EP
EP4 prostanoid receptor antagonist (Prostaglandin E2 receptor EP4 subtype antagonist).[3]

GW627368 (GW627368 GW627368 (GW627368 X) (10 μM) inhibits 100% of U-46619 (EC100)-induced GW627368 (GW627368 [1]. GW627368 (GW627368

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GW627368X is a potent and selective competitive antagonist of prostanoid EP₄ receptors, with additional affinity for human TP receptors. It shows no agonism at EP₂, EP₃, FP, or IP receptors. In HEK293 cells expressing recombinant human EP₄ receptors, it right-shifts PGE₂ concentration-effect curves (pKb = 7.9 ± 0.4). In piglet saphenous vein, it antagonizes PGE₂-induced relaxation (pKb = 9.2 ± 0.2). It inhibits U-46619-induced human platelet aggregation (100% inhibition at 10 μM). No activity was observed at rabbit, guinea-pig, or piglet TP receptors. [1]
In cAMP assays, GW627368X reduced basal cAMP levels in HEK-EP₄ cells (pIC₅₀ = 6.3 ± 0.1), an effect enhanced by indomethacin. [1]
In competition binding assays, GW627368X displaced radioligands from human EP₄ (pKi = 7.0 ± 0.3) and TP (pKi = 6.8) receptors, with <50% displacement at other prostanoid receptors at 10 μM. [1]

GW627368 (GW627368 Animal model: 6-8 weeks old Swiss albino mice [3] Dosage: 0 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 15 mg/kg Administration method: Orally, once every other day, continuously 28-day results: showed potential for anti-tumor and anti-proliferative effects in sarcoma 180-bearing mice.

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Oral administration of GW627368X (at 5, 10, and 15 mg/kg body weight, every alternate day for 28 days) to Swiss albino mice bearing subcutaneous Sarcoma 180 (S180) tumors resulted in significant dose-dependent anti-tumor effects. The highest dose group (15 mg/kg) showed tumor volume regression to 202.3 ± 18.8 mm³ compared to 8148.3 ± 76.98 mm³ in the control group, and a significant reduction in final tumor mass. [3]
TUNEL assay on tumor sections revealed a dose-dependent induction of apoptosis. The mean fluorescence intensity (indicating DNA fragmentation) increased from 1.588 ± 0.4363 in the control to 36.34 ± 4.047 in the high-dose group. Flow cytometry analysis of cells from intraperitoneal ascitic tumors showed an increase in the percentage of apoptotic cells from 2.7% in the control to 9.8% in the high-dose group. [3]
Immunohistochemistry and western blot analysis demonstrated that GW627368X treatment led to a significant decrease in the expression of the proliferation marker Ki67 and the angiogenesis marker CD31 within tumors. [3]
Western blot analysis of tumor tissues showed a significant increase in the expression of pro-apoptotic proteins (AIF and Bax) and a decrease in anti-apoptotic proteins (Mcl-1 and Bcl-2) with increasing drug dosage. [3]
GW627368X treatment significantly downregulated the expression of EP4 and COX-2 proteins in tumor tissues. It also reduced the phosphorylation levels of key signaling proteins including p-EGFR, p-VEGFR, p-Akt, and p-MAPK, while total protein levels remained unchanged. [3]
Enzyme immunoassay (ELISA) showed that GW627368X treatment drastically reduced prostaglandin E2 (PGE2) levels in both plasma and tumor tissue. Plasma PGE2 decreased from 720.7 ± 11.11 pg/ml in the tumor-bearing control to 200 ± 0.71 pg/ml in the high-dose group (normal mouse level: 248.8 ± 16.40 pg/ml). Tissue PGE2 also showed a significant decrease. [3]
Concomitantly, plasma VEGF levels were markedly reduced from 482.1 ± 17.57 pg/ml in the control to 250.5 ± 14.62 pg/ml in the high-dose group, approaching the level in healthy animals (177.3 ± 14.97 pg/ml). [3]

Competition radioligand binding assays were performed using membranes from cells expressing recombinant human prostanoid receptors. Membranes were incubated with radioligands ([³H]-PGE₂ for EP receptors, [³H]-SQ29548 for TP) and increasing concentrations of GW627368X in HEPES buffer (pH 7.4) for 120–180 min at room temperature. Binding was terminated by filtration, and radioactivity was measured by scintillation counting. Data were analyzed using Cheng–Prusoff correction to estimate pKi values. [1]
Saturation binding assays were conducted to determine Kd and Bmax for each receptor. Membranes were incubated with increasing concentrations of radioligand, and specific binding was calculated as total minus non-specific binding. [1]

cAMP assays were performed in HEK293 cells expressing human EP₄ receptors. Cells were incubated with assay medium containing IBMX and indomethacin, then treated with GW627368X or PGE₂ for 15 min at 37°C. Reactions were stopped with cold ethanol, and cAMP levels were measured using a scintillation proximity assay kit. [1]
Calcium influx assays were conducted in CHO cells expressing prostanoid receptors. Cells were loaded with Fluo-4 AM and incubated with GW627368X before agonist addition. Fluorescence changes were monitored using a FLIPR. [1]
Platelet aggregation assays used washed human platelets. Platelets were incubated with GW627368X (0.1–10 μM) for 2 min before adding U-46619 (EC₁₀₀). Aggregation was measured using an aggregometer. [1]

6-8 weeks Swiss albino mice
0 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 15 mg/kg
Oral administration, every alternate day for 28 days

