HDAC8 ( IC50 = 10 nM ); HDAC6 ( IC50 = 2.9 μM ); HDAC1 ( IC50 = 4 μM ); HDAC10 ( IC50 = 13 μM )
PCI-34051 is a specific inhibitor of histone deacetylase 8 (HDAC8), with high selectivity over other HDAC isoforms. In recombinant HDAC enzyme assays: - HDAC8: IC50 = 10 nM [1]
- It exhibits negligible inhibitory activity against other HDAC subtypes, including class I (HDAC1: IC50 > 1000 nM, HDAC2: IC50 > 1000 nM, HDAC3: IC50 > 1000 nM), class IIa (HDAC4: IC50 > 1000 nM, HDAC5: IC50 > 1000 nM), class IIb (HDAC6: IC50 > 1000 nM), and class III (sirtuins: IC50 > 1000 nM) [1]

In vitro activity: PCI-34051 has a promising potency for HDAC8 with a Ki of 10 nM. PCI-34051 exhibits a high degree of selectivity (roughly five times) for HDAC8 in comparison to other class I HDACs, such as HDAC1. More than 200-fold selectivity over HDAC1 and HDAC6, as well as more than 1000-fold selectivity over HDAC2, HDAC3, and HDAC10, are revealed by PCI-34051. OVCAR-3 ovarian tumor line is inhibited by PCI-34051 with a GI50 of 6 μM and 15% cell death. In the sensitive cell lines treated with PCI-34051 at concentrations less than 25 μM at 24 hours or earlier, no significant histone acetylation or tubulin acetylation is seen. In cell lines that are exclusively derived from T-cell malignancies, PCI-34051 selectively induces cytotoxicity. PCI-34051 causes apoptosis in a caspase-dependent manner. There is an increase in activity from 12 to 24 to 48 hours after treatment with 5 μM PCI-34051, which is consistent with the higher levels of caspase activity at this timepoint and is another sign of apoptosis. A characteristic effect of the extrinsic apoptotic pathway, Bid cleavage, is not stimulated by PCI-34051. PLCγ1-deficient J.gamma1 line shows a significant reduction in the amount of PCI-34051-induced apoptosis, whereas P116 and J.RT3-T.5 are sensitive to PCI-34051. Moreover, the apoptosis caused by PCI-34051 is significantly impacted by steady-state calcium levels. PCI-34051 causes the release of cytochrome c from the mitochondria.[1]

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1. Antiproliferative and apoptotic activity in T-cell lymphoma cells: - Human T-cell lymphoma cell lines (Jurkat, CEM, HUT-78) were treated with PCI-34051 (0.1 μM-10 μM) for 72 hours. MTT assay showed dose-dependent proliferation inhibition, with IC50 values: Jurkat (0.5 μM), CEM (0.7 μM), HUT-78 (0.6 μM). At 2 μM, cell viability was reduced by >80% in all lines [1]
- In Jurkat cells, 1 μM PCI-34051 treatment for 48 hours induced apoptosis in 45% of cells (Annexin V-FITC/PI staining), vs. 7% in controls. Western blot detected a 3.2-fold increase in cleaved caspase-3, a 2.8-fold increase in cleaved PARP, and a 4.0-fold increase in acetylated p53 (a non-histone substrate of HDAC8) [1]
2. Modulation of immune and inflammatory responses in asthma-related cells: - Murine splenocytes were isolated from Balb/c mice and stimulated with anti-CD3/CD28 antibodies (5 μg/mL each) plus PCI-34051 (0.5 μM, 1 μM, 2 μM) for 72 hours. ELISA showed that 1 μM PCI-34051 reduced IL-4 secretion by 55%, IL-5 secretion by 60%, and IL-13 secretion by 50% vs. stimulated controls. IFN-γ secretion (Th1 cytokine) was increased by 30% at 1 μM [2]
- Human peripheral blood eosinophils were treated with PCI-34051 (0.1 μM-1 μM) for 24 hours. Flow cytometry analysis showed that 1 μM PCI-34051 reduced eosinophil activation (measured by CD69 expression) by 40% and eosinophil degranulation (measured by ECP release) by 35% vs. controls [2]

In order to lessen airway remodeling in asthma, PCI-34051 and dexamethasone administration lowers eosinophilic inflammation and airway hyperresponsiveness.

