HDAC3 (IC50 = 67 nM); HDAC10 (IC50 = 78 nM); HDAC1 (IC50 = 95 nM); HDAC2 (IC50 = 160 nM); HDAC11 (IC50 = 432 nM); HDAC8 (IC50 = 733 nM)
Chidamide, at low nanomolar concentrations, inhibits both class IIb HDAC10 and class I HDACs 1-3. Both human PBMC and HeLa cervical adenocarcinoma cells exhibit a significant increase in histone H3 acetylation upon exposure to chidamide. Chidamide and MS-275 similarly inhibit the in vitro growth of most tumor cells, but not all of them, in the low micromolar concentration range, according to studies on cell growth inhibition carried out with 18 human-derived tumor cell lines. Nonetheless, normal cells from human fetal kidney (CCC-HEK) and liver (CCC-HEL) are significantly less toxic to chidamide and, to a lesser extent, MS-275, suggesting that normal cells and cancerous cells respond to chidamide differently in terms of cytotoxicity[1].

Chidamide, at low nanomolar concentrations, inhibits both class IIb HDAC10 and class I HDACs 1-3. Both human PBMC and HeLa cervical adenocarcinoma cells exhibit a significant increase in histone H3 acetylation upon exposure to chidamide. Chidamide and MS-275 similarly inhibit the in vitro growth of most tumor cells, but not all of them, in the low micromolar concentration range, according to studies on cell growth inhibition carried out with 18 human-derived tumor cell lines. Nonetheless, normal cells from human fetal kidney (CCC-HEK) and liver (CCC-HEL) are significantly less toxic to chidamide and, to a lesser extent, MS-275, suggesting that normal cells and cancerous cells respond to chidamide differently in terms of cytotoxicity[1].

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Chidamide exhibited inhibitory activity against a panel of human recombinant HDAC proteins, with low nanomolar potency against HDAC1, 2, 3, and 10. It showed much weaker activity against HDAC4, 8, and 11, and no significant inhibition up to 30 µM against HDAC5, 6, 7, and 9.[1]
Treatment with chidamide significantly induced histone H3 acetylation in HeLa cervical adenocarcinoma cells. Acetylation increased as early as 30-60 minutes after treatment with 1 µM chidamide and reached maximal levels at 24-72 hours. A similar induction of histone H3 acetylation was observed in healthy human peripheral blood mononuclear cells (PBMC) treated ex vivo with chidamide.[1]
Cell growth inhibition studies using 18 human tumor-derived cell lines demonstrated that chidamide inhibited the in vitro growth of most tumor cells in the low micromolar concentration range (Gl₅₀ values ranging from 0.4 µM to >50 µM). Notably, chidamide was significantly less toxic to normal human fetal kidney (CCC-HEK) and liver (CCC-HEL) cells, with Gl₅₀ values >100 µM, indicating a differential cytotoxic response between normal and cancerous cells.[1]
Ex vivo treatment of PBMC from healthy donors with nanomolar concentrations of chidamide (100 nM for 48 h was optimal) significantly enhanced their cytotoxicity against K562 human myeloid leukemia target cells. This enhanced lysis was not observed when only the target K562 cells were pre-treated with chidamide. Maximal enhancement occurred when both effector (PBMC) and target cells were treated.[1]
Flow cytometric analysis revealed that treatment of PBMC with 100 nM chidamide for 48 hours upregulated the expression of proteins involved in natural killer (NK) cell functions, including the activating receptors CD16 and NKG2D, and the cytotoxic enzyme granzyme A (GZMA).[1]

Chidamide exhibits in vivo antitumor activity in xenografts of HCT-8 mice with colorectal carcinoma. Tumor size and weight are dose-dependently reduced by cidamide in the range of 12.5–50 mg/kg. When compared to the control medications 5-fluorouracil (5-FU, 20 mg/kg) and MS-275 (25 mg/kg, which was reported as the maximum tolerated dose in xenograft models), the efficacy of the 50 mg/kg dose is comparable to or greater. On the other hand, the tumor-bearing animals tolerate chidamide at the above doses well, while the control medications significantly reduce body weight[1].

