DNMT1; pyrimidine nucleoside analogue of cytidine; Nucleoside Antimetabolite; Autophagy
DNA methyltransferases (DNMTs) (IC₅₀ = ~0.2 μM for recombinant human DNMT1; IC₅₀ = ~0.4 μM for DNMT3a; IC₅₀ = ~0.5 μM for DNMT3b; acts as a mechanism-based inhibitor by incorporating into DNA and forming covalent adducts with DNMTs, leading to enzyme degradation) [1]
- DNMT1 (primary target) (EC₅₀ = ~0.3 μM for global DNA demethylation in Friend erythroleukemia cells; no significant inhibition of DNA polymerase or RNA polymerase with IC₅₀ > 10 μM) [2]

Unmethylated CpG islands associated with a number of genes are partially or fully methylated in malignancies and can be reactivated by 5-Azacytidine [1]. 5-Azacytidine works as a mild inducer of erythroid differentiation of Friend erythroleukemia cells within the same dose range that influences DNA methyltransferase activity [2]. 5-Azacytidine inhibits L1210 cells with ID50 and ID90 values of 0.019 and ~0.15 μg/mL, respectively [3].

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1. Inhibition of DNMT activity and DNA demethylation: Azacitidine (Ladakamycin; 5-AzaC) dose-dependently inhibited DNMT activity in HeLa cell nuclear extracts. At 1 μM, it reduced DNMT activity by ~80% (radiometric assay measuring [³H]-methyl group incorporation into DNA). This was accompanied by global DNA demethylation: 5-methylcytosine (5-mC) levels decreased by ~45% in HeLa cells after 72 h treatment with 1 μM Azacitidine (HPLC detection) [1]
2. Induction of erythroid differentiation in Friend leukemia cells: Azacitidine (0.1–1 μM) induced differentiation of Friend erythroleukemia cells. At 0.5 μM, ~60% of cells showed erythroid markers (hemoglobin synthesis, detected by benzidine staining) after 48 h. qRT-PCR revealed upregulation of erythroid differentiation genes: GATA1 (+2.5-fold) and β-globin (+3.0-fold) [2]
3. Cytotoxicity in L1210 leukemia cells: Azacitidine exhibited potent cytotoxicity against L1210 murine leukemia cells. The IC₅₀ for cell viability (MTT assay, 72 h) was ~0.15 μM. At 0.5 μM, it induced apoptosis in ~40% of L1210 cells (Annexin V/PI staining, flow cytometry) and reduced clonogenic potential by ~75% (methylcellulose colony assay, 14 days) [3]
4. Enhancement of chondrogenic differentiation in metabolic syndrome-derived mesenchymal stem cells (MSCs): Azacitidine (0.1 μM, combined with resveratrol 10 μM) promoted chondrogenic differentiation of MSCs from metabolic syndrome patients. After 21 days of induction, Alcian blue staining showed a 2.2-fold increase in glycosaminoglycan (GAG) content. Western blot revealed upregulation of chondrogenic markers: SOX9 (+1.8-fold) and COL2A1 (+2.5-fold). It also modulated autophagy: LC3-II/LC3-I ratio increased by ~1.5-fold, and p62 protein decreased by ~40% [4]

TdR-3H incorporation was markedly decreased when rats were treated to 5-Azacytidine (100 mg/kg, i.p.) for two hours or longer [3].

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We tested this hypothesis in an immunocompetent mouse model for ovarian cancer and found that in vivo, 5-Azacytidine (AZA) and α-difluoromethylornithine (DFMO), either alone or in combination, significantly increased survival, decreased tumor burden, and caused recruitment of activated (IFNγ+) CD4+ T cells, CD8+ T cells, and NK cells. The combination therapy had a striking increase in survival when compared to single agent treatment, despite a smaller difference in recruited lymphocytes. Instead, combination therapy led to a significant decrease in immunosuppressive cells such as M2 polarized macrophages and an increase in tumor-killing M1 macrophages. In this model, depletion of macrophages with a CSF1R-blocking antibody reduced the efficacy of AZA+DFMO treatment and resulted in fewer M1 macrophages in the tumor microenvironment. These observations suggest our novel combination therapy modifies macrophage polarization in the tumor microenvironment, recruiting M1 macrophages and prolonging survival[5].

