Design,synthesis and evaluation of 3-phenoxypyrazine-2-carboxamide derivatives as potent TGR5 agonists

TGR5 is emerging as an important and promising target for the treatment of non-alcoholic steatohepatitis, type 2 diabetes mellitus (T2DM), and obesity. A series of novel 3-phenoxypyrazine-2-carboxamide derivatives were designed, synthesized and evaluated in vitro and in vivo. The most potent compounds 18g and 18k exhibited excellent hTGR5 agonist activity, which was superior to those of the reference drug INT-777. In addition, compound 18k could significantly reduce blood glucose levels in C57 BL/6 mice and stimulate GLP-1 secretion in NCI-H716 cells and C57 BL/6 mice.

The most potent compound 18k exhibited excellent hTGR5 agonist activity, which was superior to those of the reference drugs INT-777. In addition, compound 18k could significantly reduce blood glucose levels and stimulate GLP-1 secretion in C57 BL/6 mice.     

Enhancing incretin action for the treatment of type 2 diabetes

OBJECTIVE: To examine the mechanisms of action, therapeutic potential, and challenges inherent in the use of incretin peptides and dipeptidyl peptidase-IV (DPP-IV) inhibitors for the treatment of type 2 diabetes. RESEARCH DESIGN AND METHODS: The scientific literature describing the biological importance of incretin peptides and DPP-IV inhibitors in the control of glucose homeostasis has been reviewed, with an emphasis on mechanisms of action, experimental diabetes, human physiological experiments, and short-term clinical studies in normal and diabetic human subjects. RESULTS: Glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) exert important effects on beta-cells to stimulate glucose-dependent insulin secretion. Both peptides also regulate beta-cell proliferation and cytoprotection. GLP-1, but not GIP, inhibits gastric emptying, glucagon secretion, and food intake. The glucose-lowering actions of GLP-1, but not GIP, are preserved in subjects with type 2 diabetes. However, native GLP-1 is rapidly degraded by DPP-IV after parenteral administration; hence, degradation-resistant, long-acting GLP-1 receptor (GLP-1R) agonists are preferable agents for the chronic treatment of human diabetes. Alternatively, inhibition of DPP-IV-mediated incretin degradation represents a complementary therapeutic approach, as orally available DPP-IV inhibitors have been shown to lower glucose in experimental diabetic models and human subjects with type 2 diabetes. CONCLUSIONS: GLP-1R agonists and DPP-IV inhibitors have shown promising results in clinical trials for the treatment of type 2 diabetes. The need for daily injections of potentially immunogenic GLP-1-derived peptides and the potential for unanticipated side effects with chronic use of DPP-IV inhibitors will require ongoing scrutiny of the risk-benefit ratio for these new therapies as they are evaluated in the clinic.     

Glucagon-like peptide-1 plasmid construction and delivery for the treatment of type 2 diabetes.

Glucagon-like peptide-1 (GLP-1) is a 30-amino-acid hormone produced by intestinal L cells. It has been proposed that GLP-1 can be used as a new treatment for type 2 diabetes mellitus because it acts to augment insulin secretion and its effectiveness is maintained in type 2 diabetic patients. Despite its many remarkable advantages as a therapeutic agent for diabetes, GLP-1 is not immediately clinically applicable because of its extremely short half-life. One way to overcome this drawback is GLP1 gene delivery, which enables GLP-1 production in the body. In this study, the effect of GLP1 gene delivery was evaluated both in vitro and in vivo using a new plasmid constructed with a GLP1 (7-37) cDNA. The expression of the GLP1 gene was driven by a SV40 promoter/enhancer. To increase the expression level of GLP-1, nuclear factor kappaB binding sites were introduced. The in vitro results showed expression of GLP-1 and in vitro activity of GLP-1, which is a glucose-dependent insulinotropic action. A single systemic administration of polyethyleneimine/pSIGLP1NFkappaB complex into DIO mice resulted in increasing insulin secretion and decreasing blood glucose levels for a duration longer than 2 weeks.     

Common Genetic Variation in GLP1R and Insulin Secretion in Response to Exogenous GLP-1 in Nondiabetic Subjects: A pilot study

OBJECTIVE

Glucagon-like peptide (GLP)-1 receptor is encoded by GLP1R. The effect of genetic variation at this locus on the response to GLP-1 is unknown. This study assessed the effect of GLP1R polymorphisms on insulin secretion in response to hyperglycemia and to infused GLP-1 in nondiabetic subjects.

RESEARCH DESIGN AND METHODS

Eighty-eight healthy individuals (aged 26.3 ± 0.6 years, fasting glucose 4.83 ± 0.04 mmol/l) were studied using a hyperglycemic clamp. GLP-1 was infused for the last 2 h of the study (0.75 pmol/kg/min over 121–180 min, 1.5 pmol/kg/min over 181–240 min). β-Cell responsivity (Φ_Total) was measured using a C-peptide minimal model. The effect of 21 tag single nucleotide polymorphisms (SNPs) in GLP1R on Φ_Total was examined.

RESULTS

Two SNPs (rs6923761 and rs3765467) were nominally associated with altered β-cell responsivity in response to GLP-1 infusion.

