<i>In vivo</i> inhibition of hepatic ¹²⁵I-glucagon binding in the hGCGR mouse following (A) acute and (B) chronic dosing with GRA1.

<p>The data are mean (±SEM) percent reductions in liver ¹²⁵I-glucagon content measured (A) 1, 3, 5, and 8 h after a single oral dose of 3 mg/kg GRA1, and (B) after treatment for 30 days with control diet or food/drug admixtures that provided 3, 6, 10, or 30 mg/kg·day GRA1. Pharmacokinetic analysis performed during the experiment in (A) determined that mean plasma GRA1 concentrations were 0.5, 0.6, 0.5, and 0.7 µM at 1, 3, 5, and 8 h postdose, respectively.</p

The diabetic phenotype of the hGCGR.<i>ob/ob</i> mouse.

<p>The data are means ± SEM. In all comparisons, the difference between hGCGR.<i>ob/ob</i> mice and littermate controls was significant at p<0.001.</p

Plasma measurements in DIO hGCGR mice treated for 10 weeks with GRA1 administered as a food admixture.

<p>All measurements were made in terminal plasma. Data are expressed as mean ± SEM; *p<0.05 and **p<0.01 in comparisons with the group on the control diet.</p

GRA1 lowers glucose in hGCGR HFD/STZ mice and further enhances the efficacy of a DPP-4 inhibitor.

<p>(A) Mean (±SEM) blood glucose hGCGR HFD/STZ mice treated with a single dose of 1, 3, or 10 mg/kg GRA1. (B) Non-fasted blood glucose concentrations in hGCGR HFD/STZ mice treated for 6 weeks with 10 mg/kg·day GRA1, 200 mg/kg·day des-fluoro-sitagliptin (des-F-sita), or the two agents in combination. (Additional data from this study are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049572#pone-0049572-t004" target="_blank">Table 4</a>.) *p<0.05, **p<0.01, and ***p<0.001 versus HFD/STZ controls; ^#non-significant (p>0.05) versus non-STZ controls; n = 8–15 animals per group.</p

Plasma and tissue measurements from hGCGR HFD/STZ mice treated for 6 weeks with 10 mg/kg GRA1, des-fluoro-sitagliptin (des-F-sita), or GRA1 and des-fluoro-sitagliptin in combination.

a<p>Fasting blood glucose and HbA1c were measured after 5 weeks of treatment; all other measurements were made in terminal plasma and necropsy tissue. The data are means ± SEM; *p<0.05, **p<0.01 and ***p<0.001 for comparisons made with the hGCGR HFD/STZ group fed the control (drug-free) diet.</p

Genes involved in amino acid and glucose metabolism that were expressed differentially in rhesus monkey liver depending or whether animals received 30 mg/kg GRA1 or vehicle (yogurt without drug) twice daily for 6 days (n = 5 per group).

<p>ns = not significant (p≥0.05).</p><p>The data are expressed as fold differences between treatment groups with negative values indicating reduced expression in animals treated with GRA1.</p>*<p>p<0.05,</p>**<p>p<0.01,</p>***<p>p<0.001.</p

Blunting of glucagon-induced blood glucose excursions by GRA1 treatment in the (A) hGCGR mouse and (B) rhesus monkey.

<p>In (A), mice (n = 8) were administered vehicle or 3, 10 or 30 mg/kg GRA1, then challenged 1 h later (time 0) by intraperitoneal injection of 15 µg/kg glucagon or vehicle. Pharmacokinetic analysis determined that these animals had mean plasma GRA1 concentrations of 0.4, 0.9, and 4.7 µM, respectively, at 1 h postdose. In (B), monkeys (n = 4) were administered 1 mg/kg GRA1 4 h prior to intramuscular injection of 15 µg/kg glucagon or vehicle. At 1 h postdose, the mean plasma GRA1 concentration in these animals was 0.2 µM. *p<0.05; **p<0.01; and ***p<0.001 vs. glucagon-treated control animals.</p

Fasted plasma glucagon, glucose and amino acids in rhesus monkeys treated once daily with vehicle or 30 mg/kg GRA1.

<p>Data expressed as mean ± SEM; *p<0.05, **p<0.01 and ***p<0.001 vs. vehicle-treated animals at indicated days.</p

Discovery of Potent and Orally Bioavailable Dihydropyrazole GPR40 Agonists

G protein-coupled receptor 40 (GPR40) has become an attractive target for the treatment of diabetes since it was shown clinically to promote glucose-stimulated insulin secretion. Herein, we report our efforts to develop highly selective and potent GPR40 agonists with a dual mechanism of action, promoting both glucose-dependent insulin and incretin secretion. Employing strategies to increase polarity and the ratio of sp³/sp² character of the chemotype, we identified BMS-986118 (compound <b>4</b>), which showed potent and selective GPR40 agonist activity <i>in vitro</i>. <i>In vivo</i>, compound <b>4</b> demonstrated insulinotropic efficacy and GLP-1 secretory effects resulting in improved glucose control in acute animal models