Recently, discovery of an incretin hormone that can efficiently regulate glucose homeostasis brought about a new therapeutic target in diabetes research. Glucagon-Like-Peptide-1 (GLP-1) is an incretin hormone that is secreted from small intestine in response to glucose levels in plasma. After binding to its cognate receptor (GLP-1R), GLP-1 stimulates release of insulin from pancreatic islets that starts the insulin-signaling cascade and ends up with storage of excess glucose as glycogen. Apart from its glucose lowering properties, GLP-1 also induces satiety after meals and slows down digestion by ceasing gastric emptying which both prevent overeating. This aspect of the peptide makes it even a more potent target as a therapeutic due to possible uses in obesity treatment. GLP-1 itself is also well controlled in the body. First, it is strictly glucose-dependent and therefore secreted when there is an increase in glucose levels. Secretion proceeds in a dose-dependent manner. Second, immediately after GLP-1 reaches circulation, a ubiquitous protease dipeptidyl peptidase IV (DPP-IV) hydrolyses the N-terminus end, rendering it inactive. Although this second feedback mechanism plays an important role in glucose homeostasis in healthy individuals, it constitutes a challenge for using GLP-1 as a therapeutic compound for diabetic patients due to stability problems.

It should be mentioned here that GLP-1 is not the only peptide drug option for diabetes. Amylin is another gastrointestinal peptide that has been identified as a potential therapeutic target. It is secreted after food intake from pancreatic cells, along with insulin. Advanced diabetic patients lack amylin due to loss of β-islets. Although mechanism of action is not well understood, it was shown to suppress glucagon secretion in a glucose-dependent manner and delay gastric emptying. Pramlintide is a synthetic amylin analog that has been approved by the FDA in 2005 for treatment of both Type I and Type II diabetes.

Challenges and drawbacks of peptide drugs

Among many reasons that peptides have been held back from being developed as drugs, low bioavailability is significant. Administration is generally intravenous injection in order to reach the target quickly without getting degraded along the way. Due to numerous proteolytic enzymes in the digestive tract and harsh gastric acid, oral administration is not applicable for peptides. Blood plasma also contains proteases that are responsible for degradation of any immunogenic peptide, therefore shortening the half-life of peptide drugs. Clearance can also be very rapid (mins) via hepatic (liver) and renal (kidneys) routes. Hydrophilicity of a peptide chain can also make it difficult to pass through biological membranes and reach to target organs. Therefore, even for an injectable peptide drug, chemical elaborations of the peptide structure are required to overcome these problems. Compared to protein and antibody drugs, peptides have more potential to be modified and mutated without compromising their activity. Several modifications that are commonly used are amino acid sequence mutations, PEGylation, cyclisation and glycosylation.

In order to enhance the plasma half-life of peptide drugs, chemical alterations such as C-terminal amidation and N-terminal acetylations, and amino acid substitution at protease sensitive sites are often utilized. Most of the time, poor protease stability of α-peptides cannot be improved by using natural L-α-amino acids. In order to maintain a longer plasma life, unnatural amino acids such as D-α-amino acids, side chain modified L-α-amino acids and amino acids with unnatural backbone (β- or γ-amino acids) are used.

Cyclization is another method for increasing proteolytic stability. It can be done via disulfide bond between two cysteine residues or head-to-tail cyclization via amide bond.

Attachment of polyethylene glycol (PEG) increases plasma resistance of the peptides and improves the solubility of larger proteins without affecting their activity. For example, PEGylation of Interferon-α has been shown to extent its serum life. In another case, high molecular weight PEG attachment to trichosantin has yielded low immunogenicty in the new conjugate, due to transfer of non-immunogenic properties of the PEG to the new compound.

Glycosylation is also shown to have dramatic effects on half-life and the immunogenicity of protein drugs. The half-life of the native erythropoietin23 has been improved by extra glycosylations on the protein.

Reference:

Diren Pamuk, Design and engineering of new glucagonlike-peptide-1 analogues.

GLP-1, another incretin hormone, was identified after the cloning and sequencing of the cDNA and genes encoding human proglucagon. The proglucagon gene encodes two peptides that share about 50% sequence homology to glucagon. The two peptides were named glucagon-like peptide-1 (GLP-1) and glucogan-like peptide-2 (GLP-2). After tissue-specific proteolytically posttranslational processing, GLP-1 is released from L cell of lower intestine and colon in response to nutrient ingestion. GLP-1 stimulates glucose-dependent insulin secretion in isolated islets as well as in human, whereas GLP-2 was unable to stimulate insulin secretion and is not an incretin hormone.

