足叶乙苷长循环脂质体制备及抑瘤性研究
|
摘要
癌症,是严重威胁人类健康的疑难疾病之一。由于其早期确诊和发现的困难,相当数量的癌症病人必须依赖化学治疗,因此进行抗癌药物的研究非常重要。自从1946年氮芥用于治疗恶性淋巴瘤以来,抗癌药物研究已得到飞速发展,并且日益受到临床上的重视。抗癌药物在肿瘤治疗、延长肿瘤患者生命和提高生活质量方面,具有重要的意义和作用。但是,这些化疗药物缺乏特异性,作用于癌细胞的同时也损伤了正常细胞,毒副作用不可轻视,如骨髓抑制、胃肠道反应等。
脂质体具有良好的生理相容性,可赋予药物良好的靶向性能,改变药物分布规律,提高药物的疗效和治疗指数,是抗肿瘤药物的理想传输载体。然而普通脂质体易于渗漏,血浆中内源性物质易对其造成破坏,存在稳定性差、在血液中半衰期较短等问题,限制了脂质体的开发和应用。近年来的研究发现,将脂质体表面进行亲水基团修饰,可改善脂质体的稳定性,改变脂质体的体内命运。脂质体表面聚乙二醇修饰通常称为长循环脂质体,能够降低巨噬细胞系统(MPS)和血浆中的成分对脂质膜的破坏,明显延长药物在体内的循环时间,增加药物在肿瘤新生血管的渗漏,具有促进药物在肿瘤组织渗透和滞留效应,研究和应用受到越来越多的重视。
足叶乙苷,中药鬼臼有效成分鬼臼毒素的衍生物,通过抑制哺乳类动物DNA拓扑异构酶Ⅱ的活性发挥抗肿瘤效应,具有广谱抗癌活性,是目前临床上应用广泛的一线抗癌药。主要用于治疗小细胞肺癌、恶性淋巴瘤、恶性生殖细胞瘤、白血病,对神经母细胞瘤、横纹肌肉瘤、卵巢癌、非小细胞肺癌、胃癌和食管癌等也有一定疗效。目前,市场上已有注射剂和软胶囊剂型,由于药物存在水溶性差、生物利用度不高,临床应用时毒副作用明显,病人的顺应性较差。
本课题研究设计的长循环脂质体制剂,在脂质体表面进行PEG修饰,形成致密的构象云和厚的立体位阻层,一方面增加脂质体的溶剂化作用,另一方面有效阻止脂质体表面的调理作用,降低MPS对脂质体的亲和力而减少了脂质体在血液循环中破坏,延长血液中的滞留时间。同时,形成的PEG极性长链类似一层保护膜,使脂质体微粒间具有稳定的Zeta电位,脂质体微粒不会融合、沉降,在血浆中长时间保持完整原型,易于通过通透性大的肿瘤新生血管,从而释放药物和实现肿瘤部位的靶向作用。因此,本课题设想通过长循环脂质体载体的包封,增加足叶乙苷药物的稳定性,延长药物在血液中的半衰期,降低毒副作用,提高药物的肿瘤部位靶向性能。
基于以上目的,本文主要进行以下几个方面的研究。
首先进行足叶乙苷处方前研究,包括建立足叶乙苷HPLC含量测定方法、油水分配系数和包封率测定方法。在此基础上,进行足叶乙苷长循环脂质体的处方设计和制备工艺研究,考察了磷脂材料、缓冲液介质、PEG修饰材料的用量、抗氧化剂以及脂质浓度后,确定处方;通过几种常见的脂质体制备方法筛选,以包封率和性状特征指标,最终确定采用乙醇注入法制备脂质体,并通过单因素优选和正交试验,优选最佳工艺条件。结果表明,乙醇注入法避免使用有毒有机溶剂,产品性能稳定,包封率、粒径大小适中并符合正态分布。
对足叶乙苷长循环脂质体的理化性质进行系统考察,主要包括粒径、形态学、Zeta电位、体外释放度、含量测定、包封率测定及稳定性考察等。结果表明,足叶乙苷长循环脂质体外观呈半透明略带乳光的悬液,色泽均匀,流动性好。通过透射电镜观察,脂质体微粒大小均匀,圆整,分散性好。脂质体样品的粒径分布范围较窄,平均粒径112nm,呈单峰分布;用凝胶柱分离法测定包封率,测定结果在80%以上;氧化指数低,化学稳定性考察结果表明氧化程度小;样品在pH7.4磷酸盐缓冲液和大鼠血浆中24h渗漏率分别为(38.27±3.15)和(46.72±2.20)%,说明足叶乙苷长循环脂质体物理稳定性良好;放样稳定性结果表明,足叶乙苷长循环脂质体在4℃条件下放置3月仍能保持稳定;稀释稳定性表明,将其按临床用法稀释,可以保持稳定,没有药物的渗漏和沉淀。
进一步观察足叶乙苷长循环脂质体的长循环特性,采用KM种小鼠进行药物代谢动力学和组织分布试验。在建立血浆和组织中药物测定方法的基础上,按一定的时间间隔目眦静脉采血,处理样品进行HPLC分析。结果表明,足叶乙苷长循环脂质体的t_(1/2,α)、t_(1/2,β)和AUC分别是普通注射液的10、17和3.5倍,普通脂质体的t_(1/2,α)、t_(1/2,β)和AUC分别是普通注射液的4、2.5和1.4倍。说明在相同剂量下,注射剂的分布和消除都比长循环脂质体快,长循环脂质体在体内能够维持较长循环时间。普通注射液的C1为长循环脂质体的3.5倍,长循环脂质体制剂体内消除较慢。组织分布测定结果表明,相对于足叶乙苷注射液和普通脂质体,足叶乙苷长循环脂质体在心脏、肝脏中的药物分布明显减少,在血浆中的分布明显增多。
对足叶乙苷长循环脂质体进行急性毒性试验,并用寇氏法计算LD_(50)值。测定结果表明,足叶乙苷注射液的LD_(50)值为86.6mg/kg,足叶乙苷长循环脂质体的LD_(50)值179.1 mg/kg,是注射液组的2倍多,说明足叶乙苷制成脂质体后,可以显著降低毒副作用,增加用药的安全性。
为预测足叶乙苷长循环脂质体的治疗效果,通过C_(57)BL/6J小鼠接种Lewis肺癌,建立肿瘤模型后给药,以16日的小鼠肿瘤重量为指标,进行抑瘤率考察。结果表明,足叶乙苷长循环脂质体组的抑瘤率为64.15%,明显高于足叶乙苷注射液组的47.92%、普通脂质体组的58.49%。肿瘤组织病理切片的结果显示,足叶乙苷长循环脂质体组的肿瘤组织出现明显的核凝固坏死现象,且肿瘤的肺转移不明显。
综上所述,本课题采用乙醇注入法制备了符合药典要求的、具有一定靶向性能的足叶乙苷长循环脂质体,制备工艺稳定,粒径、包封率、载药量和渗漏率等指标皆符合要求。该制剂不仅可以延长药物在体内的循环时间,并在肿瘤组织浓集,具有一定的抑瘤效果。
To date,cancer is still threatening the quality of human life seriously.Because of the difficulties in early-diagnosis and discovery,a considerable number of cancer patients have to rely on chemical treatment,so reseach on anti-cancer drug is very important.Since 1946,nitrogen mustard is used in the treatment of malignant lymphoma,and then research on anti-cancer drugs has been rapidly developed and improved.As an efficient method of tumor treatment,anti-cancer drugs can extend the lives of cancer patients and improve the quality of life.However,lacking of specificity, several anti-cancer agents are utilized limitedly in clinic because of their severe side effects,and they not only act on the cancer cells but also damage normal cells.Side effects should not be ignored,such as bone marrow suppression,gastrointestinal reactions.
