EP2 ( IC50 = 16 nM )

>PF-04418948 (10 mg/kg, p.o.) in rats decreases AUC0–60 by 61% and the mean cutaneous blood flow peak response by 41%, respectively.[1]

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In dog bronchiole and mouse trachea, PF-04418948 produced parallel rightward shifts of the PGE(2)-induced relaxation curve with a K(B) of 2.5 nM and an apparent K(B) of 1.3 nM respectively. Reversal of the PGE(2)-induced relaxation in the mouse trachea by PF-04418948 produced an IC(50) value of 2.7 nM. Given orally, PF-04418948 attenuated the butaprost-induced cutaneous blood flow response in rats. PF-04418948 was selective for EP(2) receptors over homologous and unrelated receptors, enzymes and channels. Conclusions and implications: PF-04418948 is an orally active, potent and selective surmountable EP(2) receptor antagonist that should aid further elaboration of EP(2) receptor function.[1]

Selectivity and specificity [1]
Characterization of EP1 and EP3 receptor activity and broad spectrum specificity screening of PF-04418948 was carried out by Cerep SA (http://www.cerep.com). For these as well as other selectivity data cited in Table 1, they represent n = 1 or n = 2 as it is the practice within Pfizer not to generate large numbers of replicate data for compounds, where there is a greater than 100-fold difference with the primary potency. Furthermore, PF-04419848 emerged from an extensive screening campaign for which many more than one example was screened for selectivity, and in all cases this class of compounds were without significant activity at other prostanoid receptors. The IC50 values, EC50 values and Hill coefficients (nH) were determined by non-linear regression analysis of the concentration–response curves using Hill equation curve fitting. In each experiment, the respective reference compound was tested concurrently with PF-04418948 in order to assess the assay suitability and the data were compared with historical values determined at Cerep SA.

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Binding assays [1]
Binding selectivity assays for FP, BLT1 (LTB4 receptor) and TP receptors were performed at Cerep SA. Human embryonic kidneys expressing the human IP receptor were generated by Pfizer. Briefly, cells were grown in DMEM, 10% FBS, 600 µg·mL−1 Geneticin, 2 mM L-glutamine and 1 mM sodium pyruvate and harvested in 10 mM HEPES, 1 mM EDTA pH 7.4 at 4°C. Membranes were prepared by Dounce homogenization in 10 mM HEPES, 1 mM EDTA (pH 7.4, 4°C). Membrane aliquots were stored at −80°C before use and diluted in assay buffer to 62.5 µg·mL−1, to give a final assay concentration of 10 µg per well. Compounds were tested at 10 µM [0.1% DMSO in assay buffer (10 mM HEPES, 10 mM MgCl2·6H2O, 1 mM EDTA, pH 7.4)] as the highest assay concentration. Assay buffer containing 0.5% (w/v) polyethyleneimine (50 µL per well) was added to Packard GF/B filter plates, 20 µL of test compound or 20 µL assay buffer/1% DMSO (totals), or 20 µL of carbacyclin (10 µM in 0.1% (v/v) DMSO, non-specific binding) added manually. [3H] Iloprost (20 µL of 100 nM stock) and finally 160 µL of 62.5 µg·mL−1 membrane were added to start the binding reaction. The plates were centrifuged briefly to bring all the contents to the bottom of the wells (30 s, 100×g), covered and incubated at room temperature for 2 h with shaking. The binding assay was stopped rapidly by filtering through GF/B Unifilter plates using the Brandell harvester. The filters were washed with 3 × 1 mL 4°C assay buffer and then dried at 45°C for approximately 40 min in a drying oven. The bottom of the filter plates was sealed and Microscint ‘0’ (50 µL per well) added and incubated for at least 30 min before reading on an NXT Topcount.

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KB methodology [1]
PGE2 was serially diluted in buffer (PBS/0.05% (v/v) Pluronic F-127). PF-04418948 was prepared in diluent (PBS/0.05% (v/v) Pluronic F-127/2.5% DMSO) to 300, 100, 30 and 10 nM. For each concentration of PF-04418948 tested, 5 µL was added to the assay plate in duplicate. Diluent (5 µL) was added to all agonist curves and zero percentage effect control wells. Cells were prepared as described in the general cAMP assay protocol. Cell suspension (5 µL) was added to all wells of the assay plate. The plate was centrifuged at 400×g (1 min) in a benchtop centrifuge prior to incubation for 30 min in a humidified 37°C 5% (v/v) CO2 incubator. Then 5 µL of the agonist concentration–response curve were added manually. The plate was centrifuged at 400×g (1 min), before incubation for 90 min in a humidified 37°C 5% (v/v) CO2 incubator. The plate was placed at −80°C to lyse the cells and the DiscoveRx assay performed as above.

