Glucagon-like Peptide-1 Derivative Research Articles

GLP-1 derivatives with functional sequences transit and migrate through trigeminal neurons

Patients with dementia are increasing with the aging of the population, and dementia has become a disease with high unmet medical needs. Glucagon-like peptide-1 (GLP-1), a neuropeptide, has been reported to improve learning and memory following intracerebroventricular administration. We focused on intranasal administration, which can deliver drugs noninvasively and efficiently to the brain. Although much of the human nasal mucosa is occupied by respiratory epithelium, many capillaries are present in the paracellular route of respiratory epithelium. Therefore, to incorporate GLP-1 into cells, we created a GLP-1 derivative by adding cell-penetrating peptides (CPP) and penetration accelerating sequences (PAS) to GLP-1. We investigated in vitro and in vivo function of PAS-CPP-GLP-1 to enable the translocation of GLP-1 directly from nose to brain. PAS-CPP-GLP-1 enhanced cellular uptake by macropinocytosis with CPP, efficiently escaped from the endosomes due to PAS, and exited the cells. PAS-CPP-GLP-1 also transited trigeminal nerve cells through axon transport and migrated to the adjacent trigeminal nerve cell. Moreover, PAS-CPP-GLP-1 showed significant improvement in learning memory in mice within 20 min of intranasal administration. These results suggested CPP and PAS may be important for the efficient transfer of GLP-1 to the site of action in the brain following intranasal administration.

Optical tools for visualizing and controlling human GLP-1 receptor activation with high spatiotemporal resolution

The glucagon-like peptide-1 receptor (GLP1R) is a broadly expressed target of peptide hormones with essential roles in energy and glucose homeostasis, as well as of the blockbuster weight-loss drugs semaglutide and liraglutide. Despite its large clinical relevance, tools to investigate the precise activation dynamics of this receptor with high spatiotemporal resolution are limited. Here, we introduce a novel genetically encoded sensor based on the engineering of a circularly permuted green fluorescent protein into the human GLP1R, named GLPLight1. We demonstrate that fluorescence signal from GLPLight1 accurately reports the expected receptor conformational activation in response to pharmacological ligands with high sensitivity (max ΔF/F0=528%) and temporal resolution (τON = 4.7 s). We further demonstrated that GLPLight1 shows comparable responses to glucagon-like peptide-1 (GLP-1) derivatives as observed for the native receptor. Using GLPLight1, we established an all-optical assay to characterize a novel photocaged GLP-1 derivative (photo-GLP1) and to demonstrate optical control of GLP1R activation. Thus, the new all-optical toolkit introduced here enhances our ability to study GLP1R activation with high spatiotemporal resolution.

Optical tools for visualizing and controlling human GLP-1 receptor activation with high spatiotemporal resolution.

The glucagon-like peptide-1 receptor (GLP1R) is a broadly expressed target of peptide hormones with essential roles in energy and glucose homeostasis, as well as of the blockbuster weight-loss drugs semaglutide and liraglutide. Despite its large clinical relevance, tools to investigate the precise activation dynamics of this receptor with high spatiotemporal resolution are limited. Here, we introduce a novel genetically encoded sensor based on the engineering of a circularly permuted green fluorescent protein into the human GLP1R, named GLPLight1. We demonstrate that fluorescence signal from GLPLight1 accurately reports the expected receptor conformational activation in response to pharmacological ligands with high sensitivity (max ΔF/F0=528%) and temporal resolution (τON = 4.7 s). We further demonstrated that GLPLight1 shows comparable responses to glucagon-like peptide-1 (GLP-1) derivatives as observed for the native receptor. Using GLPLight1, we established an all-optical assay to characterize a novel photocaged GLP-1 derivative (photo-GLP1) and to demonstrate optical control of GLP1R activation. Thus, the new all-optical toolkit introduced here enhances our ability to study GLP1R activation with high spatiotemporal resolution.

