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 )

ITF2357 (1–10 mg/kg) significantly lowers LPS-induced serum TNFα and IFNγ levels by over 50%. ITF2357 does not inhibit the release of anti-CD3-induced cytokines from mouse circulation-derived PBMCs in mice. ITF2357 (1 or 5 mg/kg) has been shown to significantly reduce liver damage in hepatitis induced by concanavalin A.[1] When severe combined immunodeficient mice are injected with the AML-PS in vivo passaged cell line, ITF2357 (10 mg/kg) considerably increases their survival time.[2] ITF2357 (10 mg/kg) reduces lesion volume, induces glial apoptosis, improves neurobehavioral recovery, and lessens neuronal degeneration in a mouse model of closed head injury (CHI).[4]

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Con A Model of Acute Hepatitis[1]
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.

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Givinostat (ITF-2357) prolongs survival of leukemia bearing SCID mice [2]
In order to demonstrate therapeutic activity of ITF2357, an in vivo model of AML was set up. AML-PS is a cell line derived from an AML patient and established by in vivo passage in SCID mice after intraperitoneal injection.28, 29 When injected intravenously (i.v.), AML-PS cells home in blood, spleen, bone marrow and liver and lead to death of animals in 35–40 days. Groups of 7–10 SCID mice were inoculated i.v. with 5 × 106 AML-PS cells, and daily oral treatment with different doses of ITF2357 was initiated after 4 days. Survival of animals was recorded. The results demonstrate that ITF2357 had no significant effect at 1 mg/kg (P=0.36), but showed clear therapeutic activity at the intermediate dose of 10 mg/kg, with median survival of 46 compared to 40 days for the control group (P=0.0057). The therapeutic effect was even greater at 100 mg/kg, with a median survival time of 50 days (P<0.0001 compared to controls; Figure 7). All animals died of tumor as shown by necropsy of the animals and by immunophenotypic analysis of randomly selected cases (CD45 and CD33) (data not shown).

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Givinostat (ITF-2357) Prevents the Onset of Hyperglycemia and Decreases Serum Nitrite Levels in a Mouse Model of STZ-Induced β-Cell Toxicity [3]
C57BL/6 mice received an oral dose of 1.25, 2.5 or 5.0 mg/kg ITF2357 or water (0.1 mL, by gavage), 12 and 24 h before a single injection of STZ (225 mg/kg, i.p.), and then again at 12, 24 and 36 h post-STZ. Forty-eight h after STZ injection, glucose levels were determined, glucose tolerance test (GTT) was performed and serum collected for nitrite levels. The spleens were removed for ex vivo splenocyte stimulation. As shown in Figure 1A, a reduction of blood glucose was observed with each of the three oral doses of ITF2357, with an optimal response at 2.5 mg/kg (from 348 ± 64 mg/dL to 120 ± 16 mg/dL, mean ± SE, P = 0.039). Doubling of the dose of ITF2357 to 5 mg/kg reduced glucose levels to 200 ± 37 mg/dL.

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.

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Maize HDAC Assays [1]
HD2, 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.

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Cellular Crude Extract for Total HDAC Activity and Protein Acetylation Determinations [1]
Human 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.
Crude 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.

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Total HDAC Activity Assay [1]
The 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.

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Acetylation of Proteins [1]
Acetylation 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.

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Enzymatic Assay for HDAC Inhibitory Activity of Synthetic Compounds [1]
The 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).

The isolated PBMCs are cleaned, then resuspended at 5×10⁶/mL in RPMI with 5% FCS, transferred to a 50-mL conical polypropylene tube, and kept at 4 °C for the night. A 96-well flat microtiter plate is filled with 100 μL of PBMCs after they have been resuspended the previous morning. The plates are then incubated at 37 °C for an hour after adding ITF2357 for the inhibition studies. The cells are then stimulated with LPS or other stimulants in a final volume of 200 μL per well. After being incubated for 24 hours at 37 °C, the supernatants are removed and frozen at -80 °C until they are tested for cytokines.

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Cytotoxicity assays [2]
Cytotoxicity assays were performed using the alamar blue vital dye essentially as described.27 Briefly, cell lines or MSCs were plated in the presence or absence of 0.1–1 μ M Givinostat (ITF2357) or SAHA. After 2 days of culture, alamar blue solution was added. After overnight incubation, the plates were read in a fluorimeter with excitation at 535 nm and emission at 590 nm.

