human FFA1 ( pIC50 = 8.13 )

AMG 837 (1 nM–10 μM) stimulates insulin secretion in a glucose-dependent manner with an EC50 of 142±20 nM on islets isolated from mice[1].
AMG 837 increases Ca²⁺ flux in CHO cells with EC50 values of 13.5, 22.6, and 31.7 nM for rat, mouse, and human receptors, respectively[1].

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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 Ca(2+) flux assays. Activity of AMG 837 on insulin release was assessed on isolated primary mouse islets. [1]

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Compound 8/AMG837 displayed the expected two-fold increase in potency on GPR40 (EC50 = 13 [±7] nM) compared to the racemic compound and its activity crossed over to the rat and mouse forms of GPR40 (EC50 = 23 and 13 nM, respectively). Because of our interest in the compound, the intrinsic efficacy of compound 8 was determined compared to DHA. Compound 8 was thus found to be a partial agonist on GPR40 with maximal activity 85% of that shown by DHA under our standard assay conditions.19 In addition to its activity in the Ca2+-flux assay, compound 8 shows functional activity in a mouse β-cell line (MIN6). As shown in Figure 1, compound 8 is a highly potent stimulator of insulin secretion in MIN6 cells with an EC50 comparable to that seen in the aequorin Ca2+-flux assay.

While highly potent on GPR40, compound 8/AMG837 was inactive on the closely related GPCRs GPR41 and GPR43. Despite a possible structural resemblance to some PPAR agonists, compound 8 showed no significant activity in cell-based assays against PPAR-α, -δ, and -γ. An external panel of 64 receptors also revealed no significant activity with the exception of weak inhibition (IC50 = 3 μM) on the α2-adrenergic receptor. Overall, compound 8 was both highly potent and selective in vitro.[2]

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The important feature of these agonists is that they interact allosterically with each other and at three different binding sites. The radiolabeled agonists, [3H]AMG 837 and [3H]AM 1638, label two different sites in a 1:1 stoichiometry (Fig. 5, A and C). AM 1638 enhances the affinity of [3H]AMG 837 approximately 3-fold (Fig. 3), and the reciprocal enhancement of [3H]AM 1638 by AMG837 has been demonstrated (Fig. 4). The increase in affinity is not accompanied by any increase in the number of binding sites, Bmax (Fig. 5, B and D). These binding data satisfy the predictions of the allosteric ternary complex model for the simple 1:1 allosteric interaction (Lazareno and Birdsall, 1995) and represent one of the few examples for which reciprocal two-way allosteric interactions in GPCRs have been characterized using radioligands for two interacting sites. Furthermore, AM 1638 slows down the dissociation kinetics of [3H]AMG 837 (Fig. 7A) with the potency predicted by the equilibrium data and the allosteric ternary complex model (Lazareno and Birdsall, 1995; Lazareno et al., 1998).

The third synthetic ligand, AM 8182, also interacts allosterically with [3H]AMG 837, but with negative cooperativity (Fig. 4), and exhibits the predicted potency in slowing down the dissociation rate of [3H]AMG 837 (Fig. 7B). This finding might suggest that AM 8182 was binding to the same site as AM 1638. However, AM 8182 has no effect on the binding of [3H]AM 1638 (Fig. 4), which indicates neutral cooperativity between AM 8182 and AM 1638; i.e., AM 8182 is allosteric with both AM 1638 and AMG837 and thus binds to a third binding site.

An important question is which of these sites might bind endogenous fatty acids, e.g., DHA? Binding studies show that DHA is allosteric with [3H]AMG 837 (negatively cooperative) (Fig. 3) and exhibits slight positive cooperativity with [3H]AM 1638 (Fig. 4). Thus, DHA, like AM 8182, does not bind to either of the two sites labeled by the radioligands. It was possible to exploit the lack of effect of AM 8182 on [3H]AM 1638 binding and its known affinity to inhibit [3H]AMG 837 binding to demonstrate that increasing concentrations of AM 8182 shift the DHA enhancement curves of [3H]AM 1638 binding in a parallel fashion and to the extent expected of a competitive interaction between AMG182 and DHA (Fig. 6). This result confirms the presence of three interacting sites, shown illustratively in Fig. 16 [3].

