GLP-1 receptor

The therapeutic success of peptidic GLP-1 receptor agonists for treatment of type 2 diabetes mellitus (T2DM) motivated our search for orally bioavailable small molecules that can activate the GLP-1 receptor (GLP-1R) as a well-validated target for T2DM. Here, the discovery and characterization of a potent and selective positive allosteric modulator (PAM) for GLP-1R based on a 3,4,5,6-tetrahydro-1H-1,5-epiminoazocino[4,5-b]indole scaffold is reported. Optimization of this series from HTS was supported by a GLP-1R ligand binding model. Biological in vitro testing revealed favorable ADME and pharmacological profiles for the best compound 19. Characterization by in vivo pharmacokinetic and pharmacological studies demonstrated that 19 activates GLP-1R as positive allosteric modulator (PAM) in the presence of the much less active endogenous degradation product GLP1(9-36)NH2 of the potent endogenous ligand GLP-1(7-36)NH2. While these data suggest the potential of small molecule GLP-1R PAMs for T2DM treatment, further optimization is still required towards a clinical candidate [2].

Agonism of the glucagon-like peptide-1 receptor (GLP-1R) results in glycemic lowering and body weight loss and is a therapeutic strategy to treat type 2 diabetes (T2D) and obesity. We developed danuglipron (PF-06882961), an oral small-molecule GLP-1R agonist and found it had comparable efficacy to injectable peptidic GLP-1R agonists in a humanized mouse model. We then completed a placebo-controlled, randomized, double-blind, multiple ascending-dose phase 1 study ( NCT03538743 ), in which we enrolled 98 patients with T2D on background metformin and randomized them to receive multiple ascending doses of danuglipron or placebo for 28 d, across eight cohorts. The primary outcomes were assessment of adverse events (AEs), safety laboratory tests, vital signs and 12-lead electrocardiograms. Most AEs were mild, with nausea, dyspepsia and vomiting most commonly reported. There were no clinically meaningful AEs in laboratory values across groups. Heart rate generally increased with danuglipron treatment at day 28, but no heart-rate AEs were reported. Systolic blood pressure was slightly decreased and changes in diastolic blood pressure were similar with danuglipron treatment at day 28, compared with placebo. There were no clinically meaningful electrocardiogram findings. In this study in T2D, danuglipron was generally well tolerated, with a safety profile consistent with the mechanism of action of GLP-1R agonism [4].

Biological Assays. Min6 Ca2+ Mobilization Assay[3]
MIN6-c4 cells were plated at a density of 5 × 104 cells per well into black 96-well plates and cultured for 20–24 h at 37 °C and 5% CO2. For cell loading, culture supernatants were aspirated and 100 μL of assay buffer (Krebs–Ringer buffer, 10 mM HEPES, 0.1% BSA, 2.5 mM glucose) and an equal volume of calcium 6 dye (FLIPR calcium 6 assay kit, Molecular Devices, R8191) dissolved in the same buffer according to instructions of the manufacturer were added to each well. Cells were incubated for 70 min at 37 °C/5% CO2 and equilibrated for an additional 10 min at room temperature in the dark. To assess the effect of test compounds on glucose-mediated increase in intracellular Ca2+, a volume of 50 μL assay buffer containing 75 mM glucose (resulting in a final concentration of 15 mM glucose) and test compounds or DMSO was added per well during detection on a FLIPR Tetra instrument (molecular devices). For low glucose controls, 50 μL of starvation buffer without additional glucose was added to keep the final glucose concentration at 2.5 mM. Calcium flux was quantified by calculating the area under the curve of fluorescence readings from 3 s to 372 s.