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FACS analyses [2]
Apoptosis was measured by Annexin V-PE/ 7AAD double staining using fluorescence-activated cell sorter (FACS) analysis. Caspase 3 activation was measured using AlexaFluor488-labeled anti-cleaved caspase 3 antibody, according to the manufacturer's instructions and FACS analysis. For measurement of α-tubulin acetylation, cells were treated for 1 h with Givinostat (ITF2357) or medium, permeabilized and stained with anti-tubulin or acetyl-tubulin antibodies followed by FITC-labeled rabbit anti-mouse polyclonal antibody.

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Colony assays [2]
Three thousand MM or AML cells were added to 3 ml methylcellulose and plated in duplicate in 55 mm dishes. Plates were incubated at 37°C for 15–21 days and colonies counted under an inverted microscope.

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Co-culture of leukemic cells with MSCs [2]
For all co-cultures, MSCs were plated at 5000/well in 96-well plates and incubation was carried on for 48–72 h at 37°C in order to reach confluence. CMA-03 cells were then added at 5000/well and AML cells at 1 × 105 cells/well in medium in the presence or absence of MSCs or 10 ng/ml rhIL-6 and with different concentrations of Givinostat (ITF2357). Medium was replaced twice weekly. After different time intervals, non-adherent cells were collected and stained with propidium iodide (5 μg/ml, Sigma) and FITC-conjugated anti-CD138 (for CMA-03) or anti-CD33 MAb (for AML). Stained cells were analyzed on the FACS to determine cell viability and identity. Total number of live cells was also counted by Trypan blue exclusion.

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Immunoblotting [3]
Five-hundred randomly picked islets were cultured for 2–3 h and pre-exposed to Givinostat (ITF2357) or vehicle for 1 h. IL-1β (160 pg/mL) plus IFNγ (5 ng/mL) was added for 6 h and islets were then lysed and the protein content measured by the Bradford method. Lysates were subjected to gel electrophoresis as described.

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Morphological Analysis [3]
Three-hundred thousand INS-1 cells were cultured for 2 d prior to cytokine treatment. On the day of the experiment, the medium was replaced and Givinostat (ITF2357) was added 30 min preceding the addition of IL-1β (250 pg/mL) and IFNγ (10 ng/mL). After 24 h at 37°C, the cells were rinsed in PBS, fixed in 1% paraformaldehyde overnight at 4°C and incubated with DAPI stain.

Mice: For a minimum of five days prior to usage, C57BL/6 mice are kept in the animal facility. Givinostat (ITF2357) is injected intraperitoneally for the comparison study, and it is also given orally at a dose of 10 mg/kg. 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.[1]

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In vivo model [2]
In vivo passaged AML-PS cells (5 × 106) were inoculated in the tail vein of 5-week-old severe combined immunodeficient (SCID) mice. Mice were randomized and divided into four groups: Vehicle (ten mice), Givinostat (ITF2357) 1 mg/kg (nine mice), ITF2357 10 mg/kg (ten mice) and ITF2357 100 mg/kg (seven mice). On the 4th day after tumor cells injection, the drug treatment was started and maintained until day 55. ITF 2357 was suspended in 5% methylcellulose and administered daily by gavage in a volume of 0.2 ml/mouse. Survival of the animals was recorded daily and necropsy was performed on all animals. The presence of CD33+ tumor cells was confirmed in several animals by immunophenotypic analysis of spleen cells.

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In Vivo STZ Model [3]
STZ was reconstituted in cold sodium-citrate buffer pH 4.3 immediately before use. Mice were injected intraperitoneally (i.p.) with STZ (225 mg/kg). Givinostat (ITF2357) (1.25, 2.5 and 5 mg/kg) or water (vehicle) was administered by gavage (0.1 mL), 12 h and 4 h prior to STZ, and every 12 h thereafter. Forty-eight h after STZ injection, β-cell function was assessed by glucose challenge and serum was collected for nitrite levels, as described below.

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Drug treatment protocol[4]
Givinostat (ITF2357) was dissolved in DMSO. Prior to use, the compound was diluted in sterile saline, heated to boiling for complete dissolution, and cooled to ∼30°C before injection. Control for these studies was 0.5% DMSO saline solution. ITF2357 solutions were freshly prepared for each experiment. To initially substantiate a functional effect of ITF2357 on neurobehavioral outcome after brain trauma, the drug was given either as pretreatment (a single injection at 30 min prior to the induction of injury) or administered following the impact. Postinjury injections were carried out at either at 1 or 24 h after trauma. In each group of mice, animals with a similar initial severity of injury (NSS at 1 h after trauma; n≥9 mice/group) were dosed i.p. with 100 μl containing either 10 mg/kg of ITF2357 or vehicle. This dose was selected in accordance with previous studies utilizing ITF2357 in mice, and no adverse effects or mortality were observed among treated mice in all experiments included in the current report.

