鬼臼毒素脂质体壳聚糖涂膜剂的制备及动物实验研究
摘要
目的：
一、制备鬼臼毒素脂质体混悬液并观察粒径、包封率、稳定性等指标，探讨其中的关系。
二、制备和优化壳聚糖涂膜并观察其对鬼臼毒素脂质体的稳定性。
三、观察鬼臼毒素脂质体壳聚糖涂膜剂对家兔阴道的刺激性。
四、检测鬼臼毒素脂质体壳聚糖涂膜剂宫颈给药后的系统吸收情况。
五、建立HPLC测定家兔血液中鬼臼毒素的方法。
方法：
一、旋转薄膜蒸发+超声法制备鬼臼毒素脂质体混悬液，HPLC法测定鬼臼毒素包封率，壳聚糖和3％稀醋酸按6g：100ml的比例制成涂膜。制成的鬼臼毒素脂质体涂膜剂与鬼臼毒素酊剂对照行家兔阴道刺激试验。
二、40只新西兰大白兔随机分为4组，每组10只。分为一个观察组和3个对照组。观察组涂鬼臼毒素脂质体涂膜剂，对照组分别涂鬼臼毒素酊剂、鬼臼毒素软膏和鬼臼毒素壳聚糖涂膜剂。宫颈涂药后各个时间点抽血，二氯甲烷萃取血液中的鬼臼毒素，HPLC法测定鬼臼毒素含量，数据用spss10.0进行统计学分析。
结果：
一、旋转薄膜蒸发+超声法制备的鬼臼毒素脂质体为单层脂质体，粒径多数在0.2～1.0μm之间，鬼臼毒素包封率在70％～80％之间，平均包封率为76.3％。鬼臼毒素脂质体壳聚糖涂膜剂在室温下保存6个月性状未见明显变化。
二、0.5％鬼臼毒素脂质体壳聚糖涂膜剂对家兔阴道刺激性低于0.5％鬼臼毒素酊剂，对家兔阴道上皮粘着性好。
三、HPLC法测定的鬼臼毒素脂质体壳聚糖涂膜剂外用家兔宫颈后血液中鬼臼毒素浓度始终低于对照组浓度。观察组鬼臼毒素的血药浓度峰值为175±43.957ng/ml，对照组鬼臼毒素的血药浓度峰值分别为893.8±52.548ng/ml，499.4±44.1694ng/ml和
458.6士45.3338ng加1。观察组与对照组的血药浓度峰值比较有显著
性差异(p<0.001)。对照组血液中鬼臼毒素AUC分别是观察组的
4.83、2.63和2.58倍。
结论:
一、壳聚糖涂膜剂增加了鬼臼毒素脂质体的稳定性，对家兔阴道刺激较
轻，适合在家兔宫颈上皮给药。
二、鬼臼毒素脂质体壳聚糖涂膜剂外用家兔宫颈后鬼臼毒素吸收量小于
其它剂型的药物，表明脂质体制剂可以减少鬼臼毒素的全身吸收而
降低毒副作用。
三、HPLC法测定血液中的鬼臼毒素，方法精确、简便、可靠。是测定家
兔血液中鬼臼毒素浓度的一种有效方法。
Objective:
1. Prepare the podophyllotoxin liposomes liquid and observe its diameter,
embedding ratio and stability, etc, and discuss the relation among them.
2. Prepare the chitosan film and observe the stability to podophyllotoxin
liposomes.
3. Observe the suscitation of podophyllotoxin liposomes chitosan film to
rabbits' vagina.
4. Study the systemic absorption after topical applying podophyllotoxin
liposomes chitosan film to rabbits' cervix.
5. Set up the method of detecting podophyllotoxin in rabbits' blood by
HPLC.
Methods:
1. The podophyllotoxin liposomes liquid was prepared by rotary film
evaporation and ultrasonic wave. The embedding ratio of podophyllotoxin
was determined by HPLC. Film agent were made of chitosan and 3% Acetic
acid (6g: 100 ml). The podophyllotoxin liposomes chitosan film and the
podophyllotoxin tincture were contrasted in the suscitation experiment.