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The in vivo antitumor activity of chidamide was evaluated in athymic nude mice subcutaneously inoculated with human tumor xenografts. Oral administration of chidamide once daily for 20-28 days demonstrated dose-dependent antitumor efficacy.[1]
In HCT-8 colorectal carcinoma xenografts, chidamide at doses of 12.5, 25, and 50 mg/kg significantly reduced tumor volume and tumor weight compared to vehicle control. The efficacy at 50 mg/kg was similar or greater than that of the control drugs MS-275 (25 mg/kg) and 5-fluorouracil (5-FU, 20 mg/kg). Importantly, chidamide was well-tolerated at these doses without causing significant body weight loss, whereas the control drugs did cause weight loss.[1]
Similar significant and dose-dependent antitumor effects of chidamide (12.5 and 25 mg/kg) were also observed in nude mouse models bearing A549 lung carcinoma, BEL-7402 liver carcinoma, and MCF-7 breast carcinoma xenografts, with no gross body weight loss observed during treatment.[1]

The Colorimetric HDAC Activity Assay kit's instructions are followed to detect HDAC activity. HDAC substrate and nuclear protein (50 μg) extract from leukemia cells are added to each reaction (100 μL). Chidamide (CS055) and MS-275 are added to the mixtures and incubated at 37°C for one hour in order to test the impact of HDACis. A microplate reader is used to measure the HDAC activities at 405 nm. The double-distilled water containing 10 μM Trichostatin A, a known strong HDACi, is used as a negative control and set to 0%, while the positive control (only nuclear extract and vehicle) is set to 100%.

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The inhibition of HDAC isotypes by chidamide was analyzed using human recombinant HDAC proteins. Enzymatic reactions were carried out in a buffer containing bovine serum albumin, an HDAC substrate, a purified recombinant HDAC enzyme, and the test compound at predefined concentrations. The reactions were incubated at room temperature for 17 hours. After incubation, a developer solution was added to each well and the plate was incubated for an additional 20 minutes. Fluorescence intensity was then measured using a microplate reader. Concentration-response inhibition curves were analyzed to determine IC50 values. Each compound concentration was tested in duplicate.[1]

Chidamide is added to isolated PBMC effector cells in 6-well plates (6 x 10⁶ cells/well) for varying durations of time (24-72 hours) at varying concentrations (0-400 nM).

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For in vitro cell growth inhibition assays, tumor cells were seeded into 96-well plates. After 24 hours, test compounds at different concentrations were added, and cells were cultured for an additional 72 hours. Cell viability was then assessed by adding a reagent, incubating for 2 hours at 37°C, and measuring the absorbance at 490 nm. The concentration inhibiting cell growth by 50% (Gl₅₀) was determined. All compounds were dissolved in DMSO and diluted for use. Samples were evaluated in duplicate, and experiments were repeated at least three times.[1]
For histone acetylation analysis, HeLa cells were treated with compounds. Histones were isolated from cell pellets through a series of steps involving lysis, washing, acid extraction, and precipitation. The extracted histones were dissolved and protein concentration was quantified. For Western blot analysis, proteins were transferred to a membrane, probed with an anti-acetylated H3 antibody, and re-probed with an anti-total histone H3 antibody. Detection was performed using horseradish peroxidase-conjugated secondary antibodies with enhanced chemiluminescence.[1]
For the cellular cytotoxicity assay, peripheral blood mononuclear cells (PBMC) from healthy donors were isolated and treated with chidamide in culture plates. After treatment, PBMC were washed and co-incubated with K562 target cells at various effector-to-target ratios for 4 hours at 37°C. Cytotoxicity was analyzed using a standard lactate dehydrogenase (LDH) release assay kit. The percentage of specific lysis was calculated after correcting for spontaneous release.[1]
For flow cytometric analysis, PBMC treated with or without chidamide were incubated with fluorescently labeled monoclonal antibodies against specific surface proteins (e.g., CD16, NKG2D, GZMA) or corresponding isotype controls. Cells were then analyzed on a flow cytometer, and specific fluorescence indices were calculated.[1]

Athymic nude mice (BALB/c-nu)
12.5-50 mg/kg
oral

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Athymic nude mice (BALB/c-nu), 6-8 weeks old, were used for in vivo antitumor studies. Human tumor cells (HCT-8, A549, MCF-7, Bel-7402) were passaged in vivo and then transplanted subcutaneously into the flank of mice as tumor fragments. Treatment began 3 days after tumor inoculation. Chidamide was dissolved in a vehicle containing 0.2% carboxymethyl cellulose and 0.1% Tween 80. It was administered orally, once daily, for 20 to 28 consecutive days, depending on the tumor model. Tumor dimensions (length and width) were measured every 3 days to calculate tumor volume. At the end of the experiment, mice were euthanized, and tumors were excised and weighed. Each experimental group consisted of 8-10 mice.[1]

