盐酸洛拉曲克长循环脂质体的制备及其对肝癌细胞的体外毒性
陈斯泽, 汪森明, 张积仁, 南方医科大学附属珠江医院肿瘤中心
广东省广州市 510282
全军医药卫生科研指令性课题, No. 04T101
通讯作者: 张积仁, 510282, 广东省广州市工业大道253号, 南方医科大学附属珠江医院肿瘤中心. zhangjiren@hotmail.com
电话: 020-61643200 传真: 020-61643200
收稿日期: 2005-04-04 接受日期: 2005-04-09
摘要
, http://www.100md.com
目的: 研制出具有缓释作用的盐酸洛拉曲克长循环脂质体并考察其理化特性以及在体外的抗瘤效应.
方法: 用两亲性聚乙二醇-二硬脂酰磷脂酰乙醇胺(PEG-DSPE)对脂质体膜进行修饰,以薄膜挤压-硫酸铵梯度法制备盐酸洛拉曲克长循环脂质体;用MTT法比较盐酸洛拉曲克长循环脂质体与盐酸洛拉曲克普通脂质体及游离的盐酸洛拉曲克的体外细胞毒性.
结果: 制备的盐酸洛拉曲克隐形脂质体包封率达68%左右,粒径110 nm左右;与普通脂质体及游离的盐酸洛拉曲克相对照,长循环脂质体显示了较好长效的毒性作用,2 h组长循环脂质体的ID50明显高于另外两组,而48 h组的ID50则与另外两组无显著差别.
结论: 新制备的盐酸洛拉曲克长循环脂质体在体外显示了其较好的缓释效应,可以弥补盐酸洛拉曲克在体内半衰期比较短的缺点.
, 百拇医药
陈斯泽, 汪森明, 张积仁. 盐酸洛拉曲克长循环脂质体的制备及其对肝癌细胞的体外毒性. 世界华人消化杂志 2005;13(11):1346-1348
(PDF) 盐酸洛拉曲克长循环脂质体.A: 粒径分布; B: 透射电镜相片(×20K).
3 讨论
为了提高盐酸洛拉曲克的疗效,采用两亲性DSPE-PEG2000制备LCL-AG337.由于DSPE-PEG2000的PEG部分提供了脂质体亲水表面,交错重叠覆盖于脂质体表面,形成致密的“蘑菇云”.这种立体位阻降低了血浆蛋白与细胞表面配体的相互作用,其立体位阻可同时屏蔽RES系统对脂质体的识别,明显延长在血液中驻留时间,有利于肿瘤组织的有效吸收.PEG链的长度和分子质量与脂质体的稳定性关系密切,对于胶态脂质体,分子质量在1-2 ku之间的PEG长循环效果最好[13].
, http://www.100md.com
应用长循环脂质体避免RES细胞摄取,可以靶向到血管外肿瘤.许多实质性肿瘤血液供应比正常组织丰富,血管通透性高,粒径小于100 nm的隐形脂质体能从毛细血管渗漏出来,并集中于肿瘤组织的内部间隙,而正常组织其完整的毛细血管使得大部分脂质体不能渗透[14].但我们获得的LCL-AG337的粒径为110±21 nm,仅小部分可以靶向血管外肿瘤,是否确实还有待进一步的体内试验.我们下一步准备采用亲肿瘤活性小分子作为靶向装置,掺入LCL-AG337,提高其趋肿瘤靶向性,使到达肿瘤的药物增加,提高治愈率.
由表1的结果,可以推测出由于DSPE-PEG2000的加入,LCL-AG337较CL-AG337有较好的稳定性和缓释性.2 h组的LCL-AG337由于稳定性较好,释放较少的药物,故其IC50比较大;而CL-AG337由于稳定性没有LCL-AG337好,释放较多的药物出来,故其IC50比较接近F-AG337.48 h组的LCL- AG337由于经历时间较长,药物基本已释放出来,故三者的IC50较为接近.目前的这些资料仅仅显示了LCL-AG337在体外具有较好的稳定性和缓释性,但尚缺体内的情况,这是我们下一步的工作重点.