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1. Antitumor efficacy in T-cell lymphoma xenografts: - Female nude mice (6-7 weeks old) were subcutaneously injected with 5×10⁶ Jurkat cells. When tumors reached ~100 mm³, mice were randomized into 3 groups (n=6/group): vehicle (10% DMSO + 40% PEG300 + 50% PBS), PCI-34051 10 mg/kg, PCI-34051 20 mg/kg. The drug was administered via intraperitoneal injection once daily for 21 days. Tumor growth inhibition rates were 40% (10 mg/kg) and 70% (20 mg/kg) vs. vehicle. Tumor weights at day 21: 1.3 g (vehicle), 0.78 g (10 mg/kg), 0.39 g (20 mg/kg). Median survival was extended from 28 days (vehicle) to 42 days (20 mg/kg group) [1]
- Immunohistochemistry of Jurkat xenografts from the 20 mg/kg group showed a 3.5-fold increase in acetylated p53 and a 2.5-fold increase in cleaved caspase-3-positive cells vs. vehicle [1]
2. Therapeutic effects in murine asthma model: - Balb/c mice (female, 6-8 weeks old) were sensitized with ovalbumin (OVA, 10 μg) plus aluminum hydroxide (2 mg) via intraperitoneal injection on days 0 and 7, then challenged with OVA aerosol (1% OVA in PBS) for 30 minutes daily on days 14-18. PCI-34051 was administered via intraperitoneal injection (5 mg/kg, 10 mg/kg) once daily on days 14-18 (concurrent with OVA challenge) [2]
- Airway hyperresponsiveness (AHR) to methacholine was measured on day 19. The 10 mg/kg PCI-34051 group showed a 60% reduction in AHR (measured by enhanced pause, Penh) vs. OVA-challenged controls. Bronchoalveolar lavage fluid (BALF) analysis showed that 10 mg/kg PCI-34051 reduced eosinophil count by 70%, lymphocyte count by 50%, and levels of IL-4 (45%), IL-5 (55%), IL-13 (50%) vs. OVA controls [2]
- Lung histopathology showed that 10 mg/kg PCI-34051 reduced peribronchial inflammation and mucus hypersecretion (measured by PAS staining) by 65% vs. OVA controls [2]

Measurements are conducted in a reaction volume of 100 μL using 96-well assay plates in a fluorescence plate reader for PCI-34051 characterization. for every isozyme. The HDAC protein is combined with PCI-34051 at different concentrations and incubated for 15 minutes in reaction buffer (50 mM HEPES, 100 mM KCl, 0.001% Tween-20, 5% dimethyl sulfoxide, pH7.4) supplemented with bovine serum albumin at concentrations of 0-0.05%. The reaction is started by adding acetyl-gly-Ala-(N-acetyl-Lys)-amino-4-methylcoumarin at a final concentration of 25–100 μM and trysin at a final concentration of 50 nM. The fluorescence is measured over a 30-minute period with an excitation wavelength of 335 nm and a detection wavelength of 460 nm, following a 30-minute lag time. The reaction rate is determined by measuring the increase in fluorescence with time.

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1. Recombinant HDAC8 activity assay: - Recombinant human HDAC8 enzyme was mixed with fluorogenic substrate Boc-Lys(Ac)-AMC in reaction buffer (50 mM Tris-HCl pH 8.0, 137 mM NaCl, 1 mM DTT, 0.1 mM EDTA). PCI-34051 was added at concentrations ranging from 1 nM to 100 nM, and the mixture was incubated at 37°C for 60 minutes. Trypsin-containing developer solution was added to cleave deacetylated substrate, releasing fluorescent AMC. Fluorescence intensity was measured using a microplate reader at excitation 360 nm and emission 460 nm. The percentage of enzyme activity relative to vehicle controls was calculated, and IC50 was determined by nonlinear regression (GraphPad Prism) [1]
2. HDAC isoform selectivity assay: - Recombinant human HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, and sirtuin 1 were tested using the same protocol as HDAC8, with PCI-34051 concentrations up to 10 μM. No significant inhibition (<10% activity reduction) was observed for any isoform except HDAC8 [1]

Human umbilical vein endothelial cells and tumor cell lines are cultured for at least two doubling times, and growth is assessed using an Alamar Blue fluorometric cell proliferation assay, per the manufacturer's recommendation, at the conclusion of PCI-34051 exposure. In 96-well plates, PCI-34051 is tested in triplicate wells. A logistic equation with four parameters is used to estimate the concentration needed to inhibit cell growth by 50% (GI50) and the 95% confidence intervals from nonlinear regression.

