【作者】 王硕；

【导师】 梁文权；

【作者基本信息】 浙江大学 ， 药剂学， 2009， 博士

【摘要】 增加难溶性药物的溶解度是药剂学研究的热点之一,而油水均不溶性药物给药系统的研究更是其中的难点问题。本课题将纳米粒和水包油（O/W）亚微乳相结合,制成新型的水包油包纳米粒（N/O/W）亚微乳给药系统,将纳米粒包载在亚微乳的油相中,以达到增加制剂中难溶性药物载药量的目的。制剂中采用生物相容性好的辅料,安全性好,可实现难溶性药物的静脉注射给药。N/O/W亚微乳可进一步制成干乳剂,提高稳定性不好的药物在制剂中的稳定性。双氢青蒿素（Dihydroartemisinin,DHA）是青蒿素经还原而成的半缩醛化合物,具有良好的抗疟作用,近代研究表明DHA对多种人类和动物肿瘤细胞均有杀伤作用,且治疗浓度下对正常细胞几乎无毒性作用,有希望发展成为新型的抗肿瘤药物。DHA为难溶性药物,分子结构中存在特殊的过氧基团,稳定性差,胃肠道降解严重,生物利用度低。本研究以双氢青蒿素为模型药物,采用生物相容性好的辅料制备了注射用DHAN/O/W亚微乳剂,可实现DHA的静脉注射给药。同时针对DHA稳定性不好的特点,进一步制成干乳剂,提高DHA在制剂中的稳定性。本课题建立了DHA的HPLC含量测定法,考察了DHA在溶液中异构体的转化平衡情况,α-DHA与β-DHA在溶液中达到平衡所需时间约为10h,平衡后二者的比例不再变化,比例约为2.3（α/β）。在转化过程中DHA异构体总峰面积基本保持恒定,对DHA的HPLC分析可用α-DHA与β-DHA峰面积之和与浓度进行线性回归,计算DHA的含量。测定了DHA在不同溶媒中的溶解度,DHA在水和大豆油中的表观溶解度分别为0.119±0.021和1.24±0.01mg/mL,在水和大豆油中加入不同乳化剂后,均能提高DHA的溶解度,其中以大豆油/大豆磷脂/胆固醇最为显著。以注射用大豆油、大豆磷脂、胆固醇和稳定剂C为油相,Poloxamer188、甘油为水相,采用微射流技术制备DHA N/O/W亚微乳。乳剂外观为乳白色液体,无油花,不挂壁,用水稀释后有淡蓝色乳光。平均粒径为152±18nm（PDI0.078±0.018）,Zeta电位为-33.28±2.07mV,pH值为7.18±0.03,符合注射要求。DHA N/O/W亚微乳中药物含量为2.98±0.03mg/mL,载药量约为DHA O/W亚微乳最高载药量的3倍。以DHAO/W亚微乳作为对照,分别在光学显微镜、透射电镜、冷冻蚀刻透射电镜下观察不同粒径DHAN/O/W亚微乳的形态。结果显示,DHAN/O/W亚微乳呈椭圆状,边缘光滑,乳滴大小较均匀,乳滴中包含有颗粒状物质,DHA O/W亚微乳中则未观察到有颗粒状物质存在。冷冻蚀刻透射电镜可见DHAN/O/W亚微乳乳滴断面不平整,具有类似于层纹或壳状的结构存在,而DHA O/W亚微乳的乳滴断面较光滑平坦。以多种可代表DHA纳米粒不同状态的DHA制剂和DHA纯品为对照,进行DHA N/O/W亚微乳的DSC分析试验。与DHA纯品相比,DHA N/O/W亚微乳剂和DHA油混悬液中DHA的熔点下降了接近10℃。而DHA水混悬液和模拟DHA分散在N/O/W亚微乳水相的制剂中DHA熔点与DHA纯品的熔点相近。通过DHA的熔点变化情况判断,DHAN/O/W亚微乳剂中DHA的存在位置应在制剂的油相中,与显微镜的结构观察结果相符。考察了DHA在N/O/W亚微乳中的分布情况。经测定DHA N/O/W亚微乳剂约有8.06%的药物溶解在水相中,91.9%的药物存在在油相中。其中有13.8%的药物溶解在油相中,其余78.1%的DHA存在于界面膜和油相的纳米粒中。以甘露醇:蔗糖（4:1）为冻干保护剂制备了DHA N/O/W干乳剂,干乳中冻干保护剂与油相的比例为2:1（w/w）。冻干过程为DHA N/O/W亚微乳与冻干保护剂混合均匀后,在-40℃迅速冷却预冻6小时,然后在-40℃真空干燥4小时,-40℃～0℃真空干燥8小时,升温至20℃持续4小时去除残余水分,冻于过程中压力保持在30-50mTorr,冷凝器温度控制在-80℃。建立了DHA有关物质的HPLC分析法,采用强制破坏法制备了在强酸、强碱、光照、高温和氧化条件下破坏的样品,找到了7个DHA的降解产物。DHA的降解产物在本分析方法下均能达到良好分离,本法可用于DHA制剂的稳定性研究。考察了DHA、DHAN/O/W亚微乳剂和DHAN/O/W干乳剂的稳定性。结果表明,DHA对光照和高温敏感,在湿度较高的环境下含量也有改变,故应低温干燥避光保存。考察了DHA在溶液中的稳定情况。与DHA溶液相比,DHA N/O/W亚微乳的稳定性已有提高,但对温度仍然很敏感,在光照条件下不稳定。DHAN/O/W亚微乳需放置在4℃,充氮避光密闭保存。对DHA N/O/W干乳剂进行了加速试验和长期留样观察。DHA N/O/W干乳经25℃、60%RH加速试验6个月,含量下降4%,复溶时间略微延长,粒径分布、Zeta电位和pH值没有显著变化。4℃长期留样观察9个月内制剂性状、药物含量等均无明显变化。在此条件下DHAN/O/W干乳剂质量稳定,长期试验仍在进行中。通过异常毒性检查、血管刺激性试验以及溶血性试验对DHA N/O/W亚微乳剂的安全性进行了初步评价。DHA N/O/W亚微乳剂对小鼠的异常毒性试验合格,制剂在生产过程中未带来异常的毒性。对家兔耳缘静脉无明显刺激作用,对家兔红细胞无明显体外溶血及致凝集作用,DHA N/O/W亚微乳剂用于静脉注射是安全的。建立了体内样品中DHA含量测定的HPLC-MS分析方法。方法专属性好,内源性杂质对样品测定无干扰,样品处理简单,精密度、回收率均符合规定,可用于DHA的体内分析。以DHA和DHA O/W亚微乳剂为对照,考察了DHAN/O/W亚微乳剂与家兔、大鼠和人血浆的血浆蛋白结合情况。在本试验研究的浓度范围内,不同浓度的DHA制剂在同种血浆中的蛋白结合率无显著差异,蛋白结合率与药物浓度无关。不同制剂在同种血浆中的蛋白结合率也没有显著差异。不同种血浆当中,DHA与家兔的血浆蛋白结合率较高,其次为大鼠和人的,DHA与血浆蛋白具有中等强度的结合,平均血浆蛋白结合率分别76.36%、66.34%和63.94%。家兔体内药动学研究结果显示,DHA N/O/W亚微乳和DHA O/W亚微乳可以显著延长DHA的体内循环时间,提高血浆中药物浓度。DHA N/O/W亚微乳和DHA O/W亚微乳的MRT分别为DHA溶液剂的的10.39和3.42倍,DHAN/O/W亚微乳为DHA O/W亚微乳的3.03倍。DHA N/O/W亚微乳和DHA O/W亚微乳的AUC分别为DHA溶液剂的3.01和1.80倍,DHA N/O/W亚微乳为DHAO/W亚微乳的1.67倍。小鼠组织分布研究表明,DHA在体内迅速转化成一未知成分,在血液样品中可观察到转变情况,各组织中仅检测到该成分的存在。经固相萃取分离提取,HPLC-MS分析得到该成分的质谱图,该成分具有m/z 267.1的碎片离子峰提示其结构中仍有过氧基团的存在,应仍具有与DHA类似的药效。考察了该成分在小鼠体内的消除分布规律,研究DHA N/O/W亚微乳剂的组织分布趋势。结果表明,DHA亚微乳剂可延长药物在体内组织脏器中的滞留时间,在肝的摄取最高。DHA N/O/W亚微乳剂和DHA O/W亚微乳剂的体内分布趋势有所不同,DHA N/O/W亚微乳剂在心脏的分布降低,而在血中的滞留时间和在胃、脑中的分布增加。以H₂₂荷瘤小鼠模型进行DHA N/O/W亚微乳剂抗肿瘤药效学的研究。