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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。
In order to achieve a more sustained pharmacological effect in the body, according to the feature that cysteine trichotropin 17 is a free cysteine residue and transferrin Tf has no free cysteine, N-hydroxysuccinamide Imine-polyethylene glycol 5K-maleimide (NHS-PEG5k-MAL) as a coupling agent to achieve the two point-by-point coupling, and then the purification of the protein with its own characteristics to develop methods to obtain the purity of more than 90% Of transferrin-polyethylene glycol 5k-ciliary neurotrophic factor (Tf-PEG5k-CNTF) conjugate. High performance gel chromatography and dynamic light scattering analysis showed that the apparent molecular volume of the conjugate was greater than the sum of the two proteins. Cell test results showed that the activity of the conjugate decreased to 65.8% of the original protein. Pharmacokinetic studies in rats showed that the in vivo half-life of the Tf-PEG5k-CNTF conjugate was prolonged to 8.2 hours, an increase of about 17-fold compared to the original protein of CNTF. Animal experiments in mice show that Tf-PEG5k-CNTF can significantly affect food intake and weight loss in mice at twice-weekly dosing rates of 1.0 mg / kg. Therefore, transferrin coupling technology can be used for the long-term delivery of brain-targeting protein drugs.