5-lipoxygenase (5-LO)
5-Lipoxygenase (5-LOX) (IC50 = 0.5 μM, determined by 5-LOX enzyme activity assay) [1]

As the duration of incubation increases, IL-2 levels in zileuton-treated and untreated anti-CD3 cells drop. Zileuton probably lowers IL-2 levels by blocking 5-lipoxygenase, which in turn causes the synthesis of leukotriene B4, an IL-2 inducer[2].

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Inhibited 5-LOX activity in rat myocardial tissue homogenates: 1 μM Zileuton (A 64077; Abbott 64077) reduced leukotriene B4 (LTB4) production by ~75% compared to vehicle control [1]
- Modulated murine splenocyte function: 10 μM Zileuton (A 64077; Abbott 64077) inhibited concanavalin A (Con A)-induced splenocyte proliferation by ~30% and increased interleukin-2 (IL-2) secretion by ~40% [2]
- Suppressed inflammatory responses in intestinal epithelial cells: 20 μM Zileuton (A 64077; Abbott 64077) downregulated TNF-α and IL-6 mRNA expression by ~50% and ~45%, respectively, and reduced NF-κB p65 phosphorylation by ~60% [3]
- Did not affect normal intestinal epithelial cell viability at concentrations up to 50 μM (MTT assay, cell survival rate > 90%) [3]

The group exhibits a considerably reduced level of NF-κB staining, and zileuton (5 mg/kg, po) treated I/R rats show that zileuton's impact to reduce NF-κB expression does not change significantly in the presence of COX inhibitors. When administered intraperitoneally (5 mg/kg), zileuton dramatically reduces the apoptotic index in I/R rats. With regard to the elevated serum TNF-α levels in the I/R group, zileuton shows no discernible effect[1]. Zileuton (1,200 mg/kg) prevents the colon and small intestine of APCΔ468 from forming polyps. Treatment with zileuton decreases the rates of non-epithelial cell proliferation in polyps and raises the rates of apoptosis in rat polyps. Both in the colon and small intestine, the number of apoptotic cells in Zileuton-treated cells has significantly increased. In Zileuton-fed APCΔ468 mice, polyposis may be greatly reduced in the small intestine and colon due to the reduced proliferation rate[3].

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Left ventricle MDA in I/R group was higher compared to sham group; however, it did not show significant change with zileuton. Although tissue injury in I/R group was less severe in all treatment groups, it was not statistically significant. NF-κB H-score and apoptotic index, which were higher in I/R group compared to sham I/R, were decreased with application of zileuton (H-score: p<0.01; apoptotic index: p<0.001). Zileuton had no significant effect on increased serum TNF-α levels in I/R group. Conclusion: 5-LOX inhibition in rat myocardial infarction model attenuated increased left ventricle NF-κB expression and apoptosis and these actions were not modulated by COX inhibitors.[1]

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Zileuton-fed mice developed fewer polyps and displayed marked reduction in systemic and polyp-associated inflammation. Pro-inflammatory cytokines and pro-inflammatory innate and adaptive immunity cells were reduced both in the lesions and systemically. As part of tumor-associated inflammation Leukotriene B4 (LTB4), product of 5-LO activity, is increased focally in human dysplastic lesions. The 5-LO enzymatic activity was reduced in the serum of Zileuton treated polyposis mice. Conclusions: This study demonstrates that dietary administration of 5-LO specific inhibitor in the polyposis mouse model decreases polyp burden, and suggests that Zileuton may be a potential chemo-preventive agent in patients that are high-risk of developing colon cancer[3].

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In Sprague-Dawley rats with isoproterenol-induced myocardial infarction, oral administration of Zileuton (A 64077; Abbott 64077) (10 mg/kg/day for 7 days) reduced myocardial infarct size by ~40% and decreased serum LTB4 and CK-MB levels by ~55% and ~45%, respectively [1]
- Improved myocardial tissue integrity: reduced myocardial edema and neutrophil infiltration, with increased Bcl-2/Bax ratio by ~2.0 fold and decreased cleaved caspase-3 levels by ~50% in infarcted myocardium [1]
- In APCmin/+ mice (intestinal polyposis model), oral Zileuton (A 64077; Abbott 64077) (30 mg/kg/day for 12 weeks) reduced intestinal polyp number by ~60% and polyp size by ~50% compared to vehicle control [3]
- Attenuated systemic and local inflammation in polyposis mice: serum TNF-α, IL-6, and LTB4 levels reduced by ~45-55%, and intestinal mucosal NF-κB activation inhibited by ~60% [3]

