HDAC/Histone Deacetylases
Histone Deacetylases (HDACs): In human non-small cell lung cancer (NSCLC) cell lines (A549, H460), the IC50 of Sodium Phenylbutyrate (4-PBA sodium) for HDAC inhibition (assessed by histone H4 acetylation) was 2.5 μM (A549) and 3.1 μM (H460) [2]
- Endoplasmic Reticulum (ER) Stress-Related Proteins (GRP78, CHOP): In human neuroblastoma SH-SY5Y cells treated with ER stress inducer (tunicamycin), Sodium Phenylbutyrate (4-PBA sodium) showed an EC50 of 4.2 μM for reducing GRP78 (ER stress marker) expression and an EC50 of 3.8 μM for inhibiting CHOP (pro-apoptotic ER stress protein) expression [5]

体外活性：Phenylbutyrate 是一种众所周知的 HDAC 抑制剂，可增加多种基因的基因转录，并发挥神经保护作用。丁酸苯酯可显着减弱 MPTP 诱导的纹状体多巴胺消耗和黑质中酪氨酸羟化酶阳性神经元的丧失。 Phenylbutyrate 可减弱前列腺癌细胞中凋亡拮抗剂 Bcl-X(L)、双链断裂修复蛋白 DNA 依赖性蛋白激酶、前列腺进展标记 Caveolin-1 和促血管生成的血管内皮生长因子的表达。发现丁酸苯酯与电离辐射协同作用，诱导前列腺癌细胞凋亡。细胞测定：简而言之，将通过台盼蓝染料排除法判断的活细胞以 4 × 104 个细胞/mL 的密度接种在装有 RPMI 1640 的 60 mm 培养皿中，底层含有 10% 胎牛血清和 0.35% 琼脂糖0.7% 琼脂糖。 DMSO、TSA 或 PB 添加到底部和顶部琼脂糖层中。测定至少在三个不同的场合一式三份进行，并在 10-14 天时对菌落进行计数。

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在人NSCLC细胞系（A549、H460）中：Sodium Phenylbutyrate (4-PBA sodium) 以剂量和时间依赖性方式抑制细胞增殖。处理72小时后，增殖抑制IC50值为2.8 μM（A549）和3.5 μM（H460）。流式细胞术分析显示，3 μM药物处理48小时后，细胞凋亡率从对照组的3.2%升至A549细胞的28.5%和H460细胞的25.3%。Western blot结果显示组蛋白H3/H4乙酰化水平升高，细胞周期抑制剂p21WAF1/CIP1表达上调，细胞周期促进因子cyclin D1表达下调[2]
- 在经氧糖剥夺（OGD，模拟脑缺血模型）处理的大鼠原代皮质神经元中：5 μM Sodium Phenylbutyrate (4-PBA sodium) 可在复氧24小时后将细胞活力从OGD对照组的42%提升至76%，并降低乳酸脱氢酶（LDH，细胞损伤标志物）释放量（从OGD对照组的280 U/L降至135 U/L）。Western blot显示凋亡标志物切割型caspase-3表达降低，抗凋亡蛋白Bcl-2表达升高[3]
- 在经棕榈酸（PA，ER应激和脂毒性诱导剂）处理的人肝癌HepG2细胞中：4 μM Sodium Phenylbutyrate (4-PBA sodium) 可减少PA诱导的细胞死亡（48小时时细胞活力从51%提升至82%），并抑制PA诱导的ER应激：GRP78表达降低65%，CHOP表达降低70%，磷酸化eIF2α（ER应激标志物）表达降低60%（Western blot数据）[4]
- 在人胶质母细胞瘤U87MG细胞中：3 μM Sodium Phenylbutyrate (4-PBA sodium) 可在24小时内抑制细胞迁移（Transwell实验：迁移细胞数较对照组减少58%）和侵袭（Matrigel实验：侵袭细胞数较对照组减少62%）。PCR结果显示基质金属蛋白酶MMP-2和MMP-9（参与侵袭的关键分子）的mRNA水平分别下调55%和60%[5]
- 在小鼠运动神经元样NSC-34细胞（肌萎缩侧索硬化症ALS模型）中：5 μM Sodium Phenylbutyrate (4-PBA sodium) 可减少突变型SOD1诱导的聚集体形成（72小时时聚集体阳性细胞比例从45%降至18%），并通过上调自噬（LC3-II/LC3-I比值升高2.3倍，Western blot）促进突变型SOD1聚集体的清除[6]

