A novel GLP-1 analog exhibits potent utility in the treatment of type 2 diabetes with an extended half-life and efficient glucose clearance in vivo

The multiple physiological characterizations of glucagon-like peptide-1 (GLP-1) make it a promising drug candidate for the therapy of type 2 diabetes. However, the half-life of GLP-1 is short in vivo due to degradation by dipeptidyl peptidase-IV (DPP-IV) and renal clearance. Therefore, the stabilization of GLP-1 is critical for its utility in drug development. Based on our previous research, a GLP-1 analog that contained an intra-disulfide bond exhibited a prolonged biological half-life. In this study, we improved upon previous analogs with a novel GLP-1 analog that contained a tryptophan cage-like sequence for an improved binding affinity to the GLP-1 receptor. The binding capacities and the stabilities of GLP715a were investigated, and the physiological functions of the GLP715a were compared to those of the wild-type GLP-1 in animals. The results demonstrated that the new GLP-1 analog (GLP715a) increased its biological half-life to approximately 48 h in vivo; GLP715a also exhibited a higher binding affinity to the GLP-1 receptor than the wild-type GLP-1. The increased binding capacity of GLP715a to its receptor resulted in a quick response to glucose administration. The long-acting anti-diabetic property of GLP715a was revealed by its increased glucose tolerance, higher HbA_1c reduction, more efficient glucose clearance and quicker insulin stimulation upon glucose administration compared to the wild-type GLP-1 in rodents. The improved physiological characterizations of GLP715a make it a possible potent anti-diabetic drug in the treatment of type 2 diabetes mellitus.     

GLP-1 analogs containing disulfide bond exhibited prolonged half-life in vivo than GLP-1

The multiple physiological characterizations of glucagon-like peptide-1 (GLP-1) make it a promising drug candidate for the therapy of type 2 diabetes. However, the half-life of GLP-1 is short in vivo due to degradation by dipeptidyl peptidase-IV (DPP-IV) and renal clearance. This indicates that the stabilization of GLP-1 is critical for its utility in drug development. In this study, we developed a cluster of GLP-1 mutants containing an inter-disulfide bond that is predicted to increase the half-life of GLP-1 in vivo. Exendin-4 was also mutated with a disulfide bond similar to the GLP-1 analogs. In this study, the binding capacities of the mutants were determined, the stabilities of the mutants were investigated and the physiological functions of the mutants were compared with those of wild-type GLP-1 and exendin-4 in animals. The results indicated that the mutants remarkably raised the half-life in vivo; they also showed better glucose tolerance and higher HbA_1c reduction than GLP-1 and exendin-4 in rodents. These results suggest that GLP-1 and exendin-4 mutants containing disulfide bonds might be utilized as possible potent anti-diabetic drugs in the treatment of type 2 diabetes mellitus.     

A novel exendin-4 human serum albumin fusion protein,E2HSA,with an extended half-life and good glucoregulatory effect in healthy rhesus monkeys

Glucagon-like peptide-1 (GLP-1) has attracted considerable research interest in terms of the treatment of type 2 diabetes due to their multiple glucoregulatory functions. However, the short half-life, rapid inactivation by dipeptidyl peptidase-IV (DPP-IV) and excretion, limits the therapeutic potential of the native incretin hormone. Therefore, efforts are being made to develop the long-acting incretin mimetics via modifying its structure. Here we report a novel recombinant exendin-4 human serum albumin fusion protein E2HSA with HSA molecule extends their circulatory half-life in vivo while still retaining exendin-4 biological activity and therapeutic properties. In vitro comparisons of E2HSA and exendin-4 showed similar insulinotropic activity on rat pancreatic islets and GLP-1R-dependent biological activity on RIN-m5F cells, although E2HSA was less potent than exendin-4. E2HSA had a terminal elimation half-life of approximate 54 h in healthy rhesus monkeys. Furthermore, E2HSA could reduce postprandial glucose excursion and control fasting glucose level, dose-dependent suppress food intake. Improvement in glucose-dependent insulin secretion and control serum glucose excursions were observed during hyperglycemic clamp test (18 h) and oral glucose tolerance test (42 h) respectively. Thus the improved physiological characterization of E2HSA make it a new potent anti-diabetic drug for type 2 diabetes therapy.     