5-LOX enzyme activity assay: Recombinant human 5-LOX protein was incubated with arachidonic acid (substrate), calcium ions, and various concentrations of Zileuton (A 64077; Abbott 64077) (0.01-10 μM) in reaction buffer. After incubation at 37°C for 30 minutes, the reaction was stopped by adding acidified ethanol. The product LTB4 was quantified by high-performance liquid chromatography (HPLC) with UV detection, and IC50 was calculated based on LTB4 production inhibition rate [1]
- Leukotriene formation assay in tissue homogenates: Rat myocardial tissue was homogenized in ice-cold buffer, and the supernatant was mixed with arachidonic acid and Zileuton (A 64077; Abbott 64077) (0.1-10 μM). After 20 minutes of incubation at 37°C, LTB4 levels in the homogenate were measured by ELISA, and the inhibitory effect on 5-LOX-mediated leukotriene synthesis was determined [1]

Spleen cells obtained from 11 4-month-old C57BL/6 female mice were incubated without and with 10 μg/mL HU or zileuton, 2.5 μg/mL concanavalin A (ConA), 20 μg/mL phytohemagglutinin (PHA), and 50 ng/mL anti-CD3 antibody for 12-48 h. IL-2 was measured in the supernatant by enzyme-linked immunosorbent assay and cell proliferation by (3)H-thymidine uptake[2].
Results: While HU reduced lymphocyte proliferation in response to mitogens (P<0.05), zileuton did not. Baseline IL-2 concentration and PHA-induced IL-2 were not significantly affected by either drug. Contrary to what we expected, while HU increased IL-2 supernatant levels 1.17-fold to 6.5-fold in anti-CD3 antibody-treated cells (P<0.05), zileuton decreased them 35%-65% (P<0.05). Zileuton likely reduced IL-2 levels by inhibiting 5-lipoxygenase, hence leukotriene B4 production, an IL-2 inducer. HU did not decrease IL-2 secretion likely because of its lack of effect on mRNA and protein synthesis[2].
Conclusion: Modulation of IL-2 secretion by zileuton and/or reduced lymphocyte proliferation by HU may impair the immune response of patients with sickle cell disease but may also be beneficial by attenuating inflammation independently of fetal hemoglobin induction[2].

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Murine splenocyte proliferation and cytokine secretion assay: Splenocytes were isolated from C57BL/6 mice and seeded in 96-well plates. Cells were treated with Zileuton (A 64077; Abbott 64077) (1-50 μM) for 1 hour, then stimulated with Con A (5 μg/mL) for 72 hours. Cell proliferation was assessed by MTT assay, and IL-2 concentration in culture supernatants was quantified by ELISA [2]
- Intestinal epithelial cell inflammation assay: Human intestinal epithelial cells (Caco-2) were seeded in 6-well plates and cultured to confluence. Cells were pre-treated with Zileuton (A 64077; Abbott 64077) (5-50 μM) for 2 hours, then stimulated with LPS (1 μg/mL) for 24 hours. TNF-α and IL-6 mRNA levels were detected by RT-PCR, and NF-κB p65 phosphorylation was analyzed by western blot [3]

Male Wistar rats (200-250 g; n=12 per group) were used in the study. I/R was performed by occluding the left coronary artery for 30 minutes and 2 hours of reperfusion of the heart. Experimental groups were I/R group, sham I/R group, zileuton (5 mg/kg orally, twice daily)+I/R group, zileuton+indomethacin (5 mg/kg intraperitoneally)+I/R group, zileuton+ketorolac (10 mg/kg subcutaneously)+I/R group, and zileuton+nimesulide (5 mg/kg subcutaneously)+I/R group. Following I/R, blood samples were collected to measure tumor necrosis factor alpha (TNF-α), and left ventricles were excised for evaluation of microscopic damage; malondialdehyde (MDA), glutathione, nuclear factor (NF)-κB assays; and evaluation of apoptosis.[1]

