溶瘤病毒药物HSV-1/hPD-1在食蟹猴体内生物分布研究

doi:10.19803/j.1672-8629.20220181

中国药物警戒 ›› 2023, Vol. 20 ›› Issue (1): 12-18.
DOI: 10.19803/j.1672-8629.20220181

• 基因治疗产品安全性评价专栏 • 上一篇下一篇

溶瘤病毒药物HSV-1/hPD-1在食蟹猴体内生物分布研究

王欣¹, 孙立¹, 王超¹, 李路路¹, 王三龙¹, 刘家家², 田超², 李小鹏^2#, 耿兴超^1,*

¹中国食品药品检定研究院国家药物安全评价监测中心,药物非临床安全评价研究北京市重点实验室,北京100176;
²北京唯源立康生物科技有限公司,北京100085

收稿日期:2022-04-22 发布日期:2023-01-19
通讯作者: ^*耿兴超,男,博士,研究员,药物安全评价。E-mail：gengxch@nifdc.org.cn
^#为共同通信作者。
作者简介:王欣,女,硕士,副研究员,新药临床前安全性评价。
基金资助:
国家重点研发计划（2021YFA1101602）; 国家科技重大专项重大新药创制（2018ZX09201-017）

Biodistribution of oncolytic virus HSV-1 expressing human PD-1 antibody in cynomolgus macaques

WANG Xin¹, SUN Li¹, WANG Chao¹, LI Lulu¹, WANG Sanlong¹, LIU Jiajia², TIAN Chao², LI Xiaopeng^2#, GENG Xingchao^1,*

¹National Center for Safety Evaluation of Drugs, National Institutes for Food and Drug Control, Key Laboratory of Beijing for Nonclinical Safety Evaluation Research of Drugs, Beijing 100176, China;
²Beijing WellGene Company Ltd., Beijing 100085, China

Received:2022-04-22 Published:2023-01-19

摘要/Abstract

摘要： 目的拟建立检测溶瘤病毒药物HSV-1/hPD-1在小鼠和食蟹猴各组织器官中生物分布的实时定量核酸扩增检测（qPCR）试验方法,并利用该方法对食蟹猴重复给药毒性研究中伴随生物分布（含排泄）研究收集的样本进行检测。方法针对药物的特异性基因序列,设计合成引物对和探针,摸索适宜的PCR反应条件,构建检测用标准曲线并进行方法学验证。对食蟹猴重复给药HSV-1/hPD-1长毒试验中收集的组织脏器,包括血液、性腺（睾丸/附睾或子宫/卵巢）、肾、肝脏、肺、心脏、脑、脾脏、肠系膜淋巴结、腹股沟淋巴结、注射部位、泪腺、唾液腺、背根神经节、尿、粪进行qPCR检测,测定药物在各组织器官中拷贝数。结果成功建立标准曲线pMD18T-1093,可用于定量检测药物在小鼠或食蟹猴基因组中分布拷贝数,是通用型标准曲线。食蟹猴组织检测结果显示,药物主要分布在给药部位,少量分布至肺、脑,经过尿液排泄至体外,在其他组织脏器无分布。靶器官分布数量呈剂量依赖性增加,会伴随时间延长减少至消除,不会在体内蓄积。脑组织检出药物分布不排除与样本污染有关。结论本研究成功建立了在非临床阶段开展生物分布研究的检测技术方法。试验方法的建立过程和研究思路对于其他基因治疗药物开展生物分布研究具有一定的借鉴意义。

关键词: 溶瘤病毒, 基因治疗, 生物分布, 实时定量核酸扩增检测技术, 重复给药毒性, 安全性评价, 食蟹猴

Abstract: Objective To establish a real-time quantitative PCR (qPCR) analysis method to investigate the biodistribution of oncolytic virus product HSV-1/hPD-1 in mice and cynomolgus macaques, and to detect samples collected during the concomitant biodistribution (shedding included) study in the process of investigating the repeat-dose toxicity in cynomolgus macaques. Methods According to the specific genetic sequences of drugs, primers and probes were designed and a proper PCR reaction system was established. Standard curves for detection were constructed and verified methodologically. Tissues and organs collected during repeat-dose toxicity experiments, including blood, gonads, kidneys, livers, lungs, hearts, brains, spleens, lymph nodes (mesenteric and inguinal), injection sites (skeletal muscle),tear gland, salivary gland, dorsal root ganglion, urine and feces, were subjected to qPCR detection. Results A universal standard curve named pMD18T-1093 was constructed, which could be used to quantitatively determine the copy number of the tested drugs in different tissues of mice and cynomolgus macaques. The tested drugs predominately remained at the injection sites and were sparsely distributed to the lung and brain before being cleared away by urine. Drug distribution was not detected in any other tissues of cynomolgus macaques. The amount of drugs located in target organs increased in a dose-dependent manner and decreased gradually with time rather than being accumulated in the body. Drugs detected in the brain might have been due to sample pollution. Conclusion A qPCR analysis method for preclinical biodistribution studies has been established in the study, which is expected to facilitate future studies on biodistribution of other drugs for gene therapy.

Key words: oncolytic virus, gene therapy, biodistribution, real-time quantitative PCR technique, repeat-dose toxicity, safety evaluation, cynomolgus macaques

中图分类号:

R969.1

王欣, 孙立, 王超, 李路路, 王三龙, 刘家家, 田超, 李小鹏, 耿兴超. 溶瘤病毒药物HSV-1/hPD-1在食蟹猴体内生物分布研究[J]. 中国药物警戒, 2023, 20(1): 12-18.

WANG Xin, SUN Li, WANG Chao, LI Lulu, WANG Sanlong, LIU Jiajia, TIAN Chao, LI Xiaopeng, GENG Xingchao. Biodistribution of oncolytic virus HSV-1 expressing human PD-1 antibody in cynomolgus macaques[J]. Chinese Journal of Pharmacovigilance, 2023, 20(1): 12-18.

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溶瘤病毒药物HSV-1/hPD-1在食蟹猴体内生物分布研究

Biodistribution of oncolytic virus HSV-1 expressing human PD-1 antibody in cynomolgus macaques

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