鬼臼毒素二棕榈酰磷脂酰胆碱（DPPC）前体脂质体的研制
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摘要
目的：
尖锐湿疣(CA)是一种性传播疾病，传染性强，发病率高，是欧美国家最常见的性病之一，也是我国最主要的性病之一，其发病率逐年增加，其年增长率超过100％，居各类性病之首。尖锐湿疣的病因是人类乳头瘤病毒(HPV)感染，它感染人体后寄居于基底细胞等角质形成细胞的核内，部分位于核外，而位于核内的HPV可长期潜伏存在，是CA复发的根源所在。鬼臼毒素(PPT)，是1990年世界卫生组织推荐为治疗生殖器疣的一线药物。鬼臼毒素外用时通过抑制受人乳头瘤病毒(HPV)感染细胞的分裂增殖过程，使之坏死脱落从而起到治疗尖锐湿疣的作用。鬼臼毒素是目前临床治疗尖锐湿疣最有效的一线药物，常用的是0.5％的鬼臼毒素酊剂。但它只能用于肉眼可见的皮损，大面积地使用会产生严重的毒副作用，因此只能局部小量地应用。脂质体是最近几年研究较多的新型控缓释药物载体，将PPT制成脂质体制剂，会在一定程度上改善PPT的药代动力学和药效学，对尖锐湿疣的抗复发治疗有更好的疗效。相关研究显示二棕榈酰磷脂酰胆碱(DPPC)脂质体在皮肤外用药物的脂质体制剂中效果最好。但普通脂质体为液体制剂，易发生粒子聚集沉降、磷脂氧化分解、包封药物渗漏等问题，导致脂质体不够稳定。而前体脂质体(PL)是一种固体制剂，是指脂质体和冻干保护剂(亦称赋形剂)的脱水形式，它加水后能重建形成脂质体，解决了脂质体以液体制剂贮存的氧化、水解、聚集、分层、药物的渗漏及高温灭菌等问题，即克服了脂质体以溶液形式长期储存的稳定性问题，这是脂质体走向工业化生产和商品化的关键。经查阅相关文献资料，结合前期筛选出的合适配方，我们制备了鬼臼毒素二棕榈酰磷脂酰胆碱前体脂质体(PPT-DPPC-PL)，并进行了动物实验和初步临床观察。方法：
1、以逆相蒸发法(reverse-phase evaporation vesicles，REV)
制备鬼臼毒素DPPC脂质体(PPT一DPPC一L)悬液(普通型)，采用
冷冻干燥法，选择海藻糖做冻干保护剂制备PPT一DPPc一PL，并考
察P盯一DPPc一PL水合后的形态、粒径分布、包封率。分别观察在
4种不同强度的物理外力(手摇、摇床、超声、超声针)作用下，
PPT一DPPC一PL水合后的稳定性;分别将密封于安瓶中的
P盯一DPPC一PL贮存在4℃、20℃、40℃条件下，贮存1、3、6个月
后取样，经水合后分别考察其脂质体形态、粒径及包封率有无变
化。
2、选择10只母乳猪，分为2组，分别涂以相同浓度的
P盯一DPPC一L和水合后的P叮一DPPC一PL即重建型鬼臼毒素DPPC
脂质体(R一PPT一DPPC一L)，在不同的时间段抽血测定血中鬼臼毒素
的含量。
3、选择自愿受试的复发性尖锐湿疵患者进行PPT一DPPC一PL
临床疗效的初步观察。
结果:
1、制备的PPT一DPPc一PL的形态为疏松、洁白的粉末，水合后
的形态与普通型PPr一DPPC一L相同。经测定PPT一DPPC一PL的包封
率为723%，普通型PPT-DPPC一L的包封率为73.1%，二者无明显
差异，即冷冻干燥处理前后PPT一DPPC一L的包封率几乎不变。四种
不同强度的物理外力作用后测得的粒径大小无显著性差异，证明
P盯一DPPC一PL对外力稳定性良好，可在各种常见外力作用下水合
后使用。经反复测定，在不同的温度、时间等条件下PPT一DPPC一PL
的粒径分布、包封率均未发生明显变化。
2、动物体内血液中鬼臼毒素浓度的比较结果提示，二种
PPT一DPPC一L经局部外用后动物体内血药浓度无显著统计学差异，
即机体对二种脂质体制剂中的鬼臼毒素吸收程度接近。二种脂质
体制剂的血药浓度趋势图基本一致。
3、0.5%R一PPT一PPC一L治复发性尖锐湿庆，一疗程治愈率为
61%，二疗程总治愈率为85%，三疗程总治愈率为94%，副作用
较轻微。
结论:
1、冷冻干燥法不会破坏PPT一DPPC一L的物理化学性质。
2、冷冻干燥法制备的P盯一PPC一PL的包封率高，粒径分布均
匀，对外力的稳定性好，可在各种常见外力作用下水合后使用，
比普通PPT一DPPC一L有更好的稳定性。P件一DPPC一PL的制备方法
简便易行，费用低，效果好，是可取的工业化生产方法。
3、海藻糖对P盯一DPPC一L有很好的保护作用，可用于制备
PPT一DPPC一PL。
4、冷冻干燥法不会改变PPT一DPPC一L对机体的透皮吸收特
性。
5、R一PPT一DPPC一L外用治疗复发性尖锐湿虎疗效显著、安全
性高、使用简便、复发率低、再次复发患者再次治疗仍然有效。
本临床应用初步观察取得了预期的效果，显示了良好的研究和推
广应用前景。
Objective:
Condyloma acuminatum(CA) is a kind of sexually transmitted disease(STDs), with the progressive incidence,it is now in the second place of STDs. Which is human papillomavirus(HPV), a kind of virus, that leads to CA. This kind of virus live autoeciously in the nucleolus of basal cells,while a few of them in the outside of the cells. HPV inside is the main source of relapse and can exist latently in a long period. 0.5% podophyllotoxin tincture is an effective drug advocated by WHO.Podophyllotoxin can be used to treat CA for it has the effect of restraining the division growth process of these cells infected by HPV,and leading putrescence and amotic of these cells.Podophyllotoxin is recommended by WHO as valid for CA,and 0.5% podophyllotoxin tincture is commonly used in treatment.However,0.5% podophyllotoxin tincture can only be used to the warts that can be seen with eyes, and if it is used extensive,serious side-effects will take place. Liposome is the neotype carrier that can controll and delay release of dru
