摘要
目的分析注射用A型肉毒毒素和注射用A型肉毒神经毒素活性药用成分(active pharmaceutical ingredient,API)的纯度、N-末端氨基酸序列和相对分子质量,并通过多个非临床研究试验,评价两种制品在药理、毒理特性等方面的一致性。方法采用SDS-PAGE法检测注射用A型肉毒毒素(衡力~,Lantox~,以下简称复合物)和注射用A型肉毒神经毒素(Chintox~,以下简称神经毒素)的纯度;对其N-末端氨基酸序列进行测序,并将测序结果与NCBI blast数据库中A型肉毒梭菌Hall株的氨基酸序列比对,确证其各组分的成分;毛细管凝胶电泳(capillary gel electrophoresis,CGE)检测其在非还原及还原条件下各组分的相对分子质量;单次肌肉注射、静脉注射、灌胃给予大鼠及单次肌肉注射给予食蟹猴的试验评价复合物和神经毒素的急性毒性;重复肌肉注射给予食蟹猴39周和恢复期12周的药理毒理试验评价复合物和神经毒素的长期毒性;家兔红细胞的体外溶血试验评价两者的溶血性;肌肉注射给予大鼠Ⅰ、Ⅱ段生殖毒性试验评价肉毒毒素的生殖毒性。结果神经毒素组:非还原条件下,神经毒素相对分子质量约152 000,N-末端氨基酸序列ALNDLQINVN,为完整的A型肉毒神经毒素;还原条件下,由相对分子质量约101 000,N-末端氨基酸序列ALNDLQINVN的重链和相对分子质量约51 000、N-末端氨基酸序列PFVNKQFNYK的轻链组成;复合物组:非还原条件下,含A型肉毒神经毒素(Mr 152 000)、非毒素非血凝素蛋白(non-toxic non-HA,NTNH)(Mr 136 000)、HA70组分(Mr 57 000、17 000)、HA33组分(Mr 30 000、28 000)和HA17组分(Mr 15 000),还原条件下,则含有重链、轻链、NTNH、HA70、HA33和HA17组分。注射用A型肉毒毒素、注射用A型肉毒神经毒素单次肌肉注射和静脉注射给予大鼠,肌肉注射的最大耐受剂量≥100 U/kg,静脉注射的最大耐受剂量≥30 U/kg;单次肌肉注射给予食蟹猴所产生的急性中毒反应与死亡情况一致,最大耐受剂量均为20 U/kg,近似致死剂量为40 U/kg;反复肌肉注射给予食蟹猴的试验中,未见明显毒性作用剂量(no observed adverse effect level,NOAEL)均为16 U/kg;在体外对家兔红细胞无溶血作用,不引起红细胞凝聚;Ⅰ段生殖毒性试验对雄鼠生育力的NOAEL为4U/kg,对雌鼠生育力的NOAEL为8 U/kg,对孕鼠早期胚胎发育的NOAEL为16 U/kg;Ⅱ段生殖毒性试验对孕鼠的NOAEL为1 U/kg,对胚胎-胎仔毒性和致畸性的NOAEL为16 U/kg。结论注射用A型肉毒毒素和注射用A型肉毒神经毒素在食蟹猴和大鼠的急性毒性、长期毒性等试验中,剂量、动物病理解剖结果一致,进一步说明A型肉毒毒素和A型肉毒神经毒素的药理毒理特性是相同的,且在反复肌肉注射给予食蟹猴的长期毒性试验中发现,A型肉毒神经毒素更不易产生抗体。在神经毒素灌胃试验中半数致死剂量高于复合物,表明其安全性更好。
Objective To analyze the purity, N-terminal amino acid sequence and relative molecular mass of active pharmaceutical ingredients of botulinum toxin type A and botulinum neurotoxin type A, and evaluate the consistence of the two products in pharmacological and toxicological properties by several nonclinical studies. Methods Botulinum toxin type A（Lantox）and botulinum neurotoxin type A（Chintox）were determined for purity by SDS-PAGE, of which the N-terminal amino acids were sequenced, and the results were analyzed and compared with that of Hall strain in NCBIblast database to co nfirm the ingredients of various components. The relative molecular masses of various components under reduced and non-reduced conditions were determined by capillary gel electrophoresis（CGE）. The acute toxicity of botulinum toxin type A and botulinum neurotoxin type A were evaluated by administration in rats by single intramuscular injection, intravenous injection and gavage as well as in cynomolgus monkeys by single intramuscular injection. Long-term toxicity was evaluated by pharmacological and toxicological tests after repeated intramuscular injection in cynomolgus monkeys for 39 weeks and a recovery period of 12 weeks. The hemolysis was evaluated by in vitro and in vivo hemolysis tests in rabbits. The reproductive toxicity of botulinum toxin type A was evaluated by reproductive toxicity tests of stages I and II by intramuscular injection in rats. Results The profiles