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For the main efficacy study, exponentially growing S180 mouse sarcoma cells (3×10⁶ cells) were injected subcutaneously into the right flank of Swiss albino mice. After 7 days of tumor growth, animals were randomized into groups (n=10 per group): Control (vehicle), Low dose (5 mg/kg), Moderate dose (10 mg/kg), High dose (15 mg/kg), and a Recovery group (15 mg/kg). [3]
GW627368X was suspended in deionized water. The control group received polysorbate dissolved in deionized water. Treatment groups received the drug solution orally by gavage, every alternate day for 28 days. Tumor volume was measured regularly, and body weight was monitored. [3]
In a separate experiment to assess behavioral/physiological changes and confirm efficacy in a different model, ascitic tumors were induced in another group of 20 mice by intraperitoneal injection of 3×10⁶ S180 cells. These mice were similarly subdivided into control and treatment groups (5, 10, 15 mg/kg) and treated via oral gavage every alternate day. Cells from these mice were later harvested for flow cytometric cell cycle analysis. [3]
At the end of the 28-day treatment, animals were sacrificed. Tumors were excised, weighed, and preserved for analysis (IHC, western blot, TUNEL). Blood was collected for hematological and biochemical tests. Major organs (liver, heart, kidney, stomach, spleen) were dissected, weighed, and processed for histopathology (H&E staining) or functional assays (splenocyte proliferation). [3]
The Recovery group was observed for an additional 8 days after withdrawal of treatment to check for reversibility of any toxic symptoms. [3]

28-day toxicity study was conducted in tumor-bearing S180 mice, which were orally administered GW627368X every other day at doses of 5, 10, and 15 mg/kg. Apart from a slight decrease in appetite after treatment and suspected mild splenomegaly in some treatment groups, no clinical symptoms, significant behavioral changes, or significant changes in organ weight/appearance were observed. [3] Serum biochemistry showed no signs of hepatotoxicity: SGPT, SGOT, ALP, and bilirubin levels were normal in all groups. Renal function indicators (creatinine, inorganic phosphate) were stable, but uric acid levels decreased slightly with increasing dose. [3] Histopathological examination of HE-stained cardiac sections showed that the myocardial fiber structure was slightly blurred in the medium- and high-dose groups, but serum troponin levels were negative in all groups, indicating no cardiotoxicity. Mild ulcers were observed in gastric sections in the medium- and high-dose groups. [3]
Serious electrolytes (sodium, potassium), blood lipids (triglycerides, cholesterol), and protein levels (total protein, albumin, globulin) were not affected by drug treatment. [3]
Hematological parameters (hemoglobin, platelet count, white blood cell count, differential count, reticulocyte count) were all within the normal range, and no significant drug-related changes were observed. [3]
Ex vivo spleen cell proliferation assays showed a slight increase in proliferation in the low-dose group compared to the control group, but returned to normal at the highest dose (15 mg/kg), indicating that no significant immunosuppression was observed. [3]

[1]. GW627368X ((N-{2-[4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetyl} benzene sulphonamide): a novel, potent and selective prostanoid EP4 receptor antagonist. Br J Pharmacol. 2006 Jun;148(3

[2]. Molecular and pharmacological blockade of the EP4 receptor selectively inhibits both proliferation and invasion of human inflammatory breast cancer cells. J Exp Ther Oncol. 2008;7(4):299-312.

[3]. Molecular inhibition of prostaglandin E2 with GW627368X: Therapeutic potential and preclinical safety assessment in mouse sarcoma model. Cancer Biol Ther. 2015;16(6):922-32.

GW627368X (4-(4,9-diethoxy-1,3-dihydro-1-oxo-2H-benzo[f]isoindole-2-yl)-N-(benzenesulfonyl)-phenylacetamide) is a highly selective prostaglandin E2 receptor EP4 subtype antagonist. [3] This study proposes that GW627368X exerts its antitumor effect by blocking the EP4 receptor, thereby interrupting the positive feedback loop of PGE2 enhancing COX-2 expression through EP4 and further promoting PGE2 synthesis. This leads to reduced angiogenesis (by reducing VEGF production) and inhibition of multiple pro-survival and proliferative signaling pathways downstream of EP4, including the PI3K/Akt, MAPK/ERK, and trans-activated EGFR pathways. Its mechanism of action includes inducing apoptosis, inhibiting cell proliferation, and inhibiting tumor angiogenesis. [3]
This study concluded that GW627368X, at doses up to 15 mg/kg, demonstrated significant anticancer potential and good preclinical safety in a mouse sarcoma model, supporting EP4 as a promising cancer therapeutic target. [3]

C30H28N2O6S

544.6181

544.166

C, 66.16; H, 5.18; N, 5.14; O, 17.63; S, 5.89

439288-66-1

5312130

White to off-white solid powder

1.3±0.1 g/cm3

1.645

4.95

1

6

9

39

953

0

S(C1C([H])=C([H])C([H])=C([H])C=1[H])(N([H])C(C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])N1C(C2=C(C3=C([H])C([H])=C([H])C([H])=C3C(=C2C1([H])[H])OC([H])([H])C([H])([H])[H])OC([H])([H])C([H])([H])[H])=O)=O)(=O)=O

XREWXJVMYAXCJV-UHFFFAOYSA-N

InChI=1S/C30H28N2O6S/c1-3-37-28-23-12-8-9-13-24(23)29(38-4-2)27-25(28)19-32(30(27)34)21-16-14-20(15-17-21)18-26(33)31-39(35,36)22-10-6-5-7-11-22/h5-17H,3-4,18-19H2,1-2H3,(H,31,33)

N-(benzenesulfonyl)-2-[4-(4,9-diethoxy-3-oxo-1H-benzo[f]isoindol-2-yl)phenyl]acetamide

GW 627368X; GW 627368; GW627368X; GW627368; GW-627368X; GW-627368

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 (~183.6 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.59 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 2: ≥ 2.5 mg/mL (4.59 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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 (Please use freshly prepared in vivo formulations for optimal results.)