[1]. 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.

[2]. The histone deacetylase inhibitor ITF2357 has anti-leukemic activity in vitro and in vivo and inhibits IL-6 and VEGF production by stromal cells. Leukemia. 2007 Sep;21(9):1892-900.

[3]. The oral histone deacetylase inhibitor ITF2357 reduces cytokines and protects islet β cells in vivo and in vitro. Mol Med. 2011 May-Jun;17(5-6):369-77.

[4]. Histone deacetylase inhibitor ITF2357 is neuroprotective, improves functional recovery, and induces glial apoptosis following experimental traumatic brain injury. FASEB J. 2009 Dec;23(12):4266-75.

Givinostat hydrochloride monohydrate is the monohydrate form of givinostat hydrochloride. It is a histone deacetylase inhibitor indicated for the treatment of Duchenne muscular dystrophy in patients 6 years of age and older. It has a role as an angiogenesis inhibitor, an anti-inflammatory agent, an antineoplastic agent, an apoptosis inducer and an EC 3.5.1.98 (histone deacetylase) inhibitor. It contains a givinostat hydrochloride.
See also: Givinostat (annotation moved to); Givinostat hydrochloride (annotation moved to).

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We studied inhibition of histone deacetylases (HDACs), which results in the unraveling of chromatin, facilitating increased gene expression. ITF2357, an orally active, synthetic inhibitor of HDACs, was evaluated as an anti-inflammatory agent. In lipopolysaccharide (LPS)-stimulated cultured human peripheral blood mononuclear cells (PBMCs), ITF2357 reduced by 50% the release of tumor necrosis factor-alpha (TNFalpha) at 10 to 22 nM, the release of intracellular interleukin (IL)-1alpha at 12 nM, the secretion of IL-1beta at 12.5 to 25 nM, and the production of interferon-gamma (IFNgamma) at 25 nM. There was no reduction in IL-8 in these same cultures. Using the combination of IL-12 plus IL-18, IFNgamma and IL-6 production was reduced by 50% at 12.5 to 25 nM, independent of decreased IL-1 or TNFalpha. There was no evidence of cell death in LPS-stimulated PBMCs at 100 nM ITF2357, using assays for DNA degradation, annexin V, and caspase-3/7. By Northern blotting of PBMCs, there was a 50% to 90% reduction in LPS-induced steady-state levels of TNFalpha and IFNgamma mRNA but no effect on IL-1beta or IL-8 levels. Real-time PCR confirmed the reduction in TNFalpha RNA by ITF2357. Oral administration of 1.0 to 10 mg/kg ITF2357 to mice reduced LPS-induced serum TNFalpha and IFNgamma by more than 50%. Anti-CD3-induced cytokines were not suppressed by ITF2357 in PBMCs either in vitro or in the circulation in mice. In concanavalin-A-induced hepatitis, 1 or 5 mg/kg of oral ITF2357 significantly reduced liver damage. Thus, low, nonapoptotic concentrations of the HDAC inhibitor ITF2357 reduce pro-inflammatory cytokine production in primary cells in vitro and exhibit anti-inflammatory effects in vivo.[1]

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We have investigated the activity of ITF2357, a novel hydroxamate histone deacetylase inhibitor, on multiple myeloma (MM) and acute myelogenous leukemia (AML) cells in vitro and in vivo. ITF2357 induced apoptosis in 8/9 MM and 6/7 AML cell lines, as well as 4/4 MM and 18/20 AML freshly isolated cases, with a mean IC(50) of 0.2 microM. ITF2357 activated the intrinsic apoptotic pathway, upregulated p21 and downmodulated Bcl-2 and Mcl-1. The drug induced hyperacetylation of histone H3, H4 and tubulin. When studied in more physiological conditions, ITF2357 was still strongly cytotoxic for the interleukin-6 (IL-6)-dependent MM cell line CMA-03, or for AML samples maximally stimulated by co-culture on mesenchymal stromal cells (MSCs), but not for the MSCs themselves. Interestingly, ITF2357 inhibited the production of IL-6, vascular endothelial growth factor (VEGF) and interferon-gamma by MSCs by 80-95%. Finally, the drug significantly prolonged survival of severe combined immunodeficient mice inoculated with the AML-PS in vivo passaged cell line already at the 10 mg/kg oral dose. These data demonstrate that ITF2357 has potent anti-neoplastic activity in vitro and in vivo through direct induction of leukemic cell apoptosis. Furthermore, the drug inhibits production of growth and angiogenic factors by bone marrow stromal cells, in particular IL-6 and VEGF.[2]

