Human neutrophil elastase
Even at 100 μM, sivelestat (ONO-5046) does not inhibit chymotrypsin, cathepsin G, pancreatic kallikrein, thrombin, plasmin, plasma kallikrein, or plasma kallikrein[1]. The IC50 values of Sivelestat (ONO-5046) for human, rabbit, rat, hamster, and mouse neutrophil elastase are 44 nM, 36 nM, 19 nM, 37 nM, and 49 nM, respectively[1].

Even at 100 μM, sivelestat (ONO-5046) does not inhibit chymotrypsin, cathepsin G, pancreatic kallikrein, thrombin, plasmin, plasma kallikrein, or plasma kallikrein[1]. The IC50 values of Sivelestat (ONO-5046) for human, rabbit, rat, hamster, and mouse neutrophil elastase are 44 nM, 36 nM, 19 nM, 37 nM, and 49 nM, respectively[1].

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Sivelestat sodium tetrahydrate (ONO-5046) potently inhibited human neutrophil elastase (HNE) activity, with an IC50 of 0.044 ± 0.003 µM (using the synthetic substrate suc-Ala-Pro-Ala-p-nitroanilide) and a Ki of 0.20 ± 0.02 µM. The inhibition was determined to be competitive via Lineweaver-Burk plot analysis. [1]
When using the macromolecular substrate elastin-congo red, the IC50 was 7.0 ± 0.1 µM. The difference was attributed to the much higher enzyme concentration required for the elastin-congo red assay. [1]
The compound demonstrated broad cross-species activity, effectively inhibiting neutrophil elastase extracted from rabbits, rats, hamsters, and mice with IC50 values similar to that for HNE. [1]
It exhibited high specificity for neutrophil elastases over other serine proteases, showing no inhibition up to 100 µM against trypsin, thrombin, plasmin, plasma and pancreatic kallikrein, chymotrypsin, and cathepsin G. [1]
It inhibited porcine pancreatic elastase with an IC50 of 5.6 ± 0.2 µM, which is about 130 times higher than its IC50 for human neutrophil elastase, underscoring its selectivity for neutrophil-derived enzymes. [1]

Human neutrophil elastase induces both the suppression of lung hemorrhage in hamsters (ID50 = 82 pg/kg) and the increase of skin capillary permeability in guinea pigs (ID50 = 9.6 mg/kg) when sedelestat (ONO-5046, 0.021-2.1 mg/kg, intratracheally) is administered intravenously[1]. In rats, sivelestat (10 mg/kg) infused via the tail vein reduces lung damage following hemorrhagic shock[2]. In the rat bladder, ivelestat (15, 60 mg/kg, ip) prevents ischemia-reperfusion injury[3].

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Hemorrhagic shock followed by resuscitation (HSR) causes neutrophil sequestration in the lung which leads to acute lung injury (ALI). Neutrophil elastase (NE) is thought to play a pivotal role in the pathogenesis of ALI. This study investigated whether sivelestat, a specific NE inhibitor, can attenuate ALI induced by HSR in rats. Male Sprague-Dawley rats were subjected to hemorrhagic shock by withdrawing blood so as to maintain a mean arterial blood pressure of 30+/-5 mm Hg for 60 min followed by resuscitation with the shed blood. HSR-treated animals received a bolus injection of sivelestat (10 mg/kg) intravenously at the start of resuscitation followed by continuous infusion for 60 min (10 mg/kg/h) during the resuscitation phase, or the vehicle. Lung injury was assessed by pulmonary histology, lung wet-weight to dry-weight (W/D) ratio, myeloperoxidase (MPO) activity, gene expression of tumor necrosis factor (TNF)-alpha and inducible nitric oxide synthase (iNOS), DNA binding activity of nuclear factor (NF)-kappaB, and immunohistochemical analysis of intercellular adhesion molecule (ICAM)-1. HSR treatment induced lung injury, as demonstrated by pulmonary edema with infiltration of neutrophils, the increase in lung W/D ratio, MPO activity, gene expression of TNF-alpha and iNOS, and DNA-binding activity of NF-kappaB, and enhanced expression of ICAM-1. In contrast, sivelestat treatment significantly ameliorated the HSR-induced lung injury, as judged by the marked improvement in all these indices. These results indicate that sivelestat attenuated HSR-induced lung injury at least in part through an inhibition of the inflammatory signaling pathway, in addition to the direct inhibitory effect on NE.[2]

