Histone Deacetylases (HDACs); endoplasmic reticulum (ER) stress

浓度为 2 mM 时，HDAC 抑制剂 4-苯基丁酸 (4-PBA) 可阻止 NSCLC 细胞系的生长。苯丁酸和环格列酮一起可以提高癌细胞生长的抑制作用[1]。苯丁酸 (0–5 mM) 以剂量依赖性方式抑制 4-ASFV 感染。除了防止 ASFV 诱导的 H3K9/K14 低乙酰化外，苯并丁酸还抑制晚期蛋白质合成。苯丁酸和恩诺沙星一起防止 ASFV 复制 [2]。当添加巴弗洛霉素A1时，LC3II积累；然而，4-苯基丁酸显着减少了这种积累。苯丁酸可以抵消 LPS 刺激引起的 p62 水平 48 小时下降。 48小时后，LPS诱导的AVO细胞百分比上升，而4-苯基丁酸则显着降低该百分比。特别是，用苯基丁酸处理后，表现出AVO的细胞比例从61.6％下降到53.1％，表明4-苯基丁酸抑制脂多糖（LPS）诱导的自噬。本研究中使用的自噬抑制阳性对照是巴弗洛霉素 A1。巴弗洛霉素 A1 处理降低了 LPS 诱导的 AVO 细胞的百分比。在 ATG7 敲低中，苯丁酸处理没有引起 OC 面积或融合指数下降。当使用 BAY 11-7082 和 JSH23 抑制 NF-κB 时，苯丁酸对 LPS 诱导效应的抑制作用被完全消除，这也会降低 LPS 刺激后的 LC3 II 水平 [3]。

与单独使用 PBS 相比，LPS 显着降低了骨体积 (BV/TV)、小梁厚度 (Tb.Th) 和骨矿物质密度 (BMD)。小梁间隙 (Tb. Sp.) 增加。 4-苯基丁酸 (4-PBA) 可减少 LPS 引起的骨质流失。 4-BMD、BV/TV 和 Tb。苯丁酸处理后Th均升高。与单独的 LPS 相比，除了降低 Tb 的升高之外。 Sp.，但是当单独给小鼠施用苯丁酸时，没有看到任何变化。通过 TRAP 染色测量，苯丁酸处理 LPS 处理的小鼠也导致 OC.S/BS 显着降低。然而，在用 LPS 和苯丁酸治疗的小鼠中，OC.N/BS 趋于下降，尽管没有统计学上显着的方式。根据这些发现，苯丁酸导致经 LPS 处理的小鼠的 OC 体积缩小，而不是数量增加。与这些结果一致，注射 LPS 的小鼠的苯丁酸治疗导致血液 CTX-1 减少，CTX-1 是体内骨吸收的标志物，LPS 治疗增强了这种作用。与单独使用脂多糖相比，苯丁酸治疗并没有显着改变骨钙素和碱性磷酸酶（体内骨形成的两个指标）的血清水平。此外，苯丁酸可以减轻LPS引起的血清MCP-1升高，表明它可以减轻LPS引起的全身炎症[3]。

非洲猪瘟病毒（ASFV）在猪身上引起一种高度致命的疾病，既没有疫苗也没有治疗方法。最近，一类新的抑制组蛋白脱乙酰酶（HDAC）的药物作为抗病毒药物受到了越来越多的关注。考虑到其他人的研究表明，丙戊酸是一种HDAC抑制剂（HDACi），可阻断包膜病毒的复制，ASFV通过促进异染色质化和I类HDAC向病毒细胞质工厂的募集来调节宿主细胞的表观遗传学状态，本研究评估了四种HDACi对ASFV的抗病毒活性。结果显示，苯丁酸钠完全消除了ASFV的复制，而丙戊酸在感染后48小时显著减少了病毒子代（-73.9%，p=0.046），正如测试的两种泛HDAC抑制剂（曲霉菌素a:-82.2%，p=0.043；伏诺司他：73.9%，p=0.043）。进一步的评估表明，NaPB的保护作用是剂量依赖性的，干扰晚期病毒基因的表达，逆转ASFV诱导的组蛋白H3赖氨酸9和14（H3K9K14）低乙酰化状态，与开放染色质状态兼容，并可能使宿主基因的表达对感染进展不利。此外，当NaPB与ASFV拓扑异构酶II毒素（恩诺沙星）联合使用时，检测到协同抗病毒作用。总之，我们的研究结果强烈表明，细胞HDAC参与了ASFV感染的建立，并强调需要进一步的体内研究来更好地了解HDAC抑制剂的抗病毒活性[2]。