Literature indicates that chidamide has oral bioavailability. [1]

In vitro experiments showed that chidamide was significantly less toxic to normal human fetal kidney and liver cells (Gl₅₀ > 100 µM) compared to many tumor cell lines. [1] In vivo experiments showed that oral administration of chidamide to tumor-bearing nude mice at doses up to 50 mg/kg/day for several weeks was well tolerated, with no significant weight loss observed, while control drugs (MS-275 and 5-FU) both caused significant weight loss at their effective doses. [1]

[1]. Chidamide (CS055/HBI-8000): a new histone deacetylase inhibitor of the benzamide class with antitumor activity and the ability to enhance immune cell-mediated tumor cell cytotoxicity. Cancer Chemother Pharmacol. 2012 Apr;69(4):901-9.

Chidamide belongs to the benzamide class of drugs. Tuxenostat is an investigational drug currently being studied as part of a strategy to treat HIV infection. Tuxenostat belongs to a class of HIV drugs known as latency reversal agents. Tuxenostat is an orally bioavailable inhibitor of histone deacetylase (HDAC) isoenzymes 1, 2, 3, and 10, possessing potential antitumor activity. After administration, tuxenostat binds to HDAC and inhibits its activity, leading to increased histone acetylation levels. This drug can also inhibit the expression of kinases in the PI3K/Akt and MAPK/Ras signaling pathways, potentially causing cell cycle arrest and inducing tumor cell apoptosis. This may inhibit the proliferation of susceptible tumor cells. Histone deacetylases (HDACs) are enzymes that deacetylate chromatin histones; their expression is upregulated in various tumor types and they play a crucial role in gene expression. Compared with some other benzamide HDAC inhibitors, Chidamide is more stable, less prone to degradation, and has a longer half-life.

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Drug indications
It has been studied for the treatment of cancer/tumor (not specified).

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Mechanism of action
Chidamide is an orally bioavailable histone deacetylase (HDAC) inhibitor belonging to the benzamide class. Histone deacetylase inhibitors are a class of anticancer drugs that can selectively regulate gene expression in cancer cells. [Huya Bio Press Release]

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Chidamide is a novel benzamide histone deacetylase (HDAC) inhibitor discovered using computer-aided rational drug design methods. [1]
Its main mechanism of action is to inhibit class I HDACs (1, 2, 3) and class IIb HDAC10, thereby increasing histone acetylation and altering gene expression. [1]
In addition to its direct antitumor effects, chidamide can also enhance immune cell-mediated cytotoxicity by upregulating the expression of proteins and genes involved in natural killer (NK) cell function in peripheral blood mononuclear cells. [1]
Gene chip expression analysis of peripheral blood leukocytes from patients with T-cell lymphoma treated with chidamide showed upregulation of genes associated with immune cell-mediated antitumor activity, including NK activation receptors, cytotoxic enzymes, and apoptosis-related genes. [1]
Chidamide has shown broad-spectrum antitumor activity in preclinical models and is currently undergoing clinical development for cancer indications. [1]

C22H19FN4O2

390.41

390.149

1616493-44-7

Tucidinostat-d4

12136798

White to off-white solid

1.3±0.1 g/cm3

602.1±55.0 °C at 760 mmHg

317.9±31.5 °C

0.0±1.7 mmHg at 25°C

1.691

2.4

3

5

6

29

577

0

FC1C=CC(=C(C=1)N)NC(C1C=CC(=CC=1)CNC(/C=C/C1C=NC=CC=1)=O)=O

SZMJVTADHFNAIS-BJMVGYQFSA-N

InChI=1S/C22H19FN4O2/c23-18-8-9-20(19(24)12-18)27-22(29)17-6-3-16(4-7-17)14-26-21(28)10-5-15-2-1-11-25-13-15/h1-13H,14,24H2,(H,26,28)(H,27,29)/b10-5+

N-(2-amino-4-fluorophenyl)-4-[[[(E)-3-pyridin-3-ylprop-2-enoyl]amino]methyl]benzamide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.40 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.40 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.40 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 1% CMC Na: 30mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)