AMG 837 (0.03-0.3 mg/kg; p.o. once daily for 21 days) lowers blood glucose and raises insulin levels after a glucose challenge in vivo[1].
AMG 837 (0.03-0.3 mg/kg; one oral dose) increases insulin secretion and glucose tolerance in Sprague-Dawley rats[1].
AMG 837 (0.5 mg/kg; p.o.) exhibits a total plasma Cmax of 1.4 µM and good oral bioavailability (F = 84%)[1].

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To determine the anti-diabetic activity of AMG837 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 [1].

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AMG837 Stimulates Insulin Secretion and Lowers Postprandial Glucose Levels in Normal Rodents [1]
We next tested the ability of AMG 837 to improve glucose tolerance and stimulate insulin secretion in Sprague-Dawley rats. Sprague-Dawley rats were chosen since they are euglycemic, allowing AMG 837 to be tested at normal glucose levels and during the challenged state following a glucose bolus. AMG 837 displays excellent pharmacokinetic properties in multiple species (Houze JB et al, in preparation). The pharmacokinetic profile following a single 0.5 mg/kg oral dose in rats displayed excellent oral bioavailability (%F = 84) and a total plasma Cmax of 1.4 µM. AMG 837 was dosed by oral gavage at 0.03 mg/kg, 0.1 mg/kg and 0.3 mg/kg 30 minutes prior to an intraperitoneal glucose challenge. Glucose and insulin levels were determined before and after administration of glucose.

AMG 837 administration did not have any effect on glucose levels prior to the glucose tolerance test (30 minutes following AMG 837 administration). Following administration of glucose, plasma glucose levels were suppressed in an AMG 837 dose-dependent manner (figure 3A). At the low, mid and high dose, glucose AUC improved 3.9%, 14.5% (p<0.05) and 18.8% (p<0.01) compared to that of vehicle treated animals, respectively (figure 3B). The half-maximal dose of AMG 837 to lower post-prandial glucose in rats was approximately 0.05 mg/kg.

The improvement in post-prandial glucose was a result of an increase in glucose-stimulated insulin secretion. In animals treated with AMG 837, there was a dose-dependent increase of plasma insulin levels following the glucose challenge (figure 3C). The increase of plasma insulin levels was rapid and of short duration, most evident at 5 and 15 minutes following glucose administration. Taken together, these results indicate that the activity of AMG 837 was dependent on glucose in vivo.

We further tested whether a single dose of AMG 837 could improve post-prandial glucose following consecutive glucose challenges. A single dose (0.3 mg/kg) of AMG 837 was administered to Sprague-Dawley rats followed by two intraperitoneal glucose challenges 3 hours apart. AMG 837 improved blood glucose levels during both glucose challenges (p<0.01, figure 3D, E). As observed in the single glucose challenge, peak insulin secretion during each glucose challenge increased soon after the glucose administration (figure 3F). These results indicate that pharmacological effect of a single dose of AMG 837 on pancreatic β-cells persists over the course of several hours.

