GPR40 partial agonists and AgoPAMs: Differentiating effects on glucose and hormonal secretions in the rodent

<div><p>GPR40 agonists are effective antidiabetic agents believed to lower glucose through direct effects on the beta cell to increase glucose stimulated insulin secretion. However, not all GPR40 agonists are the same. Partial agonists lower glucose through direct effects on the pancreas, whereas GPR40 AgoPAMs may incorporate additional therapeutic effects through increases in insulinotrophic incretins secreted by the gut. Here we describe how GPR40 AgoPAMs stimulate both insulin and incretin secretion in vivo over time in diabetic GK rats. We also describe effects of AgoPAMs in vivo to lower glucose and body weight beyond what is seen with partial GPR40 agonists in both the acute and chronic setting. Further comparisons of the glucose lowering profile of AgoPAMs suggest these compounds may possess greater glucose control even in the presence of elevated glucagon secretion, an unexpected feature observed with both acute and chronic treatment with AgoPAMs. Together these studies highlight the complexity of GPR40 pharmacology and the potential additional benefits AgoPAMs may possess above partial agonists for the diabetic patient.</p></div

GLP-1R^-/- mice vs. wild type mice during an IPGTT.

<p>After treatment with MK-2305 at 10 mg/kg or AP1 at 30 mg/kg and a dextrose challenge, blood glucose excursion was reduced to similar levels in both the knockout and wild type mice compared to the respective vehicle groups (A, B). AP1 administered at a maximally efficacious dose increased insulin following the dextrose challenge in both knockout and wild type mice, but a much greater effect was seen in the wild type mice (C, D). The partial agonist MK-2305 administered at a maximally efficacious dose showed no effect in insulin for either the knockout or wild type mice. Data are mean ± SEM with analysis via ANOVA followed by Tukey’s posttest. ^ <i>P < 0</i>.<i>05</i> vs. GLP-1R^-/- Vehicle; * <i>P < 0</i>.<i>05</i> vs. WT Vehicle; ⁺ <i>P < 0</i>.<i>05</i> vs. GLP-1R^-/- AP1.</p

Acute treatment with GPR40 partial agonists and AgoPAMs in GK rats.

<p>One hour post-acute treatment with vehicle, TAK-875 at 100 mg/kg, AP1 at 30 mg/kg, or AP3 at 10 mg/kg, blood glucose dropped to its lowest levels (A) at the same time insulin peaked (B) in GK rats. All compounds were dosed at maximal efficacious doses. Acute treatment with the AgoPAMs produced a significant 24 hr increase in glucagon, while the glucagon increase of TAK-875 was not significant and was of a shorter duration (C). Data are mean ± SEM with analysis via ANOVA followed by Tukey’s posttest. * <i>P < 0</i>.<i>05</i> vs. Vehicle, ⁺ <i>P < 0</i>.<i>05</i> vs. TAK-875.</p

Design and Synthesis of Novel, Selective GPR40 AgoPAMs

GPR40 is a G-protein-coupled receptor expressed primarily in pancreatic islets and intestinal L-cells that has been a target of significant recent therapeutic interest for type II diabetes. Activation of GPR40 by partial agonists elicits insulin secretion only in the presence of elevated blood glucose levels, minimizing the risk of hypoglycemia. GPR40 agoPAMs have shown superior efficacy to partial agonists as assessed in a glucose tolerability test (GTT). Herein, we report the discovery and optimization of a series of potent, selective GPR40 agoPAMs. Compound <b>24</b> demonstrated sustained glucose lowering in a chronic study of Goto Kakizaki rats, showing no signs of tachyphylaxis for this mechanism

Summary of [3H]L358 and [3H]AP9 ([3H]25,) binding to WT human GPR40.

<p>Each compound was profiled multiple times (n = 2–13). N.A. specifies that a compound augmented the binding of specified radioligand in line with the positive cooperativity between the partial agonist and AgoPAM binding sites.</p

Chemical structures and measurement of IP accumulation in stable cell lines expressing human, rat, or mouse GPR40.

<p>(A) Chemical structures of partial agonists MK-8666, MK-2305, TAK-875, AMG-837; and AgoPAMs AM-1638, AP1, and AP3. Dose-response curves for partial agonists (MK-8666, MK-2305, TAK-875, and AMG-837) and AgoPAMs (AM-1638, AP1, and AP3) were generated in human GPR40/CHO-K1 (B), rat GPR40/HEK293 (C), or mouse GPR40/HEK293 (D) cells. Data are expressed as a percentage of the control response of an in-house partial agonist and fitted to a standard 4-parameter non-linear regression model. EC50’s were determined using a custom in-house developed software package. Each compound was profiled multiple times (n = 2–20) with representative graphs shown. The mean parameters of these are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186033#pone.0186033.t001" target="_blank">Table 1</a>.</p

GK rat incretin hormones are affected by 28 days treatment with AgoPAMs or partial agonist.

<p>Both AgoPAMs significantly increased total (A) and active (B) GLP-1 throughout the 4 weeks of treatment. Other gut hormones, GIP (C) and PYY (D), were significantly elevated by the AgoPAM treatments. In contrast, MK-8666 had no effect on gut hormones. Data are mean ± SEM with analysis via ANOVA followed by Tukey’s posttest. * <i>P < 0</i>.<i>05</i> vs. Vehicle, ⁺ <i>P < 0</i>.<i>05</i> vs. MK-8666.</p

Functional properties of partial agonists and AgoPAMs in human, rat, and mouse GPR40 IP accumulation assays.

<p>Functional properties of partial agonists and AgoPAMs in human, rat, and mouse GPR40 IP accumulation assays.</p

Skeletal muscle 13C-glycogen levels following a 13C-GTT in GK rats chronically treated with GPR40 partial agonist (MK-8666) or AgoPAM (AP1).

<p>Data are mean ± SEM with analysis via ANOVA followed by Tukey’s posttest. * <i>P < 0</i>.<i>05</i> vs. Vehicle; ⁺ <i>P < 0</i>.<i>05</i> vs. MK-8666.</p

Total endogenous glucose production (EGP) and contributing fluxes in GK rats following chronic treatment with GPR40 partial agonist (MK-8666) or AgoPAMs (AP1 and AP3).

<p>Data are mean ± SEM with analysis via ANOVA followed by Tukey’s posttest. * <i>P < 0</i>.<i>05</i> vs. Vehicle; ⁺ <i>P < 0</i>.<i>05</i> vs. MK-8666.</p