g from the proparation, which has been studied by many scientist in recent years.Making PPT into Liposome can improve its curative effect of anti-relapse of CA by ameliorating its Pharmacokinetics and pharmacodynamics to the certain degree.As literature described, that better effects can be achieved when some drug is incapsuled in DPPC liposome for skin use. But general Liposome is inadequate tranquilization for liquid Liposome medicine have the matter of getting together of particle,and oxidize and decomposition of the phospholipid,and the leakage of the medicine enveloped in Liposome,etc.Proliposome is a solid praeparatum and the dehydration format of the liposome ingredient and the protectant for protectant.lt can re-establish into liposome in
medium. Proliposome can solve the matter of oxidizing, hydrolysis, aggregation, delamination, seepaging of medicine,and pyro-degerming,etc.which can conquer the problem of stability when the liquid liposome be long-term deposited.And it is the key of the industrialization and the commercialization of the liposome. Based on the related literature and appropriate medical formula sifted in advance, PPT-DPPC-PL was prepared. The animal experiment and the primary clinical trial was carried out.
Methods:
Reverse-phase evaporation vesicles (REV) method was used to prepare PPT-DPPC liposomes(ordinary PPT-DPPC liposomes). Freeze-drying method was used to prepare PPT-DPPC proliposomes(PPT-DPPC-PL).The particle morphology, the size range, the encapsulation efficiency and the stability of PPT-DPPC liposomes were investigated.The PPT-DPPC-PL's stability was studied by mensurating its grain size in water under four kinds of different intension physics outside force. The proliposomes were stored at 4 ~40 for 1~6 months remained. The particle morphology, the size range, the encapsulation efficiency and the stability of PPT-DPPC liposomes were investigated after PPT-DPPC-PL was stored at 4~40 for 1-6 months and was hydrated.
2 Ten porkets were chosed and separated into 2 groups,then 2 two kinds of podophyllotoxin liposome were applied on the skin. The porkets' blood were sampled and drug concentrations were examined at different time.
3 PPT-DPPC-PL was used to treat the patients suffering from CA.And the primary clinical effect of the preparations volunteers was observed.
Results:
1 The PPT-DPPC-PL is a loosen and lily powder. The form of the
PPT-DPPC-PL after hydrated isn't different from the form of the ordinary PPT-DPPC-L.After PPT-DPPC-PL was hydrated , PPT-DPPC liposomes appeared multivesicular under electron microscope. The particle were distributed homogenously. The average particle size was 1.45+0.38 H m.The encapsulation efficiency of PPT was 72.3%.The proliposomes were stored at 4~40 for 1-6 months and were stable.