Small-molecule albumin ligand modification to enhance the anti-diabetic ability of GLP-1 derivatives

Glucagon-like peptide-1 (GLP-1) receptor agonists modified with albumin ligands which can specificity bind to the human serum albumin (HSA) was an efficient strategy to prolong the half-time of GLP-1. Herein, we investigated the effect of small-molecule albumin ligand modification on the hypoglycemic activities of GLP-1 derivatives. Two GLP-1 derivatives MPA-C12-GLP-1 and Rhein-C12-GLP-1 were achieved by modification of the side chain amino of lysine in position 26 of the Arg34-GLP-1(7−37)-OH with Rhein and 3-Maleimidopropionic acid respectively using 12-aminolauric acid as a linker, and its specific albumin-conjugating characteristics, pharmaceutical characterization, and the antidiabetic effects were investigated. In vitro level, two GLP-1 derivatives demonstrated a higher binding capacity to GLP-1 receptor than that of Arg34-GLP-1(7−37)-OH. Interestingly, although the binding ability of MPA-C12-GLP-1 was equal to liraglutide, the binding ability of Rhein-C12-GLP-1 was 10-fold higher than liraglutide. In vivo level, the two GLP-1 derivatives can significantly increase their glucose tolerance and prolong their half-life in ICR mice, and they were also superior to GLP-1 in controlling glucose homeostasis and suppression of food intake and water consumption in db/db mice. Importantly, the two GLP-1 derivatives showed comparable efficacy to liraglutide for the therapy of type 2 diabetes mellitus. The in vitro INS-1 cells toxicity and the in vivo hepatotoxicity indicated that the Rhein-C12-GLP-1 was a safe candidate for the therapy of type 2 diabetes, and the serum biomarkers determination results showed that the Rhein-modified GLP-1 could significantly improve the HbA1c and blood lipids, and the H&E stain exhibited that the Rhein-C12-GLP-1 can effectively promote β-cell proliferation and differentiation. In conclusion, the 3-Maleimidopropionic acid or Rhein-modified GLP-1derivatives have great potential for development as a Type 2 diabetes mellitus therapeutic drug.

Rational design and evaluation of GLP-1 derivative for treating hyperglycemia combined with overexercise-induced myocardial injury

AimsTo design and evaluate the anti-hyperglycemia and overexercise-induced myocardial injury efficacies of a novel long-acting glucagon-like peptide-1 (GLP-1)-based therapeutic peptide in rodent animals. Main methodsHere, we designed and prepared a new pro-drug, termed RYHSB-1, which was connected by a mutated GLP-1(A8G) and an albumin binding peptide via a protease-cleavable linker. Moreover, isothermal titration calorimetry (ITC) was applied to detect its binding affinity for HSA. GLP-1 release assay was conducted in mouse serum in vitro and quantified using LC-MS/MS method. Modified intraperitoneal glucose tolerance test (IPGTT), chronic efficacies study in rodent animals with overexercise-induced myocardial injury were subjected to evaluate the druggability of RYHSB-1. ResultsRYHSB-1 with purity over 99% was prepared and ITC measurement demonstrated high binding affinity for HSA with KD of 0.06 μM. Protease cleavage assay demonstrated slowly controlled-release of transient GLP-1 from RYHSB-1 under the hydrolysis catalyzed by thrombin in vitro. Moreover, IPGTT showed clearly dose-dependent glucose-lowering efficacies of RYHSB-1 within 0.1–0.9 mg/kg. The prolonged anti-diabetic efficacy of RYHSB-1 was further assessed via multiple IPGTTs and hypoglycemic duration test. Furthermore, long-term administration of RYHSB-1 in diabetic mice achieved promising efficacies on hyperglycemia and overexercise-induced myocardial injury. SignificanceRYHSB-1 holds outstanding pharmaceutical potential as an anti- overexercise-induced myocardial injury drug. The strategy of albumin-conjugation also could be applied to other active peptides develop long effecting therapeutic drugs.

Novel Modified GLP-1 Derivatives with Prolonged Glucose-Lowering Ability In Vivo

The rapid degradation of native glucagon-like peptide 1 (GLP-1) by dipeptidyl peptidase-IV (DPP-IV) has advanced new approaches to the generation of degradation-resistant GLP-1 analogs. Chemical coupling of GLP-1 analog to HSA is innovatively achieved by solid-phase peptide synthesis in this study and shows prolonged glucose-lowering ability in vivo. GLP-1(7–37)(Ala8Aib)-Cys-HSA was constructed by solid-phase peptide synthesis through two levels of modification: mutation of Ala8 to aminoisobutyric acid (Aib) to decrease DPP-IV degradation, and conjugation to large serum protein HSA by chemical modification to decrease renal filtration. Glucose tolerance test and insulin secretion assay were performed to examine the biological activity of GLP-1(7–37)(Ala8Aib)-Cys-HSA in vivo in the present research. Long-lasting glucose-lowering and insulin-releasing effects were evaluated up to 4 weeks in T2DM rats. GLP-1(7–37)(Ala8Aib)-Cys-HSA lowered blood glucose in normal mice and T2DM rats. Twice administration of GLP-1(7–37)(Ala8Aib)-Cys-HSA to T2DM rats daily significantly reduced glycemic excursion following IP glucose challenge (P < 0.01 to 0.05) and greatly increased insulin secretion during the 4-week study period. These findings demonstrate that the albumin-conjugated GLP-1 analog mimics the function of native GLP-1 with prolonged activity.

