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目的探讨体内外不同修饰度的聚乙二醇(PEG)化重组人白细胞介素-6(rhIL-6)的药效。方法以相对分子质量20000的PEG2-NHS修饰剂化学耦合修饰rhIL-6,利用毛细管电泳仪分离得单修饰PEG-rhIL-6(mono-PEG-rhIL-6)和双修饰PEG-rhIL-6(di-PEG-rhIL-6),采用标准方法(即7TD1细胞/MTT比色法)测定体外IL-6的生物学活性,参考品为已标定的rhIL-6。体内实验:小鼠腹腔注射环磷酰胺构建BALB/c小鼠模型,分为模型组、阳性对照组、mono-PEG-rhIL-6高剂量组、mono-PEG-rhIL-6低剂量组、di-PEG-rhIL-6高剂量组和di-PEG-rhIL-6低剂量组。各组按上述剂量1次/d皮下注射给药,连续5d,于给药的第3天各组小鼠同时腹腔注射环磷酰胺,每日1次,连续3d。于给药的第8、11、14、17天切尾采血测定各小鼠血小板数并进行统计学处理。结果体外生物活性检测表明,mono-PEG-rhIL-6与di-PEG-rhIL-6相差不大,均比rhIL-6低10倍;体内活性测定表明,阳性对照组、mono-PEG-rhIL-6和di-PEG-rhIL-6的高、低剂量组小鼠血小板数回升速度较同时期模型组快,它们之间的差异具有统计学意义(P<0.05)。结论 PEG单修饰和多修饰(≥2)的rhIL-6在体外均具有促7TD1细胞增殖的活性;在体内均具有抗环磷酰胺致小鼠血小板减少的作用。mono-PEG-rhIL-6和di-PEG-rhIL-6均是药物的主要成分。
Objective To investigate the pharmacodynamics of recombinant human interleukin-6 (rhIL-6) with different degrees of modification in vivo and in vitro. Methods Monolayer modified mono-PEG-rhIL-6 (PEG-rhIL-6) and PEG-rhIL-6 (double-modified PEG-rhIL-6) were modified by PEG-NHS with molecular weight of 20,000 by chemical coupling. di-PEG-rhIL-6). The biological activity of IL-6 in vitro was determined by standard method (ie 7TD1 cells / MTT colorimetric method). The reference material was rhIL-6. In vivo, BALB / c mice model was established by intraperitoneal injection of cyclophosphamide and divided into model group, positive control group, mono-PEG-rhIL-6 high dose group, mono-PEG-rhIL-6 low dose group, di -PEG-rhIL-6 high-dose group and di-PEG-rhIL-6 low dose group. Each group was administered subcutaneously once a day for 5 days. On the 3rd day of administration, each group mice were given intraperitoneal injection of cyclophosphamide once a day for 3 consecutive days. On the 8th, 11th, 14th, and 17th days of administration, tail blood samples were collected and the number of each platelet was counted and statistically analyzed. Results The in vitro biological activity assay showed that the mono-PEG-rhIL-6 and di-PEG-rhIL-6 were less than 10 times lower than rhIL-6. 6 and di-PEG-rhIL-6 high and low dose group mice platelet number rise faster than the model group at the same period, the difference between them was statistically significant (P <0.05). CONCLUSION: PEG-modified and multi-modified (≥2) rhIL-6 cells have the activity of promoting the proliferation of 7TD1 cells in vitro and the anti-cyclophosphamide-induced thrombocytopenia in mice. Both mono-PEG-rhIL-6 and di-PEG-rhIL-6 are the major components of the drug.