Lee Sang-Heon, Lee Seulki, Youn Yu Seok, Na Dong Hee, Chae Su Young, Byun Youngro, Lee Kang Choon
Drug Targeting Laboratory, College of Pharmacy, SungKyunKwan University, 300 Chonchon-dong, Jangan-ku, Suwon City 440-746, Korea.
Bioconjug Chem. 2005 Mar-Apr;16(2):377-82. doi: 10.1021/bc049735+.
Glucagon-like peptide-1-(7-36) (GLP-1) is a hormone derived from the proglucagon molecule, which is considered a highly desirable antidiabetic agent mainly due to its unique glucose-dependent stimulation of insulin secretion profiles. However, the development of a GLP-1-based pharmaceutical agent has a severe limitation due to its very short half-life in plasma, being primarily degraded by dipeptidyl peptidase IV (DPP-IV) enzyme. To overcome this limitation, in this article we propose a novel and potent DPP-IV-resistant form of a poly(ethylene glycol)-conjugated GLP-1 preparation and its pharmacokinetic evaluation in rats. Two series of mono-PEGylated GLP-1, (i) N-terminally modified PEG(2k)-N(ter)-GLP-1 and (ii) isomers of Lys(26), Lys(34) modified PEG(2k)-Lys-GLP-1, were prepared by using mPEG-aldehyde and mPEG-succinimidyl propionate, respectively. To determine the optimized condition for PEGylation, the reactions were monitored at different pH buffer and time intervals by RP-HPLC and MALDI-TOF-MS. The in vitro insulinotropic effect of PEG(2k)-Lys-GLP-1 showed comparable biological activity with native GLP-1 (P = 0.11) in stimulating insulin secretion in isolated rat pancreatic islet and was significantly more potent than the PEG(2k)-N(ter)-GLP-1 (P < 0.05) that showed a marked reduced potency. Furthermore, PEG(2k)-Lys-GLP-1 was clearly resistant to purified DPP-IV in buffer with 50-fold increased half-life compared to unmodified GLP-1. When PEG(2k)-Lys-GLP-1 was administered intravenously and subcutaneously into rats, PEGylation improved the half-life, which resulted in substantial improvement of the mean plasma residence time as a 16-fold increase for iv and a 3.2-fold increase for sc. These preliminary results suggest a site specifically mono-PEGylated GLP-1 greatly improved the pharmacological profiles; thus, we anticipated that it could serve as potential candidate as an antidiabetic agent for the treatment of non-insulin-dependent diabetes patients.
胰高血糖素样肽-1-(7-36)（GLP-1）是一种源自胰高血糖素原分子的激素，主要因其独特的葡萄糖依赖性胰岛素分泌刺激特性，被视为一种非常理想的抗糖尿病药物。然而，基于GLP-1的药物制剂开发存在严重限制，因为其在血浆中的半衰期极短，主要被二肽基肽酶IV（DPP-IV）酶降解。为克服这一限制，在本文中我们提出了一种新型且有效的聚乙二醇（PEG）共轭GLP-1制剂的DPP-IV抗性形式，并对其在大鼠中的药代动力学进行评估。通过分别使用甲氧基聚乙二醇醛和甲氧基聚乙二醇琥珀酰亚胺丙酸酯制备了两个系列的单PEG化GLP-1，（i）N端修饰的PEG(2k)-N(ter)-GLP-1和（ii）赖氨酸（Lys）(26)、赖氨酸(34)修饰的PEG(2k)-Lys-GLP-1异构体。为确定PEG化的优化条件，通过反相高效液相色谱（RP-HPLC）和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）在不同pH缓冲液和时间间隔下监测反应。PEG(2k)-Lys-GLP-1的体外促胰岛素作用在刺激分离的大鼠胰岛胰岛素分泌方面显示出与天然GLP-1相当的生物活性（P = 0.11），且比活性显著降低的PEG(2k)-N(ter)-GLP-1更有效（P < 0.05）。此外，与未修饰的GLP-1相比，PEG(2k)-Lys-GLP-1在缓冲液中对纯化的DPP-IV具有明显抗性，半衰期增加了50倍。当将PEG(2k)-Lys-GLP-1静脉内和皮下注射给大鼠时，PEG化改善了半衰期，导致平均血浆驻留时间大幅提高，静脉注射增加了16倍，皮下注射增加了3.2倍。这些初步结果表明，位点特异性单PEG化的GLP-1极大地改善了药理学特性；因此，我们预计它可作为治疗非胰岛素依赖型糖尿病患者的抗糖尿病药物的潜在候选物。