2 In our experiments, the podophyllotoxin of two kinds of PPT-DPPC-L concentrations in animals' blood were no difference. The trend of the podophyllotoxin of two kinds of PPT-DPPC-L concentrations in animals' blood were no difference..
3
尖锐湿疣(CA)是一种性传播疾病，传染性强，发病率高，是欧美国家最常见的性病之一，也是我国最主要的性病之一，其发病率逐年增加，其年增长率超过100％，居各类性病之首。尖锐湿疣的病因是人类乳头瘤病毒(HPV)感染，它感染人体后寄居于基底细胞等角质形成细胞的核内，部分位于核外，而位于核内的HPV可长期潜伏存在，是CA复发的根源所在。鬼臼毒素(PPT)，是1990年世界卫生组织推荐为治疗生殖器疣的一线药物。鬼臼毒素外用时通过抑制受人乳头瘤病毒(HPV)感染细胞的分裂增殖过程，使之坏死脱落从而起到治疗尖锐湿疣的作用。鬼臼毒素是目前临床治疗尖锐湿疣最有效的一线药物，常用的是0.5％的鬼臼毒素酊剂。但它只能用于肉眼可见的皮损，大面积地使用会产生严重的毒副作用，因此只能局部小量地应用。脂质体是最近几年研究较多的新型控缓释药物载体，将PPT制成脂质体制剂，会在一定程度上改善PPT的药代动力学和药效学，对尖锐湿疣的抗复发治疗有更好的疗效。相关研究显示二棕榈酰磷脂酰胆碱(DPPC)脂质体在皮肤外用药物的脂质体制剂中效果最好。但普通脂质体为液体制剂，易发生粒子聚集沉降、磷脂氧化分解、包封药物渗漏等问题，导致脂质体不够稳定。而前体脂质体(PL)是一种固体制剂，是指脂质体和冻干保护剂(亦称赋形剂)的脱水形式，它加水后能重建形成脂质体，解决了脂质体以液体制剂贮存的氧化、水解、聚集、分层、药物的渗漏及高温灭菌等问题，即克服了脂质体以溶液形式长期储存的稳定性问题，这是脂质体走向工业化生产和商品化的关键。经查阅相关文献资料，结合前期筛选出的合适配方，我们制备了鬼臼毒素二棕榈酰磷脂酰胆碱前体脂质体(PPT-DPPC-PL)，并进行了动物实验和初步临床观察。方法：
1、以逆相蒸发法(reverse-phase evaporation vesicles，REV)
制备鬼臼毒素DPPC脂质体(PPT一DPPC一L)悬液(普通型)，采用
冷冻干燥法，选择海藻糖做冻干保护剂制备PPT一DPPc一PL，并考
察P盯一DPPc一PL水合后的形态、粒径分布、包封率。分别观察在
4种不同强度的物理外力(手摇、摇床、超声、超声针)作用下，
PPT一DPPC一PL水合后的稳定性;分别将密封于安瓶中的
P盯一DPPC一PL贮存在4℃、20℃、40℃条件下，贮存1、3、6个月
后取样，经水合后分别考察其脂质体形态、粒径及包封率有无变
化。
2、选择10只母乳猪，分为2组，分别涂以相同浓度的
P盯一DPPC一L和水合后的P叮一DPPC一PL即重建型鬼臼毒素DPPC
脂质体(R一PPT一DPPC一L)，在不同的时间段抽血测定血中鬼臼毒素
的含量。
3、选择自愿受试的复发性尖锐湿疵患者进行PPT一DPPC一PL
临床疗效的初步观察。
结果:
1、制备的PPT一DPPc一PL的形态为疏松、洁白的粉末，水合后
的形态与普通型PPr一DPPC一L相同。经测定PPT一DPPC一PL的包封
率为723%，普通型PPT-DPPC一L的包封率为73.1%，二者无明显
差异，即冷冻干燥处理前后PPT一DPPC一L的包封率几乎不变。四种
不同强度的物理外力作用后测得的粒径大小无显著性差异，证明
P盯一DPPC一PL对外力稳定性良好，可在各种常见外力作用下水合
后使用。经反复测定，在不同的温度、时间等条件下PPT一DPPC一PL
的粒径分布、包封率均未发生明显变化。
2、动物体内血液中鬼臼毒素浓度的比较结果提示，二种
PPT一DPPC一L经局部外用后动物体内血药浓度无显著统计学差异，
即机体对二种脂质体制剂中的鬼臼毒素吸收程度接近。二种脂质
体制剂的血药浓度趋势图基本一致。
3、0.5%R一PPT一PPC一L治复发性尖锐湿庆，一疗程治愈率为
61%，二疗程总治愈率为85%，三疗程总治愈率为94%，副作用
较轻微。
结论:
1、冷冻干燥法不会破坏PPT一DPPC一L的物理化学性质。
2、冷冻干燥法制备的P盯一PPC一PL的包封率高，粒径分布均
匀，对外力的稳定性好，可在各种常见外力作用下水合后使用，
比普通PPT一DPPC一L有更好的稳定性。P件一DPPC一PL的制备方法
简便易行，费用低，效果好，是可取的工业化生产方法。
3、海藻糖对P盯一DPPC一L有很好的保护作用，可用于制备
PPT一DPPC一PL。
4、冷冻干燥法不会改变PPT一DPPC一L对机体的透皮吸收特
性。
5、R一PPT一DPPC一L外用治疗复发性尖锐湿虎疗效显著、安全
性高、使用简便、复发率低、再次复发患者再次治疗仍然有效。
本临床应用初步观察取得了预期的效果，显示了良好的研究和推
广应用前景。
Objective:
Condyloma acuminatum(CA) is a kind of sexually transmitted disease(STDs), with the progressive incidence,it is now in the second place of STDs. Which is human papillomavirus(HPV), a kind of virus, that leads to CA. This kind of virus live autoeciously in the nucleolus of basal cells,while a few of them in the outside of the cells. HPV inside is the main source of relapse and can exist latently in a long period. 0.5% podophyllotoxin tincture is an effective drug advocated by WHO.Podophyllotoxin can be used to treat CA for it has the effect of restraining the division growth process of these cells infected by HPV,and leading putrescence and amotic of these cells.Podophyllotoxin is recommended by WHO as valid for CA,and 0.5% podophyllotoxin tincture is commonly used in treatment.However,0.5% podophyllotoxin tincture can only be used to the warts that can be seen with eyes, and if it is used extensive,serious side-effects will take place. Liposome is the neotype carrier that can controll and delay release of dru