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In the present study, we evaluated the effect of neutrophil elastase inhibitor, sivelestat sodium hydrate on ischemia-reperfusion injury in the rat bladder. Rat abdominal aorta was clamping with a small clip to induce ischemia-reperfusion injury in the bladder. Eight-week-old male Sprague Dawley rats were divided into four groups; sham-operated control rats, 30 min ischemia-60 min reperfusion (IR) rats, and IR rats treated with 15 or 60 mg/kg of sivelestat sodium hydrate. Sixty minutes prior to induction of ischemia, sivelestat sodium hydrate was administrated intraperitoneally. Real-time monitoring of blood flow and nitric oxide (NO) release were measured simultaneously with a laser Doppler flowmeter and an NO-selective electrode, respectively. The NO2-NO3 and malonaldehyde (MDA) concentrations were measured in the experimental urinary bladders. Clamping of the abdominal aorta, blood flow was rapidly decreased and NO release was gradually increased. After removing the clip, blood flow was rapidly increased and NO release was gradually returned to the basal level. These movements of blood flow and NO release were inhibited by treatment with sivelestat sodium hydrate in a dose-dependent manner. Both NO2-NO3 and MDA concentrations in the bladder were increased by induction of IR, and NO2-NO3 and MDA concentrations were decreased by treatment with high dose of sivelestat sodium hydrate significantly. Our data indicated that sivelestat sodium hydrate could inhibit increasing NO2-NO3 and MDA concentrations by IR, and it has potentiality protective effects on IR injury in the rat urinary bladder.[3]

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In a hamster model of HNE-induced lung hemorrhage, intratracheal administration of Sivelestat sodium tetrahydrate (ONO-5046) 5 minutes prior to HNE instillation significantly and dose-dependently suppressed hemorrhage. The ID50 was calculated to be 82 µg/kg. [1]
In a guinea pig model of HNE-induced increase in skin capillary permeability, intravenous administration of Sivelestat sodium tetrahydrate (ONO-5046) 30 seconds prior to HNE injection significantly and dose-dependently suppressed dye leakage. The ID50 was calculated to be 9.6 mg/kg. This effect was specific to HNE, as the compound did not suppress trypsin-induced permeability increases. [1]

ONO-5046, N-[2-[4-(2,2-Dimethylpropionyloxy)phenylsulfonylamino] aminoacetic acid, competitively inhibited human neutrophil elastase (IC50 = 0.044 microM, Ki = 0.2 microM). It also inhibited leukocyte elastase obtained from rabbit, rat, hamster and mouse. However, ONO-5046 did not inhibit trypsin, thrombin, plasmin, plasma kallikrein, pancreas kallikrein, chymotrypsin and cathepsin G even at 100 microM[1].

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The inhibitory activity against human neutrophil elastase (HNE) was determined using two substrates. Method 1 (synthetic substrate): HNE was incubated with the synthetic substrate suc-Ala-Pro-Ala-p-nitroanilide in Tris-HCl buffer (pH 8.0) containing sodium chloride. After incubation at 37°C, the release of p-nitroaniline was measured spectrophotometrically at 405 nm. [1]
Method 2 (macromolecular substrate): HNE was incubated with the substrate elastin-congo red in Tris-HCl buffer (pH 8.0) containing sodium chloride. After incubation at 37°C, the absorbance of the supernatant was measured at 500 nm to assess elastin degradation. [1]
Inhibition constants (IC50, Ki) were derived from these assays. The inhibition type was determined by Lineweaver-Burk plot analysis using the synthetic substrate. [1]
Inhibitory activity against other proteases (trypsin, thrombin, plasmin, kallikreins, chymotrypsin, cathepsin G) was assessed using their respective chromogenic or fluorogenic substrates under appropriate buffer conditions. [1]
Inhibitory activity against porcine pancreatic elastase was assessed using the synthetic substrate succinyl-Ala-Ala-Ala-p-nitroanilide in HEPES-NaOH buffer containing calcium chloride, measuring absorbance at 405 nm. [1]
Neutrophil elastase from other species (rabbit, rat, hamster, mouse) was obtained from peritoneal neutrophils. The cells were lysed by sonication, and the lysate was used as the enzyme source. Inhibitory activity was tested using the synthetic substrate suc-Ala-Pro-Ala-p-nitroanilide as described for HNE. [1]

Animal/Disease Models: Male Golden hamsters, weighing 90 to 110 g[1].
Doses: 0.021-2.1 mg/kg.
Route of Administration: Intratracheally five min before HNE injection.
Experimental Results: Dramatically and dosedependently suppressed the lung hemorrhage.

