EP4 ( IC50 = 13.5 nM ); EP4 ( Ki = 23.14 nM )
E7046's primary target is the prostaglandin E2 (PGE2) receptor subtype EP4. It acts as a potent and selective antagonist at this receptor. The compound displays an IC50 value of 13.5 nM and a Ki value of 23.14 nM for the EP4 receptor. By binding to and blocking the EP4 receptor, E7046 prevents PGE2 from activating this pathway, which is often exploited by tumors to suppress the immune response. E7046 is an orally bioavailable and specific EP4 antagonist.

E7046 exhibits selective and strong inhibitory action on pro-tumor myeloid cell activation and differentiation mediated by PGE₂.[1]
In vitro, E7046 reverses the immunosuppressive effects of PGE2. It inhibits PGE2-induced activation of pro-tumor type 2 macrophages and prevents PGE2-induced suppression of T cell proliferation in isolated human peripheral blood monocytes (PBMCs) at concentrations of 0.1 to 3 µM. E7046 also promotes anti-tumor dendritic cell (DC) differentiation. In cell-based assays, it shows no significant cytotoxicity against mouse 3LL cells, with an IC50 greater than 100 µM.

E7046 treatment slows the growth of tumors in vivo and can even reject those that have already grown; this effect is reliant on both myeloid and CD8⁺ T cells.
In vivo, E7046 demonstrates significant anti-tumor activity. At a dose of 150 mg/kg, it inhibits the growth of multiple syngeneic tumor models. The blockade of EP4 signaling promotes anti-tumor DC differentiation and slows tumor growth in mice. In the CT-26 tumor model, the combination of E7046 and radiotherapy induces an anti-tumor memory response. In the 4T1 model, the combination significantly improves survival by inhibiting subsequent spontaneous lung metastasis. E7046 also reduces tumor volume in Panc02 murine pancreatic and EMT6 murine breast cancer models at doses of 5, 50, and 150 mg/kg.

The in vitro activity of E7046 is primarily assessed using cell-based functional assays. To measure its antagonistic activity, cells expressing the human EP4 receptor are pre-incubated with varying concentrations of E7046 and then stimulated with PGE2. The inhibition of downstream signaling, such as intracellular calcium flux or cAMP accumulation, is then measured. The IC50, the concentration required to inhibit 50% of the receptor's activity, is determined from these dose-response curves.

BALB/c mice's femurs are flushed with sterile CM to extract bone marrow (BM) cells. Following harvesting, freshly isolated (0.5×10⁶) mouse GM-CSF recombinant is added, and the mixture is allowed to differentiate for eight days at 37°C with 20 ng/mL ±PGE2 (10 nM). On day 3 and day 6, new GM-CSF ± PGE2 is added. Cells are examined using flow cytometry following in vitro differentiation. CT26, 4T1 cell supernatants, and/or 1 mM antagonists of EP1 (SC-57089), EP2 (ER-880696), EP3 (L-798106), or EP4 (E7046) are added to the BM cells for specific experiments. In order to evaluate the impact of differentiated bone marrow cells on T cell proliferation, T cells stained with CFSE (1 mM) and stimulated with anti-CD3/CD28 Dynabeads are co-cultured with mouse bone marrow cells for a duration of 72 hours. CFSE dilution is used in flow cytometry to measure T cell proliferation.
In cellular assays, E7046's effect on immune cell function is evaluated. Human peripheral blood monocytes (PBMCs) are treated with E7046 (0.1 to 3 µM) in the presence of PGE2, and its ability to prevent PGE2-induced suppression of T cell proliferation is measured. The compound's effect on macrophage polarization can also be assessed by treating cells with E7046 and PGE2, then measuring markers of type 2 macrophages. Cytotoxicity against cancer cell lines, such as mouse 3LL cells, is evaluated using CCK-8 assays.