纳摩尔浓度的曲霉菌素A在七种NSCLC细胞系中的五种中诱导生长停滞，而苯基丁酸钠（PB）的效力明显较弱。在腺癌中，曲霉菌素A上调一般分化标志物（凝胶蛋白、Mad和p21/WAF1），下调II型肺细胞祖细胞谱系的标志物（MUC1和SP-A），表明表型更成熟。PB也有类似的效果。PPARγ配体和PB同时治疗增强了腺癌的生长抑制，但没有增强非腺癌的增长抑制。生长停滞伴有细胞周期蛋白D1表达显著降低，但分化不增强。

Female 10-week-old C57BL/6J mice were housed in the pathogen-free animal facility of IRC. All mice were handled in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Immunomodulation Research Center (IRC), University of Ulsan. All animal procedures were approved by the IACUC of IRC. The approval ID for this study is # UOU-2014-014. Animals were randomized into the following 4 groups: vehicle control (n = 5), vehicle + 4-PBA (n = 6), LPS (n = 6), and LPS + 4-PBA (n = 6). Mice were treated with LPS in 200 μL phosphate-buffered saline (PBS) (or with PBS as a vehicle) once a week (5mg/kg, i.p.) for 3 weeks as described [19]. 4-PBA solution was prepared by titrating equimolecular amounts of 4-PBA and sodium hydroxide to reach pH 7.4; mice were injected daily intraperitoneally in 200 μL PBS (or with PBS as a vehicle) at a dose of 240 mg/kg for 3 weeks. Mice were sacrificed by CO2 asphyxiation. To determine the bone mineral density (BMD) and microarchitecture of the long bone, the right femur was scanned in a high-resolution Micro CT (μCT) SkyScan 1176 System. Scans were performed with an effective detector pixel size of 6.9 μm and a threshold of 77–255 mg/cc. Trabecular bone was analyzed in a region 1.6 mm in length and located 0.1 mm below the distal femur growth plate. A total of 75–125 tomographic slices were acquired; 3 D analyses were performed with CT volume software. The structural parameters such as bone volume fraction (BV/TV), trabecular thickness (Tb. Th), and trabecular space (Tb. Sp.) were analyzed. In vivo markers of bone resorption were measured according to the manufacturer’s directions; serum collagen-type I fragments (CTX-1) were assessed using a RatLaps EIA assay. Serum osteocalcin was assessed using an osteocalcin EIA kit, and alkaline phosphatase (ALP) was quantitated using a colorimetric kinetic determination kit. Serum MCP-1 was quantitated by sandwich ELISA using the recommended Abs according to manufacturer’s instruction[3].

Absorption, Distribution and Excretion
Following oral administration of a single 5g dose of sodium phenylbutyrate, the Cmax was 195-218 µg/mL under fasting conditions and the Tmax was one hour. The effect of food on drug absorption is unknown.
Approximately 80–100% of the dose was excreted by the kidneys within 24 hours as the conjugation product, phenylacetylglutamine. For each gram of sodium phenylbutyrate administered, it is estimated that between 0.12–0.15 grams of phenylacetylglutamine nitrogen are produced.

