Limits...
Novel small molecule glucagon-like peptide-1 receptor agonist stimulates insulin secretion in rodents and from human islets.

Sloop KW, Willard FS, Brenner MB, Ficorilli J, Valasek K, Showalter AD, Farb TB, Cao JX, Cox AL, Michael MD, Gutierrez Sanfeliciano SM, Tebbe MJ, Coghlan MJ - Diabetes (2010)

Bottom Line: These molecules induce GLP-1 receptor-mediated cAMP signaling in HEK293 cells expressing the GLP-1 receptor and increase insulin secretion from rodent islets in a dose-dependent manner.In vivo studies using the IVGTT and the hyperglycemic clamp in Sprague Dawley rats demonstrate increased insulin secretion in compound-treated animals.These studies characterize the insulinotropic effects of an early-stage, small molecule GLP-1 receptor agonist and provide compelling evidence to support pharmaceutical optimization.

View Article: PubMed Central - PubMed

Affiliation: Endocrine Discovery, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana, USA. sloop_kyle_w@lilly.com

ABSTRACT

Objective: The clinical effectiveness of parenterally-administered glucagon-like peptide-1 (GLP-1) mimetics to improve glucose control in patients suffering from type 2 diabetes strongly supports discovery pursuits aimed at identifying and developing orally active, small molecule GLP-1 receptor agonists. The purpose of these studies was to identify and characterize novel nonpeptide agonists of the GLP-1 receptor.

Research design and methods: Screening using cells expressing the GLP-1 receptor and insulin secretion assays with rodent and human islets were used to identify novel molecules. The intravenous glucose tolerance test (IVGTT) and hyperglycemic clamp characterized the insulinotropic effects of compounds in vivo.

Results: Novel low molecular weight pyrimidine-based compounds that activate the GLP-1 receptor and stimulate glucose-dependent insulin secretion are described. These molecules induce GLP-1 receptor-mediated cAMP signaling in HEK293 cells expressing the GLP-1 receptor and increase insulin secretion from rodent islets in a dose-dependent manner. The compounds activate GLP-1 receptor signaling, both alone or in an additive fashion when combined with the endogenous GLP-1 peptide; however, these agonists do not compete with radiolabeled GLP-1 in receptor-binding assays. In vivo studies using the IVGTT and the hyperglycemic clamp in Sprague Dawley rats demonstrate increased insulin secretion in compound-treated animals. Further, perifusion assays with human islets isolated from a donor with type 2 diabetes show near-normalization of insulin secretion upon compound treatment.

Conclusions: These studies characterize the insulinotropic effects of an early-stage, small molecule GLP-1 receptor agonist and provide compelling evidence to support pharmaceutical optimization.

Show MeSH

Related in: MedlinePlus

Compound B increases plasma insulin levels in the SD rat hyperglycemic clamp model. A: Intravenous dosing with vehicle (■) or 10 mg/kg compound B (○) occurred immediately before intravenous infusion of glucose. Glucose levels measured from venous blood every 5 min are shown. Results are expressed as mean ± SEM. B: Blood glucose concentrations of ∼250 mg/dl were maintained throughout the experiment by varying the glucose infusion rates. C: Time course of plasma insulin concentrations in fasted animals treated with either vehicle (■) or compound B (○). Results are expressed as mean ± SEM. D: AUC20–60 min of the insulin secretion for vehicle (filled bar) versus compound B-treated (open bar) animals, *P < 0.05.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2992771&req=5

Figure 6: Compound B increases plasma insulin levels in the SD rat hyperglycemic clamp model. A: Intravenous dosing with vehicle (■) or 10 mg/kg compound B (○) occurred immediately before intravenous infusion of glucose. Glucose levels measured from venous blood every 5 min are shown. Results are expressed as mean ± SEM. B: Blood glucose concentrations of ∼250 mg/dl were maintained throughout the experiment by varying the glucose infusion rates. C: Time course of plasma insulin concentrations in fasted animals treated with either vehicle (■) or compound B (○). Results are expressed as mean ± SEM. D: AUC20–60 min of the insulin secretion for vehicle (filled bar) versus compound B-treated (open bar) animals, *P < 0.05.

