Glucagon receptor (GCGR)

PF-06291874 is a potent and selective competitive antagonist of the human GCGR [equilibrium dissociation constant, 140 nM (70.5 ng/ml)]. Kinetic analysis using the quantitative Motulsky method 16 showed that PF-06291874 has a fast on and off rate. PF-06291874 is highly bound to human plasma protein, with a mean free fraction of ∼0.55%. [3]
PF-06291874 exposure has a half-life of roughly 19.7–22.7 hours and is roughly dose-proportional. The rate of PF-06291874 is quick on and off. PF-06291874 has a mean free fraction of approximately 0.55% and is strongly bound to human plasma protein[3].

Exposure to PF-06291874 is approximately dose-proportional, with a half-life of ∼19.7-22.7 hours. PF-06291874 has fast opening and closing rates. PF-06291874 is highly bound to human plasma proteins with an average free fraction of ∼0.55% [3].
PF-06291874 exposure was approximately dose-proportional with a half-life of ∼19.7-22.7 h. Day 14 fasting plasma glucose and mean daily glucose values were reduced from baseline in a dose-dependent manner, with placebo-corrected decreases of 34.3 and 42.4 mg/dl, respectively, at the 150 mg dose. After the MMTT, dose-dependent increases in glucagon and total glucagon-like peptide-1 (GLP-1) were observed, although no meaningful changes were noted in insulin, C-peptide or active GLP-1 levels. Small dose-dependent increases in LDL cholesterol were observed, along with reversible increases in serum aminotransferases that were largely within the laboratory reference range. An increase in circulating gluconeogenic amino acids was also observed on days 2 and 14. All dose levels of PF-06291874 were well tolerated. Conclusion: PF-06291874 was well tolerated, has a pharmacokinetic profile suitable for once-daily dosing, and results in reductions in glucose with minimal risk of hypoglycaemia.

MDpocket Detects Pockets in MD Trajectories [1]
MDpocket is an open-source tool used to detect potential binding pockets in MD simulation trajectories. Before using MDpocket, it is necessary to extract a PDB file every 100 ps from the processed MD trajectory using GROMACS 2020. PDB structure ensembles were then detected by MDpocket to output pockets present throughout trajectories, which are able to be observed by the visualization software PyMOL (http://www.pymol.org). This analysis was conducted along five trajectories of the GCGR/glucagon system. MDpocket also allows us to calculate the volume of the selected pockets.

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Molecular Docking [1]
To investigate the interaction modes between known active molecules and GCGR, flexible docking was performed using the AutoDock Tools package. The receptor and ligand were optimized to generate the corresponding lower energy 3D conformation and the corresponding ionization state (pH 7.0), respectively. The binding sites found from the dynamic conformation and crystal structure were used as the docking grid center, and the residues around the pocket were set to be flipped. The prepared compounds were then docked to the GCGR pockets and the first 20 conformations of each ligand were exported. The most appropriate binding conformations were selected based on the interaction energy and visual inspection. All results were analyzed and visualized using PyMOL (http://www.pymol.org). To simulate a more realistic physiological environment, POPC phospholipid molecules were added around Pocket 2, Pocket 4, and Pocket 5.

This randomized, double-blind, placebo-controlled, multiple dose-escalating study of oral PF-06291874 (ClinicalTrials.gov: NCT01856595) was conducted at three clinical research centres in the USA. Part A was conducted in cohorts of patients receiving either 5, 15, 50, 100 or 150 mg of PF-06291874 or placebo once-daily; and Part B, in cohorts of patients receiving either 15 or 30 mg of PF-06291874 or placebo once-daily. Patients in the 5-, 15-, 50- and 150-mg cohorts in Part A, and the 15-mg cohort in Part B received PF-06291874 for 14 days in an inpatient setting. Patients in the 100-mg cohort (Part A) and the 30-mg cohort (Part B) received PF-06291874 for 28 days (14 days inpatient; 14 days outpatient). The daily dose of 150 mg in Part A of the study was expected to achieve plasma concentrations resulting in mean receptor antagonism of 90% over the course of the day, based on a functional in vitro binding constant in cells expressing the human glucagon receptor. The 30-mg dose was selected for Part B after the assessment of safety and glucose-lowering data from the 15-mg cohort in Part B. PF-06291874 was administered daily before breakfast. Standardized meals were provided while in the clinical research unit. A mixed-meal tolerance test (MMTT; 700-kcal Ensure Plus®) was given in place of breakfast on days −1, 14 and 28 (in applicable cohorts) to assess attenuation by PF-06291874 of glucose excursion, and to measure other biomarkers after controlled intake of nutrients and energy. Doses of PF-06291874 or placebo were administered on days 14 and 28 immediately before the MMTT. Escalation to the next dose level in both parts occurred in the absence of dose-limiting toxicity or other significant safety findings.[3]

