In vitro, Ethyl Pyruvate (10 mM) significantly suppressed LPS (1 μg/mL, 4 h) and ATP (5 mM, 1 h)-induced NLRP3 inflammasome activation in N9 microglial cells, decreasing active caspase-1, IL-1β and IL-18 secretion, and reducing pyroptotic cell death. [2]
EP (10 mM) reduced intracellular and mitochondrial ROS production and restored mitochondrial membrane potential disrupted by LPS/ATP in N9 cells. [2]
EP (10 mM) decreased HMGB1 upregulation and nuclear NF-κB translocation, and reversed the downregulation of miR-223 expression induced by LPS/ATP in N9 cells. [2]
In RAW 264.7 murine macrophage-like cells, EP inhibited LPS-induced TNF release and decreased TNF mRNA levels, blocked NF-κB DNA binding, and downregulated phosphorylation of p38 MAPK. [1]
In LPS-stimulated RAW 264.7 cells, EP inhibited HMGB1 release. [1]
In Caco-2 human enterocyte-like cell monolayers, EP (concentrations not specified, but up to 10 mM range) prevented cytomix (IFN-γ, IL-1β, TNF)-induced hyperpermeability, whereas sodium pyruvate did not. [1]
In primary rat microglia cells, EP (5-20 mM) significantly inhibited LPS-induced nitric oxide production, while sodium pyruvate had no effect. [1]
In PC12 cells, EP protected against dopamine-induced apoptosis. [1]

In a murine model of hemorrhagic shock, resuscitation with EP solution (instead of Ringer's lactate) markedly improved 24-h survival, decreased NF-κB activation in liver and colonic mucosa, and reduced expression of iNOS, TNF, COX-2, and IL-6. [1]
In a rat model of LPS-induced shock, resuscitation with EP solution (volume titrated to maintain MAP >60 mmHg, total 7 mL/kg) prolonged survival time, decreased circulating nitrite/nitrate and IL-6, and increased IL-10. [1]
In mice challenged with a lethal dose of LPS, pretreatment with EP significantly improved survival and decreased circulating TNF. Treatment with EP 4 h after LPS injection also improved survival. [1]
In a mouse cecal ligation and puncture (CLP) model of severe sepsis, treatment with EP (40 mg/kg per dose, intraperitoneal, five doses at 24, 30, 36, 42, 48 h after CLP) significantly improved survival (P<0.005). EP also decreased circulating HMGB1 levels. [1]
In aged mice with CLP-induced sepsis, a single intraperitoneal dose of EP immediately after surgery inhibited acute renal failure; benefit was still apparent when treatment was delayed until 12 h after infection. EP decreased kidney expression of TNF, tissue factor, and plasminogen activator inhibitor-1. [1]
In a porcine model of sustained endotoxemia, delayed treatment with continuous infusion of EP (beginning 12 h after LPS) significantly increased PaO2/FiO2 ratio (measure of acute lung injury), increased diuresis, ameliorated metabolic acidosis, and decreased lipid peroxidation. [1]
In a rat model of permanent middle cerebral artery occlusion (pMCAO, stroke), EP (40 mg/kg IP) administered 30 min before or 4, 12, or even 24 h after occlusion reduced infarct volume and suppressed neurological deficits. Sodium pyruvate (500 mg/kg) was ineffective when delayed until 4 h. [1]
In mice, EP (40 mg/kg IP) attenuated kainic acid-induced hippocampal neuronal cell death. [1]
In a mouse model of colitis (IL-10 deficient mice), EP ameliorated gut mucosal damage. [1]
In a rat model of mesenteric ischemia/reperfusion, EP solution (28 mmol/L) preserved ileal mucosal architecture better than equimolar pyruvate. [1]

