Drug Development of GLP-1 receptor agonists

当存在2型糖尿病时，制造和分泌胰岛素的胰腺β细胞不像非糖尿病受试者的β细胞那样响应。具体地，患有2型糖尿病的受试者响应于葡萄糖而具有第一时相的钝化或甚至绝对损失和严重钝化的第二时相胰岛素释放。与此同时，尽管目前有所有治疗糖尿病的方法，β细胞功能仍会随着时间的推移而恶化。根据英国前瞻性糖尿病研究（9月11日）的数据，1998年，这一点被更有力地带回家。尽管对参与研究的患者进行了持续监测，但由于β细胞功能下降，即使采用强化治疗也无法维持正常。我们已经研究GLP-1一段时间了，GLP-1是一种天然存在的肽，在食物反应中从肠道产生和释放。释放的量取决于摄入的葡萄糖和脂肪的量。在其血浆水平增加后，GLP-1与β细胞上的GLP-1受体（GLP-1 R）结合，并因cAMP生成而增加PKA活性。在PKA活性增加的下游，胰岛素释放也以葡萄糖依赖性方式发生，血糖由于进餐而升高。最终结果是血糖恢复至基线水平。因此，GLP-1类似物和GLP-1 R激动剂作为2型糖尿病的治疗正在进行深入研究。一种天然存在的GLP-1 R激动剂exendin-4现在可用于治疗。它与GLP-1 R的结合亲和力是天然GLP-1的10倍。它含有许多与GLP-1同源的氨基酸，并且有趣的是，含有独特的9个氨基酸C-末端。我们已经通过研究当将其添加到GLP-1的C-末端时对GLP-1的影响以及通过从毒蜥外泌肽-4顺序地和以三联体删除9个氨基酸来广泛地研究了C-末端的功能意义。将其添加到GLP-1中可提高其与自身受体的结合亲和力，将其从exendin-4中删除可消除其对GLP-1 R的亲和力增加。因此，对于毒蜥外泌肽-4对GLP-1 R的超激动剂性质，需要9个氨基酸的C-末端。最近，我们一直在研究GLP-1融合蛋白，其中GLP-1融合到人转铁蛋白，从而延长其半衰期，在体外，几天。融合蛋白通过重组技术在酵母中合成，并分泌到从中提取融合蛋白的酵母肉汤中。使用GLP-1本身的一个主要障碍是它的半衰期只有几分钟，因为它通过二肽基肽酶1V去除其N-末端的前两个氨基酸而迅速失活。我们修饰了融合蛋白中的GLP-1，使其不是二肽基肽酶1V的底物。我们已经在小型和大型动物中进行了体内工作，以描绘融合蛋白的动力学和给药方案，并且FDA已经批准了人体给药方案。此外，我们现在已经将exendin-4融合到转铁蛋白上，发现与单独的exendin-4相比，啮齿动物中exendin-4的活性没有损失。我们希望获得FDA批准用于人体研究。 另一种促胰岛素肽GIP也在进食后从肠道释放，但是，根据BLSA数据，一旦血糖超过126 mg/dl，胰腺的β细胞就会对其作用产生抵抗。此外，使用外源性GIP的人体内实验方案已经显示，β细胞不会响应于外源性人GIP而增加胰岛素分泌。GIP也会被二肽基肽酶1V快速分解。因此，我们目前正在2型糖尿病患者中测试二肽基肽酶1V抗性GIP肽是否可以成功增加β细胞的胰岛素分泌。这项研究涉及24名受试者，其中增加浓度的肽已给予最高20 ng/kg/min。这项协议将在今年完成。

Beta cells of the pancreas, which make and secrete insulin, do not respond like those of non-diabetic subjects when type 2 diabetes is present. Specifically, subjects suffering from type 2 diabetes have a blunted or even absolute loss of first phase and a severely blunted second phase insulin release in response to glucose. In conjunction with this, and despite all treatments currently available to treat diabetes, beta cell function continues to deteriorate over time. With the data now available from the United Kingdom Prospective Diabetes Study (Sept. 1998) this point was brought home even more forcefully. Despite continual monitoring of patients enrolled in the study, euglycemia could not be maintained even with intensive therapy, because of declining beta cell function. We have been working for some time with GLP-1, a naturally occurring peptide produced and released from the gut in response to food. The amount released depends on the amount of glucose and fat that has been ingested. After its plasma levels increase, GLP-1 binds to the GLP-1 receptor (GLP-1R) on beta cells, and increases PKA activity because of cAMP generation. Downstream of the increased PKA activity, insulin release occurs also in a glucose-dependent manner, plasma glucose having risen because of the meal. The end result is a restoration of plasma glucose back to baseline. Consequently, GLP-1 analogs and GLP-1R agonists are under intense study as treatments for type 2 diabetes. A naturally occurring GLP-1R agonist, exendin-4, is now available for treatment. It binds to GLP-1R with ten times more avidity than does native GLP-1. It contains many amino acids homologous to GLP-1 and, interestingly, contains a unique 9 amino acid C-terminus. We have extensively investigated the functional significance of the C-terminus by studying the effects on GLP-1 when it is added to the C-terminus of GLP-1 and by deleting the 9 amino acids sequentially and in triads from exendin-4. Its addition to GLP-1 improved its binding affinity to its own receptor and its deletion from exendin-4 abrogated its increased affinity for GLP-1R. Therefore, for superagonist properties of exendin-4 to GLP-1R the 9 amino acid C-terminus is required. More recently, we have been working on a GLP-1 fusion protein whereby GLP-1 is fused to human transferrin, thus extending its half-life, in vitro, by several days. The fusion protein is synthesised in yeast by recombinant technology and secreted into the yeast broth from which it is extracted. A major impediment to using GLP-1 itself is that its half-life is only a few minutes because it is rapidly inactivated by removel of its first two amino acids at its N-terminus by dipeptidyl peptidase 1V. We have modified the GLP-1 in the fusion protein so that it is not a substrate for dipeptidyl peptidase 1V. We have carried out in vivo work in small and large animals to delineate kinetics and dosing schedules of the fusion protein and FDA approval has been granted for a dosing schedule in humans. Additionally, we have now fused exendin-4 to transferrin and found there is no loss of exendin-4 activity in rodents, when compared to exendin-4 alone. We hope to receive FDA approval for its study in humans. Another insulinotropic peptide, GIP, is also released from the gut after eating, but, based on BLSA data, beta cells of the pancreas appear resistant to its effects once blood glucose exceed 126 mg/dl. Additionally, in vivo experimental protocols in humans using exogenous GIP have shown that beta cells do not increase insulin secretion in response to exogenous human GIP. GIP is also subject to rapid breakdown by dipeptidyl peptidase 1V. Therefore, we are currently testing in type 2 diabetic humans if a dipeptidyl peptidase 1V-resistant GIP peptide might be successful at increasing insulin secretion from beta cells. This study involves 24 subjects to which increasing concentrations of the peptide have been given up to a maximum of 20 ng/kg/min. This protocol will be finished this year.