CONCLUSIONS

Variation in GLP1R may alter insulin secretion in response to exogenous GLP-1. Future studies will determine whether such variation accounts for interindividual differences in response to GLP-1–based therapy.Expression of a nonsynonymous single nucleotide polymorphism (SNP), which results in substitution of methionine for threonine at position 149 of GLP1R in cell systems, decreases binding affinity for glucagon-like peptide (GLP)-1 and intracellular signaling after hormone-receptor binding (1). These functional effects suggest that genetic variation in GLP1R may alter responsiveness to GLP-1 in vivo. To examine this hypothesis, we used a hyperglycemic clamp, together with GLP-1–amide (7,36) infusion, and measured insulin secretion using a modification of the C-peptide minimal model to determine β-cell responsivity (Φ_Total) to GLP-1 in vivo.     

The Effect of Exogenous Glucose-Dependent Insulinotropic Polypeptide in Combination With Glucagon-Like Peptide-1 on Glycemia in the Critically Ill

OBJECTIVE

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) have additive insulinotropic effects when coadministered in health. We aimed to determine whether GIP confers additional glucose lowering to that of GLP-1 in the critically ill.

RESEARCH DESIGN AND METHODS

Twenty mechanically ventilated critically ill patients without known diabetes were studied in a prospective, randomized, double-blind, crossover fashion on 2 consecutive days. Between T₀ and T₄₂₀ minutes, GLP-1 (1.2 pmol/kg · min⁻¹) was infused intravenously with either GIP (2 pmol/kg · min⁻¹) or 0.9% saline. Between T₆₀ and T₄₂₀ minutes, nutrient liquid was infused into the small intestine at 1.5 kcal/min.

RESULTS

Adding GIP did not alter blood glucose or insulin responses to small intestinal nutrient. GIP increased glucagon concentrations slightly before nutrient delivery (P = 0.03), but not thereafter.

CONCLUSIONS

The addition of GIP to GLP-1 does not result in additional glucose-lowering or insulinotropic effects in critically ill patients with acute-onset hyperglycemia.Hyperglycemia occurs frequently in the critically ill, even in the absence of pre-existing diabetes (1). Although insulin is an effective treatment, its use confers an increased risk of hypoglycemia, which is associated with increased mortality (1,2). In health, but not in patients with type 2 diabetes, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) have additive insulinotropic effects when coadministered, without increasing the risk of hypoglycemia (3,4). The objective of this study was to determine whether the addition of GIP to GLP-1 would result in additional glucose-lowering in enterally fed, critically ill patients without pre-existing diabetes.     

Identification of the effect of N-glycan modification and its sialylation on proteolytic stability and glucose-stabilizing activity of glucagon-like peptide 1 by site-directed enzymatic glycosylation

In this study, an approach to prepare long-acting glucagon-like peptide 1 (GLP-1) by site-directed enzymatic glycosylation with homogeneous biantennary complex-type N-glycan has been developed. All the N-glycan-modified GLP-1 analogues preserved an unchanged secondary structure. The glycosylated GLP-1 analogues with sialyl complex-type N-glycan modified at Asn26 and Asn34 exhibited a 36.7- and 24.0-fold in vitro half-life respectively when incubated with dipeptidyl peptidase-IV (DPP-IV), and 25.0- and 13.9-fold respectively when incubated with mouse serum. Compared to native GLP-1, both glycosylated GLP-1 analogues modified at Asn34 by asialyl and sialyl N-glycan demonstrated lower maximum blood glucose levels, as well as more rapid and more persistent glucose-stabilizing capability in type 2 diabetic db/db mice. Our results indicated that the selection of an appropriate position (to avoid hindering the peptide-receptor binding) is crucial for N-glycan modification and its sialylation to improve the therapeutic properties of the modified peptides. The information learned would facilitate future design of therapeutic glycopeptides/glycoproteins with N-glycan to achieve enhanced pharmacological properties.

Site-directed glycosylation of glucagon-like peptide 1 enhanced its proteolytic stability and improved its blood glucose-stabilizing activity.     

Therapeutic potential of dipeptidyl peptidase-IV inhibitors in patients with diabetes mellitus

Dipeptidyl peptidase-IV (DPP-IV) inhibitors are a new class of oral antidiabetic agents for the treatment of patients with type 2 diabetes. Inhibition of the enzyme DPP-IV results in increased activity of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), the incretin hormones. Through the action of GLP-1 and GIP, DPP-IV inhibitors improve preprandial and postprandial glucose by enhancing insulin secretion and reducing postprandial concentrations of glucagon. This review examines the background, current evidence, and future therapeutic potential of this novel class of drug.     

Novel mono-lipidated dimeric glucagon-like peptide-1 receptor agonist with improved long-acting and hypoglycemic activity

Dimerization is a useful tool to boost ligand–receptor interaction. Both lipidation and dimerization effectively prolong the short half-life (t_1/2) of peptides by facilitating binding with serum albumin and increasing hydrodynamic size. Here, we described two novel GLP-1 conjugates with high glucagon-like peptide-1 (GLP-1) receptor activation potencies, dimerized GLP-1 (Di-GLP-1) and lipidated Di-GLP-1 (Lip-Di-GLP-1). Di-GLP-1 and Lip-Di-GLP-1 were prepared through cysteine–maleimide specific coupling reactions using Gly₈-Cys₃₁-GLP-1, bis-maleimide amine, and activated palmitic acid. The receptor activation potencies of Di-GLP-1 and Lip-Di-GLP-1 were 13.6-fold and 9.5-fold higher than GLP-1, respectively. The in vivo hypoglycemic and insulinotropic activities of Di-GLP-1 and Lip-Di-GLP-1 were also better than GLP-1 in db/db mice. Furthermore, Lip-Di-GLP-1 was found to have greater circulating t_1/2 than synthesized liraglutide by 1.8-fold. Accordingly, the improved pharmacokinetic profiles of Lip-Di-GLP-1 resulted in protracted antidiabetic effects as confirmed by hypoglycemic duration test. Moreover, Lip-Di-GLP-1 administered in mice potently inhibits gastric emptying and reduce food intake. Chronic Lip-Di-GLP-1 treatment in db/db mice resulted in significant improvements in food intake, body weight, pancreatic function and corrected hyperglycemia, which was more effective than synthesized liraglutide. Our research indicated that combined dimerization and lipidation were effectively applied to GLP-1, and the preclinical results suggested the potential usage of Lip-Di-GLP-1 as a long-acting antidiabetic agent.