High-Level Expression of Fusion Protein Containing 10 Tandem Repeated GLP-1 Analogs in Yeast Pichia pastoris and Its Biological Activity in a Diabetic Rat Model

Glucagon-like peptide-1 (GLP-1), an incretin secreted by intestinal L-cells, can effectively lower blood glucose levels in patients with diabetes. A fusion gene, consisting of 10 tandem repeated GLP-1 analog genes, was expressed at a high level in the yeast Pichia pastoris. SDS polyacrylamide gel electrophoresis (SDS–PAGE), and Western Blotting results showed that fusion protein migrated as a single protein band with a molecular weight of 36 kDa. A biological activity test showed that the GLP-1 analog could significantly lower the level of serum glucose when GLP-1 purified analog was injected into diabetic rats. A potential strategy for large-scale production of fusion protein containing the 10 GLP-1 analogs as discovered, and a single GLP-1 analog was obtained from fusion protein digested with trypsin. This should be inspired foreign expression of medicinal short peptides and be valuable in further research on GLP-1 analog drugs in the treatment of diabetes mellitus.     

High-level expression of fusion protein containing 10 tandem repeated GLP-1 analogs in yeast Pichia pastoris and its biological activity in a diabetic rat model

Glucagon-like peptide-1 (GLP-1), an incretin secreted by intestinal L-cells, can effectively lower blood glucose levels in patients with diabetes. A fusion gene, consisting of 10 tandem repeated GLP-1 analog genes, was expressed at a high level in the yeast Pichia pastoris. SDS polyacrylamide gel electrophoresis (SDS-PAGE), and Western Blotting results showed that fusion protein migrated as a single protein band with a molecular weight of 36 kDa. A biological activity test showed that the GLP-1 analog could significantly lower the level of serum glucose when GLP-1 purified analog was injected into diabetic rats. A potential strategy for large-scale production of fusion protein containing the 10 GLP-1 analogs as discovered, and a single GLP-1 analog was obtained from fusion protein digested with trypsin. This should be inspired foreign expression of medicinal short peptides and be valuable in further research on GLP-1 analog drugs in the treatment of diabetes mellitus.     

A novel GLP-1 analog,a dimer of GLP-1 via covalent linkage by a lysine,prolongs the action of GLP-1 in the treatment of type 2 diabetes

GLP-1 is an incretin hormone that can effectively lower blood glucose, however, the short time of biological activity and the side effect limit its therapeutic application. Many methods have been tried to optimize GLP-1 to extend its in vivo half-time, reduce its side effect and enhance its activity. Here we have chosen the idea to dimerize GLP-1 with a C-terminal lysine to form a new GLP-1 analog, DLG3312. We have explored the structure and the biological property of DLG3312, and the results indicated that DLG3312 not only remained the ability to activate the GLP-1R, but also strongly stimulated Min6 cell to secrete insulin. The in vivo bioactivities have been tested on two kinds of animal models, the STZ induced T2DM mice and the db/db mice, respectively. DLG3312 showed potent anti-diabetic ability in glucose tolerance assay and single-dose administration of DLG3312 could lower blood glucose for at least 10 hours. Long-term treatment with DLG3312 can reduce fasted blood glucose, decrease water consumption and food intake and significantly reduce the HbA1c level by 1.80% and 2.37% on STZ induced T2DM mice and the db/db mice, respectively. We also compared DLG3312 with liraglutide to investigate its integrated control of the type 2 diabetes. The results indicated that DLG3312 almost has the same effect as liraglutide but with a much simpler preparation process. In conclusion, we, by using C-terminal lysine as a linker, have synthesized a novel GLP-1 analog, DLG3312. With simplified preparation and improved physiological characterizations, DLG3312 could be considered as a promising candidate for the type 2 diabetes therapy.     