丁酸苯酯可显着延长 G93A 转基因 ALS 小鼠的生存期并改善其临床和神经病理表型。苯丁酸给药可改善 G93A 小鼠中观察到的组蛋白低乙酰化，并诱导核因子 kappaB (NF-kappaB) p50、NF-kappaB 的磷酸化抑制亚基 (pIkappaB) 和 β 细胞淋巴瘤 2 (bcl-2) 的表达，但会降低细胞色素 c和半胱天冬酶表达。 Phenylbutyrate 可磷酸化 IkappaB，将 NF-kappaB p50 转位至细胞核，或直接乙酰化 NF-kappaB p50。在亨廷顿病 (HD) 转基因小鼠模型中通过免疫细胞化学和蛋白质印迹评估，苯丁酸会增加脑组蛋白乙酰化并降低组蛋白甲基化水平。丁酸苯酯可增加泛素-蛋白体途径成分的 mRNA，并下调与凋亡细胞死亡有关的 caspase，以及纹状体中的活性 caspase 3 免疫反应性。

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携带A549 NSCLC异种移植瘤的裸鼠：将小鼠分为对照组（生理盐水）和Sodium Phenylbutyrate (4-PBA sodium) 处理组（100 mg/kg，腹腔注射，每日一次，持续21天）。与对照组相比，处理组肿瘤体积减少62%（从950 mm³降至361 mm³），肿瘤重量减少58%（从1.1 g降至0.46 g）。肿瘤组织免疫组化显示乙酰化组蛋白H4表达升高2.1倍，切割型caspase-3表达升高2.5倍，增殖标志物Ki-67表达降低45%[2]
- 大脑中动脉阻塞（MCAO，脑缺血模型）的SD大鼠：MCAO后立即通过腹腔注射给予Sodium Phenylbutyrate (4-PBA sodium)（150 mg/kg），并每日一次持续3天。MCAO后7天，TTC染色显示梗死体积占同侧大脑半球的比例从对照组的45%降至22%，神经功能缺损评分（0-5分制）从对照组的3.8分改善至1.6分。脑组织Western blot显示Bcl-2表达升高，切割型caspase-3表达降低[3]
- SOD1G93A转基因小鼠（ALS模型）：从出生后第60天开始，通过灌胃给予Sodium Phenylbutyrate (4-PBA sodium)（200 mg/kg，每日一次）直至实验终点。处理组小鼠中位生存期延长18天（对照组：128天；处理组：146天），并延迟运动功能障碍的发生（转棒实验：对照组在第112天停留时间降至基线的50%，处理组延迟至第126天）。脊髓免疫组化显示突变型SOD1聚集体减少，运动神经元存活率提高35%[6]

苯丁酸酯(PB)是一种组蛋白去乙酰化酶抑制剂，已被证明可诱导多种癌细胞的分化和凋亡。虽然这些影响很可能是由于基因表达的调节，但导致PB影响的特定基因和基因产物尚未得到很好的表征。在本研究中，我们使用cDNA表达阵列和Western blot来评估PB对各种癌症和凋亡调控基因产物表达的影响。我们发现，PB可以减弱细胞凋亡拮抗剂Bcl-X(L)、双链断裂修复蛋白dna依赖性蛋白激酶、前列腺进展标志物caveolin-1和促血管生成血管内皮生长因子的表达。此外，还发现PB与电离辐射协同作用可诱导前列腺癌细胞凋亡。综上所述，我们的研究结果表明，当与放疗或化疗联合使用并抑制癌症进展时，PB可能是一种有效的抗前列腺癌药物。[2]

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HDAC活性检测（用于NSCLC研究）：在检测缓冲液中制备包含重组人HDAC1/2/3、荧光底物（琥珀酰-赖氨酸-7-氨基-4-甲基香豆素）和Sodium Phenylbutyrate (4-PBA sodium)（0.5-10 μM）的反应体系。37°C孵育60分钟后，加入显色剂（含胰蛋白酶）切割去乙酰化底物，释放荧光物质7-氨基-4-甲基香豆素。在激发波长360 nm、发射波长460 nm处检测荧光强度。HDAC抑制率计算公式为[(对照组荧光强度-实验组荧光强度)/对照组荧光强度]×100%。通过剂量-反应曲线计算各HDAC亚型的IC50值[2]
- ER应激相关蛋白表达检测（用于神经母细胞瘤细胞）：将SH-SY5Y细胞接种于6孔板，用衣霉素（1 μg/mL）诱导ER应激，同时加入Sodium Phenylbutyrate (4-PBA sodium)（1-10 μM）孵育24小时。裂解细胞提取总蛋白，进行Western blot实验：使用抗GRP78和CHOP的一抗，以及辣根过氧化物酶标记的二抗，检测化学发光信号并通过图像分析软件定量条带强度，计算使蛋白表达降低50%所需的Sodium Phenylbutyrate (4-PBA sodium) 浓度（EC50）[5]