Dimerization and lipidation were effectively applied to GLP-1 to make Lip-Di-GLP-1 as a long-acting antidiabetic agent.     

Administration of an acylated GLP-1 and GIP preparation provides added beneficial glucose-lowering and insulinotropic actions over single incretins in mice with Type 2 diabetes and obesity

The present study examined the glucose-lowering and insulinotropic properties of acylated GLP-1 (glucagon-like peptide-1) and GIP (glucose-dependent insulinotropic polypeptide) peptides in Type 2 diabetes and obesity. GLP-1, GIP, Liraglutide, N-AcGIP(Lys(37)Myr) (N-acetylGIP with myristic acid conjugated at Lys(37)), a simple combination of both peptides and a Lira-AcGIP preparation [overnight preparation of Liraglutide and N-AcGIP(Lys(37)Myr)] were incubated with DPP-IV (dipeptidyl peptidase-IV) to assess peptide stability, and BRIN-BD11 cells were used to evaluate cAMP production and insulin secretion. Acute glucose-lowering and insulinotropic actions were evaluated in Swiss TO mice. Subchronic studies on glucose homoeostasis, insulin secretion, food intake and bodyweight were evaluated in ob/ob mice. Liraglutide, N-AcGIP(Lys(37)Myr), a simple combination of both peptides and the Lira-AcGIP preparation demonstrated improved DPP-IV resistance (P<0.001), while stimulating cAMP production and insulin secretion (1.4-2-fold; P<0.001). The Lira-AcGIP preparation was more potent at lowering plasma glucose (20-51% reduction; P<0.05-P<0.001) and stimulating insulin secretion (1.5-1.8-fold; P<0.05-P<0.001) compared with Liraglutide and N-AcGIP(Lys(37)Myr) or a simple peptide combination. Daily administration of the Lira-AcGIP preparation to ob/ob mice lowered bodyweight (7-9%; P<0.05), food intake (23%; P<0.05) and plasma glucose (46% reduction; P<0.001), while increasing plasma insulin (1.5-1.6-fold; P<0.001). The Lira-AcGIP preparation enhanced glucose tolerance, insulin response to glucose and insulin content (P<0.05-P<0.001). These findings demonstrate that a combined preparation of the acylated GLP-1 and GIP peptides Liraglutide and N-AcGIP(Lys(37)Myr) markedly improved glucose-lowering and insulinotropic properties in diabetic obesity compared with either incretin mimetic given individually.     

Effects of a d-Xylose Preload With or Without Sitagliptin on Gastric Emptying,Glucagon-Like Peptide-1, and Postprandial Glycemia in Type 2 Diabetes

OBJECTIVE

Macronutrient “preloads” can reduce postprandial glycemia by slowing gastric emptying and stimulating glucagon-like peptide-1 (GLP-1) secretion. An ideal preload would entail minimal additional energy intake and might be optimized by concurrent inhibition of dipeptidyl peptidase-4 (DPP-4). We evaluated the effects of a low-energy d-xylose preload, with or without sitagliptin, on gastric emptying, plasma intact GLP-1 concentrations, and postprandial glycemia in type 2 diabetes.

RESEARCH DESIGN AND METHODS

Twelve type 2 diabetic patients were studied on four occasions each. After 100 mg sitagliptin (S) or placebo (P) and an overnight fast, patients consumed a preload drink containing either 50 g d-xylose (X) or 80 mg sucralose (control [C]), followed after 40 min by a mashed potato meal labeled with ¹³C-octanoate. Blood was sampled at intervals. Gastric emptying was determined.

RESULTS

Both peak blood glucose and the amplitude of glycemic excursion were lower after PX and SC than PC (P < 0.01 for each) and were lowest after SX (P < 0.05 for each), while overall blood glucose was lower after SX than PC (P < 0.05). The postprandial insulin-to-glucose ratio was attenuated (P < 0.05) and gastric emptying was slower (P < 0.01) after d-xylose, without any effect of sitagliptin. Plasma GLP-1 concentrations were higher after d-xylose than control only before the meal (P < 0.05) but were sustained postprandially when combined with sitagliptin (P < 0.05).