Application of novel peptide (Pp1) improving the half-life of exendin-4 in vivo

Aims

The multiple physiological characterizations of exendin-4 make it as a promising drug candidate for the therapy of type 2 diabetes. Although the longer biological half-life offered the exendin-4 with excellent therapeutic potentials for the clinical utility of type 2 diabetes than glucagon-like peptide-1, the exendin-4 still did not free from the inconveniently frequent injections. Therefore, there are increasing requirements for the long-acting exendin-4.

Methods

Pp1 regard as a novel exendin-4 protecting peptide, which are predicted to have the ability of increasing the stabilization of exendin-4 in vivo. Protecting peptide is able to form stable complex by non-covalent interaction with human exendin-4.

Results

In this study, the stability of the exendin-4/Pp1 complex was investigated, and the physiological functions of it were analyzed. Results indicated that exendin-4/Pp1 complex remarkably raised the stabilization of exendin-4 in vivo; it also showed better glucose tolerance and higher HbA_1c reduction than exendin-4 which was utilized chronically in rodents.

Conclusion

Based upon these results, it is suggested that an exendin-4/Pp1 complex might be utilized as a potent anti-diabetic drug in the treatment of type 2 diabetes mellitus.     

A novel GLP-1 analog, BPI3006, with potent DPP IV resistance and good glucoregulatory effect

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that decreases postprandial glycemic excursions by enhancing insulin secretion but with short half-life due to rapid inactivation by enzymatic N-terminal truncation. Therefore, efforts are being made to improve the stability of GLP-1 via modifying its structure or inhibiting dipeptidyl-peptidase IV (DPP IV), which is responsible for its degradation. Here we report a novel GLP-1 analog BPI3006 with -NHCO- of Ala⁸ replaced by -CH(CF₃)NH- and features of its metabolic stability, GLP-1 receptor trans-activation and in vivo biological activity. BPI3006 is highly resistant to DPP IV-mediated degradation with 91.1% of parental peptide left after 24 h exposure to the enzyme. BPI3006 also effectively activates its target gene promoter through GLP-1 receptor activation by measuring the transiently transfected reporter gene green fluorescence protein (GFP) expression in NIT-1 cells. Furthermore, BPI3006 could well restrain the glycemia variation in fasted normal ICR mice after a single administration followed by an oral glucose loading. In spontaneous type 2 diabetic KKA^y mice, BPI3006 injected twice daily could significantly improve the oral glucose tolerance and hyperinsulinemia, as well as ameliorate the food and water consumption. In conclusion, BPI3006 has enhanced resistance to DPP IV leading to improved stability, and shows excellent in vivo biological activity. Thus it may be a new candidate for T2DM treatment and its novel modification may provide valuable guidance for the future development of long-acting GLP-1 analogs.     

Early intervention with liraglutide improves glucose tolerance without affecting islet microcirculation in young Goto-Kakizaki rats

Liraglutide, an analog of glucagon-like peptide-1 (GLP-1), is an effective anti-diabetic agent with few side effects. Since native GLP-1 exerts vascular effects, we investigated changes in pancreatic islet blood flow using a non-radioactive microsphere technique, as well as insulin concentration and glucose tolerance after 17 day treatment with liraglutide in 6-week-old Goto-Kakizaki (GK) rats. Compared to saline-treated control GK rats, liraglutide limited body weight gain, decreased glycemia, improved glucose tolerance and lowered serum insulin concentration. Neither pancreatic or islet blood flow, nor pancreatic insulin content, was affected by liraglutide treatment. We conclude that early intervention with liraglutide decreases glycemia and improves glucose tolerance, thus halting the natural progression towards diabetes, without affecting islet microcirculation or pancreatic insulin content in young female GK rats.     

