生物活性

Entinostat inhibits HDAC1 and HDAC3 with IC50 of 0.51 μM and 1.7 μM. Entinostat shows inhibitory to HDACs by 2'-amino group. Entinostat induces accumulation of p21WAF1/CIP1 and gelsolin in K562 cell. Entinostat could reduce S-phase cells and induce G1-phase cells in A2780 cell. Inhibits the proliferation of human tumor cell lines including A2780, Calu-3, HL-60, K562, St-4, HT-29, KB-3-1, Capan-1, 4-1St and HCT-15 with IC50 from 41.5 nM to 4.71 μM, which due to HAD-inhibition. Entinostat is not sensitive to other HDACs (4, 6, 8 and 10) with IC50 about/above 100 μM. It shows great inhibition to human leukemia and lymphoma cells, including U937, HL-60, K562, and Jurkat. Entinostat also decreases expression of cyclin D1 and the antiapoptotic proteins Mcl-1 and XIAP.

Inhibition of recombinant human histonedeacetylases (HDACs)

Cell viability

体外研究

体内研究

激酶实验

HDAC inhibition assay[1]

The HDAC inhibition assay was performed intriplicate. Briefly, the respective human recombinant HDAC enzymes wereincubated in absence and/or in presence of various concentrations MS-275 andthe pro-fluorogenic substrate at room temperature for 60 min. Next, thedeacetylation reaction was stopped with the addition of the HDAC Stop Solution(6 mg/mL trypsin, 0.3 mM SAHA) in all wells and the plate was incubated at 37°C for 20 min. The release of the fluorescent 7-amino-4-methylcoumarin wasmonitored by measuring the fluorescence atλem = 460 nmand λ ex = 390 nm using a Synergy H1 plate reader. The fluorescence value ofthe background wells was subtracted from the fluorescence of the positive control,blank and inhibitor wells. Nonlinear regression was used to fit the data to thelog (inhibitor) versus response curve using GraphPad Prism.

细胞实验

Materials[6]

Estrogen receptor (ER)α-negativeMDA-MB-231, BT549, and Hs578T cells were obtained and cultured in Dulbecco ModifiedEagle Medium (DMEM) containing 5% FBS and penicillin/ streptomycin solution(P/S; 10,000 IU each). Entinostat for in vitro use was prepared in DMSO as a 0.1mol/Lstock and further diluted in95% ethanol. Treatment with entinostat subjectedcells to less than 0.5% of vehicle.

Flowcytometry

TNBC cells were treated with entinostat (1μmol/L)for 72 hours. The cells were harvested and resuspended in DPBS containing 2%BSA. One million cells were pelleted and resuspended with a cocktail ofAPC-anti human CD44, FITC-anti human CD24 and nearinfrared viability dye. Cellswere incubated on ice for 40 minutes. Following incubation, cells were washedwith DPBS containing 2% BSA and resuspended in DPBS containing2% BSA. Afterresuspension, data were acquired on FACS Canto with appropriate compensationcontrols. At least 10,000 viable cells were acquired and data plotted. Datawere analyzed using FlowJo software.

动物实验

TumorMouse Xenograft Models[7]

Murine lung metastases were established in6- to 8-wk-old NOD.Cg-Rag1tm1MomPrf1tm1Sdz/SzJ mice by injecting5×105HT1080.eGFP-FFLuc tumor cells via tail vein. Mice were treatedwith 752ng entinostat via intraperitoneal administration (in 0.1 mL PBS), or8×106IL-2-activated NK cells (in 0.2 mL PBS) by tail-vein injection.Mice received entinostat, NK cells, or both on days 4, 11, and 18. The controlgroup received no treatment.

BioluminescentImaging (BLI)

Tumor volume was assessed by imagingisofluoraneanesthetized mice using the IVIS system. Images were obtained 15 minafter intraperitoneal injection of 1.5 mg (approx. 75 mg/kg) D-luciferin in 0.1mL of PBS. The light emitted by luciferase-expressing tumors was quantifiedusing Living Image Version 2.50. A heatmap image corresponding tophotons/second/cm2/steradian (blue lowest and red highest) wassuperimposed over the reflected-light image of the animals for tumorlocalization. A rectangular region of interest (ROI) encompassing the mousehead and body was replicated between images, images were adjusted to identicalluminescent scales, and bioluminescence reported as total flux for the regionin photons/sec. Animals were imaged on day 12, 15, 18, 23, and 27.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Leus NG, van den Bosch T, van der Wouden PE, et al. HDAC1-3 inhibitor MS-275 enhances IL10 expression in RAW264.7 macrophages and reduces cigarette smoke-induced airway inflammation in mice. Sci Rep. 2017;7:45047.
[2] Takayuki Tatamiya AS TS, Osamu Nakanishi. Isozyme-seIective activity of the HDAC inhibitor MS-275. American Association for Cancer Research. 2004;64(7):567.
[3] Altmann A, Eisenhut M, Bauder-Wust U, et al. Therapy of thyroid carcinoma with the histone deacetylase inhibitor MS-275. Eur J Nucl Med Mol Imaging. 2010;37(12):2286-2297.
[more]

体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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