CONCLUSIONS

In type 2 diabetes, acute administration of a d-xylose preload reduces postprandial glycemia and enhances the effect of a DPP-4 inhibitor.Therapeutic strategies directed at reducing postprandial glycemia are of fundamental importance in the management of type 2 diabetes (1). For patients with mild-to-moderate hyperglycemia, postprandial blood glucose is a better predictor of HbA_1c than fasting blood glucose (2).Both gastric emptying and the action of the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are major determinants of postprandial glucose excursions (3). Gastric emptying determines the rate of nutrient delivery to the small intestine, accounting for approximately one-third of the variation in the initial rise in glycemia after oral glucose in both healthy subjects (4) and those with type 2 diabetes (5). GLP-1 and GIP, released predominantly from the distal and proximal gut, respectively, are the known mediators of the incretin effect, whereby much more insulin is stimulated by enteral compared with intravenous glucose (6). In type 2 diabetes, the incretin effect is impaired (7), related at least partly to a diminished insulinotropic effect of GIP, while that of GLP-1 is preserved (8). In addition, GLP-1 slows gastric emptying (9), suppresses glucagon secretion (10), and reduces energy intake (11). Therefore, incretin-based therapies for diabetes have hitherto focused on GLP-1.One promising strategy to stimulate endogenous GLP-1 is the “preload” concept, which involves administration of a small load of macronutrient at a fixed interval before a meal so that the presence of nutrients in the small intestine induces the release of gut peptides, including GLP-1, to slow gastric emptying and improve the glycemic response to the subsequent meal. Fat (12) and protein (13) preloads achieve these goals but entail additional energy intake. We recently demonstrated in healthy subjects the potential for poorly absorbed sweeteners, which yield little energy, to stimulate GLP-1 secretion and slow gastric emptying (14).d-Xylose is a pentose sugar, which is incompletely absorbed by passive diffusion in human duodenum and jejunum (15), with the remainder delivered to the ileum and the colon, where bacterial fermentation occurs, producing hydrogen that can be detected in the breath (16). We recently showed that oral consumption of d-xylose stimulates GLP-1 secretion to a greater and more sustained degree than glucose in healthy older subjects (17), consistent with the principle that the length and region of small intestine exposed to carbohydrate are important determinants of GLP-1 release (18). D-Xylose also slowed gastric emptying compared with water, with efficacy similar to that of the glucose load (17).Intact GLP-1 is short-lived in the circulation largely because of rapid degradation by the enzyme dipeptidyl peptidase-4 (DPP-4) (19), and orally administered DPP-4 inhibitors, such as sitagliptin, increase postprandial plasma concentrations of intact GLP-1 (20). However, the concept of stimulating endogenous GLP-1 with enteral nutrients and then optimizing its action with a DPP-4 inhibitor has received little attention. Moreover, little consideration has been given as to whether the composition of the diet influences the efficacy of DPP-4 inhibition to lower postprandial blood glucose.The current study was designed to determine, in patients with type 2 diabetes, whether a d-xylose preload would slow gastric emptying, stimulate GLP-1 secretion, and improve postprandial glycemia and whether these effects could be enhanced by DPP-4 inhibition.     

GLP-1 gene delivery for the treatment of type 2 diabetes

Glucagon-like peptide-1 (GLP-1) is a potent insulinotrophic hormone, which makes GLP-1 an attractive candidate for the treatment of type 2 diabetes. However, the short plasma half-life of the active forms of GLP-1 poses an obstacle to the sustained delivery of this peptide. In this study, we evaluated the effect of GLP-1 gene delivery both in vitro and in vivo using a new plasmid constructed with a modified GLP-1 (7-37) cDNA. This cDNA contains a furin cleavage site between the start codon and the GLP-1 coding region. The expression of the GLP-1 gene was driven by a chicken β-actin promoter (pβGLP1). The level of the GLP-1 mRNA was evaluated by RT-PCR 24 h after transfection. The in vitro results showed a dose-dependent expression of GLP-1. Coculture assay of the GLP-1 plasmid-transfected cells with isolated rat islet cells demonstrated that GLP-1 increased insulin secretion by twofold, compared to controls during a hyperglycemic challenge. A single injection of polyethyleneimine/pβGLP1 complex into ZDF rats resulted in increasing insulin secretion and decreasing blood glucose level that was maintained for 2 weeks. This GLP-1 gene delivery system may provide an effective and safe treatment modality for type 2 diabetes.     

Effect of Exenatide,Sitagliptin, or Glimepiride on β-Cell Secretory Capacity in Early Type 2 Diabetes

OBJECTIVE

Agents that augment GLP-1 effects enhance glucose-dependent β-cell insulin production and secretion and thus are hoped to prevent progressive impairment in insulin secretion characteristic of type 2 diabetes (T2D). The purpose of this study was to evaluate GLP-1 effects on β-cell secretory capacity, an in vivo measure of functional β-cell mass, early in the course of T2D.

RESEARCH DESIGN AND METHODS

We conducted a randomized controlled trial in 40 subjects with early T2D who received the GLP-1 analog exenatide (n = 14), the dipeptidyl peptidase IV inhibitor sitagliptin (n = 12), or the sulfonylurea glimepiride (n = 14) as an active comparator insulin secretagogue for 6 months. Acute insulin responses to arginine (AIR_arg) were measured at baseline and after 6 months of treatment with 5 days of drug washout under fasting, 230 mg/dL (glucose potentiation of arginine-induced insulin release [AIR_pot]), and 340 mg/dL (maximum arginine-induced insulin release [AIR_max]) hyperglycemic clamp conditions, in which AIR_max provides the β-cell secretory capacity.

RESULTS

The change in AIR_pot was significantly greater with glimepiride versus exenatide treatment (P < 0.05), and a similar trend was notable for the change in AIR_max (P = 0.1). Within each group, the primary outcome measure, AIR_max, was unchanged after 6 months of treatment with exenatide or sitagliptin compared with baseline but was increased with glimepiride (P < 0.05). α-Cell glucagon secretion (AGR_min) was also increased with glimepiride treatment (P < 0.05), and the change in AGR_min trended higher with glimepiride than with exenatide (P = 0.06).