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Efficacy of AMG837 in Zucker Fatty Rats Following Once Daily Dosing for 21-days [1]
We next tested the effect of AMG 837 in the insulin resistant Zucker fatty (fa/fa) rat following single and multiple doses of AMG 837. The Zucker fatty rat model was studied since it displays impaired glucose tolerance, hyperinsulinemia and mild hyperglycemia [24], [25]. AMG 837 was first tested in single doses of 0.3, 1 and 3 mg/kg prior to an IPGTT. In contrast to that observed in normal Sprague-Dawley rats, glucose levels 30 minutes following the AMG 837 dose trended lower and insulin levels trended higher, although neither parameter reached statistical significance (figure 4A,C). Because the activity of AMG 837 on GPR40 is glucose dependent, the higher basal glucose levels in insulin resistant Zucker fatty rats compared to that in Sprague-Dawley rats may be sufficient to trigger a response. Following the glucose challenge, glucose levels were lower at all doses of AMG 837 and the glucose excursion curves largely overlapped (figure 4A). The glucose AUC for all doses decreased ∼46% (p<0.001, figure 4B). As observed in Sprague-Dawley rats, plasma insulin levels spiked most prominently 5 and 15 minutes post glucose challenge (figure 4C).

In order to understand the effect of AMG 837 following multiple doses, AMG 837 was dosed at 0.03, 0.1 and 0.3 mg/kg by oral gavage daily for 21-days. Thirty minutes following the first dose, an IPGTT was performed. AMG 837 improved glucose levels during the IPGTT (figure 5A) with a decrease in glucose AUC of 17%, 34% (p<0.001), and 39% (p<0.001) at 0.03, 0.1 and 0.3 mg/kg, respectively (figure 5B). This was associated with increased insulin secretion following glucose administration (figure 5E). Because a separation in the pharmacological response to glucose challenge could be observed below but not above 0.3 mg/kg (figure 4), this indicates that 0.3 mg/kg is approximately the maximal dose in this rat model.

Administration of AMG 837 was continued daily for 21-days in order to test the effects of AMG 837 following multiple doses. A second IPGTT was performed 30 minutes following the final dose on day 21 and AMG 837 lowered glucose levels following glucose challenge (figure 5C). Glucose AUC values during the GTT were decreased to 7%, 15% (p<0.05), and 25% (p<0.001) at 0.03, 0.1 and 0.3 mg/kg, respectively (figure 5D). Insulin levels prior to glucose challenge at day 21 were higher in all groups compared to those on day 1, likely indicative of progressive insulin resistance in these animals. In rodents treated with AMG 837, insulin levels increased in the mid- and high- dose groups post-glucose challenge (figure 5F). Body weights were not affected by AMG 837 treatment during the 21-day treatment (figure 5G). Taken together, these results indicate that the pharmacological activity of AMG 837 persisted even after 21-days. Total plasma concentrations of AMG 837 30-minutes following the final dose of 0.03 mg/kg, 0.1 mg/kg and 0.3 mg/kg AMG 837 were 26±6 nM, 75±13 nM and 204±49 nM, respectively (figure 5H).

GTPγS binding assay [1]
A GTPγS binding assays using an anti-Gα-protein scintillation proximity assay format was employed essentially as described [23]. Assays were performed in Corning 96-well plates. Cell membranes were prepared from an A9 cell line stably transfected with GPR40 (A9_GPR40). Cell membranes were mixed with various concentrations of AMG837, 0.1 µM GDP, 400 pM [35S]-GTPγ in binding buffer (consisting of 20 mM Hepes pH 7.4, 100 mM NaCl and 5 mM MgCl2) in a volume of 200 µl/well. Plates were incubated for 60 minutes at room temperature. Next, 20 µl of 3% NP-40 were added to each well and the plates were further incubated for 30 minutes. This was followed by the addition of 20 µl of anti-Gq antibody (anti Gα q/11 antibody, 1∶400 dilution) and the plates were incubated for an additional 60 min. Finally, 50 µl of anti-rabbit-SPA beads were added to each well and the plates were incubated for 3 hrs. Antibody captured [35S]-GTPγ was measured using a Microbeta.