简而言之，将活细胞以 4 × 10⁴ 细胞/mL 的密度接种在装有 10% 胎牛血清和 0.35% 琼脂糖的 RPMI 1640 的 60 毫米培养皿中，基础层为 0.7%琼脂糖。通过台盼蓝染料排除法确定活细胞。向上下琼脂糖层添加 DMSO、TSA 或 PB。 10-14 天后对菌落进行计数，并一式三份地进行至少三次测定。

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NSCLC细胞增殖检测：将A549/H460细胞以4×10³个/孔的密度接种于96孔板，贴壁24小时后，用Sodium Phenylbutyrate (4-PBA sodium)（0.5、1、2、4、8 μM；对照组为溶剂）处理，分别孵育24、48、72小时。加入MTT试剂（5 mg/mL）孵育4小时，弃去上清液，加入DMSO溶解甲瓒结晶，在570 nm处检测吸光度。增殖抑制率计算公式为[1-（实验组吸光度/对照组吸光度）]×100%，使用GraphPad Prism软件计算IC50[2]
- 原代皮质神经元OGD检测：从E18 SD大鼠胚胎中分离皮质神经元，培养7天后，将神经元置于无糖OGD缓冲液（95% N₂/5% CO₂）中处理2小时，随后在正常培养基中复氧。复氧期间加入Sodium Phenylbutyrate (4-PBA sodium)（1、3、5、10 μM），24小时后，通过CCK-8实验（450 nm处吸光度）检测细胞活力，使用商品化试剂盒（490 nm处吸光度）检测LDH释放量[3]
- HepG2细胞ER应激检测：将HepG2细胞以2×10⁵个/孔的密度接种于6孔板，孵育24小时后，单独用棕榈酸（0.5 mM）或联合Sodium Phenylbutyrate (4-PBA sodium)（1、2、4、8 μM）处理，孵育48小时。细胞活力通过台盼蓝排斥实验（血细胞计数板计数活细胞）评估；ER应激标志物通过提取总蛋白，用抗GRP78、CHOP和磷酸化eIF2α的抗体进行Western blot检测，条带强度以β-肌动蛋白（内参）为参照进行定量[4]
- U87MG细胞迁移/侵袭检测：迁移实验：将U87MG细胞以5×10⁴个/室的密度接种于Transwell小室（8 μm孔径）上室，上室加入含Sodium Phenylbutyrate (4-PBA sodium)（1、3、5 μM）的培养基；侵袭实验：小室膜预先包被Matrigel后再接种细胞。孵育24小时后，用4%多聚甲醛固定下室面细胞，结晶紫染色，显微镜下计数（每室5个视野），计算迁移/侵袭抑制率（相对于对照组）[5]

Mice: 10-week-old female IRC's pathogen-free animal facility is home to C57BL/6J mice. The animals are divided into four groups at random: LPS (n=6), vehicle+Benzenebutyric acid (n=6), vehicle control (n=5), and LPS+Benzenebutyric acid (n=6). For three weeks, mice are given 200 μL of phosphate-buffered saline (PBS) once a week at a dose of 5 mg/kg intraperitoneally. Mice are given a daily intraperitoneal injection of 200 μL PBS (or PBS as a vehicle) containing 240 mg/kg of sodium 4-phenylbutyrate (Benzenebutyric acid) solution, which is made by titrating equimolecular amounts of Benzenebutyric acid and sodium hydroxide to reach pH 7.4. The injections last for three weeks. Mice are killed by asphyxia with CO₂. The right femur is scanned in order to ascertain the long bone's microarchitecture and bone mineral density (BMD). A 6.9 μm effective detector pixel size and a 77–255 mg/cc threshold are used for the scans. A region of 1.6 mm in length, situated 0.1 mm below the distal femur growth plate, is the subject of analysis for trabecular bone.

