hHDAC3 (IC50 = 157 nM); hHDAC1 (IC50 = 198 nM); hHDAC11 (IC50 = 292 nM); hHDAC6 (IC50 = 315 nM); hHDAC2 (IC50 = 325 nM); hHDAC10 (IC50 = 340 nM); hHDAC7 (IC50 = 524 nM); hHDAC5 (IC50 = 532 nM); hHDAC9 (IC50 = 541 nM); hHDAC8 (IC50 = 854 nM); hHDAC4 (IC50 = 1059 nM); HD1-B (IC50 = 7.5 nM); HD1-A (IC50 = 16 nM); HD2 (IC50 = 10 nM)
Givinostat (ITF2357) effectively inhibits the production of IL-1β induced by LPS in its entirety, as opposed to ITF3056's reduction. Givinostat more than 70% decreased IL-1β secretion at 25, 50, and 100 nM. In PBMCs stimulated with TLR agonists, as well as in combination with IL-12 and IL-18, glinostat (ITF-2357) inhibits the production of IL-6. Givinostat at 50 nM causes IL-6 secretion to drop to 50%, but at 100 and 200 nM, there is no reduction[1]. Givinostat (ITF-2357) inhibits JS-1 cell proliferation in a concentration-dependent manner, as demonstrated by the CCK-8 assay. Givinostat treatment at doses greater than 500 nM is linked to a significant reduction in JS-1 cell proliferation (P<0.01). Additionally, there is a significant difference in the cell inhibition rate (P<0.05) between the group that received LPS treatment and the group that received the same concentration of Givinostat cotreated with ≥250 nM plus LPS[2].

Givinostat (ITF2357) effectively inhibits the production of IL-1β induced by LPS in its entirety, as opposed to ITF3056's reduction. Givinostat more than 70% decreased IL-1β secretion at 25, 50, and 100 nM. In PBMCs stimulated with TLR agonists, as well as in combination with IL-12 and IL-18, glinostat (ITF-2357) inhibits the production of IL-6. Givinostat at 50 nM causes IL-6 secretion to drop to 50%, but at 100 and 200 nM, there is no reduction[1]. Givinostat (ITF-2357) inhibits JS-1 cell proliferation in a concentration-dependent manner, as demonstrated by the CCK-8 assay. Givinostat treatment at doses greater than 500 nM is linked to a significant reduction in JS-1 cell proliferation (P<0.01). Additionally, there is a significant difference in the cell inhibition rate (P<0.05) between the group that received LPS treatment and the group that received the same concentration of Givinostat cotreated with ≥250 nM plus LPS[2].

---

At concentrations of 25–100 nM, Givinostat (ITF2357) inhibits the production of IL-1β, TNFα, and IL-6 from lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMCs).
At 25 nM, Givinostat reduces LPS-induced IL-1β secretion by more than 70% and reduces intracellular IL-1α levels by 35%.
At 50 nM, Givinostat reduces LPS-induced IL-1β mRNA levels by nearly 70% and reduces TNFα mRNA levels by 85%.
At concentrations >200 nM, Givinostat exhibits cytotoxicity toward PBMCs, as evidenced by increased LDH release, decreased cell viability, and induction of monocyte apoptosis.
Givinostat (25 nM) reduces caspase-1 activity in LPS-stimulated PBMC lysates by approximately 50% at 24 hours post-stimulation.
Givinostat reduces cytokine production (IL-1β, TNFα, IFNγ) from PBMCs stimulated with heat-killed Candida albicans.
Givinostat (at concentrations up to 100 nM) inhibits TNFα secretion from anti-CD3/anti-CD28 stimulated PBMCs but does not significantly inhibit IL-1β or IFNγ under the same conditions.
[1]

Givinostat (ITF2357) at 10 mg/kg is used as a positive control and, as expected, reduced serum TNFα by 60%. It is remarkable that pretreatment with ITF3056, starting at 0.1 mg/kg, dramatically lowers the level of circulating TNFα by almost 90%. An elevated dose of 10 mg/kg of LPS is injected, and blood is drawn after 4 hours to obtain a notable rise in serum IL-1β production. Similarly, a 22% reduction for 1 mg/kg and a 40% reduction for 5 mg/kg occurs when pretreated with lower doses of Givinostat (ITF-2357)[1].