Liposomes is a promising and effective vector in tumor treatment.Liposomal antineoplastic agents are characterized by targeting tumor and controlled-release,and thus they can increase therapeutic index(TI),improve clinical effect and reduce toxicity. However,the general ease of liposome leakage,and plasma-derived material,the existence of poor stability,short half-life in the blood and so on,restricting the development and applications of liposomes.Recent reseach showed that the surface-modified liposomes can improve the stability of liposomes and change the fate of liposomes in vivo.PEG-modified liposome surface is often named as long-circulating liposomes(LCL),which can reduce macro-phagocyte system(MPS) and prevent destruction of plasma membrane lipid composition on the the drug.LCL can significantly prolong the cycle time in the body and increase the release ratio of drug in tumor site,play EPR(enhanced permeability and retention) effect.For the above reasons,research and application of LCL have been paid on attention.
In this paper,we selected etoposide,one of semisynthetic derivatives of the podophyllotoxins from Podophyllum peltatum and Podophyllus emodii Wall.etoposide is clinical drug of small cell lung and ovarian cancer in the first line,by inhibiting DNA topoisomeraseⅡto play the anti-tumor effect of activity,with broad-spectrum anti-tumor activity,which is mainly acted as the treatment of small cell lung cancer, malignant lymphoma,malignant germ cell tumors,leukemia,neuroblastoma, rhabdomyosarcoma,ovarian cancer,non-small cell lung cancer,gastric cancer,esophageal cancer and so on.The injection preparation and oral soft capsule of etoposide had been manufactured,but they have some shortcomings,such as low bioavailability and poor stability,which restrict the application of its use.
To overcome these problems,we design the long-circulating liposomes preparations containing etoposide.PEG modifying in the surface of liposomes,forming a compact conformation of the three-dimensional clouds and thick steric layer,not only enhance the role of the solvent,but prevent the surface of liposome opsonization.In conclusion, we desire that adoption of long-circulating liposome vectors as etoposide,to increase drug stability,extend the drug half-life in the blood,reduce side effects and low toxicity and improve tumor-targeted drug properties.
Around these objectives,this paper carried out the reseach as follows.
Firstly,we research on the pre-prescription of etoposide,such as establishment of etoposide by HPLC analysis method,oil-water distribution coefficient and determination method of encapsulation efficiency.Base on these reseaches,screen on etoposide long-circulating liposome prescription and research process.In this study,we research on the phospholipid material,the buffer medium,PEG-modified material usage,as well as the lipid concentration of antioxidants,and then ensure these parameters.After choosing several common methods of preparation of liposomes,such as film dispersion method,freezing dryness method and ethanol injection method, encapsulation efficiency and trait characteristics as indicators,we finalize the adoption of ethanol injection method as preparation method.Through a single factor and orthogonal design optimization,we achieve the best preparation process.Ethanol injection method is a good process to avoid using of toxic organic solvents,and the products have shown characteristic and good stability,fit encapsulation efficiency, appropriate particle size and be in accordance with the normal distribution.
To observe on etoposide long-circulating liposome's physical and chemical properties systematically,including particle size,morphology,Zeta potential,in vitro release rate,determination of encapsulation efficiency,stability reseach and so on.The appearance of etoposide long-circulating liposomes was slightly translucent opalescence,color uniformity,good mobility.Through the TEM observation,liposomes have uniform particle size,good dispersion,internal space of the water-based nanoparticles.Liposome samples have narrow particle size distribution,with an average diameter of 112nm,showed single-peak distribution.Entrapment efficiency is over 80%.Resarch on chemical stability show that oxidation index is low.Physical stability test results show that percent ratio of leakage for 24h were(38.27±3.15) and(46.72±2.20)%in pH7.4 phosphate buffer and rat plasma,show that etoposide long-circulating liposomes have good physical stability.Reseach on lofting stability results show that etoposide long-circulating liposome situated at 0～4℃place for three months to maintain stability.Dilution tests show that by forms of clinical use dilution,ETP-LCL remain stable,and there is no any drugs leakage and precipitation.
Furthermore,we observe on the pharmacokinetic behavior and distribution of etoposide long-circulating liposomes.Both the two formulations and free etoposide were administered via tail vein at a dose of 10mg/kg.Etoposide was separated from the plasma component by solvent extract.The t_(1/2,α),t_(1/2,β) and the AUC values of etoposide long-circulating liposomes are 10,17 and 3.5 times as ordinary etoposide injection.The t_(1/2,α),t_(1/2,β) and AUC values of conventional liposomes are 4,2.5 and 1.4 folds as etoposide injection in thesame dose.The idistribution and elimination rate of the of long-circulating liposome is slower than the etoposide injection and conventional liposomes.Long-circulating liposome can be maintained in the body and keep longer cycle time,to have more opportunities to reach the target site,target to achieve the role played by EPR effect.Cl value of etoposide injection is 3.5 times that the long-circulating liposome,which show that in vivo elimination of etoposide is very slow.Determination of tissue distribution results showed that compared with etoposide and ordinary injection liposome,etoposide long-circulating liposomes in the liver, spleen drug distribution in a significant decrease in the distribution in the plasma increased.
To carry out acute toxicity tests of etoposide long-circulating liposome,and acquire LD_(50) values by Karber method.Measurement results show that the LD_(50) value of etoposide injection is 86.6mg/kg,LD_(50) value of etoposide long-circulating liposome is 179.1 mg/kg,is 2 times more than the injection group,which shows that etoposide liposomes have the low toxicity and more drug safety.
For predicting tumor inhibition ratio of ETP-LCL,use the C_(57)BL/6J mice inoculated by Lewis lung cancer cell(LCC),after the administration of the establishment of the tumor model,and study the inhibition ratio.The results show that inhibition ratio of etoposide injection group was 47.92%,conventional liposomes group was 58.49%,ETP-LCL group was 64.15%.Biopsy of tumor tissue showed that etoposide long-circulating liposome group shows a visible tumor necrosis and rupture of nuclear solidification phenomenon dissolved.
In summary,using ethanol injection method,we have successfully prepared long-circulating liposome containing etoposide and the method is repeatable with satisfactory results.Physical and chemical stability,particle size,encapsulation efficiency,drug loading and the leakage rate of the products meet the requirements and so on.It was demonstrated that ETP-LCL not only prolonged the resident time of the drug in blood circulation,but also showed a higher anti-neoplastic activity.
