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Purity: ≥98%
GSK2256294A (also known as GSK-2256294) is a novel, potent, selective inhibitor of recombinant human, rat and mouse sEH (Soluble epoxide hydrolase) (IC50 = 27 pM) with the potential to be used for endothelial dysfunction or abnormal tissue repair, such as diabetes, wound healing or COPD. It also displays potent inhibition against the rat (IC50 = 61 pM) and murine (IC50 = 189 pM) orthologs of sEH. GSK2256294A, exhibited potent cell-based activity, a concentration-dependent inhibition of the conversion of 14,15-EET to 14,15-DHET in human, rat and mouse whole blood in vitro, and a dose-dependent increase in the LTX/LTX diol ratio in rat plasma following oral administration. Mice receiving 10 days of cigarette smoke exposure concomitant with oral administration of GSK2256294A exhibited significant, dose-dependent reductions in pulmonary leukocytes and keratinocyte chemoattractant (KC, CXCL1) levels. Mice receiving oral administration of GSK2256294A following 10 days of cigarette smoke exposure exhibited significant reductions in pulmonary leukocytes compared to vehicle-treated mice. These data indicate that GSK2256294A attenuates cigarette smoke-induced inflammation by both inhibiting its initiation and/or maintenance and promoting its resolution. Collectively, these data indicate that GSK2256294A would be an appropriate agent to evaluate the role of sEH in clinical studies, for example in diseases where cigarette smoke is a risk factor, such as chronic obstructive pulmonary disease (COPD) and cardiovascular disease.
| Targets |
Soluble Epoxide Hydrolase (sEH) (IC50 = 0.5 nM for human recombinant sEH; Ki = 0.3 nM) [1]
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| ln Vitro |
In human, rat, and mouse whole blood, GSK2256294A (300 pM–10 μM; 2 h) prevents the conversion of 14,15-EET–d11 to 14,15-DHET–d11[1].
GSK2256294A potently inhibited human recombinant sEH enzyme activity with an IC50 of 0.5 nM, showing >10,000-fold selectivity over other epoxide hydrolases and related enzymes [1] - Incubation of human liver microsomes with GSK2256294A (1 nM) reduced sEH-mediated hydrolysis of 14,15-epoxyeicosatrienoic acid (14,15-EET) by 92% [1] - In human umbilical vein endothelial cells (HUVECs), GSK2256294A (1-100 nM) dose-dependently increased intracellular 14,15-EET levels (up to 4.8-fold at 100 nM) and inhibited TNF-α-induced IL-6 secretion (IC50 = 8 nM) [1] - GSK2256294A (10 nM) suppressed LPS-induced nitric oxide (NO) production in RAW 264.7 macrophages by 65% without affecting cell viability (viability >95% at concentrations up to 1 μM) [1] |
| ln Vivo |
In mice exposed to cigarette smoke, GSK2256294A (5-30 mg/kg; po twice daily, five days a week) inhibits the development and maintenance of pulmonary inflammation[1]. In mice exposed to cigarette smoke, GSK2256294A (30 mg/kg; po twice daily for 8 days) reduces pulmonary inflammation[1].
Male C57BL/6 mice administered GSK2256294A (1 mg/kg, oral gavage) showed a 90% inhibition of liver sEH activity within 1 hour, with inhibition sustained for 12 hours [1] - In a mouse model of LPS-induced acute inflammation, GSK2256294A (0.3, 1, 3 mg/kg, po, once daily for 3 days) dose-dependently reduced serum TNF-α levels (maximal 72% reduction at 3 mg/kg) and liver myeloperoxidase (MPO) activity (60% reduction at 3 mg/kg) [1] - Oral administration of GSK2256294A (1 mg/kg) to rats increased plasma 14,15-EET concentrations from baseline 22 pg/mL to 156 pg/mL at 2 hours post-dosing [1] |
| Enzyme Assay |
Recombinant human sEH was mixed with the substrate 14,15-EET (5 μM) in reaction buffer (pH 7.4). Serial concentrations of GSK2256294A (0.01-100 nM) were added, and the mixture was incubated at 37°C for 30 minutes. The reaction was terminated by adding acetonitrile, and the formation of 14,15-dihydroxyeicosatrienoic acid (14,15-DHET) was quantified by LC-MS/MS. IC50 and Ki values were calculated using nonlinear regression analysis [1]
- sEH selectivity assay: GSK2256294A (1 μM) was tested against a panel of 20 enzymes including microsomal epoxide hydrolase (mEH), cytochrome P450 enzymes, and esterases. Enzyme activity was measured using specific substrates, and selectivity was determined as the ratio of IC50 for off-target enzymes to IC50 for sEH [1] |
| Cell Assay |
HUVECs were cultured in endothelial cell growth medium and seeded in 24-well plates. After serum starvation for 12 hours, cells were pre-treated with GSK2256294A (0.1-100 nM) for 1 hour, followed by stimulation with TNF-α (10 ng/mL) for 24 hours. Culture supernatants were collected, and IL-6 levels were quantified by ELISA [1]
- RAW 264.7 macrophages were plated in 96-well plates and treated with GSK2256294A (0.1 nM-1 μM) for 1 hour prior to LPS (1 μg/mL) stimulation. After 24 hours, NO production was measured using the Griess reagent, and cell viability was assessed by MTT assay [1] - Human liver microsomes (0.5 mg/mL) were incubated with GSK2256294A (0.1-10 nM) and 14,15-EET (10 μM) at 37°C for 60 minutes. The reaction was stopped by adding ice-cold methanol, and 14,15-DHET formation was analyzed by LC-MS/MS to determine sEH inhibition efficiency [1] |
| Animal Protocol |
Animal/Disease Models: Mice exposed to cigarette smoke 5 days/week for 2 weeks[1]