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Animal/Disease Models: Male SD (Sprague-Dawley) rats weighing 350-400 g[2].
Doses: 10 mg/kg.
Route of Administration: Continuous infusion via the tail vein at 10 mg/kg/h for 60 min during the resuscitation phase.
Experimental Results: Greatly suppressed lung injury, as revealed by the decreased histological damage. Dramatically ameliorated HSR-induced lung injury. Markedly diminished the levels of TNF-α and iNOS gene.

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Animal/Disease Models: Male Sprague Dawley rats, 8 weeks old and weighing 250-320 g[3].
Doses: 15 mg/kg or 60 mg/kg.
Route of Administration: IP.
Experimental Results: diminished the blood flow in the bladder during reperfusion phase compared to the IR group.

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For the lung hemorrhage model in hamsters: Male Golden hamsters (90-110 g) were anesthetized. Human neutrophil elastase (HNE, 0.3 U in 150 µl PBS) was instilled intratracheally via a catheter. Sivelestat sodium tetrahydrate (ONO-5046) was administered intratracheally as a solution/suspension (vehicle not specified in detail) 5 minutes before HNE injection. One hour after HNE, animals were sacrificed, lungs were homogenized in water, and hemoglobin content in the supernatant was measured at 412 nm as an index of hemorrhage. [1]
For the skin capillary permeability model in guinea pigs: Male Hartley guinea pigs (250-350 g) received an intradermal injection of HNE (20 x 10^-3 U in 100 µl PBS) on the shaved back. Evans blue dye was then injected intravenously. Sivelestat sodium tetrahydrate (ONO-5046) was administered intravenously (dissolved in saline, 1 ml/kg as vehicle control volume) 30 seconds before HNE injection. Thirty minutes after dye injection, animals were sacrificed, the blue skin areas were excised, solubilized, and the extracted dye was quantified spectrophotometrically at 620 nm. [1]

The manuscript reported that intravenous administration of 30 mg/kg of cefoperazone sodium tetrahydrate (ONO-5046) had no significant effect on the blood pressure of guinea pigs. [1]

[1]. ONO-5046, a novel inhibitor of human neutrophil elastase. Biochem Biophys Res Commun. 1991 Jun 14;177(2):814-20.

[2]. A neutrophil elastase inhibitor, sivelestat, ameliorates lung injury after hemorrhagic shock in rats. Int J Mol Med. 2007 Feb;19(2):237-43.

[3]. Neutrophil elastase inhibitor, sivelestat sodium hydrate prevents ischemia-reperfusion injury in the rat bladder. Mol Cell Biochem. 2008 Apr;311(1-2):87-92.

[4]. Neutrophil elastase inhibitor (sivelestat) may be a promising therapeutic option for management of acute lung injury/acute respiratory distress syndrome or disseminated intravascular coagulation in COVID-19. J Clin Pharm Ther. 2020 Dec;45(6):1515-1519.

Sivelestat sodium hydrate is an N-acylglycine with functions similar to N-benzoylglycine. ONO-5046, N-[2-[4-(2,2-dimethylpropionyloxy)benzenesulfonylamino]aminoacetic acid], competitively inhibits human neutrophil elastase (IC50 = 0.044 μM, Ki = 0.2 μM). It also inhibits leukocyte elastase in rabbits, rats, hamsters, and mice. However, even at concentrations up to 100 μM, ONO-5046 did not inhibit trypsin, thrombin, plasmin, plasma kallikrein, pancreatic kallikrein, chymotrypsin, or cathepsin G. In in vivo studies, intratracheal administration of ONO-5046 inhibited pulmonary hemorrhage in hamsters (ID50 = 82 μg/kg) and intravenous administration increased capillary permeability in guinea pigs (ID50 = 9.6 mg/kg), both induced by human neutrophil elastase. [1]