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Metabolism / Metabolites
The major sites for metabolism of sodium phenylbutyrate are the liver and kidney. Phenylbutyric acid is rapidly metabolized to phenylacetate via beta-oxidation. Phenylacetate is conjugated with phenylacetyl-CoA, which in turn combines with glutamine via acetylation to form phenylacetylglutamine.

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Biological Half-Life
Following oral administration of a single 5g dose of sodium phenylbutyrate, the elimination half-life of phenylbutyric acid ranged from 0.76 to 0.77 hours.

Hepatotoxicity
While the urea cycle disorders are caused by deficiencies of hepatic enzymes responsible for the elimination of nitrogen, patients generally present with hyperammonemia without other features or biochemical evidence of hepatic injury. Thus, serum aminotransferase, alkaline phosphatase and bilirubin levels are generally normal or only mildly elevated. Newborns presenting with hyperammonemia may have hepatomegaly but other, non-urea cycle, liver function is normal as is hepatic histology. Phenylbutyrate can help to lower ammonia levels acutely and manage to keep them in the normal or near normal range, but generally does not affect other liver functions. In open label studies, a small proportion of patients (particularly with ornithine transcarbamylase [OTC] deficiency) have had ALT or AST elevations, but these have generally been attributed to the underlying condition or its complications. Phenylbutyrate has not been linked to instances of clinically apparent liver injury with jaundice.
Likelihood score: E (unlikely cause of clinically apparent liver injury, but experience with its use is limited).

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Protein Binding
When co-administered with tauroursodeoxycholic acid as a combination product, the _in vitro_ plasma protein binding of phenylbutyric acid is 82%.

[1]. Enhanced growth inhibition by combination differentiation therapy with ligands of peroxisome proliferator-activated receptor-gamma and inhibitors of histone deacetylase in adenocarcinoma of the lung. Clin Cancer Res. 2002 Apr;8(4):1206-12.

[2]. Sodium phenylbutyrate abrogates African swine fever virus replication by disrupting the virus-induced hypoacetylation status of histone H3K9/K14. Virus Res. 2017 Oct 15;242:24-29.

[3]. 4-Phenylbutyric acid protects against lipopolysaccharide-induced bone loss by modulating autophagy in osteoclasts. Biochem Pharmacol. 2018 May;151:9-17.

4-phenylbutyric acid is a monocarboxylic acid the structure of which is that of butyric acid substituted with a phenyl group at C-4. It is a histone deacetylase inhibitor that displays anticancer activity. It inhibits cell proliferation, invasion and migration and induces apoptosis in glioma cells. It also inhibits protein isoprenylation, depletes plasma glutamine, increases production of foetal haemoglobin through transcriptional activation of the gamma-globin gene and affects hPPARgamma activation. It has a role as an EC 3.5.1.98 (histone deacetylase) inhibitor, an antineoplastic agent, an apoptosis inducer and a prodrug. It is functionally related to a butyric acid. It is a conjugate acid of a 4-phenylbutyrate.
Phenylbutyric acid is a fatty acid and a derivative of [butyric acid] naturally produced by colonic bacteria fermentation. It demonstrates a number of cellular and biological effects, such as relieving inflammation and acting as a chemical chaperone. It is used to treat genetic metabolic syndromes, neuropathies, and urea cycle disorders.
Phenylbutyric acid is a Nitrogen Binding Agent. The mechanism of action of phenylbutyric acid is as an Ammonium Ion Binding Activity.
Phenylbutyrate and sodium benzoate are orphan drugs approved for the treatment of hyperammonemia in patients with urea cycle disorders, a series of at least 8 rare genetic enzyme deficiencies. The urea cycle is the major pathway of elimination of excess nitrogen including ammonia, and absence of one of the urea cycle enzymes often causes elevations in serum ammonia which can be severe, life-threatening and result in permanent neurologic damage and cognitive deficiencies. Both phenylbutyrate and sodium benzoate act by promoting an alternative pathway of nitrogen elimination. Neither phenylbutyrate nor sodium benzoate have been linked to cases of liver injury either in the form of serum enzyme elevations during therapy or clinically apparent acute liver injury.
4-Phenylbutyric acid has been reported in Streptomyces with data available.
See also: Sodium Phenylbutyrate (active moiety of); Glycerol Phenylbutyrate (is active moiety of).