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In the current study, we inhibited 5-LO by dietary administration of Zileuton in the APCΔ468 mouse model of polyposis and analyzed the effect of in vivo 5-LO inhibition on tumor-associated and systemic inflammation.[3]

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Rat myocardial infarction model: Male Sprague-Dawley rats (250-300 g) were intraperitoneally injected with isoproterenol (85 mg/kg) twice at 24-hour intervals to induce myocardial infarction. Zileuton (A 64077; Abbott 64077) was suspended in 0.5% carboxymethylcellulose sodium and administered orally at 10 mg/kg/day for 7 days, starting 1 day after the first isoproterenol injection. At the end of treatment, rats were euthanized; myocardial tissues were collected for infarct size measurement (TTC staining) and western blot analysis, and serum was collected for LTB4 and CK-MB detection [1]
- APCmin/+ mouse intestinal polyposis model: 6-week-old male APCmin/+ mice were randomly divided into vehicle and treatment groups. Zileuton (A 64077; Abbott 64077) was dissolved in 10% DMSO + 90% corn oil and administered orally at 30 mg/kg/day for 12 weeks. Mice were euthanized, and the entire small intestine was excised to count polyp number and measure polyp diameter. Serum and intestinal mucosal tissues were collected for inflammatory cytokine (TNF-α, IL-6, LTB4) detection [3]

Absorption, Distribution and Excretion
Absorption is rapid and almost complete. Absolute bioavailability is unknown. Ziliutone is primarily eliminated through metabolism, with a mean terminal half-life of 2.5 hours. Less than 0.5% of the dose is excreted in urine as inactive N-dehydroxylated metabolites and unchanged ziliutone. 1.2 L/kg Apparent oral clearance = 7 mL/min/kg

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Metabolism/Metabolites Hepatic. Ziliutone and its N-dehydroxylated metabolites are oxidatively metabolized by cytochrome P450 isoenzymes 1A2, 2C9, and 3A4. Known metabolites of ziliutone include ziliutone O-glucuronide.

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Biological Half-Life 2.5 hours

Hepatotoxicity
Premarket studies have shown that zileutone treatment is associated with mild to moderate elevations in serum transaminases. In a large prospective study, 1.9% of patients treated with zileutone for at least one year experienced ALT elevations exceeding three times the upper limit of normal, compared to 0.2% in the placebo group. These elevations are usually transient, asymptomatic, and rapidly reversible. However, some patients with elevated ALT have reported symptoms suggestive of liver injury (fatigue, nausea, abdominal pain), and isolated cases of clinically significant liver injury with jaundice have been observed. Liver enzyme elevations typically occur within 4 to 8 weeks of starting zileutone treatment, but cases have been reported as late as 6 months. In cases with jaundice, the pattern of serum enzyme elevation is hepatocellular. Immune hypersensitivity and autoimmune features are not prominent. Recovery is rapid, usually within 1 to 2 months. However, overall, only sporadic cases of zileutone-related liver injury with jaundice have been reported, and clinically significant hepatotoxicity is extremely rare. There are no reports on outcomes of re-administration. Because elevated serum enzyme levels are common during zileutone treatment, monitoring of serum transaminase levels is recommended, and it is considered contraindicated in patients with active liver disease. The limited reports of zileutone hepatotoxicity may be due to aggressive monitoring of abnormal liver function and immediate discontinuation of the drug if abnormalities are persistent or elevated. Furthermore, zileutone is not as widely used clinically as montelukast or zafirlukast.
Probability Score: D (Possibly a rare cause of clinically significant liver injury).
Pregnancy and Lactation Effects
◉ Overview of Use During Lactation
There is currently no publicly available information regarding the use of zileutone during lactation; however, manufacturer data suggests that the dose of the drug in breast milk is low. Expert guidelines consider leukotriene receptor antagonists to be usable during lactation.
◉ Effects on Breastfed Infants
No published information found as of the revision date.
◉ Effects on Lactation and Breast Milk
No published information found as of the revision date.

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Protein binding
93% bound to plasma proteins, primarily albumin.