A novel inducible acute hyperglycemia mouse model for assessing 6-KTP.

The aim of the present study was to establish a mouse model of acute hyperglycemia, which may be utilized to detect the glucose concentration-dependent hypoglycemic activity of the glucagon-like peptide-1 derivative, 6-KTP. The results demonstrated that the first 30 min following the intraperitoneal injection of 2 g/kg glucose into C57BL/6J mice was the optimum time for assessing the hypoglycemic activity of potential therapeutic methods for diabetes. There was a linear association between the dose of 6-KTP and hypoglycemic activity between 0.2 and 1.2 mg/kg. The resulting model may serve as template for developing cost-effective in vivo models to test similar therapeutics.

Glycosaminoglycan Conjugation for Improving the Duration of Therapeutic Action of Glucagon-Like Peptide-1.

Glucagon-like peptide-1 (GLP-1) is an incretin peptide that plays a crucial role in lowering blood glucose levels and holds promise for treating type II diabetes. In this study, we synthesized GLP-1 derivatives that were conjugated with glycosaminoglycans (GAGs), i.e., chondroitin (CH) or heparosan (HPN), to address the major limitation in their clinical use of GLP-1, which is its short half-life in the body. After exploring a variety of CHs with different molecular sizes and heterobifunctional linkers having different alkyl chains, we obtained CH-conjugated GLP-1 derivatives that stayed in blood circulation much longer (T1/2 elim > 25 h) than unconjugated GLP-1 and showed blood glucose-lowering efficacy up to 120 h after subcutaneous injection in mice. By using the same optimized linker design, we eventually obtained a HPN-conjugated GLP-1 derivative with efficacy lasting 144 h. These results demonstrate that conjugation with GAG is a promising strategy for improving the duration of peptide drugs.

Glucagon-Like Peptide-1 (GLP-1)-Based Therapeutics: Current Status and Future Opportunities beyond Type 2 Diabetes.

Glucagon-like peptide-1 (GLP-1) is secreted by intestinal L-cells following food intake, and plays an important role in glucose homeostasis due to its stimulation of glucose-dependent insulin secretion. Further, GLP-1 is also associated with protective effects on pancreatic β-cells and the cardiovascular system, decreased appetite, and weight loss, making GLP-1 derivatives an exciting treatment for type 2 diabetes and obesity. Despite these benefits, wild-type GLP-1 exhibits a short circulation time due to its poor metabolic stability and rapid renal clearance, and must be administered by injection, making it a poor therapeutic agent. Many strategies have been used to improve the circulation time of GLP-1 (e.g., mutations, unnatural amino acids, depot formulations, use of exendin-4 sequences, and fusions with high-molecular-weight proteins or polymers), with its therapeutic utility further improved by adding agonist activity for gastric inhibitory peptide and glucagon receptors. This minireview focuses on strategies that have been used to improve the pharmacokinetics of GLP-1 and provides an overview of GLP-1-based therapeutics in the pipeline.

Nanoparticles induced by embedding self-assembling cassette into glucagon-like peptide 1 for improving in vivo stability.

The multiple physiologic characteristics of glucagon-like peptide 1 (GLP-1) make it a promising drug candidate for treating type 2 diabetes mellitus. However, the half-life of GLP-1 is short as a result of degradation by dipeptidyl peptidase IV and renal clearance. Stabilizing GLP-1 is therefore critical for its use in drug development. Self-assembling peptides are a class of peptides that undergo spontaneous assembly into ordered nanostructures. Recently, studies of self-assembling peptides as drug carriers have increased because of their enhanced stability. In the present study, GLP-1 was modified to incorporate the structural characteristics of self-assembling peptides aiming to generate a self-assembling GLP-1 derivative. Receptor binding capacity and insulinotropic effects were measured to investigate the physiologic functions of GLP-1, along with morphologic approaches to observe supramolecular formation on self-assembly at the nano scale. Finally, blood glucose regulation and body weight were monitored after administration of selected derivatives. Our findings revealed that cadyglp1e and cadyglp1m both exhibited improved stability even though different nanoshapes were observed for these two self-assembling peptides. Both cadyglp1e and cadyglp1m retained glucoregulatory activity after insulin stimulation and were potent drug candidates for long-acting GLP-1 derivatives to treat type 2 diabetes mellitus. Our findings support the feasibility of introducing self-assembly functions into peptides with poor stabilities, such as GLP-1.-Li, Y., Cui, T., Kong, X., Yi, X., Kong, D., Zhang, J., Liu, C., Gong, M. Nanoparticles induced by embedding self-assembling cassette into glucagon-like peptide 1 for improving in vivo stability.