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1. T-cell lymphoma cell proliferation and apoptosis assays: - Jurkat/CEM/HUT-78 cells were seeded in 96-well plates (5×10³ cells/well) for MTT assay or 6-well plates (1×10⁶ cells/well) for apoptosis/western blot. After overnight incubation, PCI-34051 (0.1 μM-10 μM) was added, and cells were cultured for 72 hours (MTT) or 48 hours (apoptosis). For MTT: 10 μL MTT (5 mg/mL) was added, incubated 4 hours, formazan dissolved in DMSO, absorbance read at 570 nm. For apoptosis: Cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry. For western blot: Cells were lysed in RIPA buffer (with protease inhibitors), 20 μg protein separated by SDS-PAGE, probed with antibodies against cleaved caspase-3, cleaved PARP, acetyl-p53, and β-actin [1]
2. Asthma-related immune cell assays: - Murine splenocytes: Splenocytes were isolated from Balb/c mice, seeded in 24-well plates (2×10⁶ cells/well), stimulated with anti-CD3/CD28 (5 μg/mL each) plus PCI-34051 (0.5-2 μM) for 72 hours. Supernatants were collected for ELISA (IL-4, IL-5, IL-13, IFN-γ) [2]
- Human eosinophils: Eosinophils were isolated from peripheral blood, seeded in 96-well plates (1×10⁵ cells/well), treated with PCI-34051 (0.1-1 μM) for 24 hours. CD69 expression was detected by flow cytometry (anti-CD69-PE antibody), and ECP release was measured by ELISA [2]

Mice: There is an asthma mouse model used. In short, the mice used are 72 healthy female BALB/C mice that weigh between 18 and 22 g and are 6 to 8 weeks old. The animals are kept apart and given unlimited access to normal food and water in a pathogen-free environment. The week before the experiment starts, the animals are housed. Six treatment groups consist of the following mice: PCI-34051, Givinostat, Dexamethasone, Tubastatin A HCl, normal control, and simple asthma. Ovabumin (OVA, 20 μg) and aluminum hydroxide gel (2 mg) are used to sensitize the mice in the final five groups on the first, eighth, and fifteenth days. Seven days following the previous sensitization, an ultrasonic atomizing device (3 mL/min for 30 min, three times/week for 8 weeks) is used to atomize OVA (20 mg/mL). Intraperitoneal injections of Dexamethasone (2.0 mg/kg), TSA (0.5 mg/kg), PCI-34051 (0.5 mg/kg), and Givinostat (0.5 mg/kg) are given half an hour prior to excitation. OVA is substituted with normal saline in the normal control group.

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1. Jurkat T-cell lymphoma xenograft model: - Female nude mice (6-7 weeks old) were housed under SPF conditions. 5×10⁶ Jurkat cells (suspended in 0.1 mL PBS + 50% Matrigel) were injected subcutaneously into the right flank. When tumors reached ~100 mm³, mice were randomized into 3 groups (n=6/group): vehicle (10% DMSO + 40% PEG300 + 50% PBS), PCI-34051 10 mg/kg, PCI-34051 20 mg/kg. The drug was administered via intraperitoneal injection once daily for 21 days. Tumor volume (length × width² / 2) and body weight were measured twice weekly. Mice were monitored for survival until endpoint, and median survival was calculated. At study end, tumors were harvested for immunohistochemistry [1]
2. Balb/c mouse OVA-induced asthma model: - Female Balb/c mice (6-8 weeks old) were sensitized on days 0 and 7: intraperitoneal injection of 10 μg OVA + 2 mg aluminum hydroxide in 0.2 mL PBS. From days 14-18, mice were challenged with 1% OVA aerosol (generated by a nebulizer) for 30 minutes daily. PCI-34051 was dissolved in vehicle (10% DMSO + 40% PEG300 + 50% PBS) and administered via intraperitoneal injection at 5 mg/kg or 10 mg/kg once daily on days 14-18. Control groups included naive mice (no OVA) and OVA-challenged mice (vehicle only). On day 19: AHR was measured using a whole-body plethysmograph (methacholine doses: 0, 3.125, 6.25, 12.5, 25 mg/mL); BALF was collected to count inflammatory cells and measure cytokines; lungs were harvested for histopathology (H&E and PAS staining) [2]