Functional cAMP assays in recombinant CHO cells [1]
Cells were suspended in DMEM at 1 × 106 cells·mL−1. Stocks of PGE2, BW245C and PF-04418948 (4 mM) were prepared in 100% DMSO and diluted in compound buffer (PBS; 2.5% (v/v) DMSO, 0.15% (v/v) pluronic F-127) to 2 µM final assay concentration. PGE2 was further diluted to 5 nM in PBS. PF-04418948 stock was serially diluted in 100% DMSO. Complete inhibition was determined by 10 µM of a relevant standard (data not shown). Each independent experiment was run on a separate day with freshly processed compounds. Compounds were diluted 1:40 in compound buffer, and 5 µL transferred to a Lumitrac 200 white plate. Prepared cells (5 µL) were added and incubated at 37°C for 30 min. Agonist (5 µL) was added and incubated for 90 min at 37°C. The plates were then placed at −80°C to lyse the cells. Plates were thawed at 37°C for 15 min. Pre-warmed DiscoveRx assay reagents were added according to the manufacturer's protocol. Plates were incubated in the dark for 4–20 h and read on a LJL Analyst plate reader with visible light. The agonist assay protocol was as the antagonist protocol, except that 5 µL compound buffer was used instead of the agonist stimulation. The CRTH2 receptor antagonist assay was carried out in the DiscoveRx platform as described above, but cells were stimulated with 8 µM forskolin.

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PF-04418948 was profiled on a range of isolated tissues to assess its EP receptor potency and selectivity: ONO-DI-004-induced contraction of guinea pig trachea (EP(1)); ONO-AE1-259 and PGE(2)- induced relaxation of mouse and guinea pig trachea (EP(2)); PGE(2)-induced depolarization of guinea pig isolated vagus (EP(3)); PGE(2)-induced relaxation of human and rat trachea (EP(4)). PF-04418948 was also profiled in functional murine TP, IP, DP and FP receptor assays.[2]

Dog bronchiole [1]
Male and female beagle dogs, aged 7–18 months, were killed by an overdose of pentobarbitone. The lungs were excised and placed in oxygenated Krebs buffer (as above with the addition of 1 µM propanolol, 10 µM phentolamine) at room temperature and transported to the laboratory. Slices of lung (3–5 mm) were cut with a scalpel, from which bronchiole rings were carefully dissected. The rings were attached to triangle hooks and connected to force displacement isometric transducers in standard 5 mL organ baths under 1 g tension. Tissues were continuously perfused with modified oxygenated Krebs buffer at 37°C for 60 min. Then, 80 mM KCl was added to the baths and the tissues allowed to contract to plateau (∼5 min). Tissues were then washed for 30 min. Carbachol (1 µM) was added and after 15 min test, concentrations of PF-04418948 (3–100 nM) or vehicle were added to the tissues. After 1 h equilibration, tissues with a stable contraction were challenged with cumulatively increasing concentrations of PGE2. At the end of the experiment, nifedipine (10 µM) was added to each bath to determine the maximum relaxation.

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Mouse trachea [1]
Male C57BL/6 mice, aged 8–10 weeks, from Charles River, were killed using an overdose of pentobarbitone. The trachea was removed using blunt dissection of the tracheal pipe and placed in oxygenated Krebs buffer at room temperature. The connective tissue was removed and each trachea divided into two (about 2 mm in length). Tracheal rings were mounted in standard 5 mL tension myograph baths containing Krebs buffer aerated with 95% O2/5% CO2 at 37°C, and connected to force displacement isometric transducer heads, under a resting tension of 0.35 g. After 30 min equilibration, carbachol (10 µM) was added and following response plateau (∼35 min), tissues were washed. After 40 min the tracheal rings were again contracted with carbachol (10 µM). Upon attaining a stable plateau, half-log concentrations of PGE2 or vehicle were added cumulatively to the bath to obtain a final assay concentration range of 0.1 nM–30 µM. Following the first cumulative concentration–response curve, the tissues were washed for 1 h. Test concentrations of PF-04418948 (3 nM) or DMSO were equilibrated for 30 min prior to repeating the cumulative PGE2 relaxation curve following pre-contraction with carbachol as described above. At the end of the experiment, forskolin (10 µM) was added to each bath to determine the maximum relaxation.