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cAMP Stimulation Assay in PSC-HEK293 Cell Line Stably Expressing Human GLP-1R[3]
In vitro cellular assays for GLP-1R agonists and positive allosteric modulators were conducted in 1536-well plates using thaw-and-use frozen cells. Prior to use, frozen cells were thawed quickly at 37 °C and washed (5 min at 900 rpm) with 20 mL of cell buffer (1× HBSS; 20 mM HEPES, 0.1% BSA). Cells were resuspended in assay buffer (cell buffer plus 2 mM IBMX) and adjusted to a cell density of 1 million cells/mL. To a 1536-well microtiter plate, an amount of 2 μL of cells is added (final 2000 cells/well) and 2 μL compound for an agonist assay. For a PAM assay, two assay formats were applied, namely, (a) an enhancer assay with 1 μL of different doses of the compound and 1 μL of a fixed concentration (EC20) of GLP1(9–36)NH2 and (b) a shift assay with 1 μL of different doses of GLP1(9–36)NH2 and 1 μL of 10 μM and 3 μM compound. The mixtures containing 2 μL of each cells and compounds were incubated for 30 min at room temperature.
The cAMP content of cells was determined using a kit from Cisbio Corp. (catalog no. 62AM4PEC) based on HTRF (homogeneous time resolved fluorescence). After addition of HTRF reagents diluted in lysis buffer (kit components), plates were incubated for 1 h, followed by measurement of the fluorescence ratio at 665/620 nm. Dose–response results were calculated with the internal software Biost@t-Speed version 2.0 HTS using a four-parameter logistic model.

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cAMP Stimulation Assay in the Human Pancreatic β-Cell Line 1.1B4[3]
In vitro cellular assays of GLP-1(7–36)NH2, GLP1(9–36)NH2, and test compounds were conducted using the human pancreatic β-cell line 1.1B4. Upon GLP-1R activation, the 1.1B4 cells accumulate intracellular cyclic adenosine monophosphate (cAMP). Cyclic AMP formation was measured using a commercial immunoassay technology with HTRF readout. In these experiments, all reagents necessary for quantification of cAMP were supplied in a kit (catalog no. 62AM4PEC from Cisbio Corp., France) and applied according to protocols supplied by the vendor. Two assay formats were applied, namely, (a) an enhancer assay with a compound concentration–response curve and a fixed concentration of 10 nM GLP1(9–36)NH2 and (b) a shift assay with a GLP1(9–36)NH2 concentration–response curve and a fixed concentration of 1 μM compound. 20 000 cells were seeded into a 96-well microtiter plate. Following overnight culturing, cells were washed twice, and serial dilutions of GLP-1R ligand or test compound with or without the respective fixed concentration of either test compound or GLP1(9–36)NH2 were transferred to the cells. After incubation for 30 min with the test agents, the cells were lysed and prepared for cAMP determination according to the manufacturer’s description. Data points were obtained by fluorescence measurement at 665 and 620 nm, calculation of 665/620 nm ratio, and expression in percentage of effect relative to negative (0%) and positive (100%) controls. The negative control was assay buffer (1× HBSS, 0.1% BSA, 1 mM IBMX), and the positive control was GLP-1(7–36)NH2. Concentration–response results were calculated with internal software Biost@t-Speed version 2.0 LTS using a four-parameter logistic model. The adjustment was obtained by nonlinear regression using the Marquardt algorithm in SAS version 9.1.3.

HEK293 cells that express hGLP-1R fused to green fluorescent protein (GFP) steadily (400,000 cells/well) are grown on 6-well plates for a full day and then stimulated for half a minute with PF-06882961. For these investigations, an agonist concentration of 1 μM that has been shown to cause maximal internalization is used. To test the endocytosis process' reversibility, cells are placed in specific wells, rinsed three times with PBS containing 0.1% BSA, and then incubated for a further two hours at 37 °C. After fixing the cells for 15 minutes at room temperature with 4% paraformaldehyde, the cells are cleaned three times using PBS containing 0.1% BSA.

male cynomolgus monkeys
1 mg/kg, 5 mg/kg, 100 mg/kg
IV, Oral gavage

[1]. 20th SCI/RSC Medicinal Chemistry Symposium.

[2]. Design, Synthesis, and Pharmacological Evaluation of Potent\nPositive Allosteric Modulators of the Glucagon-like Peptide-1 Receptor\n(GLP-1R). J Med Chem. 2020 Mar 12;63(5):2292-2307..