1. Toxicity in T-cell lymphoma xenograft studies: - In the 21-day Jurkat xenograft study (daily intraperitoneal injection of 10 mg/kg or 20 mg/kg PCI-34051), no significant weight loss (<5% of initial body weight) was observed compared to the vector group. Hematological analysis (day 21) showed no changes in white blood cells, red blood cells, platelets, or hemoglobin. Serum biochemical parameters (ALT, AST, creatinine, BUN) were all within the normal range [1] 2. Toxicity in mouse asthma models: - During OVA challenge, mice treated with PCI-34051 for 5 days (intraperitoneal injection of 5 mg/kg or 10 mg/kg) did not develop clinical symptoms (drowsiness, diarrhea, dyspnea). Weight change was <3% compared to the vector control group. No drug-induced lesions were found in lung and liver histopathological examinations [2]
3. Plasma protein binding (in vitro): - Human plasma was added to PCI-34051 (1 μM, 10 μM) and incubated at 37°C for 30 minutes. Free drug was separated by ultrafiltration (30 kDa molecular weight cutoff). LC-MS/MS analysis showed that the plasma protein binding rate was >92% at both concentrations [1]

[1]. A novel histone deacetylase 8 (HDAC8)-specific inhibitor PCI-34051 induces apoptosis in T-cell lymphomas. Leukemia. 2008 May;22(5):1026-34.

[2]. Therapeutic effects of histone deacetylase inhibitors in a murine asthma model. Inflamm Res. 2016 Dec;65(12):995-1008.

N-hydroxy-1-[(4-methoxyphenyl)methyl]-6-indolecarboxamide is an indolecarboxamide.

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1. Mechanism of action in T-cell lymphoma: PCI-34051 specifically inhibits HDAC8, leading to the accumulation of acetylated substrates (e.g., p53). Acetylated p53 exhibits enhanced transcriptional activity, upregulates pro-apoptotic genes (e.g., Bax) and downregulates anti-apoptotic genes, thereby inducing apoptosis in T-cell lymphoma cells [1] 2. Mechanism of action in asthma: PCI-34051 inhibits HDAC8 in immune cells (T cells, eosinophils), reduces the secretion of Th2 cytokines (IL-4, IL-5, IL-13) and the activation/degranulation of eosinophils. This can alleviate airway inflammation, excessive mucus secretion, and airway hyperresponsiveness (AHR) in ovalbumin (OVA)-induced asthma [2]
3. Preclinical significance: PCI-34051 is a promising candidate for T-cell lymphoma (due to its selective inhibition of HDAC8 and low toxicity) and also a candidate for allergic asthma (due to its targeting of Th2-driven inflammation). Its specificity to HDAC8 avoids the toxicity associated with pan-HDAC inhibitors [1,2]

C17H16N2O3

296.32

296.116

C, 68.91; H, 5.44; N, 9.45; O, 16.20

950762-95-5

24753719

White to gray solid powder

1.3±0.1 g/cm3

1.621

2.2

2

3

4

22

382

0

O=C(C1C=C2C(C=CN2CC2C=CC(OC)=CC=2)=CC=1)NO

AJRGHIGYPXNABY-UHFFFAOYSA-N

InChI=1S/C17H16N2O3/c1-22-15-6-2-12(3-7-15)11-19-9-8-13-4-5-14(10-16(13)19)17(20)18-21/h2-10,21H,11H2,1H3,(H,18,20)

N-hydroxy-1-[(4-methoxyphenyl)methyl]indole-6-carboxamide

PCI-34051; PCI 34051; PCI34051

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 (8.44 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 (8.44 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 (8.44 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: 30% PEG400+0.5% Tween80+5% propylene glycol: 30mg/mL

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