脂质体具有良好的生理相容性,可赋予药物良好的靶向性能,改变药物分布规律,提高药物的疗效和治疗指数,是抗肿瘤药物的理想传输载体。然而普通脂质体易于渗漏,血浆中内源性物质易对其造成破坏,存在稳定性差、在血液中半衰期较短等问题,限制了脂质体的开发和应用。近年来的研究发现,将脂质体表面进行亲水基团修饰,可改善脂质体的稳定性,改变脂质体的体内命运。脂质体表面聚乙二醇修饰通常称为长循环脂质体,能够降低巨噬细胞系统(MPS)和血浆中的成分对脂质膜的破坏,明显延长药物在体内的循环时间,增加药物在肿瘤新生血管的渗漏,具有促进药物在肿瘤组织渗透和滞留效应,研究和应用受到越来越多的重视。
足叶乙苷,中药鬼臼有效成分鬼臼毒素的衍生物,通过抑制哺乳类动物DNA拓扑异构酶Ⅱ的活性发挥抗肿瘤效应,具有广谱抗癌活性,是目前临床上应用广泛的一线抗癌药。主要用于治疗小细胞肺癌、恶性淋巴瘤、恶性生殖细胞瘤、白血病,对神经母细胞瘤、横纹肌肉瘤、卵巢癌、非小细胞肺癌、胃癌和食管癌等也有一定疗效。目前,市场上已有注射剂和软胶囊剂型,由于药物存在水溶性差、生物利用度不高,临床应用时毒副作用明显,病人的顺应性较差。
本课题研究设计的长循环脂质体制剂,在脂质体表面进行PEG修饰,形成致密的构象云和厚的立体位阻层,一方面增加脂质体的溶剂化作用,另一方面有效阻止脂质体表面的调理作用,降低MPS对脂质体的亲和力而减少了脂质体在血液循环中破坏,延长血液中的滞留时间。同时,形成的PEG极性长链类似一层保护膜,使脂质体微粒间具有稳定的Zeta电位,脂质体微粒不会融合、沉降,在血浆中长时间保持完整原型,易于通过通透性大的肿瘤新生血管,从而释放药物和实现肿瘤部位的靶向作用。因此,本课题设想通过长循环脂质体载体的包封,增加足叶乙苷药物的稳定性,延长药物在血液中的半衰期,降低毒副作用,提高药物的肿瘤部位靶向性能。
基于以上目的,本文主要进行以下几个方面的研究。
首先进行足叶乙苷处方前研究,包括建立足叶乙苷HPLC含量测定方法、油水分配系数和包封率测定方法。在此基础上,进行足叶乙苷长循环脂质体的处方设计和制备工艺研究,考察了磷脂材料、缓冲液介质、PEG修饰材料的用量、抗氧化剂以及脂质浓度后,确定处方;通过几种常见的脂质体制备方法筛选,以包封率和性状特征指标,最终确定采用乙醇注入法制备脂质体,并通过单因素优选和正交试验,优选最佳工艺条件。结果表明,乙醇注入法避免使用有毒有机溶剂,产品性能稳定,包封率、粒径大小适中并符合正态分布。
对足叶乙苷长循环脂质体的理化性质进行系统考察,主要包括粒径、形态学、Zeta电位、体外释放度、含量测定、包封率测定及稳定性考察等。结果表明,足叶乙苷长循环脂质体外观呈半透明略带乳光的悬液,色泽均匀,流动性好。通过透射电镜观察,脂质体微粒大小均匀,圆整,分散性好。脂质体样品的粒径分布范围较窄,平均粒径112nm,呈单峰分布;用凝胶柱分离法测定包封率,测定结果在80%以上;氧化指数低,化学稳定性考察结果表明氧化程度小;样品在pH7.4磷酸盐缓冲液和大鼠血浆中24h渗漏率分别为(38.27±3.15)和(46.72±2.20)%,说明足叶乙苷长循环脂质体物理稳定性良好;放样稳定性结果表明,足叶乙苷长循环脂质体在4℃条件下放置3月仍能保持稳定;稀释稳定性表明,将其按临床用法稀释,可以保持稳定,没有药物的渗漏和沉淀。
进一步观察足叶乙苷长循环脂质体的长循环特性,采用KM种小鼠进行药物代谢动力学和组织分布试验。在建立血浆和组织中药物测定方法的基础上,按一定的时间间隔目眦静脉采血,处理样品进行HPLC分析。结果表明,足叶乙苷长循环脂质体的t_(1/2,α)、t_(1/2,β)和AUC分别是普通注射液的10、17和3.5倍,普通脂质体的t_(1/2,α)、t_(1/2,β)和AUC分别是普通注射液的4、2.5和1.4倍。说明在相同剂量下,注射剂的分布和消除都比长循环脂质体快,长循环脂质体在体内能够维持较长循环时间。普通注射液的C1为长循环脂质体的3.5倍,长循环脂质体制剂体内消除较慢。组织分布测定结果表明,相对于足叶乙苷注射液和普通脂质体,足叶乙苷长循环脂质体在心脏、肝脏中的药物分布明显减少,在血浆中的分布明显增多。
对足叶乙苷长循环脂质体进行急性毒性试验,并用寇氏法计算LD_(50)值。测定结果表明,足叶乙苷注射液的LD_(50)值为86.6mg/kg,足叶乙苷长循环脂质体的LD_(50)值179.1 mg/kg,是注射液组的2倍多,说明足叶乙苷制成脂质体后,可以显著降低毒副作用,增加用药的安全性。
为预测足叶乙苷长循环脂质体的治疗效果,通过C_(57)BL/6J小鼠接种Lewis肺癌,建立肿瘤模型后给药,以16日的小鼠肿瘤重量为指标,进行抑瘤率考察。结果表明,足叶乙苷长循环脂质体组的抑瘤率为64.15%,明显高于足叶乙苷注射液组的47.92%、普通脂质体组的58.49%。肿瘤组织病理切片的结果显示,足叶乙苷长循环脂质体组的肿瘤组织出现明显的核凝固坏死现象,且肿瘤的肺转移不明显。
综上所述,本课题采用乙醇注入法制备了符合药典要求的、具有一定靶向性能的足叶乙苷长循环脂质体,制备工艺稳定,粒径、包封率、载药量和渗漏率等指标皆符合要求。该制剂不仅可以延长药物在体内的循环时间,并在肿瘤组织浓集,具有一定的抑瘤效果。
To date,cancer is still threatening the quality of human life seriously.Because of the difficulties in early-diagnosis and discovery,a considerable number of cancer patients have to rely on chemical treatment,so reseach on anti-cancer drug is very important.Since 1946,nitrogen mustard is used in the treatment of malignant lymphoma,and then research on anti-cancer drugs has been rapidly developed and improved.As an efficient method of tumor treatment,anti-cancer drugs can extend the lives of cancer patients and improve the quality of life.However,lacking of specificity, several anti-cancer agents are utilized limitedly in clinic because of their severe side effects,and they not only act on the cancer cells but also damage normal cells.Side effects should not be ignored,such as bone marrow suppression,gastrointestinal reactions.
Liposomes is a promising and effective vector in tumor treatment.Liposomal antineoplastic agents are characterized by targeting tumor and controlled-release,and thus they can increase therapeutic index(TI),improve clinical effect and reduce toxicity. However,the general ease of liposome leakage,and plasma-derived material,the existence of poor stability,short half-life in the blood and so on,restricting the development and applications of liposomes.Recent reseach showed that the surface-modified liposomes can improve the stability of liposomes and change the fate of liposomes in vivo.PEG-modified liposome surface is often named as long-circulating liposomes(LCL),which can reduce macro-phagocyte system(MPS) and prevent destruction of plasma membrane lipid composition on the the drug.LCL can significantly prolong the cycle time in the body and increase the release ratio of drug in tumor site,play EPR(enhanced permeability and retention) effect.For the above reasons,research and application of LCL have been paid on attention.
In this paper,we selected etoposide,one of semisynthetic derivatives of the podophyllotoxins from Podophyllum peltatum and Podophyllus emodii Wall.etoposide is clinical drug of small cell lung and ovarian cancer in the first line,by inhibiting DNA topoisomeraseⅡto play the anti-tumor effect of activity,with broad-spectrum anti-tumor activity,which is mainly acted as the treatment of small cell lung cancer, malignant lymphoma,malignant germ cell tumors,leukemia,neuroblastoma, rhabdomyosarcoma,ovarian cancer,non-small cell lung cancer,gastric cancer,esophageal cancer and so on.The injection preparation and oral soft capsule of etoposide had been manufactured,but they have some shortcomings,such as low bioavailability and poor stability,which restrict the application of its use.
To overcome these problems,we design the long-circulating liposomes preparations containing etoposide.PEG modifying in the surface of liposomes,forming a compact conformation of the three-dimensional clouds and thick steric layer,not only enhance the role of the solvent,but prevent the surface of liposome opsonization.In conclusion, we desire that adoption of long-circulating liposome vectors as etoposide,to increase drug stability,extend the drug half-life in the blood,reduce side effects and low toxicity and improve tumor-targeted drug properties.
Around these objectives,this paper carried out the reseach as follows.
Firstly,we research on the pre-prescription of etoposide,such as establishment of etoposide by HPLC analysis method,oil-water distribution coefficient and determination method of encapsulation efficiency.Base on these reseaches,screen on etoposide long-circulating liposome prescription and research process.In this study,we research on the phospholipid material,the buffer medium,PEG-modified material usage,as well as the lipid concentration of antioxidants,and then ensure these parameters.After choosing several common methods of preparation of liposomes,such as film dispersion method,freezing dryness method and ethanol injection method, encapsulation efficiency and trait characteristics as indicators,we finalize the adoption of ethanol injection method as preparation method.Through a single factor and orthogonal design optimization,we achieve the best preparation process.Ethanol injection method is a good process to avoid using of toxic organic solvents,and the products have shown characteristic and good stability,fit encapsulation efficiency, appropriate particle size and be in accordance with the normal distribution.