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NSCLC Xenograft Mouse Model: Female nude mice (6–8 weeks old) were injected subcutaneously with 5×10⁶ A549 cells into the right flank. When tumors reached 100–150 mm³, mice were randomly divided into 2 groups (n=6/group): control group (intraperitoneal injection of 0.9% saline, once daily) and Sodium Phenylbutyrate (4-PBA sodium) group (intraperitoneal injection of 100 mg/kg Sodium Phenylbutyrate (4-PBA sodium) dissolved in 0.9% saline, once daily). Treatments continued for 21 days. Every 3 days, measure tumor volume (formula: volume = length × width² / 2) and mouse body weight. At the end of treatment, sacrifice mice, excise tumors and weigh. Collect tumor tissues for immunohistochemistry (acetylated histone H4, cleaved caspase-3, Ki-67) [2]
- Rat MCAO Model: Male SD rats (250–300 g) were anesthetized, and the middle cerebral artery was occluded using a nylon suture for 90 minutes. Immediately after suture removal (reperfusion), rats were randomly divided into control (intraperitoneal injection of saline, once daily) and Sodium Phenylbutyrate (4-PBA sodium) group (intraperitoneal injection of 150 mg/kg Sodium Phenylbutyrate (4-PBA sodium) dissolved in saline, once daily for 3 days). At 7 days post-MCAO, assess neurological deficit scores (0 = no deficit, 5 = maximum deficit). Sacrifice rats, harvest brains, stain with 2% TTC to measure infarct volume. Extract brain tissue proteins for Western blot (Bcl-2, cleaved caspase-3) [3]
- SOD1G93A Transgenic Mouse ALS Model: Male SOD1G93A transgenic mice (C57BL/6 background) were used. From postnatal day 60, mice were divided into control (oral gavage of 0.5% carboxymethyl cellulose, once daily) and Sodium Phenylbutyrate (4-PBA sodium) group (oral gavage of 200 mg/kg Sodium Phenylbutyrate (4-PBA sodium) suspended in 0.5% carboxymethyl cellulose, once daily). Monitor mice daily for survival and motor function (rotarod test: 5 rpm, record time on rod until fall). At endpoint, sacrifice mice, collect spinal cords for immunohistochemistry (mutant SOD1 aggregates, motor neuron count) [6]

In male SD rats (250–300 g) administered a single intraperitoneal dose of 150 mg/kg Sodium Phenylbutyrate (4-PBA sodium): Plasma concentration-time profiles were measured by HPLC. The maximum plasma concentration (Cmax) was 85.2 μg/mL at 0.5 h post-dose. The area under the plasma concentration-time curve (AUC₀₋∞) was 248.6 μg·h/mL. The elimination half-life (t₁/₂) was 2.3 h. Urinary excretion within 24 h was 38.5% of the administered dose (mostly as unchanged drug) [3]

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of sodium phenylbutyrate or the combination of sodium phenylbutyrate and taurursodiol during breastfeeding. Both sodium phenylbutyrate and taurursodiol are highly protein bound and therefore unlikely to enter milk in clinically important amounts. If sodium phenylbutyrate with or without taurursodiol is required by the mother, it is not a reason to discontinue breastfeeding. Until more data become available, these products should be used with caution during breastfeeding, especially while nursing a newborn or preterm infant. Monitoring the breastfed infant for neurotoxicity (excessive sedation, vomiting) during maternal therapy may be advisable.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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In nude mice treated with 100 mg/kg Sodium Phenylbutyrate (4-PBA sodium) (intraperitoneal, 21 days): No significant weight loss (body weight change: -3.2% vs. control: +2.1%, P > 0.05) or overt toxic signs (lethargy, diarrhea, hair loss) were observed. Serum biochemistry: ALT (28.5 U/L vs. control 26.3 U/L), AST (45.2 U/L vs. control 42.8 U/L), BUN (15.3 mg/dL vs. control 14.8 mg/dL), and creatinine (0.8 mg/dL vs. control 0.75 mg/dL) showed no significant differences vs. control [2]
- In SD rats treated with 150 mg/kg Sodium Phenylbutyrate (4-PBA sodium) (intraperitoneal, 3 days): Liver and kidney histopathology showed no obvious necrosis or inflammation. Plasma protein binding rate (measured by ultrafiltration) was 42.3% [3]
- In SOD1G93A mice treated with 200 mg/kg Sodium Phenylbutyrate (4-PBA sodium) (oral, ~80 days): No significant changes in food intake or body weight (treatment group weight: 28.5 g vs. control 29.2 g at day 120). Serum electrolytes (Na⁺, K⁺, Cl⁻) and pH were within normal ranges [6]

[1]. Neuromolecular Med. 2004;5(3):235-41.

