HDAC6 82 nM (IC50) HDAC 27 nM (IC50, Hela cell)
Belinostat targets histone deacetylases (HDACs), specifically inhibiting enzymes from Class I (HDAC1, HDAC2, HDAC3) and Class II (HDAC6). The compound has an IC50 of 27 nM against HDAC enzymes in cell-free assays. By inhibiting HDACs, belinostat promotes the acetylation of histone and nonhistone proteins, which leads to the re-expression of tumor suppressor genes, cell cycle regulators (such as p21WAF1), and pro-apoptotic factors. The inhibition of HDACs also affects the immune response and other signaling pathways, including p27 and caspase 3 activation. Belinostat is a pan-HDAC inhibitor, meaning it is not highly selective for a single HDAC isoform, which contributes to its broad activity but also to its side effect profile.

In tumor cell lines, belinostat (PXD101) causes an increase in the acetylation of histone H4 that is concentration-dependent (0.2–5 μM). A clonogenic study revealed that belinostat is cytotoxic in vitro in a variety of tumor cell lines, causing apoptosis with IC50s in the 0.2–3.4 μM range. Many human tumor cell lines are inhibited in vitro by belinostat, whose IC50s, as measured by a clonogenic experiment, fall between 0.2 and 3.4 μM[1]. The enzymatic activity of pure recombinant HDAC6 (IC50 of 82 nM) is potently inhibited by belinostat (PXD101), a strong histone deacetylase (HDAC) inhibitor[2].

---

Belinostat has demonstrated potent in vitro activity against a wide range of cancer cell lines, including those derived from melanoma, prostate, breast, lung, colon, and ovarian cancers. In cell-free assays, belinostat inhibits HDAC enzymes with an IC50 of 27 nM. The compound induces cell cycle arrest at the G1/S and G2/M phases by upregulating cyclin-dependent kinase inhibitors such as p21WAF1. It also promotes apoptosis through the activation of caspase 3 and the degradation of poly (ADP-ribose) polymerase-1 (PARP-1). Belinostat has been shown to increase the acetylation of histones H3 and H4 in treated cells, confirming its mechanism of action as an HDAC inhibitor. The compound demonstrates cytotoxic activity with IC50 values in the low micromolar to nanomolar range across various cancer cell lines, with selectivity for tumor cells over normal cells.

For seven days, Belinostat (10–40 mg/kg/day ip) was given intraperitoneally to nude mice carrying human ovarian and colon tumor xenografts. This treatment significantly delayed the animals's growth without showing any overt evidence of harm. Cells resistant to cisplatin found in ovarian tumor xenografts also show growth retardation. Three hours following treatment with belinostat (PXD101), a significant rise in acetylation of H4 is found in the blood and tumor of mice. mice's human tumor xenografts are grown less rapidly, and there is no visible toxicity[1]. When used in combination with carboplatin therapy, belinostat (PXD101) exhibits increased single-agent antitumor efficacy on human A2780 ovarian cancer sc xenografts[2].

---

In preclinical in vivo models, belinostat has shown antitumor efficacy in xenograft models of various cancer types, including solid tumors and hematological malignancies. In patients with advanced solid tumors, intravenous administration of belinostat was well tolerated and showed promising antitumor activity. The compound has been evaluated in multiple Phase I and II clinical trials, demonstrating clinical activity in both solid and hematological cancers. In the pivotal Phase 2 trial that led to FDA approval, belinostat demonstrated an overall response rate of 25.8% in patients with relapsed or refractory PTCL. The drug has also been evaluated in combination with other anticancer agents, including chemotherapy and other targeted therapies, to enhance efficacy and overcome resistance.

The in vitro enzyme activity of belinostat can be assessed using fluorogenic HDAC activity assays. In a typical assay, purified recombinant HDAC enzymes (individual isoforms or a mixture) are incubated with a fluorogenic peptide substrate that contains an acetylated lysine residue. Upon deacetylation by the HDAC enzyme, the substrate becomes susceptible to cleavage by a developer, releasing a fluorophore that can be quantified using a fluorescence plate reader. Belinostat is added to the reaction at varying concentrations, and the decrease in fluorescence signal is measured to determine the half-maximal inhibitory concentration (IC50). The assay can be performed with HDAC isoforms from Class I (HDAC1, HDAC2, HDAC3) and Class II (HDAC6) to assess the isoform selectivity of the compound. The IC50 of belinostat in this cell-free assay is 27 nM.