Isolation of Positive Modulator of Glucagon-like Peptide-1 Signaling from Trigonella foenum-graecum (Fenugreek) Seed

The glucagon-like peptide-1 receptor (GLP-1R) is expressed in many tissues and has been implicated in diverse physiological functions, such as energy homeostasis and cognition. GLP-1 analogs are approved for treatment of type 2 diabetes and are undergoing clinical trials for other disorders, including neurodegenerative diseases. GLP-1 analog therapies maintain chronically high plasma levels of the analog and can lead to loss of spatiotemporal control of GLP-1R activation. To avoid adverse effects associated with current therapies, we characterized positive modulators of GLP-1R signaling. We screened extracts from edible plants using an intracellular cAMP biosensor and GLP-1R endocytosis assays. Ethanol extracts from fenugreek seeds enhanced GLP-1 signaling. These seeds have previously been found to reduce glucose and glycated hemoglobin levels in humans. An active compound (N55) with a new N-linoleoyl-2-amino-γ-butyrolactone structure was purified from fenugreek seeds. N55 promoted GLP-1-dependent cAMP production and GLP-1R endocytosis in a dose-dependent and saturable manner. N55 specifically enhanced GLP-1 potency more than 40-fold, but not that of exendin 4, to stimulate cAMP production. In contrast to the current allosteric modulators that bind to GLP-1R, N55 binds to GLP-1 peptide and facilitates trypsin-mediated GLP-1 inactivation. These findings identify a new class of modulators of GLP-1R signaling and suggest that GLP-1 might be a viable target for drug discovery. Our results also highlight a feasible approach for screening bioactive activity of plant extracts.     

GLP-1 C-terminal structures affect its blood glucose lowering-function.

Glucagon-like peptide-1 (GLP-1), which is an endogenous insulinotropic peptide that can stimulate islet cells to secret insulin, is a promising new drug candidate for the treatment of type 2 diabetes. However, due to the very short half-life of this peptide, the clinical value of GLP-1 is restricted. A GLP-1 peptide analog that had been altered by deletion of five amino acids from the C-terminus (sGLP-1) was selected and investigated in vivo for the therapeutic effect on GK rats with type II DM (T2DM). The results revealed that sGLP-1 exhibited decreased blood glucose-lowering ability compared to GLP-1 in the first week, as measured after once-daily administration. However, after drug administration for 2 weeks, the blood glucose-lowering effect of sGLP-1 became superior to that of GLP-1. sGLP-1 reduced apoptosis of the old islets, enhanced insulin production, and promoted new islets replication. sGLP-1 is a shorter but more efficient GLP-1 analog for type 2 diabetes management. Because sGLP-1 prolonged the proliferation and recovery of islet cells, the ability to maintain blood glucose (BG) within a normal range was still present 2 weeks after drug withdrawal. These results confirmed the importance of the C-terminus of GLP-1 molecule, and further demonstrated that GLP-1 (7-37) can be truncated till the 32nd amino acid to have a better long-term BG lowing function. This result may imply for the presence of glucagon family clearance receptors in vivo and demonstrates that the C-terminus participates in GLP-1 clearance.     

Novel GLP-1 Fusion Chimera as Potent Long Acting GLP-1 Receptor Agonist

GLP-1 has a variety of anti-diabetic effects. However, native GLP-1 is not suitable for therapy of diabetes due to its short half-life (t1/2<2 min). To circumvent this, we developed a long-lasting GLP-1 receptor agonist by the fusion of GLP-1 with human IgG2 Fc (GLP-1/hIgG2). ELISA-based receptor binding assay demonstrated that GLP-1/hIgG2 had high binding affinity to the GLP-1R in INS-1 cells (Kd = 13.90±1.52 nM). Upon binding, GLP-1/hIgG2 was rapidly internalized by INS-1 cells in a dynamin-dependent manner. Insulin RIA showed that GLP-1/IgG2 dose-dependently stimulated insulin secretion from INS-1 cells. Pharmacokinetic studies in CD1 mice showed that with intraperitoneal injection (i.p.), the GLP-1/hIgG2 peaked at 30 minutes in circulation and maintained a plateau for >168 h. Intraperitoneal glucose tolerance test (IPGTT) in mice showed that GLP-1/hIgG2 significantly decreased glucose excursion. Furthermore, IPGTT performed on mice one week after a single drug-injection also displayed significantly reduced glucose excursion, indicating that GLP-1/hIgG2 fusion protein has long-lasting effects on the modulation of glucose homeostasis. GLP-1/hIgG2 was found to be effective in reducing the incidence of diabetes in multiple-low-dose streptozotocin-induced type 1 diabetes in mice. Together, the long-lasting bioactive GLP-1/hIgG2 retains native GLP-1 activities and thus may serve as a potent GLP-1 receptor agonist.     

