rEP2 ( IC50 = 50 nM )

To further characterize the potential mast cell (MC) inhibitory effect of the non-prostanoid EP2 agonist Evatanepag (CP-533536) , an in vivo study was performed in HDM-sensitized mice. HDM-sensitized BALB/c mice were exposed i.n. to Evatanepag (CP-533536) . Airway reactivity, inflammation, and LMC activity were assessed. [2]

Fig. 10A depicts airway reactivity to methacholine in mice sensitized to HDM aeroallergens for 10 days and exposed i.n. to the EP2 selective agonist Evatanepag (CP-533536)  from days −1 to 4 of sensitization. Mice sensitized and challenged with HDM exhibited a significant increase in airway reactivity to methacholine. Local administration of CP-533536 at 0.3 mg·kg−1 prevented aeroallergen-driven increased RL, which was approximately half of the reactivity measured in non-treated mice. This effect, however, did not occur at the 3 mg·kg−1 dose. [2]

Differential inflammatory cell recruitment in the airways was also assessed in mice exposed to Evatanepag (CP-533536)  (Fig. 10B). Strong eosinophilic recruitment was induced in HDM-sensitized mice. Differential airway inflammatory cell count was not altered by pre-treatment with CP-533536. [2]

Finally, HDM-induced LMC activation was evaluated by measuring lung mMCP-1 concentrations. Fig. 10C shows mMCP-1 concentration normalized to total protein in lung extract homogenates. The mMCP-1 was overexpressed locally by a factor of 5.4 in HDM-exposed vs. non-exposed mice. Treatment with Evatanepag (CP-533536)  did not produce a statistically significant change in mMCP-1, but the 3 mg·kg−1 dose prevented the enhanced MC activity by approximately 48% (P = 0.13). [2]

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Evatanepag (0.3-3.0 mg/kg, directly injected into the marrow cavity of the tibia) stimulates the growth of bones[1]. Evatanepag (0.3, 3.0 mg/kg, intranasal administration, from day1 to day4) lessens the increased RL response to methacholine caused by HDM aeroallergens in mice[2]. Evatanepag (1 mg/kg, intravenous injection) has a brief half-life (t1/2: 0.33 h) and a high i.v. clearance (Cl: 56 mL/min/kg)[1].

Evatanepag (CP-533536) is a prostaglandin E2 (PGE2) agonist selective for EP2 receptors that, at an EC50 of 0.3 nM, stimulates local bone formation. CP-533536 is a potent and selective EP2agonist. In rat models of fracture healing, CP-533536 shows promise as a localized single dose treatment for fractures. CP-533536 demonstrates excellent in vitro potency against EP2 and selectivity against a broad panel of other targets.

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Screening for EP2 Selective Agonists. [3]
Compounds were categorized for further characterization on the basis of their ability to selectively bind the EP2 receptor. EP2 receptor agonism was defined by the ability of compounds to selectively bind to the EP2 receptor and increase intracellular cAMP levels.

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Receptor Binding. [3]
Membranes were prepared from stably transfected HEK-293 cells expressing PGE2 EP1, -2, -3, or -4 receptor subtypes as well as those for prostaglandin D2, prostaglandin F2α, prostacyclin, and thromboxane. Receptor binding was measured as described by Castleberry et al.

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Determination of cAMP. [3]
Cellular cAMP levels in the HEK-293/EP2 line were determined after pretreatment of 2 × 105 cells with 1 mM 3-isobutyl-1-methylxanthine for 10 min at 37°C followed by treatment with the indicated concentrations of test compounds for 12 min at 37°C. cAMP was quantitated by using an RIA kit according to the manufacturer's instructions.

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Analysis of Evatanepag (CP-533536)  in Plasma. [3]
Plasma samples were thawed, and 20 ×l of each sample was injected into a PE-Sciex API 3000 triple quadrapole mass spectrometer with a turbo ionspray source. A Luna phenyl-hexyl (4.6 × 50 mm × 3 ×m) column was used for separation. Evatanepag (CP-533536)  and the internal standard were determined in negative ion mode by using multireaction monitoring by following the mass transitions of 467.3/303.2 m/z and 388.3/198.1 m/z, respectively. The linear dynamic range of the assay was from 1 to 2,000 ng/ml. The mean accuracy of the assay characterized with quality control standards was 80–116%. [2]

