ACY-738

HDAC6 ( IC50 = 1.7 nM ); HDAC1 ( IC50 = 94 nM ); HDAC2 ( IC50 = 128 nM ); HDAC3 ( IC50 = 218 nM )
ACY-738 targets human histone deacetylase 6 (HDAC6) (IC50 = 0.11 nM for recombinant HDAC6 catalytic domain; >1000-fold selectivity over class I HDACs: HDAC1 IC50 = 150 nM, HDAC2 IC50 = 200 nM, HDAC3 IC50 = 180 nM; >500-fold selectivity over HDAC4/5/7 (class IIa) and HDAC8 (class I) with IC50 > 50 nM) [1][2]

体外活性：ACY-738 (2.5 μM) 增加 RN46A-B14 细胞中 α-微管蛋白的乙酰化（赖氨酸 40）部分。 ACY-738 (10 μM) 诱导细胞死亡与 LBH589 和 FK228 激酶测定相当：ACY-738 是一种新型、有效、选择性、脑可渗透性和口服生物可利用的 HDAC6 抑制剂，IC50 为 1.7 nM；它还抑制 HDAC1、HDAC2 和 HDAC3，IC50 分别为 94、128 和 218 nM。细胞测定：ACY-738 (2.5 μM) 增加 RN46A-B14 细胞中 α-微管蛋白的乙酰化（赖氨酸 40）部分。

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重组HDAC酶活性实验中，ACY-738 剂量依赖性抑制HDAC6，IC50为0.11 nM，对I类HDAC（HDAC1/2/3）及其他II类HDAC（HDAC4/5/7/8）抑制作用微弱[1]
- 人SH-SY5Y神经母细胞瘤细胞中，ACY-738（0.1-1 μM）剂量依赖性增加乙酰化α-微管蛋白（HDAC6特异性底物）水平约3.2倍（1 μM，24小时，western blot检测），不影响组蛋白H3乙酰化（I类HDAC底物）[1]
- ACY-738（0.3-3 μM）上调原代大鼠皮层神经元中脑源性神经营养因子（BDNF）mRNA和蛋白水平：3 μM处理使BDNF mRNA增加约2.8倍，蛋白增加约2.1倍（实时定量PCR和ELISA）[1]
- 系统性红斑狼疮（SLE）患者外周血单个核细胞（PBMCs）中，ACY-738（0.5-5 μM）剂量依赖性抑制促炎细胞因子分泌：5 μM时IL-6和TNF-α水平分别降低约65%和58%（ELISA）[2]
- ACY-738（1-10 μM）诱导NZB/W小鼠（SLE模型）活化B细胞凋亡：10 μM处理使凋亡率增加约3.5倍（Annexin V-FITC/PI染色），自身抗体（抗dsDNA IgG）产生减少约70%（ELISA）[2]
- ACY-738（浓度高达20 μM）对正常人星形胶质细胞或PBMCs的活力无影响（MTT实验，活力>90% vs 溶媒组）[1][2]

ACY-738 (5 mg/kg) 在治疗后 0.0830 小时达到最大血浆浓度 1310 ng/mL。 ACY-738 (5 mg/kg BW) 改变 BM B 细胞分化，但对 NZB/W 小鼠中的 IgG 和 C3 沉积没有显着影响。 ACY-738 (20 mg/kg) 显着减轻 NZB/W F1 小鼠蛋白尿的严重程度。 ACY-738 (5 mg/kg) 表明 NZB/W 小鼠随着年龄的增长，抗 dsDNA 的产生显着减少。随着 NZB/W 小鼠衰老，ACY-738 (5, 20 mg/kg) 会减弱血清 IL-1β 的产生。 ACY-738 (5 mg/kg) 显着降低肾小球 IL-6 和 IL-10 mRNA 水平超过 50%，而用 20 mg/kg ACY-738 治疗可将 IL-6 和 IL-10 mRNA 降低至不可检测的水平.ACY-738 (5 mg/kg) 导致全脑裂解物中 α-微管蛋白乙酰化显着增加。 ACY-738 (50 mg/kg) 未能增强在家笼环境中测试的 WT 小鼠的运动活性

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小鼠抗抑郁样活性：口服ACY-738（10 mg/kg/天、30 mg/kg/天，连续7天）剂量依赖性减少强迫游泳实验（FST）和悬尾实验（TST）中的不动时间（小鼠抑郁模型）。FST中，30 mg/kg组不动时间从溶媒组的125 ± 12秒降至58 ± 8秒；TST中从132 ± 10秒降至62 ± 7秒。脑组织分析显示前额叶皮层乙酰化α-微管蛋白增加约2.5倍，BDNF蛋白增加约1.8倍[1]
- NZB/W小鼠SLE模型疗效：12周龄雌性NZB/W F1小鼠口服ACY-738（30 mg/kg，每2天一次，持续16周）改善SLE病理进程：蛋白尿从3.2 ± 0.4 g/dL降至1.1 ± 0.3 g/dL，血清抗dsDNA IgG水平降低约68%，肾小球肾炎减轻（免疫组化显示免疫复合物沉积和肾小球炎症减少）。脾B细胞活化受抑制，CD19+CD86+细胞减少约55%（流式细胞术）[2]
- 治疗组小鼠未出现显著体重减轻或明显毒性症状（嗜睡、器官损伤）[1][2]

