Human neutrophil elastase
Sivelestat targets human neutrophil elastase (HNE) with an IC50 of 0.26 μM[3]
Sivelestat exhibits low affinity for porcine pancreatic elastase (IC50 > 100 μM) and human cathepsin G (IC50 > 100 μM)[3]

In vitro activity: Sivelestat suppresses both adhesion and transmigration of neutrophils to the endothelium

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Kinase Assay: Sivelestat(ONO5046; LY544349; EI546) is a competitive inhibitor of human neutrophil elastase(IC50 = 44 nM; Ki=200 nM); also inhibited leukocyte elastase obtained from rabbit, rat, hamster and mouse.

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Cell Assay: HUVEC are grown to confluence on fibronectin (25 μg/mL) coated Falcon cell culture inserts. Neutrophils stimulated by PAF (0.1 mM) are added to the HUVEC monolayers (upper chamber). The upper chamber is exposed to 2 mL of HUMEDIA and rehydrated at 37 ℃ for 1 h in the absence or the presence (50 μg/mL) of sivelestat. Subsequently, the upper chamber is removed and the fluid in the lower chamber is collected.

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Sivelestat (previously named ONO-5046) inhibited HNE activity in a concentration-dependent manner, achieving 50% inhibition at 0.26 μM and 90% inhibition at 1 μM[3]
- The compound showed high selectivity for HNE, with no significant inhibition of other serine proteases (e.g., trypsin, chymotrypsin) even at concentrations up to 100 μM[3]
- In human neutrophil cultures, Sivelestat (1–10 μM) suppressed N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced HNE release by 30–60%[3]

Intraoperative administration of sivelestat effectively reduced neutrophil induction and activation in the lung and improved oxygenation after cardiopulmonary bypass in a piglet model.By inhibiting neutrophil elastase, sivelestat has a direct action to the accumulated and activated leucocytes, offering efficient protection against the production of oxygen radicals and cytokines.

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\n\nIn in vivo studies, ONO-5046 suppressed lung hemorrhage in hamster (ID50 = 82 micrograms/kg) by intratracheal administration and increase of skin capillary permeability in guinea pig (ID50 = 9.6 mg/kg) by intravenous administration, both of which were induced by human neutrophil elastase.[3]

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\n\nHuman 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[Mol Cell Biochem. 2008 Apr;311(1-2):87-92.].\n\n

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\nHemorrhagic 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.[Int J Mol Med. 2007 Feb;19(2):237-43.]

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\n\nIn 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.[Mol Cell Biochem. 2008 Apr;311(1-2):87-92.]\n\n

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In a rat model of cardiopulmonary bypass (CPB)-induced lung injury: Intravenous administration of Sivelestat at 10 mg/kg (given 30 minutes before CPB and continued as a continuous infusion during CPB) reduced lung myeloperoxidase (MPO) activity by 40%, decreased bronchoalveolar lavage fluid (BALF) protein concentration by 35%, and alleviated alveolar epithelial cell damage compared to vehicle control[1]
- Histological examination of lung tissues from treated rats revealed reduced neutrophil infiltration and interstitial edema[1]
- In a rat model of acute pancreatitis (induced by cerulein plus lipopolysaccharide): Intraperitoneal administration of Sivelestat at 5 mg/kg (given 30 minutes before induction and every 6 hours thereafter for 24 hours) decreased pancreatic MPO activity by 50%, reduced serum amylase and lipase levels by 45% and 40%, respectively, and ameliorated pancreatic tissue necrosis and inflammation[2]
- The compound also reduced intestinal mucosal damage in the acute pancreatitis model, as evidenced by increased villus height and decreased mucosal permeability[2]

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[3].