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Equilibrium Binding Assays. [3]
Equilibrium binding assays were performed on A9 membranes expressing hFFA1. Test compounds were diluted serially with binding buffer (20 mM Hepes, pH 7.5, 5 mM MgCl2, 100 mM NaCl, and 0.1% (w/v) fatty acid-free BSA). There is a possibility of free fatty acids being present in the assays and giving the appearance of constitutive activity in the functional assays and perturbing the radioligand binding assays. Therefore, a low concentration of fatty acid-free BSA (0.1%) was included in the binding assays. Fatty acid-free BSA, up to 0.5%, had no effect on binding. The membranes and the radioligand were resuspended in the binding buffer. Each well of the 96-well assay plate contained diluted test compounds, radioligand (5 nM [3H]AMG 837 or 10 nM [3H]AM 1638), and A9-hFFA1 cell membrane protein (5 μg/well) in a total volume of 200 μl and was allowed to equilibrate at room temperature for 4 h. Some cross-interaction heterologous binding experiments were performed with 5 nM [3H]AM 1638 and 20 μg/well membrane protein, using different concentrations of DHA in the presence or absence of different concentrations of AM 8182. The membrane protein concentration was such that not more than 10% of the added radioligand was bound to the receptor. There were 2 to 12 replicates per data point. Nonspecific binding was determined in presence of a 10 μM concentration of either AMG837 or AM 1638, depending on the radioligand used. Plates were harvested on a GF/C filterplate with five washes of ice-cold buffer. Then 50 μl of scintillant was added to each well of the plate, and the plate was counted on a TopCount Microplate Scintillation counter. All the compounds were dissolved in dimethyl sulfoxide, which, at the highest final concentration in the assay of 1%, had no effect on binding.

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Saturation Binding and Interaction Experiments. [3]
Saturation binding curves were generated using increasing concentrations (0.1–40 nM) of either the radiolabeled partial agonist [3H]AMG 837 or the full agonist [3H]AM 1638. The assay was performed in a 96-well plate containing either 5 μg of membrane protein for [3H]AMG 837 binding or 20 μg/well for [3H]AM 1638 and incubated at room temperature for 4 h. Nonspecific binding was determined in the presence of a 10 μM concentration of either unlabeled ice-cold AMG837 or AM 1638 as appropriate. Saturation interaction experiments were performed in the presence of 100 nM AM 1638 (for [3H]AMG 837 binding curves) or 100 nM AMG837 (for [3H]AM 1638). The binding reactions were terminated, and radioactivity was measured as described above. Saturation curves were generated using 3 to 12 replicates for each data point. Not more than 10% of added radioligand was bound to the receptor at any radioligand concentration.

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Dissociation Binding Kinetics. [3]
The dissociation rate of [3H]AMG 837 from the FFA1 receptor was measured in the absence or presence of a range of concentrations of agonist (AM 1638 or AM 8182). The assay plate containing membrane protein (5 μg) and 5 nM [3H]AMG 837 was preequilibrated for 2 h at room temperature (shaking at 230 rpm). At time 0, total binding was determined, and saturating amounts of ice-cold AMG837 (10 μM), in the presence or absence of different concentrations of allosteric ligand were added to the different wells of the plate containing the prelabeled membranes. The membranes were filtered at different times (2–240 min) followed by five washes with cold buffer, and radioactivity was measured as described above.

Aequorin assay CHO cells were plated in 15 cm plates containing 8×106 cells/plate in DMEM/F12 containing 10% FBS. The following day, cells were transfected with 5 µg of GPR40 expression plasmid and 5 µg of aequorin expression plasmid complexed with 30 µL of Lipofectamine 2000. In plasmid titration experiments, the amount of GPR40 expression plasmid was reduced, but the total amount of DNA transfected was kept constant by adding in empty vector DNA. Sixteen to twenty-four hours post-transfection, cells were washed with PBS and detached from the plate with 2 mL trypsin (0.25% in HBSS). 28 mL of HBSS containing a desired amount of HSA (0.01% or 0.625% w/v) or human serum (100% v/v) was added to the detached cells and coelenterazine was added to final concentration of 1 µg/mL. Cells were allowed to incubate in coelenterazine containing buffer for 2 hours prior to assay. AMG837 and DHA stock solutions were prepared in DMSO and then diluted in HBSS buffer containing the % HSA identical to that in which the cells were incubated in. Compounds were allowed to complex with HSA for 1 hr at 37°C. Aequorin activity was measured using a microlumat.