2. 40 rabbits were divided into four groups randomly, which including one
test group and three control groups, each group ten rabbits. The test rabbits
were applied with podophyllotoxin liposomes chitosan film and the control
rabbits were applied with podophyllotoxin tincture, podophyllotoxin
ointment and podophyllotoxin chitosan film respectively. Rabbits' blood was
drew out in regular time and was extracted by dichlormethane and was
determined by HPLC and the data was analysed by spss10.0.
Result:
1. The podophyllotoxin liposomes prepared by rotary film evaporation and
ultrasonic wave are all unilaminar vesicles. The diameter is from 0.2 μM to
1.0μ m and the embedding ratio is between 70% to 80% and the average
embedding ratio is 76.3%. The podophyllotoxin liposomes chitosan film
under the room temperature didn't appear obvious variety in six months.
2. The suscitation of 0.5% podophyllotoxin liposomes chitosan film to
rabbits' vagina is lighter than that of 0.5% podophyllotoxin tincture and the
film agent is good adherent to rabbits' cervix.
3. The podophyllotoxin concentration of test group is always lower than that
of three control groups, the highest podophyllotoxin concentration value of
test group is 175±43.957ng/ml and that of control groups is respective 893.8
±52.548 ng/ml, 499.4±44.1694 ng/ml and 458.6±45.3338ng/ml ( p<0.001).
The AUC of podophyllotoxin of the control groups are respective 4.83-fold ,
2.63-fold and 2.58-fold higher than that of the test group.
Conclusion:
1. The stability of podophyllotoxin liposomes was improved by the chitosan
film and the suscitation to rabbits' vagina is lighter than that of the
podophyllotoxin tincture and is good fit with rabbits' cervix.
2. The systemic absorption of podophyllotoxin of the test group is lower than
that of the control groups, It is sure that the side effect of podophyllotoxin
can be decreased by liposomes agent.
3. The HPLC method is precise, facile and reliable, and it can be used with
determining the podophyllotoxin concentration of animals' blood.
一、制备鬼臼毒素脂质体混悬液并观察粒径、包封率、稳定性等指标，探讨其中的关系。
二、制备和优化壳聚糖涂膜并观察其对鬼臼毒素脂质体的稳定性。
三、观察鬼臼毒素脂质体壳聚糖涂膜剂对家兔阴道的刺激性。
四、检测鬼臼毒素脂质体壳聚糖涂膜剂宫颈给药后的系统吸收情况。
五、建立HPLC测定家兔血液中鬼臼毒素的方法。
方法：
一、旋转薄膜蒸发+超声法制备鬼臼毒素脂质体混悬液，HPLC法测定鬼臼毒素包封率，壳聚糖和3％稀醋酸按6g：100ml的比例制成涂膜。制成的鬼臼毒素脂质体涂膜剂与鬼臼毒素酊剂对照行家兔阴道刺激试验。
二、40只新西兰大白兔随机分为4组，每组10只。分为一个观察组和3个对照组。观察组涂鬼臼毒素脂质体涂膜剂，对照组分别涂鬼臼毒素酊剂、鬼臼毒素软膏和鬼臼毒素壳聚糖涂膜剂。宫颈涂药后各个时间点抽血，二氯甲烷萃取血液中的鬼臼毒素，HPLC法测定鬼臼毒素含量，数据用spss10.0进行统计学分析。
结果：
一、旋转薄膜蒸发+超声法制备的鬼臼毒素脂质体为单层脂质体，粒径多数在0.2～1.0μm之间，鬼臼毒素包封率在70％～80％之间，平均包封率为76.3％。鬼臼毒素脂质体壳聚糖涂膜剂在室温下保存6个月性状未见明显变化。
二、0.5％鬼臼毒素脂质体壳聚糖涂膜剂对家兔阴道刺激性低于0.5％鬼臼毒素酊剂，对家兔阴道上皮粘着性好。
三、HPLC法测定的鬼臼毒素脂质体壳聚糖涂膜剂外用家兔宫颈后血液中鬼臼毒素浓度始终低于对照组浓度。观察组鬼臼毒素的血药浓度峰值为175±43.957ng/ml，对照组鬼臼毒素的血药浓度峰值分别为893.8±52.548ng/ml，499.4±44.1694ng/ml和
458.6士45.3338ng加1。观察组与对照组的血药浓度峰值比较有显著
性差异(p<0.001)。对照组血液中鬼臼毒素AUC分别是观察组的
4.83、2.63和2.58倍。
结论:
一、壳聚糖涂膜剂增加了鬼臼毒素脂质体的稳定性，对家兔阴道刺激较
轻，适合在家兔宫颈上皮给药。
二、鬼臼毒素脂质体壳聚糖涂膜剂外用家兔宫颈后鬼臼毒素吸收量小于
其它剂型的药物，表明脂质体制剂可以减少鬼臼毒素的全身吸收而
降低毒副作用。
三、HPLC法测定血液中的鬼臼毒素，方法精确、简便、可靠。是测定家
兔血液中鬼臼毒素浓度的一种有效方法。
Objective:
1. Prepare the podophyllotoxin liposomes liquid and observe its diameter,
embedding ratio and stability, etc, and discuss the relation among them.