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Known information and purpose: This article summarizes the effects of sivelestat on acute lung injury/acute respiratory distress syndrome (ALI/ARDS) or ARDS with coagulopathy, both of which are common in COVID-19 patients. Comment: COVID-19 patients are more prone to thromboembolic events, including disseminated intravascular coagulation (DIC). Several studies have highlighted the role of neutrophil elastase (NE) in the development of DIC in patients with ARDS and sepsis. Studies have shown that sivelestat can alleviate acute lung injury (ALI) by inhibiting neutrophil elastase (NE), and its mechanism includes improving alveolar epithelial and vascular endothelial damage, as well as reversing neutrophil-mediated vascular permeability. Recent progress and conclusions: Sivelestat is a selective NE inhibitor, but its potential therapeutic effect on SARS-CoV-2 infection has not been evaluated. Given its good efficacy in COVID-19 complications, sivelestat is expected to become an effective means of treating COVID-19-induced ALI/ARDS or coagulation dysfunction. Keywords: COVID-19; acute lung injury/acute respiratory distress syndrome; coagulation dysfunction; neutrophil elastase inhibitor sivelestat [4]

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Sivelestat sodium tetrahydrate (referred to in the literature by its research number ONO-5046) has the chemical name N-[2-[4-(2,2-dimethylpropionyloxy)benzenesulfonylamino]benzoyl]aminoacetic acid. It is a low molecular weight synthetic inhibitor. [1]
This compound was identified as a potent, specific, and systemically active (intravenous) neutrophil elastase inhibitor, distinguishing it from many similar inhibitors that exert their effects primarily through local (e.g., intratracheal) administration. [1]
It has the advantage of being able to enter and inhibit elastase in the tight contact area between neutrophils and the extracellular matrix, areas that macromolecular inhibitors may be unable to reach. [1]
This study demonstrates that this approach can serve as a pharmacological tool for investigating the role of neutrophil elastase in animal models of diseases such as emphysema, adult respiratory distress syndrome (ARDS), and septic shock. [1]

C20H21N2O7S.4H2O.NA

528.51

528.138

C, 45.45; H, 5.53; N, 5.30; Na, 4.35; O, 33.30; S, 6.07

201677-61-4

Sivelestat;127373-66-4;Sivelestat sodium;150374-95-1

23663985

Typically exists as white to off-white solids at room temperature

1.4±0.1 g/cm3

1.598

2.206

6

12

9

35

738

0

S(C1C([H])=C([H])C(=C([H])C=1[H])OC(C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])=O)(N([H])C1=C([H])C([H])=C([H])C([H])=C1C(N([H])C([H])([H])C(=O)[O-])=O)(=O)=O.[Na+].O([H])[H].O([H])[H].O([H])[H].O([H])[H]

PLHREJBSQUSUCW-UHFFFAOYSA-M

InChI=1S/C20H22N2O7S.Na.4H2O/c1-20(2,3)19(26)29-13-8-10-14(11-9-13)30(27,28)22-16-7-5-4-6-15(16)18(25)21-12-17(23)24;;;;;/h4-11,22H,12H2,1-3H3,(H,21,25)(H,23,24);;4*1H2/q;+1;;;;/p-1

sodium (2-((4-(pivaloyloxy)phenyl)sulfonamido)benzoyl)glycinate tetrahydrate.

Sivelestat sodium tetrahydrate; Sivelestat Sodium Hydrate; Sivelestat sodium, ONO5046-Na, Sodium sivelestat, EI546 sodium, LY544349 sodium; ONO5046, LY544349, EI546; ONO 5046; 201677-61-4; Sivelestat sodium tetrahydrate; sivelestat sodium hydrate; Elaspol; ONO-5046.Na; LY544349 Sodium Hydrate; Sivelestat (sodium tetrahydrate); Sivelestat sodium [USAN]; ONO5046; ONO-5046; LY544349; LY-544349; LY 544349; EI 546 sodium salt hydrate, Elaspol sodium salt hydrate, LY 544349 sodium salt hydrate, Trade name: Elaspol.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.73 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: ≥ 2.08 mg/mL (3.94 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.94 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 20.8 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.)