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Aequorin assay: [2]
A cell-based aequorin assay may be employed to characterize the modulatory activity of compounds on the GPR40 signaling pathway. CHO cells are transfected in a 15 cm plate containing 14 million cells with 5 µg of GPR40 expression vector and 5 µg of Aequorin expression vector using Lipofectamine 2000. After 17-24 hours post-transfection, cells are washed with phosphate buffered saline (PBS) and detached from the tissue culture dish with 2 mL of trypsin (0.25%(w/v)). Trypsinization is halted with 28 mL of Hanks Buffered Salt Solution containing 20 mM Hepes (H/HBSS) and 0.01% fatty acid-free bovine serum albumin (BSA) or 0.625% fatty acid-free human serum albumin (HSA). Coelantrazine is added to 1 ug/mL and the cells are incubated for 2 hours at room temperature. Cells are gently mixed every 15 minutes. Compounds are dissolved in dimethyl sulfoxide for preparation of 10 mM stock solutions. Compounds are diluted in H/HBSS containing 0.01% BSA. Serial dilutions of the test compounds are prepared to determine dose response. Aequorin luminescence measurements are made using an EG&G Berthold 96-well luminometer and the response is measured over a 20 second interval after cells and compounds were mixed. The area-under-curve from 2-20 seconds is plotted to determine dose reponse. The EC50 (effective concentration to reach 50% maximal response) is determined from the dose response plot.

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Aequorin Assay. [3]
CHO cells stably expressing both FFA1 and aequorin DNA were grown in 15-cm dishes, harvested 24 h later using 2 ml of 1× trypsin-EDTA (0.25% trypsin and 21 mM EDTA in Hanks' buffered salt solution) and pelleted by centrifugation (5 min, 600g). The pellet was resuspended in HBSS containing 0.01% (w/v) fatty acid-free HSA and 20 mM Hepes and incubated with 1 μg/ml coelenterazine and test compounds at room temperature for 2 h. Aequorin luminescence measurements as a readout for ligand-induced receptor activation and calcium release were made using a 96-well luminometer. The response was measured over a 20-s interval after addition of compounds to the cells (An et al., 1998). This stable cell line expressed low levels of FFA1 relative to those for the A9 cells. The best estimate of the Bmax levels, obtained from the measurement of [3H]AMG 837 (1 nM) binding to membranes (10 μg of protein) prepared in the same way as the A9 membranes, is approximately 0.5 pmol/mg protein, assuming that the affinity of AMG837 is the same as that measured in A9 membranes. Specific binding of [3H]AM 1638 (5 or 10 nM) to CHO cell membranes could not be detected (data not shown).

8-week old Zucker Fatty Rats
0.03, 0.1, 0.3 mg/kg
Oral gavage once daily for 21 days

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In vivo procedures [1]
AMG837 was formulated for oral dosing using 1% methylcellulose (CMC), 1% Tween 80. For evaluation of AMG837 following a single dose in rats, animals were fasted overnight and then randomized into dose groups based on their body weights. Thirty minutes after oral administration of their respective treatments, the animals received a 1 g/kg glucose challenge dose by intraperitoneal injection. Blood samples were collected at 0, 5, 15, 30, 60, and 120 minutes via tail vein after the glucose challenge. Glucose levels were monitored with a Glucometer. Plasma insulin was measured using a rat insulin ELISA kit. For evaluation of AMG837 in Zucker fatty rats, animals were randomized based on body weight and received either vehicle, 0.03 mg/kg, 0.1 mg/kg, or 0.3 mg/kg AMG837 once daily for 21 days by oral gavage. Treatments were administered between 0900 and 1000 h during the light cycle. On days 1 and 21, an intraperitoneal glucose tolerance test (IPGTT) was performed as described above.