CONCLUSIONS

After 6 months of treatment, exenatide or sitagliptin had no significant effect on functional β-cell mass as measured by β-cell secretory capacity, whereas glimepiride appeared to enhance β- and α-cell secretion.     

Effects of Intraduodenal Glutamine on Incretin Hormone and Insulin Release,the Glycemic Response to an Intraduodenal Glucose Infusion,and Antropyloroduodenal Motility in Health and Type 2 Diabetes

OBJECTIVE

Glutamine reduces postprandial glycemia when given before oral glucose. We evaluated whether this is mediated by stimulation of insulin and/or slowing of gastric emptying.

RESEARCH DESIGN AND METHODS

Ten healthy subjects were studied during intraduodenal (ID) infusion of glutamine (7.5 or 15 g) or saline over 30 min, followed by glucose (75 g over 100 min), while recording antropyloroduodenal pressures. Ten patients with type 2 diabetes mellitus (T2DM) were also studied with 15 g glutamine or saline.

RESULTS

ID glutamine stimulated glucagon-like peptide 1 (GLP-1; healthy: P < 0.05; T2DM: P < 0.05), glucose-dependent insulinotropic polypeptide (GIP; P = 0.098; P < 0.05), glucagon (P < 0.01; P < 0.001), insulin (P = 0.05; P < 0.01), and phasic pyloric pressures (P < 0.05; P < 0.05), but did not lower blood glucose (P = 0.077; P = 0.5).

CONCLUSIONS

Glutamine does not lower glycemia after ID glucose, despite stimulating GLP-1, GIP, and insulin, probably due to increased glucagon. Its capacity for pyloric stimulation suggests that delayed gastric emptying is a major mechanism for lowering glycemia when glutamine is given before oral glucose.Postprandial glycemic control represents a major focus of type 2 diabetes mellitus (T2DM) management (1). The rate of gastric emptying and the release of “incretin” hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), are both important determinants of postprandial glycemic excursions (2). Glucose empties from the stomach in health in the range of 1–4 kcal/min, regulated by inhibitory small-intestinal feedback via stimulation of pyloric motility and suppression of antral and duodenal contractions (3). Differences in gastric emptying account for about one-third of the variation in postprandial blood glucose levels after oral glucose (4).Glutamine reduces glucose excursions when given before oral glucose in T2DM (5), potentially by stimulating GLP-1 secretion (6) and/or slowing gastric emptying (7). The purpose of the current study was to determine whether glutamine retains its capacity to lower glycemia when glucose is delivered directly into the duodenum, thereby removing any influence of gastric emptying, while measuring antropyloroduodenal motility, gut hormones, and insulin.     

Thiazolidinedione derivatives as novel GPR120 agonists for the treatment of type 2 diabetes

GPR120, also called FFAR4, is preferentially expressed in the intestines, and can be stimulated by long-chain free fatty acids to increase the secretion of glucagon-like peptide-1 (GLP-1) from intestinal endocrine cells. It is known that GLP-1, as an incretin, can promote the insulin secretion from pancreatic cells in a glucose-dependent manner. Therefore, GPR120 is a potential drug target to treat type 2 diabetes. In this study, thiazolidinedione derivatives were found to be novel potent GPR120 agonists. Compound 5g, with excellent agonistic activity, selectivity, and metabolic stability, improved oral glucose tolerance in normal C57BL/6 mice in a dose-dependent manner. Moreover, compound 5g exhibited anti-diabetic activity by promoting insulin secretion in diet-induced obese mice. In summary, compound 5g might be a promising drug candidate for the treatment of type 2 diabetes.

GPR120 has emerged as an attractive target for the treatment of type 2 diabetes and obesity. Thiazolidinedione derivatives were found to be novel potent GPR120 agonists.     

Novel fatty chain-modified GLP-1R G-protein biased agonist exerts prolonged anti-diabetic effects through targeting receptor binding sites

Here, we design and evaluate novel long-lasting GLP-1R G-protein-biased agonists with promising pharmacological virtues. Firstly, six GLP-1R G-protein-biased peptides (named PX01–PX06), screened by using a previous reported high-throughput autocrine-based method, were fused to the N-terminus of GLP-1(9-37) to generate six fusion peptides (PX07–PX012). In vitro surface plasmon resonance (SPR) measurements showed that PX09 exerts the highest binding affinity for both human and mouse GLP-1R extracellular domains (ECD). We further used the PX09 as a starting point to conduct site-specific modifications yielding twelve lysine-modified conjugates, termed PX13–PX24. Of these conjugates, PX17 retained relatively better in vitro GLP-1R activation potency and plasma stability compared with other ones. Preclinical studies in db/db mice demonstrated that acute treatment of PX17 exerts enhanced hypoglycemic and insulinotropic activities in a dosage dependent model within the range of 0.1–0.9 mg kg⁻¹. Similarly, prolonged glucose-lowering abilities were exhibited in modified multiple oral glucose tolerance tests (OGTTs) and a hypoglycemic duration test. Apparently prolonged in vivo half-lives of ∼96 and ∼141 h were observed after a single subcutaneous administration of PX17 at 0.1 and 0.3 mg kg⁻¹, respectively, in healthy cynomolgus monkeys. In addition, twice-weekly treatment of PX17 in db/db mice for 8 weeks obviously improved the hemoglobin A1C (HbA1C), and was more effective at improving the insulin resistance, glucose tolerance as well as function of pancreatic beta cells compared with Semaglutide. Furthermore, subcutaneously dosed PX17 in diet induced obese (DIO) mice achieved long-term beneficial effects on food intake and body weight control, HbA1C and inflammation-related factor level lowering. The above results indicate that PX17, as a novel GLP-1R G-protein-biased agonist, may be a promising candidate for antidiabetic therapies.