Mentions: To explore the in vivo insulinotropic effects of compound B, glucose-stimulated insulin secretion was measured in compound-treated SD rats undergoing an IVGTT and a hyperglycemic clamp. In both models, compound B induced insulin secretion over the time course of the glucose challenge (Figs. 5 and 6). In the IVGTT, fasted-SD rats implanted with dual cannulas were anesthetized and administered via a jugular vein cannula either vehicle, GLP-1 (dosed at 10 μg/kg), or compound B (dosed at 10 mg/kg) immediately followed by a bolus of glucose (administered at 0.5 g/kg). Blood samples were drawn from the carotid artery cannula for measurement of glucose (Fig. 5A) and insulin (Fig. 5B) levels at times 0, 2, 4, 6, 10, and 20 min. Both GLP-1 and compound B displayed insulin secretagogue activity in this experimental model (Fig. 5B). Insulin output is reported as incremental area under the curve (AUC) of plasma insulin concentrations during the 20-min time course. Compared with vehicle, animals dosed with compound B exhibited a threefold elevation in insulin AUC, whereas GLP-1 treatment increased insulin by fourfold (Fig. 5C). Although the IVGTT model demonstrated significant increases in circulating levels of insulin, robust changes in plasma glucose were not induced by compound B treatment. This result is likely caused by anesthesia-induced insulin resistance in this model in which typically only very potent insulinotropic molecules, such as the GLP-1 peptide, induce a statistically significant change in glucose (unpublished data). Therefore, we further assessed the glucodynamic effects of compound B in the more sensitive (nonanesthetized SD rats) hyperglycemic clamp model that enables evaluation of changes in insulin and glucose over a longer time course. Compound B (dosed at 10 mg/kg) or vehicle was administered to animals as a single intravenous dose at the beginning of the experiment. Intravenous infusion of glucose maintained blood glucose concentrations at ∼250 mg/dl (Fig. 6A). Compound B-treated animals required ∼20% higher glucose infusion rates to maintain this glucose level (Fig. 6B). Measurement of serum insulin concentrations over the next 60 min demonstrated higher insulin levels in compound B-treated animals compared with vehicle controls (Fig. 6C), with the AUC20–60 min calculation showing an 83% increase (Fig. 6D). The insulin secretory response induced by compound B observed using both the IVGTT and hyperglycemic clamp methods is consistent with pharmacology demonstrated by peptide GLP-1 receptor agonists (29) and supports the results obtained from the islet assays showing that compound B is a glucose-dependent insulin secretagogue. Because oral dosing of compound B failed to show insulinotropic effects similar to those achieved by intravenous administration, additional optimization of compound B is required to improve the pharmacokinetic properties of the molecule to enable longer-term in vivo testing that will allow evaluation of efficacy, including effects on food intake and body weight, and toxicity.


Novel small molecule glucagon-like peptide-1 receptor agonist stimulates insulin secretion in rodents and from human islets.

Sloop KW, Willard FS, Brenner MB, Ficorilli J, Valasek K, Showalter AD, Farb TB, Cao JX, Cox AL, Michael MD, Gutierrez Sanfeliciano SM, Tebbe MJ, Coghlan MJ - Diabetes (2010)

Compound B increases plasma insulin levels in the SD rat hyperglycemic clamp model. A: Intravenous dosing with vehicle (■) or 10 mg/kg compound B (○) occurred immediately before intravenous infusion of glucose. Glucose levels measured from venous blood every 5 min are shown. Results are expressed as mean ± SEM. B: Blood glucose concentrations of ∼250 mg/dl were maintained throughout the experiment by varying the glucose infusion rates. C: Time course of plasma insulin concentrations in fasted animals treated with either vehicle (■) or compound B (○). Results are expressed as mean ± SEM. D: AUC20–60 min of the insulin secretion for vehicle (filled bar) versus compound B-treated (open bar) animals, *P < 0.05.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2992771&req=5