To observe on etoposide long-circulating liposome's physical and chemical properties systematically,including particle size,morphology,Zeta potential,in vitro release rate,determination of encapsulation efficiency,stability reseach and so on.The appearance of etoposide long-circulating liposomes was slightly translucent opalescence,color uniformity,good mobility.Through the TEM observation,liposomes have uniform particle size,good dispersion,internal space of the water-based nanoparticles.Liposome samples have narrow particle size distribution,with an average diameter of 112nm,showed single-peak distribution.Entrapment efficiency is over 80%.Resarch on chemical stability show that oxidation index is low.Physical stability test results show that percent ratio of leakage for 24h were(38.27±3.15) and(46.72±2.20)%in pH7.4 phosphate buffer and rat plasma,show that etoposide long-circulating liposomes have good physical stability.Reseach on lofting stability results show that etoposide long-circulating liposome situated at 0～4℃place for three months to maintain stability.Dilution tests show that by forms of clinical use dilution,ETP-LCL remain stable,and there is no any drugs leakage and precipitation.
Furthermore,we observe on the pharmacokinetic behavior and distribution of etoposide long-circulating liposomes.Both the two formulations and free etoposide were administered via tail vein at a dose of 10mg/kg.Etoposide was separated from the plasma component by solvent extract.The t_(1/2,α),t_(1/2,β) and the AUC values of etoposide long-circulating liposomes are 10,17 and 3.5 times as ordinary etoposide injection.The t_(1/2,α),t_(1/2,β) and AUC values of conventional liposomes are 4,2.5 and 1.4 folds as etoposide injection in thesame dose.The idistribution and elimination rate of the of long-circulating liposome is slower than the etoposide injection and conventional liposomes.Long-circulating liposome can be maintained in the body and keep longer cycle time,to have more opportunities to reach the target site,target to achieve the role played by EPR effect.Cl value of etoposide injection is 3.5 times that the long-circulating liposome,which show that in vivo elimination of etoposide is very slow.Determination of tissue distribution results showed that compared with etoposide and ordinary injection liposome,etoposide long-circulating liposomes in the liver, spleen drug distribution in a significant decrease in the distribution in the plasma increased.
To carry out acute toxicity tests of etoposide long-circulating liposome,and acquire LD_(50) values by Karber method.Measurement results show that the LD_(50) value of etoposide injection is 86.6mg/kg,LD_(50) value of etoposide long-circulating liposome is 179.1 mg/kg,is 2 times more than the injection group,which shows that etoposide liposomes have the low toxicity and more drug safety.
For predicting tumor inhibition ratio of ETP-LCL,use the C_(57)BL/6J mice inoculated by Lewis lung cancer cell(LCC),after the administration of the establishment of the tumor model,and study the inhibition ratio.The results show that inhibition ratio of etoposide injection group was 47.92%,conventional liposomes group was 58.49%,ETP-LCL group was 64.15%.Biopsy of tumor tissue showed that etoposide long-circulating liposome group shows a visible tumor necrosis and rupture of nuclear solidification phenomenon dissolved.
In summary,using ethanol injection method,we have successfully prepared long-circulating liposome containing etoposide and the method is repeatable with satisfactory results.Physical and chemical stability,particle size,encapsulation efficiency,drug loading and the leakage rate of the products meet the requirements and so on.It was demonstrated that ETP-LCL not only prolonged the resident time of the drug in blood circulation,but also showed a higher anti-neoplastic activity.
引文
1.Bangham A.D.,Standish M.M.,Warkine J.C.Diffusion of univalent ions of swollen phospholipids.J.Mol.Biol.1965,13,238-252
2.Peter W.,Sosaku I.Enzymes inside lipid vesicles:preparation,reactivity and application.Biomolecular Engineering.2001,18:143-177
3.Schatzlein A.,Cevc G.Phospholipids characterization,Metabolism,and Novel Biological Applications.Champaign,Ⅱ:AOCS Press,1995:319-325
4.邓英杰主编.脂质体制备技术基础[M].北京:人民卫生出版社,2007
5.Wagner A,Vorauer-Uhl K,Kreismayr G,Katinger H.The crossflow injection technique-an improvement of the ethanol injection method.Journal of Liposome Research.2002,12(3):259-270
6.Evans J.R.,Fildes F.J.T.,Oliver J.E.Process for preparing freeze-dried liposome compositions.US Pat.4311712,1982
7.Szoka F.,Olson F.,Heath T.et al.Preparation of unilamellar liposomes of intermediate size(0.1～0.2μm) by a combination of reverse phase evaporation and extrusion through polycarbonate membranes.BBA Biomembranes,1980,661:559-571
8.Mayer L.D.,Bally M.D.,Cullis P.R.Uptake of adriamycin into large unilamellar vesicles in response to a pH gradient.Biochim Biophys Acta,1986,857(1):123-126
9.Torchilin V.P.Recent advances with liposomes as pharmaceutical carriers.Nat Rev Drug Discov.2005,4:145-160
10.Patel H.M.,Rayman B.E.Orally administered liposomally entrapped insulin.Biochemistry Society Transactions 1977;5:1739-1741
11.Lasic D.,Papahadjopoulos.Medical Application of Liposomes.Elsevier Press,1998,1-7
12.李富春,邓英杰.国外部分公司脂质体药物研发现状.中国药剂学杂志.2004,2(31:73-78
13.姜殿君,王俊平,赵丽妮.抗癌药物脂质体及其研究进展.中国现代化医药杂志.2007,9(4):152
14.张晶晶,方芸,谈恒山等.中药脂质体的制备及应用.南京中医药大学学报,2006(3):202-204
15.张跃珍,林军.天然药物脂质体制备研究进展.中国药房,2007,18(21):1671-1675
16.黄义昆,张志荣,梁建成等.盐酸川芎嗪脂质体的制备及其在小鼠体内分布的研究.中国药学杂志.2006,41(2):129-131
17.陈卫东,刘晓东.抗肿瘤药紫杉醇脂质体的研究进展.中国药物与临床杂 志.2005,5(10):776-779
18.Woodle M.C.Sterically stabilized liposome therapeutics.Adv.Drug Deliv.Rev.1995,16:249-265
19.Torchilin V,Weissing V.liposomes:a pratical approach.2 nd edn.Oxford University Press.Oxford.2003,3-16
20.Manju R.,Deependra S.,Saraf S.et al.Nanocarriers:Promising Vehicle for Bioactive Drugs.[J].Biol Pharm Bull,2006,29(9):1790-1798
21.Moghimi SM.Hunter AC.Recognition by macrophage and liver cells of opsonized phospholipids vesicles and phospholipids headgroups.Pharm Res.2008,18:1
22.Lian T.,Ho RJY.Trends and developments in liposome drug delivery systems.J.Pharm Sci.2001,90:667
23.Verma A.,misra U.,Mishra N.,et al.Liposomes as carrier systems.Inpharm Communique.2009,2(2):20-24
24.Shoko Yokoyama,Tadahiro Takeda,Tadashi Tsunoda et al.Membrane properties of mixed dipalmitoylphosphatidylglycerol/ganglioside GM3 liposomes in the presence of bovine serum albumin.Biointerfaces,2003,27,(2):141-146
25.Allen,T.M.and Chonn,A.Large unilamellar liposomes with low uptake into reticuloendothe lial system.FEBS Lett.,1987,223,42-46.