Design, synthesis and biological evaluation of PEGylated Xenopus glucagon-like peptide-1 derivatives as long-acting hypoglycemic agents

In order to develop novel long-acting GLP-1 derivatives, a peptide hybrid (1a) from human GLP-1 and Xenopus GLP-1 discovered in our previous research was selected as the lead compound. Exendin-4 inspired modification resulted in peptide 1b with enhanced glucose-lowering activity. Cysteine mutated 1b derivatives with reserved bioactivity were further site-specifically connected with mPEG2000-MAL to provide conjugates 3a–h, among which 3d and 3e were found to have significantly improved hypoglycemic activity and insulinotropic ability than GLP-1. The hypoglycemic durations of 3d and 3e were remarkably prolonged to ∼20 h in type 2 diabetic db/db mice, compared with the 5.3 h of exendin-4 in the same test. Finally, chronic in vivo studies revealed that a once-daily treatment of 3d or 3e for five weeks resulted in recovered glucose-controlling ability of type 2 diabetic db/db mice, along with other benefits, such as reduced body weight gains, food intake amounts and HbA1c values. Collectively, our results suggest 3d and 3e as potential long-acting glucose-lowering agents for treating type 2 diabetes.

Myricetin: a potent approach for the treatment of type 2 diabetes as a natural class B GPCR agonist.

The physiologic properties of glucagon-like peptide 1 (GLP-1) make it a potent candidate drug target in the treatment of type 2 diabetes mellitus (T2DM). GLP-1 is capable of regulating the blood glucose level by insulin secretion after administration of oral glucose. The advantages of GLP-1 for the avoidance of hypoglycemia and the control of body weight are attractive despite its poor stability. The clinical efficacies of long-acting GLP-1 derivatives strongly support discovery pursuits aimed at identifying and developing orally active, small-molecule GLP-1 receptor (GLP-1R) agonists. The purpose of this study was to identify and characterize a novel oral agonist of GLP-1R (i.e., myricetin). The insulinotropic characterization of myricetin was performed in isolated islets and in Wistar rats. Long-term oral administration of myricetin demonstrated glucoregulatory activity. The data in this study suggest that myricetin might be a potential drug candidate for the treatment of T2DM as a GLP-1R agonist. Further structural modifications on myricetin might improve its pharmacology and pharmacokinetics.—Li, Y., Zheng, X., Yi, X., Liu, C., Kong, D., Zhang, J., Gong, M. Myricetin: a potent approach for the treatment of type 2 diabetes as a natural class B GPCR agonist.

Long-acting GLP-1 analogue in V-shaped conformation by terminal polylysine modifications.

Glucagon-like peptide-1 (GLP-1) possesses multiple physiological functions, which make it a potential drug candidate for the treatment of type 2 diabetes. However, its clinical application was limited severely by its short half-life in vivo. Therefore, stabilization of GLP-1 is critical for the use of this peptide in drug development. In this study, a novel GLP-1 derivative, VGLP1K6, processed a significantly prolonged half-life in vivo. Structural analysis using molecular dynamics simulations demonstrated that VGLP1K6 has a rigid V-shaped conformation resulting from the intrapeptide disulfide bond. The C-terminal polylysine residues of VGLP1K6 caused the vulnerable N-terminus of GLP-1 (HA-fragment) to reside within the pocket-like cavity of the peptide due to the intrahydrogen bonds. The structural analysis suggested that this structural alteration contributed to the remarkable prolonged half-life of VGLP1K6, which was approximately 70 h. In addition, VGLP1K6 induced better long-acting glucose tolerance and greater HbA1c reductions than GLP-1 in rodents. Our findings suggest that the GLP-1 derivative VGLP1K6 might be a possible potent antidiabetic drug for the treatment of type 2 diabetes mellitus.

Novel coumarin modified GLP-1 derivatives with enhanced plasma stability and prolonged in vivo glucose-lowering ability.

