| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| Other Sizes |
| Targets |
Ki: 12.2 nM (Neutrophil elastase)[3]
Freselestat selectively targets human neutrophil elastase (HNE), a serine protease released from activated neutrophils during inflammation. HNE is involved in the degradation of extracellular matrix components such as elastin, collagen, and proteoglycans, and contributes to tissue damage in various inflammatory diseases. By inhibiting HNE with high potency (Ki 12.2 nM), Freselestat reduces elastase-mediated tissue destruction and inflammation. |
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| ln Vitro |
In order to create a simulation of extracorporeal circulation, 3.75 U/mL of freshly heparinized human blood are circulated for 120 minutes using a roller pump and membrane oxygenator, both with and without 1.0 μM of Freselestat (ONO-6818). The Freselestat group exhibits significantly reduced levels of neutrophil elastase. Freselestat dramatically lowers the synthesis of C5b-9 and interleukin 8. L-selectin and CD11b alterations during recirculation are not modulated by frecelsestat[3].
In vitro, Freselestat (1.0 uM) significantly reduces neutrophil elastase levels in a simulated extracorporeal circulation model using fresh heparinized human blood circulated for 120 minutes. Freselestat treatment significantly decreases C5b-9 and interleukin-8 (IL-8) synthesis, indicating anti-inflammatory activity. The compound shows no effect on L-selectin and CD11b expression during recirculation, confirming its specific action on elastase rather than general neutrophil activation. |
| ln Vivo |
Freselestat (ONO-6818; oral; 10-100 mg/kg; once day; 8 weeks) Treatment reduces hemoglobin, neutrophil count, and lung myeloperoxidase activity increases in bronchoalveolar lavage fluid that are dose-dependently produced by HNE. ONO-6818 reduces lung bleeding and neutrophil buildup in the lungs to prevent acute lung damage brought on by HNE[1].
In vivo, Freselestat administered orally at doses of 10-100 mg/kg once daily for 8 weeks dose-dependently reduces HNE-induced increases in lung myeloperoxidase activity, hemoglobin levels, and neutrophil count in bronchoalveolar lavage fluid. Freselestat decreases lung hemorrhage and pulmonary neutrophil accumulation, preventing HNE-induced acute lung injury in male Wistar rats. These results demonstrate its potential for treating inflammatory lung diseases. |
| Enzyme Assay |
For enzymatic inhibition assays, incubate purified human neutrophil elastase (0.1-1 nM) with increasing concentrations of Freselestat quarterhydrate (0.1-100 nM) in assay buffer (100 mM HEPES, 500 mM NaCl, 0.05% Brij-35, pH 7.5) for 10-15 minutes at 25degC. Add the fluorogenic substrate MeOSuc-Ala-Ala-Pro-Val-AMC (10-100 uM). Measure fluorescence at excitation 380 nm, emission 460 nm. Calculate inhibition constants (Ki) using the Cheng-Prusoff equation from IC50 values determined by nonlinear regression.
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| Cell Assay |
Neutrophil elastase activity assays are typically cell-free. For cellular studies, culture human neutrophils isolated from fresh whole blood or cell lines such as HL-60 differentiated with DMSO. Plate cells (1 × 10^5 cells/well) and treat with Freselestat (1 nM-10 uM) for 30 minutes at 37degC. Activate neutrophils with PMA (100 ng/mL) or fMLP (1 uM) to induce elastase release. Collect supernatant, add MeOSuc-Ala-Ala-Pro-Val-AMC substrate, and measure fluorescence. Assess cell viability by trypan blue exclusion to confirm that observed effects are not due to cytotoxicity.
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| Animal Protocol |
Animal/Disease Models: Male Wistar rats (228 g) induced by human neutrophil elastase (HNE)[1]
Doses: 10 mg/kg, 100 mg/kg Route of Administration: Oral administration; daily; for 8 weeks Experimental Results: Attenuated dose-dependently HNE-induced increases in lung myeloperoxidase activity, hemoglobin, and neutrophil count in bronchoalveolar lavage fluid. For HNE-induced acute lung injury models, use male Wistar rats (approximately 228 g). Administer Freselestat quarterhydrate by oral gavage at doses of 10 or 100 mg/kg once daily for 8 weeks. Induce lung injury by intratracheal instillation of human neutrophil elastase (HNE) or vehicle control. After treatment, collect bronchoalveolar lavage fluid (BALF) to measure neutrophil count, hemoglobin, and myeloperoxidase activity. Harvest lung tissues for histopathological analysis. Assess lung hemorrhage by Evans blue dye extravasation or histological scoring. |
| ADME/Pharmacokinetics |
Pharmacokinetic properties of Freselestat quarterhydrate have been characterized in preclinical species. The compound is orally active, indicating good oral bioavailability. Following oral administration, Freselestat achieves sufficient systemic exposure to inhibit neutrophil elastase in lung tissue. The quarterhydrate formulation enhances stability and solubility. Molecular weight is 457.03, and the compound is soluble in DMSO. Storage conditions: Powder at -20degC for up to 3 years, or 4degC for 2 years; in solvent at -80degC for 6 months or -20degC for 1 month, protected from light and moisture.
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| Toxicity/Toxicokinetics |
Freselestat quarterhydrate has a favorable toxicity profile based on preclinical studies. At the effective oral dose of 10-100 mg/kg, no significant acute toxicity was reported in rats. The compound is highly selective for neutrophil elastase with >100-fold selectivity over other proteases, reducing the risk of off-target toxicity. No genotoxicity or carcinogenicity data are publicly available. Standard laboratory safety precautions should be used. Avoid inhalation of fine powder. Use personal protective equipment (gloves, lab coat, safety glasses).
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| References | |
| Additional Infomation |
Freselestat quarterhydrate is also known by synonyms ONO-6818 quarterhydrate and ONO-PO-736 quarterhydrate. The compound was developed by ONO Pharmaceutical Co. as a therapeutic candidate for inflammatory lung diseases such as acute lung injury, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. The quarterhydrate form contains 0.25 equivalents of water per Freselestat molecule, providing improved crystallinity. The compound is exclusively for research use and not for human therapeutic applications.
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| Molecular Formula |
C23H28N6O4.1/4H2O
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| Molecular Weight |
457.03
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| Related CAS # |
Freselestat;208848-19-5
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| Appearance |
Typically exists as solid at room temperature
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.1880 mL | 10.9402 mL | 21.8804 mL | |
| 5 mM | 0.4376 mL | 2.1880 mL | 4.3761 mL | |
| 10 mM | 0.2188 mL | 1.0940 mL | 2.1880 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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