g from the proparation, which has been studied by many scientist in recent years.Making PPT into Liposome can improve its curative effect of anti-relapse of CA by ameliorating its Pharmacokinetics and pharmacodynamics to the certain degree.As literature described, that better effects can be achieved when some drug is incapsuled in DPPC liposome for skin use. But general Liposome is inadequate tranquilization for liquid Liposome medicine have the matter of getting together of particle,and oxidize and decomposition of the phospholipid,and the leakage of the medicine enveloped in Liposome,etc.Proliposome is a solid praeparatum and the dehydration format of the liposome ingredient and the protectant for protectant.lt can re-establish into liposome in
medium. Proliposome can solve the matter of oxidizing, hydrolysis, aggregation, delamination, seepaging of medicine,and pyro-degerming,etc.which can conquer the problem of stability when the liquid liposome be long-term deposited.And it is the key of the industrialization and the commercialization of the liposome. Based on the related literature and appropriate medical formula sifted in advance, PPT-DPPC-PL was prepared. The animal experiment and the primary clinical trial was carried out.
Methods:
Reverse-phase evaporation vesicles (REV) method was used to prepare PPT-DPPC liposomes(ordinary PPT-DPPC liposomes). Freeze-drying method was used to prepare PPT-DPPC proliposomes(PPT-DPPC-PL).The particle morphology, the size range, the encapsulation efficiency and the stability of PPT-DPPC liposomes were investigated.The PPT-DPPC-PL's stability was studied by mensurating its grain size in water under four kinds of different intension physics outside force. The proliposomes were stored at 4 ~40 for 1~6 months remained. The particle morphology, the size range, the encapsulation efficiency and the stability of PPT-DPPC liposomes were investigated after PPT-DPPC-PL was stored at 4~40 for 1-6 months and was hydrated.
2 Ten porkets were chosed and separated into 2 groups,then 2 two kinds of podophyllotoxin liposome were applied on the skin. The porkets' blood were sampled and drug concentrations were examined at different time.
3 PPT-DPPC-PL was used to treat the patients suffering from CA.And the primary clinical effect of the preparations volunteers was observed.
Results:
1 The PPT-DPPC-PL is a loosen and lily powder. The form of the
PPT-DPPC-PL after hydrated isn't different from the form of the ordinary PPT-DPPC-L.After PPT-DPPC-PL was hydrated , PPT-DPPC liposomes appeared multivesicular under electron microscope. The particle were distributed homogenously. The average particle size was 1.45+0.38 H m.The encapsulation efficiency of PPT was 72.3%.The proliposomes were stored at 4~40 for 1-6 months and were stable.