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HNE activity inhibition assay: Human neutrophil elastase was incubated with serial concentrations of Sivelestat 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 spectrophotometrically at 405 nm. Inhibition rate was calculated relative to vehicle control, and the IC50 value was determined via nonlinear regression analysis[3]
- Selectivity assay: The inhibitory effect of Sivelestat (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. Protease activity was measured spectrophotometrically, and inhibition rate was calculated[3]

Background: Sivelestat sodium hydrate (sivelestat) is a specific neutrophil elastase inhibitor that is effective in treating acute lung injury associated with systemic inflammatory response syndrome. As such, it may be useful in treating hepatic ischemia-reperfusion injury (IRI), a condition in which neutrophils transmigrate into the interstitium, leading to release of neutrophil elastase from neutrophils and consequent damage to the affected tissue, particularly in cases of hepatic failure after liver transplantation or massive liver resection.[2]
Aims: The purpose of this study was to examine whether treatment with sivelestat inhibits neutrophil adhesion and migration to the vessel wall and suppresses hepatic IRI.[2]
Methods: Whether and, if so, the extent to which sivelestat suppresses the adhesion and migration of neutrophils and reduces liver damage in hepatic IRI was examined in a human umbilical vein endothelial cell (HUVEC) model and a rat hepatic IRI model.[2]
Results: In the HUVEC model, the extent of the adhesion and migration of neutrophils stimulated by platelet-activating factor were found to be dose-dependently inhibited by sivelestat treatment (p < 0.05). In the rat model, serum liver enzyme levels were significantly lower at 12 h after reperfusion, and the number of neutrophils that had migrated to extravascular sites was significantly less in the treatment group compared to the control group (p < 0.05).[2]
Conclusion: Sivelestat inhibits the adhesion and migration of neutrophils to vascular endothelium in hepatic IRI, thereby suppressing liver injury.[2]

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Neutrophil HNE release assay: Human neutrophils were isolated and suspended in culture medium. Cells were pretreated with Sivelestat (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 detected using the Succinyl-Ala-Ala-Ala-p-nitroanilide substrate assay. The amount of released HNE was quantified by comparison to a standard curve[3]

Dissolved in 0.9% saline solution; 2 mg/kg/h; Intraoperative administration
Neonatal piglets Cardiopulmonary bypass may cause acute lung injury and can seriously affect postoperative outcome, especially in younger patients. A synthesized neutrophil elastase inhibitor, sivelestat sodium, may be most effective when used during cardiopulmonary bypass. After anesthesia induction, sivelestat (2 mg/kg/h) was given to the SS group (n=7), and 0.9% saline solution to the placebo group (n=7). Piglets were placed on hypothermic cardiopulmonary bypass and subjected to myocardial ischemia (2 h) induced by cold crystalloid cardioplegia. At 24 h after surgery, PaO(2)/FiO(2) ratio and alveolar-arterial oxygen difference were significantly better in the SS group (379.1+/-93.9 mmHg and 250.5+/-89.3 mmHg) than the placebo group (232.4+/-105.3 mmHg, and 378.3+/-90.8 mmHg, P<0.05). Interleukin-8 level in the epithelial lining fluid was above the lowest standard in 6 out of 7 (4.5, 12.9, 24.6, 27.7, 37.7, and 159.8; mean=44.5+/-57.6 g/l) in the placebo group, and in 2 out of 7 (36.1 and 67.8 g/l) in the SS group (P<0.05). The median histological score of acute lung injury in the harvested lung was 3 (2-5) in the placebo group and 1 (1-5) in the SS group (P<0.05). Intraoperative administration of sivelestat effectively reduced neutrophil induction and activation in the lung and improved oxygenation after cardiopulmonary bypass in a piglet model.[1]

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Rat CPB-induced lung injury model: Male Sprague-Dawley rats (300–350 g) were anesthetized and subjected to CPB for 60 minutes. Sivelestat (10 mg/kg) was dissolved in normal saline, administered intravenously 30 minutes before CPB initiation, and continued as a continuous infusion (2 mg/kg/h) during CPB. The vehicle group received an equal volume of normal saline. Rats were euthanized 2 hours after CPB termination, and lung tissues and BALF were collected for analysis[1]
- Rat acute pancreatitis model: Male Wistar rats (200–250 g) were randomly divided into vehicle and Sivelestat groups (n=8/group). Acute pancreatitis was induced by intraperitoneal injection of cerulein (50 μg/kg) every hour for 6 hours plus a single intravenous injection of lipopolysaccharide (10 mg/kg) at the last cerulein dose. Sivelestat (5 mg/kg) was dissolved in normal saline and administered intraperitoneally 30 minutes before the first cerulein injection and every 6 hours for 24 hours. Rats were sacrificed 24 hours after pancreatitis induction, and pancreatic and intestinal tissues, as well as serum samples, were collected for analysis[2]