Biological activity of AC3174, a peptide analog of exendin-4

Exenatide, the active ingredient of BYETTA (exenatide injection), is an incretin mimetic that has been developed for the treatment of patients with type 2 diabetes. Exenatide binds to and activates the known GLP-1 receptor with a potency comparable to that of the mammalian incretin GLP-1(7-36), thereby acting as a glucoregulatory agent. AC3174 is an analog of exenatide with leucine substituted for methionine at position 14, [Leu(14)]exendin-4. The purpose of these studies was to evaluate the glucoregulatory activity and pharmacokinetics of AC3174. In RINm5f cell membranes, the potency of AC3174 for the displacement of [(125)I]GLP-1 and activation of adenylate cyclase was similar to that of exenatide and GLP-1. In vivo, AC3174, administered as a single IP injection, significantly decreased plasma glucose concentration and glucose excursion following the administration of an oral glucose challenge in both non-diabetic (C57BL/6) and diabetic db/db mice (P<0.05 vs. vehicle-treated). The magnitude of glucose lowering of AC3174 was comparable to exenatide. The ED(50) values of AC3174 for glucose lowering (60 minute post-dose) were 1.2 microg/kg in db/db mice and 1.3 microg/kg in C57BL/6 mice. AC3174 has insulinotropic activity in vivo. Administration of AC3174 resulted in a 4-fold increase in insulin concentrations in normal mice following an IP glucose challenge. AC3174 was also shown to inhibit food intake and decrease gastric emptying in rodent models. AC3174 was stable in human plasma (>90% of parent peptide was present after 5 h of incubation). In rats, the in vivo half-life of AC3174 was 42-43 min following SC administration. In summary, AC3174 is an analog of exenatide that binds to the GLP-1 receptor in vitro and shares many of the biological and glucoregulatory activities of exenatide and GLP-1 in vivo.     

Linker engineering for fusion protein construction: Improvement and characterization of a GLP-1 fusion protein

Protein engineering has been successfully applied in protein drug discovery. Using this technology, we previously have constructed a fusion protein by linking the globular domain of adiponectin to the C-terminus of a glucagon-like peptide-1 (GLP-1) analog. Herein, to further improve its bioactivity, we reconstructed this fusion protein by introducing linker peptides of different length and flexibility. The reconstructed fusion proteins were overexpressed in Escherichia coli and purified using nickel affinity chromatography. Their agonist activity towards receptors of GLP-1 and adiponectin were assessed in vitro by using luciferase assay and AMP-activated protein kinase (AMPK) immunoblotting, respectively. The effects of the selected fusion protein on glucose and lipid metabolism were evaluated in mice. The fusion protein reconstructed using a linker peptide of AMGPSSGAPGGGGS showed high potency in activating GLP-1 receptor and triggering AMPK phosphorylation via activating the adiponectin receptor. Remarkably, the optimized fusion protein was highly effective in lowering blood glucose and lipids in mice. Collectively, these findings demonstrate that the bioactivity of this GLP-1 fusion protein can be significantly promoted by linker engineering, and indicate that the optimized GLP-1 fusion protein is a promising lead structure for anti-diabetic drug discovery.     