26.Russo R.,Nigro L.C.,Minniti S.et al.Visceral leishmaniasis in HIV infected patients:Treatment with high dose liposomal amphotericin B(AmBisome).Journal of Infection.1996,32(2) 133-137
27.Hashim F.A.,Khalil E.A.et al.Apparently successful treatment of two cases of post kala-azar dermal leishmaniasis with liposomal Amphotericin B.Transactions of the Royal Society of Tropical Medicine and Hygiene,1995,89(4):440
28.吕万良,齐宪荣,孙华东,等.阿霉素隐形脂质体的研制及其在小鼠体内的组织分布[J].中国药学杂志,1999,34(5):310-312
29.Yasuyuki S.,Sadao H.Physical properties and tissue distribution of adriamycin encapsulated in polyethyleneglycol-coated liposomes.Advanced Drug Delivery Reviews 1997,24:257-263
30.Conrad R.Lewanski,Simon S.Pegylated liposomal adriamycin:a review of current and future applications,research focus.1999,2(12):473-477
31.Peter G.,John A.Blessing et alo Evaluation of pegylated liposomal doxorubicin (Doxil) as second-line chemotherapy of squamous cell carcinoma of the cervix:A phase Ⅱ study of the Gynecologic Oncology Group.Gynecologic Oncology.2006,102(2):210-213
32.Maeda H.,Fang J.,Inuzuki T.et al.Vascular permeability enhancement in solid tumors:various factors,mechanisms involved and its implications.[J].Int Immumpharmacol,2003,3(3):319
33.Gerish K.,Fang J.,Inuzuki T.et al.Macromolecular therapeutics,advantage and prospects with special emphasis on solid tumor targeting.[J].Clin Pharmocokinet.2003,42(3):1089
34.Huang SK,Lee KD,Hong K.et al.Microscopic localization of sterically stabilized liposomes in colon carcinoma-bearing mice.[J].Cancer Res,1992,52;5135-43
35.Hande KR.Etoposide:four decades of development of a topoisome-rase Ⅱ inhibitor.Eur J Cancer.1998,34:1514-21
36.Smit E.F.,Carney D.N.,Harford P.,Sleijfer D.T.,Postmus P.E.A phase Ⅱ study of oral etoposide in elderly patients with small cell lung cancer.Thorax,1989,44(8):631-633
37.Holthuis,J.J.M.et al,Analytical Profiles of Drug Substances,1989,18:121
38.USPDI.Volume 1.Drug Information for the Health Care Professional.22nd ed.Englewood,Colorado:Micromedex,INc;2002:1426-30.
39.Aisner J,Lee EJ.Etoposide:Current and future status [J].Cancer,1991,67(Suppl):215
40.Repchinsky C,BSP.Compendium of Pharmaceuticals and Specialties;2005.74
41.Ogilvie GK,Cockburn CAJranquilli WJ,et al.Hypotension an PMID-2972235,Hypotension and cutaneous reactions associated with intravenous administration of etoposide in the dog.Am J Vet Res.,1988,49,1367-1370
42.Kim Chi.Personal Communication.Medical Oncologist,GU Tumour Group Designate,BC Cancer Agency 2005;21 April.
43.Sengupta S,Tyagi P,Chandra S,Kochupillai V,Gupta SK.Etoposide encapsulated in positively charged liposomes:pharmacokinetic studies in mice and formulation stability studies.Phamacological Res.,2000,42(5):459-464.
44.Lakkireddy H.R.Rakesh K.S.,Krishna C,et al.Etoposide-incorporated tripalmitin nanoparticles with difference with different surface charge:formulation,characterizeation,radiolabeling,and biodistribution studies.The AAPS Journal,2004,6(3),1-10.
45.姚瑶,陶昱斐,丁燕飞等．依托泊苷明胶微球的制备．中国现代应用药学杂志．2005,22(3),219-221
1.Hauser H.In:Cevc G,editor:Phospholipids handbook.New York:Marcel Dekker,1993:603-607
2.Martin FJ.Clinical pharmacology and antitumour efficacy of DOXIL(pegylated liposomal doxorubicin).In:Medical applications of liposomes.Lasic DD,Papahadjopoulos D,eds.New York:Elsevier Science BV,1998,635-88.
3.Igor V.Zhigaltsev,N.,Quet-Fah A.Liposome-encapsulated vincristine,vinblastine and vinorelbine:A comparative study of drug loading and retention.Journal of Controlled Release,2005,104(1),103-111
4.国家药典委员会编.2005年版《中国药典》,二部.化学工业出版社.204-206
5.张虹,陈立仁,蔡文斌等.肺小细胞癌患者低剂量口服鬼臼乙叉苷软胶囊药物动力学.[J].中国医院药学杂志.1999,19(11):647-48
6.Azin FH.,Bahrami G.,Stewart DJ.et al.A rapid reversed phase high performance liquid chromatographic method for determination of etoposide(VP-16) in human plasma.[J].J Pharm Biomed Anal.2001,25(3):353-6
7.Robieux I,Aita P,Sorio R.et al.Determination of unbound etoposide concentration in ultrafilitered plasma by high-performance liquid chromatography with fluorimetric detection.[J].J Chromatogr B.1996,686:35-41
8.Carneiro A.,Andrade S.Production of liposomes in a multitubular system useful for scaling up of processes.Progress in colloid & polymer science.[J].2004,128,273-277
9.Tatsuya T.,Atsuomi K.,Yong-Zhong D.,Partition coefficients in a Liposome/Water system methods for determination and error analysis.[J].Analytical Science.1992,8:761-765
10.Van Balen GR,Martinet CM.,Caron G.et al.Liposomes/water lipophilicity:methods,information content,and pharmaceutical applications.[J].Med Res Rev,2004,24(3):299-324
11.叶兆伟,承伟.脂质体包封率测定方法及影响因素.中国生物制品学杂志.2007,20(10):789-792
12.王汀,李文秀,邓英杰.微柱离心-药脂比测定脂质体药物包封率.沈阳药科大学学报,2008,25(1):10-13
13.Gunter KK,Gunter TE,Jarkowski A,Rosier RN.A method of resuspending small vesicles separated from suspension by protamine aggregation and centrifugation.Anal Biochem.1982 Feb;120(1):113-24
14.孙维彤,桂华,叶杰胜,等.鱼精蛋白凝聚法测定脂质体和纳米脂质体包封率中国药学杂志,2006,41,22(15):
15.高晓黎,季兴梅.葡聚糖凝胶柱色谱法测定脂质体包封率的条件筛选.中国药学 杂志.2007,38(7):515-517
16.Zuidam NJ.Crommelin DJ.J Pharm Sci.1995,84:1113
17.Asayama K.,Aramak Y.Yoshida T.et al.Liposome Res.1992,2:275
18.Chunlei Li,Yingjie Deng.A novel method for the preparation of liposomes:Freeze drying of monophase solutions.Journal of Pharmaceutical Sciences.[J].2004,93(6):1403-1414
19.Wagner A,Vorauer-Uhl K,Kreismayr G,Katinger H.The crossflow injection technique-an improvement of the ethanol injection method.Journal of Liposome Research.[J].2002,12(3):259-270
20.王健,李明轩.冷冻干燥对提高脂质体稳定性的研究概况.中国医药工业杂志.[J].2005,36(9):576-581
21.Koynova R.,Caffrey M.Phases and phase transitions of the phosphatidylcholines.Biochim Biophys Acta.[J].1998,1376(1):91-145
22.秦晶;陈大为;胡海洋等.醋酸钙梯度法制备阿魏酸脂质体的研究及体外评价.中国药学杂志,2008,43(1):40-44
23.靳浩,吴诚,梅兴国.多指标综合评价法优选阿霉素微球的制备工艺及体内的初步考察.中国药学杂志,2006,41(22):1723-25
24.Lu(?)sa C.,Otto C.,Wim J.G.et al.Oyen Intravenous administration of superoxide dismutase entrapped in long circulating liposomes:Ⅱ.In vivo fate in a rat model of adjuvant arthritis.[J].Biochimica et Biophysica Acta(BBA)-Biomembranes.,1999,1419(2),325-334
25.赵蓉,朱家壁,李颖寰等.全反式维甲酸立体稳定脂质体家兔体内药动学研究[J].中国药学杂志,2001,36(10):680-682
26.李唐棣,郝丽梅,梅兴国.脂质体包封率的研究进展.国外医学(药学分册),2006,03.224-28
27.于波涛,张志荣,刘文胜.提高脂质体包封率的方法及其研究进展.中国医药工业杂志.2002,(11):564-568
28.中国药典编纂委员会.中华人民共和国药典2005版.化学工业出版社.附录ⅦE:204-206
1.Barbosa C.,Stebbing D.,Cheung E.,et al.Determination of detailed liposome particle size distributions[J].AAPS PharmSci Tech.2002,3(2):1-7
2.Zheng-hong WU,Qi-neng PING,Yi WEI,Jia-ming LAI.Hypoglycemic efficacy of chitosan-coated insulin liposomes after oral administration in mice[J].Acta Pharmacol Sin.2004,25(7):966-972
3.Heurtault B.,Saulnier P.,Pech B.et al.Physico-chemical stability of colloidal lipid particles.[J].Biomaticrials,2003,24:4283-4300
4.Allen T M.Long-circulating(sterically stabilized) liposomes for targeted drug delivery.[J].Trends Pharm Sci,1994,15:215-220
5.赵海英,王思玲,苏德森.促肝细胞生长素冻干脂质体的制备与物理化学性质研究.[JJ.沈阳药科大学学报,2001,18(6):391-394
6.Bac S.J.,Kim N.H.A study of stabilization effect of α-tocopherol incorporated into liposomal phospholipid membrane.[J].Archives of Pharmacal Research.1990,13(1):64-68
7.Chris L.,Joellen B.,Peter D.Liposomes enriched in oleic acid are less susceptible to oxidation and have less proinflammatory activity when exposed to oxidizing conditions.[J].The Journal of Lipid Research,1998,39(6),1239-1247
8.Awashi V.D.Garcia D.Goins B.et al.Circulation and biodistribution profiles of long-circulating PEG-liposomes of various sizes in rabbits.International journal of pharmaceutics.2003,253(1):121-132
9.国家药典委员会编.2005年版中国药典,二部.化学工业出版社.附录ⅦE 204-206
1.Martin FJ.Clinical pharmacology and antitumour efficacy of DOXIL(pegylated liposomal doxorubicin).In:Medical applications of liposomes.Lasic DD,Papahadjopoulos D,eds.New York:Elsevier Science BV,1998,635-88.