乳剂给药剂量为45mg/kg时,抑瘤率达51.8%（P＜0.01）,给药剂量为11.25mg/kg时,抑瘤率为23.0%,低剂量乳剂组药效略高于同等剂量的溶液组,高剂量的溶液对照组给药后溶媒毒性超过了动物的耐受剂量,因此未得到高剂量溶液组的数据。结果表明,45mg/kg的DHA N/O/W亚微乳剂对H₂₂肿瘤细胞具有显著抑制效果。对小鼠肿瘤的免疫组化结果分析表明,DHA可下调VEGF的表达,高剂量乳剂组尤为明显,对各组组织切片的MVD观察结果表明,MVD与VEGF表达结果趋势相同,DHA可能是通过抑制肿瘤细胞的血管生成来抑制肿瘤的生长。通过DHAN/O/W亚微乳的处方与工艺筛选、制剂特征、稳定性、安全性考察,动物组织分布、体内药物动力学、以及抗肿瘤作用的研究,表明N/O/W亚微乳具有以下优点:（1）可提高油水均不溶性药物的载药量;（2）使用生物相容性好的辅料,制剂安全性好,可适用于静脉注射给药;（3）以亚微乳作为纳米粒的载体,可增加纳米制剂的物理稳定性;（4）可进一步制成干乳剂,增加不稳定药物的化学稳定性;（5）能够改变药物在体内的组织分布和药物动力学性质,有可能进一步开发成靶向制剂;（6）易于工业化生产。本课题为N/O/W亚微乳新剂型的研究奠定了基础,为难溶性药物给药系统的研发提供新思路。DHA N/O/W亚微乳的研究可为疟疾重症患者提供急救用药,同时也可为研发双氢青蒿素抗肿瘤作用新用途提供制剂。更多还原

【Abstract】 The way to increase the solubility of poorly soluble drugs is concerned in recent years.Both poorly water-soluble and poorly oil-soluble drugs are faced with a major challenge in dosage form development.In this study,nanoparticle and emulsion were designed and put together to prepare a novel emulsion which have both the advantages of nanoparticle and emulsion.An nanoparticle-in-oil-in-water（N/O/W） submicron emulsion was prepared with physiological excipients and achieved intravenous administration of poorly soluble drugs.Nanoparticles were incorporated in the oil phase of O/W emulsion to enhance the drug loading capacity of N/O/W emulsion.Furthermore,this novel emulsion could be converted into dry emulsion to increase the stablity of unstable drugs.Dihydroartemisinin（DHA）is the lactol reduction product of artemisinin and found to be effective against acute malaria and chloroquine resistant strains of falciparum malaria.In recent years,DHA has been proven to have potent antitumor activity, well-tolerated and relative non-toxicity in vitro and in vivo.DHA kills tumor cells under the therapeutic dose while common cells partly increased.These findings show that DHA offer great potential and a promising approach in the treatment of cancer, particularly in view of the relative non-toxicity.DHA is a poorly soluble drug in both water and oil and unstable under various conditions.DHA would be serious degraded in gastrointestinal tract.These properties restricts the application of its administration. In this study,DHA was used as a model drug and DHA N/O/W emulsion was prepared for intraveous administration of DHA.Furthermore,DHA N/O/W dry emulsion was prepared to increase the stablity of DHA.The method of high performance liquid chromatography（HPLC）was set up to determine the concentration of DHA.DHA exsist as a mixture of a andβanomers in solution and the ratio of a versusβremained constant after the equilibrium of the andβanomers had been reached.In our study,the ratio of a versusβremained constant around 2.3（α/β）and did not vary over a period of 10 h.The peak area of DHA could be calculated as the sum of a andβanomer to assure the correct results. The solubility of DHA in different solvents were also determined.The apparent solubility of DHA in distilled water and soybean oil was 0.119±0.021 and 1.24±0.01 mg/ml,respectively.The solubility could be enhanced by adding different emulsifiers into systems.The most effective one is Soybean Oil/SPL/Chol.DHA N/O/W emulsion containing