For N9 microglial cells: Cells were seeded at 5×10^3 cells/well in 96-well plates for viability, or 1×10^6 cells/flask for other assays. Ethyl Pyruvate (10 mM) was pretreated for 1 hour, then cells were treated with LPS (1 μg/mL) for 4 hours, followed by ATP (5 mM) for 1 hour. Cell viability was measured using Presto Blue reagent (excitation 560 nm, emission 590 nm). Pyroptotic cell death was assessed by propidium iodide (50 μg/mL) staining and fluorescence microscopy. ASC speck formation was visualized by confocal microscopy after fixing with 4% paraformaldehyde, permeabilizing with 0.5% Triton X-100, blocking with 10% FBS, and incubating with ASC primary antibody (1:100) followed by Alexa Fluor 594-conjugated secondary antibody (1:1000). Intracellular ROS was measured using CM-H2DCFDA dye (15 min incubation, fluorometric reading). Mitochondrial ROS was measured using MitoSOX (5 μM, 15 min). Mitochondrial membrane potential was analyzed using JC-1 stain (2.5 μg/mL) by flow cytometry (488 nm laser). Western blotting was performed on cell lysates (RIPA buffer) and nuclear/cytosolic fractions (NE-PER). Proteins were separated by 10% SDS-PAGE, transferred to PVDF membranes, blocked with 5% milk, and probed with primary antibodies against IL-1β, caspase-1, NLRP3, HMGB1, NF-κB p65, IκBα, β-actin, lamin A/C, followed by HRP-conjugated secondary antibodies, and visualized by chemiluminescence. qPCR was performed for IL-1β, IL-18, NLRP3 (SYBR green, normalized to GAPDH) and for miR-223 (TaqMan, normalized to U6). For miRNA inhibition, cells were transfected with miR-223 antagomiR or negative control (50 nM) using HiPerFect transfection reagent for 24 h before EP and inflammasome activation. [2]
For RAW 264.7 cells: Cells were stimulated with LPS and incubated with EP; TNF release was measured, TNF mRNA by Northern blot, NF-κB DNA binding by EMSA, p38 MAPK phosphorylation by Western blot. [1]
For Caco-2 cells: Monolayers were incubated with cytomix (IFN-γ, IL-1β, TNF) with or without EP; permeability was assessed by transepithelial flux of FITC-dextran (4 kDa) added to apical compartments. [1]

In the mouse CLP model: Sepsis was induced by cecal ligation and puncture. Ethyl Pyruvate was administered intraperitoneally at doses of 0.4, 4, or 40 mg/kg per dose, with five doses given at 24, 30, 36, 42, and 48 hours after CLP. Survival was monitored. [1]
In the rat pMCAO stroke model: Permanent middle cerebral artery occlusion was performed. EP (40 mg/kg) was injected intraperitoneally 30 minutes before, or 4, 12, or 24 hours after occlusion. Infarct volume was measured by 2,3,5-triphenyl tetrazolium chloride staining at 2 days post-occlusion. [1]
In the porcine endotoxemia model: Anesthetized pigs received LPS infusion. Delayed treatment with a continuous infusion of EP was started 12 hours after LPS challenge; the infusion rate and volume were titrated to maintain MAP. PaO2/FiO2 ratio, diuresis, metabolic acidosis, and lipid peroxidation were measured. [1]
In the rat hemorrhagic shock model: Resuscitation with Ringer's ethyl pyruvate solution (containing 28 mmol/L EP) was compared to Ringer's lactate. Volume was titrated to maintain MAP. [1]
In the mouse LPS-induced lethality model: Mice were injected with a lethal dose of LPS. EP was administered either as pretreatment (before LPS) or 4 hours after LPS injection. Survival was recorded. [1]

[1]. Fink MP. Ethyl pyruvate: a novel anti-inflammatory agent. J Intern Med. 2007 Apr;261(4):349-62.

[2]. Ethyl Pyruvate Attenuates Microglial NLRP3 Inflammasome Activation via Inhibition of HMGB1/NF-κB/miR-223 Signaling. Antioxidants (Basel). 2021;10(5):745.