[3]. POSSIBLE SIDE EFFECTS AND RISKS OF THE STUDY DRUG AND PROCEDURES.

[4].Nat Med. 2021 Jun;27(6):1079-1087.

The therapeutic success of peptidic GLP-1 receptor agonists for treatment of type 2 diabetes mellitus (T2DM) motivated our search for orally bioavailable small molecules that can activate the GLP-1 receptor (GLP-1R) as a well-validated target for T2DM. Here, the discovery and characterization of a potent and selective positive allosteric modulator (PAM) for GLP-1R based on a 3,4,5,6-tetrahydro-1H-1,5-epiminoazocino[4,5-b]indole scaffold is reported. Optimization of this series from HTS was supported by a GLP-1R ligand binding model. Biological in vitro testing revealed favorable ADME and pharmacological profiles for the best compound 19. Characterization by in vivo pharmacokinetic and pharmacological studies demonstrated that 19 activates GLP-1R as positive allosteric modulator (PAM) in the presence of the much less active endogenous degradation product GLP1(9-36)NH2 of the potent endogenous ligand GLP-1(7-36)NH2. While these data suggest the potential of small molecule GLP-1R PAMs for T2DM treatment, further optimization is still required towards a clinical candidate.[2]

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Agonism of the glucagon-like peptide-1 receptor (GLP-1R) results in glycemic lowering and body weight loss and is a therapeutic strategy to treat type 2 diabetes (T2D) and obesity. We developed danuglipron (PF-06882961), an oral small-molecule GLP-1R agonist and found it had comparable efficacy to injectable peptidic GLP-1R agonists in a humanized mouse model. We then completed a placebo-controlled, randomized, double-blind, multiple ascending-dose phase 1 study ( NCT03538743 ), in which we enrolled 98 patients with T2D on background metformin and randomized them to receive multiple ascending doses of danuglipron or placebo for 28 d, across eight cohorts. The primary outcomes were assessment of adverse events (AEs), safety laboratory tests, vital signs and 12-lead electrocardiograms. Most AEs were mild, with nausea, dyspepsia and vomiting most commonly reported. There were no clinically meaningful AEs in laboratory values across groups. Heart rate generally increased with danuglipron treatment at day 28, but no heart-rate AEs were reported. Systolic blood pressure was slightly decreased and changes in diastolic blood pressure were similar with danuglipron treatment at day 28, compared with placebo. There were no clinically meaningful electrocardiogram findings. In this study in T2D, danuglipron was generally well tolerated, with a safety profile consistent with the mechanism of action of GLP-1R agonism.[4]

C35H41FN6O7

676.7345

555.23

C, 62.12; H, 6.11; F, 2.81; N, 12.42; O, 16.55

2230198-03-3

2230198-02-2 (free acid);2230198-03-3 (tris);

154702463

Solid powder

5

13

12

49

995

1

C1CO[C@@H]1CN2C3=C(C=CC(=C3)C(=O)O)N=C2CN4CCC(CC4)C5=NC(=CC=C5)OCC6=C(C=C(C=C6)C#N)F.C(C(CO)(CO)N)O

JEJPAGACGZQFHN-JIDHJSLPSA-N

InChI=1S/C31H30FN5O4.C4H11NO3/c32-25-14-20(16-33)4-5-23(25)19-41-30-3-1-2-26(35-30)21-8-11-36(12-9-21)18-29-34-27-7-6-22(31(38)39)15-28(27)37(29)17-24-10-13-40-245-4(1-6,2-7)3-8/h1-7,14-15,21,24H,8-13,17-19H2,(H,38,39)6-8H,1-3,5H2/t24-/m0./s1

(S)-2-((4-(6-((4-cyano-2-fluorobenzyl)oxy)pyridin-2-yl)piperidin-1-yl)methyl)-1-(oxetan-2-ylmethyl)-1H-benzo[d]imidazole-6-carboxylic acid 2-amino-2-(hydroxymethyl)-1,3-propanediol

PF-06882961 tris Danuglipron PF 06882961 PF06882961 PF-06882961 Tris salt

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO: >10 mM

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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