DHA,soybean oil,SPL,stabilizer C and cholesterol as oily phase and Poloxamer 188 and glycerol as aqueous phase were prepared by microfluid technology.The DHA N/O/W emulsion were characterized and its droplet size,zeta potential and pH were 152±18 nm（PDI 0.078±0.018）, -33.28±2.07 mV,and 7.18±0.03,respectively.The drug loading capacity of DHA N/O/W emulsion was 2.98±0.03 mg/ml and was nearly 3 times more than the highest concentration of DHA O/W emulsion.Light microscopy,Transmission electron microscopy（TEM）and Freeze-fracture transmission electron microscopy（FF-TEM） were used for the observation of DHA N/O/W emulsion structure and compared with DHA O/W emulsion.There were some particles existed in the oil droplets of N/O/W emulsion while not in O/W emulsion.We also used FF-TEM method to observe the internal structure of DHA N/O/W emulsion and compared with DHA O/W emulsion. Thermal analysis was performed to study the characterization of N/O/W emulsion. Compared with DHA powder,the endothermic peak of DHA N/O/W emulsion and DHA oil nanosuspension were decreased nearly 10 centi-degree,while the endothermic peak of DHA aqueous nanosuspension and the mixture of DHA aqueous nanosuspension and blank N/O/W emulsion were similar to DHA powder’s.These results indicated that DHA nanoparticles were exsited in the oil phase of DHA N/O/W emulsion,which was conformed to the microscopy micrographs of DHA N/O/W emulsion.About 91.94%of DHA located in oil phase of DHA N/O/W emulsion. About 13.83%of DHA dissolved in oil phase and 8.06%dissolved in aqueous phase. Most of DHA distributed in the interface and nanoparticles（～78.11%）.The lyophilized emulsion was developed to improve the stability of DHA N/O/W emulsion.Screening from various cryopotectants,Sucrose-mannitol（4:1）was optimized as a mixture of cryopotectants for DHA N/O/W emulsion.The ratio of cryopotectant and oil phase was 2:1（w/w）.The emulsions were freezed at -40℃for 6 h,primary drying at -40℃for 4 hours,from-40℃to 0℃for 8 hours,and secondary drying at 20℃for 4 hours to allow for a complete solidification.The chamber pressure was maintained at 30-50 mTorr while the condenser temperature remained at -80℃during the drying process.A HPLC method was established to determine DHA and its related substances by optimizing the mobile phase.The samples in destroy tests of acid,alkali,strong light, heat and oxygen were prepared.We found and detected 7 decomposable compounds under different destroy conditions.This method was simple,sensitive and suitable for the determation of the related substances of DHA for its stability study.The stability of DHA,DHA N/O/W emulsion and DHA N/O/W dry emulsion were investigated.The chemical raw medicine of DHA was extremly unstable under strong light and heat condition.It was also not stable in high humidity environment and should be stored in a dry and light-proof enviroment with low temperature.The stability of DHA N/O/W emulsion was better than DHA solution,while it was still sensitive with strong light and heat.It should be stored at 4℃and fullfilled with N₂. The accelerated test of DHA N/O/W dry emulsion at 25℃,60%RH and long stability