利用家蚕蛹生物反应器生产的人粒细胞/巨噬细胞集落刺激因子口服给药的临床前安全性评价

开发了用家蚕蛹表达的人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)的口服药物。试验评价了猕猴、大鼠和小鼠口服rhGM-CSF毒性情况,包括遗传毒性、单剂量和重复剂量的全身毒性以及生殖毒性。毒理学试验显示:静脉给药在NIH小鼠中最大耐受剂量为3 300μg/kg,LD50为2 020μg/kg,在SD大鼠中LD50为3 660μg/kg。在猕猴和大鼠的重复剂量毒性试验结果中显示:口服rhGM-CSF后,除了雌性SD大鼠的血糖含量外,其余各项指标包括白细胞、红细胞、血红蛋白含量、血小板数量以及血细胞凝集等都正常。雌性大鼠每天口服剂量为1 250μg/kg时,给药后血糖浓度显著增加(P<0.001),但在恢复期间可恢复到正常水平。微核实验、CHL染色体畸变试验和埃姆斯测验法都表明无论是在体内还是体外服用rhGM-CSF均未见遗传毒性,普通的生殖毒性试验和围产期及致畸敏感期的毒性试验均未见异常现象,表明口服rhGM-CSF无明显生殖毒性。临床前毒性试验表明口服超过临床剂量10倍的rhGM-CSF与严重的慢性中毒没有联系,其在药理学有效剂量的范围内是很安全的。

Preclinical Safety Evaluation of Oral Drug of Human Granulocyte-Macrophage Colony-Stimulating Factor Expressed in Silkworm Pupae As Bioreactor

We developed an oral drug of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) expressed in silkworm pupae. Our goal in this study was to determine the toxicity of oral drug of rhGM-CSF in monkeys (Macaca mulatto), rats and mice, including genetic toxicology, single-and repeat-dose systemic toxicity and reproductive toxicity. Toxicology tests showed that the maximum tolerated dose (I.v) was 3 300 μg/kg, and that the LD50 (I.v) was 2 020 μg/kg in NIH mice, and 3.660 μg/kg in SD rats respectively. Repeat-dose toxicity test of oral drug of rhGM-CSF in monkeys and rats showed that all indices (including white blood cell, red blood cell, haematoglobin content, platelet number, and hemagglutination) were normal, with the exception of glucose content in female rats, which increased significantly (P＜0.001) after oral administration of the high-dose 1 250 μg·kg-1·d-1 rhGM-CSF while it reversibly was normal during recovery. It was found that rhGM-CSF oral drug showed no genotoxicity either in vitro or in vivo through the micronucleus test, CHL chromosome aberration test and Ames test. No abnormality was found in general reproductive toxicity test, and toxicity test in perinatal stage and sensitive period to teratogenic agent, which suggests little reproductive toxicity. Preclinical toxicity indicated administration of the rhGM-CSF oral drug in excess of ten times as many as clinical dosages was not associated with severe chronic toxicities and it is very safe at the range of pharmacologically functional dose. This study represents the first report of detailed toxicological evaluation of human protein drug expressed in silkworm using baculovirus expression system.