Design, synthesis and biological evaluation of double fatty chain-modified glucagon-like peptide-1 conjugates.

Twelve double fatty chains and Aib8-Arg34-GLP-1 (7-37) were designed and obtained by microwave-assisted solid-phase synthesis. Then, twelve conjugates of Aib8-Arg34-GLP-1 (7-37) were synthesized in 1% triethylamine aqueous solution. Conjugates 2, 3, 6, 7, 10 and 11 showed better GLP-1 receptor activation potency than semaglutide. However, conjugates 2, 6 and 10 showed slightly worse glucose-lowering effects in vivo than semaglutide but better effects than conjugates 3, 7 and 11. The CD spectra of conjugates 2, 6 and 10 indicated that they had the same secondary structure as liraglutide and semaglutide. The receptor affinity results for conjugates 2, 6 and 10 measured by SPR (surface plasmon resonance) showed that conjugate 2 had higher receptor affinity than conjugates 6 and 10. In addition, albumin binding assays indicated that double fatty acid chains had obvious synergistic effects compared with single fatty acid chains. In conclusion, the structure-activity relationship of different side chains was summarized and one candidate, conjugate 2, was screened.

The Design of a GLP-1/PYY Dual Acting Agonist.

The two gut hormones GLP-1 and PYY3-36 , which are both secreted from the L-cells upon food stimuli, have a stronger inhibitory effect on food intake when they are combined, compared to their individual effects as single agonists. Although they are not homologous and share no sequence similarity, we show that a GLP-1 analogue can be designed to exhibit potent activity on both the Y2 and GLP-1 receptors. Dual acting hybrid analogues were realized by designing truncated and potent Y2 receptor PYY analogues, followed by integrating the critical residues into GLP-1. In this study, we show that one of these dual acting agonists acutely reduces food intake significantly more than the respective mono-agonist counterparts.

Automated radiosynthesis of [18F]FBEM, a sulfhydryl site specific labeling agent for peptides and proteins.

In the process of developing [18F]FBEM coupled target peptide, we have instituted a robust automated synthesis of [18F]FBEM, a sulfhydryl (-SH) site specific agent for radiolabeling of peptides and proteins. The radiosynthesis generated 1.67-3.89 GBq (45.1-105.1 mCi, 7.5-18.8% non-decay corrected yield) of [18F]FBEM from 22.2 GBq (600 mCi) of starting [18F]fluoride with molar activity of 31.8 ±â€¯5.3 GBq/µmol (0.86 ±â€¯0.14 mCi/nmol) (n = 3) at the end of synthesis. Radiochemical purity was greater than 98%, and total synthesis time was ~90 min.

Novel fatty acid chain modified GLP-1 derivatives with prolonged in vivo glucose-lowering ability and balanced glucoregulatory activity.

Glucagon-like peptide-1 is a potent hypoglycemic hormone with beneficial properties for the treatment of diabetes. However, its half-life is short because the rapid metabolic degradation. This study aims to prolong the half-life of glucagon-like peptide-1 through conjugation with the fatty acid side chain which helps the conjugates to interact with the albumin. Firstly, we chose two optimized polypeptide chains which have tremendous hypoglycemic effect named Cys17-Gly8-GLP-1(7-36)-NH2 and Cys37-Gly8-GLP-1(7-37)-NH2, and various fatty acid chains were modified. All conjugates preserved relatively strong GLP-1R activation and I-6 behaved best in glucose-lowering ability. The prolonged antidiabetic effects of I-6 were further confirmed by hypoglycemic efficacy test in vivo. Meanwhile, once daily injection of I-6 to diabetic mice achieved long-term beneficial effects on glucose tolerance, body weight and blood chemistry. It is concluded that I-6 is a promising agent for further investigation of its potential to treat obese patients with diabetes.

Preparation and Pharmaceutical Characterizations of Lipidated Dimeric Xenopus Glucagon-Like Peptide-1 Conjugates.

