A Potent Class of GPR40 Full Agonists Engages the EnteroInsular Axis to Promote Glucose Control in Rodents

<div><p>Type 2 diabetes is characterized by impaired glucose homeostasis due to defects in insulin secretion, insulin resistance and the incretin response. GPR40 (FFAR1 or FFA1) is a G-protein-coupled receptor (GPCR), primarily expressed in insulin-producing pancreatic β-cells and incretin-producing enteroendocrine cells of the small intestine. Several GPR40 agonists, including AMG 837 and TAK-875, have been disclosed, but no GPR40 synthetic agonists have been reported that engage both the insulinogenic and incretinogenic axes. In this report we provide a molecular explanation and describe the discovery of a unique and potent class of GPR40 full agonists that engages the enteroinsular axis to promote dramatic improvement in glucose control in rodents. GPR40 full agonists AM-1638 and AM-6226 stimulate GLP-1 and GIP secretion from intestinal enteroendocrine cells and increase GSIS from pancreatic islets, leading to enhanced glucose control in the high fat fed, streptozotocin treated and NONcNZO10/LtJ mouse models of type 2 diabetes. The improvement in hyperglycemia by AM-1638 was reduced in the presence of the GLP-1 receptor antagonist Ex(9–39)NH₂.</p> </div

Specificity of AM-1638 to GPR40 (FFAR1) <i>in vivo</i> and effect of the GLP-1R antagonist GLP-1(9–39)NH₂.

<p>An OGTT was performed in (A) wild type or (B) GPR40 null mice following a single oral dose of AM-1638 or sitagliptin. Glucose was dosed 1-hr post drug treatment. (C) Glucose AUC during OGTT. (D) GLP-1 secretion following a single oral dose of AM-1638 in wild type or GPR40 null mice. AM-1638 (60 mg/kg) was tested in an IPGTT in the presence or absence of the GLP-1R antagonist GLP-1(9–39)NH₂ (300 µg/kg) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046300#s4" target="_blank">Materials and Methods</a> section. (E) Plasma glucose levels (F) Glucose AUC and (G) plasma insulin levels at the indicated timepoints during the experiment. Statistical significance compared to vehicle treatment is denoted by *(p<0.05), **(p<0.01), ***(p<0.001) and ****(p<0.0001), as determined by one-way or two-way ANOVA, and are color-coded to the treatment in the figure legends.</p

<i>In vitro</i> characterization of AM-1638 and AM-6226 and comparison to AMG 837.

<p>(A) Aequorin Ca²⁺ assay comparing AMG 837 to natural fatty acid ligands DHA, α-LNN and arachidonic acid. (B) Chemical structures of the key compounds synthesized during the medicinal chemistry effort that led to the discovery of AM-1638 and AM-6226. (C) Aequorin Ca²⁺ flux with key synthetic agonists and fatty acids. (D) Inositol phosphate assay with key synthetic agonists and fatty acids. (E–G) Plasmid titration experiments to examine agonist activity under conditions with reduced receptor levels, where either 5000 ng (E), 500 ng (F) or 50 ng (G) of GPR40 (FFAR1) expression plasmid was co-transfected with aequorin expression plasmids into CHO cells. (H) Competition binding experiment with ³H-AMG 837. (I) Competition binding experiment with ³H-AM-1638.</p

During an OGTT in NONcNZO10/LtJ type 2 diabetic mice, AM-1638 lowers blood glucose levels through an increase in insulin and incretin secretion.

<p>Vehicle (purple, n = 8) or 60 mg/kg AM-1638 (green, n = 8) was administered 1-hour prior to an oral glucose bolus. (A) Glucose levels at various timepoints. (B) Glucose AUC values. (C) Plasma insulin levels (D) GLP-1 levels and (E) GIP levels at baseline (-60 minutes) and 15 minutes after glucose challenge. Statistical significance compared to vehicle treatment is denoted by *(p<0.05), **(p<0.01), ***(p<0.001) and ****(p<0.0001), as determined by two-way ANOVA or student’s t-test.</p

Engagement of the enteroendocrine axis in HF/STZ type 2 diabetic mice by AM-1638.

<p>Following administration of a single dose of the indicated treatments at t = 0 minutes in HF/STZ mice, measurements of (A) GLP-1 (B) insulin (C) glucose and (D) GIP were taken at various timepoints. Statistical significance compared to vehicle treatment is denoted by *(p<0.05), **(p<0.01), ***(p<0.001) and ****(p<0.0001), as determined by one-way or two-way ANOVA, and are color-coded to the treatment in the figure legends.</p

Model depicting the dual mechanism-of-action of GPR40 (FFAR1) full agonists to lower blood glucose levels.

