AMG 837: A Novel GPR40/FFA1 Agonist that Enhances Insulin Secretion and Lowers Glucose Levels in Rodents

Agonists of GPR40 (FFA1) have been proposed as a means to treat type 2 diabetes. Through lead optimization of a high throughput screening hit, we have identified a novel GPR40 agonist called AMG 837. The objective of these studies was to understand the preclinical pharmacological properties of AMG 837. The activity of AMG 837 on GPR40 was characterized through GTPγS binding, inositol phosphate accumulation and Ca2+ flux assays. Activity of AMG 837 on insulin release was assessed on isolated primary mouse islets. To determine the anti-diabetic activity of AMG 837 in vivo, we tested AMG 837 using a glucose tolerance test in normal Sprague-Dawley rats and obese Zucker fatty rats. AMG 837 was a potent partial agonist in the calcium flux assay on the GPR40 receptor and potentiated glucose stimulated insulin secretion in vitro and in vivo. Acute administration of AMG 837 lowered glucose excursions and increased glucose stimulated insulin secretion during glucose tolerance tests in both normal and Zucker fatty rats. The improvement in glucose excursions persisted following daily dosing of AMG 837 for 21-days in Zucker fatty rats. Preclinical studies demonstrated that AMG 837 was a potent GPR40 partial agonist which lowered post-prandial glucose levels. These studies support the potential utility of AMG 837 for the treatment of type 2 diabetes

Application of Virtual Screening to the Identification of New LpxC Inhibitor Chemotypes, Oxazolidinone and Isoxazoline

This report summarizes the identification and synthesis of novel LpxC inhibitors aided by computational methods that leveraged numerous crystal structures. This effort led to the identification of oxazolidinone and isoxazoline inhibitors with potent in vitro activity against P. aeruginosa and other Gram-negative bacteria. Representative compound 13f demonstrated efficacy against P. aeruginosa in a mouse neutropenic thigh infection model. The antibacterial activity against K. pneumoniae could be potentiated by Gram-positive antibiotics rifampicin (RIF) and vancomycin (VAN) in both in vitro and in vivo models

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

Efficacy of AMG 837 in Zucker fatty (<i>fa/fa</i>) rats following daily dosing for 21-days.

<p>8-week old Zucker fatty rats were administered a single bolus of AMG 837 (at 0.03, 0.1 and 0.3 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 = 0.03 mg/kg AMG 837, green diamond = 0.1 mg/kg AMG 837 and purple square = 0.3 mg/kg AMG 837). (B) Glucose AUC (from −30 to 120 minutes) during the IPGTT. (E) Plasma insulin levels during the IPGTT (black bar = vehicle, blue bar = 0.03 mg/kg AMG 837, green bar = 0.1 mg/kg AMG 837 and purple bar = 0.3 mg/kg AMG 837). Once daily dosing was continued for 21-days. On day 21, an IPGTT was performed in an identical manner to that on day 1. (C) Blood glucose (D) glucose AUC (from −30 to 120 minutes) and (F) plasma insulin levels were measured from the day 21 IPGTT. Figure legends are identical to those of the day 1 figures. (G) Body weights of the animals were followed through the course of the 21-day study; no difference in BW was observed between the groups. (H) Total plasma concentration of AMG 837 30-minutes following the final dose on day 21. Statistical significance compared to vehicle treated animals is denoted by * (p<0.5), ** (p<0.01) and *** (p<0.001) as determined by one-way or two-way ANOVA.</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

<i>In vitro</i> characterization of AMG 837.

<p>(A) The chemical structure of AMG 837 is shown. (B–D) The activity of AMG 837 in various GPCR assays was assessed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027270#s4" target="_blank">Materials and Methods</a>. Dose response relationships of AMG 837 in GTPγS binding (B), inositol phosphate accumulation (C) and aequorin Ca²⁺ flux assays (D) in cell lines overexpressing GPR40/FFA1 were determined. (D–G) In order to compare the activity of AMG 837 to fatty acids, plasmid titration experiments where either 5000 ng (D), 500 ng (E), 50 ng (F) or 5 ng (G) of GPR40 expression plasmid was co-transfected with aequorin expression plasmids into CHO cells. Activity of AMG 837 (blue diamond) was compared to the naturally occurring GPR40/FFA1 ligand docosahexaenoic acid (DHA, green square) in aequorin Ca²⁺ flux. (H) The activity of AMG 837 in the aequorin Ca²⁺ flux assays in the presence of 0.01% (v/v) purified human serum albumin (HSA, blue diamond), 0.625% (w/v) HSA (green square) or human serum (100% v/v, black circle) was determined.</p

Aequorin Ca²⁺ Flux Activity (EC₅₀, nM) of AMG 837 on Various Receptors.

<p>CHO cells were co-transfected with expression plasmids of a given receptor along with the Ca²⁺ sensitive bioluminescent reporter aequorin, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027270#s4" target="_blank">Materials and Methods</a>. Response to AMG 837 was measured with a luminometer and the EC₅₀ (nM) ± SEM was determined.</p

Optimization of GPR40 Agonists for Type 2 Diabetes

GPR40 (FFA1 and FFAR1) has gained significant interest as a target for the treatment of type 2 diabetes. TAK-875 (<b>1</b>), a GPR40 agonist, lowered hemoglobin A1c (HbA1c) and lowered both postprandial and fasting blood glucose levels in type 2 diabetic patients in phase II clinical trials. We optimized phenylpropanoic acid derivatives as GPR40 agonists and identified AMG 837 (<b>2</b>) as a clinical candidate. Here we report our efforts in searching for structurally distinct back-ups for AMG 837. These efforts led to the identification of more polar GPR40 agonists, such as AM-4668 (<b>10</b>), that have improved potency, excellent pharmacokinetic properties across species, and minimum central nervous system (CNS) penetration