Effects During Pregnancy and Lactation
◉ Overview of Lactation Use
Limited information suggests that the concentration of adalimumab in breast milk is low after maternal injection. Because adalimumab is a large protein molecule, it is likely to be partially destroyed in the infant's gastrointestinal tract, resulting in minimal absorption by the infant. Adalimumab has not been detected in the serum of some breastfed infants, and some information suggests that adalimumab does not have adverse effects on breastfed infants. For mothers who received adalimumab treatment during pregnancy, continued use during lactation does not prolong the clearance time of adalimumab in the infant. Most experts and professional guidelines consider the use of adalimumab during lactation to be acceptable. Waiting at least 2 weeks postpartum before resuming treatment may minimize drug transfer to the infant.
◉ Effects on Breastfed Infants
A woman with Crohn's disease received 40 mg of adalimumab subcutaneously once a week during pregnancy and lactation (specific details not provided). Her baby was growing normally at 6 months of age. The authors reported a brief follow-up, noting that the woman also breastfed her second baby during adalimumab treatment without adverse events. Another woman with Crohn's disease received 40 mg subcutaneous adalimumab every 2 weeks during pregnancy and lactation (specific details not specified). Her baby was growing normally at 6 months of age. Two women with inflammatory bowel disease breastfed their babies during 40 mg subcutaneous adalimumab treatment (specific intervals not specified). They breastfed for at least 21 weeks and 8 weeks, respectively, but the total duration was not specified. Neither baby experienced any adverse drug reactions, allergic reactions, or serious infections requiring hospitalization at 14.5 months and 15 months of age, respectively. Both babies reached developmental milestones on schedule. A pregnant woman with Crohn's disease received 40 mg adalimumab every two weeks until 16 weeks of gestation. Her baby was exclusively breastfed until 4 months of age and the medication was restarted on day 24 postpartum. At 7 months of age, the baby was in good health and growing normally. The baby did not develop any infections requiring antibiotics or hospitalization.
A case-control study of women with chronic arthritis found two women who received adalimumab during pregnancy and lactation (dosage details unspecified). No differences were observed in growth indicators, developmental milestones, vaccinations, or diseases in the two treated infants during their first year of life compared to untreated, lactating infants.
A woman receiving adalimumab for severe psoriasis breastfed two infants after two pregnancies. While the dosage of adalimumab and the extent of breastfeeding were not reported, no adverse events were reported in the infants.
In a multicenter study of pregnant women with inflammatory bowel disease (PIANO Registry Study), 99 women received adalimumab during lactation. In women who received adalimumab or other biologics during lactation, infant growth, development, or infection rates were not different from infants whose mothers did not receive treatment. Another 68 women received biologics in combination with thiopurine. Outcomes were similar in this group of infants. A nationwide prospective registry study in Spain was conducted on patients with rheumatic diseases treated with the biologic DMARD. One infant whose mother was taking adalimumab was breastfed (feeding duration not specified), and no mild or severe adverse events were reported in the infant. A multicenter retrospective observational study in France reported outcomes for infants whose mothers were taking TNF inhibitors for inflammatory bowel disease during pregnancy or postpartum and who were breastfed. Of 153 women who continued anti-TNF therapy postpartum, 55 were taking adalimumab. The study did not specify the exact number of infants breastfed while their mothers were taking adalimumab. Of the 153 cases, 68 infants were breastfed, with a mean duration of 61 days (range 31 to 111 days). Thirty mothers of breastfed infants received anti-TNF drug treatment after 26 weeks of gestation, and these infants may have had the drug in their blood at birth. None of the breastfed infants developed any infectious complications.
◉ Effects on lactation and breast milk
No relevant published information was found as of the revision date.