2.赵国琴,许健健,张海燕.HPLC法测定皮下植入依托泊苷的血浆浓度.中国新药杂志.2003,12(3):197-199
3.熊雯,蒋心惠,张丹.盐酸阿霉素及其脂质体在大鼠体内的相关药动学参数比较.华西药学杂志,2004,19(2):97-99
4.黄义昆,张志荣,梁建成等.盐酸川芎嗪脂质体的制备及其在小鼠体内分布的研究.中国药学杂志.2006,41(2):129-131
5.王健松,朱家壁,吕瑞勤等.肺靶向阿奇霉素脂质体的制备及其在小鼠体内的分布.药学学报,2005,40(3):274-278
6.解建勋,李玉珍,王佩.高效液相色谱法测定人血清中鬼臼乙叉苷浓度[J].色谱,1998,16(1):74-77
7.Azin FH,Bahrami G,Stewart DJ,et al.A rapid reversed phase high performance liquid chromatographic method for determination of etoposide(VP-16) in human plasma[J].J Pharm Biomed Anal,2001,25(3):353-356
8.Robieux I,Aita P,Sorio R,et al.Determination of unbound etopside concentration in ultrafiltered plasma by high performance liquid chromatography with fluorimetric detection[J].J Chromatogr B,1996,686:35-41.
9.李安良,吴艳芬主译.Leon Shargel,Susanna Wu-Pong,Andrew B.C.Yu.Applied Biopharmaceutics & Pharmacokinetics.化学工业出版社.2006.4-6
10.Crosasso,P.,Ceruti,M.,Brusa,P.,Arpicco,S.,Cattel,L.Preparation,characterization and properties of sterically stabilized paclitaxel-containing,liposomes.J.Control.Release 2000,63(1),19-30.
11.邓意辉,徐晖主译.V.P.Torchilin.V.Weissig Lipososomes:A Practical Approach.化学工业出版社.2007.252-258
1.陈奇.中药药理研究方法学.第一版[M].北京:人民卫生出版社.1993:113-115.
2.魏永燕,沈秀,刘培勋.中药仙鹤复方提取物急性毒性试验及对小鼠移植性肿瘤H_(22)的抗肿瘤作用研究[J].海峡药学,2007,19(2):27-29.
3.Material Safety Data Sheet.Toposar~(?) Injection.http://www.newsicor.com.2005,12:P5
4.王治乔,袁伯峻.新药临床前安全性评价与实践[M].北京:军事医学科学出版社,1997:23
1.Qing Zhang,Keiko Furukawa,Ho-Hsiang Chen et al.Metastatic Potential of Mouse Lewis Lung Cancer Cells Is Regulated via Ganglioside GM1 by Modulating the Matrix Metalloprotease-9 Localization in Lipid Rafts.[J].J.Biol.Chem.2006,281(26),18145-55
2.李春艳,王颖,刘浩等.C57BL/6近交系小鼠Lewis肺癌动物模型:建设性描述肺癌损害程度的指标.中国临床康复.2003,7(26):3582-83
3.Tsuchida K.Aoyagi Y.Odani S.Isolation of a novel collagen-binding protein from the mushroom,Hypsizigus marmoreus,which inhibits the lewis lung carcinoma cell adhesion to type Ⅳ collagen,270(4):1481-84
4.蔡卫斌,程锐,徐祖敏.人纤溶酶原K_5体内抑制Lewis肺癌肿瘤生长与转移.中山大学学报(医学科学版).2008,29(5),380-83
5.Maca RD.Enhancement of etoposide and methotrexate sensitivity by indomethacin in vitro.[J].Anticancer Drug Des.1991,6(5):453-66
6.Maeda H,Fang J,Inuzuki T,et al.Vascular permeability enhancement in solid tumors:various factors,mechanisms involved and its implications.[J].Int Immunpharmacol,2003,3(3):319
7.Greish K,Fang J,Inutsuka T,et al.Macromolecular therapeutics,advantage and prospects with special emphasis on solid tumor targeting.[J].Clin Pharmacokinet.,2003,42(13):1089
2.Peter W.,Sosaku I.Enzymes inside lipid vesicles:preparation,reactivity and application.Biomolecular Engineering.2001,18:143-177
3.Schatzlein A.,Cevc G.Phospholipids characterization,Metabolism,and Novel Biological Applications.Champaign,Ⅱ:AOCS Press,1995:319-325
4.邓英杰主编.脂质体制备技术基础[M].北京:人民卫生出版社,2007
5.Wagner A,Vorauer-Uhl K,Kreismayr G,Katinger H.The crossflow injection technique-an improvement of the ethanol injection method.Journal of Liposome Research.2002,12(3):259-270
6.Evans J.R.,Fildes F.J.T.,Oliver J.E.Process for preparing freeze-dried liposome compositions.US Pat.4311712,1982
7.Szoka F.,Olson F.,Heath T.et al.Preparation of unilamellar liposomes of intermediate size(0.1～0.2μm) by a combination of reverse phase evaporation and extrusion through polycarbonate membranes.BBA Biomembranes,1980,661:559-571
8.Mayer L.D.,Bally M.D.,Cullis P.R.Uptake of adriamycin into large unilamellar vesicles in response to a pH gradient.Biochim Biophys Acta,1986,857(1):123-126
9.Torchilin V.P.Recent advances with liposomes as pharmaceutical carriers.Nat Rev Drug Discov.2005,4:145-160
10.Patel H.M.,Rayman B.E.Orally administered liposomally entrapped insulin.Biochemistry Society Transactions 1977;5:1739-1741
11.Lasic D.,Papahadjopoulos.Medical Application of Liposomes.Elsevier Press,1998,1-7
12.李富春,邓英杰.国外部分公司脂质体药物研发现状.中国药剂学杂志.2004,2(31:73-78
13.姜殿君,王俊平,赵丽妮.抗癌药物脂质体及其研究进展.中国现代化医药杂志.2007,9(4):152
14.张晶晶,方芸,谈恒山等.中药脂质体的制备及应用.南京中医药大学学报,2006(3):202-204
15.张跃珍,林军.天然药物脂质体制备研究进展.中国药房,2007,18(21):1671-1675
16.黄义昆,张志荣,梁建成等.盐酸川芎嗪脂质体的制备及其在小鼠体内分布的研究.中国药学杂志.2006,41(2):129-131
17.陈卫东,刘晓东.抗肿瘤药紫杉醇脂质体的研究进展.中国药物与临床杂 志.2005,5(10):776-779
18.Woodle M.C.Sterically stabilized liposome therapeutics.Adv.Drug Deliv.Rev.1995,16:249-265
19.Torchilin V,Weissing V.liposomes:a pratical approach.2 nd edn.Oxford University Press.Oxford.2003,3-16
20.Manju R.,Deependra S.,Saraf S.et al.Nanocarriers:Promising Vehicle for Bioactive Drugs.[J].Biol Pharm Bull,2006,29(9):1790-1798
21.Moghimi SM.Hunter AC.Recognition by macrophage and liver cells of opsonized phospholipids vesicles and phospholipids headgroups.Pharm Res.2008,18:1
22.Lian T.,Ho RJY.Trends and developments in liposome drug delivery systems.J.Pharm Sci.2001,90:667
23.Verma A.,misra U.,Mishra N.,et al.Liposomes as carrier systems.Inpharm Communique.2009,2(2):20-24
24.Shoko Yokoyama,Tadahiro Takeda,Tadashi Tsunoda et al.Membrane properties of mixed dipalmitoylphosphatidylglycerol/ganglioside GM3 liposomes in the presence of bovine serum albumin.Biointerfaces,2003,27,(2):141-146
25.Allen,T.M.and Chonn,A.Large unilamellar liposomes with low uptake into reticuloendothe lial system.FEBS Lett.,1987,223,42-46.