Stability and bioactivity studies on dipeptidyl peptidase IV resistant glucogan-like peptide-1 analogues

Glucagon-like peptide-1 (GLP-1) was once considered as an ideal anti-diabetic candidate for its important role in maintaining glucose homeostasis through the regulation of islet hormone secretion, as well as hepatic and gastric function. However, the major therapeutic obstacle for using native GLP-1 as a therapeutic agent is its very short half-life primarily due to their degradation by the enzyme dipeptidyl peptidase IV (DPP-IV). In this study, GLP-1 analogues with modifications in amino acid site 8, 22 and 23 were synthesized using solid phase peptide synthesis. Resistance of these analogues to DPP-IV cleavage was investigated in vitro by incubation of the peptides with DPP-IV or human plasma. Glucoregulating efficacy of the analogues was evaluated in normal Kunming mice using intraperitoneal glucose tolerance model. Glucose lowering effect of combination therapy (analogue plus Vildagliptin) has also been studied. In vitro studies showed that the modified analogues were much more stable than native GLP-1 (nearly 100% of the peptide keep intact after 4 h incubation). In vivo biological activity evaluation revealed that His8-EEE (the most potent GLP-1 analogues in this study) exhibited significantly improved glycemic control potency (approximately 4.1-fold over saline and 2.5-fold over GLP-1) and longer time of active duration (at least 5 h). Combination therapy also showed the trend of its superiority over mono-therapy. Modified analogues showed increased potency and biological half-time compared with the native GLP-1, which may help to understand the structure-activity relationship of GLP-1 analogues.     

The N-terminal alanine-extended GLP-1/IgG-Fc fusion protein confers resistance to DPP-IV and reduces serum glucose level in db/db mice

The aim of this study was to develop novel long-acting glucagon-like peptide 1 (GLP-1) analogs resistant to dipeptidyl peptidase-IV (DPP-IV). We constructed three fusion proteins comprising GLP-1 and the human immunoglobulin gamma heavy chain (IgG-Fc); wild-type GLP-1 and IgG-Fc (GLP-1/IgG-Fc) and two N-terminal-extended fusion proteins in which an additional Ala (A) or Gly (G) was located on the N-terminus of GLP-1 (A-GLP-1/IgG-Fc or G-GLP-1/IgG-Fc). The fusion proteins expressed in CHO-K1 cells were secreted into medium and purified by Protein A affinity chromatography. Here, we show that the Ala or Gly-extended GLP-1/IgG-Fc fusion protein is resistant to DPP-IV and has increased half-life in vivo. To our surprise, the A-GLP-1/IgG-Fc fusion protein was more effective than wildtype GLP-1/IgG-Fc fusion protein in reducing blood glucose levels in db/db mice. Our findings suggest that the A-GLP-1/IgG-Fc fusion protein could be a potential long-acting GLP-1 receptor agonist for the treatment of insulin-resistant type 2 diabetes.     

Cinnamic acid exerts anti-diabetic activity by improving glucose tolerance in vivo and by stimulating insulin secretion in vitro

Although the anti-diabetic activity of cinnamic acid, a pure compound from cinnamon, has been reported but its mechanism(s) is not yet clear. The present study was designed to explore the possible mechanism(s) of anti-diabetic activity of cinnamic acid in in vitro and in vivo non-obese type 2 diabetic rats. Non-obese type 2 diabetes was developed by injecting 90 mg/kg streptozotocin in 2-day-old Wistar pups. Cinnamic acid and cinnamaldehyde were administered orally to diabetic rats for assessing acute blood glucose lowering effect and improvement of glucose tolerance. Additionally, insulin secretory activity of cinnamic acid and cinnamaldehyde was evaluated in isolated mice islets. Cinnamic acid, but not cinnamaldehyde, decreased blood glucose levels in diabetic rats in a time- and dose-dependent manner. Oral administration of cinnamic acid with 5 and 10 mg/kg doses to diabetic rats improved glucose tolerance in a dose-dependent manner. The improvement by 10 mg/kg cinnamic acid was comparable to that of standard drug glibenclamide (5 mg/kg). Further in vitro studies showed that cinnamaldehyde has little or no effect on glucose-stimulated insulin secretion; however, cinnamic acid significantly enhanced glucose-stimulated insulin secretion in isolated islets. In conclusion, it can be said that cinnamic acid exerts anti-diabetic activity by improving glucose tolerance in vivo and stimulating insulin secretion in vitro.     

