HDAC1 ( IC50 = 243 nM ); HDAC3 ( IC50 = 248 nM ); HDAC2 ( IC50 = 453 nM )

Synthetic benzamide derivatives were investigated for their ability to inhibit histone deacetylase (HDA). In this study, one of the most active benzamide derivatives, MS-27-275, was examined with regard to its biological properties and antitumor efficacy. MS-27-275 inhibited partially purified human HDA and caused hyperacetylation of nuclear histones in various tumor cell lines. It behaved in a manner similar to other HDA inhibitors, such as sodium butyrate and trichostatin A; MS-27-275 induced p21(WAF1/CIP1) and gelsolin and changed the cell cycle distribution, decrease of S-phase cells, and increase of G1-phase cells. The in vitro sensitivity spectrum of MS-27-275 against various human tumor cell lines showed a pattern different than that of a commonly used antitumor agent, 5-fluorouracil, and, of interest, the accumulation of p21(WAF1/CIP1) tended to be faster and greater in the cell lines sensitive to MS-27-275. MS-27-275 administered orally strongly inhibited the growth in seven of eight tumor lines implanted into nude mice, although most of these did not respond to 5-fluorouracil. A structurally analogous compound to MS-27-275 without HDA-inhibiting activity showed neither the biological effects in cell culture nor the in vivo therapeutic efficacy. These results suggest that MS-27-275 acts as an antitumor agent through HDA inhibition and may provide a novel chemotherapeutic strategy for cancers insensitive to traditional antitumor agents.[3]

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Experimental autoimmune neuritis (EAN) is a T cell-mediated autoimmune inflammatory demyelinating disease of the peripheral nervous system and serves as the animal model of human inflammatory demyelinating polyradiculoneuropathies. MS-275, a potent histone deacetylase inhibitor currently undergoing clinical investigations for various malignancies, has been reported to demonstrate promising anti-inflammatory activities. In our present study, MS-275 administration (3.5 mg/kg i.p.) to EAN rats once daily from the appearance of first neurological signs greatly reduced the severity and duration of EAN and attenuated local accumulation of macrophages, T cells and B cells, and demyelination of sciatic nerves. Further, significant reduction of mRNA levels of pro-inflammatory interleukin-1beta, interferon-gamma, interleukine-17, inducible nitric oxide synthase and matrix metalloproteinase-9 was observed in sciatic nerves of MS-275 treated EAN rats. In lymph nodes, MS-275 depressed pro-inflammatory cytokines as well, but increased expression of anti-inflammatory cytokine interleukine-10 and of foxhead box protein3 (Foxp3), a unique transcription factor of regulatory T cells. In addition, MS-275 treatment increased proportion of infiltrated Foxp3(+) cells and anti-inflammatory M2 macrophages in sciatic nerves of EAN rats. In summary, our data demonstrated that MS-275 could effectively suppress inflammation in EAN, through suppressing inflammatory T cells, macrophages and cytokines, and inducing anti-inflammatory immune cells and molecules, suggesting MS-275 as a potent candidate for treatment of autoimmune neuropathies.[4]

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Entinostat (MS-27-275) at 49 mg/kg shows marked antitumor effects against the tumor lines KB-3-1, 4-1St, and St-4, and a moderate effect against the Capan-1 tumor. There are notable results when using entinostat at doses of 24.5 mg/kg and 12.3 mg/kg in treating these tumors. Furthermore, 4–24 hours after oral Entinostat administration, HT-29 tumor xenografts appear to have higher levels of histone acetylation[3]. When MS-275 (3.5 mg/kg i.p.) is given once daily to rats with experimental autoimmune neuritis (EAN) starting at the earliest neurological symptoms, it significantly lessens the severity and duration of EAN, as well as the local buildup of macrophages, T cells, and B cells, as well as the demyelination of sciatic nerves. Furthermore, in the sciatic nerves of EAN rats, MS-275 treatment raises the percentage of infiltrated Foxp3+ cells and anti-inflammatory M2 macrophages[4].