of SDS-PAGE and CGE showed a single band under nonreducing condition, which was confirmed as a full neurotoxin with a relative molecular mass of about 152 000 and a N-terminal amino acid sequence of ALNDLQINVN. However, under reduced condition, the neurotoxin was cleaved to two fragments, i. e. the heavy chain with a relative molecular mass of about 101 000 and a N-terminal amino acid sequence of ALNDLQINVN and the light chain with a relative molecular mass of about 51 000 and a N-terminal amino acid sequence of PFVNKQFNYK. The botulinum toxin type A consisted of botulinum neurotoxin type A（Mr 152 000）, non-toxic nonHA（NTNH）（Mr 136 000）, HA70（Mr 57 000, 17 000）, HA33（Mr 30 000, 28 000）and the HA17（Mr 15 000）under non-reduced condition. However, under reduced condition, the complex consisted of heavy chain, light chain, NTNH,HA70, HA33 and HA17. The maximum tolerated dose of botulinum toxin type A and botulinum neurotoxin type A were not less than 100 U/kg for intramuscular injection, and not less than 30 U/kg for intravenous injection, in rats. Both the acute poisoning reactions and deaths in cynomogus monkeys after a single intramuscular injection with botulinum toxin type A and botulinum neurotoxin type A were in agreement. Both the maximum tolerated doses were 20 U/kg, while the approximate lethal doses were 40 U/kg. However, both the no observed adverse effect level（NOAEL）doses to cynomolgus monkeys after repeat intramuscular injection were 16 U/kg. No hemolysis or agglutination of rabbit red blood cells was observed in vitro. In the stage I reproductive toxicity test, the NOAEL dose was 4 U/kg to the fertility of male rats, 8 U/kg to that of female rats, and 16 U/kg to the early embryos development of pregnant rats. However, in the stage II reproductive toxicity test, the NOAEL dose was 1 U/kg to pregnant rats, and 16 U/kg to the toxicity and teratogenicity of embryo-fetus. Conclusion The dosage and animal pathological anatomy results of botulinum toxin type A were consistent with those of botulinum neurotoxin type A in acute and chronic toxicity tests in cynomolgus monkeys and rats, which further confirmed that pharmacological and toxicological properties of botulinum toxin type A were identical to those of botulinum neurotoxin type A. However, a long time toxicity test in cynomolgus monkeys by repeat intramuscular injection found that antibodies were not easily to be induced by botulinum neurotoxin type A. Furthermore, the median lethal dose of botulinum neurotoxin type A administered by gavage was higher than that of botulinum toxin type A, indicating a higher safety.
出处
《中国生物制品学杂志》
CAS
CSCD
2017年第7期710-717,共8页
Chinese Journal of Biologicals
关键词
A型肉毒毒素
A型肉毒神经毒素
非临床安全性评价
Botulinum toxin type A
Botulinum neurotoxin type A
Nonclinical evaluation on safety