The in vitro cellular activity of belinostat is assessed using a panel of cancer cell lines representing different tumor types, including PTCL, other hematological malignancies, and solid tumors. Cells are seeded in 96-well plates and treated with escalating concentrations of belinostat for 48 to 72 hours. Cell viability is measured using MTT, XTT, or CellTiter-Glo assays, and the half-maximal inhibitory concentration (IC50) values are calculated from dose-response curves. Apoptosis is assessed by flow cytometry using annexin V/propidium iodide staining or by measuring caspase-3/7 activity. Cell cycle analysis is performed using propidium iodide staining followed by flow cytometry to determine the proportion of cells in G1, S, and G2/M phases. Histone acetylation is measured by Western blotting using antibodies specific for acetylated histone H3 or H4. The effects on downstream target genes, such as p21WAF1, are also assessed by quantitative PCR or Western blot.

Belinostat has been evaluated in numerous clinical trials in patients with various cancers. In the Phase 2 clinical trial that supported FDA approval, belinostat was administered intravenously at a dose of 1000 mg/m² once daily on days 1-5 of a 21-day cycle. The primary endpoint was overall response rate (ORR), and secondary endpoints included duration of response, progression-free survival (PFS), and overall survival (OS). In Phase 1 studies, belinostat was administered to patients with advanced solid tumors to determine the maximum tolerated dose (MTD), dose-limiting toxicities (DLTs), and pharmacokinetic and pharmacodynamic profiles. The recommended Phase 2 dose was established based on these studies. Belinostat has also been evaluated in combination with other anticancer agents, including carboplatin, paclitaxel, and bortezomib, in various clinical trials.

Absorption, Distribution and Excretion
Approximately 40% of the dose of Belinostat is excreted by the kidneys, primarily as metabolites, with less than 2% of the total dose recovered unchanged. Volume of distribution: 409 ± 76.7 L. Outflow rate: 1240 mL/min Metabolism/Metabolites Primarily metabolized by hepatic UGT1A1. Potent UGT1A1 inhibitors are expected to increase Belinostat exposure. Belinostat can also be metabolized by hepatic CYP2A6, CYP2C9, and CYP3A4 enzymes to Belinostatamide and Belinostatic acid. The enzymes responsible for the formation of methylbenilinostat and 3-(anilinesulfonyl)benzoic acid (3-ASBA) are unknown. Biological half-life Exhibits three-compartment pharmacokinetics with an elimination half-life of 1.1 hours.

---

After intravenous administration, belinostat exhibits dose-dependent pharmacokinetics. The compound has a brief elimination half-life and undergoes substantial first-pass metabolism in the liver, primarily via glucuronidation by UGT1A1. The pharmacokinetic parameters, including maximum plasma concentration (Cmax), area under the curve (AUC), clearance, and volume of distribution, have been characterized in patients. Belinostat is rapidly cleared from the plasma, with a terminal half-life of approximately 1-2 hours. The compound is metabolized to several metabolites, including belinostat acid, methyl belinostat, and belinostat glucuronide. The pharmacokinetics of belinostat are not significantly affected by age, gender, or renal function, but may be influenced by hepatic function due to the role of UGT1A1 in its metabolism. Drug-drug interactions may occur with inhibitors or inducers of UGT1A1.

Hepatotoxicity
In clinical trials of Belinostat in patients with PTCL, the incidence of elevated serum enzymes during treatment was generally less than 5%, with only 1% to 2% of patients experiencing serum enzyme levels exceeding five times the upper limit of normal. In an open-label trial of Belinostat monotherapy in 120 patients with PTCL, a case of severe acute liver injury leading to liver failure and death was reported. This liver injury occurred after 10 cycles of treatment and continued to progress despite discontinuation of the drug. No specific details were provided. Two cases of cholestatic liver injury were reported in another clinical trial, but similarly, no specific details were provided. Therefore, Belinostat is considered a rare cause of acute liver injury, but its onset time, related characteristics, clinical course, and prognosis remain unclear. Probability score: D (likely a clinically significant cause of liver injury). Protein Binding Belinostat has a protein binding rate of 92.9% to 95.8%.

---

Belinostat is generally well-tolerated, but it is associated with a range of adverse events typical of HDAC inhibitors. Common side effects include fatigue, nausea, vomiting, diarrhea, and hematological abnormalities such as thrombocytopenia, anemia, and neutropenia. Other reported adverse events include electrocardiogram (ECG) changes (QT prolongation), infusion site reactions, and liver enzyme elevations. The toxicity profile of belinostat is manageable with supportive care, dose adjustments, and treatment interruptions. The compound has been shown to have a certain level of cytotoxicity specific to tumor cells, which contributes to its therapeutic window. Long-term toxicity data from clinical trials has been collected, and the safety profile has been deemed acceptable for the approved indication of relapsed or refractory PTCL. Belinostat is contraindicated in patients with severe hepatic impairment due to the risk of increased toxicity.