HDAC activity biochemical assays are performed by Nanosyn in 384-well microplates with a reaction volume of 10 μL. Five microliters of a 2× HDAC inhibitor (such as Entinostat), four microliters of 2.5× enzyme, and one microliter of 10× substrate are combined with assay buffer (100 mM HEPES, pH 7.5, 25 mM KCl, 0.1% BSA, 0.01% Triton X-100, 1% DMSO) in a typical enzymatic reaction. In the enzymatic assays, the final concentration of each HDAC ranges from 0.5 to 5 nM. In every experiment, a final substrate concentration of 1 μM FAM-RHKK(Ac)-NH₂ or FAM-RHKK(trifluoroacetyl)-NH₂ is employed, and it is discovered to be lower than the calculated K_m,app for every enzyme[1].

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Enzymatic HDAC Activity Assays [1]
Biochemical assays of HDAC activity were carried out by Nanosyn in a reaction volume of 10 μl in 384-well microplates. A standard enzymatic reaction contained 5 μl of 2× HDAC inhibitor, 4 μl of 2.5× enzyme, and 1 μl of 10× substrate in assay buffer (100 mm HEPES, pH 7.5, 25 mm KCl, 0.1% BSA, 0.01% Triton X-100, 1% DMSO). Final concentration of all HDACs in the enzymatic assays was between 0.5 and 5 nm. A final substrate concentration of 1 μm FAM-RHKK(Ac)-NH2 or FAM-RHKK(trifluoroacetyl)-NH2 was used in all assays and found to be below the determined Km, app for each enzyme. All inhibitors were serially diluted in DMSO prior to cross-dilution in assay buffer and were incubated with enzyme for 15 min prior to initiating the reaction by the addition of substrate. After incubation for 3 h, the reaction was terminated by the addition of EDTA and SDS to final concentrations of 24 mm and 0.04%, respectively. The product and substrate in each reaction were separated using a 12-sipper microfluidic chip run on a Caliper LC3000®. The separation conditions used a downstream voltage of −800V, an upstream voltage of −3000 V, and a screening pressure of −1.4 p.s.i. The product and substrate fluorescence was excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electrophoregram using HTS Well Analyzer software.

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Assay for Histone Deacetylase. [3]
HDA was partially purified as described by Yoshida et al. with slight modifications. K562 cells (2.5 × 108) were disrupted in 15 ml of HDA buffer (15 mM potassium phosphate, pH 7.5/5% glycerol/0.2 mM EDTA). Nuclei of the cells were collected by centrifugation (35,000 × g, 10 min) and were resuspended in 15 ml of HDA buffer containing 1 M (NH4)2SO4. After sonication to reduce viscosity, the supernatant was collected by centrifugation, solid (NH4)2SO4 was added to the supernatant to make the final concentration 3.5 M, and was stirred for 1 h at 0°C. The precipitates collected by centrifugation were dissolved again with 4 ml of HDA buffer and were dialyzed against 2 liters of HDA buffer. The dialysate was loaded onto MonoQ HR5/5 (Amersham Pharmacia) equilibrated with HDA buffer, and the proteins were eluted with a linear gradient of 0–1 M NaCl in 30 ml of HDA buffer. A single peak of HDA activity was eluted at 0.4 M NaCl, and the fraction was stored at −80°C until use. Nuclear histones were labeled by incubation of K562 cells (108 cells) in a 25 ml of growth medium containing 0.5 mCi/ml [3H]sodium acetate (152.8 GBq/mmol; NEN) and 5 mM NaBu at 37°C for 1 h. Histones were extracted as described.
HDA-inhibitory activity of the compound was estimated in 50 μl of reaction mixture containing 2 μl of the above HDA fraction, 100 μg/ml of [3H]acetylated histones, and 5 μl of the compound dissolved in HDA buffer at 37°C for 10 min. [3H]acetic acid released by the reaction was extracted with 50 μl of 1M HCl and 0.55 ml of ethyl acetate, and the radioactivity in the solvent layer was measured by liquid-scintillation counting. To assess in vivo HDA inhibition, cellular histones were extracted and examined by acid/urea/Triton X-100 PAGE followed by staining with Coomasie brilliant blue R-250, as described.