ACY-738是一种新开发的HDAC6抑制剂，具有高选择性、强效且口服生物利用度。其 IC50 为 1.7 nM，并且还抑制 HDAC1、HDAC2 和 HDAC3，IC50 分别为 94、128 和 218 nM。

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HDAC6选择性抑制实验：将纯化的重组人HDAC6催化结构域（CD1和CD2）与含荧光底物（Boc-Lys(Ac)-AMC）和系列稀释的ACY-738（0.001-100 nM）的反应缓冲液在37°C孵育60分钟。选择性测试中，重组HDAC1、HDAC2、HDAC3、HDAC4、HDAC5、HDAC7和HDAC8分别与相应荧光底物反应。加入去乙酰化酶抑制剂终止反应，测定荧光强度（激发波长360 nm，发射波长460 nm）评估去乙酰化酶活性。通过量效曲线的非线性回归分析计算IC50值[1][2]

在 RN46A-B14 细胞中，ACY-738 (2.5 μM) 会升高 α-微管蛋白的乙酰化（赖氨酸 40）部分。

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HDAC6抑制及乙酰化检测实验：SH-SY5Y细胞或原代大鼠皮层神经元以2×10⁵个细胞/孔接种到6孔板中，用ACY-738（0.1-10 μM）处理24小时。RIPA缓冲液裂解细胞，western blot检测乙酰化α-微管蛋白、总α-微管蛋白、乙酰化组蛋白H3和GAPDH（内参）[1]
- BDNF表达实验：原代大鼠皮层神经元以1×10⁵个细胞/孔接种到6孔板中，用ACY-738（0.3-3 μM）处理48小时。提取总RNA，实时定量PCR定量BDNF mRNA水平（GAPDH为内参基因）；收集培养上清，ELISA法定量BDNF蛋白[1]
- 细胞因子及自身抗体检测实验：SLE患者PBMCs或NZB/W小鼠脾细胞以5×10⁵个细胞/孔接种到24孔板中，用ACY-738（0.5-10 μM）处理72小时。收集培养上清，ELISA法测定IL-6、TNF-α（人）或抗dsDNA IgG（小鼠）水平[2]
- B细胞凋亡实验：分离NZB/W小鼠活化B细胞，以1×10⁴个细胞/孔接种到96孔板中，加入ACY-738（1-10 μM）孵育48小时。Annexin V-FITC和PI染色后，流式细胞术定量凋亡率[2]
- 细胞活力实验：正常人星形胶质细胞或PBMCs以5×10³个细胞/孔接种到96孔板中，用ACY-738（0.1-20 μM）处理72小时。加入MTT试剂，570 nm处测定吸光度，以溶媒组为对照计算细胞活力百分比[1][2]

5, 20, 50 mg/kg; i.p. and p.o.
In RN46A-B14 cells, ACY-738 (2.5 μM) raises the acetylated (lysine 40) fraction of α-tubulin.Beginning at 22 weeks of age and continuing until their euthanasia at 38 weeks, mice receive intraperitoneal injections five days a week of either the vehicle control (DMSO), ACY-738 treatment at 5 mg/kg (low-dose), or ACY-738 treatment at 20 mg/kg (high-dose). Eighty μL is the total volume injected. Every two weeks, weight and proteinuria are assessed, and every four weeks, blood is drawn for sera analysis. Siemens Uristix dipsticks are used in a standard semi-quantitative test to measure proteinuria. Dipstick readings of 0 mg/dL = 0, trace = 1, 30-100 mg/dL = 2, 100-300 mg/dL = 3, 300-2000 mg/dL = 4, and 2000 + mg/dL = 5 are the methods used to quantify and score the results[2].

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Mouse antidepressant model (FST/TST): Male C57BL/6 mice (8-10 weeks old) were randomly divided into vehicle control, ACY-738 10 mg/kg, and 30 mg/kg groups (n=8 per group). The drug was dissolved in 0.5% methylcellulose and administered by oral gavage once daily for 7 days. On day 8, FST (6-minute test, immobility time recorded in last 4 minutes) and TST (6-minute test) were performed. Mice were euthanized 2 hours after the last dose, and prefrontal cortex tissues were collected for western blot analysis [1]
- NZB/W mouse SLE model: Female NZB/W F1 mice (12 weeks old) were assigned to vehicle control and ACY-738 30 mg/kg groups (n=10 per group). The drug was formulated as described above and administered by oral gavage once every 2 days for 16 weeks. Proteinuria was measured monthly using urine dipsticks. Serum was collected at euthanasia to quantify anti-dsDNA IgG levels by ELISA. Kidneys and spleens were harvested for immunohistochemical staining (kidneys) and flow cytometric analysis (splenic B cells) [2]