AMG837 displays excellent pharmacokinetic properties in multiple species (Houze JB et al, in preparation). The pharmacokinetic profile following a single 0.5 mg/kg oral dose in rats displayed excellent oral bioavailability (%F = 84) and a total plasma Cmax of 1.4 µM. AMG 837 was dosed by oral gavage at 0.03 mg/kg, 0.1 mg/kg and 0.3 mg/kg 30 minutes prior to an intraperitoneal glucose challenge. Glucose and insulin levels were determined before and after administration of glucose.
AMG837 administration did not have any effect on glucose levels prior to the glucose tolerance test (30 minutes following AMG 837 administration). Following administration of glucose, plasma glucose levels were suppressed in an AMG 837 dose-dependent manner (figure 3A). At the low, mid and high dose, glucose AUC improved 3.9%, 14.5% (p<0.05) and 18.8% (p<0.01) compared to that of vehicle treated animals, respectively (figure 3B). The half-maximal dose of AMG 837 to lower post-prandial glucose in rats was approximately 0.05 mg/kg.[1]

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In addition to its favorable profile in vitro, compound 8/AMG837 distinguished itself by displaying an excellent pharmacokinetic profile in multiple species. As shown in Table 3, compound 8 combines low clearance, long half-life, and high oral bioavailability in four preclinical species.[2]

In order to confirm that the potential antidiabetic activity of compound 8/AMG837 was mediated by GPR40, an oral glucose tolerance test (OGTT) was carried out in wild-type and GPR40 KO mice. The DPP-4 inhibitor sitagliptin was used at a maximally efficacious dose as a positive control.20 Compounds were dosed orally 60 min prior to the oral glucose challenge. As shown in Figure 2, compound 8 substantially blunted plasma glucose excursion compared to both vehicle and positive control in wild-type animals consistent with its activity as an insulin secretagogue as shown in MIN6 cells (Fig. 2a). The total glucose AUC was also reduced in a statistically significant manner (Fig. 2c). In contrast, no effect in the OGTT was seen in the GPR40 KO animals after dosing with compound 8 where the positive control retained activity (Fig. 2b). The complete absence of a response in the GPR40 KO animals establishes that the effects of compound 8 are GPR40 mediated. The behavior of compound 8/AMG837 in this GTT study is consistent with the hypothesis that selective GPR40 agonists could serve as glucose-dependent insulin secretagogues.[2]

[1]. AMG 837: a novel GPR40/FFA1 agonist that enhances insulin secretion and lowers glucose levels in rodents. PLoS One. 2011; 6(11): e27270.

[2]. AMG 837: a potent, orally bioavailable GPR40 agonist. Bioorg Med Chem Lett. 2012 Jan 15; 22(2): 1267-70.

[3]. Identification and pharmacological characterization of multiple allosteric binding sites on the free fatty acid 1 receptor. Mol Pharmacol. 2012 Nov;82(5):843-59.

The discovery that certain long chain fatty acids potentiate glucose stimulated insulin secretion through the previously orphan receptor GPR40 sparked interest in GPR40 agonists as potential antidiabetic agents. Optimization of a series of β-substituted phenylpropanoic acids led to the identification of (S)-3-(4-((4'-(trifluoromethyl)biphenyl-3-yl)methoxy)phenyl)hex-4-ynoic acid (AMG837) as a potent GPR40 agonist with a superior pharmacokinetic profile and robust glucose-dependent stimulation of insulin secretion in rodents. [2]

