转铁蛋白-PEG-睫状神经营养因子的制备及其生物活性评价

Preparation of Transferrin Conjugated Ciliary Neurotrophic Factor and Evaluation of Biological Activity

为了实现在体内更持久的药效作用,根据睫状神经营养因子CNTF第17位为游离半胱氨酸残基,而转铁蛋白Tf无游离半胱氨酸的特点,采用N-羟基琥珀酰亚胺-聚乙二醇5K-马来酰亚胺(NHS-PEG5k-MAL)作为偶联剂,实现了两者定点偶联,然后结合蛋白自身特性制定纯化方法,制备获得纯度高于90%的转铁蛋白-聚乙二醇5k-睫状神经营养因子(Tf-PEG5k-CNTF)耦合物。高效凝胶色谱和动态光散射分析显示耦合物的表观分子体积大于两蛋白之和。细胞试验结果显示耦合物的活性下降至原蛋白的65.8%。大鼠药代动力学试验显示Tf-PEG5k-CNTF耦合物在体内的代谢半衰期延长至8.2小时,与CNTF原蛋白相比提高了约17倍。小鼠动物试验显示在每周2次的给药频率,每次1.0 mg/kg的剂量下,Tf-PEG5k-CNTF能更为显著地影响小鼠对食物摄入量和减轻体重。因此,转铁蛋白偶联技术可用于脑部靶向蛋白药物的长效递送。

To achieve a persistent in vivo efficacy,based on the characteristic that ciliary neurotrophic factor has a free cysteine residue while transferrin does not,transferrin was coupled to ciliary neurotrophic factor by using NHS-PEG5k-MAL.Combining with the purification method of each module,transferrin-PEG5k-CNTF conjugate was successfully prepared with a purity of above 90%.High performance size exclusion chromatography and dynamic light scattering analysis showed the apparent molecular volume of Tf-PEG5k-CNTF was larger than CNTF and transferrin together.The survival activity of TF-1（CN5α-1）cells showed that the bioactivity of TfPEG5k-CNTF was decreased to 65.8% of the CNTF.Pharmacokinetics study showed that the in vivo half-life of Tf-PEG5k-CNTF conjugate extended from about 28 minutes to 8.2 hours,increasing by about 17 times compared with CNTF.Pharmacodynamics showed that Tf-PEG5k-CNTF impacted more significantly on reducing food intake and losing weight in mice with twice weekly administration of 1.0 mg/kg drugs.Thus,transferrin coupling technique could be used for long-acting protein drugs delivery to the brain.