Synthesis and biological properties of insulin - Deoxycholic acid chemical conjugates
Synthesis and biological properties of insulin - Deoxycholic acid chemical conjugates
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DOI：
10.1021/bc049871e
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发表时间：
2005-05-01
影响因子：
4.7
通讯作者：
Byun, Y
中科院分区：
文献类型：
--
作者：
Lee, S;Kim, K;Byun, Y
胆汁酸由于其天然的化学和生物学性质，被认为在新药物的制备中非常有用，并且最近在肽和蛋白质药物的制备中非常有用。在这项研究中，我们通过共价连接脱氧胆酸（DOCA）衍生物来修饰重组人胰岛素，以合成口服活性胰岛素类似物。制备DOCA衍生物，即琥珀酰亚胺脱氧胆酸盐和琥珀酰亚胺双脱氧胆酰-L-赖氨酸，并在胰岛素的Lys（B19）处位点特异性缀合。研究了所得胰岛素缀合物[N-B29-脱氧胆酰基]胰岛素（Ins-DOCA）和[N-B29-双脱氧胆酰基-L-lysil]胰岛素（Ins-bisDOCA）的化学、结构和生物学性质。通过HPLC、MALDIT-OF质谱和动态光散射测定了它们的化学性质。随着标记胆汁酸数目的增加，胰岛素结合物的亲脂性和自聚集行为增强。远紫外区的圆二色性光谱表明，由于共轭作用，胰岛素在水溶液中的三级结构没有明显变化。与HepG 2细胞的竞争性胰岛素结合试验表明，单取代的胰岛素缀合物仍然保留了对胰岛素受体的高结合亲和力。当将胰岛素缀合物静脉内施用（0.33 IU/kg）至链脲佐菌素（STZ）诱导的糖尿病大鼠时，与天然胰岛素相比，缀合物显示持续较长时间的生物活性，具有相似的最低血糖水平（葡萄糖最低值）。在进一步的研究中，将研究所得的新胰岛素缀合物作为用于治疗糖尿病患者的长效胰岛素制剂的口服效率。
Bile acids have been considered very useful in the preparation of new pharmaceuticals, and more recently in the preparation of peptide and protein drugs because of their natural chemical and biological properties. In this study, we modified recombinant human insulin by covalently attaching deoxycholic acid (DOCA) derivatives in order to synthesize orally active insulin analogues. DOCA derivatives, namely succinimido deoxycholate and succinimido bisdeoxycholyl-L-lysine were prepared and site specifically conjugated at Lys(B19) of insulin. The resultant insulin conjugates, [N-B29-deoxycholyl] insulin (Ins-DOCA) and [N-B29-bisdeoxycholyl-L-lysil] insulin (Ins-bisDOCA), were studied for their chemical, structural, and biological properties. Their chemical properties were determined by HPLC, MALDIT-OF mass spectroscopy, and dynamic light scattering. Lipophilicity and self-aggregation behavior of insulin conjugates were enhanced with increasing number of labeled bile acid. The far-ultraviolet region of circular dichroism spectra showed no significant change of the tertiary structure of insulin in aqueous solution due to conjugation. Competitive insulin binding assay with HepG2 cells revealed that monosubstituted insulin conjugates still retained high binding affinity to the insulin receptor. When the insulin conjugates were intravenously administered (0.33 IU/kg) to streptozotocin (STZ)-induced diabetic rats, the conjugates showed sustained biological activity for a longer period with the similar lowest blood glucose level (glucose nadir), compared to native insulin. In further studies, the resulting new insulin conjugates will be investigated for their oral efficiency as a long-acting insulin formulation for the treatment of diabetic patients.