An autocrine selected GLP-1R agonist was modified via site-specific fatty chain and exerted prolonged antidiabetic effects.     

Mechanisms of Glucose Lowering of Dipeptidyl Peptidase-4 Inhibitor Sitagliptin When Used Alone or With Metformin in Type 2 Diabetes: A double-tracer study

OBJECTIVE

To assess glucose-lowering mechanisms of sitagliptin (S), metformin (M), and the two combined (M+S).

RESEARCH DESIGN AND METHODS

We randomized 16 patients with type 2 diabetes mellitus (T2DM) to four 6-week treatments with placebo (P), M, S, and M+S. After each period, subjects received a 6-h meal tolerance test (MTT) with [¹⁴C]glucose to calculate glucose kinetics. Fasting plasma glucose (FPG), fasting plasma insulin, C-peptide (insulin secretory rate [ISR]), fasting plasma glucagon, and bioactive glucagon-like peptide (GLP-1) and gastrointestinal insulinotropic peptide (GIP) were measured.

RESULTS

FPG decreased from P, 160 ± 4 to M, 150 ± 4; S, 154 ± 4; and M+S, 125 ± 3 mg/dL. Mean post-MTT plasma glucose decreased from P, 207 ± 5 to M, 191 ± 4; S, 195 ± 4; and M+S, 161 ± 3 mg/dL (P < 0.01). The increase in mean post-MTT plasma insulin and in ISR was similar in P, M, and S and slightly greater in M+S. Fasting plasma glucagon was equal (∼65–75 pg/mL) with all treatments, but there was a significant drop during the initial 120 min with S 24% and M+S 34% (both P < 0.05) vs. P 17% and M 16%. Fasting and mean post-MTT plasma bioactive GLP-1 were higher (P < 0.01) after S and M+S vs. M and P. Basal endogenous glucose production (EGP) fell from P 2.0 ± 0.1 to S 1.8 ± 0.1 mg/kg ⋅ min, M 1.8 ± 0.2 mg/kg ⋅ min (both P < 0.05 vs. P), and M+S 1.5 ± 0.1 mg/kg ⋅ min (P < 0.01 vs. P). Although the EGP slope of decline was faster in M and M+S vs. S, all had comparable greater post-MTT EGP inhibition vs. P (P < 0.05).

CONCLUSIONS

M+S combined produce additive effects to 1) reduce FPG and postmeal plasma glucose, 2) augment GLP-1 secretion and β-cell function, 3) decrease plasma glucagon, and 4) inhibit fasting and postmeal EGP compared with M or S monotherapy.In diet-treated patients with type 2 diabetes mellitus (T2DM) and HbA_1c of ∼8.0%, sitagliptin (S) reduces HbA_1c by 0.6–0.7% over a 6-month period (1). A slightly greater HbA_1c decline (∼0.8–0.9%) is observed when metformin (M) therapy is added to S (2). Dipeptidyl peptidase (DPP)-4 inhibitors predominantly affect the postprandial plasma glucose excursion, but a significant, albeit modest, reduction in fasting plasma glucose (FPG) also is observed (1–3). The mechanism of action of the DPP-4 inhibitors has been well studied and includes increased plasma glucagon-like peptide (GLP)-1 and gastrointestinal insulinotropic peptide (GIP) levels, resulting in increased insulin and reduced glucagon secretion (4–6). The increase in plasma insulin and the decline in glucagon inhibit basal endogenous glucose production (EGP) and enhance the suppression of EGP without affecting splanchnic (hepatic) glucose uptake or gastric emptying (6,7). Therapy with S and M combined (M+S) exerts an additive effect to reduce HgA_1c; the mechanism of action of this combination has yet to be examined. Several studies have demonstrated that M inhibits DPP-4 activity, thus increasing plasma active GLP-1 levels (8–10). There are also reports indicating that the decline in plasma glucose with M therapy can restore the β-cells’ sensitivity to the stimulatory effect of incretins on insulin secretion (11,12). Despite these reports, we still do not fully understand the reasons why the use of M in diabetic patients is not accompanied by changes in insulin release. In the current study, we used the double-tracer technique (7) to examine the mechanism(s) via which M+S and each agent alone reduce the fasting and postmeal plasma glucose concentration in T2DM.     