2. Prepare the chitosan film and observe the stability to podophyllotoxin
liposomes.
3. Observe the suscitation of podophyllotoxin liposomes chitosan film to
rabbits' vagina.
4. Study the systemic absorption after topical applying podophyllotoxin
liposomes chitosan film to rabbits' cervix.
5. Set up the method of detecting podophyllotoxin in rabbits' blood by
HPLC.
Methods:
1. The podophyllotoxin liposomes liquid was prepared by rotary film
evaporation and ultrasonic wave. The embedding ratio of podophyllotoxin
was determined by HPLC. Film agent were made of chitosan and 3% Acetic
acid (6g: 100 ml). The podophyllotoxin liposomes chitosan film and the
podophyllotoxin tincture were contrasted in the suscitation experiment.
2. 40 rabbits were divided into four groups randomly, which including one
test group and three control groups, each group ten rabbits. The test rabbits
were applied with podophyllotoxin liposomes chitosan film and the control
rabbits were applied with podophyllotoxin tincture, podophyllotoxin
ointment and podophyllotoxin chitosan film respectively. Rabbits' blood was
drew out in regular time and was extracted by dichlormethane and was
determined by HPLC and the data was analysed by spss10.0.
Result:
1. The podophyllotoxin liposomes prepared by rotary film evaporation and
ultrasonic wave are all unilaminar vesicles. The diameter is from 0.2 μM to
1.0μ m and the embedding ratio is between 70% to 80% and the average
embedding ratio is 76.3%. The podophyllotoxin liposomes chitosan film
under the room temperature didn't appear obvious variety in six months.
2. The suscitation of 0.5% podophyllotoxin liposomes chitosan film to
rabbits' vagina is lighter than that of 0.5% podophyllotoxin tincture and the
film agent is good adherent to rabbits' cervix.
3. The podophyllotoxin concentration of test group is always lower than that
of three control groups, the highest podophyllotoxin concentration value of
test group is 175±43.957ng/ml and that of control groups is respective 893.8
±52.548 ng/ml, 499.4±44.1694 ng/ml and 458.6±45.3338ng/ml ( p<0.001).
The AUC of podophyllotoxin of the control groups are respective 4.83-fold ,
2.63-fold and 2.58-fold higher than that of the test group.
Conclusion:
1. The stability of podophyllotoxin liposomes was improved by the chitosan
film and the suscitation to rabbits' vagina is lighter than that of the
podophyllotoxin tincture and is good fit with rabbits' cervix.
2. The systemic absorption of podophyllotoxin of the test group is lower than
that of the control groups, It is sure that the side effect of podophyllotoxin
can be decreased by liposomes agent.
3. The HPLC method is precise, facile and reliable, and it can be used with
determining the podophyllotoxin concentration of animals' blood.
引文
1、Wallin-KL et al. Type-specific persistence of human papillomavirus DNA before the development of invasive cervical cancer. N-Engl-J-Med. 1999 Nov25; 341(22): 1633-1638.
2、Tjiong MY, Out TA, Ter Schegget J, et al. Epidemiologic and mucosal immunologic aspects of HPV infection and HPV-related cervical neoplasia in the lower female genital tract: a review. Int J Gynecol Cancer, 2001,11(1): 9-17.