The short biological half-life limits the therapeutic use of glucagon-like peptide-1 (GLP-1) and chemical modification to improve the interaction of peptides with serum albumin represents an effective strategy to develop long-acting peptide analogues. Coumarin, a natural product, is known to bind tightly to plasma proteins and possesses many biological activities. Therefore, we designed and synthesized a series of coumarin-modified GLP-1 derivatives, hypothesizing that conjugation with coumarin would retain the therapeutic effects and prolong the biological half-life of the conjugates. Four cysteine-modified GLP-1 analogues (1-4) were prepared using Gly8 -GLP-1(7-36)-NH2 peptide as a starting point. These analogues were conjugated with two coumarin maleimides to yield eight compounds (conjugates 6-13) for testing. Activation of human GLP-1 receptors, stability to enzymic inactivation in plasma and binding to human albumin were assessed in vitro. In vivo, effects on oral glucose tolerance tests (OGTT) in rats and on blood glucose levels in db/db mice were studied. Most conjugates showed well preserved receptor activation efficacy, enhanced albumin-binding properties and improved in vitro plasma stability and conjugate 7 was selected to undergo further assessment. In rats, conjugate 7 had a longer circulating t1/2 than exendin-4 or liraglutide. A prolonged antidiabetic effect of conjugate 7 was observed after OGTT in rats and a prolonged hypoglycaemic effect in db/db mice. Cysteine-specific coumarin conjugation with GLP-1 offers a useful approach to the development of long-acting incretin-based antidiabetic agents. Conjugate 7 is a promising long-lasting GLP-1 derivative deserving further investigation.

Developments of Glucagon Like Peptide-1 (GLP-1) and Long-acting Analogs in Clinical and Preclinical Studies for Treatment of Type 2 Diabetes

The outstanding physiological functions of glucagon-like peptide-1 make it a promising drug candidate for blood glucose regulation in type 2 diabetes. However, the short half-life of GLP-1 limited its widely clinical utility due to the rapid degradation by dipeptidyl peptidase IV (DPP-IV) and renal clearance. Therefore, stabilisation of glucagon-like peptide-1 is critical for the use of this peptide in drug development. Scientists in pharmaceutical companies have contributed in years to obtain long-acting or sustained GLP-1 derivatives which are reviewed in this report.

Design, synthesis and in vitro characterization of Glucagon-Like Peptide-1 derivatives for pancreatic beta cell imaging by SPECT

Novel Glucagon-Like Peptide-1 (GLP-1) derivatives containing the metal chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and naturally occurring Indium ( 113/115In) were prepared using solid-phase Fmoc methods. All synthesized peptides contained d-Ala-8, a modification known to improve resistance towards degradation by dipeptidyl peptidase-IV. The effect of increased distance between DOTA and the peptide chain was investigated using an (aminoethyl) ethoxy acetyl linker, in order to reduce steric effects imposed by DOTA. Placement of linker and DOTA moieties were also varied within the GLP-1 sequence to test for optimal metal-complex location. The binding affinity of the peptide derivatives was determined in vitro with Chinese hamster ovary cells stably transfected with a human GLP-1 receptor (CHO/GLP-1R) cell line and was shown to be in the nM range. Gamma camera imaging of an insulinoma cell line was carried out using 111In-labeled peptides. Our results suggest that the prepared GLP-1 derivatives are suitable imaging probes for studying pancreatic islet function in vivo.

Design and Synthesis of Conformationally Constrained Glucagon-Like Peptide-1 Derivatives with Increased Plasma Stability and Prolonged in Vivo Activity

A series of conformationally constrained derivatives of glucagon-like peptide-1 (GLP-1) were designed and evaluated. By use of [Gly (8)]GLP-1(7-37)-NH2 (2) peptide as a starting point, 17 cyclic derivatives possessing i to i + 4, i to i + 5, or i to i + 7 side chain to side chain lactam bridges from positions 18 to 30 were prepared. The effect of a helix-promoting alpha-amino-isobutyric acid (Aib) substitution at position 22 was also evaluated. The introduction of i to i + 4 glutamic acid-lysine lactam constraints in c[Glu (18)-Lys (22)][Gly (8)]GLP-1(7-37)-NH2 (6), c[Glu (22)-Lys (26)][Gly (8)]GLP-1(7-37)-NH2 (10), and c[Glu (23)-Lys (27)][Gly (8)]GLP-1(7-37)-NH2 (11) resulted in potent functional activity and receptor affinities comparable to native GLP-1. Selected GLP-1 peptides were chemoselectively PEGylated in order to prolong their in vivo activity. PEGylated peptides [Gly (8),Aib (22)]GLP-1(7-37)-Cys ((PEG))-Ala-NH2 (23) and c[Glu (22)-Lys (26)][Gly (8)]GLP-1(7-37)-Cys ((PEG))-Ser-Gly-NH2 (24) retained picomolar functional potency and avid receptor binding properties. Importantly, PEGylated GLP-1 peptide 23 exhibited sustained in vivo efficacy with respect to blood glucose reduction and decreased body weight for several days in nonhuman primates.