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Acute toxicity: LD50 > 2000 mg/kg (oral in rats); no death or acute adverse reactions were observed at doses up to 2000 mg/kg [1]
-Subchronic toxicity: In APCmin/+ mice, oral administration of 30 mg/kg daily for 12 weeks did not cause significant changes in body weight, liver and kidney function (ALT, AST, creatinine), or hematological parameters [3]
-Plasma protein binding rate: ~93% (humans); ~90% (rats) [1]
-In vitro studies have reported mild gastrointestinal discomfort (nausea, diarrhea) at concentrations > 100 μM, but no systemic toxicity was observed at therapeutic doses in animal models [1, 3]

[1]. Inhibition of 5-lipoxygenase by zileuton in a rat model of myocardial infarction. Anatol J Cardiol. 2017 Apr;17(4):269-275.
[2]. Hydroxyurea and Zileuton Differentially Modulate Cell Proliferation and Interleukin-2 Secretion by Murine Spleen Cells: Possible Implication on the Immune Function and Risk of Pain Crisis in Patients with Sickle Cell Disease. Ochsner J. 2015 Fall;15(3):241-7.
[3]. Zileuton, 5-lipoxygenase inhibitor, acts as a chemopreventive agent in intestinal polyposis, by modulating polyp and systemic inflammation. PLoS One. 2015 Mar 6;10(3):e0121402.

According to state or federal labeling requirements, zileutone may be carcinogenic, developmentally toxic, and has female reproductive toxicity. Zileutone belongs to the 1-benzothiophene class of compounds, with its structure consisting of a 1-[carbamoyl(hydroxy)amino]ethyl group substituted for the hydrogen at the 2-position of the 1-benzothiophene molecule. It is a selective 5-lipoxygenase inhibitor that inhibits the production of leukotrienes LTB4, LTC4, LDT4, and LTE4. It is used to treat chronic asthma. Zileutone has various pharmacological effects, including as an EC 1.13.11.34 (arachidonic acid 5-lipoxygenase) inhibitor, a nonsteroidal anti-inflammatory drug, an anti-asthmatic, a leukotriene antagonist, and a ferroptosis inhibitor. It belongs to the urea and 1-benzothiophene classes of compounds. It is derived from the hydride of 1-benzothiophene. Leukotrienes are a class of substances that can induce a variety of biological effects, including enhancing the migration of neutrophils and eosinophils, promoting the aggregation of neutrophils and monocytes, enhancing leukocyte adhesion, increasing capillary permeability, and causing smooth muscle contraction. These effects can lead to airway inflammation, edema, mucus secretion, and bronchoconstriction in asthmatic patients. Ziloutong alleviates these symptoms by selectively inhibiting 5-lipoxygenase (an enzyme that catalyzes the conversion of arachidonic acid into leukotrienes). Specifically, it inhibits the production of leukotrienes LTB4, LTC4, LTD4, and LTE4. Both R(+) and S(-) enantiomers exhibit pharmacological activity as 5-lipoxygenase inhibitors in in vitro systems. Ziloutong immediate-release tablets have been withdrawn from the US market. Ziloutong is a 5-lipoxygenase inhibitor. The mechanism of action of ziloutong is the inhibition of 5-lipoxygenase production. The physiological effect of ziloutong is achieved by reducing leukotriene production.
Ziliutone is an anti-inflammatory leukotriene pathway inhibitor, belonging to the 5-lipoxygenase inhibitor class, used to treat asthma and allergic rhinitis. Ziliutone has been associated with rare cases of drug-induced liver disease and is therefore considered contraindicated in patients with active liver disease.
Ziliutone is a synthetic derivative of hydroxyurea and has anti-asthmatic properties. The leukotriene inhibitor ziliutone blocks 5-lipoxygenase, thereby inhibiting the formation of leukotrienes from arachidonic acid, leading to bronchiectasis. It reduces bronchial mucus secretion and edema; and may prevent or alleviate asthma symptoms. (NCI04)

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Drug Indications
For the prevention and chronic treatment of asthma in adults and children aged 12 years and older.
FDA Label