SH-SY5Y cells are split twice a week and kept in a humidified incubator with 5% CO₂ at 37°C under standard culture conditions. After plating cells at a density of 2500 cells per well in a 20-μL volume of DMEM/F-12 culture media supplemented with 10% FBS, the cells are left to adhere for the entire night in black 384-well plates. After being serially diluted in 100% DMSO the next day, HDAC inhibitors (such as Entinostat) are then cross-diluted into culture media. To achieve the desired inhibitor final concentration (e.g., 0.1% DMSO), 5 μL of the compound (e.g., Entinostat) diluted in media is added to the appropriate well of the cell plate. Cellular ATP levels are quantified using CellTiter-Glo reagents after treated cells are incubated for 6, 24, 48, 72, or 96 hours under standard tissue culture conditions. Similarly, media from different cell plates are aspirated after 6 hours of incubation with HDAC inhibitors (such as Entinostat), and cells are once again washed with media free of inhibitors. After 24, 48, 72, or 96 hours of incubation, the cells are given 25 μL of media supplemented with 10% FBS and 0.1% DMSO (no inhibitors), and the levels of cellular ATP are measured using CellTiter-Glo. An Envision Instrument with a 0.1 s count time is used to measure luminosity at each time point[1].

Mice: Subcutaneous injection of A2780 cells (9×10⁶) in PBS suspension is administered subcutaneously into the flank of a naked mouse. For the remaining tumor lines, KB-3-1, HCT-15, 4-1St, Calu-3, St-4, Capan-1, and HT-29, the tumors are passaged multiple times prior to initiating in vivo antitumor testing. A trocar needle is used to implant a tumor lump, measuring 2-3 mm in diameter, subcutaneously into the flank of a nude mouse. Once the tumors are confirmed to have grown in the body (tumor size, 20-100 mm³), treatment with the drugs is initiated in four or five mice per experimental group. For four weeks, one oral dose of entinostat is given five days a week. Tumor width and length are measured twice a week, and the volume of the tumor is computed.

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Rats: Male Lewis rats (weight: 170-200 g, 8-10 weeks) are kept in a 12-hour light/dark cycle with unrestricted access to food and drink. Six rats per group receive an intraperitoneal injection of EntinostatMS-275 (3.5 mg/kg) every day from day 10 to day 14 as part of a therapeutic treatment. EntinostatMS-275 is dissolved in phosphate buffered saline (PBS) for injection, and control rats are administered the same volume (1 mL) of PBS.

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In Vivo Antitumor Activity. [3]
A2780 cells (9 × 106) grown in vitro were suspended in PBS and were injected subcutaneously into the flank of nude mouse. For the other tumor lines, KB-3-1, HCT-15, 4-1St, Calu-3, St-4, Capan-1, and HT-29, tumors were passaged several times before starting in vivo antitumor testing, and a tumor lump (2–3 mm in diameter) was transplanted subcutaneously into the flank of a nude mouse by using a trocar needle. Treatment (four or five mice in each experimental group) with the drugs was started after the tumors were confirmed to have grown in the body (tumor size, 20–100 mm3). Entinostat/MS-27-275 and compound 2, both dissolved with 0.05 N HCl, 0.1% Tween 80, and 5-fluorouracil (5-FU) and diluted with physiological saline, were administered orally once daily 5 days per week for 4 weeks. Tumor length and width were monitored twice weekly, and tumor volume was calculated as described.

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EAN induction and Entinostat/MS-275 treatment [4]
EAN was induced as described (Zhang et al., 2008a). Briefly, rats were immunized by s.c. injection at the basal part of tails with 100 μL of an inoculum containing 100 μg of synthetic neuritogenic P2 peptide 57–81. Neurological scores of EAN were evaluated every day as follows: 0=normal, 1=reduced tonus of tail, 2=limp tail, impaired righting, 3=absent righting, 4=gait ataxia, 5=mild paresis of the hind limbs, 6=moderate paraparesis, 7=severe paraparesis or paraplegia of the hind limbs, 8=tetraparesis, 9=moribund, and 10=death. For therapeutic treatment, EAN rats received i.p. injection of Entinostat/MS-275 (3.5 mg/kg) daily from day 10 to day 14 (six rats/group). For injection, EntinostatMS-275 was suspended in phosphate buffered saline (PBS) and the same volume (1 ml) of PBS was given to control rats.

