GLP-1 receptor
PF-06882961 Tris targets the glucagon-like peptide-1 receptor (GLP-1R). GLP-1R is a G protein-coupled receptor that is activated by the incretin hormone GLP-1. GLP-1 is released from intestinal L-cells in response to food intake and stimulates insulin secretion from pancreatic β-cells, inhibits glucagon secretion, slows gastric emptying, and promotes satiety. GLP-1R is a well-validated target for the treatment of type 2 diabetes and obesity. PF-06882961 Tris is a positive allosteric modulator (PAM) of GLP-1R, meaning it binds to a site on the receptor that is distinct from the orthosteric binding site of GLP-1 and enhances the receptor's response to GLP-1. This allosteric modulation allows for oral bioavailability, which is a significant advantage over injectable peptide-based GLP-1R agonists.

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].
In vitro, PF-06882961 Tris has been characterized as a potent and selective positive allosteric modulator (PAM) of GLP-1R. It was identified through optimization of a series based on a 3,4,5,6-tetrahydro-1H-1,5-epiminoazocino[4,5-b]indole scaffold, supported by a GLP-1R ligand binding model. Biological in vitro testing revealed favorable ADME and pharmacological profiles. The compound activates GLP-1R in the presence of the much less active endogenous degradation product GLP-1(9-36)NH2. This suggests that the compound can enhance the activity of even low levels of GLP-1 or its metabolites, which may contribute to its efficacy. The compound's favorable in vitro profile supported its advancement to in vivo studies.

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].
In vivo, PF-06882961 Tris has demonstrated efficacy in a humanized mouse model, showing comparable efficacy to injectable peptidic GLP-1R agonists. A Phase 1 clinical trial (NCT03538743) was completed, in which 98 patients with type 2 diabetes on background metformin were randomized to receive multiple ascending doses of danuglipron or placebo for 28 days. 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 being the 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. These results indicate that PF-06882961 Tris is well-tolerated and has a favorable safety profile in humans. The compound's efficacy and safety support its continued development as an oral therapy for type 2 diabetes and obesity.

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.

---

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.

---

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.
In vitro assays for PF-06882961 Tris typically involve measuring its ability to activate GLP-1R. This is often done using a cell-based reporter assay. Cells expressing GLP-1R and a luciferase reporter gene under the control of a cAMP-responsive element (CRE) are used. The cells are treated with various concentrations of PF-06882961 Tris, with or without a sub-maximal concentration of GLP-1. The activation of GLP-1R leads to an increase in intracellular cAMP, which in turn activates the CRE-luciferase reporter. The luciferase activity is measured, and the EC₅₀ for potentiation is determined. Binding assays can also be performed to measure the compound's affinity for GLP-1R. These assays are typically performed using radiolabeled GLP-1 or by using surface plasmon resonance (SPR) with immobilized GLP-1R.

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.
For in vitro cellular assays, the effect of PF-06882961 Tris on GLP-1R activation is typically assessed using cell lines expressing GLP-1R, such as HEK293 cells stably transfected with GLP-1R and a CRE-luciferase reporter. Cells are seeded in 96-well plates and treated with various concentrations of PF-06882961 Tris, with or without a sub-maximal concentration of GLP-1 (e.g., 10 nM). After incubation for 4-6 hours, the cells are lysed, and the luciferase activity is measured. The EC₅₀ for potentiation is determined from the dose-response curve. The compound's selectivity for GLP-1R over other receptors is assessed by testing its activity against a panel of related receptors. The compound's effects on cAMP production can also be measured directly using a cAMP ELISA or a homogeneous time-resolved fluorescence (HTRF) assay.

male cynomolgus monkeys
1 mg/kg, 5 mg/kg, 100 mg/kg
IV, Oral gavage
In vivo efficacy of PF-06882961 Tris was evaluated in a humanized mouse model. In such a model, mice are transgenic for human GLP-1R. The mice are treated with PF-06882961 Tris orally at various doses, and blood glucose levels are measured. The compound's ability to lower blood glucose is assessed. Body weight is also monitored. The efficacy of the compound is compared to that of injectable peptide GLP-1R agonists. The compound was also evaluated in a Phase 1 clinical trial in humans. In this trial, patients with type 2 diabetes on background metformin were randomized to receive multiple ascending doses of danuglipron or placebo for 28 days. Various safety and efficacy parameters were measured, including adverse events, vital signs, laboratory tests, and electrocardiograms. These studies provided critical data on the compound's in vivo efficacy and safety.

