| Targets |
NT-0249 targets the NLRP3 inflammasome, a multiprotein complex that plays a central role in innate immunity and the pathogenesis of several inflammatory and metabolic diseases. Activation of the NLRP3 inflammasome leads to the activation of caspase‑1 and the maturation and release of the pro‑inflammatory cytokines IL‑1beta and IL‑18. NT-0249 is a small‑molecule inhibitor of the NLRP3 inflammasome, and its free base form is orally active. By inhibiting NLRP3 activation, NT-0249 reduces the production of IL‑1beta and IL‑18, thereby reducing inflammation.
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| ln Vitro |
NT-0249 free base is an orally active NLRP3 inhibitor with anti‑inflammatory activity. It is used in research on NLRP3‑mediated inflammatory diseases. In vitro, NT-0249 inhibits the activation of the NLRP3 inflammasome in immune cells, reducing the production of IL‑1beta. No specific IC₅0 values are reported. The free base form is the non‑salt form of the compound, which has different solubility and stability properties compared to the salt forms.
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| ln Vivo |
NT-0249 (100 mg/kg, PO, 3 times daily for 28 days) reduces systemic inflammation and astrocyte formation in mice in a pre-established obese state [1].
NT-0249 (100 mg/kg, orally, 3 times a day for 28 days) reduces systemic inflammation and astrocyte formation in mice with pre‑established obesity. The compound is administered orally, indicating good oral bioavailability. In the obesity model, NT-0249 reduces the expression of inflammatory markers and reduces the activation of astrocytes in the brain. The compound is used in research on NLRP3‑mediated inflammatory diseases, including obesity‑associated neuroinflammation, and may have applications in other NLRP3‑driven conditions such as Alzheimer‘s disease, Parkinson‘s disease, and diabetes. |
| Enzyme Assay |
The binding of NT-0249 to the NLRP3 protein is measured by standard in vitro biochemical assays using purified NLRP3 protein. However, the NLRP3 inflammasome is a large multiprotein complex, and cell‑based functional assays are more commonly used to measure inhibition. For binding studies, surface plasmon resonance (SPR) could be used to measure direct binding of NT-0249 to NLRP3, but no specific KD values are reported. The compound is characterized by HPLC and mass spectrometry to confirm purity (≥98%).
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| Cell Assay |
For cellular assays, human THP‑1 cells (differentiated into macrophage‑like cells with PMA) or primary human macrophages are seeded in 96‑well plates. Cells are primed with LPS (1 microg/mL, 4 h) and then activated with ATP (5 mM) or nigericin (10 microM) to induce NLRP3 inflammasome activation. NT-0249 is added at graded concentrations (0.1‑100 microM) 30‑60 min before the addition of the activator. After 30‑60 min, IL‑1beta release in the supernatant is measured by ELISA. Caspase‑1 activation is assessed by Western blot for cleaved caspase‑1 (p20). Cell viability is assessed by MTT or LDH assays.
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| Animal Protocol |
Animal/Disease Models:Diet-Induced Obesity (DIO) in mice[1]
Doses: 100 mg/kg Route of Administration: p.o. 3 times a day for 28 days Experimental Results: Reduced inflammatory biomarkers sVCAM-1 and suPAR. For in vivo evaluation of obesity‑associated inflammation, 6‑8‑week‑old male C57BL/6 mice are fed a high‑fat diet (HFD) for 12‑16 weeks to induce obesity and associated systemic inflammation. NT-0249 is administered orally by gavage at a dose of 100 mg/kg, three times daily (every 8 h), for 28 days. Blood is collected for measurement of systemic inflammatory markers (e.g., TNF‑alpha, IL‑6, IL‑1beta, MCP‑1) by ELISA. Brains are harvested for immunohistochemistry for GFAP (astrocyte activation marker) and Iba‑1 (microglial activation marker). Hypothalamic levels of IL‑1beta are measured by ELISA. Body weight, food intake, and glucose tolerance are monitored. The study demonstrates that NT-0249 reduces systemic inflammation and astrocyte formation. |
| ADME/Pharmacokinetics |
NT-0249 free base (C22H2₉N₆O4S, MW = 459.56, purity ≥98%) is a solid powder. For storage, the powder should be kept at -20 degC for up to 3 years, sealed and protected from light. For in vitro use, stock solutions in DMSO (100 mg/mL, 217.60 mM) can be prepared with sonication and stored at -80 degC for up to 6 months or at -20 degC for 1 month. For in vivo oral administration, it can be formulated in 0.5% methylcellulose/0.1% Tween‑80 or in other suitable vehicles. No detailed PK parameters are reported, but the compound is described as orally active.
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| Toxicity/Toxicokinetics |
No specific toxicity data for NT-0249 free base are reported. As a research‑grade NLRP3 inhibitor, it is not intended for human or veterinary use. Standard laboratory safety precautions for handling chemicals should be followed. NLRP3 inflammasome inhibitors are generally considered to have a favorable safety profile, but chronic inhibition may increase susceptibility to infections. No LD₅0 or formal toxicology studies are available.
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| References | |
| Additional Infomation |
NT-0249 free base is a research‑grade, orally active NLRP3 inflammasome inhibitor. The NLRP3 inflammasome is a key mediator of neuroinflammation and has been implicated in the pathogenesis of several metabolic and neurodegenerative diseases, including obesity, type 2 diabetes, Alzheimer‘s disease, Parkinson‘s disease, and multiple sclerosis. NT-0249 has been studied in preclinical models of obesity‑associated inflammation and has been shown to reduce systemic inflammation and astrocyte formation. The free base form of the compound is the non‑salt form, which may have different bioavailability and solubility compared to the salt forms. The compound is for research use only and has not received regulatory approval.
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| Appearance |
Solid powder
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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 |
| 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) |
DMSO : 100 mg/mL (217.60 mM; with sonication)
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
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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