The pharmacokinetic summary of MS-275 is shown in Table 5. In Part I, due to the once-weekly dosing and the initial sampling point being after the time to peak concentration (tmax), the early characteristics of the maximum plasma concentration (Cmax) and AUC were not clearly defined. Figure 1 shows the plasma concentration-time curves after single doses of 2, 4, and 6 mg/m². The maximum plasma concentration of MS-275 was reached within 1 hour after both single and repeated dosing regimens. The mean Cmax after the first dose increased almost proportionally with the dose. In Parts II and III of the study, the plasma concentration of MS-275 decreased to approximately 4% of Cmax within 4 hours after a single dose, at which point Cmax was clearly defined, indicating rapid distribution of the drug to tissues. A slower secondary distribution phase followed the initial distribution phase. However, because the perceived linear portion of the curves was less than two half-lives for most patients, the terminal half-life value could not be accurately determined. Therefore, the calculated terminal half-life, AUC, and apparent clearance values are estimates and can be used as approximate reference values. When the perceived linear portion of the curve persists for a relatively long time (approximately 168 hours after administration), the estimated terminal half-life is between 60 and 150 hours, regardless of dose or dosing regimen. Due to dosing regimen deviations, such as missed doses and dose reductions due to toxicity, assessments of drug accumulation in plasma under once-weekly and twice-weekly dosing regimens are limited. Based on limited data, plasma drug concentrations appear to continue increasing until the last dose of cycle 1, indicating that steady state has not yet been reached in the twice-weekly treatment group. No significant signs of drug accumulation were observed after once-weekly treatment. https://pubmed.ncbi.nlm.nih.gov/18579665/

Hematologic Toxicity and Laboratory Abnormalities [https://pubmed.ncbi.nlm.nih.gov/18579665/]
Table 3 lists the number of cycles in which hematologic toxicity occurred. Overall, hematologic toxicity was rare and none of the cases reached dose limits. Grade 3 hemoglobin abnormalities occurred in 5 out of 149 cycles (3%): 1 case occurred in the 2 mg/m² twice-weekly dosing regimen and 4 cases occurred in the 5 mg/m² weekly dosing regimen. Grade 3 or 4 neutropenia occurred in 6 out of 149 cycles (4%): 2 cases occurred in the 2 mg/m² dosing regimen, 1 case occurred in the 4 mg/m² every two weeks dosing regimen and 3 cases occurred in the 5 mg/m² weekly dosing regimen. No grade 3 or 4 thrombocytopenia was observed. Myelosuppression was not associated with prior chemotherapy or radiotherapy, and no complications such as fever, serious infection, or bleeding occurred in any of the cases.

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Non-hematologic toxicities [https://pubmed.ncbi.nlm.nih.gov/18579665/]
Non-hematologic toxicities are shown in Table 4. The most common adverse events were nausea and fatigue [occurring in 48 and 47 of 149 treatment cycles, respectively (32%)]. The second most common adverse event was anorexia, occurring in 23 of 149 treatment cycles (15%). The most common grade 3 or 4 adverse event was fatigue, occurring in 7 of 149 treatment cycles (5%).
Asymptomatic hypophosphatemia was relatively common, constituting a dose-limiting toxicity (DLT) in two patients. Hypophosphatemia was not associated with other toxicities such as renal insufficiency or other electrolyte disturbances, and no serious complications were observed. One patient with metastatic colon cancer developed grade 3 hyponatremia concurrently with massive ascites and dehydration (associated with rapid tumor progression and reduced oral intake). The patient had no prior history of kidney disease, electrolyte disturbances, or chemotherapy known to cause electrolyte disturbances. Following therapeutic paracentesis, intravenous infusion, and colloid infusion, the patient's dehydration and electrolyte disturbances were corrected within 72 hours. Patients with ≥ grade 2 hypophosphatemia underwent urinalysis and serum electrolyte analysis, received daily oral phosphorus supplementation, and were monitored at least weekly until serum phosphorus levels stabilized within the normal range. Other histone deacetylase inhibitors (HDAC inhibitors), including hydroxamic acid derivatives, have been cited as a potential cause of arrhythmias and myocardial infarction, including QTc interval prolongation and T-wave and ST-wave abnormalities, particularly in preclinical models. In this study, patients in the bi-weekly dosing group underwent electrocardiograms (ECGs) at baseline, at the start of the first treatment cycle, at the beginning of the second treatment cycle, and at the end of the study; patients in the twice-weekly and once-weekly dosing groups underwent ECGs at baseline and as clinically necessary. Patients in the every-weekly dosing group underwent MUGA scans at baseline, before the second and fourth treatment cycles, and every 6 weeks after the fourth treatment cycle; patients in the twice-weekly and once-weekly dosing groups underwent MUGA scans at baseline and as clinically necessary. Following baseline assessment, electrocardiograms were performed in 18 patients: 10 in the every-weekly dosing group, 3 in the twice-weekly dosing group, and 5 in the once-weekly dosing group. Ten patients completed consecutive MUGA scans: 6 in the every-weekly dosing group, 2 in the twice-weekly dosing group, and 2 in the once-weekly dosing group. No significant electrocardiographic or MUGA abnormalities at least possibly related to MS-275 were found.