[1]. Pharmacodynamic response and inhibition of growth of human tumor xenografts by the novel histonedeacetylase inhibitor PXD101. Mol Cancer Ther. 2003 Aug;2(8):721-8.

[2]. Activity of PXD101, a histone deacetylase inhibitor, in preclinical ovarian cancer studies. Mol Cancer Ther. 2006 Aug;5(8):2086-95.

[3]. Phenotype-driven precision oncology as a guide for clinical decisions one patient at a time. Nat Commun. 2017 Sep 5;8(1):435.

Belinostat is a hydroxamic acid histone deacetylase (HDAC) inhibitor with antitumor activity. It is both an antitumor drug and an EC 3.5.1.98 (histone deacetylase) inhibitor. It is a hydroxamic acid, sulfonamide, and olefin compound. Belinostat is a novel drug with a sulfonamide-hydroxamic acid structure that inhibits histone deacetylase (HDAC). Developed by TopoTarget, it is intended as an orphan drug for the treatment of hematologic malignancies and solid tumors. The safety and efficacy of Belinostat in combination with conventional first-line therapy for the treatment of peripheral T-cell lymphoma (PTCL) are currently being evaluated. The drug is administered intravenously under the brand name Beleodaq and can be used as monotherapy in a 21-day cycle. Belinostat was approved in the United States in July 2014 for the treatment of relapsed or refractory peripheral T-cell lymphoma. Belinostat is a histone deacetylase inhibitor. Belinostat's mechanism of action is the inhibition of histone deacetylase. Belinostat is an intravenously administered histone deacetylase inhibitor and antitumor drug approved for the treatment of refractory or relapsed peripheral T-cell lymphoma. Belinostat can cause moderate to mild elevations in serum enzymes during treatment, and there are reports of clinically significant and potentially fatal acute liver injury. Belinostat is a novel hydroxamic acid histone deacetylase (HDAC) inhibitor with antitumor activity. Belinostat targets HDAC enzymes, thereby inhibiting tumor cell proliferation, inducing apoptosis, promoting cell differentiation, and inhibiting angiogenesis. This drug may enhance the sensitivity of drug-resistant tumor cells to other antitumor drugs by downregulating thymidylate synthase.

---

Drug Indications
Belinostat is indicated for the treatment of patients with relapsed or refractory peripheral T-cell lymphoma (PTCL) with a good safety profile. For patients who have not responded well to first-line PTCL therapies, Belinostat is a potential alternative therapy. This drug can also be used in patients with baseline thrombocytopenia.
FDA Label

---

Mechanism of Action
Belisitar inhibits the activity of histone deacetylase (HDAC), thereby preventing the removal of acetyl groups from histone and certain non-histone lysine residues. In vitro studies have shown that belisitar leads to the accumulation of acetylated histones and other proteins, and increases the expression of tumor suppressor genes. It ultimately induces cell cycle arrest, inhibits angiogenesis, and/or causes apoptosis in certain transformed cells.

---

Belinostat (Beleodaq, PXD101, PX105684) is a pan-HDAC inhibitor with a sulfonamide group, approved by the FDA for relapsed or refractory peripheral T-cell lymphoma (PTCL). The drug was discovered by TopoTarget and is marketed by Acrotech Biopharma. It received accelerated approval based on a Phase 2 trial showing an overall response rate of 25.8%. The mechanism of action involves inhibition of Class I and II HDACs, leading to histone hyperacetylation, cell cycle arrest, and apoptosis. The pharmacokinetics are characterized by a short half-life and UGT1A1-mediated metabolism. Belinostat is administered intravenously at 1000 mg/m² on days 1-5 of a 21-day cycle. The drug has been studied in various cancers, including solid tumors and hematological malignancies. Analytical methods for belinostat quantification include UPLC-MS/MS, HPLC-UV, FTIR, TLC, NMR, and ESI-MS.

C15H14N2O4S

318.35

318.067

866323-14-0

866323-14-0; 414864-00-9;

6918638

White to off-white solid powder

160 °C(dec.)

4

3

5

5

22

492

0

C1=CC=C(C=C1)NS(=O)(=O)C2=CC=CC(=C2)/C=C/C(=O)NO

NCNRHFGMJRPRSK-MDZDMXLPSA-N

InChI=1S/C15H14N2O4S/c18-15(16-19)10-9-12-5-4-8-14(11-12)22(20,21)17-13-6-2-1-3-7-13/h1-11,17,19H,(H,16,18)/b10-9+

(E)-N-hydroxy-3-[3-(phenylsulfamoyl)phenyl]prop-2-enamide

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)

DMSO : 100 mg/mL (314.12 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.85 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.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (6.53 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 20.8 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.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (6.53 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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