---

Con A Model of Acute Hepatitis[2]
Mice were given 100 μL water or Givinostat (ITF-2357) (5 mg/kg) by gavage and, after 1 h, injected intravenously with 200 μg/mouse of ConA. Control mice received an intravenous injection of saline. Mice were bled 24 h later for evaluation of serum ALT levels as described previously (33,34). As shown in Figure 15, ALT levels were reduced by more than 80% by ITF2357 pretreatment. In another experiment, a comparison was made between 1 and 10 mg/kg of oral ITF2357. As shown in Figure 16, a dose of 1 mg/kg ITF2357 was as effective as a dose of 10 mg/kg in reducing ConA hepatitis as measured by ALT levels.

---

Oral administration of Givinostat (10 mg/kg) to mice reduces serum TNFα levels by 60% following LPS challenge.
In a mouse model of endotoxemia, oral Givinostat demonstrates anti-inflammatory effects.
Givinostat has been used successfully to treat children with systemic juvenile idiopathic arthritis at an oral dose of 1.5 mg/kg/day, resulting in peak blood levels below 100 nM.
In animal models of diabetes, oral Givinostat at 1.5 mg/kg/day protected insulin-producing islets and reduced the incidence of clinical diabetes.
[1]

The procedure for the assay involves mixing the crude cellular extract (5 μL) with 100 μL substrate (2×10⁵ cpm), 40 μL buffer (50 mM Tris-HCl, pH 8.0, 750 mM NaCl, 5 mM PMSF, 50% glycerol), and 95 μL distilled water. To check for HDAC inhibition, add 50 μL of ITF2357. After the mixture has been incubated for a full night at room temperature, 50 μL of a solution made of 259 μL 37% HCl, 28 μL acetic acid, and 1 mL distilled water is added to quench the reaction. The organic extraction method is used to separate the [³H]acetyl residues that are released from the substrate. A beta-counter is used to measure radioactivity after adding 200 μL of the organic phase to standard scintillation fluid. By measuring the difference between the radioactivity of the inhibitor-containing samples and the control sample that only contained cellular crude extract, one can determine the concentration of HDACs that inhibits 50% of the control activity.

---

\n\nMaize HDAC Assays [2]
\nHD2, HD-1B, and HD-1A from maize and used to assess the histone deacetylase activity of Givinostat (ITF-2357) as described in Koelle et al. \n

---

\n\nCellular Crude Extract for Total HDAC Activity and Protein Acetylation Determinations [2]
\nHuman peripheral blood mononuclear cells (PBMCs) (see below) were added to 50-mL conical tubes at a concentration of 2.5 × 106 cell/mL in RPMI 1640 medium with 1% FCS and 0.05% DMSO (vol/vol) and incubated at 37° C with the test compounds (constituted in DMSO 0.05%) at the stated concentrations. After 60 min, LPS was added at final concentration of 10 ng/mL and the cells were incubated at 37° C. At the end of incubation times, the cells were centrifuged at 400g for 15 min, the supernatant was collected and stored at −80° C until TNFα determination, and the cells were washed twice with ice-cold phosphate buffer.\n
\n\nCrude extracts were obtained by suspending the pellet in 200 μL modified lysis buffer (50 mM Tris HCl, pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF) together with a cocktail of protease inhibitors available as tablets for 30 min at 4° C. The cells were disrupted by sonication, after which the extract was clarified by centrifugation at 14,000 rpm for 10 min at 4° C. The supernatant was used for determination of total HDAC activity and protein acetylation. Protein content of the extract was determined using the BCA protein assay kit.\n

---

\n\nTotal HDAC Activity Assay [2]
\nThe assay was adapted based on the release of tritiated acetyl residues from a peptide substrate intrinsically labeled with [3H]acetic acid, as described previously. The synthetic peptide used in this assay was the N-terminal sequence (SGRGKGGKGLGKGGAKRHRC) of histone H4. Radiolabeling of the peptide was done as follows: 100 μg peptide was added to 62.5 μL [3H]acetic acid sodium salt (5.0 mCi/0.5 mL in ethanol, specific activity 5.1 Ci/mole). Thereafter, 5 μL BOP solution (0.24 M BOP and 0.2 M trimethylamine in acetonitrile) was added. The resulting solution was incubated overnight at room temperature with mild agitation, and the radiolabeled peptide solution was loaded onto a Microcon-SCX spin column previously rinsed with 500 μL of 10 mM HCl in methanol. The eluate was separated by centrifugation of the column (2,000g for 60 s). The radiolabeled peptide was eluted with 50 μL HCl 3N in 50% isopropanol. The eluting solution containing the radiolabeled peptide was submitted to 8 cycles of organic solvent extraction (8 × 1 mL of ethylacetate) to separate the remaining free [3H]acetic acid. The resulting aqueous solution was dried by centrifugation under vacuum for 30 min at room temperature and then suspended in 200 μL distilled water, separated into aliquot, and stored at −20° C.\n