26.Russo R.,Nigro L.C.,Minniti S.et al.Visceral leishmaniasis in HIV infected patients:Treatment with high dose liposomal amphotericin B(AmBisome).Journal of Infection.1996,32(2) 133-137
27.Hashim F.A.,Khalil E.A.et al.Apparently successful treatment of two cases of post kala-azar dermal leishmaniasis with liposomal Amphotericin B.Transactions of the Royal Society of Tropical Medicine and Hygiene,1995,89(4):440
28.吕万良,齐宪荣,孙华东,等.阿霉素隐形脂质体的研制及其在小鼠体内的组织分布[J].中国药学杂志,1999,34(5):310-312
29.Yasuyuki S.,Sadao H.Physical properties and tissue distribution of adriamycin encapsulated in polyethyleneglycol-coated liposomes.Advanced Drug Delivery Reviews 1997,24:257-263
30.Conrad R.Lewanski,Simon S.Pegylated liposomal adriamycin:a review of current and future applications,research focus.1999,2(12):473-477
31.Peter G.,John A.Blessing et alo Evaluation of pegylated liposomal doxorubicin (Doxil) as second-line chemotherapy of squamous cell carcinoma of the cervix:A phase Ⅱ study of the Gynecologic Oncology Group.Gynecologic Oncology.2006,102(2):210-213
32.Maeda H.,Fang J.,Inuzuki T.et al.Vascular permeability enhancement in solid tumors:various factors,mechanisms involved and its implications.[J].Int Immumpharmacol,2003,3(3):319
33.Gerish K.,Fang J.,Inuzuki T.et al.Macromolecular therapeutics,advantage and prospects with special emphasis on solid tumor targeting.[J].Clin Pharmocokinet.2003,42(3):1089
34.Huang SK,Lee KD,Hong K.et al.Microscopic localization of sterically stabilized liposomes in colon carcinoma-bearing mice.[J].Cancer Res,1992,52;5135-43
35.Hande KR.Etoposide:four decades of development of a topoisome-rase Ⅱ inhibitor.Eur J Cancer.1998,34:1514-21
36.Smit E.F.,Carney D.N.,Harford P.,Sleijfer D.T.,Postmus P.E.A phase Ⅱ study of oral etoposide in elderly patients with small cell lung cancer.Thorax,1989,44(8):631-633
37.Holthuis,J.J.M.et al,Analytical Profiles of Drug Substances,1989,18:121
38.USPDI.Volume 1.Drug Information for the Health Care Professional.22nd ed.Englewood,Colorado:Micromedex,INc;2002:1426-30.
39.Aisner J,Lee EJ.Etoposide:Current and future status [J].Cancer,1991,67(Suppl):215
40.Repchinsky C,BSP.Compendium of Pharmaceuticals and Specialties;2005.74
41.Ogilvie GK,Cockburn CAJranquilli WJ,et al.Hypotension an PMID-2972235,Hypotension and cutaneous reactions associated with intravenous administration of etoposide in the dog.Am J Vet Res.,1988,49,1367-1370
42.Kim Chi.Personal Communication.Medical Oncologist,GU Tumour Group Designate,BC Cancer Agency 2005;21 April.
43.Sengupta S,Tyagi P,Chandra S,Kochupillai V,Gupta SK.Etoposide encapsulated in positively charged liposomes:pharmacokinetic studies in mice and formulation stability studies.Phamacological Res.,2000,42(5):459-464.
44.Lakkireddy H.R.Rakesh K.S.,Krishna C,et al.Etoposide-incorporated tripalmitin nanoparticles with difference with different surface charge:formulation,characterizeation,radiolabeling,and biodistribution studies.The AAPS Journal,2004,6(3),1-10.
45.姚瑶,陶昱斐,丁燕飞等．依托泊苷明胶微球的制备．中国现代应用药学杂志．2005,22(3),219-221
1.Hauser H.In:Cevc G,editor:Phospholipids handbook.New York:Marcel Dekker,1993:603-607
2.Martin FJ.Clinical pharmacology and antitumour efficacy of DOXIL(pegylated liposomal doxorubicin).In:Medical applications of liposomes.Lasic DD,Papahadjopoulos D,eds.New York:Elsevier Science BV,1998,635-88.
