鬼臼毒素-固体脂质纳米粒与鬼臼毒素酊经皮外用的安全性比较

Safety of podophyllotoxin-solid lipid nanoparticles versus podophyllotoxin tincture applied on skins

目的：为了克服鬼臼毒素酊剂在临床应用的局限性,进行了鬼臼毒素-固体脂质纳米粒与鬼臼毒素酊经皮外用安全性比较的实验。方法：实验于2006-09/2007-08在南方医科大学药学部实验室完成。①取豚鼠32只,按照随机数字表法分成0.5%鬼臼毒素-固体脂质纳米粒完整皮肤组和破损皮肤组、0.5%鬼臼毒素酊剂完整皮肤组和破损皮肤组,每组4只,分别进行单次和多次给药皮肤刺激试验。单次给药方法：豚鼠背部两侧对称脱毛后用手术刀片作#字划痕制作皮肤破损模型,采用同体左右侧自身对照法,左侧为受试区,分别取0.5%鬼臼毒素-固体脂质纳米粒混悬液及0.5%鬼臼毒素酊剂各0.1mL均匀涂布于受试区,再以自制单层塑料薄膜和双层纱布封包受试区。分别于去除敷料和清洗受试物后1,24,48h观察涂药部位有无红斑和水肿等情况,以及上述变化的恢复情况与时间。多次给药方法：皮肤处理方法、观察指标及评价指标同上,每日给药1次,连续给药14d。②取大鼠100只,按照随机数字表法分成0.5%鬼臼毒素-固体脂质纳米粒、0.5%鬼臼毒素酊剂完整皮肤组和破损皮肤组及正常对照组,每组10只,分别进行急性和长期皮肤毒性试验。急性毒性试验方法：脱毛后,分别取0.5%鬼臼毒素固体-脂质纳米粒、0.5%鬼臼毒素酊剂1mL均匀涂布于脱毛区,连续观察14d,每日观察大鼠体质量、进食量、呼吸、体态、眼、中枢神经系统、四肢活动、粪便性状及死亡情况。长期毒性试验方法：皮肤处理方法及观察指标同上,每日给药1次,药量均为0.1mL,连续给药30d,末次给药后24h麻醉后处死动物,取血3.0~4.0mL进行血液学、血液生化学检查,然后切取各组大鼠涂药中心区域的皮肤组织行皮肤病理检查及激光共聚焦显微镜扫描。结果：纳入大鼠100只、豚鼠32只,均进入结果分析。①单次和多次给予鬼臼毒素-固体脂质纳米粒对豚鼠完整和破损皮肤均无刺激作用。②单次大剂量给予0.5%鬼臼毒素酊剂对豚鼠完整皮肤无刺激性,对破损皮肤有较强的刺激性;每日小剂量给予0.5%鬼臼毒素酊剂对豚鼠完整皮肤、破损皮肤均有较强的刺激性,其中破损皮肤组豚鼠皮肤刺激性出现的最早和最严重。③单次大剂量应用0.5%鬼臼毒素-固体脂质纳米粒和0.5%鬼臼毒素酊剂时大鼠容易出现短期的系统吸收毒性,尤以皮肤破损组症状较明显,其中0.5%鬼臼毒素酊剂组大鼠中毒症状持续时间均较0.5%鬼臼毒素-固体脂质纳米粒组持久。④每日小剂量长期应用0.5%鬼臼毒素-固体脂质纳米粒和鬼臼毒素酊剂对大鼠比较安全无系统吸收毒性。⑤每日小剂量给予0.5%鬼臼毒素-固体脂质纳米粒组大鼠于给药后的18~25d,均出现轻度的皮肤红斑、水肿、糜烂和结痂等炎症反应及局部毛发生长稀疏、缓慢或无毛发生长,皮肤组织病理学检查可见以表皮为主的急性炎症反应。激光共聚焦显微镜扫描可见以红色荧光显像的药物主要富集于表皮层及毛囊而真皮及皮下组织药物含量相对较少。⑥每日小剂量给予0.5%鬼臼毒素酊剂组大鼠于给药后的4~7d,均出现程度逐渐加重的皮肤红斑、水肿、糜烂、坏死、结痂及明显的毛发生长障碍,皮肤组织病理学检查可见皮肤全层急性炎症反应,激光共聚焦显微镜扫描可见表皮全层坏死,以红色荧光显像的药物主要富集于真皮、皮下组织、毛囊及其周围,荧光强度相对较0.5%鬼臼毒素-固体脂质纳米粒组强。结论：在有效观察时间和一定浓度范围内,鬼臼毒素-固体脂质纳米粒、鬼臼毒素酊剂对实验动物均无严重系统吸收毒性,但鬼臼毒素-固体脂质纳米粒与鬼臼毒素酊剂相比具有局部不良反应少,不良反应程度轻,不良反应出现迟,皮肤靶向性好等优点。