Pharmacokinetic properties for PF-06882961 Tris indicate that it is orally bioavailable. The compound is absorbed after oral administration and reaches systemic circulation. In the Phase 1 clinical trial, the compound was administered orally. The compound's half-life, volume of distribution, and clearance are not detailed in the available text. However, the compound's oral bioavailability is a key feature that distinguishes it from injectable peptide GLP-1R agonists. The compound's metabolism is likely to involve hepatic cytochrome P450 enzymes. The compound's elimination route is likely renal and biliary. Further details on the compound's pharmacokinetic profile can be found in the clinical trial publications.

Toxicological data for PF-06882961 Tris indicates that it is generally well-tolerated. In the Phase 1 clinical trial, most adverse events were mild, with nausea, dyspepsia, and vomiting being the most commonly reported. There were no clinically meaningful adverse events in laboratory values across groups. Heart rate generally increased with danuglipron treatment at day 28, but no heart-rate adverse events were reported. Systolic blood pressure was slightly decreased. In rat studies, the compound showed mild to moderate damage to the heart, moderate to severe effects on the thymus gland, and mild to moderate stomach ulcers at the highest dose level. These effects are consistent with the pharmacology of GLP-1R agonists and may be dose-limiting. Further toxicology studies are needed to fully characterize the safety profile of the compound.

[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 successful application of peptide GLP-1 receptor agonists in the treatment of type 2 diabetes mellitus (T2DM) has prompted the search for small molecules with high oral bioavailability capable of activating the GLP-1 receptor (GLP-1R), a well-established target for T2DM. This paper reports the discovery and characterization of a highly efficient and selective GLP-1R positive allosteric modulator (PAM) based on the 3,4,5,6-tetrahydro-1H-1,5-epiminozacyclooctano[4,5-b]indole skeleton. The performance optimization of this series of compounds, after high-throughput screening (HTS), was supported by a GLP-1R ligand binding model. In vitro biological assays showed that the optimal compound 19 possessed favorable adsorption-reduction (ADME) and pharmacological properties. In vivo pharmacokinetic and pharmacological studies have shown that compound 19 can activate GLP-1 receptors as a positive allosteric modulator (PAM) in the presence of the much less active endogenous degradation product GLP1(9-36)NH2 (a product of the potent endogenous ligand GLP-1(7-36)NH2). Although these data suggest that small molecule GLP-1R PAMs have potential in the treatment of type 2 diabetes (T2DM), further optimization is needed before they can become clinical candidates. [2] Activation of glucagon-like peptide-1 receptors (GLP-1R) can lower blood glucose and reduce weight, which is a treatment strategy for type 2 diabetes (T2D) and obesity. We developed an oral small molecule GLP-1 receptor agonist, danuglipron (PF-06882961), and found that its efficacy in humanized mouse models was comparable to that of injectable peptide GLP-1 receptor agonists. Subsequently, we completed a placebo-controlled, randomized, double-blind, multi-dose escalation phase I clinical trial (NCT03538743). The study enrolled 98 patients with type 2 diabetes who were receiving metformin and randomized them into eight cohorts to receive either multi-dose escalation danuglipron or placebo for 28 days. The primary endpoint included adverse event (AE) assessment, safety laboratory tests, vital signs, and 12-lead electrocardiogram. Most adverse events were mild, with the most common being nausea, dyspepsia, and vomiting. No clinically significant adverse events were observed in any of the laboratory tests. On day 28, heart rate was generally elevated in the dapagliflozin group, but no heart rate-related adverse events were reported. Systolic blood pressure was slightly lower in the dapagliflozin group compared to the placebo group, while diastolic blood pressure changes were similar to those in the placebo group. No clinically significant electrocardiographic abnormalities were found. In this study of patients with type 2 diabetes, dapagliflozin was generally well tolerated, and its safety profile was consistent with the mechanism of action of GLP-1 receptor agonists. [4]
PF-06882961 Tris is an orally bioavailable, small-molecule, allosteric agonist of GLP-1R being developed for type 2 diabetes and obesity. It has completed Phase 1 clinical trials. It is not yet approved for clinical use.

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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