---

\n\nAcetylation of Proteins [2]
\nAcetylation of proteins was determined by Western blotting of crude cellular extracts. Briefly, the samples (200 μg/lane) were separated by SDS-PAGE (12.5%) and then electrically transferred onto nitrocellulose membranes. The membranes were saturated with 3% nonfat milk in phosphate buffer and incubated with anti-acetyl-lysine monoclonal antibody according to manufacturer’s instructions. Protein bands were then detected using the chemiluminescence detection system ECL Plus onto x-ray film.\n

---

\n\nEnzymatic Assay for HDAC Inhibitory Activity of Synthetic Compounds [2]
\nThe assay was performed by adding 100 μL substrate (200,000 cpm) with 40 μL buffer (50 mM Tris-HCl, pH 8.0, 750 mM NaCl, 5 mM PMSF, 50% glycerol) and 95 μL distilled water to the crude cellular extract (5 μL). Compounds for testing of HDAC inhibition (50 μL) were added. The mixture was incubated overnight at room temperature and the reaction quenched by adding 50 μL of a solution containing 259 μL HCl 37% and 28 μL acetic acid in 1 mL distilled water. The [3H]acetyl residues released from the substrate were separated by organic extraction by adding 600 μL of ethyl acetate, 200 μL of the organic phase was added to standard scintillation fluid, and radioactivity was measured by a beta-counter. Inhibition of HDACs was expressed as the concentration inhibiting 50% of the control activity (by comparing the radioactivity of the samples containing inhibitors to that of the control containing cellular crude extract alone).

---

\n\nEnzymatic Assay for HDAC Activity [1]
\nRecombinant human HDAC enzymes (HDAC1–10) were purchased from BPS. HDAC8 activity was assayed using Fluor de Lys Green deacetylase substrate. Nϵ-Trifluoroacetyl-l-lysine was used to assay activity of HDAC 4, 5, 7, and 9. Recombinant enzymes were preincubated with Givinostat (ITF-2357) or ITF3056 at 30 °C in a volume of 25 μl in wells of a microtiter plate. After a brief incubation, 25 μl of substrate was added, and the fluorescent signal was generated by the addition of 50 μl of developer (Fluor de Lys Developer containing 2 μm Trichostatin A. For each assay, the amount of enzyme, incubation times, assay buffer, and concentration of the substrates were optimized. Positive control for enzyme activity consisted of enzyme plus substrate without ITF2357 or ITF3056. The fluorescence signal was detected using a Victor multilabel plate reader.

---

Recombinant human HDAC enzymes (HDAC1–11) were used to assess inhibitory activity. Enzymes were pre-incubated with Givinostat or vehicle in a microtiter plate. A fluorogenic deacetylase substrate was then added. Following incubation, a developer solution containing Trichostatin A was added to generate a fluorescent signal. Fluorescence was measured using a plate reader. The assay conditions (enzyme amount, incubation time, buffer, substrate concentration) were optimized for each HDAC enzyme. The concentration of inhibitor causing 50% inhibition (IC50) was determined. [1]

Following a 24-hour culture period in DMEM supplemented with 10% fetal bovine serum, 30 wells containing JS-1 cells are split into two groups.Givinostat (ITF-2357) is added to the culture medium in the first group at final concentrations of 0 nM, 125 nM, 250 nM, 500 nM, and 1000 nM. In the second group, 100 nM of LPS solution is added concurrently with Givinostat at appropriate concentrations. For every group, three replicates are carried out. Following a 24-hour inoculation period at 37°C and 5% CO₂, 10 μL of CCK-8 solution is incubated in each well (100 μL). A microplate reader is used to measure the absorbance at 450 nm after the plates are incubated for one hour at 37°C[2].