[3]. Ethyl pyruvate administration inhibits hepatic tumor growth. J Leukoc Biol. 2009 Sep;86(3):599-607.

[4]. Ethyl pyruvate confers protection against endotoxemia and sepsis by inhibiting caspase-11-dependent cell pyroptosis. Int Immunopharmacol. 2020 Jan;78:106016.

[5]. Protective effects of ethyl pyruvate treatment on paraquat-intoxicated rats. Hum Exp Toxicol. 2008 Jan;27(1):49-54.

Ethyl pyruvate is a carbonyl carboxylic acid. CTI-01 (ethyl pyruvate) is a novel anti-inflammatory drug used to treat severe inflammatory diseases. CTI-01 has shown potent anti-inflammatory and tissue-protective activity in various animal disease models, including pancreatitis, ischemia-reperfusion injury, sepsis, kidney injury, and endotoxemia. Ethyl pyruvate has been reported to be found in Zanthoxylum schinifolium, and relevant data exist. Drug Indications It has been studied for the treatment of burns and burn infections, cardiac surgery, inflammatory diseases (not specified), ischemia-reperfusion injury, and sepsis and septicemia. Mechanism of Action CTI-01 inhibits the systemic release of cytokines such as TNF-α and HMGB1, which promote the body's inflammatory response. Overexpression of these cytokines is associated with diseases occurring in intensive care settings, such as severe organ damage following cardiopulmonary bypass (CPB) and postoperative bowel obstruction following abdominal surgery.

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In a Phase I multiple-dose safety study in healthy human volunteers, a proprietary formulation of EP (CTI-01) was safe and well-tolerated at doses exceeding the therapeutic range seen in preclinical models. [1]
In a double-blind, randomized, placebo-controlled clinical trial in patients undergoing cardiac surgery with cardiopulmonary bypass (n=102, planned 150), EP showed no significant safety concerns; the adverse event profile was similar to placebo. The trial was stopped early due to packaging problems, and no efficacy signal was observed. [1]
EP has been tested in a wide range of preclinical models including sepsis, hemorrhagic shock, stroke, acute lung injury, acute pancreatitis, mesenteric ischemia/reperfusion, hepatic ischemia/reperfusion, myocardial ischemia, necrotizing pancreatitis, alcohol-induced liver injury, common bile duct ligation, burn injury, and colitis, generally showing beneficial effects. Negative results were reported in two porcine models of hemorrhagic shock. [1]
EP is a lipophilic ester of pyruvic acid with calculated log P of 0.58 (pyruvic acid log P = -0.68), suggesting better membrane permeability than pyruvate. Its anti-inflammatory mechanism involves covalent modification of Cys38 in the p65 subunit of NF-κB, inhibiting DNA binding. EP also scavenges H2O2 and other ROS, but its anti-inflammatory effects are not solely due to antioxidant activity. EP enolizes to form ethyl-2-hydroxypropenoate, a Michael acceptor that may alkylate target proteins. [1]
EP suppresses NLRP3 inflammasome activation in microglial cells via upregulation of miR-223 and inhibition of HMGB1/NF-κB signaling. [2]

116.047

617-35-6

Ethyl pyruvate-d3;66966-38-9

12041

Colorless to light yellow liquid

1.0±0.1 g/cm3

155.0±0.0 °C at 760 mmHg

-58 °C

45 ºC

3.1±0.2 mmHg at 25°C

1.399

0.05

0

3

3

8

106

0

XXRCUYVCPSWGCC-UHFFFAOYSA-N

InChI=1S/C5H8O3/c1-3-8-5(7)4(2)6/h3H2,1-2H3

ethyl 2-oxopropanoate

CTI-01 CTI 01 CTI01

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)

H2O : ~100 mg/mL (~861.18 mM)
DMSO : ~100 mg/mL (~861.18 mM)

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.)