生物活性

5-Azacytidine (5-AZA), a cytidine analog,has been approved for the treatment of patients with acute myeloid leukemia(AML) and myelodyplastic syndrome (MDS). 5-Azacytidine is a potent growth inhibitor and a cytotoxic agent. DNA methyltransferase inhibitor. 5-Azacytidine acts as a demethylating agent by inhibiting DNA methyltransferase (Dnmt), forming covalent adducts with cellular DNMT1 depleting enzyme activity. Improves the efficiency of reprogramming of stem cells; induces differentiation of mesenchymal stem cells into cardiomyocytes.

体外研究

体内研究

30% Propylene glycol, 5% Tween 80, 65% D5W(5%葡萄糖水溶液)

激酶实验

细胞实验

Cell culture[1]

The cells were cultured in DMEM with theF-12 Ham’s nutrient supplemented with 10% of foetal bovine serum (FBS) and 1%P/S/A solution. During the experiment, the cells were cultured under asepticand constant conditions in an incubator (37°C, 5% CO₂ and 95%humidity). The media were changed every 2 days and the cells were passagedusing trypsin solution according to manufacturers’ instructions after reaching80% confluence.

Pre-treatmentof ASCs with 5-AZA

Prior experiment ASCs were seeded onto24-well plates at the density of 20 x 10³ per well. After cell attachmentculture medium was replaced with medium containing 5-AZA (1μM).After total pre-treatment (48 h) with 5-AZA, cells were maintained in standardculture medium again. Cells cultured in standard medium (DMEM, 10% of FBS, 1% P/S/A)without addition of 5-AZA served as a control group. Non-treated cells servedas a control for comparison with the test culture. All experiments wereperformed after seventh day of experiment.

Cellviability, population doubling time and colony forming unit-fibroblasts (CFU-fs)assay

Cell proliferation rate was evaluated using10% resazurin-based dye-TOX-8 following manufacturer’s protocols. To performthe test, culturemedia were removed and replaced with culture medium containing 10% of the dye.The cells were then incubated with the dye in the CO₂ incubator,37°C for 2 hrs. Supernatants were collected and transferred to 96-well plate toperform the spectrophotometric assay. The absorbance of the supernatants wasmeasured at a wavelength of 600 nm for resazurin, and 690 nm referencewavelength. Each measurement included a blank sample, containing medium withoutcells. The number of cells was estimated on the basis of standard curve,generated during the experiment. To prepare the curve, cells were seeded at thedensity of 20x 10³, 40 x 10³, 80 x10³ per welland dye absorbance was measured in relation to certain cells number. Lineartrendline equation allowed estimating cells number throughout the experiment.

To evaluate the ability of cells to formcolonies, ASCs were seeded in six-well plates at a density of 100 cells perwell and inoculated into a culture medium. The medium was changed every 2 daysand cultures were maintained for 7 days. After fixation in 4% ice-coldparaformaldehyde, cells were stained with pararosaniline solution and coloniesof more than 50 cells were counted and documented by a Power Shot Camera. Theefficiency of colony forming was calculated using the formula presented below.

动物实验

Animals[2]

The C57BL/6 J mice (male, 6–8 weeks old,weighing 20–25 g) were fed with diet and water ad libitum under specificpathogen-free conditions and 12 h dark/light cycle.

APAP-inducedtoxic hepatitis

Toxic hepatitis was induced byintraperitoneal injection of APAP (350 mg/kg, dissolved in warm phosphatebuffered saline) in mice, vehicle or various doses of 5-AZA (1.25 mg/kg,2.5mg/kg, 5mg/kg, 10mg/kg, dissolved in normal saline, i.p.) was administered0.5 h prior to APAP exposure (n =8 per group). The animals were sacrificed by decapitationat 8 h after APAP exposure, blood and liver samples were harvested for furtherexperiments.

Survivalanalysis

To determine the effect of 5-AZA on themortality of APAP-exposed mice, lethal toxic hepatitis was induced byintraperitoneal injection of APAP (450 mg/kg), vehicle or 5-AZA (5 mg/kg) wasadministered 0.5 h prior to APAP exposure (n= 20 per group). The survival rateof these animals was assessed every 6 h for at least 7 days.

Histologicalanalysis

Formalin-fixed specimens were embedded inparaffin. Sections (4μm) were prepared and stained with hematoxylin & eosinroutinely for morphological evaluation under light microscope.

Determinationof aminotransferases

The ALT and AST activities in plasma wereassessed using the detection kits according to the manufacturer's instructions.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Kornicka K, Marycz K, Maredziak M, Tomaszewski KA, Nicpon J. The effects of the DNA methyltranfserases inhibitor 5-Azacitidine on ageing, oxidative stress and DNA methylation of adipose derived stem cells. J Cell Mol Med. 2017;21(2):387-401.
[2] Yang C, Yi J, Gong X, et al. Anti-oxidative and anti-inflammatory benefits of the ribonucleoside analogue 5-azacitidine in mice with acetaminophen-induced toxic hepatitis. Int Immunopharmacol. 2017;48:91-95.
[3] Broday, L., et al.: Mol. Cell Biol., 19, 3198 (1999)
[4] Qian, X., et al.: Am. J. Pathol., 153, 1475 (1999)

体内溶解度
24 mg/mL

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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