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Potency estimation of PF-04418948 using functional inhibition curve method [1]
The tissues were primed and pre-contracted with carbachol as described above. Upon obtaining a stable contraction, 700 nM PGE2 was added. Once a new plateau was reached, PF-04418948 or vehicle was added to the bath in cumulatively increasing half-log concentrations (0.1 nM–1 µM) or a single concentration of 100 nM PF-04418948 .

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Cutaneous blood flow measurements in vivo [1]
Technical problems associated with cutaneous blood flow studies in the mouse forced these in vivo studies to be performed in the rat. Sprague Dawley rats (weight range during study 442–515 g) were dosed orally with either PF-04418948 or vehicle (0.5% w/v methylcellulose + 0.1% v/v Tween 80 in purified water), dose volume 1 mL·kg(1. PF-04418948 exhibited a low clearance (0.3 mL·min−1·kg−1) and a low volume of distribution (0.1 L·kg−1) in the rat, resulting in a terminal half-life of 8.8 h. Oral absorption was high with an oral bioavailability of 78%. Plasma protein binding was observed to be very high in rat (unbound fraction of 0.0003). Approximately 1 h 25 min post dose, at the approximate peak of PF-04418948 exposure, rats were anaesthetized with 5% isoflurane + 2 L·min−1 oxygen and maintained on 2.5% isoflurane + 2 L·min−1 oxygen. Rats were briefly removed from the anaesthetic to have the abdomen shaved. Baseline scanning laser Doppler recordings were taken over a 2.5 × 2.5 cm area of the abdomen every 5 min for a total of 35 min. Following these baseline recordings, 10 µL of 3 µg·mL−1 s.c. of PGE2 (10% ethanol + 90% saline) or 10 µL of 10, 30, 100 or 1000 µg·mL−1 s.c. of butaprost (10% ethanol + 90% saline) was injected into the centre of the scan area. Laser Doppler recordings were taken for a further 60 min.

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Formulated in 0.5% w/v methylcellulose + 0.1% v/v Tween 80 in purified water; 10 mg/kg; p.o.

Sprague Dawley rats

[1]. Br J Pharmacol. 2011 Dec;164(7):1847-56.

[2]. Br J Pharmacol. 2013 Jan;168(1):129-38.

[3]. Am J Respir Crit Care Med. 2016 Jan 15;193(2):186-97.

Pf 04418948 is under investigation in clinical trial NCT01002963 (A Study To Investigate The Safety And Toleration Of A Single Dose Of PF-04418948 In Healthy Volunteers).

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Background and purpose: Studies of the role of the prostaglandin EP(2) receptor) have been limited by the availability of potent and selective antagonist tools. Here we describe the in vitro/in vivo pharmacological characterization of a novel EP(2) receptor antagonist, PF-04418948 (1-(4-fluorobenzoyl)-3-{[(6-methoxy-2-naphthyl)oxy]methyl} azetidine-3-carboxylic acid). Experimental approach: Functional antagonist potency was assessed in cell-based systems expressing human EP(2) receptors and native tissue preparations from human, dog and mouse. The selectivity of PF-04418948 was assessed against related receptors and a panel of GPCRs, ion channels and enzymes. The ability of PF-04418948 to pharmacologically block EP(2) receptor function in vivo was tested in rats. Key results: PF-04418948 inhibited prostaglandin E(2)(PGE(2))-induced increase in cAMP in cells expressing EP(2) receptors with a functional K(B) value of 1.8 nM. In human myometrium, PF-04418948 produced a parallel, rightward shift of the butaprost-induced inhibition of the contractions induced by electrical field stimulation with an apparent K(B) of 5.4 nM. In dog bronchiole and mouse trachea, PF-04418948 produced parallel rightward shifts of the PGE(2)-induced relaxation curve with a K(B) of 2.5 nM and an apparent K(B) of 1.3 nM respectively. Reversal of the PGE(2)-induced relaxation in the mouse trachea by PF-04418948 produced an IC(50) value of 2.7 nM. Given orally, PF-04418948 attenuated the butaprost-induced cutaneous blood flow response in rats. PF-04418948 was selective for EP(2) receptors over homologous and unrelated receptors, enzymes and channels. Conclusions and implications: PF-04418948 is an orally active, potent and selective surmountable EP(2) receptor antagonist that should aid further elaboration of EP(2) receptor function.[1]