MC stimulation and release assay [2]
Murine cells (C57.1, PDMC, and LMC) were sensitized with 1 μg·mL−1 DNP-specific IgE for 2 hours in SCF- and IL-3-free media. After sensitization, cells were washed and resuspended in HEPES buffer (10 mM HEPES [pH 7.4], 137 mM NaCl, 2.7 mM KCl, 0.4 mM Na2HPO4_7H2O, 5.6 mM glucose, 1.8 mM CaCl2_2H2O, and 1.3 mM MgSO4_7H2O) with 0.04% BSA. Cells were seeded on a V-bottom 96-well plate, with 50,000 cells in a final volume of 100 μL, and treated with 10−5 M butaprost, 10−5 M PGE2, or vehicle (PBS with 0.1% DMSO) for 30 minutes and 10−5 M AH6809 (LMC) or vehicle (PS with 20% ethanol) for 1 hour at 37°C with 5% v/v CO2. PDMCs from wild-type (WT) BALB/c were also treated with increasing concentrations of butaprost (10−6 M, 3·10−6 M, and 3·10−5 M). Cells were stimulated with 50 ng·mL−1 DNP-HSA as an antigen (Ag) for 30 minutes. LAD2 MCs were sensitized with 100 ng·mL−1 for 2 hours in SCF- and IL-3-free media. RS-ATL8 cells were sensitized with 500 ng·mL−1 biotinylated hIgE for 16 hours. PDMCs from FcεRI−/−hFcεRI+ BALB/c mice were sensitized with 100 ng·mL−1 chimeric hIgE anti-NP for 16 hours. After sensitization, cells were washed, resuspended with HEPES buffer with 0.04% BSA, and seeded on a V-bottom 96-well plate, with 150,000 cells in a final volume of 300 μL (LAD2) or 200,000 cells in a final volume of 320 μL (PDMCs). Two days before the release assay, 50,000 RS-ATL8 cells were cultured in a final volume of 100 μL to obtain 100,000 adhering cells. Cells were treated for 2 hours and 15 minutes at 37°C with 5% v/v CO2 as follows: increasing concentrations of butaprost and Evatanepag (CP-533536)  (10−7 M, 3·10−7 M, 10−6 M, 3·10−6 M, 10−5 M, 3·10−5 M, 10−4 M, or 3·10−4 M) or vehicle (PBS with 0.1% DMSO) in LAD2; Evatanepag (CP-533536)  (10−12 M, 10−11 M, 10−10 M, 10−9 M, 10−8 M, 10−7 M, 10−6 M, 10−5 M, or 10−4 M) in RS-ATL8; and butaprost (10−6 M, 3·10−6 M, or 10−5 M) in PDMCs from FcεRI−/−hFcεRI+ mice. Cells were challenged with 100 ng·mL−1 SA (LAD2), 1,000 ng·mL−1 SA (RS-ATL8), or 50 ng·mL−1 NP-BSA (PDMC from FcεRI−/−hFcεRI+ mice) for 30 minutes at 37°C with 5% v/v CO2. Degranulation was stopped by placing the cells in iced water, and the cell suspension was centrifuged for 10 minutes at 4°C at 1,500 rpm.

Treatment with the EP2 agonist Evatanepag (CP-533536)  [2]
Sensitized mice were treated with the EP2 agonist CP-533536 (Fig. 2). CP-533536 was administered i.n. (0.3 mg·kg−1 or 3 mg·kg−1) 1 hour before exposure to the HDM extract, starting 1 day before initiating sensitization (day −1) and continuing through the first 4 days of sensitization (day 4). HDM-sensitized untreated mice received i.n. PBS in 0.1% DMSO.

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Rat Experiments.[3]
All animal experiments were conducted in accordance with relevant institutional guidelines for animal research. Forty male rats at 6 weeks of age were injected with 10 ×l of either vehicle (5% ethanol in sterile injection water, n = 10) or Evatanepag (CP-533536)  at 0.3, 1, and 3 mg/kg (n = 10 per dose) into the marrow cavity of the proximal tibial metaphysis underneath the secondary spongiosa. At 7 days postinjection, the animals were necropsied, and tibial injection sites were analyzed cross-sectionally by using peripheral quantitative computerized tomography, as described by Ke et al. Briefly, a 1-mm-thick cross section of the injection site was analyzed with a voxel size of 0.10 mm. Total bone area, bone mineral content, and total bone mineral density were determined as percent changes compared with vehicle-treated controls.

