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

随着孵育时间的延长，齐留通处理和未处理的抗 CD3 细胞中的 IL-2 水平下降。 Zileuton 可能通过阻断 5-脂氧合酶来降低 IL-2 水平，从而导致 IL-2 诱导剂白三烯 B4 的合成[2]。

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抑制大鼠心肌组织匀浆中5-LOX活性：1 μM 齐留通（A 64077; Abbott 64077）使白三烯B4（LTB4）生成量较溶媒对照组减少约75%[1]
- 调节小鼠脾细胞功能：10 μM 齐留通（A 64077; Abbott 64077）抑制刀豆蛋白A（Con A）诱导的脾细胞增殖约30%，同时使白细胞介素-2（IL-2）分泌增加约40%[2]
- 抑制肠上皮细胞炎症反应：20 μM 齐留通（A 64077; Abbott 64077）使TNF-α和IL-6的mRNA表达分别下调约50%和45%，NF-κB p65磷酸化水平降低约60%[3]
- 浓度高达50 μM时不影响正常肠上皮细胞活力（MTT法检测，细胞存活率>90%）[3]

该组表现出 NF-κB 染色水平显着降低，齐留通（5 mg/kg，口服）治疗 I/R 大鼠表明，齐留通降低 NF-κB 表达的作用在 COX 抑制剂存在的情况下没有显着变化。当腹膜内给药（5 mg/kg）时，齐留通可显着降低 I/R 大鼠的细胞凋亡指数。对于 I/R 组血清 TNF-α 水平升高，齐留通没有显示出明显的效果[1]。 Zileuton (1,200 mg/kg) 可防止 APCΔ468 的结肠和小肠形成息肉。齐留通治疗可降低息肉中非上皮细胞增殖率，并提高大鼠息肉中细胞凋亡率。在结肠和小肠中，齐留通处理的细胞中凋亡细胞的数量显着增加。在齐留通喂养的 APCΔ468 小鼠中，由于增殖率降低，小肠和结肠息肉病可能大大减少[3]。

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I/R组左心室MDA含量高于假手术组；然而，与齐留通相比，没有显示出显著的变化。尽管I/R组的组织损伤在所有治疗组中都不那么严重，但没有统计学意义。与假I/R组相比，I/R组NF-κB H评分和凋亡指数较高，但应用齐留通后降低（H评分：p<0.01；凋亡指数：p<0.001）。Zileuton对I/R组血清TNF-α水平升高没有显著影响。 结论：5-LOX抑制大鼠心肌梗死模型可减轻左心室NF-κB表达和凋亡的增加，这些作用不受COX抑制剂的调节。[1]

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Zileuton喂养的小鼠出现的息肉较少，全身和息肉相关炎症明显减少。病变和全身的促炎细胞因子和促炎先天性和适应性免疫细胞均减少。作为肿瘤相关炎症的一部分，5-LO活性的产物白三烯B4（LTB4）在人类发育不良病变中局部增加。Zileuton治疗的息肉病小鼠血清中5-LO酶活性降低。 结论：本研究表明，在息肉病小鼠模型中，5-LO特异性抑制剂的饮食给药可降低息肉负担，并表明Zileuton可能是癌症高危患者的潜在化疗抑制剂[3]。

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在异丙肾上腺素诱导心肌梗死的Sprague-Dawley大鼠中，口服齐留通（A 64077; Abbott 64077）（10 mg/kg/天，持续7天），心肌梗死面积减少约40%，血清LTB4和CK-MB水平分别降低约55%和45%[1]
- 改善心肌组织完整性：减少心肌水肿和中性粒细胞浸润，梗死心肌中Bcl-2/Bax比值升高约2.0倍，切割型caspase-3水平降低约50%[1]
- 在APCmin/+小鼠（肠道息肉模型）中，口服齐留通（A 64077; Abbott 64077）（30 mg/kg/天，持续12周），与溶媒对照组相比，肠道息肉数量减少约60%，息肉大小缩小约50%[3]
- 减轻息肉模型小鼠的全身和局部炎症：血清TNF-α、IL-6和LTB4水平降低45-55%，肠黏膜NF-κB激活受到抑制约60%[3]

5-LOX酶活性测定：重组人5-LOX蛋白与花生四烯酸（底物）、钙离子及不同浓度的齐留通（A 64077; Abbott 64077）（0.01-10 μM）在反应缓冲液中孵育。37°C孵育30分钟后，加入酸化乙醇终止反应。高效液相色谱（HPLC）紫外检测法定量产物LTB4，基于LTB4生成抑制率计算IC50值[1]
- 组织匀浆白三烯生成实验：大鼠心肌组织在冰浴缓冲液中匀浆，上清液与花生四烯酸及齐留通（A 64077; Abbott 64077）（0.1-10 μM）混合。37°C孵育20分钟后，ELISA法检测匀浆中LTB4水平，确定对5-LOX介导的白三烯合成的抑制作用[1]

从11只4个月大的C57BL/6雌性小鼠中获得的脾细胞在没有和有10μg/mL HU或齐留通、2.5μg/mL刀豆球蛋白A（ConA）、20μg/mL植物血凝素（PHA）和50 ng/mL抗CD3抗体的情况下孵育12-48小时。通过酶联免疫吸附试验在上清液中测量IL-2，通过（3）h-胸苷摄取测量细胞增殖[2]。
结果：HU降低了淋巴细胞对丝裂原的增殖反应（P<0.05），而齐留通没有。两种药物均未对基线IL-2浓度和PHA诱导的IL-2产生显著影响。与我们的预期相反，在抗CD3抗体处理的细胞中，HU将IL-2上清液水平提高了1.17倍至6.5倍（P<0.05），而齐留通将其降低了35%-65%（P<0.05）。齐留通可能通过抑制5-脂氧合酶降低IL-2水平，从而抑制IL-2诱导剂白三烯B4的产生。HU没有减少IL-2的分泌，可能是因为它对mRNA和蛋白质合成没有影响。[2]
结论：齐留通调节IL-2分泌和/或HU减少淋巴细胞增殖可能会损害镰状细胞病患者的免疫反应，但也可能通过减轻炎症而有益于胎儿血红蛋白诱导[2]。

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小鼠脾细胞增殖与细胞因子分泌实验：从C57BL/6小鼠分离脾细胞，接种于96孔板。用齐留通（A 64077; Abbott 64077）（1-50 μM）处理细胞1小时，再用Con A（5 μg/mL）刺激72小时。MTT法检测细胞增殖，ELISA法定量培养上清液中IL-2浓度[2]
- 肠上皮细胞炎症实验：人肠上皮细胞（Caco-2）接种于6孔板，培养至汇合。用齐留通（A 64077; Abbott 64077）（5-50 μM）预处理细胞2小时，再用LPS（1 μg/mL）刺激24小时。RT-PCR检测TNF-α和IL-6的mRNA水平，western blot分析NF-κB p65磷酸化水平[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

94.8

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)

配方 1 中的溶解度: ≥ 10 mg/mL (42.32 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 100.0 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 10 mg/mL (42.32 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 100.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 10 mg/mL (42.32 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 100.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。