Pharmacokinetic Results [3]
All patients who received PF-06291874, except two (one with insufficient samples collected owing to study discontinuation on day 14, and one dosing error in which the patient received PF-06291874 instead of placebo for ∼5 days), were included in the pharmacokinetic analysis. PF-06291874 plasma exposure (Cmax and AUCτ (area under the concentration-time profile from time zero to time tau (τ)) increased proportionally throughout the dose range studied (Table S1). Dose-normalized pharmacokinetic variables were similar between patients receiving PF-06291874 on a background of metformin, or metformin and sulphonylurea. Accumulation ratios of plasma exposure after 14 days of dosing ranged from 1.5- to 2-fold relative to day 1; and steady-state concentrations were reached within 7 days of first dose. The median time to maximum drug concentration ranged from 4 to 6 h; and the mean apparent terminal elimination t½ on day 14 ranged from 19.7 to 22.7 h and was independent of dose. Urinary recovery of unchanged PF-06291874 was minimal (<0.3% of the administered dose when quantifiable).

Safety and Tolerability [3]
PF-06291874 was generally well tolerated; there were no deaths, severe AEs, permanent discontinuations, dose reductions because of treatment-emergent AEs, or clinically significant variations in vital signs (Table S5) or ECG variables. There was one temporary discontinuation as a result of chromatopsia (the patient discontinued on day 8 and resumed PF-06291874, 30 mg, on day 11), which was considered mild in severity by the investigator. In patients on background metformin, the incidence of hypoglycaemia (fasting blood glucose ≤70 mg/dl) was approximately the same across all doses of PF-06291874 and similar to that in the placebo group. In patients on background metformin and sulphonylurea, the incidence was similar in subjects receiving placebo and 15 mg PF-06291874, but higher in patients receiving 30 mg PF-06291874. None of the hypoglycaemia AEs were considered severe and recovery was rapid. With the exception of hypoglycaemia AEs, there was no dose-related increase in AE frequency. The most commonly reported AEs were diarrhoea, nausea, upper respiratory infection and headache. All were judged to be mild by the investigator. After 14 days of dosing, there appeared to be a dose-dependent increase in mean fasting LDL cholesterol values. Patients who received the 150-mg dose had a mean increase from baseline of ∼10% (∼20% compared with placebo-treated subjects; Figure 2). An increase of similar magnitude in total cholesterol was observed; however, changes in HDL cholesterol, triglycerides and apolipoprotein B100 were more variable (Table S6). Small, dose-related, reversible increases in mean alanine aminotransferase (ALT) and aspartate aminotransferase (AST) values were observed (Figure 3) within the first few days of dosing, remaining constant or decreasing slightly over 14/28 days of dosing. The majority of observed aminotransferase increases remained within the laboratory reference range and returned to baseline values after discontinuation of dosing. None of these changes was associated with an increase in bilirubin. Three of the 86 patients who received PF-06291874 had ALT or AST elevations >3× ULN. One patient who received 100 mg on background metformin had three consecutive ALT values of >3× ULN (peak value 105 IU/l) beginning 7 days after the start of dosing. The ALT values returned to approximate baseline values by day 21, while the subject continued to receive PF-06291874. A second subject with an ALT value of >3× ULN had received 30 mg in combination with metformin/sulphonylurea. After 28 days of dosing, the subject's ALT value was 81 IU/l (baseline 49 IU/l). At the follow-up visit, 8 days after the last dose, the subject's ALT value remained elevated at 87 IU/l. A third subject who received 15 mg in combination with metformin/sulphonylurea had a single AST value >3× ULN (105 IU/l) on day 14. This value represented a slight increase from the baseline value of 87 IU/l. A repeat assessment was performed on day 17 (3 days after the final dose), at which time the AST value was 52 IU/l.

[1]. Identification of a novel conformationally constrained glucagon receptor antagonist. Bioorg Med Chem Lett, 2014 Feb 1, 24(3):839-44.

[2]. Glucagon receptor as a drug target: A witches' brew of eye of newt (peptides) and toe of frog (receptors). Diabetes Obes Metab. 2018 Feb;20(2):233-237.

[3]. Effects of multiple ascending doses of the glucagon receptor antagonist PF-06291874 in patients with type 2 diabetes mellitus. Diabetes Obes Metab. 2016 Aug;18(8):795-802.