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Drug Indication
Phenylbutyric acid is used for the treatment of various conditions, including urea cycle disorders, neonatal-onset deficiency, late-onset deficiency disease in patients with a history of hyperammonemic encephalopathy. Phenylbutyric acid must be combined with dietary protein restriction and, in some cases, essential amino acid supplementation. Phenylbutyric acid, as sodium phenylbutyrate, is used in combination with [tauroursodeoxycholic acid] to treat amyotrophic lateral sclerosis (ALS) in adults.

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Mechanism of Action
Sodium phenylbutyrate is the most commonly used salt used in drug products of phenylbutyric acid. Sodium phenylbutyrate is a pro-drug that rapidly metabolizes to phenylacetate. Phenylacetate is conjugated with phenylacetyl-CoA, which in turn combines with glutamine via acetylation to form phenylacetylglutamine. Phenylacetylglutamine is then excreted by the kidneys, thus providing an alternate mechanism of waste nitrogen excretion to the urea cycle. Phenylacetylglutamine is comparable to urea, as each molecule contains two moles of nitrogen.

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Pharmacodynamics
Phenylbutyric acid decreases elevated plasma ammonia glutamine levels in patients with urea cycle disorders. It increases waste nitrogen excretion in the form of phenylacetylglutamine. In the intestines, phenylbutyric acid was shown to reduce mucosal inflammation, regulate transepithelial fluid transport, and improve oxidative status. Some studies report antineoplastic properties of phenylbutyric acid, showing that phenylbutyric acid can promote growth arrest and apoptosis of cancer cells. It is suggested that phenylbutyric acid can act as an ammonia scavenger, chemical chaperone, and histone deacetylase inhibitor.

C10H12O2

164.2

164.083

C, 73.15; H, 7.37; O, 19.49

1821-12-1

Sodium 4-phenylbutyrate;1716-12-7;4-Phenylbutyric acid-d11;358730-86-6;4-Phenylbutyric acid-d5;64138-52-9;4-Phenylbutyric acid-d2;461391-24-2

4775

White to off-white solid powder

1.1±0.1 g/cm3

290.7±9.0 °C at 760 mmHg

49-52ºC

187.9±13.9 °C

0.0±0.6 mmHg at 25°C

1.535

2.42

37.3

1

2

4

12

137

0

O([H])C(C([H])([H])C([H])([H])C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])=O

OBKXEAXTFZPCHS-UHFFFAOYSA-N

nChI=1S/C10H12O2/c11-10(12)8-4-7-9-5-2-1-3-6-9/h1-3,5-6H,4,7-8H2,(H,11,12)

4-Phenylbutyric acid

4-Phenylbutyric acid; AI3 12065; 4-PHENYLBUTYRIC ACID; 4-Phenylbutanoic acid; 1821-12-1; Benzenebutanoic acid; Benzenebutyric acid; Phenylbutyrate; Phenylbutyric acid; gamma-Phenylbutyric acid; AI312065; AI3-12065

2934.99.03.00

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)

DMSO : ~100 mg/mL (~609.01 mM)
H2O : ~2 mg/mL (~12.18 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (15.23 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 25.0 mg/mL澄清DMSO储备液加入到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: ≥ 2.5 mg/mL (15.23 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 25.0 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: ≥ 2.5 mg/mL (15.23 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 25.0 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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配方 4 中的溶解度: 33.33 mg/mL (202.98 mM) in 20% HP-β-CD in Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*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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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。