A pair of glucagon-like peptide-1 (GLP-1) analogs (1 and 2) were synthesized by hybridizing the key sequences of GLP-1, exendin-4, lixisenatide, and xenGLP-1B (Xenopus GLP-1 analog). To achieve long-acting hypoglycemic effects and to further improve their anti-diabetic potencies, lipidization and dimerization strategies were used to afford two lipidated dimeric conjugates (9 and 11). Conjugates 9 and 11 showed stronger receptor activation potency than GLP-1 and exendin-4 in vitro. Moreover, 9 and 11 exhibited superior hypoglycemic and insulinotropic activities to liraglutide in type 2 diabetic C57BL/6J-m+/+ Leprdb (db/db) mice. Pharmacokinetic studies revealed that the circulating half-lives (t1/2) of 9 and 11 were 2.3- and 1.7-fold longer than that of liraglutide. The improved pharmacokinetic profiles led to significantly protracted in vivo anti-diabetic effects as confirmed by multiple oral glucose tolerance tests and hypoglycemic duration tests. Most importantly, chronic treatment studies found that once daily administration of 9 or 11 in db/db mice achieved more beneficial effects on HbA1c reduction and glucose tolerance normalization than liraglutide. Our research demonstrated lipidization and dimerization as useful tools for the development of novel GLP-1 receptor agonists. The preclinical studies suggested the potential of 9 and 11 to be developed as novel anti-diabetic agents.

Bioactivity of a modified human Glucagon-like peptide-1.

Diabetes has become the third largest cause of death in humans worldwide. Therefore, effective treatment for this disease remains a critical issue. Glucagon-like peptide-1 (GLP-1) plays an important role in glucose homeostasis, and therefore represents a promising candidate to use for the treatment of diabetes. Native GLP-1, however, is quickly degraded in in the circulatory system; which limits its clinical application. In the present study, a chemically-synthesized, modified analogue of human GLP-1 (mGLP-1) was designed. Our analyses indicated that, relative to native GLP-1, mGLP-1 is more resistant to trypsin and pancreatin degradation. mGLP-1 promotes mouse pancreatic ß-cell proliferation by up-regulating the expression level of cyclin E, CDK2, Bcl-2 and down-regulating Bax, p21, and stimulates insulin secretion. An oral glucose tolerance test indicated that mGLP-1 significantly improved glucose tolerance in mice. Intraperitoneal injections of mGLP-1 into streptozotocin (STZ)-induced type 2 diabetic mice significantly reduced blood sugar levels and stimulated insulin secretion. Oral gavages of mGLP-1 in diabetic mice did not result in significant hypoglycemic activity.

Rational design of α-helix-stabilized exendin-4 analogues.

Exendin-4 (Ex4) is a potent glucagon-like peptide-1 receptor agonist, a drug regulating the plasma glucose level of patients suffering from type 2 diabetes. The molecule's poor solubility and its readiness to form aggregates increase the likelihood of unwanted side effects. Therefore, we designed Ex4 analogues with improved structural characteristics and better water solubility. Rational design was started from the parent 20-amino acid, well-folded Trp cage (TC) miniprotein and involved the step-by-step N-terminal elongation of the TC head, resulting in the 39-amino acid Ex4 analogue, E19. Helical propensity coupled to tertiary structure compactness was monitored and quantitatively analyzed by electronic circular dichroism and nuclear magnetic resonance (NMR) spectroscopy for the 14 peptides of different lengths. Both (15)N relaxation- and diffusion-ordered NMR measurements were established to investigate the inherent mobility and self-association propensity of Ex4 and E19. Our designed E19 molecule has the same tertiary structure as Ex4 but is more helical than Ex4 under all studied conditions; it is less prone to oligomerization and has preserved biological activity. These conditions make E19 a perfect lead compound for further drug discovery. We believe that this structural study improves our understanding of the relationship between local molecular features and global physicochemical properties such as water solubility and could help in the development of more potent Ex4 analogues with improved pharmacokinetic properties.

The C-terminal extension of exendin-4 provides additional metabolic stability when added to GLP-1, while there is minimal effect of truncating exendin-4 in anaesthetized pigs.