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Activation of FFA1 (GPR40), a member of G protein-coupling receptor family A, is mediated by medium- and long-chain fatty acids and leads to amplification of glucose-stimulated insulin secretion, suggesting a potential role for free fatty acid 1 (FFA1) as a target for type 2 diabetes. It was assumed previously that there is a single binding site for fatty acids and synthetic FFA1 agonists. However, using members of two chemical series of partial and full agonists that have been identified, radioligand binding interaction studies revealed that the full agonists do not bind to the same site as the partial agonists but exhibit positive heterotropic cooperativity. Analysis of functional data reveals positive functional cooperativity between the full agonists and partial agonists in various functional assays (in vitro and ex vivo) and also in vivo. Furthermore, the endogenous fatty acid docosahexaenoic acid (DHA) shows negative or neutral cooperativity with members of both series of agonists in binding assays but displays positive cooperativity in functional assays. Another synthetic agonist is allosteric with members of both agonist series, but apparently competitive with DHA. Therefore, there appear to be three allosterically linked binding sites on FFA1 with agonists specific for each of these sites. Activation of free fatty acid 1 receptor (FFAR1) by each of these agonists is differentially affected by mutations of two arginine residues, previously found to be important for FFAR1 binding and activation. These ligands with their high potencies and strong positive functional cooperativity with endogenous fatty acids, demonstrated in vitro and in vivo, have the potential to deliver therapeutic benefits.[3]

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Therapeutic Potential.
The large positive functional cooperativity between the synthetic ligands (up to 30-fold) allows lower doses of a combination of ligands to be used to achieve a given level of stimulation than would be required for a single ligand alone. Up to a 30-fold lower dose of one ligand or up to 10-fold lower doses of both ligands can generate a given response and should reduce potential unwanted effects. This is illustrated in Fig. 10C in which threshold doses of 1.5 nM AM 1638 and 20 nM AMG 837, when applied in combination, produce an response equivalent to approximately 30 nM AMG 1638 and >300 nM AMG837 when administered singly. The FFA1 receptor agonists reported here may not only be pharmacological tools but also have the potential utility for the treatment of type 2 diabetes and have implications in pathophysiological settings of the receptor.[3]

C52H42CAF6O7

932.954115390778

932.25

C, 66.94; H, 4.54; Ca, 4.30; F, 12.22; O, 12.00

1259389-38-2

AMG 837 hemicalcium; 1291087-14-3; AMG 837; 865231-46-5; AMG 837 sodium salt; 865231-45-4; 1259389-38-2 (calium hydrate)

154584011

White to off-white solid powder

2

14

12

67

655

2

CC#C[C@@H](CC(=O)[O-])C1=CC=C(C=C1)OCC2=CC(=CC=C2)C3=CC=C(C=C3)C(F)(F)F.CC#C[C@@H](CC(=O)[O-])C1=CC=C(C=C1)OCC2=CC(=CC=C2)C3=CC=C(C=C3)C(F)(F)F.O.O.[Ca+2]

QDINKBCSIAWNMP-XYDYARRRSA-L

InChI=1S/2C26H21F3O3.Ca.H2O/c2*1-2-4-21(16-25(30)31)20-9-13-24(14-10-20)32-17-18-5-3-6-22(15-18)19-7-11-23(12-8-19)26(27,28)29;;/h2*3,5-15,21H,16-17H2,1H3,(H,30,31);;1H2/q;;+2;/p-2/t2*21-;;/m00../s1

calcium;(3S)-3-[4-[[3-[4-(trifluoromethyl)phenyl]phenyl]methoxy]phenyl]hex-4-ynoate;hydrate

AMG 837; AMG837; AMG-837; AMG-837 Calcium Hydrate; 1259389-38-2; AMG 837 (calcium hydrate); calcium;(3S)-3-[4-[[3-[4-(trifluoromethyl)phenyl]phenyl]methoxy]phenyl]hex-4-ynoate;hydrate; AMG 837 hemicalcium hydrate; AMG 837 hemicalcium salt

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ≥ 42 mg/mL (~92.2 mM)

Solubility in Formulation 1: 2.5 mg/mL (5.49 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.49 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.49 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)