Figure 6: Compound B increases plasma insulin levels in the SD rat hyperglycemic clamp model. A: Intravenous dosing with vehicle (■) or 10 mg/kg compound B (○) occurred immediately before intravenous infusion of glucose. Glucose levels measured from venous blood every 5 min are shown. Results are expressed as mean ± SEM. B: Blood glucose concentrations of ∼250 mg/dl were maintained throughout the experiment by varying the glucose infusion rates. C: Time course of plasma insulin concentrations in fasted animals treated with either vehicle (■) or compound B (○). Results are expressed as mean ± SEM. D: AUC20–60 min of the insulin secretion for vehicle (filled bar) versus compound B-treated (open bar) animals, *P < 0.05.
Mentions: To explore the in vivo insulinotropic effects of compound B, glucose-stimulated insulin secretion was measured in compound-treated SD rats undergoing an IVGTT and a hyperglycemic clamp. In both models, compound B induced insulin secretion over the time course of the glucose challenge (Figs. 5 and 6). In the IVGTT, fasted-SD rats implanted with dual cannulas were anesthetized and administered via a jugular vein cannula either vehicle, GLP-1 (dosed at 10 μg/kg), or compound B (dosed at 10 mg/kg) immediately followed by a bolus of glucose (administered at 0.5 g/kg). Blood samples were drawn from the carotid artery cannula for measurement of glucose (Fig. 5A) and insulin (Fig. 5B) levels at times 0, 2, 4, 6, 10, and 20 min. Both GLP-1 and compound B displayed insulin secretagogue activity in this experimental model (Fig. 5B). Insulin output is reported as incremental area under the curve (AUC) of plasma insulin concentrations during the 20-min time course. Compared with vehicle, animals dosed with compound B exhibited a threefold elevation in insulin AUC, whereas GLP-1 treatment increased insulin by fourfold (Fig. 5C). Although the IVGTT model demonstrated significant increases in circulating levels of insulin, robust changes in plasma glucose were not induced by compound B treatment. This result is likely caused by anesthesia-induced insulin resistance in this model in which typically only very potent insulinotropic molecules, such as the GLP-1 peptide, induce a statistically significant change in glucose (unpublished data). Therefore, we further assessed the glucodynamic effects of compound B in the more sensitive (nonanesthetized SD rats) hyperglycemic clamp model that enables evaluation of changes in insulin and glucose over a longer time course. Compound B (dosed at 10 mg/kg) or vehicle was administered to animals as a single intravenous dose at the beginning of the experiment. Intravenous infusion of glucose maintained blood glucose concentrations at ∼250 mg/dl (Fig. 6A). Compound B-treated animals required ∼20% higher glucose infusion rates to maintain this glucose level (Fig. 6B). Measurement of serum insulin concentrations over the next 60 min demonstrated higher insulin levels in compound B-treated animals compared with vehicle controls (Fig. 6C), with the AUC20–60 min calculation showing an 83% increase (Fig. 6D). The insulin secretory response induced by compound B observed using both the IVGTT and hyperglycemic clamp methods is consistent with pharmacology demonstrated by peptide GLP-1 receptor agonists (29) and supports the results obtained from the islet assays showing that compound B is a glucose-dependent insulin secretagogue. Because oral dosing of compound B failed to show insulinotropic effects similar to those achieved by intravenous administration, additional optimization of compound B is required to improve the pharmacokinetic properties of the molecule to enable longer-term in vivo testing that will allow evaluation of efficacy, including effects on food intake and body weight, and toxicity.

Bottom Line: These molecules induce GLP-1 receptor-mediated cAMP signaling in HEK293 cells expressing the GLP-1 receptor and increase insulin secretion from rodent islets in a dose-dependent manner.In vivo studies using the IVGTT and the hyperglycemic clamp in Sprague Dawley rats demonstrate increased insulin secretion in compound-treated animals.These studies characterize the insulinotropic effects of an early-stage, small molecule GLP-1 receptor agonist and provide compelling evidence to support pharmaceutical optimization.

View Article: PubMed Central - PubMed

Affiliation: Endocrine Discovery, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana, USA. sloop_kyle_w@lilly.com

ABSTRACT

Objective: The clinical effectiveness of parenterally-administered glucagon-like peptide-1 (GLP-1) mimetics to improve glucose control in patients suffering from type 2 diabetes strongly supports discovery pursuits aimed at identifying and developing orally active, small molecule GLP-1 receptor agonists. The purpose of these studies was to identify and characterize novel nonpeptide agonists of the GLP-1 receptor.

Research design and methods: Screening using cells expressing the GLP-1 receptor and insulin secretion assays with rodent and human islets were used to identify novel molecules. The intravenous glucose tolerance test (IVGTT) and hyperglycemic clamp characterized the insulinotropic effects of compounds in vivo.

Results: Novel low molecular weight pyrimidine-based compounds that activate the GLP-1 receptor and stimulate glucose-dependent insulin secretion are described. These molecules induce GLP-1 receptor-mediated cAMP signaling in HEK293 cells expressing the GLP-1 receptor and increase insulin secretion from rodent islets in a dose-dependent manner. The compounds activate GLP-1 receptor signaling, both alone or in an additive fashion when combined with the endogenous GLP-1 peptide; however, these agonists do not compete with radiolabeled GLP-1 in receptor-binding assays. In vivo studies using the IVGTT and the hyperglycemic clamp in Sprague Dawley rats demonstrate increased insulin secretion in compound-treated animals. Further, perifusion assays with human islets isolated from a donor with type 2 diabetes show near-normalization of insulin secretion upon compound treatment.

Conclusions: These studies characterize the insulinotropic effects of an early-stage, small molecule GLP-1 receptor agonist and provide compelling evidence to support pharmaceutical optimization.

Show MeSH
Related in: MedlinePlus