The most striking sequence difference between glucagon-like peptide-1 (GLP-1)(2) and the longer-acting GLP-1 receptor agonist, exendin-4 (Ex-4),(3) is the nine-amino acid COOH-terminal extension of Ex-4. We investigated the contribution of this extension to the survival time of Ex-4. We assessed the overall metabolism of GLP-1, Ex-4, a COOH-terminally extended GLP-1 peptide (GLP-1+Ex(31-39); GLP-Ex),(4) and a COOH-terminally truncated exendin peptide (Ex(1-30)) in anaesthetized, catheterized pigs, with focus on the extraction across the kidneys and a peripheral tissue (a hindleg, representing muscle, adipose- and connective tissue). Peptide analysis was carried out with assays against the mid-region of the peptides, whereby the role of dipeptidyl peptidase-4 (DPP-4)(5) mediated NH(2)-terminal degradation could be disregarded. The half-life of GLP-1 was significantly increased when the COOH-terminal extension of Ex-4 was added (GLP-1 4.8±3.3min; GLP-Ex 19.5±3.3min). In contrast, there was no effect of truncating Ex-4 (Ex-4 32.4±4.1min; Ex(1-30) 28.4±1.7min). Ex-4 and Ex(1-30) were cleared solely by the kidneys at rates corresponding to the glomerular filtration rate (GFR),(6) while GLP-1 and GLP-Ex were cleared by both the kidneys and peripheral tissues. Both extraction rates were, however, significantly reduced with GLP-Ex compared to GLP-1. The renal clearance rate of GLP-1 greatly exceeded GFR, while GLP-Ex was cleared at a rate resembling GFR. In conclusion, the COOH-terminal extension of Ex-4 contributes minimally to the increased survival time of Ex-4, while addition of this sequence to GLP-1 significantly reduces its clearance.

(Val(8))GLP-1-Glu-PAL: a GLP-1 agonist that improves hippocampal neurogenesis, glucose homeostasis, and ß-cell function in high-fat-fed mice.

This study examined the biological properties of a novel GLP-1 peptide, (Val(8))GLP-1-Glu-PAL, engineered with an Ala(8)→Val(8) substitution and additional incorporation of a C(16) fatty acid moiety at Lys(26) via a glutamic acid linker. GLP-1 underwent 75 % degradation by DPP-IV over 8 h, whereas (Val(8))GLP-1 and (Val(8))GLP-1-Glu-PAL remained intact. All GLP-1 peptides significantly stimulated insulin secretion at 5.6 mM (1.3- to 4.9-fold, p<0.01 to p<0.001) and 16.7 mM glucose (1.5- to 2.3-fold, p<0.001). At higher concentrations (Val(8))GLP-1-Glu-PAL was significantly more potent at stimulating insulin secretion (1.2- to 1.3-fold, p<0.05). In high-fat-fed mice, all GLP-1 peptides significantly lowered plasma glucose concentrations (41-66 % decrease, p<0.05 to p<0.001), with (Val(8))GLP-1-Glu-PAL eliciting protracted glucose-lowering actions (32-59 % decrease, p<0.05 to p<0.01) when administered 8 h prior to a glucose load. Twice-daily administration of (Val(8))GLP-1-Glu-PAL in high-fat-fed mice for 21 days had no effect on bodyweight or food intake, but significantly lowered non-fasting plasma glucose (43-46 % decrease, p<0.05). (Val(8))GLP-1-Glu-PAL markedly decreased glycemic excursion following intraperitoneal glucose (32-48 % decrease, p<0.05), enhanced insulin response to glucose (2- to 2.3-fold, p<0.05 to p<0.01), and improved insulin sensitivity (25-38 % decrease in plasma glucose, p<0.05). O(2) consumption, CO(2) production, RER, and energy expenditure were not altered by (Val(8))GLP-1-Glu-PAL therapy. Treatment with (Val(8))GLP-1-Glu-PAL resulted in a significant increase in BrdU-positive cells (1.3-fold, p<0.05) in the granule cell layer of the dentate gyrus. These data demonstrate that (Val(8))GLP-1-Glu-PAL is a long-acting GLP-1 peptide that significantly improves hippocampal neurogenesis, glucose homeostasis, and insulin secretion in high-fat-fed mice.