<p>GPR40 full agonists engage both the enteroendocrine axis as well as the pancreatic β-cell axis. These pathways both lead to augmentation of glucose stimulated insulin secretion on the pancreatic β-cell. Additionally, GLP-1 has multiple physiological and pharmacological roles, such as inhibition of glucagon secretion, that could further benefit type 2 diabetics. GPR40 partial agonists such as AMG 837 engage only the pancreatic pancreatic β-cell axis <i>in vivo</i>.</p

Activity of AM-1638 and AM-6226 in primary cells.

<p>(A) GSIS assay in mouse islets incubated with a dose response of compounds in the presence of 16.7 mM glucose. (B) Mouse islet perifusion assay. The glucose concentration was raised from 3 mM to 16.7 mM at t = 20 minutes and returned to 3 mM glucose at t = 30 minutes. 10 µM compound was perifused through the entire experiment. (C) GSIS assay with islets from wild type or GPR40 knock-out mice. (D) Inositol phosphate assay with dispersed human islet cells. (E) GSIS assay with human islets incubated with GPR40 agonists and 12.5 mM glucose. (F) GLP-1 secretion assay with fetal rat intestinal cells. (G) GIP-1 secretion assay with fetal rat intestinal cells. (H) Inositol phosphate accumulation assay using the mouse GLUTag enteroendocrine L-cell line. Statistical significance is denoted by *(p<0.05), **(p<0.01), ***(p<0.001) and ****(p<0.0001), as determined by one-way or two-way ANOVA, and are color-coded to treatment in the figure legends. For (A), (B), (F), (G), and (H) statistical comparisons were made to AMG 837 treatment. For (C) and (E) statistical comparisons were made to vehicle treatment.</p

Enhanced <i>in vivo</i> efficacy of AM-1638 compared to AMG 837 in HF/STZ type 2 diabetic mice.

<p>Drug treatment was administered 1-hour prior to an oral glucose bolus. (A) Glucose levels during an OGTT in high-fat fed, streptozotocin treated type 2 diabetic mice. (B) Glucose AUC values. (C) Change in plasma insulin levels from baseline during an OGTT (D) Insulin AUC (E) Unbound (free) plasma drug concentration in plasma 1-hour following drug dose, as determined by MS. Statistical significance compared to vehicle treatment is denoted by *(p<0.05), **(p<0.01), ***(p<0.001) and ****(p<0.0001), as determined by one-way or two-way ANOVA, and are color-coded to the treatment in the figure legends.</p

Efficacy of AMG 837 in Zucker fatty (<i>fa/fa</i>) rats following a single dose.

<p>8-week old Zucker fatty rats were administered a single bolus of AMG 837 (at 0.3, 1 and 3 mg/kg, n = 6/group) by oral gavage 30-minutes prior to an intraperitoneal glucose challenge at t = 0 minutes. (A) Blood glucose during the IPGTT (black circle = vehicle, blue triangle = 0.3 mg/kg AMG 837, green diamond = 1 mg/kg AMG 837 and purple square = 3 mg/kg AMG 837) (B) Glucose AUC (from −30 to 120 minutes) during the IPGTT. (C) Plasma insulin levels during the IPGTT (black circle = vehicle, blue triangle = 0.3 mg/kg AMG 837, green diamond = 1 mg/kg AMG 837 and purple square = 3 mg/kg AMG 837). Statistical significance compared to vehicle treated animals is denoted by * (p<0.5), ** (p<0.01), *** (p<0.001) and **** (p<0.001) as determined by one-way or two-way ANOVA and colors match the corresponding groups in the figure legend.</p

AMG 837 Potentiates Insulin Secretion from Islets.

<p>Islets were isolated from mice and the activity of AMG 837 on insulin secretion was determined. (A) The dose response relationship of AMG 837 and insulin secretion on mouse islets at 16.7 mM glucose was evaluated. (B) In order to determine whether the activity of AMG 837 was GPR40/FFA1 dependent, islets were isolated from GPR40 null mice (<i>gpr40^−/−</i>). AMG 837 potentiated glucose stimulated insulin secretion from wild type islets (black bar), but not <i>gpr40^−/−</i> islets (blue bar). (C) Glucose dependence of AMG 837 on glucose stimulated insulin secretion was determined by incubating islets in buffer containing either 0.1% DMSO (black bar) or 1 µM AMG 837 in 0.1% DMSO (blue bar) in the presence of increasing concentrations of glucose. Statistical significance is denoted by * (p<0.5), ** (p<0.01) and *** (p<0.001) as determined by one-way or two-way ANOVA.</p