[1]. Histone deacetylase (HDAC) inhibitor kinetic rate constants correlate with cellular histone acetylation but not transcription and cell viability. J Biol Chem. 2013 Sep 13;288(37):26926-43.

[2]. The histone deacetylase inhibitor MS-275 promotes differentiation or apoptosis in human leukemia cells through a process regulated by generation of reactive oxygen species and induction of p21CIP1/WAF1 1. Cancer Res. 2003 Jul 1;63(13):3637-45.

[3].A synthetic inhibitor of histone deacetylase, MS-27-275, with marked in vivo antitumor activity against human tumors. Proc Natl Acad Sci U S A, 1999, 96(8), 4592-4597.

[4]. MS-275, an histone deacetylase inhibitor, reduces the inflammatory reaction in rat experimental autoimmune neuritis. Neurosci, 2010, 169, 370-377.

Entinostat belongs to the benzamide class of compounds and is formed by the condensation of the carboxyl group of a pyridin-3-ylmethylcarbamate derivative of p-(aminomethyl)benzoic acid with the amino group of phenyl-1,2-diamine. It is an inhibitor of histone deacetylase 1 (HDAC1) and 3 (HDAC3). It has multiple functions, including as a histone deacetylase inhibitor (EC 3.5.1.98), an antitumor drug, and an inducer of apoptosis. Enteninostat belongs to the pyridine, carbamate, substituted aniline, primary amine, and benzamide classes. Its function is similar to that of 1,2-phenylenediamine. Enteninostat is currently under investigation for the treatment of volunteers, breast cancer patients, human volunteers, and healthy volunteers. Enteninostat has been investigated for the treatment of non-small cell lung cancer and for epigenetic therapy. Enteninostat is a synthetic benzamide derivative with potential antitumor activity. Entenoxat binds to and inhibits histone deacetylases, enzymes that regulate chromatin structure and gene transcription. This drug appears to have a dose-dependent effect on human leukemia cells, including inducing cyclin-dependent kinase inhibitor 1A (p21/CIP1/WAF1)-dependent growth arrest and differentiation at low concentrations; significantly inducing reactive oxygen species (ROS) generation; mitochondrial damage; caspase activation; and inducing apoptosis at high concentrations. In normal cells, cyclin-dependent kinase inhibitor 1A expression is associated with cell cycle exit and differentiation. We investigated the effects of the histone deacetylase (HDAC) inhibitor MS-275 on differentiation and apoptosis in human leukemia and lymphoma cells (U937, HL-60, K562, and Jurkat cells) and primary acute myeloid leukemia blasts. MS-275 showed a dose-dependent effect in each cell line. When administered at low concentrations (e.g., 1 μM), MS-275 exhibits potent antiproliferative activity, inducing p21 (CIP1/WAF1)-mediated growth arrest and expression of the differentiation marker (CD11b) in U937 cells. These events are accompanied by an increase in hypophosphorylated retinoblastoma proteins and a downregulation of cell cycle-related proteins, including cyclin D1. However, at high concentrations (e.g., 5 μM), MS-275 effectively induces cell death, resulting in approximately 70% apoptosis within 48 hours. Unlike other HDAC inhibitors (e.g., aspirin), the exogenous receptor-mediated pathway plays a minimal role in the lethal effect of MS-275. However, MS-275 rapidly (e.g., within 2 hours) effectively induces an increase in reactive oxygen species (ROS) levels, subsequently leading to loss of mitochondrial membrane potential (Δψm) and cytochrome c release. These events ultimately activated the caspase cascade, manifested as the degradation of poly(ADP-ribose) polymerase, p21(CIP1/WAF1), p27(KIP), Bcl-2, and retinoblastoma proteins. MS-275 exposure also led to decreased expression of cyclin D1 and the anti-apoptotic proteins Mcl-1 and XIAP. Administration of the free radical scavenger LN-acetylcysteine blocked MS-275-mediated mitochondrial damage and apoptosis, indicating that reactive oxygen species (ROS) generation plays a major role in MS-275-related cell death. Finally, U937 cells stably expressing the p21(CIP1/WAF1) antisense construct were significantly more sensitive to MS-275-mediated apoptosis than the control group, but their differentiation response was impaired. These findings collectively indicate that MS-275 has a dose-dependent effect on human leukemia cells, namely, at low drug concentrations it induces p21(CIP1/WAF1)-dependent growth arrest and differentiation, while at higher concentrations it significantly induces reactive oxygen species (ROS) generation, mitochondrial damage, caspase activation and apoptosis. [2]