Optimization of co-agonism at GLP-1 and glucagon receptors to safely maximize weight reduction in DIO-rodents.

The ratio of GLP-1/glucagon receptor (GLP1R/GCGR) co-agonism that achieves maximal weight loss without evidence of hyperglycemia was determined in diet-induced obese (DIO) mice chronically treated with GLP1R/GCGR co-agonist peptides differing in their relative receptor agonism. Using glucagon-based peptides, a spectrum of receptor selectivity was achieved by a combination of selective incorporation of GLP-1 sequences, C-terminal modification, backbone lactam stapling to stabilize helical structure, and unnatural amino acid substitutions at the N-terminal dipeptide. In addition to α-amino-isobutyric acid (Aib) substitution at position two, we show that α,α'-dimethyl imidazole acetic acid (Dmia) can serve as a potent replacement for the highly conserved histidine at position one. Selective site-specific pegylation was used to further minimize enzymatic degradation and provide uniform, extended in vivo duration of action. Maximal weight loss devoid of any sign of hyperglycemia was achieved with a co-agonist comparably balanced for in vitro potency at murine GLP1R and GCGR. This peptide exhibited superior weight loss and glucose lowering compared to a structurally matched pure GLP1R agonist, and to co-agonists of relatively reduced GCGR tone. Any further enhancement of the relative GCGR agonist potency yielded increased weight loss but at the expense of elevated blood glucose. We conclude that GCGR agonism concomitant with GLP1R agonism constitutes a promising approach to treatment of the metabolic syndrome. However, the relative ratio of GLP1R/GCGR co-agonism needs to be carefully chosen for each species to maximize weight loss efficacy and minimize hyperglycemia.

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 48h 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.

Disulfide bond prolongs the half-life of therapeutic peptide-GLP-1.

The multiple physiological characterization of glucagon-like peptide-1 (GLP-1) makes 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 rapid 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 homodimeric analogs, which fused the mutated GLP-1 monomer by an intra-disulfide bridge. The stabilities of the GLP-1 homodimeric analogs were investigated and the physiological functions of the analogs were compared with those of wild-type GLP-1 in rats and human serum. Single dose glucose tolerance test was performed to investigate the administration frequency which satisfied the efficient glucose regulatory in rats. Multiple dose glucose tolerance tests were employed also to study the long-acting anti-diabetic activity of GLP-1 homodimeric analog. The results indicated that the GLP-1 homodimeric analog (hdGLP1G10C) remarkably raised the biological half-life of GLP-1; also HDGLP1G10C showed better glucose tolerance and higher HbA(1c) reduction than GLP-1 in rodents. Based upon the results in this study, it was suggested that hdGLP1G10C prolonged the stability of GLP-1 and retained the biological activity of GLP-1. The improved physiological characterization of hdGLP1G10C makes it as 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.

Development of potent glucagon-like peptide-1 agonists with high enzyme stability via introduction of multiple lactam bridges.

Glucagon-like peptide-1 (GLP-1) has the ability to lower the blood glucose level, and its regulatory functions make it an attractive therapeutic agent for the treatment of type 2 diabetes. However, its rapid degradation by enzymes like dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase (NEP) 24.11 severely compromises its effective clinical use. Whereas specific DPP-IV inhibitors have been developed, NEP 24.11 targets multiple sites in the GLP-1 sequence, which makes it difficult to block. To address this drawback, we have designed and synthesized conformationally constrained GLP-1 analogues by introducing multiple lactam bridges that stabilized both alpha-helices in the N- and C-terminal regions simultaneously. In addition to improving the receptor activation capability (up to 5-fold) by fixing the alpha-helical conformations required for optimal receptor interaction, the introduced lactam bridges provided outstanding shielding over NEP 24.11 (half-life of >96 h). These highly constrained peptides are the first examples of NEP 24.11-resistant GLP-1 analogues.