3.Igor V.Zhigaltsev,N.,Quet-Fah A.Liposome-encapsulated vincristine,vinblastine and vinorelbine:A comparative study of drug loading and retention.Journal of Controlled Release,2005,104(1),103-111
4.国家药典委员会编.2005年版《中国药典》,二部.化学工业出版社.204-206
5.张虹,陈立仁,蔡文斌等.肺小细胞癌患者低剂量口服鬼臼乙叉苷软胶囊药物动力学.[J].中国医院药学杂志.1999,19(11):647-48
6.Azin FH.,Bahrami G.,Stewart DJ.et al.A rapid reversed phase high performance liquid chromatographic method for determination of etoposide(VP-16) in human plasma.[J].J Pharm Biomed Anal.2001,25(3):353-6
7.Robieux I,Aita P,Sorio R.et al.Determination of unbound etoposide concentration in ultrafilitered plasma by high-performance liquid chromatography with fluorimetric detection.[J].J Chromatogr B.1996,686:35-41
8.Carneiro A.,Andrade S.Production of liposomes in a multitubular system useful for scaling up of processes.Progress in colloid & polymer science.[J].2004,128,273-277
9.Tatsuya T.,Atsuomi K.,Yong-Zhong D.,Partition coefficients in a Liposome/Water system methods for determination and error analysis.[J].Analytical Science.1992,8:761-765
10.Van Balen GR,Martinet CM.,Caron G.et al.Liposomes/water lipophilicity:methods,information content,and pharmaceutical applications.[J].Med Res Rev,2004,24(3):299-324
11.叶兆伟,承伟.脂质体包封率测定方法及影响因素.中国生物制品学杂志.2007,20(10):789-792
12.王汀,李文秀,邓英杰.微柱离心-药脂比测定脂质体药物包封率.沈阳药科大学学报,2008,25(1):10-13
13.Gunter KK,Gunter TE,Jarkowski A,Rosier RN.A method of resuspending small vesicles separated from suspension by protamine aggregation and centrifugation.Anal Biochem.1982 Feb;120(1):113-24
14.孙维彤,桂华,叶杰胜,等.鱼精蛋白凝聚法测定脂质体和纳米脂质体包封率中国药学杂志,2006,41,22(15):
15.高晓黎,季兴梅.葡聚糖凝胶柱色谱法测定脂质体包封率的条件筛选.中国药学 杂志.2007,38(7):515-517
16.Zuidam NJ.Crommelin DJ.J Pharm Sci.1995,84:1113
17.Asayama K.,Aramak Y.Yoshida T.et al.Liposome Res.1992,2:275
18.Chunlei Li,Yingjie Deng.A novel method for the preparation of liposomes:Freeze drying of monophase solutions.Journal of Pharmaceutical Sciences.[J].2004,93(6):1403-1414
19.Wagner A,Vorauer-Uhl K,Kreismayr G,Katinger H.The crossflow injection technique-an improvement of the ethanol injection method.Journal of Liposome Research.[J].2002,12(3):259-270
20.王健,李明轩.冷冻干燥对提高脂质体稳定性的研究概况.中国医药工业杂志.[J].2005,36(9):576-581
21.Koynova R.,Caffrey M.Phases and phase transitions of the phosphatidylcholines.Biochim Biophys Acta.[J].1998,1376(1):91-145
22.秦晶;陈大为;胡海洋等.醋酸钙梯度法制备阿魏酸脂质体的研究及体外评价.中国药学杂志,2008,43(1):40-44
23.靳浩,吴诚,梅兴国.多指标综合评价法优选阿霉素微球的制备工艺及体内的初步考察.中国药学杂志,2006,41(22):1723-25
24.Lu(?)sa C.,Otto C.,Wim J.G.et al.Oyen Intravenous administration of superoxide dismutase entrapped in long circulating liposomes:Ⅱ.In vivo fate in a rat model of adjuvant arthritis.[J].Biochimica et Biophysica Acta(BBA)-Biomembranes.,1999,1419(2),325-334
25.赵蓉,朱家壁,李颖寰等.全反式维甲酸立体稳定脂质体家兔体内药动学研究[J].中国药学杂志,2001,36(10):680-682
26.李唐棣,郝丽梅,梅兴国.脂质体包封率的研究进展.国外医学(药学分册),2006,03.224-28
27.于波涛,张志荣,刘文胜.提高脂质体包封率的方法及其研究进展.中国医药工业杂志.2002,(11):564-568
28.中国药典编纂委员会.中华人民共和国药典2005版.化学工业出版社.附录ⅦE:204-206
1.Barbosa C.,Stebbing D.,Cheung E.,et al.Determination of detailed liposome particle size distributions[J].AAPS PharmSci Tech.2002,3(2):1-7
2.Zheng-hong WU,Qi-neng PING,Yi WEI,Jia-ming LAI.Hypoglycemic efficacy of chitosan-coated insulin liposomes after oral administration in mice[J].Acta Pharmacol Sin.2004,25(7):966-972
3.Heurtault B.,Saulnier P.,Pech B.et al.Physico-chemical stability of colloidal lipid particles.[J].Biomaticrials,2003,24:4283-4300
4.Allen T M.Long-circulating(sterically stabilized) liposomes for targeted drug delivery.[J].Trends Pharm Sci,1994,15:215-220
5.赵海英,王思玲,苏德森.促肝细胞生长素冻干脂质体的制备与物理化学性质研究.[JJ.沈阳药科大学学报,2001,18(6):391-394
6.Bac S.J.,Kim N.H.A study of stabilization effect of α-tocopherol incorporated into liposomal phospholipid membrane.[J].Archives of Pharmacal Research.1990,13(1):64-68
7.Chris L.,Joellen B.,Peter D.Liposomes enriched in oleic acid are less susceptible to oxidation and have less proinflammatory activity when exposed to oxidizing conditions.[J].The Journal of Lipid Research,1998,39(6),1239-1247
8.Awashi V.D.Garcia D.Goins B.et al.Circulation and biodistribution profiles of long-circulating PEG-liposomes of various sizes in rabbits.International journal of pharmaceutics.2003,253(1):121-132
9.国家药典委员会编.2005年版中国药典,二部.化学工业出版社.附录ⅦE 204-206
1.Martin FJ.Clinical pharmacology and antitumour efficacy of DOXIL(pegylated liposomal doxorubicin).In:Medical applications of liposomes.Lasic DD,Papahadjopoulos D,eds.New York:Elsevier Science BV,1998,635-88.
2.赵国琴,许健健,张海燕.HPLC法测定皮下植入依托泊苷的血浆浓度.中国新药杂志.2003,12(3):197-199
3.熊雯,蒋心惠,张丹.盐酸阿霉素及其脂质体在大鼠体内的相关药动学参数比较.华西药学杂志,2004,19(2):97-99
4.黄义昆,张志荣,梁建成等.盐酸川芎嗪脂质体的制备及其在小鼠体内分布的研究.中国药学杂志.2006,41(2):129-131
5.王健松,朱家壁,吕瑞勤等.肺靶向阿奇霉素脂质体的制备及其在小鼠体内的分布.药学学报,2005,40(3):274-278
6.解建勋,李玉珍,王佩.高效液相色谱法测定人血清中鬼臼乙叉苷浓度[J].色谱,1998,16(1):74-77
7.Azin FH,Bahrami G,Stewart DJ,et al.A rapid reversed phase high performance liquid chromatographic method for determination of etoposide(VP-16) in human plasma[J].J Pharm Biomed Anal,2001,25(3):353-356
8.Robieux I,Aita P,Sorio R,et al.Determination of unbound etopside concentration in ultrafiltered plasma by high performance liquid chromatography with fluorimetric detection[J].J Chromatogr B,1996,686:35-41.
9.李安良,吴艳芬主译.Leon Shargel,Susanna Wu-Pong,Andrew B.C.Yu.Applied Biopharmaceutics & Pharmacokinetics.化学工业出版社.2006.4-6
10.Crosasso,P.,Ceruti,M.,Brusa,P.,Arpicco,S.,Cattel,L.Preparation,characterization and properties of sterically stabilized paclitaxel-containing,liposomes.J.Control.Release 2000,63(1),19-30.
11.邓意辉,徐晖主译.V.P.Torchilin.V.Weissig Lipososomes:A Practical Approach.化学工业出版社.2007.252-258
1.陈奇.中药药理研究方法学.第一版[M].北京:人民卫生出版社.1993:113-115.
2.魏永燕,沈秀,刘培勋.中药仙鹤复方提取物急性毒性试验及对小鼠移植性肿瘤H_(22)的抗肿瘤作用研究[J].海峡药学,2007,19(2):27-29.
3.Material Safety Data Sheet.Toposar~(?) Injection.http://www.newsicor.com.2005,12:P5
4.王治乔,袁伯峻.新药临床前安全性评价与实践[M].北京:军事医学科学出版社,1997:23
1.Qing Zhang,Keiko Furukawa,Ho-Hsiang Chen et al.Metastatic Potential of Mouse Lewis Lung Cancer Cells Is Regulated via Ganglioside GM1 by Modulating the Matrix Metalloprotease-9 Localization in Lipid Rafts.[J].J.Biol.Chem.2006,281(26),18145-55
2.李春艳,王颖,刘浩等.C57BL/6近交系小鼠Lewis肺癌动物模型:建设性描述肺癌损害程度的指标.中国临床康复.2003,7(26):3582-83
3.Tsuchida K.Aoyagi Y.Odani S.Isolation of a novel collagen-binding protein from the mushroom,Hypsizigus marmoreus,which inhibits the lewis lung carcinoma cell adhesion to type Ⅳ collagen,270(4):1481-84
4.蔡卫斌,程锐,徐祖敏.人纤溶酶原K_5体内抑制Lewis肺癌肿瘤生长与转移.中山大学学报(医学科学版).2008,29(5),380-83
5.Maca RD.Enhancement of etoposide and methotrexate sensitivity by indomethacin in vitro.[J].Anticancer Drug Des.1991,6(5):453-66
6.Maeda H,Fang J,Inuzuki T,et al.Vascular permeability enhancement in solid tumors:various factors,mechanisms involved and its implications.[J].Int Immunpharmacol,2003,3(3):319
7.Greish K,Fang J,Inutsuka T,et al.Macromolecular therapeutics,advantage and prospects with special emphasis on solid tumor targeting.[J].Clin Pharmacokinet.,2003,42(13):1089