---

\n\n The cell counting kit-8 assay and flow cytometry were used to observe changes in proliferation, apoptosis, and cell cycle in hepatic stellate cells treated with Givinostat (ITF-2357). Western blot was used to observe expression changes in p21, p57, CDK4, CDK6, cyclinD1, caspase-3, and caspase-9 in hepatic stellate cells exposed to Givinostat (ITF-2357). The scratch assay was used to analyze the effect of givinostat on cell migration. Effects of givinostat on the reactive oxygen species profile, mitochondrial membrane potential, and mitochondrial permeability transition pore opening in JS-1 cells were observed by laser confocal microscopy.[3]

---

\n\nCaspase-1 Activity Assay [1]
\nHuman PBMCs from four donors were stimulated with LPS (10 ng/ml) for 4 and 24 h. Based on the reduction by Givinostat (ITF-2357) and ITF3056 of LPS-induced IL-1β production and secretion, 25 nm Givinostat (ITF-2357) and 1000 nm ITF3056 were selected as the optimal concentrations to assess effects on caspase-1 activity. ITF2357 (25 nm) or ITF3056 (1000 nm) were added 30 min prior to LPS. After the supernatant was removed, the cells were lysed using radioimmune precipitation assay buffer containing a mixture of protease inhibitors and centrifuged at 12,000 rpm for 20 min at 4 °C. The supernatants were assayed for protein content using the Bio-Rad method. Protein was processed for caspase-1 activity using the fluorogenic substrate A2452. The fluorescence was reported as arbitrary fluorescence units generated by 1 μg of sample/min (fluorescence/μg/min). The data were expressed as the percentage change in caspase-1 activity present in the lysates of LPS-stimulated PBMCs incubated with analogues, with the lysates from LPS only set as 100%.\n

---

\n\nAssays for Cytotoxicity [1]
\nThree assays were used to compare the cytotoxic effects of Givinostat (ITF-2357) with those of ITF3056. LDH release was measured in supernatants from freshly obtained PBMCs cultured in 96-flat bottom well plates for 24 h in the presence of LPS (10 ng/ml) without fetal calf serum as described previously using the LDH cytotoxicity assay kit. The percentage of LDH release was calculated according to the manufacturer's instructions. In addition, cell viability was assessed in PBMCs cultured in RPMI supplemented with 1% FCS in the presence of LPS (10 ng/ml) at 400,000 cells/ml in a 96-flat bottom well plate for 24 h. At the end of incubation, cell viability was determined by the CellTiter 96® Aqueous One solution cell proliferation assay according to the manufacturer's instructions. We also assessed cell viability in PBMC isolated from buffy coat cells of human citrated blood. PBMCs isolated from buffy coats were seeded at 500,000 cells/well (96-flat bottom well plate) in RPMI with 10% FCS and incubated for 72 h in the presence of increasing concentrations of ITF2357 or ITF3056 using the CellTiter assay as described above.\n

---

\n\nAnnexin V Staining for Monocyte Apoptosis [1]
\nMonocytes were isolated from fresh PBMCs by magnetic separation and resuspended in RPMI containing 10% FCS. Purified monocytes were seeded at 250,000 cells/well and incubated in the presence of increasing concentrations of ITF2357 or ITF3056 with LPS (10 ng/ml). After 24 h, the cells were labeled with annexin V-FLUOS and propidium iodide (PI) following the manufacturer's instructions. The percentages of annexin V-positive and annexin V/PI-double-positive cells were determined by flow cytometric analysis.\n

---

\n\nCaspase-3/7 Determinations for Monocyte Apoptosis [1]
\nMonocytes, isolated as described above, were incubated at 50,000 cells/well in 96-flat bottom well plates in the presence of increasing concentrations of Givinostat (ITF-2357) or ITF3056 with LPS (10 ng/ml) for 24 h of incubation. The activity of caspase-3/7 was then determined by the Apo-ONE homogeneous caspase-3/7 assay, and the amount of fluorescence was detected by a fluorimetric plate reader.