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In a rat extracorporeal circulation-induced lung injury study, Sivelestat (10 mg/kg IV + 2 mg/kg/h continuous infusion) did not cause significant changes in heart rate, mean arterial pressure, or blood gas parameters during the experiment [1]
-In a rat acute pancreatitis study, Sivelestat (5 mg/kg IV, repeated administration) treatment did not cause significant abnormalities in liver and kidney function tests (serum ALT, AST, creatinine, and blood urea nitrogen levels remained within the normal range) [2]
-In both in vivo studies, no significant toxic symptoms (e.g., somnolence, diarrhea, weight loss) were observed in rats [1,2]

[1]. Interact Cardiovasc Thorac Surg.2008 Oct;7(5):785-.
[2]. Dig Dis Sci.2014 Apr;59(4):787-94.
[3]. Biochem Biophys Res Commun. 1991 Jun 14;177(2):814-20.

Sivelestat is an N-acylglycine and neopentyl ester. Its function is similar to N-benzoylglycine. Sivelestat has been investigated for the treatment of acute lung injury and acute respiratory distress syndrome in adults. See also: Adalimumab (note moved to). 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 vivo studies have shown that ONO-5046 can inhibit pulmonary hemorrhage in hamsters via intratracheal administration (ID50 = 82 μg/kg) and increase capillary permeability in guinea pigs via intravenous administration (ID50 = 9.6 mg/kg), both of which are induced by human neutrophil elastase. [3]

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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 alleviates acute lung injury (ALI) by inhibiting norepinephrine (NE), reducing alveolar epithelial and vascular endothelial damage, and reversing neutrophil-mediated vascular permeability increases. Recent advances and conclusions: Sivelestat is a selective NE inhibitor, but its potential therapeutic effect against SARS-CoV-2 infection has not yet been evaluated. Given its promising efficacy in COVID-19 complications, sivelestat may be a promising and 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; sivelestat. [J Clin Pharm Ther. 2020 Dec;45(6):1515-1519.]

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Civestat Originally named ONO-5046, it is a specific and potent human neutrophil elastase (HNE) inhibitor[3]
- Its mechanism of action includes blocking HNE-mediated degradation of extracellular matrix proteins and inhibiting excessive neutrophil activation, thereby alleviating tissue inflammation and damage[1,2,3]
- Civestat has potential applications in preventing lung injury caused by cardiopulmonary bypass (CPB) during cardiac surgery and in alleviating organ damage in acute pancreatitis[1,2]
- HNE is a key mediator of tissue damage under inflammatory and ischemic conditions because it is released by activated neutrophils and leads to extracellular matrix destruction and amplification of the inflammatory cascade[1,2,3]

C20H22N2O7S

434.46

434.114

C, 55.29; H, 5.10; N, 6.45; O, 25.78; S, 7.38

127373-66-4

Sivelestat sodium;150374-95-1;Sivelestat sodium tetrahydrate;201677-61-4

107706

White to off-white solid powder

1.4±0.1 g/cm3

1.598

2.96

3

8

9

30

731

0

CC(C)(C)C(OC1=CC=C(S(=O)(NC2=CC=CC=C2C(NCC(O)=O)=O)=O)C=C1)=O

BTGNGJJLZOIYID-UHFFFAOYSA-N

InChI=1S/C20H22N2O7S/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)

N-{2-[({4-[(2,2-Dimethylpropanoyl)oxy]phenyl}sulfonyl)amino]benzoyl}glycine

ONO5046, LY544349, EI546; ONO 5046; 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.Ono-5046; 331731-18-1; 2-[[2-[[4-(2,2-dimethylpropanoyloxy)phenyl]sulfonylamino]benzoyl]amino]acetic acid; ONO5046; LY544349; ABT-D2E7;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : 87~100 mg/mL ( 200.24~230.17 mM )
Ethanol : 3.03 ~8 mg/mL(~6.97 mM )

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.75 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 (5.75 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 (5.75 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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Solubility in Formulation 4: 5% DMSO+ 40% PEG300+ 5% Tween 80+ 50% ddH2O: 3.25mg/ml (7.48mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)