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Canine Experiments [3]
Critical Defect. Experiment 1. Male beagle dogs were surgically prepared by creating a 1.5-cm segmental critical defect in the midulna by using a pendular saw according to accepted veterinary surgical practices. The radius and remaining interosseal membrane were left intact, and the soft tissues were closed in layers. Animals were divided into three experimental groups (n = 8 per group) and treated with three (Group A), seven (Group B), or 14 (Group C) daily injections of an aqueous solution of Evatanepag (CP-533536)  [100 mg/ml in calcium magnesium-free PBS after surgery and implantation of Helistat (2.5 × 5 cm) precut collagen sponges into the defect area]. Radiographs of the forelimbs were obtained immediately after surgery and every 2 weeks until termination of the study (week 10). [3]

Experiment 2. [3]
In another set of ulnar critical defect experiments, male beagle dogs (n = 28), surgically prepared as above, were divided into four groups and treated with 1.0 ml of poly(D,L-lactide-co-glycolide) (PLGH) matrix alone (Group A), 50 mg of Evatanepag (CP-533536)  dissolved in 1.0 ml of matrix (Group B), 10 mg of CP-533,536 dissolved in 1.0 ml of matrix (Group C), or 10 mg of Evatanepag (CP-533536)  dissolved in 0.2 ml of matrix (Group D). In these experiments, no precut collagen sponge was used in the defect area. The compound was administered into the defect area in a single dose at surgery. Blood (1.0 ml) was collected from animals 30 min, 2 h, 4 h, 24 h, 72 h, and 7 days after surgery. Animals were monitored postsurgically for side effects, and radiographs of the forelimbs were obtained immediately after surgery and every week until the termination of the study (week 24). [3]

Tibial Osteotomy. [3]
For the tibial osteotomy model, male beagle dogs (n = 14) were prepared by making a surgical osteotomy on the distal portion of the dog tibia by using a Gigli saw according to accepted veterinary surgical practices. The proximal and distal bone segments were stabilized by using an AO plate. The remaining interosseal membrane was left intact. The defect site was irrigated with saline to remove bone debris and filled with PLGH matrix alone or matrix containing Evatanepag (CP-533536)  in the following manner: Group A, dogs were left untreated (n = 3); Group B, dogs were treated with 0.5 ml of matrix alone (n = 3); Group C, dogs were treated with 5 mg of CP-533,536 dissolved in 0.5 ml of matrix (n = 4); and Group D, dogs were treated with 25 mg of CP-533,536 dissolved in 0.5 ml of matrix (n = 4). Blood (1.0 ml) was collected from all animals at 30 min, 2 h, 4 h, and 24 h after surgery. Radiographs of the forelimbs were obtained immediately after surgery and every week until the termination of the study (week 8). [3]

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Administered locally as a single dose

Rats

[1]. Discovery of CP-533536: an EP2 receptor selective prostaglandin E2 (PGE2) agonist that induces local bone formation. Bioorg Med Chem Lett. 2009 Apr 1;19(7):2075-8.

[2]. In Vitro and In Vivo Validation of EP2-Receptor Agonism to Selectively Achieve Inhibition of Mast Cell Activity. Allergy Asthma Immunol Res. 2020 Jul;12(4):712-728.

[3]. An EP2 receptor-selective prostaglandin E2 agonist induces bone healing. Proc Natl Acad Sci U S A. 2003 May 27;100(11):6736-40.

Evatanepag is a monocarboxylic acid.
Evatanepag has been used in trials studying the treatment of Tibial Fractures.

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Sulfonamides, exemplified by 3a, were identified as highly selective EP(2) agonists. Lead optimization led to the identification of CP-533536, 7f, a potent and selective EP(2) agonist. CP-533536 demonstrated the ability to heal fractures when administered locally as a single dose in rat models of fracture healing. [1]

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Purpose: Agonism of the prostaglandin E2 receptor, E-prostanoid receptor 2 (EP2), may represent an alternative protective mechanism in mast cell (MC)-mediated diseases. Previous studies have suggested that activation of the MC EP2 receptor prevents pathological changes in the murine models of allergic asthma. This work aimed to analytically validate the EP2 receptor on MCs as a therapeutic target. Methods: Murine MC lines and primary cultures, and MCs bearing the human immunoglobulin E (IgE) receptor were subjected to IgE-mediated activation subsequent to incubation with selective EP2 agonists. Two molecularly unrelated agonists, butaprost and CP-533536, were tested either in vitro or in 2 in vivo models of allergy. Results: The diverse range of MC populations was consistently inhibited through selective EP2 agonism in spite of exhibiting a heterogeneous phenotype. Such inhibition occurred in both mouse and human IgE (hIgE)-mediated activation. The use of molecularly unrelated selective EP2 agonists allowed for the confirmation of the specificity of this protective mechanism. This effect was further demonstrated in 2 in vivo murine models of allergy where MCs are a key to pathological changes: cutaneous anaphylaxis in a transgenic mouse model expressing the hIgE receptor and aeroallergen-induced murine model of asthma. Conclusions: Selective EP2 agonism is a powerful pharmacological strategy to prevent MCs from being activated through IgE-mediated mechanisms and from causing deleterious effects. The MC EP2 receptor may be an effective pharmacological target in allergic and other MC-mediated conditions.[2]