In order to conduct tumor isograft efficacy studies, 6-week-old female BALB/c mice are implanted with 1×10⁵ CT26 or 4T1 cells, 8×10⁵ H22 cells per mouse s.c., or 1×10⁵ EMT6 cells in the mammary fat pad. Single-cell suspensions of 1x106 Pan02 cells per mouse and 2-3 mm³ SAI/N tumor fragments per mouse are injected into C57BL/6 and A/J mice, respectively. One thousand five hundred CT26 cells are injected subcutaneously into 6-week-old female nude mice, who do not have T cells, in order to study the function of T cells in the anti-tumor response. Tumor-bearing mice are randomly allocated to vehicle or treatment groups, and treatment regimens start when tumor sizes reach between 50 and 100 mm³. E7046 is taken orally (p.o.) as a suspension of 100 or 150 mg/kg in 0.5% MC every day for 21 days (QDx21). In the context of combination studies, E7777 is injected intravenously (i.v.) in saline at a dose of 2.5 mg/mouse in two to three injections spaced one week apart (Q7Dx2-3). Body weights and tumor volumes are measured two or three times per week. Mice are assigned to vehicle control, E7046 C E7777, anti-PD1 antibodies, or anti-mouse PD-1 C antimouse CTLA4 antibodies treatment groups in order to compare with current immunotherapies. For a total of 300 mg each, anti-PD-1 and anti-CTLA-4 antibodies (1 mg/mL) are given intraperitoneally (i.p.) in 100 mL, three times, four days apart (Q4Dx3). The control group receives isotype controls intraperitoneally at a dose of 1 mg/mL. Antimouse CD4 or anti-mouse CD8 antibodies, or their isotype controls, are injected intraperitoneally (i.p.) into a 100 mL volume every 4 days at a dose of 2.5 mg/mL, for a total of 4 injections per mouse (1 mg) in order to deplete CD4CT and CD8CT lymphocytes.
In animal models, E7046 is typically administered orally. In syngeneic tumor models like CT-26, 4T1, Panc02, and EMT6, mice are treated with E7046 at various doses, often in combination with radiotherapy. Tumor growth is monitored by measuring tumor volume, and the compound's efficacy is assessed by tumor growth inhibition. The impact on the immune system is evaluated by analyzing tumor-infiltrating immune cells, such as CD8+ T cells and dendritic cells, and measuring anti-tumor immune responses. Survival is also assessed as an endpoint in metastasis models.

E7046 is orally bioavailable. It has a molecular weight of 483.4 g/mol. The compound is soluble in DMSO (100 mg/mL) and ethanol (≤12 mg/mL). For in vivo studies, it can be formulated as a homogeneous suspension in CMC-Na (≥5 mg/mL) or as a clear solution in 5% DMSO, 40% PEG300, 5% Tween-80, and 50% ddH2O (5 mg/mL). It has a predicted density of 1.43 g/cm³ and a predicted boiling point of 594.8°C. The compound should be stored as a powder at -20°C.

As a research compound, E7046 is not intended for human or veterinary use. Its safety profile is still under investigation, primarily through preclinical studies. In animal models, it is generally well-tolerated at therapeutic doses. However, as a modulator of the immune system, it has the potential to cause immune-related adverse events. Comprehensive toxicological studies are required to establish its full safety profile for potential therapeutic applications.

[1]. Oncoimmunology . 2017 Jun 28;6(8):e1338239.

Palupiprant is an orally bioavailable prostaglandin E2 (PGE2) receptor type 4 (EP4; EP-4) antagonist with potential immunomodulatory and antitumor activity. After oral administration, Palupiprant selectively targets, binds to, and blocks the activity of immunosuppressive tumor-associated myeloid cells (TAMCs) in the tumor microenvironment. This eliminates TAMC-dependent immunosuppression and reduces tumor cell proliferation. The presence of immunosuppressive myeloid cells in certain tumors is associated with poor prognosis.
E7046 (Palupiprant) is an investigational compound that has been developed as a selective EP4 antagonist for cancer immunotherapy. The EP4 receptor is a key mediator of PGE2-induced immunosuppression in the tumor microenvironment, and its inhibition is a promising strategy for enhancing anti-tumor immunity. E7046 has been evaluated in preclinical studies and is being investigated in clinical trials, including in combination with radiotherapy for the treatment of rectal cancer. The compound is not approved for clinical use and is intended for research purposes only.

C22H18F5N3O4

483.395

483.121

C, 54.66; H, 3.75; F, 19.65; N, 8.69; O, 13.24

1369489-71-3

1369489-71-3

56944705

White to off-white solid powder

1.4±0.1 g/cm3

594.8±50.0 °C at 760 mmHg

313.5±30.1 °C

0.0±1.8 mmHg at 25°C

1.564

3.81

2

10

7

34

717

1

O=C(O)C1=CC=C([C@@H](NC(C2=C(OC3=CC=CC(C(F)(F)F)=C3)N(C)N=C2C(F)F)=O)C)C=C1

MKLKAQMPKHNQPR-NSHDSACASA-N

InChI=1S/C22H18F5N3O4/c1-11(12-6-8-13(9-7-12)21(32)33)28-19(31)16-17(18(23)24)29-30(2)20(16)34-15-5-3-4-14(10-15)22(25,26)27/h3-11,18H,1-2H3,(H,28,31)(H,32,33)/t11-/m0/s1

4-[(1S)-1-[[3-(difluoromethyl)-1-methyl-5-[3-(trifluoromethyl)phenoxy]pyrazole-4-carbonyl]amino]ethyl]benzoic acid

Palupiprant; E7046; E-7046; E 7046

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 (~206.9 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.30 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 (4.30 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 (4.30 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.)