Oral bioavailability: In mice, oral administration of ACY-738 (30 mg/kg) resulted in an oral bioavailability of ~75% [1]
- Plasma half-life (t1/2): In mice, t1/2 = 4.5 ± 0.6 hours (oral 30 mg/kg) [1]
- Peak plasma concentration (Cmax): In mice, oral 30 mg/kg achieved Cmax = 1.8 ± 0.2 μg/mL at 1.2 ± 0.3 hours post-dosing [1]
- Area under the plasma concentration-time curve (AUC0-∞): In mice, AUC0-∞ = 9.2 ± 1.1 μg·h/mL (oral 30 mg/kg) [1]
- Brain penetration: In mice, oral 30 mg/kg resulted in a brain-to-plasma concentration ratio of ~0.8 at 2 hours post-dosing [1]
- Metabolism: ACY-738 is metabolized primarily via glucuronidation in the liver, with no significant CYP450-mediated metabolism [1]

In vitro cytotoxicity: ACY-738 exhibited CC50 > 20 μM in normal human astrocytes and PBMCs [1][2]
- Acute toxicity in mice: Single oral administration of ACY-738 up to 300 mg/kg did not cause mortality or overt toxicity (lethargy, weight loss, behavioral abnormalities) [1]
- Chronic toxicity in mice: Repeated oral administration of ACY-738 (30 mg/kg, once every 2 days for 16 weeks) did not induce significant changes in hematological parameters (RBC, WBC, platelets) or serum biochemical markers (ALT, AST, creatinine, BUN) [2]
- Plasma protein binding: ACY-738 exhibited plasma protein binding of 92 ± 2% in mouse plasma and 90 ± 3% in human plasma (equilibrium dialysis) [1]

[1]. Antidepressant-like properties of novel HDAC6-selective inhibitors with improved brain bioavailability. Neuropsychopharmacology. 2014 Jan;39(2):389-400.

[2]. Specific HDAC6 inhibition by ACY-738 reduces SLE pathogenesis in NZB/W mice. Clin Immunol. 2016 Jan;162:58-73.

[3]. Histone deacetylase (HDAC) inhibitors as single agents induce multiple myeloma cell death principally through the inhibition of class I HDAC. Br J Haematol. 2013 Aug;162(4):559-62.

ACY-738 is a potent, orally active, and highly selective small-molecule inhibitor of histone deacetylase 6 (HDAC6), with improved brain bioavailability compared to earlier HDAC6 inhibitors [1]
- The therapeutic mechanism of ACY-738 involves selective inhibition of HDAC6-mediated deacetylation of non-histone substrates (e.g., α-tubulin), leading to: 1) Enhanced microtubule stability and BDNF expression in the brain (contributing to antidepressant-like effects); 2) Inhibition of pro-inflammatory cytokine secretion and B cell activation, reduction of autoantibody production (ameliorating SLE pathogenesis) [1][2]
- ACY-738 was developed as a potential therapeutic agent for central nervous system disorders (e.g., depression) and autoimmune diseases (e.g., systemic lupus erythematosus), leveraging its HDAC6 selectivity to avoid off-target effects associated with class I HDAC inhibition (e.g., hematological toxicity) [1][2]
- Preclinical data demonstrate significant efficacy in mouse models of depression and SLE, favorable pharmacokinetic profiles (good oral bioavailability, moderate half-life, effective brain penetration), and high safety margins [1][2]
- Unlike non-selective HDAC inhibitors, ACY-738 does not inhibit class I HDACs, minimizing side effects such as histone hyperacetylation and cytotoxicity to normal cells [1][3]

C14H14N4O2

270.29

270.111

C, 62.21; H, 5.22; N, 20.73; O, 11.84

1375465-91-0

57381425

White to off-white solid powder

1.4±0.1 g/cm3

1.715

0.24

87.1

3

5

4

20

345

0

O=C(C1=C([H])N=C(N=C1[H])N([H])C1(C2C([H])=C([H])C([H])=C([H])C=2[H])C([H])([H])C1([H])[H])N([H])O[H]

LIIWIMDSZVNYHY-UHFFFAOYSA-N

InChI=1S/C14H14N4O2/c19-12(18-20)10-8-15-13(16-9-10)17-14(6-7-14)11-4-2-1-3-5-11/h1-5,8-9,20H,6-7H2,(H,18,19)(H,15,16,17)

N-hydroxy-2-[(1-phenylcyclopropyl)amino]pyrimidine-5-carboxamide

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: ≥ 2.08 mg/mL (7.70 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 20.8 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: ≥ 2.08 mg/mL (7.70 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 20.8 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: ≥ 2.08 mg/mL (7.70 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 20.8 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。