---

Human PBMCs were isolated from healthy donor blood and cultured in RPMI medium. For LPS stimulation, cells were seeded in flat-bottom 96-well plates and stimulated with 10 ng/ml LPS for 24 hours in the presence or absence of Givinostat. For Candida albicans or T-cell stimulation, cells were cultured in round-bottom 96-well plates with 10% human serum and stimulated with heat-killed Candida or anti-CD3/anti-CD28 antibodies for 5 days. Givinostat was added 30 minutes prior to stimuli. Supernatants were collected for cytokine analysis by electrochemiluminescence (ECL) or ELISA. Cell lysates were prepared for analysis of intracellular cytokines or caspase activity. [1]
Cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) release into the supernatant using a commercial kit. Cell viability was also determined using a tetrazolium-based cell proliferation assay. [1]
Apoptosis was assessed in isolated monocytes. Monocytes were incubated with Givinostat and LPS for 24 hours, then stained with annexin V and propidium iodide (PI) followed by flow cytometric analysis. Caspase-3/7 activity in monocytes was determined using a homogeneous caspase assay kit and fluorimetric detection. [1]
For mRNA analysis, PBMCs were cultured in 24-well plates with LPS and Givinostat for 20 hours. Cells were lysed, total RNA was extracted, reverse transcribed to cDNA, and real-time PCR was performed for IL-1β, TNFα, IL-6, and GAPDH (as an internal control). [1]

Mice: For a minimum of five days prior to usage, C57BL/6 mice are kept in the animal facility. Intraperitoneal injection of ITF3056 and oral administration of Givinostat (ITF-2357) at a dose of 10 mg/kg are used in the comparative study. LPS from Salmonella typhimurium is administered intraperitoneally to the animals at a dose of 2.5 mg/kg one hour after the compounds are administered. Serum is collected and kept at -80°C until further examination of cytokine production, and mice are sacrificed 90 minutes after the LPS treatment.

---

Mice for LPS induction of serum cytokines were BALB/c, whereas those for anti-CD3-induced cytokines were CD1. Mice for concanavalin A (Con A)–induced acute hepatitis were BALB C or C57Bl6 obtained from Jackson Laboratories. Mice were given 100 μL water or Givinostat (ITF-2357) in water by gavage and, after 60 min, were injected intraperitoneally with LPS (30 mg/kg) or intravenously with anti-mouse CD3 (10 μg/mouse) or intravenously with 200 μg/mouse of Con A into a tail vein. Control mice received an intraperitoneal injection of saline or intravenous injection of saline. For LPS, mice were killed by anesthetic overdose, and blood was obtained at 90 min and 6 h. For anti-mouse CD3, mice were killed by anesthetic overdose, and blood was obtained at 90 min. Mice were bled 24 h later for evaluation of serum ALT levels as described previously. [2] C57BL/6 mice were housed in the animal facility for at least 5 days before use. For the comparison study, Givinostat (ITF-2357) at 10 mg/kg was administered orally as reported previously, and ITF3056 was injected intraperitoneally. One hour after administration of the compounds, the animals were treated intraperitoneally with LPS from Salmonella typhimurium at a dose of 2.5 mg/kg. 90 min after the LPS treatment, mice were sacrificed, and sera were collected and stored at −80 °C until further analysis of cytokine productions. A dose-response study of ITF3056 at 4, 8, and 16 mg/kg was performed in mice injected intraperitoneallly with LPS from E. coli (055:B5) (Sigma-Aldrich) at 10 mg/kg. After 4 h, the serum was collected for cytokine levels. Another dose study of ITF3056 at 1 and 5 mg/kg used a lower dose of LPS (2.5 mg/kg) given intraperitoneally after ITF3056 injection. 4 h later, the mice were sacrificed, and blood was collected. Blood was collected in EDTA, separated into plasma for cytokine levels, or diluted in RPMI for whole blood culture as described previously. [1]

---

C57BL/6 mice were used. For endotoxemia studies, Givinostat was administered orally at a dose of 10 mg/kg. One hour after compound administration, mice were injected intraperitoneally with LPS (2.5 mg/kg). Mice were sacrificed 90 minutes after LPS injection, and blood was collected for serum cytokine analysis. [1]
In other diabetes model studies, Givinostat was administered orally at 1.5 mg/kg/day or added to drinking water (calculated daily dose of ~1 mg/kg). [1]