3、马丁、奚玲．宫颈癌流行病学及病因学研究进展．实用妇产科杂志．2001，17(2)：61．
4、Diaz-Wrrastia-C, Arany-I, Robazetti-S-C. Clinical and molecular responses in high-grade intraepithelial neoplasia treated with toplical imiquimod 5%. Clin-Caneer-Res. 2001, 7(10): 3031-3033.
5、Koutsky LA, Ault K A, Wheeler C M, et al. A Controlled Trial of a Human Papillomavirus Type 16 Vaccine. N Engl J Med. 2002. 347: 1645-1651.
6、Kinghom GR, Mcmillan A, Mulcahy F, et al. An open, comparative study of the efficacy of 0.5% podophyllotoxin lotion and 25% podophyllin solution in the treatment of condyloma acuminate in males and females. Int J STD AIDS, 1993,4:194-199.
7、Petersen CS, Weisaman K, Kaempherol Q. An argument against the use of podophyllin?. Genitourin Med, 1995, 71(1): 92-93.
8、曾抗，李国锋，许重远，等，脂质体鬼臼毒素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．
9、江彬彬、曾抗、张三泉，等．鬼臼毒素二棕榈酸磷脂酰胆碱脂质体局部外用的实验研究．第一军医大学学报，2002，22(10)：875-877．
10、马守栋、李国峰、陈志良等．脂质体鬼臼毒素的制备及质量研究．中国药业，1997(5)：12-13．
11、李国峰、候连兵、曾抗．维甲酸脂质体的制备与临床应用．中国药
学杂志，1998，33(2)：121．
12、张灵芝．脂质体制备及其在生物医学中的应用．第1版．北京：北京医科大学、中国协和医科大学联合出版社，1998．53-54．
13、Cairnes DA. High performance liquid chromatography of podophyllotoxins and Related lignans. J Nat Prod, 1981, 44(1): 34.
14、Lim CK. High- performance liquid chromatography of aryltetrahydronaphthalene lignans. J Chromatogr, 1983,255: 247.
15、余自成，王丽平，郁人海等．HPLC 法测定广西八角莲中鬼臼毒素的含量．药物分析杂志，1997，17(1)：43．
16、王宝玺，王日升，汪青．植物桃耳七中鬼臼毒素的提取及 HPLC 分析．中国医药工业杂志，1999，30(11)：484．
17、俞培忠，姚莉韵，王丽平．HPLC 法分离和测定山荷叶中的鬼臼毒素．药物分析杂志，1999，19(1)：35．
18、童桥，聂松青．脂质体的稳定性研究．生理科学进展，1996，27(2)：125．
19、赵红，郑俊民．新型的皮肤给药系统-脂质体．沈阳药科大学学报，1997．14(2)：148-150．
20、Crowe JH, Crowe LM. Factors affecting the stability of dry liposomes. BBA, 1988,939:327-334.
21、曾科，林克椿．海藻糖对聚乙二醇融合脂质体的防护．北京医科大学学报，1991，23：271-273．
22、童桥，聂松青，林克椿．海藻糖对载药脂质体在干燥-再水化过程中的保护作用．生物化学杂志，1992，8：711-714．
23、李平，王爱勤，张建林，等．壳聚糖人工泪液的制备及应用．中国医院药学杂志，1996，16(12)：569．
24、王风山，凌沛学，张天民，等．生化药物研究．第一版。北京：人民卫生出版社，1997，357．
25、刘文辉、刘晓亚、陈明清等，壳聚糖基生物医用材料及其应用研究进展．功能高分子学报 2001，14(4)：493-496．
26、Chang LW, Yang CM, Chen CF, et al. Experimental podophyllotoxin
(bajiaolian)poisoning: Ⅱ. Effects on the liver, intestine, kidney, pancreas and testis. Biomed-Environ-Sci,1992,5(4) :293-302．
27、 Claesson U, Lassus A, Happonen H, et al. Topical treatment of venereal warts: a comparative open study of podophyllotoxin cream versus solution. Int J STD AIDS, 1996(7) : 429-34．
28、 Tyring S, Edwards L, Cherry LK, et al. Safety and efficacy of 0． 5% podofilox gel in the treatment of anogenital warts. Arch Dermatol,1998,134(1) : 33-38．
29、 Reynolds M, Fraser PA, Lacey CJN. Audits of treatment of genital warts: Closing the feedback loop. Int J STD AIDS,1996(7) :347-352．
30、 Braun-Falco O, Korting HC, Maibach HI, et al. Biodisposition of liposome-encapsulated active ingredients applied on the sky. Liposome Dermatics,1992:206-214．
