摘要
目的 开展基因治疗药物rAAV2-sTRAIL在正常鼠和荷瘤鼠2种小鼠体内的生物分布研究。方法 BALB/c小鼠随机分成3组,包括溶媒对照组,每性别8只动物;低(每只1.5×109 vg)、高(每只1.5×1010 vg)剂量组,每组每性别20只动物。采用肌肉注射给药,给药1次。每天观察动物临床症状,分别在给药后24 h,2,4,12周解剖动物。BALB/c裸鼠在右前肢腋窝皮下接种肿瘤细胞ACC-2,肿瘤造模成功后,按照瘤体体积将动物随机分成3组。包括溶媒对照组,每性别4只动物;低(每只1.5×109 vg)、高(每只1.5×1010 vg)剂量组,每组每性别10只动物。采用瘤内注射给药,给药1次。每天观察动物临床症状,分别在给药后24 h,2周解剖动物。每只动物采集组织脏器包括血液、睾丸/子宫、附睾/卵巢、肾脏、脾脏、小肠、肠系膜淋巴结、肝脏、胃、肺、心脏、脑、注射部位(肌肉或肿瘤)、骨髓。对组织样本提取DNA后,采用qPCR技术,Taqman外标绝对定量方法对样本进行测定,检测受试物在各组织器官中拷贝数。结果 正常小鼠试验结果显示,受试物分布靶器官是脾脏、小肠、肠系膜淋巴结、肝脏、肺、注射部位(肌肉)、骨髓、血液,主要富集于脾脏、肝脏和注射部位。随着时间延长药物逐渐被清除,不会在体内蓄积。荷瘤小鼠试验结果显示,受试物体内分布靶器官和代谢消除趋势,与正常小鼠生物分布特征一致。并且,未见受试物在肿瘤组织和其他靶器官中可复制性增长。结论 采用正常小鼠和荷瘤小鼠分别进行基因治疗药物rAAV2-sTRAIL体内生物分布研究,受试物在两种模型中分布特征一致,均无可复制性和体内蓄积性。本实验初步预测了受试物临床使用安全性,为临床试验设计提供了参考信息。
Abstract
OBJECTIVE To conduct biodistribution studies of gene therapy pharmaceutical rAAV2-sTRAIL in both normal and tumor bearing BALB/c mice. METHODS BALB/c mice were divided randomly into three groups including vehicle control group(0 vg) with eight animals per sex, low-dose (1.5×109 vg) and high-dose (1.5×1010 vg) groups with 20 animals per sex for each group. The mice were administered the test drug intramuscularly once and observed for clinical symptoms daily and dissected at 24 h, 2, 4,12 week to get organs. BALB/c nude mice were transplanted ACC-2 cells at armpit of right fore to form tumor block which was considered appropriately to use when most of them grew up to 100 mm3. Tumor bearing mice were sorted into three groups according to tumor size, which were vehicle control group(0 vg) with four animals per sex, low-dose (1.5×109 vg) and high-dose (1.5×1010 vg) groups with ten animals per sex for each group.The mice were administered the test drug intratumorally once and dissected at 24 h and 2week after dosing. Blood, testis (uterus), epididymis (ovary), kidney, spleen, small intestine, mesenteric glands, liver, stomach, lung, heart, brain, injection site (muscle or tumor), sternum marrow were collected from each animal of normal or tumor model. DNA of tissues was extracted and test article copies in it were detectedwith Taqman external standard method. RESULTS The normal experiment showed that the target organs were spleen, small intestine, mesenteric glands, liver, lung, muscle, sternum marrow, blood and the test drug mainly concentrated in spleen, liver and injection site. Drugs were cleared up with time extending and were not accumulated in the body. Tumor model experiment showed similar results in distribution and elimination features, and the test articles didnot exhibit reproducibility in tumor and other organs. CONCLUSION The gene therapy pharmaceutical rAAV2-sTRAIL showed similar distribution characteristic in normal and tumor bearing BALB/c mice, and were not reproducible and accumulative in the body. Our work helps to forecast rAAV2-sTRAIL safety preliminarily for clinical use and give suggestive information for clinical trial design.
关键词
rAAV2-sTRAIL /
荧光定量PCR /
实时定量核酸扩增 /
生物分布 /
安全性评价
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Key words
rAAV2-sTRAIL /
real-time quantitative PCR /
qPCR /
biodistribution /
safety evaluation
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王欣, 李伟, 郑德先, 王超, 苗玉发, 周晓冰, 王三龙, 李波, 霍艳.
基因治疗药物rAAV2-sTRAIL在小鼠体内生物分布研究[J]. 中国药学杂志, 2021, 56(13): 1076-1082 https://doi.org/10.11669/cpj.2021.13.010
WANG Xin, LI Wei, ZHENG De-xian, WANG Chao, MIAO Yu-fa, ZHOU Xiao-bing, WANG San-long, LI Bo, HUO Yan.
Biodistribution Study of Gene Therapy Pharmaceutical rAAV2-sTRAIL in Mice[J]. Chinese Pharmaceutical Journal, 2021, 56(13): 1076-1082 https://doi.org/10.11669/cpj.2021.13.010
中图分类号：
R915
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参考文献
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