Regulation of GLP-1 and GIP secretion

GLP-1 is secreted from enteroendocrine L cells in the distal intestine in response to food ingestion. Glucose and fatty acids in food are the primary physiologic stimuli for GLP-1 secretion. Administration of mixed meals or individual nutrients such as glucose or other sugars, fatty acids, essential amino acids, and dietary fiber all can stimulate GLP-1 secretion. In humans, oral but not intravenous glucose administration stimulates GLP-1 secretion. It was also reported that zein hydrolysate (a hydrolysate prepared from zein: a major corn protein) stimulated GLP-1 secretion directly in the ileum and indirectly in the duodenum in rat. It was found that nutrients stimulated GLP-1 secretion through two alternative pathways: one is via direct contact with L cells and the other is through indirect information transfer. GLP-1 secretion generally includes two phases: an early phase occurred at 10 to 15 minutes and the second phase occurred at 30 to 60 minutes after oral nutrient ingestion. It seems that the second phase of GLP-1 secretion is caused by direct contact of nutrients with L cells in the distal small intestine, while it is unlikely that the early phase of GLP-1 secretion is caused by the same mechanism since it takes more than 15 minutes for nutrients to get to the distal small intestine after oral meal uptake. This suggests that the existence of a proximal gut signal regulating GLP-1 release from the L cells of the distal small intestine.

Many agents that can directly stimulate GLP-1 secretion have been identified in various models of the intestinal L cell, including a perfused model of the rat ileum, a murine intestinal endocrine cell line, and a primary cell culture of fetal rat intestinal cells. It was found that GIP, gastrin-releasing peptide (GRP), calcitonin gene-related peptide, and agonists of acetylcholine all can contribute to the rapid GLP-1 secretion (40-42). It has also been shown that leptin significantly stimulated GLP-1 secretion (by up to 250% of the control) from fetal rat intestinal cells, a mouse L cell line (GLUTag), and a human L cell line (NCI-H716) in a dose-dependent manner. Moreover, leptin also stimulated GLP-1 secretion in rat and ob/ob mouse models. However, high fat diet induced obese and diabetic mice showed decreased GLP-1 secretion in response to oral glucose administration. It was reported that autonomic nervous system plays an important role in the regulation of nutrient-induced GLP-1 secretion. Study in an in situ model of the rat gastrointestinal system showed that both corn oil and electrical stimulation of the celiac branches of the vagus can significantly increase GLP-1 secretion, whereas bilateral subdiaphragmatic vagotomy completely inhibited fat-induced GLP-1 secretion. Recently, studies using murine GLP-1-producing enteroendocrine cell lines showed that, at the cellular level, sodium-dependent glucose co-transporters and sweet taste receptor T1R3 mediate glucose-induced GLP-1 secretion. It also reported that fatty acid receptors GPR40 and GPR120 mediate fatty acids-induced GLP-1 secretion. In addition, GLP-1 secretion is also associated with activation of a number of intracellular signaling pathways including PKA, PKC, calcium, and MAPK.

GIP secretion is triggered by the ingestion of carbohydrate or fatty acids in intestinal K cells, which are located in the duodenal and jejunal epithelia. GIP is secreted 10 to 20 minutes after oral nutrients. Because the GIP-containing K cells are predominantly located in the duodenum and proximal jejunum and have apical surfaces opening into the gut lumen, GIP secretion is believed to be triggered by a direct nutrient contact with K cells. Studies on isolated perfused rodent intestine have suggested that carbohydrate detection by K cells involves the sodium-dependent sugar uptake pathway, which is consistent with the structural requirements for activation of the intestinal Na⁺-coupled glucose transporters. In contrast, the inhibitor of sodium-dependent glucose transporter 1 phloridzin inhibited glucose-stimulated GIP secretion in rodent intestine. Because there is a lack of validated cell models for studying GIP release in vitro, little is known about how K cells respond to glucose and other stimuli at the cellular and molecular levels. Several studies in cultured canine endocrine cells suggested that activation of adenylyl cyclase, increases in intracellular calcium ion levels, K⁺-mediated depolarization, glucose, GRP, and beta-adrenergic stimulation can increase GIP secretion.

Reference:

Zhangfang,Kang. Impaired Incretin Effects in Type 2 Diabetes: Mechanism and Therapeutic Implication. The Chinese University of Hong Kong, 2012