31、 瞿光喜，邹立家，张天民．脂质体经皮给药的研究进展．中国药学 杂志，1997，32(6) ：329-332．
32、 Micheal M, Vijeyalaksnmi G. Liposome: a selective drug delivery system for the topical of administration: gel dosage form . J Pharm Pharmacol, 1982: 34(7) :473．
33、 Micheal M, Vijeyalakshmi G. Liposome: a selective drug delivery system for the topical of administration lotion dosage form, Life Sci, 1980, 26: 1473．
34、 E1-Ridy MS, Khalil RM, Free versus liposome-encapsulated lignocaine hydrochloride yopical applications. Pharmazie, 1999;54 (9) :682-684．
35、 Waldrep JC, Gilbert BE, Knight CM, et al. Pulmonary delivery of beclomethasone liposome aerosol in volunteers:tolerance and safety. Chest, 1997;111 (2) :316-323．
36、 Wyde PPR, Gilbert BE, Mehta K, Aerosol delivery of liposomal all-trans-retinoic acid to the lungs. Cancer Chemother Pharmacol, 1999;43(4) : 277-283．
37、 Perugini P, Genta I, Pavanetto F. Study on glycolic acid delivery by liposomes and microspheres. Int-J-Pharm, 2000, 196(1) : 56-60．
38、 Yarosh DB, O'Connor A, Alas L, Photoprotection by topical DNA repair enzymes: molecular correlates of clinical studies. Photochem-Photobiol, 1999, 69(2) : 136-140．
39、 Domashenko A, Gupta S, Cotsarelis G. Efficient delivery of transgenes to human hair follicle progenitor cells using topical lipoplex. Nat-Biotechnol. 2000,18(4) : 420-423．
2、Tjiong MY, Out TA, Ter Schegget J, et al. Epidemiologic and mucosal immunologic aspects of HPV infection and HPV-related cervical neoplasia in the lower female genital tract: a review. Int J Gynecol Cancer, 2001,11(1): 9-17.
3、马丁、奚玲．宫颈癌流行病学及病因学研究进展．实用妇产科杂志．2001，17(2)：61．
4、Diaz-Wrrastia-C, Arany-I, Robazetti-S-C. Clinical and molecular responses in high-grade intraepithelial neoplasia treated with toplical imiquimod 5%. Clin-Caneer-Res. 2001, 7(10): 3031-3033.
5、Koutsky LA, Ault K A, Wheeler C M, et al. A Controlled Trial of a Human Papillomavirus Type 16 Vaccine. N Engl J Med. 2002. 347: 1645-1651.
6、Kinghom GR, Mcmillan A, Mulcahy F, et al. An open, comparative study of the efficacy of 0.5% podophyllotoxin lotion and 25% podophyllin solution in the treatment of condyloma acuminate in males and females. Int J STD AIDS, 1993,4:194-199.
7、Petersen CS, Weisaman K, Kaempherol Q. An argument against the use of podophyllin?. Genitourin Med, 1995, 71(1): 92-93.
8、曾抗，李国锋，许重远，等，脂质体鬼臼毒素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．
9、江彬彬、曾抗、张三泉，等．鬼臼毒素二棕榈酸磷脂酰胆碱脂质体局部外用的实验研究．第一军医大学学报，2002，22(10)：875-877．
10、马守栋、李国峰、陈志良等．脂质体鬼臼毒素的制备及质量研究．中国药业，1997(5)：12-13．
11、李国峰、候连兵、曾抗．维甲酸脂质体的制备与临床应用．中国药
学杂志，1998，33(2)：121．
12、张灵芝．脂质体制备及其在生物医学中的应用．第1版．北京：北京医科大学、中国协和医科大学联合出版社，1998．53-54．
13、Cairnes DA. High performance liquid chromatography of podophyllotoxins and Related lignans. J Nat Prod, 1981, 44(1): 34.
14、Lim CK. High- performance liquid chromatography of aryltetrahydronaphthalene lignans. J Chromatogr, 1983,255: 247.
