Enhancement of the specific activities of PEGylated proteins by a simple procedure using a reversible amino-protective reagent

使用可逆氨基保护试剂通过简单的程序增强聚乙二醇化蛋白质的特异性活性

• 批准号: 13672385
• 负责人: TSUTSUMI Yasuo
• 金额: $ 2.3万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672385/
• 关键词: PEGylation; interleukin-6; cytokine; Drug Delivery System; ポリエチレングリコール; プロテオーム創薬; ドラッグデリバリーシステム; 創薬; トランスレーショナルリサーチ; ライフサイエンス; 医療

We developed a novel method for the chemical modification of proteins with synthetic polymers to increase the therapeutic efficacy of the former in vivo. A pH-reversible amino-protective reagent, dimethylmaleic anhydride (DMMAn), was used for modification of interleukin-6 (IL-6) with polyethylene glycol (PEG). The novel PEGylated IL-6 (DmPEG-IL-6), which had been pretreated with DMMAn before PEGylation, showed up to a 140% increase in in vitro specific activity compared with PEG-IL-6 that had been synthesized by the previous method. Moreover, DmPEG-IL-6 caused thrombopoiesis more potently in mice than PEG-IL-6. The DmPEG-IL-6, having 3-4 PEG chains attached to the cytokine, showed the strongest thrombopoietic effect among the DmPEG-IL-6s with different molecular sizes that were tested. PEG-IL-6 had a 500-fold higher potency in stimulating thrombopoiesis than native IL-6, and DmPEG-IL-6 Fr.1 achieved a threefold higher thrombopoietic effect than PEG-IL-6. In addition, side-effects, such as an increase in the plasma fibrinogen level, were not observed after injection of either PEG-IL-6s or DmPEG-IL-6s. Additionally, we also found that PEGylation of tumor necrosis factor-alpha and G-CSF using DMMAn increased their specific activities. These results suggest that PEGylation with DMMAn pretreatment may become a useful means for clinical cytokine delivery.

我们开发了一种利用合成聚合物对蛋白质进行化学修饰的新方法，以提高前者在体内的治疗效果。用pH可逆氨基保护剂二甲基马来酸酐(DMMAn)对聚乙二醇化白介素6(IL-6)进行修饰。经DMMAn处理后的新型聚乙二醇化IL-6(DmPEG-IL-6)的体外比活性比用前一种方法合成的聚乙二醇化的IL-6提高了140%。此外，与聚乙二醇IL-6相比，DmPEG-IL-6在小鼠体内的促血小板生成作用更强。在所测试的不同分子尺寸的DmPEG-IL-6中，具有3-4个聚乙二醇链连接到细胞因子上的DmPEG-IL-6具有最强的促血小板作用。PEG-IL-6的促血小板生成活性是天然IL-6的500倍，而DmPEG-IL-6 Fr.1的促血小板生成作用是PEG-IL-6的三倍。此外，注射PEG-IL-6s或DmPEG-IL-6s后都没有观察到副作用，如血浆纤维蛋白原水平的增加。此外，我们还发现，在DMMAn的作用下，肿瘤坏死因子-α和G-CSF的聚乙二醇化增强了它们的比活性。这些结果表明，聚乙二醇化和DMMAn预处理可能成为临床细胞因子输送的一种有效手段。

项目成果

Tsunoda S. et al.: "Selective enhancement of thrombopoietic activity of PEGylated interleukin 6 by a simple procedure using a reversible amino-protective reagent"Br. J. Haematol.. 112. 181-188 (2001)

Tsunoda S. 等人：“通过使用可逆氨基保护试剂的简单程序，选择性增强聚乙二醇化白细胞介素 6 的血小板生成活性”Br。

Yamamoto Y., et al.: "Creation of lysine-deficient TNF-alpha with full bloactivity using phage libraries produces a novel PEGylation system"Nature Biotechnology. (in press).

Yamamoto Y.等人：“使用噬菌体文库创建具有完全活性的赖氨酸缺陷型 TNF-α 产生了一种新型聚乙二醇化系统”《自然生物技术》。

Yamamoto Y. et al.: "Molecular design of bioconjugated cell adhesion peptide with a water-soluble polymeric modifier for enhancement of antimetastatic effect"Current Drug Targets. 3(2). 123-130 (2002)

Yamamoto Y.等人：“使用水溶性聚合物改性剂进行生物共轭细胞粘附肽的分子设计，以增强抗转移作用”当前药物靶点。

Kamada H. et al.: "Synthesis of poly(vinylpyrrolldone-co-dimethyl maleic anhydride) co-polymer and its application as a renal targeting carrier"Nat. Biotechnol.. in press.

Kamada H.等人：“聚(乙烯基吡咯酮-共-二甲基马来酸酐)共聚物的合成及其作为肾靶向载体的应用”Nat。

Yamamoto Y. et al.: "Creation of lysine-deficient TNF-a with full bioactivity using phage libraries produces a novel PEGylation system"Nat. Biotechnol.. in press.

Yamamoto Y. 等人：“使用噬菌体文库创建具有完整生物活性的赖氨酸缺陷型 TNF-a，产生了一种新型聚乙二醇化系统”Nat.