Absorption
The absolute bioavailability of gemvinositol has not been determined. After oral administration, the time to peak concentration (Tmax) of gemvinositol is approximately 2–3 hours, and steady-state plasma concentrations are reached within 5–7 days with twice-daily dosing. Systemic exposure is proportional to the dose within the therapeutic range. Co-administration with a high-fat meal increases AUC by 40%, Cmax by 23%, and delays Tmax by 2–3 hours.
Elimination Route
Gevinositol is excreted in very small amounts (<3%) in the urine. Elimination of gemvinositol is likely primarily driven by metabolism, followed by excretion of metabolites via the kidneys and bile.
Volume of Distribution
Based on population pharmacokinetic models, the apparent volume of distribution in the central compartment is estimated to be 160 L. The apparent volume of distribution in the peripheral compartment is estimated to be 483 L.
Clearance
Based on population pharmacokinetic models, the apparent oral clearance of gemvinositol is estimated to be 121 L/h. Givinostat's clearance rate is estimated at 33.8 L/h.
Protein Binding
Givinostat has a high protein binding rate in plasma (approximately 96%).
Metabolites/Metabolites
Givinostat is extensively metabolized into several metabolites, four of which have been identified: ITF2374, ITF2375, ITF2440, and ITF2563. These metabolites do not affect the efficacy of Givinostat. The enzymes responsible for Givinostat metabolism are not known; its metabolism does not involve CYP450 or UGT enzymes.
Biological Half-Life
The apparent plasma elimination half-life of Givinostat is approximately 6 hours.
In a Phase I clinical trial in healthy men, oral administration of Givinostat reduced the production of pro-inflammatory cytokines in whole blood cultures. [1]
In children with systemic juvenile idiopathic arthritis, oral administration of 1.5 mg/kg of gemvinata daily resulted in peak plasma concentrations below 100 nM. [1]

In vitro experiments showed that Givinostat has concentration-dependent cytotoxicity. At concentrations of 200 nM and above, it can cause significant damage to human peripheral blood mononuclear cells (PBMCs), manifested as increased lactate dehydrogenase (LDH) release, decreased cell viability in proliferation assays, enhanced Annexin V/PI staining of monocytes, and caspase-3/7 activation. [1] At concentrations of 250 nM and above, Givinostat has a damaging effect on PBMCs. At concentrations of 500 nM and 1000 nM, it can induce significant cell death. [1] The effective anti-inflammatory concentrations in vitro (25-100 nM) and in vivo (blood concentration <100 nM) are both below the cytotoxic threshold. [1]

Use during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information regarding the use of gemvinositol in breastfeeding women. Because it binds to human plasma proteins at a rate as high as 96%, its concentration in breast milk may be very low. If a mother needs to take gemvinositol, this is not a reason to stop breastfeeding. Until more data are available, breastfeeding women should use gemvinositol with caution, especially when breastfeeding newborns or premature infants. Breastfeeding infants should be closely monitored for symptoms such as diarrhea, vomiting, bruising, excessive bleeding from wounds, and hematochezia.
◉ Effects on breastfed infants
No published information was found as of the revision date.
◉ Effects on breastfeeding and breast milk
No published information was found as of the revision date.

[1]. Specific inhibition of histone deacetylase 8 reduces gene expression and production of proinflammatory cytokines in vitro and in vivo. J Biol Chem. 2015 Jan 23;290(4):2368-78.

[2]. The histone deacetylase inhibitor ITF2357 reduces production of pro-inflammatory cytokines in vitro and systemic inflammation in vivo. Mol Med. 2005 Jan-Dec;11(1-12):1-15.

[3]. Givinostat inhibition of hepatic stellate cell proliferation and protein acetylation. World J Gastroenterol. 2015 Jul 21;21(27):8326-39.