15、余自成，王丽平，郁人海等．HPLC 法测定广西八角莲中鬼臼毒素的含量．药物分析杂志，1997，17(1)：43．
16、王宝玺，王日升，汪青．植物桃耳七中鬼臼毒素的提取及 HPLC 分析．中国医药工业杂志，1999，30(11)：484．
17、俞培忠，姚莉韵，王丽平．HPLC 法分离和测定山荷叶中的鬼臼毒素．药物分析杂志，1999，19(1)：35．
18、童桥，聂松青．脂质体的稳定性研究．生理科学进展，1996，27(2)：125．
19、赵红，郑俊民．新型的皮肤给药系统-脂质体．沈阳药科大学学报，1997．14(2)：148-150．
20、Crowe JH, Crowe LM. Factors affecting the stability of dry liposomes. BBA, 1988,939:327-334.
21、曾科，林克椿．海藻糖对聚乙二醇融合脂质体的防护．北京医科大学学报，1991，23：271-273．
22、童桥，聂松青，林克椿．海藻糖对载药脂质体在干燥-再水化过程中的保护作用．生物化学杂志，1992，8：711-714．
23、李平，王爱勤，张建林，等．壳聚糖人工泪液的制备及应用．中国医院药学杂志，1996，16(12)：569．
24、王风山，凌沛学，张天民，等．生化药物研究．第一版。北京：人民卫生出版社，1997，357．
25、刘文辉、刘晓亚、陈明清等，壳聚糖基生物医用材料及其应用研究进展．功能高分子学报 2001，14(4)：493-496．
26、Chang LW, Yang CM, Chen CF, et al. Experimental podophyllotoxin
(bajiaolian)poisoning: Ⅱ. Effects on the liver, intestine, kidney, pancreas and testis. Biomed-Environ-Sci,1992,5(4) :293-302．
27、 Claesson U, Lassus A, Happonen H, et al. Topical treatment of venereal warts: a comparative open study of podophyllotoxin cream versus solution. Int J STD AIDS, 1996(7) : 429-34．
28、 Tyring S, Edwards L, Cherry LK, et al. Safety and efficacy of 0． 5% podofilox gel in the treatment of anogenital warts. Arch Dermatol,1998,134(1) : 33-38．
29、 Reynolds M, Fraser PA, Lacey CJN. Audits of treatment of genital warts: Closing the feedback loop. Int J STD AIDS,1996(7) :347-352．
30、 Braun-Falco O, Korting HC, Maibach HI, et al. Biodisposition of liposome-encapsulated active ingredients applied on the sky. Liposome Dermatics,1992:206-214．
31、 瞿光喜，邹立家，张天民．脂质体经皮给药的研究进展．中国药学 杂志，1997，32(6) ：329-332．
32、 Micheal M, Vijeyalaksnmi G. Liposome: a selective drug delivery system for the topical of administration: gel dosage form . J Pharm Pharmacol, 1982: 34(7) :473．
33、 Micheal M, Vijeyalakshmi G. Liposome: a selective drug delivery system for the topical of administration lotion dosage form, Life Sci, 1980, 26: 1473．
34、 E1-Ridy MS, Khalil RM, Free versus liposome-encapsulated lignocaine hydrochloride yopical applications. Pharmazie, 1999;54 (9) :682-684．
35、 Waldrep JC, Gilbert BE, Knight CM, et al. Pulmonary delivery of beclomethasone liposome aerosol in volunteers:tolerance and safety. Chest, 1997;111 (2) :316-323．
36、 Wyde PPR, Gilbert BE, Mehta K, Aerosol delivery of liposomal all-trans-retinoic acid to the lungs. Cancer Chemother Pharmacol, 1999;43(4) : 277-283．
37、 Perugini P, Genta I, Pavanetto F. Study on glycolic acid delivery by liposomes and microspheres. Int-J-Pharm, 2000, 196(1) : 56-60．
38、 Yarosh DB, O'Connor A, Alas L, Photoprotection by topical DNA repair enzymes: molecular correlates of clinical studies. Photochem-Photobiol, 1999, 69(2) : 136-140．
39、 Domashenko A, Gupta S, Cotsarelis G. Efficient delivery of transgenes to human hair follicle progenitor cells using topical lipoplex. Nat-Biotechnol. 2000,18(4) : 420-423．