test at 4℃indicated that DHA N/O/W dry emulsion should be stored in a dry and light-proof enviroment at 4℃.The safety of DHA N/O/W emulsion was evaluted by abnormally toxic test,blood vessel irritation test and hemolysis test.The results indicated that DHA N/O/W emulsion had neither serious intravenous toxicity nor undesirable irritation to veins and no stimulation on blood vessel of rabbits,no hemolysis and agglomeration on vitro body red cells of rabbit.DHA N/O/W emulsion has no hemolysis and stimulative reaction.The pharmaceutical safety of DHA N/O/W emulsion was confirmed.To further understand and reveal the case of DHA N/O/W emulsion in vivo,a HPLC-MS method for determining DHA was developed.This method was simple, sensitive and suitable for the determation of DHA in biological samples.The plasma protein binding rate of DHA was determined with ultraflitration.The plasma protein binding rate of DHA with rabbit,rat and human plasma were 76.36%,66.34%and 63.94%,respectively.The binding rate of DHA with plasma is midding strength.After intraveous administration of DHA N/O/W emulsion,DHA O/W emulsion and DHA solution to rabbit,pharmacokinetics parameters were calculated.It turned out that DHA N/O/W emulsion and O/W emulsion significantly prolonged MRT of DHA.DHA N/O/W emulsion and O/W emulsion could extend the MRT of DHA solution by 10.39 and 3.42 times,when AUC was enhanced by 3.01 and 1.80 times, respectively.The biodistribution test was carried out on ICR mice.DHA N/O/W emulsion enhanced DHA content in plasma,stomach and brain,while decreased the ditribution in heart.Experimental tumor H₂₂mice was used to observe the anti-tumor effect of DHA on tumor growth.The tumor inhibitory rate of DHA N/O/W emulsion（45 mg/kg）group is 51.8%,which was better than other DHA different doses groups（P＜0.01）.DHA N/O/W emulsion（45 mg/kg）can significantly inhibit H₂₂growth.The expression of VEGF and MVD were down regulated.Histopathological examination showed the tumor inhibition of DHA might related to the inhibition of angiogenesis in tumor.In this study,the physicochemical characteristics,stablility,safety test,and antitumor effect of DHA N/O/W emulsion were studied.The results show that this novel formulation has the following advantages:（1）Increasing drug-loaded capability of emulsion for the drugs which solubility are both poorly in oils and in water;（2） N/O/W emulsion was prepared with physiological excipients and achieved intravenous administration;（3）Submicron emulsion as carriers of nanoparticles can restrain nanoparticles aggregate with each other,and this can improve formulation’s physical stability;（4）N/O/W emulsion can be further converted into dry emulsion for unstable drugs;（5）The pharmacokinetics and biodistribution could be changed and possible to be developed as a targeted agent;（6）ease of manufacture.We provide some academic and experimental data for DHA intraveous administration and N/O/W emulsion as carrier of poor solubility drugs.The study of DHA N/O/W emulsion could provided an emergency medicine for patients with severe malaria and a promising approach in the study of cancer treatment.更多还原