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In type 1 diabetes, inflammatory and immunocompetent cells enter the islet and produce proinflammatory cytokines such as interleukin-1β (IL-1β), IL-12, tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ); each contribute to β-cell destruction, mediated in part by nitric oxide. Inhibitors of histone deacetylases (HDAC) are used commonly in humans but also possess antiinflammatory and cytokine-suppressing properties. Here we show that oral administration of the HDAC inhibitor ITF2357 to mice normalized streptozotocin (STZ)-induced hyperglycemia at the clinically relevant doses of 1.25-2.5 mg/kg. Serum nitrite levels returned to nondiabetic values, islet function improved and glucose clearance increased from 14% (STZ) to 50% (STZ + ITF2357). In vitro, at 25 and 250 nmol/L, ITF2357 increased islet cell viability, enhanced insulin secretion, inhibited MIP-1α and MIP-2 release, reduced nitric oxide production and decreased apoptosis rates from 14.3% (vehicle) to 2.6% (ITF2357). Inducible nitric oxide synthase (iNOS) levels decreased in association with reduced islet-derived nitrite levels. In peritoneal macrophages and splenocytes, ITF2357 inhibited the production of nitrite, as well as that of TNFα and IFNγ at an IC(50) of 25-50 nmol/L. In the insulin-producing INS cells challenged with the combination of IL-1β plus IFNγ, apoptosis was reduced by 50% (P < 0.01). Thus at clinically relevant doses, the orally active HDAC inhibitor ITF2357 favors β-cell survival during inflammatory conditions.[3]

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Despite efforts aimed at developing novel therapeutics for traumatic brain injury (TBI), no specific pharmacological agent is currently clinically available. Here, we show that the pan-histone deacetylase (HDAC) inhibitor ITF2357, a compound shown to be safe and effective in humans, improves functional recovery and attenuates tissue damage when administered as late as 24 h postinjury. Using a well-characterized, clinically relevant mouse model of closed head injury (CHI), we demonstrate that a single dose of ITF2357 administered 24 h postinjury improves neurobehavioral recovery from d 6 up to 14 d postinjury (improved neurological score vs. vehicle; P< or =0.05), and that this functional benefit is accompanied by decreased neuronal degeneration, reduced lesion volume (22% reduction vs. vehicle; P< or =0.01), and is preceded by increased acetylated histone H3 levels and attenuation of injury-induced decreases in cytoprotective heat-shock protein 70 kDa and phosphorylated Akt. Moreover, reduced glial accumulation and activation were observed 3 d postinjury, and total p53 levels at the area of injury and caspase-3 immunoreactivity within microglia/macrophages at the trauma area were elevated, suggesting enhanced clearance of these cells via apoptosis following treatment. Hence, our findings underscore the relevance of HDAC inhibitors for ameliorating trauma-induced functional deficits and warrant consideration of applying ITF2357 for this indication.[4]

C24H27N3O4.HCL.H2O

475.97

475.187

C, 60.56; H, 6.35; Cl, 7.45; N, 8.83; O, 16.81

732302-99-7

199657-29-9 (HCl); 497833-27-9; 732302-99-7(HCl monohydrate);

9804991

White to beige solid powder

5.75

5

6

9

33

575

0

O=C(OCC1=CC=C2C=C(CN(CC)CC)C=CC2=C1)NC3=CC=C(C(NO)=O)C=C3.[H]Cl.O

FKGKZBBDJSKCIS-UHFFFAOYSA-N

InChI=1S/C24H27N3O4.ClH.H2O/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);1H;1H2

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

ITF 2357; ITF2357; 732302-99-7; Givinostat hydrochloride monohydrate; Givinostat Hydrochloride Hydrate; Givinostat (ITF2357); ITF2357; ITF2357 (Givinostat); (6-((diethylamino)methyl)naphthalen-2-yl)methyl (4-(hydroxycarbamoyl)phenyl)carbamate hydrochloride hydrate; DUVYZAT; ITF-2357; Givinostat; gavinostat; ITF2357 HCl; ITF2357 hydrochloride; Givinostat HCl

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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: ≥ 2.17 mg/mL (4.56 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 21.7 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: ≥ 2.17 mg/mL (4.56 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 21.7 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: ≥ 2.17 mg/mL (4.56 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 21.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% propylene glycol, 5% Tween 80, 65% D5W: 30mg/mL

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