, 百拇医药
盐酸洛拉曲克为抗代谢类肿瘤化疗药物,是具有我国独立知识产权的国家一类抗肿瘤新药.目前该药在美国进行Ⅲ期临床试验[15],我国基本与美国同步.我们如能在此药尚未上市之前,研制出能弥补盐酸洛拉曲克本身缺点的长循环脂质体以至靶向长循环脂质体,则此药应用前景未可限量.
4 参考文献
1 Stout TJ, Stroud RM. The complex of the anti-cancer therapeutic,BW1843U89, with thymidylate synthase at 2. 0
A resolution: Implication for a new mode ofinhibition. Structure 1996;4:67-77
, http://www.100md.com
2 Rustum YM, Harstrick A, Cao S, Vanhoefer U, Yin MB, Wilke H, SeeberS. Thymidylate synthase inhibitors in
cancer therapy: direct and indirectinhibitors. J Clin Oncol 1997;15:389-400
3 Schiffer CA, Clifton IJ, Davisson VJ, Santi DV, Stroud RM. Crystalstructure of human thymidylate synthase: a
structural mechanism for guiding substratesinto the active site. Biochemistry 1995;34:16279-16287
, http://www.100md.com
4 Sakoff JA, Howitt IJ, Ackland SP, McCluskey A. Serine/threonineprotein phosphatase inhibition enhances the effect
of thymidylate synthase inhibition. CancerChemother Pharmacol 2004;53:225-232
5 Wells P, Aboagye E, Gunn RN, Osman S, Boddy AV, Taylor GA, Rafi I,Hughes AN, Calvert AH, Price PM, Newell DR.
2-[11C]thymidine positron emissiontomography as an indicator of thymidylate synthase inhibition in patientstreated
, 百拇医药
with AG337. J Natl Cancer Inst 2003;95:675-682
6 Mok TS, Leung TW, Lee SD, Chao Y, Chan AT, Huang A, Lui MC, Yeo W,Chak K, Johnston A, Johnson P. A
multi-centre randomized phase II study ofnolatrexed Versus doxorubicin in treatment of Chinese patients with
advanced hepatocellular carcinoma. CancerChemother Pharmacol 1999;44:307-311
7 Staurt K, Tessitore J, Rudy J, Clendinnen N, Johnston A. A phaseIItrial of nolatrexed dihydrochloride in patients
, 百拇医药
with advanced hepatocellular carcinoma. Cancer 1999;86:410-414
8 陆彬. 药物新剂型与新技术, 北京: 人民卫生出版社, 1998:133-132
9 Moghimi SM, Szebeni J. Stealth liposomes and long circulatingnanoparticles: critical issues in
pharmacokinetics, opsonization andprotein-binding properties. Prog Lipid Res 2003;42:463-478
10 Bakker-Woudenberg IA. Long-circulating sterically stabilizedliposomes as carriers of agents for treatment of infection
, 百拇医药
or for imaging infectious foci. IntJ Antimicrob Agents 2002;19:299-311
11 Kallinteri P, Fatouros D, Klepetsanis P, Antimisiaris SG. Arsenictrioxide liposomes: encapsulation efficiency and in
vitro stability. J Liposome Res 2004;14:27-38
12 Maswadeh H, Demetzos C, Dimas K, Hatziantoniou S, Georgopoulos A,Rallis M, Dallas P, Papaioannou G. Accumulation
of vinblastine into transfersomes andliposomes in response to a transmembrane ammonium sulfate gradient and
, 百拇医药
their cytotoxic/cytostatic activityin vitro. Anticancer Res 2001;21:2577-2583
13 Kenworthy AK, Simon SA, McIntosh TJ. Structure and phase behaviorof lipid suspensions containing phospholipids
with covalently attached poly(ethylene glycol). Biophys J 1995;68:1903-1920
14 Wu NZ, Da D, Rudoll TL, Needham D, Whorton AR, Dewhirst MW. Increased microvascular permeability contributes
, http://www.100md.com
to preferential accumulation ofStealth liposomes in tumor tissue. Cancer Res 1993;53:3765-3770
15 Chu E, Callender MA, FarreII MP, Schmitz JC. Thymidylate synthaseinhibitors as anticancer agents: from bench
to bedside. Cancer ChemotherPharmacol 2003;52(Suppl1):S80-89
编辑 张海宁, 百拇医药( 陈斯泽,汪森明,张积仁)
广东省广州市 510282
全军医药卫生科研指令性课题, No. 04T101
通讯作者: 张积仁, 510282, 广东省广州市工业大道253号, 南方医科大学附属珠江医院肿瘤中心. zhangjiren@hotmail.com
电话: 020-61643200 传真: 020-61643200
收稿日期: 2005-04-04 接受日期: 2005-04-09
摘要
, http://www.100md.com
目的: 研制出具有缓释作用的盐酸洛拉曲克长循环脂质体并考察其理化特性以及在体外的抗瘤效应.
