Mono-PEGylated Dimeric Exendin-4 as High Receptor Binding and Long-Acting Conjugates for Type 2 Anti-Diabetes Therapeutics
Mono-PEGylated Dimeric Exendin-4 as High Receptor Binding and Long-Acting Conjugates for Type 2 Anti-Diabetes Therapeutics
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DOI：
10.1021/bc100404x
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发表时间：
2011-04-01
影响因子：
4.7
通讯作者：
Lee, Kang Choon
中科院分区：
文献类型：
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作者：
Kim, Tae Hyung;Jiang, Hai Hua;Lee, Kang Choon
二聚化被认为是增加受体结合亲和力的最有效手段，并且二聚化和PEG化都通过延迟经由肾途径的排泄而有效地延长短寿命肽和蛋白质在体内的寿命。在这里，我们描述了两种长效毒蜥外泌肽-4（Ex 4）缀合物（二聚Ex 4（Di-Ex 4）和聚乙二醇化Di-Ex-4（PEG-Di-Ex 4））的高结合亲和力。使用Ex 4-Cys、bisMal-NH 2和活化的PEG，使用半胱氨酸和胺残基特异性偶联反应制备Di-Ex 4和PEG-Di-Ex 4。所产生的Ex 4缀合物具有高纯度（>98.5%），如通过尺寸排阻色谱法和MALDI-TOF质谱法所测定的。Di-Ex 4对RIN-m5 F细胞的受体结合亲和力比Ex 4高3.5倍，并且Di-Ex 4在2型糖尿病db/db小鼠中的体内抗高血糖功效也大于天然Ex 4。此外，发现Di-Ex 4和PEG-Di-Ex 4具有比天然Ex 4分别大2.7倍和13.7倍以及4.0倍和17.3倍的血液循环t（1/2）和AUC（inf）值。因此，在25 nmol/kg的剂量下，低血糖持续时间分别大大增加至15.0和40.1 h;（天然Ex 4 7.3 h）。本研究的结果表明，将二聚化和聚乙二醇化结合应用于Ex 4是有效的，并表明PEG-Di-Ex 4作为2型抗糖尿病药物具有相当大的潜力。
Dimerization is viewed as the most effective means of increasing receptor binding affinity, and both dimerization and PEGylation effectively prolong the life spans of short-lived peptides and proteins in vivo by delaying excretion via the renal route. Here, we describe the high binding affinities of two long-acting exendin-4 (Ex4) conjugates, dimerized Ex4 (Di-Ex4) and PEGylated Di-Ex-4 (PEG-Di-Ex4). Di-Ex4 and PEG-Di-Ex4 were prepared using cysteine and amine residue specific coupling reactions using Ex4-Cys, bisMal-NH2, and activated PEG. The Ex4 conjugates produced were of high purity (>98.5%), as determined by size-exclusion chromatography and MALDI-TOF mass spectrometry. The receptor binding affinity of Di-Ex4 on RIN-m5F cells was 3.5-fold higher than that of Ex4, and the in vivo antihyperglycemic efficacy of Di-Ex4 was also greater than that of native Ex4 in type 2 diabetic db/db mice. Furthermore, Di-Ex4 and PEG-Di-Ex4 were found to have greater blood circulating t(1/2) and AUC(inf) values than native Ex4 by 2.7- and 13.7-fold, and by 4.0- and 17.3-fold, respectively. Accordingly, hypoglycemic durations were greatly increased to 15.0 and 40.1 h, respectively, at a dose of 25 nmol/kg; (native Ex4 7.3 h). The results of this study show that combined dimerization and PEGylation are effective when applied to Ex4, and suggest that PEG-Di-Ex4 has considerable potential as a type 2 anti-diabetic agent.