PF-06291874 is under investigation in clinical trial NCT02175121 (Safety, Tolerability, Pharmacokinetics And Pharmacodynamics Study of PF-06291874 as Oral Monotherapy To Treat Adults With Type 2 Diabetes Mellitus).

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Consistent with studies in healthy subjects, plasma concentrations of PF-06291874 increased in a dose-proportional manner across the studied range and the pharmacokinetic profile supports once-daily dosing. PF-06291874 was well tolerated; the maximum tolerated dose was not identified. Although this study was not optimally designed to establish the effects of PF-06291874 on plasma lipids, there was an ∼20% (placebo-corrected) increase in plasma LDL cholesterol relative to baseline at the highest dose tested. A trend for increased plasma LDL cholesterol was also identified in the MK-0893 12-week study and in the LY2409021 28-day study, indicating that this could be a mechanism-related effect. Indeed, results from investigation of MK-0893 in a preclinical mouse model, coupled with archived clinical samples from the 12-week study of MK-0893, suggest that glucagon receptor antagonists may increase LDL cholesterol by increasing cholesterol absorption from the gut. Data from the present study, not included in this manuscript, showed that there was no change in mevalonic acid concentrations, suggesting glucagon receptor antagonism has no effect on cholesterol biosynthesis.
Similar to other glucagon receptor antagonists, small dose-related increases in ALT and AST were observed in patients treated with PF-06291874. These changes were not associated with any changes in bilirubin or alkaline phosphatase. The mechanism underlying these increases is not understood, although it did not appear to be associated with elevated plasma alanine concentrations; there were no obvious other reasons (alcohol use, concomitant medications or infections) for these observed increases. Further work will be necessary to determine whether increases in AST and ALT are a physiological adaptation to blocking glucagon signalling.
In summary, once-daily dosing with the glucagon receptor antagonist, PF-06291874, elicited reductions in fasting and postprandial glucose with a low risk of hypoglycaemia in patients on background therapy of either metformin alone, or in combination with a sulphonylurea. Mild, reversible increases in serum aminotransferase levels were observed, along with mild increases in LDL cholesterol. Administration of PF-06241874 for up to 4 weeks was generally well tolerated, supporting its suitability for continued clinical development in longer-term studies.[3]

C26H28F3N3O4

503.5134

503.203

C, 62.02; H, 5.61; F, 11.32; N, 8.35; O, 12.71

1393124-08-7

60151939

White to off-white solid powder

1.3±0.1 g/cm3

675.0±55.0 °C at 760 mmHg

362.0±31.5 °C

0.0±2.2 mmHg at 25°C

1.563

5.44

2

8

10

36

718

1

FC(C1C([H])=NN(C=1[H])C1C(C([H])([H])[H])=C([H])C(=C([H])C=1C([H])([H])[H])O[C@]([H])(C1C([H])=C([H])C(C(N([H])C([H])([H])C([H])([H])C(=O)O[H])=O)=C([H])C=1[H])C([H])([H])C([H])([H])C([H])([H])[H])(F)F

IBDYYOQKQCCSDP-QFIPXVFZSA-N

InChI=1S/C26H28F3N3O4/c1-4-5-22(18-6-8-19(9-7-18)25(35)30-11-10-23(33)34)36-21-12-16(2)24(17(3)13-21)32-15-20(14-31-32)26(27,28)29/h6-9,12-15,22H,4-5,10-11H2,1-3H3,(H,30,35)(H,33,34)/t22-/m0/s1

3-[[4-[(1S)-1-[3,5-dimethyl-4-[4-(trifluoromethyl)pyrazol-1-yl]phenoxy]butyl]benzoyl]amino]propanoic acid

PF6291874; PF-06291874; 1393124-08-7; glucagon receptor antagonists-4; PF-06291874; CGY4I8F278; (S)-3-(4-(1-(3,5-dimethyl-4-(4-(trifluoromethyl)-1H-pyrazol-1-yl)phenoxy)butyl)benzamido)propanoic acid; UNII-CGY4I8F278; CHEMBL2381848; .BETA.-ALANINE, N-(4-((1S)-1-(3,5-DIMETHYL-4-(4-(TRIFLUOROMETHYL)-1H-PYRAZOL-1-YL)PHENOXY)BUTYL)BENZOYL)-; PF-6291874; PF 6291874; PF 06291874; PF06291874

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: ~100 mg/mL (~198.6 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.13 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 20.8 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: ≥ 2.08 mg/mL (4.13 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 20.8 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: ≥ 2.08 mg/mL (4.13 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 20.8 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.)