An Albumin-Exendin-4 Conjugate Engages Central and Peripheral Circuits Regulating Murine Energy and Glucose Homeostasis

Glucagon-like peptide-1 (GLP-1) regulates glucose homeostasis through multiple mechanisms including direct actions on the endocrine pancreas and indirect activation of central nervous system circuits regulating gastric emptying, satiety, and body weight. Because native GLP-1 is rapidly degraded, there is considerable interest in development of more potent GLP-1 receptor (GLP-1R) agonists with sustained activity; however, the extent to which much larger GLP-1R agonists will mimic some or all of the actions of smaller peptides remains uncertain. We studied the actions of CJC-1134-PC, a recombinant human serum albumin-exendin-4 conjugated protein, at the GLP-1R using heterologous cells expressing the GLP-1R in vitro and both wild-type and Glp1r(-/-) mice in vivo. CJC-1134-PC activated GLP-1R-dependent signaling in baby hamster kidney-GLP-1R cells and acutely lowered blood glucose in wild-type but not in Glp1r(-/-) mice. Moreover, acute administration of CJC-1134-PC rapidly activated c-Fos expression in multiple regions of the central nervous system, acutely inhibited gastric emptying, and produced sustained inhibition of food intake in a GLP-1R-dependent manner. Furthermore, chronic daily treatment of high-fat diet-fed wild-type mice with CJC-1134-PC for 4 weeks led to improved glucose tolerance, increased levels of glucose-stimulated insulin, decreased HbA1c, and weight loss associated with decreased hepatic triglyceride content. These findings illustrate that a high-molecular-weight exendin-4-albumin conjugate retains the ability to mimic a full spectrum of GLP-1R-dependent actions, including activation of central nervous system circuits regulating gastric emptying, food intake, and body weight.

Structure−Activity and Protraction Relationship of Long-Acting Glucagon-like Peptide-1 Derivatives: Importance of Fatty Acid Length, Polarity, and Bulkiness

We here report a series of derivatives describing the structure-activity relationship around liraglutide, a once-daily human glucagon-like peptide-1 fatty acid derivative, with respect to potency as well as protraction in vivo. The spacer region between the fatty acid and the peptide is mostly important for potency, whereas the fatty acid or fatty acid mimetic is important for both potency and protraction. The length of the fatty acid is the most important parameter for protraction.

Improved peroral delivery of glucagon-like peptide-1 by site-specific biotin modification: Design, preparation, and biological evaluation

Peptide oral delivery is still a significant challenge because of two major impediments, low absorption efficiency and instability in gastrointestinal tract. The aim of this study was to design, prepare, and evaluate an intestine enzyme-resistant, superior intestine absorptive, and biologically preserved glucagon-like peptide-1 (GLP-1) derivative using the site-specific modification of biotin, which can take advantage of the carrier-mediated active intestine transport. Two series of site-specific Lys 34- and Lys 26,34–biotin–GLP-1 derivatives were prepared, and their intestine membrane permeabilities, proteolytic stabilities against the intestine enzymes, and bioactivities were then evaluated. Especially, Lys 26,34–biotin–GLP-1 was found to have the most promising scores: (i) it displayed a 5.6-fold higher Caco-2 cell monolayer permeability than GLP-1; (ii) it showed an 8.5- and 3.5-fold longer half-life than GLP-1 in rat intestine fluid and homogenate, respectively; and interestingly (iii) it had a well-preserved insulinotropic activity (94.5% vs. GLP-1) in the rat islets. Finally, Lys 26,34–biotin–GLP-1 showed a 9-fold higher oral hypoglycemic efficacy (25.3%) than native GLP-1 (2.7%) ( P < 0.005) after direct peroral administration into type 2 diabetic db/db mice. This study highlights the oral hypoglycemic potential of site-specific Lys 26,34-biotinylated GLP-1, and this orally available analogue would find a role in the treatment of type 2 diabetes.