Human neutrophil elastase (IC50 = 44 nM; Ki=200 nM)
Sivelestat sodium targets human neutrophil elastase (HNE) with an IC50 of 0.26 μM[1]
Sivelestat sodium shows low affinity for porcine pancreatic elastase (IC50 > 100 μM) and human cathepsin G (IC50 > 100 μM)[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 (formerly designated ONO-5046) inhibited HNE activity in a concentration-dependent manner, with 50% inhibition at 0.26 μM and 90% inhibition at 1 μM[1]
- The compound exhibited high selectivity for HNE, as it did not significantly inhibit other serine proteases (e.g., trypsin, chymotrypsin) even at concentrations up to 100 μM[1]
- In human neutrophil cultures, Sivelestat sodium (1–10 μM) suppressed HNE release induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) by 30–60%[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 rat model of hemorrhagic shock-induced lung injury: Intravenous administration of Sivelestat sodium at 10 mg/kg (given 30 minutes before resuscitation) reduced lung wet/dry weight ratio by 25%, decreased pulmonary vascular permeability by 30%, and lowered serum levels of TNF-α and IL-6 by 40% and 35%, respectively, compared to vehicle control[2]
- Histopathological analysis of lung tissues from treated rats showed reduced neutrophil infiltration (by 50%) and alveolar wall thickening[2]
- In a rat model of bladder ischemia-reperfusion injury: Intraperitoneal administration of Sivelestat sodium at 5 mg/kg (given 30 minutes before ischemia and again at reperfusion) decreased bladder tissue malondialdehyde (MDA) content by 45% and increased superoxide dismutase (SOD) activity by 60% compared to vehicle group[3]
- The compound ameliorated bladder tissue damage, as evidenced by reduced epithelial cell necrosis, edema, and inflammatory cell infiltration (histological score decreased by 55%)[3]

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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HNE activity inhibition assay: Human neutrophil elastase was incubated with serial concentrations of Sivelestat sodium and a synthetic substrate (Succinyl-Ala-Ala-Ala-p-nitroanilide) in assay buffer at 37°C for 60 minutes. The release of p-nitroaniline was measured by spectrophotometry at 405 nm. Inhibition rate was calculated relative to the vehicle control, and IC50 value was determined by nonlinear regression analysis[1]
- Selectivity assay: The inhibitory effect of Sivelestat sodium (100 μM) on other proteases (porcine pancreatic elastase, human cathepsin G, trypsin, chymotrypsin) was evaluated using their respective specific substrates under the same incubation conditions. The activity was measured spectrophotometrically, and inhibition rate was calculated[1]

Neutrophil HNE release assay: Human neutrophils were isolated and suspended in culture medium. Cells were pretreated with Sivelestat sodium (1, 3, 10 μM) for 30 minutes, then stimulated with fMLP (100 nM) for 1 hour. The culture supernatant was collected, and HNE activity in the supernatant was measured using the succinyl-Ala-Ala-Ala-p-nitroanilide substrate assay. The amount of released HNE was calculated by comparing to a standard curve[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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Rat hemorrhagic shock-induced lung injury model: Male Wistar rats (250–300 g) were anesthetized and subjected to hemorrhagic shock by withdrawing blood to maintain mean arterial pressure at 40 mmHg for 60 minutes. Sivelestat sodium (10 mg/kg) was dissolved in normal saline and administered intravenously 30 minutes before resuscitation. The vehicle group received an equal volume of normal saline. After resuscitation with shed blood, rats were sacrificed 4 hours later, and lung tissues and serum were collected for analysis[2]
- Rat bladder ischemia-reperfusion injury model: Male Sprague-Dawley rats (200–250 g) were anesthetized, and the bilateral internal iliac arteries were clamped for 60 minutes to induce bladder ischemia, followed by reperfusion for 24 hours. Sivelestat sodium (5 mg/kg) was dissolved in normal saline and administered intraperitoneally 30 minutes before ischemia and again at the start of reperfusion. The vehicle group received normal saline. At the end of reperfusion, rats were euthanized, and bladder tissues were collected for biochemical and histological analysis[3]

In a rat hemorrhagic shock study, intravenous administration of cetuximab sodium (10 mg/kg) did not cause significant changes in heart rate, blood pressure, or body weight during the experiment [2]. In a rat bladder ischemia-reperfusion study, intraperitoneal administration of cetuximab sodium (5 mg/kg) did not cause significant abnormalities in the gross pathological examination of major organs (liver, kidney, spleen) [3].

[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.

ONO-5046, or 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 pig skin (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 and progression of DIC in patients with ARDS and sepsis. Studies have shown that sivelestat can alleviate acute lung injury (ALI) by inhibiting neutrophil elastase (NE), with mechanisms including improving alveolar epithelial and vascular endothelial damage and reversing neutrophil-mediated vascular permeability. Recent advances 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, civestat is expected to become an effective treatment for COVID-19-induced ALI/ARDS or coagulation disorders. Keywords: COVID-19; acute lung injury/acute respiratory distress syndrome; coagulation disorders; neutrophil elastase inhibitor civestat[4]

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Civestat sodium (formerly known as ONO-5046) is a specific and potent human neutrophil elastase (HNE) inhibitor[1]
- Its mechanism of action includes blocking HNE-mediated degradation of extracellular matrix proteins and inhibiting excessive inflammatory response, thereby reducing tissue damage in inflammatory and ischemic states[2,3]
- Civestat sodium has potential application value in the treatment of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) or COVID-19-related disseminated intravascular coagulation (DIC) because HNE is involved in the pathogenesis of these diseases[4]
- HNE It is a serine protease released by activated neutrophils, and its overexpression can lead to tissue damage in various pathological conditions, including ischemia-reperfusion injury and inflammatory diseases [1,2,3]

C₂₀H₂₁N₂NAO₇S

456.44

456.096

C, 52.63; H, 4.64; N, 6.14; Na, 5.04; O, 24.54; S, 7.02

150374-95-1

Sivelestat;127373-66-4;Sivelestat sodium tetrahydrate;201677-61-4

23664980

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

2.463

2

8

9

31

738

0

[O-]C(CNC(C1=CC=CC=C1NS(C2=CC=C(OC(C(C)(C)C)=O)C=C2)(=O)=O)=O)=O.[Na+]

ZAIFANJZUGNYCK-UHFFFAOYSA-M

InChI=1S/C20H22N2O7S.Na/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);/q;+1/p-1

sodium;2-[[2-[[4-(2,2-dimethylpropanoyloxy)phenyl]sulfonylamino]benzoyl]amino]acetate

Sivelestat sodium, ONO5046-Na, Sodium sivelestat, EI546 sodium, LY544349 sodium; ONO5046, LY544349, EI546; ONO 5046; ONO5046; ONO-5046; LY544349; LY-544349; Sivelestat sodium; 150374-95-1; Sivelestat sodium salt; Sivelestat sodium anhydrous; Sivelestat (sodium); Sivelestat sodium salt hydrate; sodium;2-[[2-[[4-(2,2-dimethylpropanoyloxy)phenyl]sulfonylamino]benzoyl]amino]acetate; 0CLL4232KD; 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 (5.48 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: ≥ 2.5 mg/mL (5.48 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 25.0 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: ≥ 2.5 mg/mL (5.48 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 25.0 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.)