AIM： To compare the safety of podophyllotoxin-solid lipid nanoparticles and podophyllotoxin tincture applied on skins in order to overcome the limitation ofpodophyllotoxin tincture in clinic. METHODS： Experiments were performed at the Laboratory of Pharmacology of Southern Medical University from September 2006 to August 2007, （1）Thirty-two guinea pigs were selected and assigned into 0.5% podophyllotoxin-solid lipid nanoparticles and 0,5% podophyllotoxin tincture intact skin group and dermal injury group by random digits table method, 4 in each group. These guinea pigs received single and multiple dosing skin stimulus tests.Single dosing methods： After bilateral symmetric depilation, back of guinea pigs was cut as ＂#＂shape by operating knife blade till staxis.In homobody left and right sides own control test, left side was as test region,Self-made monolayer plastic film and double layer gauze were used to wrap the test region,Medicine-treated with or without erythema and edema was observed at hours 1, 24 and 48 after removing dressing and cleaning test region,The recovery of above-mentioned changed and time were recorded.Multiple dosing methods： treatment approach, observational index and evaluation index were the same as above-mentioned data, administering once a day for 14 days successivdy.（2）100 rats were selected and divided into 0,5% podophyllotoxin-solid lipid nanoparticles and 0,5% podophyllotoxin tincture intact skin group, dermal injury group and normal control group, with 10 in each group.These rats received acute and long-term dermal toxicity test,Acute toxicity test method： After depilation, 0.5% podophyllotoxin-solid lipid nanoparticles suspension and 0.5% podophyllotoxin tincture （1 mL） evenly spread on test region, observed for 14 days successively.Body mass, food-intake, respiration, body carriage, eye, central nervous system, activity of limbs, excrement and death of rats were recorded every day, Long-term toxicity test methods： dermal disposal method and observational index were the same as above-mentioned data, 0.1 mL dosing once a day for 30 days successively,After last administration, animal were killed 24 hours after anaesthesia and 3.0-4.0 mL blood was collected for hematology, blood biochemical analysis, and then dermal pathology tests were performed and a confocal laser scanning microscope was used to scan with skin slice fi＇om the center district of the smeared skin, RESULTS： 100 rats and 32 guinea pigs were involved in the result analysis,（1）There was no irritant dermal reaction in intact or injured skin by single or repeated applications of podophyllotoxin-solid lipid nanoparticles in guinea pigs.（2）There was no irritant dermal reaction in intact skin by single large-dose application of 0.5% podophyllotoxin tincture in guinea pigs.There was obviously irritant dermal reaction in injured skin by single large-dose application of 0.5% podophyllotoxin tincture.There was obviously irritant dermal reaction in intact and injured skin by repeated applications of 0.5% podophyUotoxin tincture,Among them stimulating emergence of a guinea pig of damaged skin groups was of the earliest stage and serious.（3）Short date of systemic absorbing toxicity was easily observed by single large-dose application of 0.5% podophyllotoxin-solid lipid nanoparticles and 0.5% podophyllotoxin tincture in rats, which were even obvious in injured skin groups.The duration of toxic symptom was longer in rats taking 0.5% podophyllotoxin tincture than those taking 0.5% podophyllotoxin-solid lipid nanoparticles. （4）No long-term systemic toxic reaction was obviously observed by low-dose everyday applications of 0.5% podophyllotoxin-solid lipid nanoparticles and 0.5% podophyllotoxin tincture in rats.（5）Mild erythema, edema, erosio, scab and hair growth obstacle appeared by low-dose everday applications in the 0.5% podophyllotoxin-solid lipid nanoparticles group 18-25 days after administration.Acute inflammatory reaction was primarily observed in the epidermis histopathology test.Medicine was mainly enriched to gather in the epidermis layer, hair follicle and its surroundings in rats of podophyllotoxin-solid lipid nanoparticles groups under the confocal laser scanning microscope, but medicine content was very few in dermis and subcutaneous tissue.（6）Increasingly serious erythema, edema, erosio, necrosis, scab and hair growth obstacle appeared by low-dose everyday applications in the 0.5% podophyllotoxin tincture group 4-7 days after administmtion.A severe whole layer skin inflammatory reaction was observed in epidermis histopathology test.A whole layer necrosis and medicine was mainly enriched to gather in the dermis layer, subcutaneous tissue, hair follicle and its surroundings in rats＇of podophyllotoxin tincture groups under the confocal laser scanning microscope, but fluorescence strength was stronger than 0.5% podophyllotoxin-solid lipid nanoparticles group. CONCLUSION： Within the certain survey period and concentration, no serious systemic absorbing toxicity to experimental animal of podophyllotoxin-solid lipid nanoparticles and podophyllotoxin tincture, but compared podophyllotoxin-solid lipid nanoparticles and podophyllotoxin tincture, the former has some advantages such as the little partial reaction, light reaction degree, late adverse reaction and good skin target.