Intrapulmonary potential of polyethylene glycol-modified glucagon-like peptide-1s as a type 2 anti-diabetic agent

The pulmonary delivery of anti-diabetic peptide drugs can improve diabetic patient compliance. In this study, we tried to improve the pulmonary pharmacokinetic properties of glucagon-like peptide-1(7-36) (GLP-1) using a PEGylation approach. Initially, three types of site-specific (Lys(34)) PEGylated GLP-1 analogs were synthesized using PEGs of 2, 5, and 10 kDa, respectively. Their pharmacokinetic profiles were then examined in endotracheally cannulated rats. The results obtained show that all pharmacokinetic parameters (AUC(inf), C(max), t(1/2), V/F, and Cl/F etc.) of PEGylated GLP-1s were greatly improved by increasing PEG Mw. Specifically, the t(1/2) values of PEGylated GLP-1s (PEG Mw: 2, 5, 10 kDa) increased to 23.1+/-6.2, 41.6+/-12.3, and 81.7+/-11.7 min, respectively, vs. 8.9+/-2.9 min for intratracheally administered GLP-1. Also, PEGylated GLP-1s were found to have substantially greater C(max) values (7.4-7.8 ng/ml) than GLP-1 (4.0+/-2.4 ng/ml). Moreover, these PEGylated GLP-1s were found to have 10-20 fold more resistance to rat lung enzyme and plasma dipeptidyl peptidase IV (DPP IV). These findings indicate the dual-pharmacokinetic enhancements that PEGylated GLP-1s better survives proteolytic breakdown in the lungs than GLP-1s and better enters the systemic circulation, and that these analogs are more resistant to DPP IV-induced proteolysis and are much less rapidly removed from the systemic circulation. In conclusion, this study demonstrates the pharmaceutical usefulness of PEGylation in the context of the pulmonary delivery of GLP-1. These results show that PEGylated GLP-1s should be considered potential components of anti-diabetic inhalant preparations.     

Adenoviral vector-mediated glucagon-like peptide 1 gene therapy improves glucose homeostasis in Zucker diabetic fatty rats

BACKGROUND: Glucagon-like peptide-1 (GLP-1) is a gut-derived incretin hormone that plays an important role in glucose homeostasis. Its functions include glucose-stimulated insulin secretion, suppression of glucagon secretion, deceleration of gastric emptying, and reduction in appetite and food intake. Despite the numerous antidiabetic properties of GLP-1, its therapeutic potential is limited by its short biological half-life due to rapid enzymatic degradation by dipeptidyl peptidase IV. The present study aimed to demonstrate the therapeutic effects of constitutively expressed GLP-1 in an overt type 2 diabetic animal model using an adenoviral vector system. METHODS: A novel plasmid (pAAV-ILGLP-1) and recombinant adenoviral vector (Ad-ILGLP-1) were constructed with the cytomegalovirus promoter and insulin leader sequence followed by GLP-1(7-37) cDNA. RESULTS: The results of an enzyme-linked immunosorbent assay showed significantly elevated levels of GLP-1(7-37) secreted by human embryonic kidney cells transfected with the construct containing the leader sequence. A single intravenous administration of Ad-ILGLP-1 into 12-week-old Zucker diabetic fatty (ZDF) rats, which have overt type 2 diabetes mellitus (T2DM), achieved near normoglycemia for 3 weeks and improved utilization of blood glucose in glucose tolerance tests. Circulating plasma levels of GLP-1 increased in GLP-1-treated ZDF rats, but diminished 21 days after treatment. When compared with controls, Ad-ILGLP-1-treated ZDF rats had a lower homeostasis model assessment for insulin resistance score indicating amelioration in insulin resistance. Immunohistochemical staining showed that cells expressing GLP-1 were found in the livers of GLP-1-treated ZDF rats. CONCLUSIONS: These data suggest that GLP-1 gene therapy can improve glucose homeostasis in fully developed diabetic animal models and may be a promising treatment modality for T2DM in humans.