2 In our experiments, the podophyllotoxin of two kinds of PPT-DPPC-L concentrations in animals' blood were no difference. The trend of the podophyllotoxin of two kinds of PPT-DPPC-L concentrations in animals' blood were no difference..
3
引文
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[17] 童桥等．生物化学杂志，1992；8(6)：711—714
[18] 吴道澄，万明习，吴红．智能化释药载体纳米凝胶的制备及其释药特性[J]．第四军医大学学报，2001，22(8)：767．
[19] 施锋，吴敏，林德明．磁性阿霉素纳米微球在磁场中磁感应的发热研究[J]．中国医学物理学杂志，2002，19(2)：125-126．
[20] HOU Gui-rong, ZENG Kang, ZHOU Zai-gao, et al. Preparation and comparative analysis of two podophyllotoxin liposomes [J]. J First Mil Med Univ, 2003, 23(3): 216-8.
[21] Guo. Novel Antifungal Drug Delivery: stable Ampho-tericin BCholesteryl Sulfate Disk. Int J Pharm, 1991, 75:45
[22] 刘占杰，华泽钊，陈建明，等．药品冷冻干燥过程中的玻璃化作用[J]．中国医药工业杂志，2000，31(8)：380-383．
[23] 陈建明，张仰眉，高申，等．维生素A前体脂质体的研制及其特性考察[J]．第二军医大学学报，2003，24(2)：207．
[24] Jenson AB. Kurman W. Lancaster WD. 1 is-sue effects of and hog respowe to human pap-M omavirus infection. Dermatd Qin 1991;9:203-209,
[25] Hfzei M. Ciilasekharam V. Liposomes-aselective drug rklivery system for the topical route of administration: gel dosage form.
J Pharm Pharmacd 1982 ;34:473.
[26] Masini V, Bonte F,Meybeck A, et al. Cutaneous bioavailability in hairless rate of retinoic acid in liposome or gel J Pharm Sci 1993:82:17-21.
[27] 颉玉胜，曾抗，江彬彬，张泉，等．不同鬼臼毒素制剂局部外用血药浓度的实验研究．中国麻风皮肤病杂志．2003：19(4)：352-354．
[28] von Krogh G. podophyllotoxin in Serum absorption subsequem to three-day repeated applications of a 0.5％ ethanolic preparation on condylomata acuminata. Sex Transm Disl982;9:26-33.
[29] 江彬彬，曾抗，张泉，等．鬼臼毒素二棕榈酸磷脂酰胆碱脂质休局部外用的实验研究．第一军医大学学报 2002：22：875-877．
[30] von Krogh G. Penile condylomata acuminata:an experimental model for evaluation of topical self-treat-ment with 0.50％-1.0％ ethanoic preparations of podophyllotoxin for three days. Sex Transm Dis, 1981,8:179.
[31] 王建琴，曾仁山．不同方法治疗尖锐湿疣时合并干扰素的疗效观察中华皮肤科杂志，1998，31：194 4．
[32] Krogh GV, Szpak E, Anderson M, et al. Selftreatment using 0.25％～0.5％ podophyllotoxinethanol solutions against penile condylomata acuminata: A place bo-controlled comparative study. Genitourin Med 1994:70:1 05
[33] 王家壁，刘跃华，曹仁烈，等．尤脱欣治疗尖锐湿疣临床疗效观察临床皮肤科杂志 1996；1：26
[34] 王建六．尖锐湿疣的诊断和治疗进展．中国医刊．2003；38(2)：17-19
[35] Pavelic Z. et al. Liposomes containing drugs for treatment of vaginal infections. Eur J Pharm Sci. 1999 Aug; 8(4): 345-51
[36] Mezei M. US Patent 4897 269; 1990
[37] 李国锋、许重远等．鬼臼毒素具有荧光性．第一军医大学学报．1996；16(3)：268-269
[38] 马守栋、李国锋等．荧光分光光度法控制鬼臼毒素脂质体的质量．药学实践杂志．1997；15(6)：365-367
[39] 马守栋、李国锋等．鬼臼毒素脂质体的制备及质量研究．中国药业．1997；(5)：12-13
[40] 曾抗、李国锋等．鬼臼毒素脂质体软膏治疗尖锐湿疣的双盲随机对照试验．第一军医大学学报．1998；18(3)：246