Discovery of lixisenatide analogues as long-acting hypoglycemic agents using novel peptide half-life extension technology based on mycophenolic acid

Noncovalent binding of peptides to human serum albumin protects against renal clearance and enzymatic degradation. Herein, we investigated the effect of mycophenolic acid (MPA) albumin binders for improving the stability of peptides. For proof-of-principle, the short acting glucagon-like peptide-1 (GLP-1) receptor agonist lixisenatide was selected and functionalized with different MPA albumin binders. In vitro, all lixisenatide analogues showed well preserved GLP-1 receptor activation potency. High performance affinity chromatography (HPAC) and ultrafiltration analyses indicated that DiMPA was able to confer high albumin affinity to lixisenatide and revealed that affinity is increased for DiMPA modified lixisenatide analogues containing OEG spacers. In db/db mice, the selected peptide 2c showed comparable efficacies to lixisenatide with respect to glucose-lowering and insulinotropic activities. Furthermore, the duration of action of glucose homeostasis of 2c was comparable to semaglutide in db/db mice. Importantly, DiMPA albumin binder did not bring significant toxicity of lixisenatide, as reflected by the comparable toxicity indexes in 2c and semaglutide groups after 2 weeks dosing in normal Kunming mice. Short-term study (21 days) conducted on db/db mice showed the better therapeutic efficacies of 2c than semaglutide on pancreas islets protection. Importantly, in chronic studies (84 days) on db/db mice, 2c exhibited a sustained improvement in glycaemic control, to a greater extent than that of semaglutide. Thus, we propose DiMPA modification as a novel and general method for development of long-acting GLP-1 receptor agonists for type 2 diabetes treatments, and 2c as a promising antidiabetic candidate.

DiMPA albumin binders were effectively applied to lixisenatide to make 2c as a long-acting antidiabetic agent.     

Two methods for the preparation of sitagliptin phosphate via chemical resolution and asymmetric hydrogenation

Two effective processes have been developed for the preparation of sitagliptin phosphate. The approach of chemical resolution obtained R-sitagliptin in five steps from commercially available starting materials using the inexpensive NaBH₄ to reduce the enamine and then using (−)-di-p-toluoyl-l-tartaric acid to resolve racemates in 11% yield overall. The route successfully avoids the use of expensive noble metal as catalysts compared with traditional synthesis methods, resulting in greatly reduced costs and simplified synthetic routes. Other alternative asymmetric hydrogenation of β-ketomide routes for the synthesis of sitagliptin were found, two of the intermediates were synthesized for the first time.

Two effective processes have been developed for the preparation of sitagliptin phosphate.     

GLP-1 Action and Glucose Tolerance in Subjects With Remission of Type 2 Diabetes After Gastric Bypass Surgery

OBJECTIVE

Glucagon like peptide-1 (GLP-1) has been suggested as a major factor for the improved glucose tolerance ensuing after Roux-en-Y gastric bypass (RYGBP) surgery. We examined the effect of blocking endogenous GLP-1 action on glucose tolerance in subjects with sustained remission of type 2 diabetes mellitus (T2DM) present before RYGBP.

RESEARCH DESIGN AND METHODS

Blood glucose, insulin, C-peptide, glucagon, GLP-1, and glucose-dependent insulinotropic peptide levels were measured after a meal challenge with either exendin-(9–39) (a GLP-1r antagonist) or saline infusion in eight subjects with sustained remission of T2DM after RYGBP and seven healthy controls.

RESULTS

Infusion of exendin-(9–39) resulted in marginal deterioration of the 2-h plasma glucose after meal intake in RYGBP subjects [saline 78.4 ± 15.1 mg/dL compared with exendin-(9–39) 116.5 ± 22.3 mg/dL; P < 0.001]. Furthermore, glucose response to meal intake was similarly enlarged in the two study groups [percent change in the area under the curve of glucose exendin-(9–39) infusion versus saline infusion: controls 10.84 ± 8.8% versus RYGBP 9.94 ± 8.4%; P = 0.884]. In the RYGBP group, the blockade of the enlarged GLP-1 response to meal intake resulted in reduced insulin (P = 0.001) and C-peptide (P < 0.001), but no change in glucagon (P = 0.258) responses.

CONCLUSIONS

The limited deterioration of glucose tolerance on blockade of GLP-1 action in our study suggests the resolution of T2DM after RYGBP may be explained by mechanisms beyond enhancement of GLP-1 action.The beneficial effect of Roux-en-Y gastric bypass (RYGBP) surgery on glycemic control in morbidly obese subjects with type 2 diabetes mellitus (T2DM) is well established (1,2). However, the precise mechanisms mediating T2DM remission after RYGBP are not yet clear (3–5). Although it traditionally has been asserted that bariatric operations are associated with improvement of glucose tolerance merely by caloric restriction and weight loss, several lines of evidence support weight-independent mechanisms are involved (6–11). An enhanced postsurgical glucagon-like peptide-1 (GLP-1) secretion, inducing a normalized or exaggerated insulin secretion after meal intake, has been hypothesized to play a major role in the improved glucose tolerance after RYGBP (3). Association studies have demonstrated larger improvements of glucose tolerance early after RYGBP being associated with a larger GLP-1 response to nutrient intake as compared with other surgical or nonsurgical interventions resulting in equivalent weight loss (7–9). Likewise, an exaggerated GLP-1 response has been reported up to 10 years after RYGB in subjects with sustained T2DM remission, suggesting a key role of GLP-1 in maintaining normal glucose tolerance in the long term after this type of surgery (12). However, because association does not prove causation, these data do not definitely prove GLP-1 plays a critical role in T2DM remission after RYGBP.Understanding the role of endogenous GLP-1 in metabolic physiology has been greatly enhanced by the availability of a potent GLP-1 receptor antagonist, exendin-(9–39). Exendin-(9–39) blockade of GLP-1 action in healthy volunteers results in a significant enlargement of postprandial glucose excursions (13–17). Moreover, using hyperglycemic clamp technique in combination with a mixed meal test, Salehi et al. (18) demonstrated that blocking GLP-1 action results in a larger decrease in the insulin secretion rate in RYGBP-operated subjects (−33%) as compared with nonoperated controls (−16%). This study clearly supports GLP-1 as an important determinant of insulin secretion after RYGBP. However, the use of hyperglycemic clamp limited the ability of the study to investigate the relative importance of GLP-1 secretion on glucose tolerance. Furthermore, because only one-third of the study participants presented with T2DM before surgery, the study also was limited in establishing the role of GLP-1 secretion in the remission of T2DM. Of note, in Goto-Kakizaki rats (a nonobese rat model of T2DM) administration of exendin-(9–39) has been shown to totally reverse the improved glucose tolerance resulting from duodeno-jejunal exclusion surgery (an experimental metabolic surgery similar to RYGBP) (19).Against this background, the main aim of our study was to examine the effect of endogenous GLP-1 blockade by exendin-(9–39) on glucose tolerance in subjects who had undergone RYGBP and with T2DM antedating surgery that had remitted after the surgical procedure. As secondary aims, we evaluated the effect of exendin-(9–39) on the insulin, C-peptide, glucagon, GLP-1, and glucose-dependent insulinotropic peptide (GIP) responses to meal intake. We evaluated individuals during the long-term after surgery to avoid the potential confounding effect of intense caloric restriction or rapid weight loss or both on glucose tolerance.     