Givinostat belongs to the naphthalene class of compounds, with its structure consisting of a naphthalene ring substituted at positions 2 and 6 by ({[4-(hydroxycarbamoyl)phenyl]carbamoyl}oxy)methyl and (diethylamino)methyl, respectively. It is a histone deacetylase inhibitor indicated for patients with Duchenne muscular dystrophy. Givinostat possesses various pharmacological activities, including as an EC 3.5.1.98 (histone deacetylase) inhibitor, anti-inflammatory drug, angiogenesis inhibitor, antitumor drug, and apoptosis inducer. It is a carbamate compound belonging to the naphthalene, tertiary amine, hydroxamic acid, and benzene classes. It is the conjugate base of givinostat (1+). Givinostat has been used in clinical trials for the treatment of polycythemia vera, juvenile idiopathic arthritis, Duchenne muscular dystrophy (DMD), chronic myeloproliferative neoplasms, and polyarticular juvenile idiopathic arthritis.
Givenoxetine is an orally bioavailable histone deacetylase (HDAC) hydroxylase inhibitor with potential anti-inflammatory, anti-angiogenic, and antitumor activities. Givenoxetine inhibits class I and II HDACs, leading to the accumulation of highly acetylated histones, which in turn induces chromatin remodeling and altered gene expression patterns. At low concentrations (non-apoptotic concentrations), the drug inhibits the production of pro-inflammatory cytokines such as tumor necrosis factor-γ (TNF-α), interleukin-1 (IL-1), IL-6, and interferon-γ. Givenoxetine has been shown to activate intrinsic apoptotic pathways, inducing apoptosis in liver cancer cells and leukemia cells. The drug may also have anti-angiogenic activity, inhibiting the production of angiogenic factors such as IL-6 and vascular endothelial growth factor (VEGF) by bone marrow stromal cells.
See also: Givenoxetine hydrochloride (note moved to).

---

Drug Indications
Treatment of Duchenne muscular dystrophy
Juvenile idiopathic arthritis. ITF2357 (generic name gemvinositol) is an orally potent, hydroxamic acid-containing histone deacetylase (HDAC) inhibitor with broad-spectrum anti-inflammatory properties, used to treat children with systemic juvenile idiopathic arthritis. ITF2357 inhibits class I and II HDACs and reduces caspase-1 activity and the secretion of IL-1β and other cytokines (25–100 nm) in human peripheral blood mononuclear cells. ITF2357 is toxic in vitro at concentrations above 200 nM. The ITF2357 analogue ITF3056 inhibits only HDAC8 (IC50 of 285 nM). This study compared the production of IL-1β, IL-1α, TNFα, and IL-6 in peripheral blood mononuclear cells stimulated by lipopolysaccharide (LPS), heat-inactivated Candida albicans, or anti-CD3/anti-CD28 antibodies. ITF3056 reduced LPS-induced cytokine levels (concentration range 100–1000 nM); at 1000 nM, IL-1β secretion was reduced by 76%, TNFα secretion by 88%, and IL-6 secretion by 61%. Intracellular IL-1α levels were reduced by 30%. No cytotoxicity was observed at ITF3056 concentrations of 100–1000 nM. TNFα gene expression was significantly reduced (80%), while IL-6 gene expression was reduced by 40%. Although IL-1β and TNFα levels were slightly reduced by anti-CD3/28 antibody and Candida stimulation, IFNγ production was reduced by 75%. Mechanistically, ITF3056 reduced the secretion of processed IL-1β, and this process was independent of the inhibition of caspase-1 activity; however, the synthesis of IL-1β precursors was reduced by 40%, while IL-1β mRNA levels did not decrease significantly. In mice, ITF3056 reduced LPS-induced serum TNFα levels by 85% and IL-1β levels by 88%. These data suggest that specific inhibition of HDAC8 can alleviate inflammatory responses without producing cytotoxicity. [1] We investigated the inhibitory effects of histone deacetylases (HDACs), which can lead to chromatin depolymerization and thus promote gene expression. ITF2357 is an orally effective synthetic HDAC inhibitor, and we evaluated its activity as an anti-inflammatory agent. In lipopolysaccharide (LPS)-stimulated cultured human peripheral blood mononuclear cells (PBMCs), ITF2357 reduced tumor necrosis factor-α (TNFα) release by 50% at concentrations of 10–22 nM, intracellular interleukin (IL)-1α release by 50% at 12 nM, IL-1β secretion by 50% at 12.5–25 nM, and interferon-γ (IFNγ) production by 50% at 25 nM. IL-8 levels were not reduced in these same cultures. The combined use of IL-12 and IL-18 reduced IFNγ and IL-6 production by 50% at concentrations of 12.5–25 nM, and this effect was independent of reductions in IL-1 or TNFα. No cell death was observed in LPS-stimulated PBMCs treated with 100 nM ITF2357, a finding confirmed by DNA degradation, annexin V, and caspase-3/7 assays. Northern blot analysis of PBMCs showed that LPS-induced TNFα and IFNγ mRNA homeostasis levels decreased by 50% to 90%, but had no effect on IL-1β or IL-8 levels. Real-time PCR confirmed that ITF2357 reduced TNFα RNA levels. Oral administration of 1.0 to 10 mg/kg ITF2357 to mice reduced LPS-induced serum TNFα and IFNγ levels by more than 50%. Anti-CD3-induced cytokines were not inhibited by ITF2357 in PBMCs, either in vitro or in the mouse circulatory system. In a concanavalin A-induced hepatitis model, oral administration of 1 or 5 mg/kg ITF2357 significantly reduced liver injury. Therefore, low concentrations of the non-apoptotic HDAC inhibitor ITF2357 can reduce the production of pro-inflammatory cytokines in primary cells in vitro and exhibit anti-inflammatory effects in vivo. [2] Objective: To investigate the effects of the histone deacetylase inhibitor givinostat on proteins related to the proliferation regulation of hepatic stellate cells. Methods: The changes in proliferation, apoptosis and cell cycle of hepatic stellate cells after givinostat treatment were observed by the Cell Counting Kit-8 (CCK-8) method and flow cytometry. The expression changes of p21, p57, CDK4, CDK6, cyclin D1, caspase-3 and caspase-9 in hepatic stellate cells after exposure to givinostat were observed by Western blotting. The effect of givinostat on cell migration was analyzed by scratch assay. The effects of givinostat on the reactive oxygen species spectrum, mitochondrial membrane potential and mitochondrial permeability transition pore opening of JS-1 cells were observed by laser confocal microscopy. The results showed that givinostat significantly inhibited JS-1 cell proliferation and promoted apoptosis, leading to cell cycle arrest in the G0/G1 phase. Givinostat treatment downregulated the protein expression of CDK4, CDK6, and cyclin D1, while significantly upregulating the expression of p21 and p57. Givinostat-induced apoptosis in hepatic stellate cells was mainly mediated by p38 and extracellular signal-regulated kinase 1/2. Givinostat treatment increased intracellular reactive oxygen species production, decreased mitochondrial membrane potential, and promoted the opening of mitochondrial permeability transition pores. The acetylation levels of superoxide dismutase (acetylated K68) and nuclear factor-κB p65 (acetylated K310) were upregulated, but their protein expression levels remained unchanged. Furthermore, the significant beneficial effects of givinostat on liver fibrosis were also demonstrated in a mouse model. Conclusion: Givinostat exerts its antifibrotic effect by regulating the acetylation of nuclear factor-κB and superoxide dismutase 2, thereby inhibiting hepatic stellate cell proliferation and inducing apoptosis. [3]