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Background and purpose: Understanding the role of the EP(2) receptor has been hampered by the lack of a selective antagonist. Recently, a selective EP(2) receptor antagonist, PF-04418948, has been discovered. The aim of this study was to demonstrate the selectivity profile of PF-04418948 for the EP(2) receptor over other EP receptors using a range of isolated tissue systems. Experimental approach: PF-04418948 was profiled on a range of isolated tissues to assess its EP receptor potency and selectivity: ONO-DI-004-induced contraction of guinea pig trachea (EP(1)); ONO-AE1-259 and PGE(2)- induced relaxation of mouse and guinea pig trachea (EP(2)); PGE(2)-induced depolarization of guinea pig isolated vagus (EP(3)); PGE(2)-induced relaxation of human and rat trachea (EP(4)). PF-04418948 was also profiled in functional murine TP, IP, DP and FP receptor assays. Key results: In bioassay systems, where assessment of potency/selectivity is made against the 'native' receptor, PF-04418948 only acted as an antagonist of EP(2) receptor-mediated events. PF-04418948 competitively inhibited relaxations of murine and guinea pig trachea induced by ONO-AE1-259 and PGE(2) respectively. However, the affinity of PF-04418948 was not equal in the two preparations. Conclusions and implications: Using a wide range of bioassay systems, we have demonstrated that PF-04418948 is a selective EP(2)-receptor antagonist. Interestingly, an atypically low affinity was found on the guinea pig trachea, questioning its utility as an EP(2) receptor assay system. Nevertheless, this compound should be an invaluable tool for investigating the biological activity of PGE(2) and the role of EP(2) receptors in health and disease.[2]

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Rationale: Autologous and allogeneic hematopoietic stem cell transplant (HSCT) patients are susceptible to pulmonary infections, including bacterial pathogens, even after hematopoietic reconstitution. We previously reported that murine bone marrow transplant (BMT) neutrophils overexpress cyclooxygenase-2, overproduce prostaglandin E2 (PGE2), and exhibit defective intracellular bacterial killing. Neutrophil extracellular traps (NETs) are DNA structures that capture and kill extracellular bacteria and other pathogens. Objectives: To determine whether NETosis was defective after transplant and if so, whether this was regulated by PGE2 signaling. Methods: Neutrophils isolated from mice and humans (both control and HSCT subjects) were analyzed for NETosis in response to various stimuli in the presence or absence of PGE2 signaling modifiers. Measurements and main results: NETs were visualized by immunofluorescence or quantified by Sytox Green fluorescence. Treatment of BMT or HSCT neutrophils with phorbol 12-myristate 13-acetate or rapamycin resulted in reduced NET formation relative to control cells. NET formation after BMT was rescued both in vitro and in vivo with cyclooxygenase inhibitors. Additionally, the EP2 receptor antagonist (PF-04418948) or the EP4 antagonist (AE3-208) restored NET formation in neutrophils isolated from BMT mice or HSCT patients. Exogenous PGE2 treatment limited NETosis of neutrophils collected from normal human volunteers and naive mice in an exchange protein activated by cAMP- and protein kinase A-dependent manner. Conclusions: Our results suggest blockade of the PGE2-EP2 or EP4 signaling pathway restores NETosis after transplantation. Furthermore, these data provide the first description of a physiologic inhibitor of NETosis.[3]

C23H20FNO5

409.41

409.132

C, 67.48; H, 4.92; F, 4.64; N, 3.42; O, 19.54

1078166-57-0

25114442

White to off-white solid powder

1.4±0.1 g/cm3

639.1±55.0 °C at 760 mmHg

340.3±31.5 °C

0.0±2.0 mmHg at 25°C

1.639

3.08

1

6

6

30

629

0

FC1C([H])=C([H])C(=C([H])C=1[H])C(N1C([H])([H])C(C(=O)O[H])(C([H])([H])OC2C([H])=C([H])C3C([H])=C(C([H])=C([H])C=3C=2[H])OC([H])([H])[H])C1([H])[H])=O

LWJGMYMNSNVCEM-UHFFFAOYSA-N

InChI=1S/C23H20FNO5/c1-29-19-8-4-17-11-20(9-5-16(17)10-19)30-14-23(22(27)28)12-25(13-23)21(26)15-2-6-18(24)7-3-15/h2-11H,12-14H2,1H3,(H,27,28)

1-(4-fluorobenzoyl)-3-[(6-methoxynaphthalen-2-yl)oxymethyl]azetidine-3-carboxylic acid

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)

Solubility in Formulation 1: ≥ 6.5 mg/mL (15.88 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: ≥ 2.6 mg/mL (6.35 mM) (saturation unknown) in 2% DMSO + 40% PEG300 + 5% Tween80 + 53% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 3: ≥ 2.5 mg/mL (6.11 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 25.0 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 4: ≥ 2.5 mg/mL (6.11 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 25.0 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 5: ≥ 2.5 mg/mL (6.11 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 6: 5% DMSO + 95% Corn oil: 0.5mg/ml (1.22mM)

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