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]
Except for two patients (one patient discontinued the study on day 14, resulting in insufficient sample collection, and another patient received PF-06291874 instead of placebo for approximately 5 days due to an incorrect dosing), all patients treated with PF-06291874 were included in the pharmacokinetic analysis. Plasma exposures (Cmax and AUCτ, i.e., area under the concentration-time curve, from time zero to time τ) of PF-06291874 increased proportionally within the studied dose range (Table S1). Dose-normalized pharmacokinetic parameters were similar in patients receiving PF-06291874 in patients taking metformin or metformin in combination with sulfonylureas. The cumulative fold of plasma exposures after 14 days of administration was 1.5 to 2 times that of day 1; steady-state concentrations were reached within 7 days of the first administration. The median time to reach maximum drug concentration was 4 to 6 hours; the mean apparent terminal elimination half-life on day 14 was 19.7 to 22.7 hours, and was dose-independent. The recovery of unmetabolized PF-06291874 in urine was extremely low (less than 0.3% of the administered dose when quantifiable).

Safety and Tolerability [3] PF-06291874 was generally well tolerated; no deaths, serious adverse events, permanent discontinuation, dose reduction due to adverse events during treatment, or clinically significant changes in vital signs (Table S5) or electrocardiogram variables occurred. One patient temporarily discontinued the drug due to color vision impairment (the patient discontinued the drug on day 8 and resumed taking PF-06291874 at a dose of 30 mg on day 11), which the researchers considered to be of mild severity. In patients taking metformin, the incidence of hypoglycemia (fasting blood glucose ≤70 mg/dl) with PF-06291874 was roughly the same across all dose groups and similar to that in the placebo group. In patients taking metformin and sulfonylureas, the incidence of hypoglycemia was similar in the placebo group and the 15 mg PF-06291874 group, but the incidence was higher in the 30 mg PF-06291874 group. All hypoglycemic adverse events were mild and resolved rapidly. No dose-related increase in the frequency of other adverse events was observed except for hypoglycemic events. The most common adverse events were diarrhea, nausea, upper respiratory tract infection, and headache. The investigators considered all adverse events to be mild. After 14 days of administration, mean fasting LDL cholesterol appeared to increase in a dose-dependent manner. Patients receiving a 150 mg dose showed an approximately 10% increase in mean fasting LDL cholesterol from baseline (approximately 20% increase compared to the placebo group; Figure 2). A similar increase in total cholesterol was observed. However, changes in HDL cholesterol, triglycerides, and apolipoprotein B100 were more variable (Table S6). In the initial days after administration, a slight, reversible dose-related increase in mean alanine aminotransferase (ALT) and aspartate aminotransferase (AST) values was observed (Figure 3), which remained stable or slightly decreased over 14 days of the 28-day administration period. Most observed aminotransferase increases were within laboratory reference ranges and returned to baseline values upon discontinuation of the drug. These changes were not related to elevated bilirubin. Among the 86 patients treated with PF-06291874, 3 had ALT or AST levels exceeding three times the upper limit of normal (ULN). One patient receiving 100 mg PF-06291874 in addition to metformin had three consecutive ALT levels exceeding three times the ULN (peak 105 IU/L) seven days after starting treatment. On day 21, the subject's ALT level returned to near baseline, and treatment with PF-06291874 continued. Another subject with an ALT level exceeding three times the ULN received 30 mg PF-06291874 in combination with metformin/sulfonylureas. After 28 days of treatment, this subject's ALT level was 81 IU/L (baseline 49 IU/L). At the 8-day follow-up after the last dose, the subject's ALT level remained elevated at 87 IU/L. The third subject received 15 mg PF-06291874 in combination with metformin/sulfonylurea. On day 14, their AST level was 3 times higher than the upper limit of normal (105 IU/L). This value was slightly higher than the baseline of 87 IU/L. A repeat assessment was performed on day 17 (3 days after the last dose), at which time the AST level 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 being investigated in the clinical trial NCT02175121 (Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics Study of PF-06291874 as Oral Monotherapy for Adults with Type 2 Diabetes). Consistent with results from studies in healthy subjects, plasma concentrations of PF-06291874 increased dose-proportionally within the study dose range, and its pharmacokinetic profile supports once-daily dosing. PF-06291874 was well tolerated; however, the maximum tolerated dose has not been determined. Although this study was not designed to fully assess the effects of PF-06291874 on lipids, at the highest test dose, plasma low-density lipoprotein cholesterol (LDL-C) increased by approximately 20% from baseline (placebo adjusted). A trend toward increased plasma LDL-C levels was observed in both the 12-week study (MK-0893) and the 28-day study (LY2409021), suggesting this may be a mechanism-related effect. In fact, results from preclinical mouse models of MK-0893, combined with archived clinical samples from the MK-0893 12-week study, suggest that glucagon receptor antagonists may increase LDL-C levels by increasing intestinal cholesterol uptake. Data from this study (not included in this article) showed no change in mevalonate concentration, indicating that glucagon receptor antagonism has no effect on cholesterol biosynthesis. Similar to other glucagon receptor antagonists, dose-related slight increases in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were observed in patients treated with PF-06291874. These changes were unrelated to any changes in bilirubin or alkaline phosphatase. The mechanism behind these increases is unclear, although it does not appear to be related to elevated plasma alanine concentrations; no other obvious cause (alcohol consumption, concomitant medication, or infection) could explain these observed increases. Further research is needed to determine whether the increases in AST and ALT are a physiological adaptation to blockade of glucagon signaling. In summary, once-daily administration of the glucagon receptor antagonist PF-06291874 reduced fasting and postprandial blood glucose levels, and the risk of hypoglycemia was low in patients receiving metformin monotherapy or metformin in combination with sulfonylureas. Mild, reversible elevations in serum transaminase levels and mild elevations in low-density lipoprotein cholesterol were observed. PF-06241874 was generally well-tolerated over a period of up to 4 weeks, supporting its continued clinical development in long-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.)