GLP-1 receptor

PF-06882961 promotes the accumulation of cAMP in CHO cells expressing the GLP-1Rs from both humans and monkeys with comparable EC50 values. On the other hand, PF-06882961 does not raise cAMP levels in GLP-1R-expressing mouse, rat, or rabbit cells.[2]

PF-06882961 decreases food intake and increases insulin release in response to glucose stimulation in monkeys.[2]

Danuglipron Is Orally Bioavailable and Efficacious in Decreasing the Level of Glucose and Food Intake in Monkeys. Following iv administration, Danuglipron demonstrated moderate to high plasma clearance (CLp) values in rats (CLp = 57.3 mL/min/kg) and monkeys [CLp = 13.8 mL/min/kg] with relatively short elimination half-lives of 1.1 and 1.9 h in rats and monkeys, respectively. The oral bioavailability of a Tris salt form of danuglipron in animals was low to moderate and increased in a dose-dependent manner, which was adequate for studying the preclinical in vivo efficacy and safety of Danuglipron delivered via oral gavage in a standard 0.5% methyl cellulose formulation containing 2% Tween 80.[3]

Because danuglipron does not activate the rodent GLP-1R, the therapeutic effects of danuglipron on insulin and glucose were examined in an intravenous glucose tolerance test (IVGTT) in cynomolgus monkeys following iv infusion and po administration. Infusion rates and po doses were projected using systemic concentrations from monkey pharmacokinetic (PK) studies that were required to achieve receptor occupancies (RO) bracketing the RO estimated from the reported human plasma exposures for liraglutide at its clinically efficacious dose (1.8 mg once daily). The iv infusion of danuglipron during the IVGTT led to an increase in the rate of insulin secretion and the rate of glucose disappearance (K value) (Figure ​Figure88B–D). Enhancement of glucose-stimulated insulin secretion by danuglipron was concentration-dependent and comparable following the po dosing and iv infusion routes (Figure ​Figure88E). Once-daily subcutaneous administration of danuglipron for 2 days also inhibited food intake compared with that of vehicle-treated monkeys (Figure ​Figure88F). The subcutaneous route of administration was chosen to reduce variability in systemic exposure noted upon po administration and was more convenient than iv administration.[3]

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

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Animal Pharmacokinetic Studies[3]
Jugular vein/carotid artery doubly cannulated male Wistar-Han rats (∼250 g), and male cynomolgus monkeys (∼7 kg) were used for these studies. Animals were fasted overnight and through the duration of the study (1.0 or 2.0 h), whereas access to water was provided ad libitum. Danuglipron was administered by slow iv bolus as a solution (1 mg/mL) in a 5:95 (v/v) polyethylene glycol 400/12% (w/v) sulfobutyl-β-cyclodextrin in water mixture or a 10:50:40 (v/v/v) DMSO/polyethylene glycol 400/water mixture via the tail vein in rats (n = 4) or the femoral vein in monkeys (n = 2) at a dose of 1.0 mg/kg in a dosing volume of 1 mL/kg. Serial blood samples were collected before dosing and 0.083, 0.25, 0.5, 1.0, 2.0, 4.0, 7.0, and 24 h after dose administration. The crystalline 2-amino-2-hydroxymethylpropane-1,3-diol (Tris) salt form of Danuglipron was also administered by oral gavage to rats (5 and 100 mg/kg at 10 mL/kg) and monkeys [5 mg/kg (5.0 mL/kg) and 100 mg/kg (10 mL/kg)] as a homogeneous suspension in a 2:98 (v/v) Tween 80/0.5% (w/v) methylcellulose A4M in distilled water mixutre. Blood samples were taken prior to po administration, and then serial samples were collected 0.083, 0.25, 0.5, 1, 2, 4, 7, and 24 h after dosing. Blood samples from the pharmacokinetic studies were centrifuged to generate plasma. All plasma samples were kept frozen until analysis. For rat and monkey samples, aliquots of plasma (20–50 μL) were transferred to 96-well blocks, and acetonitrile (150–200 μL) containing verapamil (monkeys) or terfenadine (rat) as internal standard was added to each well. The supernatant was dried under nitrogen and reconstituted with 100 μL of water without evaporation. Following extraction, the samples were then analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS), and concentrations of Danuglipron in plasma were determined by interpolation from a standard curve as described in the Supporting Information.

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Determination of Preclinical Pharmacokinetic Parameters[3]
Pharmacokinetic parameters in animals were determined using noncompartmental analysis. Maximum plasma concentrations (Cmax) of Danuglipron in plasma after po dosing in rats and monkeys were determined directly from the experimental data, with Tmax defined as the time of first occurrence of Cmax. The area under the plasma concentration–time curve from time zero to infinity (AUC0–∞) was estimated using the linear trapezoidal rule. Systemic plasma clearance (CLp) was calculated as the intravenous dose divided by AUC0–∞iv. The terminal rate constant (kel) was calculated by a linear regression of the log-linear concentration–time curve, and the terminal elimination t1/2 was calculated as 0.693/kel. The apparent steady-state distribution volume (Vdss) in animals was determined as the iv or po dose divided by the product of AUC0–∞ and kel. The absolute bioavailability (F) of the po doses in animals was calculated using the equation F = AUC0–∞po/AUC0–∞iv × doseiv/dosepo. Detailed protocols used in animal pharmacology studies are provided in the Supplementary Methods.

[1]. Endocrinol Metab (Seoul). 2021 Feb;36(1):22-29

[2]. bioRxiv. 2020 Sep 30.

[3]. J Med Chem . 2022 Jun 23;65(12):8208-8226.

[4]. J Med Chem. 2020 Mar 12;63(5):2292-2307.

PF-06882961 is under investigation in clinical trial NCT03985293 (A 16 Week Study to Evaluate the Efficacy and Safety of PF-06882961 in Adults With Type 2 Diabetes Mellitus).

C31H30FN5O4

555.599410533905

555.23

C, 67.01; H, 5.44; F, 3.42; N, 12.61; O, 11.52

2230198-02-2

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

134611040

Off-white to light yellow solid powder

1.4

1

9

9

41

941

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

HYBAKUMPISVZQP-DEOSSOPVSA-N

InChI=1S/C31H30FN5O4/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)17-24-10-13-40-24/h1-7,14-15,21,24H,8-13,17-19H2,(H,38,39)/t24-/m0/s1

2-[[4-[6-[(4-cyano-2-fluorophenyl)methoxy]pyridin-2-yl]piperidin-1-yl]methyl]-3-[[(2S)-oxetan-2-yl]methyl]benzimidazole-5-carboxylic acid

PF06882961; Danuglipron; PF 06882961; PF-06882961

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)

Ethanol: ~100 mg/mL
DMSO: ~10 mg/mL (~18 mM)

5%DMSO + 40%PEG300 + 5%Tween 80: 5.0mg/ml (9.00mM) (Please use freshly prepared in vivo formulations for optimal results.)