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Researchers investigated the ability of synthetic benzamide derivatives to inhibit histone deacetylase (HDA). This study investigated the biological characteristics and antitumor efficacy of MS-27-275, one of the most active benzamide derivatives. MS-27-275 inhibited partially purified human HDA and caused excessive acetylation of nuclear histones in various tumor cell lines. Its mechanism of action is similar to other HDA inhibitors (such as sodium butyrate and trogostatin A); MS-27-275 induced the expression of p21(WAF1/CIP1) and colloid protein and altered cell cycle distribution, resulting in a decrease in S phase cells and an increase in G1 phase cells. MS-27-275 exhibits a different in vitro sensitivity profile to various human tumor cell lines compared to the commonly used antitumor drug 5-fluorouracil. Notably, p21 (WAF1/CIP1) accumulates at a faster rate and to a greater extent in cell lines sensitive to MS-27-275. Oral administration of MS-27-275 significantly inhibited the growth of 7 out of 8 tumor cell lines transplanted into nude mice, most of which were unresponsive to 5-fluorouracil. A compound with a structure similar to MS-27-275 but without HDA inhibitory activity did not show any biological effect in cell culture or in vivo therapeutic effect. These results suggest that MS-27-275 exerts its antitumor effect by inhibiting HDA and may provide a new chemotherapy strategy for cancers that are insensitive to traditional antitumor drugs. [3]

C21H20N4O3

376.41

376.153

C, 67.01; H, 5.36; N, 14.88; O, 12.75

209783-80-2

4261

White off white solid powder

1.3±0.1 g/cm3

566.7±50.0 °C at 760 mmHg

159-160ºC

296.6±30.1 °C

0.0±1.6 mmHg at 25°C

1.672

1.46

3

5

7

28

508

0

O=C(NCC1=CC=C(C=C1)C(NC2=CC=CC=C2N)=O)OCC3=CC=CN=C3

INVTYAOGFAGBOE-UHFFFAOYSA-N

InChI=1S/C21H20N4O3/c22-18-5-1-2-6-19(18)25-20(26)17-9-7-15(8-10-17)13-24-21(27)28-14-16-4-3-11-23-12-16/h1-12H,13-14,22H2,(H,24,27)(H,25,26)

pyridin-3-ylmethyl N-[[4-[(2-aminophenyl)carbamoyl]phenyl]methyl]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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.64 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: ≥ 2.08 mg/mL (5.53 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.53 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 20.8 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 4: ≥ 2.08 mg/mL (5.53 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 corn oil and mix evenly.

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Solubility in Formulation 5: 2% DMSO+30% PEG 300: 10mg/mL

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Solubility in Formulation 6: 3% DMSO + 22% Castor oil + 75% Saline

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