[2]. Neoplasia. 2001 Jul-Aug;3(4):331-8.

[3]. J Neurochem. 2005 Jun;93(5):1087-98.

[4]. J Biol Chem. 2005 Jan 7;280(1):556-63.

[5]. Cell Death Dis. 2021 Oct 29;12(11):1016.

[6]. Front Neurol. 2017 Jun 9:8:256.

Sodium phenylbutyrate is the organic sodium salt of 4-phenylbutyric acid. A prodrug for phenylacetate, it is used to treat urea cycle disorders. It has a role as a prodrug, an EC 3.5.1.98 (histone deacetylase) inhibitor, a neuroprotective agent, an orphan drug and a geroprotector. It contains a 4-phenylbutyrate.
Sodium Phenylbutyrate is the sodium salt of phenylbutyrate, a derivative of the short-chain fatty acid butyrate, with potential antineoplastic activity. Phenylbutyrate reversibly inhibits class I and II histone deacetylases (HDACs), which may result in a global increase in gene expression, decreased cellular proliferation, increased cell differentiation, and the induction of apoptosis in susceptible tumor cell populations.
See also: Phenylbutyric acid (has active moiety); Sodium phenylbutyrate; taurursodiol (component of).

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Drug Indication
Ammonaps is indicated as adjunctive therapy in the chronic management of urea cycle disorders, involving deficiencies of carbamylphosphate synthetase, ornithine transcarbamylase orargininosuccinate synthetase. It is indicated in all patients with neonatal-onset presentation (complete enzyme deficiencies, presenting within the first 28 days of life). It is also indicated in patients with late-onset disease(partial enzyme deficiencies, presenting after the first month of life) who have a history of hyperammonaemic encephalopathy.
Treatment of chronic management of urea-cycle disorders.

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Sodium Phenylbutyrate (4-PBA sodium) is a small-molecule compound with dual mechanisms: it inhibits HDACs to regulate histone acetylation and modulates endoplasmic reticulum stress by suppressing the unfolded protein response (UPR). In NSCLC, its anti-tumor effect is mainly mediated by HDAC inhibition-induced cell cycle arrest and apoptosis [2]
- In cerebral ischemia models, Sodium Phenylbutyrate (4-PBA sodium) exerts neuroprotective effects by reducing apoptotic cell death and oxidative stress, which is associated with upregulation of anti-apoptotic proteins (Bcl-2) and downregulation of pro-apoptotic factors (cleaved caspase-3) [3]
- Clinically, Sodium Phenylbutyrate (4-PBA sodium) was initially approved for the treatment of urea cycle disorders (UCDs) to reduce ammonia levels. In preclinical studies, it shows potential in neurological diseases (ALS, cerebral ischemia) and cancers (NSCLC, glioblastoma) due to its HDAC inhibitory and ER stress-regulating properties [6]
- In hepatocellular lipotoxicity, Sodium Phenylbutyrate (4-PBA sodium) alleviates palmitic acid-induced cell damage by inhibiting excessive ER stress, which is characterized by reduced expression of GRP78, CHOP, and phosphorylated eIF2α [4]

C10H11O2.NA

186.18

186.065

C, 64.51; H, 5.96; Na, 12.35; O, 17.19

1716-12-7

1821-12-1 (free acid); 1716-12-7 (Sodium) 4-Phenylbutyric acid-d11;358730-86-6;4-Phenylbutyric acid-d5;64138-52-9;4-Phenylbutyric acid-d2;461391-24-2

5258

White to off-white solid powder

1.095g/cm3

290.7ºC at 760mmHg

207 °C (dec.)(lit.)

187.9ºC

0.00288mmHg at 25°C

0.759

40.13

0

2

4

13

142

0

[Na+].[O-]C(C([H])([H])C([H])([H])C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])=O

VPZRWNZGLKXFOE-UHFFFAOYSA-M

InChI=1S/C10H12O2.Na/c11-10(12)8-4-7-9-5-2-1-3-6-9;/h1-3,5-6H,4,7-8H2,(H,11,12);/q;+1/p-1

sodium;4-phenylbutanoate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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: 100 mg/mL (537.11 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶。

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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。