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The observed in vivo EP2-driven effect could presumably counteract diseases based on IgE-mediated MC overactivation or hyper-releasability. We thereby assessed LMC activity and airway responses in HDM-sensitized BALB/c mice, which exhibited increased airway MC activity according to mouse mMCP-1 protease determination. The selective EP2-agonist CP-533536 partially (albeit insignificantly) prevented the ability of airway MCs to release mMCP-1. The CP-533536 effect was less than that of butaprost13 as anticipated by its more limited inhibitory effect in vitro. In parallel with preventing airway MC activity, CP-533536 also reduced HDM aeroallergen-induced increased RL response to methacholine. This confirms earlier findings that butaprost suppressed airway hyper-reactivity and inflammation.13 CP-533536 produced a nonsignificant reduction in eosinophilic inflammation, which may be explained by its relatively limited ability to reduce MC activity (compared to butaprost).[2]

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The morbidity and mortality associated with impaired/delayed fracture healing remain high. Our objective was to identify a small nonpeptidyl molecule with the ability to promote fracture healing and prevent malunions. Prostaglandin E2 (PGE2) causes significant increases in bone mass and bone strength when administered systemically or locally to the skeleton. However, due to side effects, PGE2 is an unacceptable therapeutic option for fracture healing. PGE2 mediates its tissue-specific pharmacological activity via four different G protein-coupled receptor subtypes, EP1, -2, -3, and -4. The anabolic action of PGE2 in bone has been linked to an elevated level of cAMP, thereby implicating the EP2 and/or EP4 receptor subtypes in bone formation. We identified an EP2 selective agonist, CP-533,536, which has the ability to heal canine long bone segmental and fracture model defects without the objectionable side effects of PGE2, suggesting that the EP2 receptor subtype is a major contributor to PGE2's local bone anabolic activity. The potent bone anabolic activity of CP-533,536 offers a therapeutic alternative for the treatment of fractures and bone defects in patients.[3]

We have presented data that the EP2 receptor subtype is important in skeletal healing. Further, a selective and potent functional EP2 receptor agonist, CP-533,536, induced healing of critical defects in the canine ulna and dramatically accelerated healing in the tibial osteotomy model. A single injection of CP-533,536, administered in a PLGH sustained release matrix at the time the bone defect was created, was efficacious in accelerating healing. Thus, given the high unmet medical need for fracture healing therapy and the current limitations of therapeutic procedures such as autographs and allographs, the potent bone anabolic capacity of CP-533,536 offers a promising therapeutic alternative for the enhancement of bone healing and treatment of bone defects and fractures in patients.[3]

C25H28N2O5S

468.57

468.171

C, 64.08; H, 6.02; N, 5.98; O, 17.07; S, 6.84

223488-57-1

223490-49-1 (sodium); 223488-57-1 (free acid)

9890801

White to off-white solid powder

1.3±0.1 g/cm3

660.2±65.0 °C at 760 mmHg

353.0±34.3 °C

0.0±2.1 mmHg at 25°C

1.600

4.78

1

7

10

33

722

0

S(C1=C([H])N=C([H])C([H])=C1[H])(N(C([H])([H])C1C([H])=C([H])C([H])=C(C=1[H])OC([H])([H])C(=O)O[H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])(=O)=O

WOHRHWDYFNWPNG-UHFFFAOYSA-N

InChI=1S/C25H28N2O5S/c1-25(2,3)21-11-9-19(10-12-21)16-27(33(30,31)23-8-5-13-26-15-23)17-20-6-4-7-22(14-20)32-18-24(28)29/h4-15H,16-18H2,1-3H3,(H,28,29)

2-[3-[[(4-tert-butylphenyl)methyl-pyridin-3-ylsulfonylamino]methyl]phenoxy]acetic acid

Evatanepag; CP-533536; CP 533536; 223488-57-1; CP-533536 free acid; 2-[3-[[(4-tert-butylphenyl)methyl-pyridin-3-ylsulfonylamino]methyl]phenoxy]acetic acid; 2-(3-((N-(4-(tert-butyl)benzyl)pyridine-3-sulfonamido)methyl)phenoxy)acetic acid; CP533536

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)

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.

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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).

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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)

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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).

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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

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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.

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