方法: 用两亲性聚乙二醇-二硬脂酰磷脂酰乙醇胺(PEG-DSPE)对脂质体膜进行修饰,以薄膜挤压-硫酸铵梯度法制备盐酸洛拉曲克长循环脂质体;用MTT法比较盐酸洛拉曲克长循环脂质体与盐酸洛拉曲克普通脂质体及游离的盐酸洛拉曲克的体外细胞毒性.
结果: 制备的盐酸洛拉曲克隐形脂质体包封率达68%左右,粒径110 nm左右;与普通脂质体及游离的盐酸洛拉曲克相对照,长循环脂质体显示了较好长效的毒性作用,2 h组长循环脂质体的ID50明显高于另外两组,而48 h组的ID50则与另外两组无显著差别.
结论: 新制备的盐酸洛拉曲克长循环脂质体在体外显示了其较好的缓释效应,可以弥补盐酸洛拉曲克在体内半衰期比较短的缺点.
, 百拇医药
陈斯泽, 汪森明, 张积仁. 盐酸洛拉曲克长循环脂质体的制备及其对肝癌细胞的体外毒性. 世界华人消化杂志 2005;13(11):1346-1348
(PDF) 盐酸洛拉曲克长循环脂质体.A: 粒径分布; B: 透射电镜相片(×20K).
3 讨论
为了提高盐酸洛拉曲克的疗效,采用两亲性DSPE-PEG2000制备LCL-AG337.由于DSPE-PEG2000的PEG部分提供了脂质体亲水表面,交错重叠覆盖于脂质体表面,形成致密的“蘑菇云”.这种立体位阻降低了血浆蛋白与细胞表面配体的相互作用,其立体位阻可同时屏蔽RES系统对脂质体的识别,明显延长在血液中驻留时间,有利于肿瘤组织的有效吸收.PEG链的长度和分子质量与脂质体的稳定性关系密切,对于胶态脂质体,分子质量在1-2 ku之间的PEG长循环效果最好[13].
, http://www.100md.com
应用长循环脂质体避免RES细胞摄取,可以靶向到血管外肿瘤.许多实质性肿瘤血液供应比正常组织丰富,血管通透性高,粒径小于100 nm的隐形脂质体能从毛细血管渗漏出来,并集中于肿瘤组织的内部间隙,而正常组织其完整的毛细血管使得大部分脂质体不能渗透[14].但我们获得的LCL-AG337的粒径为110±21 nm,仅小部分可以靶向血管外肿瘤,是否确实还有待进一步的体内试验.我们下一步准备采用亲肿瘤活性小分子作为靶向装置,掺入LCL-AG337,提高其趋肿瘤靶向性,使到达肿瘤的药物增加,提高治愈率.
由表1的结果,可以推测出由于DSPE-PEG2000的加入,LCL-AG337较CL-AG337有较好的稳定性和缓释性.2 h组的LCL-AG337由于稳定性较好,释放较少的药物,故其IC50比较大;而CL-AG337由于稳定性没有LCL-AG337好,释放较多的药物出来,故其IC50比较接近F-AG337.48 h组的LCL- AG337由于经历时间较长,药物基本已释放出来,故三者的IC50较为接近.目前的这些资料仅仅显示了LCL-AG337在体外具有较好的稳定性和缓释性,但尚缺体内的情况,这是我们下一步的工作重点.