Synthesis and characterization of ester-based prodrugs of glucagon-like peptide 1.

Peptides represent a rich natural source of potential medicines with one notable pharmaceutical limitation being their relatively short duration of action. A particularly good example of this phenomenon is glucagon-like peptide 1 (GLP), a hormone of appreciable interest for the treatment of type II diabetes. In the native form, GLP demonstrates an extremely short half-life in plasma and a relatively narrow therapeutic index with gastrointestinal adverse pharmacology. We envisioned a prodrug of GLP as a means to extend the duration of action and broaden the therapeutic index of this peptide hormone. We designed, synthesized, and characterized ester-based prodrugs of GLP that differentially convert to the parent drug under physiological conditions driven by their inherent chemical instability. In a set of dipeptide extended GLP-analogs we explored the rate of diketopiperazine (DKP) and diketomorpholine (DMP) formation, and the release of the active peptide. The rate of cleavage was observed to be a function of the conformation of the dipeptide promoiety and the strength of the cyclization nucleophile. Through the careful selection of chemical functionality, a set of GLP ester prodrugs of variable half-lives has been identified.

Synthesis and bioactivity evaluation of dipeptidyl peptidase IV resistant glucagon-like peptide-1 analogues.

Glucagon-like peptide -1 (GLP-1) is an incretin hormone displaying glucose-dependent stimulation of insulin secretion and trophic effects on the pancreatic ß-cells. However, GLP-1 is rapidly degraded to GLP-1(9-36) by dipeptidyl peptidase-IV (DPP-IV), which removes the N-terminal dipeptide His(7)-Ala(8). The rapid inactivation of GLP-1 in the blood circulation limits its clinical application. Hence, we replaced the enzymatic hydrolyzation position Ala(8) with other natural amino acids. The GLP-1 analogues were synthesized rapidly and efficiently under microwave irradiation, using Fmoc/tBu orthogonal protection strategy. Studies on blood-glucose-lowering effect of GLP-1 analogues in vivo were undertaken using 10-week-old male Kunming mice. The metabolic stability was tested by incubation with dipeptidyl peptidase-IV (DPP-IV). Generally, Xaa(8)-GLP-1 analogues exhibit resistance to DPP-IV degradation in vitro and stronger hypoglycemic effect than GLP-1. This may help to understand the structure-activity relationship of GLP-1 analogues.

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 ((113/115)In) 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 (111)In-labeled peptides. Our results suggest that the prepared GLP-1 derivatives are suitable imaging probes for studying pancreatic islet function in vivo.

Influence of selective fluorination on the biological activity and proteolytic stability of glucagon-like peptide-1.

The relative simplicity and high specificity of peptide therapeutics has fueled recent interest. However, peptide and protein drugs generally require injection and suffer from low metabolic stability. We report here the design, synthesis, and characterization of fluorinated analogues of the gut hormone peptide, GLP-1. Overall, fluorinated GLP-1 analogues displayed higher proteolytic stability with simultaneous retention of biological activity (efficacy). Fluorinated amino acids are useful for engineering peptide drug candidates and probing ligand-receptor interactions.

Search for alpha-helical propensity in the receptor-bound conformation of glucagon-like peptide-1.

To elucidate the receptor-bound conformation of glucagon-like peptide-1 (GLP-1), a series of conformationally constrained GLP-1 analogues were synthesized by introducing lactam bridges between Lys(i) and Glu(i)(+4) to form alpha-helices at various positions. The activity and affinity of these analogues to GLP-1 receptors suggested that the receptor-bound conformation comprises two alpha-helical segments between residues 11-21 and 23-34. It is notable that the N-terminal alpha-helix is extended to Thr(11), and that Gly(22) plays a pivotal role in arranging the two alpha-helices. Based on these findings, a highly potent bicyclic GLP-1 analogue was synthesized which is the most conformationally constrained GLP-1 analogue reported to date.