridonin in heart and lung decreased quickly while the concentration of oridonin in spleen decreased slowly. On the contrary, the concentration of oridonin in liver was low soon after drug administration and increased until it reached a maximum value at the time of 0.5 h after drug administration.Lastly, this paper studied the dosage form design of oridonin. Considering the poor water solubility and oral bioavailability of oridonin and severe side effect of organic medium, we planned to develop organic medium free colloidal drug delivery system including HP-β-CD complex, submicron emulsion, liposome and solid lipid nanoparticles. This paper studied the solubilizing effect of HP-β-CD on oridonin and the action between oddoain and HP-β-CD. Though the action between oridonin and HP-β-CD was poor, HP-β- CD was a good solubilizing agent for oridonin and the concentration of oridonin in HP-β-CD solution can fit clinical requirement.Using DSC and X-ray diffraction, we confirmed the formation of oridonin-HP-β-CD complex. And with the help of UV, IR and ¹H-NMR, we got that the active radical of ofidonin（α-methylene-cyclopentanone） was not in the cavity of HP-β-CD, so it can not be protected by HP-β-CD. Though HP-β-CD can not protect oridonin for degradation, the solid state of oddonin-HP-β-CD complex is rather stable. Furthermore, powder of the complex can form clear solution when water is added, and the solution keeps clear when dilution.Pharmacokinetics behavior of oridonin-HP-β-CD complex in rat is similar with oddonin solution solubilized with organic medium. Only the distribution rate of oddonin after intravenous administration of oridonin-HP-β-CD complex increased slighty which induced a smaller AUC value.Distributioin of ofidonin after intravenous administration of oridonin-HP-β-CD complex is different from that of oridonin solution. Less distribution in heart and more in lung was got after intravenous administration of oddonin-HP-β-CD complex. Safety experiment showed the oddonin-HP-β-CD complex did not induce hemolysis or irritation and fit for parenteral drug delivery.Considering the stability and entrapment efficiency of the dosage forms as well as the chemical stability of ofidonin, we considered that liposome prepared by "freeze-drying of monophase solutions" method is for fit for the delivery of oridonin.Preparing with the optimized method mentioned above, we will get liposome with entrapment efficiency of 63％or so. The average particle size of the liposome is 119 and its distribution is narrow. After stored for 12 h, entrapment efficiency did not decreased significantly and no precipitation was found.Safety experiment showed the oridonin loaded liposome did not induce hemolysis or irritation and fit for parenteral ues.Pharmacokinetics behavior of oridonin loaded lipsome in rat was different from that of oridonin solution solubilized with’organic medium. The distribution rate of oridonin increased slighty while the terminate rate decreased significantly. The apparent elimination half-life extended to about 2 folds of that after intravenous administration of oridonin solution. The longer apparent elimination half-life induced a bigger AUC value. Distributioin of oridonin after intravenous administration of oridonin loaded liposome wass different from that of oridonin solution. Compared with oridonin solution, oridonin loaded liposome enhanced the liver distribution and reduced the distribution in heart significantly. While the distribution in spleen and lung was slightly reduced and the drug distribution in brain was increased.更多还原