, 百拇医药
盐酸洛拉曲克为抗代谢类肿瘤化疗药物,是具有我国独立知识产权的国家一类抗肿瘤新药.目前该药在美国进行Ⅲ期临床试验[15],我国基本与美国同步.我们如能在此药尚未上市之前,研制出能弥补盐酸洛拉曲克本身缺点的长循环脂质体以至靶向长循环脂质体,则此药应用前景未可限量.
4 参考文献
1 Stout TJ, Stroud RM. The complex of the anti-cancer therapeutic,BW1843U89, with thymidylate synthase at 2. 0
A resolution: Implication for a new mode ofinhibition. Structure 1996;4:67-77
, http://www.100md.com
2 Rustum YM, Harstrick A, Cao S, Vanhoefer U, Yin MB, Wilke H, SeeberS. Thymidylate synthase inhibitors in
cancer therapy: direct and indirectinhibitors. J Clin Oncol 1997;15:389-400
3 Schiffer CA, Clifton IJ, Davisson VJ, Santi DV, Stroud RM. Crystalstructure of human thymidylate synthase: a
structural mechanism for guiding substratesinto the active site. Biochemistry 1995;34:16279-16287
, http://www.100md.com
4 Sakoff JA, Howitt IJ, Ackland SP, McCluskey A. Serine/threonineprotein phosphatase inhibition enhances the effect
of thymidylate synthase inhibition. CancerChemother Pharmacol 2004;53:225-232
5 Wells P, Aboagye E, Gunn RN, Osman S, Boddy AV, Taylor GA, Rafi I,Hughes AN, Calvert AH, Price PM, Newell DR.
2-[11C]thymidine positron emissiontomography as an indicator of thymidylate synthase inhibition in patientstreated
, 百拇医药
with AG337. J Natl Cancer Inst 2003;95:675-682
6 Mok TS, Leung TW, Lee SD, Chao Y, Chan AT, Huang A, Lui MC, Yeo W,Chak K, Johnston A, Johnson P. A
multi-centre randomized phase II study ofnolatrexed Versus doxorubicin in treatment of Chinese patients with
advanced hepatocellular carcinoma. CancerChemother Pharmacol 1999;44:307-311
7 Staurt K, Tessitore J, Rudy J, Clendinnen N, Johnston A. A phaseIItrial of nolatrexed dihydrochloride in patients
, 百拇医药
with advanced hepatocellular carcinoma. Cancer 1999;86:410-414
8 陆彬. 药物新剂型与新技术, 北京: 人民卫生出版社, 1998:133-132
9 Moghimi SM, Szebeni J. Stealth liposomes and long circulatingnanoparticles: critical issues in
pharmacokinetics, opsonization andprotein-binding properties. Prog Lipid Res 2003;42:463-478
10 Bakker-Woudenberg IA. Long-circulating sterically stabilizedliposomes as carriers of agents for treatment of infection
, 百拇医药
or for imaging infectious foci. IntJ Antimicrob Agents 2002;19:299-311
11 Kallinteri P, Fatouros D, Klepetsanis P, Antimisiaris SG. Arsenictrioxide liposomes: encapsulation efficiency and in
vitro stability. J Liposome Res 2004;14:27-38
12 Maswadeh H, Demetzos C, Dimas K, Hatziantoniou S, Georgopoulos A,Rallis M, Dallas P, Papaioannou G. Accumulation
of vinblastine into transfersomes andliposomes in response to a transmembrane ammonium sulfate gradient and
, 百拇医药
their cytotoxic/cytostatic activityin vitro. Anticancer Res 2001;21:2577-2583
13 Kenworthy AK, Simon SA, McIntosh TJ. Structure and phase behaviorof lipid suspensions containing phospholipids
with covalently attached poly(ethylene glycol). Biophys J 1995;68:1903-1920
14 Wu NZ, Da D, Rudoll TL, Needham D, Whorton AR, Dewhirst MW. Increased microvascular permeability contributes
, http://www.100md.com
to preferential accumulation ofStealth liposomes in tumor tissue. Cancer Res 1993;53:3765-3770
15 Chu E, Callender MA, FarreII MP, Schmitz JC. Thymidylate synthaseinhibitors as anticancer agents: from bench
to bedside. Cancer ChemotherPharmacol 2003;52(Suppl1):S80-89
编辑 张海宁, 百拇医药( 陈斯泽,汪森明,张积仁)