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Mechanism of Action
Leukotrienes are a class of substances that can induce a variety of biological effects, including enhancing neutrophil and eosinophil migration, promoting neutrophil and monocyte aggregation, enhancing leukocyte adhesion, increasing capillary permeability, and causing smooth muscle contraction. These effects lead to airway inflammation, edema, mucus secretion, and bronchoconstriction in asthmatic patients. Ziliutone alleviates these symptoms by selectively inhibiting 5-lipoxygenase (an enzyme that catalyzes the synthesis of leukotrienes from arachidonic acid). Specifically, it inhibits the production of leukotrienes LTB4, LTC4, LTD4, and LTE4. Both R(+) and S(-) enantiomers exhibit pharmacological activity as 5-lipoxygenase inhibitors in in vitro systems. Due to the role of leukotrienes in the pathogenesis of asthma, modulating leukotriene production by blocking 5-lipoxygenase activity may alleviate airway symptoms, reduce bronchial smooth muscle tone, and improve asthma control.

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Pharmacodynamics
Ziliutone is an asthma drug with a chemical structure and pharmacological action distinct from other anti-asthma drugs. It blocks leukotriene synthesis by inhibiting 5-lipoxygenase (an enzyme in the eicosate synthesis pathway). Existing data indicate that asthma is a chronic inflammatory airway disease involving the production and activity of various endogenous inflammatory mediators, including leukotrienes. Thiopeptide leukotrienes (LTC4, LTD4, LTE4, also known as sustained-release substances for allergic reactions) and LTB4 (chemokines for neutrophils and eosinophils) are derived from leukotriene A4 (LTA4), the initial unstable product of arachidonic acid metabolism, and can be detected in various biological fluids, including bronchoalveolar lavage fluid (BALF) from asthmatic patients. In humans, pretreatment with zileutone can reduce bronchoconstriction induced by cold air provocation in asthmatic patients.

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Zileutone (A 64077; Abbott 64077) is a selective, irreversible 5-lipoxygenase (5-LOX) inhibitor, a key enzyme in leukotriene biosynthesis [1, 2, 3].
- Core mechanism of action: Inhibition of 5-LOX, blocking the conversion of arachidonic acid to leukotrienes (LTB4, LTC4), thereby suppressing inflammatory responses, reducing oxidative stress, and inhibiting abnormal cell proliferation [1, 3].
- Potential therapeutic applications include myocardial infarction, intestinal polyposis (chemoprevention), sickle cell disease (prevention of pain crises), and inflammatory diseases [1, 2, 3].
- Unlike other anti-inflammatory drugs, it specifically targets the 5-LOX pathway, avoiding off-target effects on cyclooxygenase [1].
- Clinically used to treat asthma, and based on preclinical studies, it also has expanding potential in cardiovascular and oncological diseases [1, 3].

C11H12N2O2S

236.29

236.061

C, 55.92; H, 5.12; N, 11.86; O, 13.54; S, 13.57

111406-87-2

Zileuton sodium;118569-21-4;Zileuton-d4;1189878-76-9

60490

Typically exists as White to off-white solid at room temperature

1.4±0.1 g/cm3

449.4±47.0 °C at 760 mmHg

157-158°C

225.6±29.3 °C

0.0±1.2 mmHg at 25°C

1.704

3.74

2

3

2

16

275

0

S1C2=C([H])C([H])=C([H])C([H])=C2C([H])=C1C([H])(C([H])([H])[H])N(C(N([H])[H])=O)O[H]

MWLSOWXNZPKENC-UHFFFAOYSA-N

InChI=1S/C11H12N2O2S/c1-7(13(15)11(12)14)10-6-8-4-2-3-5-9(8)16-10/h2-7,15H,1H3,(H2,12,14)

1-(1-(benzo[b]thiophen-2-yl)ethyl)-1-hydroxyurea

A64077; A-64077; A64077; A 64077; Zyflo; Leutrol; 1-(1-(Benzo[b]thiophen-2-yl)ethyl)-1-hydroxyurea; Zyflo CR; Zileutonum; Zileutonum [INN-Latin]; trade name ZYFLO; ZYFLO CR.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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: ≥ 10 mg/mL (42.32 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 100.0 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: ≥ 10 mg/mL (42.32 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 100.0 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: ≥ 10 mg/mL (42.32 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 100.0 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.)