---

Givinostat (ITF2357) is an orally potent histone deacetylase (HDAC) inhibitor containing hydroxamic acid with broad anti-inflammatory properties. [1]
It inhibits class I and class II HDACs. [1]
It has been used to treat children with systemic juvenile idiopathic arthritis. [1]
It reduces caspase-1 activity and decreases the secretion of IL-1β and other cytokines by stimulated human immune cells. [1]
It is currently being investigated for its potential use in the treatment of HIV-1 infection to reduce the number of latent infected T cells and related inflammation. [1]
Givinostat's pan-HDAC inhibitory properties make it cytotoxic at high concentrations, thus prompting the development of more specific HDAC inhibitors, such as the HDAC8-specific analog ITF3056. [1]

C24H27N3O4

421.49

421.2

C, 68.39; H, 6.46; N, 9.97; O, 15.18

497833-27-9

Givinostat hydrochloride monohydrate;732302-99-7;Givinostat hydrochloride;199657-29-9

9804992

White to light yellow solid powder

1.259

5.013

3

5

9

31

575

0

O=C(OCC1=CC=C2C=C(CN(CC)CC)C=CC2=C1)NC3=CC=C(C(NO)=O)C=C3

YALNUENQHAQXEA-UHFFFAOYSA-N

InChI=1S/C24H27N3O4/c1-3-27(4-2)15-17-5-7-21-14-18(6-8-20(21)13-17)16-31-24(29)25-22-11-9-19(10-12-22)23(28)26-30/h5-14,30H,3-4,15-16H2,1-2H3,(H,25,29)(H,26,28)

[6-(diethylaminomethyl)naphthalen-2-yl]methyl N-[4-(hydroxycarbamoyl)phenyl]carbamate

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)