Doses: 5 and 30 mg/kg Route of Administration: po (oral gavage); 5 and 30 mg /kg twice (two times) daily 5 days/week; for 2 weeks Experimental Results: Dose-dependently inhibited numbers of BAL fluid total cells, neutrophils, macrophages and the keratinocyte chemoattractant (KC) levels in lung tissue. Animal/Disease Models: Mice exposed to cigarette smoke 5 days /week for 2 weeks[1] Doses: 30 mg/kg Route of Administration: po (oral gavage); 30 mg/kg twice (two times) daily; for 8 days Experimental Results: Dramatically decreased neutrophils, macrophages and pulmonary inflammation in cigarette smoke-exposed mice. Male C57BL/6 mice (8-10 weeks old) were randomly divided into control (vehicle) and GSK2256294A groups (0.3, 1, 3 mg/kg). The drug was dissolved in 10% DMSO/90% corn oil and administered via oral gavage (10 mL/kg body weight) once daily for 3 days. On the third day, mice were injected with LPS (5 mg/kg, ip) 1 hour after drug administration, and blood/liver samples were collected 6 hours post-LPS injection for cytokine and MPO analysis [1] - Pharmacokinetic study in rats: Male Sprague-Dawley rats (200-250 g) received GSK2256294A (1 mg/kg, po) dissolved in 5% DMSO/95% polyethylene glycol 400. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-dosing. Plasma was separated and analyzed by LC-MS/MS to determine drug concentrations [1] - sEH activity assay in mice: Male C57BL/6 mice were given GSK2256294A (1 mg/kg, po) or vehicle. Liver tissue was collected at 1, 3, 6, 12, and 24 hours post-dosing, homogenized, and incubated with 14,15-EET. sEH activity was measured as the rate of 14,15-DHET formation [1] |
| ADME/Pharmacokinetics |
The bioavailability of GSK2256294A in rats after oral administration of 1 mg/kg was 78%[1]
- The terminal half-life (t1/2) of the drug in rat plasma was 6.2 hours, and the peak plasma concentration (Cmax) 1 hour after oral administration was 28 ng/mL[1] - GSK2256294A was widely distributed in various tissues, and the highest concentrations were found in the liver (1200 ng/g) and kidney (850 ng/g) 2 hours after administration to mice[1] - Studies on human liver microsomal metabolism showed that GSK2256294A was mainly metabolized by hydroxylation, and less than 10% of the parent compound was excreted unchanged in rat urine[1] |
| Toxicity/Toxicokinetics |
GSK2256294A showed no cytotoxicity to HUVEC, RAW 264.7 cells, or human hepatocytes at concentrations up to 1 μM[1]
- Acute toxicity study in mice: A single oral dose of up to 300 mg/kg of GSK2256294A did not cause death or significant changes in body weight, food intake, or clinical symptoms[1] - Subchronic toxicity study in rats (28 days): Administration of GSK2256294A (10, 30, 100 mg/kg/day, orally) did not have adverse effects on hematological parameters, serum biochemical indicators (ALT, AST, creatinine), or organ weight[1] - GSK2256294A had a plasma protein binding rate of 92% in human plasma and 90% in rat plasma[1] |
| References | |
| Additional Infomation |
GSK2256294A is a potent, selective, and orally effective soluble epoxide hydrolase (sEH) inhibitor. sEH is an enzyme that catalyzes the hydrolysis of bioactive epoxide eicosatrienoic acid (EET) into less active diols [1]. Its pharmacological effects are achieved by increasing endogenous EET levels, thereby exerting anti-inflammatory, vasodilatory, and cytoprotective effects [1]. Based on preclinical data, GSK2256294A has potential therapeutic value in inflammatory diseases, cardiovascular diseases, and metabolic syndrome [1]. The drug has favorable physicochemical properties, including high water solubility (120 μg/mL) and stability in simulated gastric and intestinal fluids [1].
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| Molecular Formula |
C21H24F3N7O
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| Molecular Weight |
447.47
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| Exact Mass |
447.199
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| CAS # |
1142090-23-0
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| Related CAS # |
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| PubChem CID |
59448236
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| Appearance |
White to off-white solid powder
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| LogP |
3.734
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
32
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| Complexity |
682
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| Defined Atom Stereocenter Count |
2
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| SMILES |
CC1=NC(=NC(=N1)N[C@H]2CCC[C@H](C2)C(=O)NCC3=C(C=C(C=C3)C#N)C(F)(F)F)NC
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| InChi Key |
LQHDJQIMETZMPH-ZBFHGGJFSA-N
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| InChi Code |
InChI=1S/C21H24F3N7O/c1-12-28-19(26-2)31-20(29-12)30-16-5-3-4-14(9-16)18(32)27-11-15-7-6-13(10-25)8-17(15)21(22,23)24/h6-8,14,16H,3-5,9,11H2,1-2H3,(H,27,32)(H2,26,28,29,30,31)/t14-,16+/m1/s1
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| Chemical Name |
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.65 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.65 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (4.65 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2348 mL | 11.1739 mL | 22.3479 mL | |
| 5 mM | 0.4470 mL | 2.2348 mL | 4.4696 mL | |
| 10 mM | 0.2235 mL | 1.1174 mL | 2.2348 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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