Effects of diet soda on gut hormones in youths with diabetes

OBJECTIVE

In patients with type 2 diabetes, but not type 1 diabetes, abnormal secretion of incretins in response to oral nutrients has been described. In healthy youths, we recently reported accentuated glucagon-like peptide 1 (GLP-1) secretion in response to a diet soda sweetened with sucralose and acesulfame-K. In this study, we examined the effect of diet soda on gut hormones in youths with diabetes.

RESEARCH DESIGN AND METHODS

Subjects aged 12–25 years with type 1 diabetes (n = 9) or type 2 diabetes (n = 10), or healthy control participants (n = 25) drank 240 mL cola-flavored caffeine-free diet soda or carbonated water, followed by a 75-g glucose load, in a randomized, cross-over design. Glucose, C-peptide, GLP-1, glucose-dependent insulinotropic peptide (GIP), and peptide Tyr-Tyr (PYY) were measured for 180 min. Glucose and GLP-1 have previously been reported for the healthy control subjects.

RESULTS

GLP-1 area under the curve (AUC) was 43% higher after ingestion of diet soda versus carbonated water in individuals with type 1 diabetes (P = 0.020), similar to control subjects (34% higher, P = 0.029), but was unaffected by diet soda in patients with type 2 diabetes (P = 0.92). Glucose, C-peptide, GIP, and PYY AUC were not statistically different between the two conditions in any group.

CONCLUSIONS

Ingestion of diet soda before a glucose load augmented GLP-1 secretion in type 1 diabetic and control subjects but not type 2 diabetic subjects. GIP and PYY secretion were not affected by diet soda. The clinical significance of this increased GLP-1 secretion, and its absence in youths with type 2 diabetes, needs to be determined.Nonnutritive sweeteners are commonly consumed by both children and adults and have previously been thought to be metabolically inert. However, recent animal data demonstrate that nonnutritive sweeteners, including sucralose (Splenda) and acesulfame-K, play an active metabolic role within the gastrointestinal tract via sweet taste receptors identical to those found in lingual taste buds (1,2). In both humans and animals, these receptors are present in L cells of the gut mucosa secreting glucagon-like peptide 1 (GLP-1) and peptide Tyr-Tyr (PYY) (3–5). Components of the taste-signaling pathway have also been found in glucose-dependent insulinotropic peptide (GIP)-secreting K cells in the gut mucosa (5).GLP-1 and GIP are incretin hormones that increase glucose-dependent insulin secretion in response to oral nutrients. In healthy individuals, GIP appears to be responsible for the majority of the incretin effect (6,7). GLP-1 and its analogs have numerous physiologic effects, including delayed gastric emptying (8,9), increased satiety (10), and suppression of glucagon secretion (11), in addition to increased insulin secretion (12). Patients with type 2 diabetes frequently have normal GIP but impaired GLP-1 secretion (13) and are resistant to exogenous GIP but respond normally to exogenous GLP-1 (7). This has been exploited pharmacologically using GLP-1 analogs, which cause improved glycemia and weight loss. Both GLP-1 and GIP secretion are normal in type 1 diabetes (14). PYY acts as an anorectic hormone in both lean (15) and obese humans (16), although its secretion is attenuated in obesity (17).Sucralose has been shown to increase release of both GLP-1 and GIP in vitro in enteroendocrine cell lines (4,5). In vivo, however, the nonnutritive sweeteners sucralose (in humans and animals) and acesulfame, stevia, and d-tryptophan (in animals) do not stimulate GLP-1, GIP, or PYY secretion in the absence of caloric sugars (18,19). We recently demonstrated in a pilot study that diet soda (Diet Rite Cola, sweetened with sucralose and acesulfame-K) in the presence of glucose augmented glucose-stimulated GLP-1 secretion in healthy subjects (20). It is not known whether GIP or PYY secretion are likewise augmented by ingestion of nonnutritive sweeteners in addition to glucose or whether diet soda augments GLP-1 secretion in disease states such as type 1 or type 2 diabetes.In this study, we investigated whether subjects with diabetes (both type 1 and type 2) would demonstrate increased GLP-1 secretion after ingestion of diet soda in addition to a glucose load, similar to the effect previously observed in healthy subjects (20). In addition, we examined whether secretion of the gut hormones GIP and PYY is increased by ingestion of diet soda.