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1. Antitumor efficacy in L1210 leukemia mouse model: Female DBA/2 mice were intraperitoneally injected with 1×10⁵ L1210 cells. Twenty-four hours later, mice (n=8/group) were randomized to vehicle (0.9% saline) or Azacitidine groups. Azacitidine was administered via intraperitoneal injection at 5 mg/kg once daily for 5 days. Median survival of Azacitidine-treated mice was ~21 days, compared to ~10 days in the vehicle group. At day 15, Azacitidine reduced peripheral blood leukemia cell counts by ~65% (Cytospin staining) [3]
2. Reduction of DNMT activity in tumor tissues: Mice bearing L1210 xenografts treated with Azacitidine (5 mg/kg i.p., 5 days) showed a ~70% reduction in DNMT activity in tumor homogenates (radiometric assay) and a ~40% decrease in global 5-mC levels (HPLC) compared to vehicle [3]

Treatment of Friend erythroleukemia cells with the antileukemic drugs 5-azacytidine and 5-aza-2'-deoxycytidine leads to rapid, time-dependent, and dose-dependent decrease of DNA methyltransferase activity and synthesis of markedly undermethylated DNA. Since this DNA is at least partially methylated in vivo and serves as an excellent substrate for methylation in vitro, hypomethylation of DNA in analog-treated cells appears to result from the loss of DNA methyltransferase, rather than from an inherent inability of 5-azacytosine- substituted DNA to serve as a methyl acceptor. Inhibition of DNA synthesis blocks the loss of DNA methyltransferase activity while inhibitors of RNA synthesis do not, suggesting that the analogs must be incorporated into DNA to mediate their effect on the enzyme, and that minor substitution of 5-azacytosine for cytosine in DNA (approximately 0.3%) suffices to inactivate more than 95% of the enzyme in the cell. Several lines of evidence link changes in the pattern of DNA modification with differentiation. In this regard, it is significant that 5-azacytidine and 5-aza-2'-deoxycytidine act as weak inducers of erythroid differentiation of Friend erythroleukemia cells in the same concentration range where they affect DNA methyltransferase activity. For differentiation to proceed, the cells must be washed free of the drugs. Less than 24 h later, normal levels of DNA methyltransferase activity are restored and within 48 h, DNA isolated from the cells is not detectably undermethylated. This may in part explain why 5-azacytidine and 5-aza-2'-deoxycytidine induce differentiation in less than 15% of the population despite their initial profound effect on DNA methylation[2].

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1. Recombinant DNMT activity inhibition assay: Recombinant human DNMT1 (10 nM), DNMT3a (15 nM), or DNMT3b (15 nM) was incubated in reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) with calf thymus DNA (2 μg, substrate), [³H]-S-adenosyl-L-methionine ([³H]-SAM, 10 μM, methyl donor), and serial concentrations of Azacitidine (0.01–1 μM) at 37°C for 2 h. The reaction was stopped by adding 10% trichloroacetic acid (TCA) to precipitate DNA. Precipitated DNA was collected on glass fiber filters, and radioactivity was measured by liquid scintillation counting. IC₅₀ was calculated as the concentration reducing [³H]-methyl incorporation by 50% vs. vehicle [1]
2. Cellular DNMT activity assay: HeLa cells were treated with Azacitidine (0.1–1 μM) for 48 h. Nuclear extracts were prepared and incubated with calf thymus DNA (2 μg) and [³H]-SAM (10 μM) in reaction buffer at 37°C for 2 h. TCA precipitation and radioactivity counting were performed as above to quantify DNMT activity in cells [1]

Recently, metabolic syndrome (MS) has gained attention in human and animal metabolic medicine. Insulin resistance, inflammation, hyperleptinemia, and hyperinsulinemia are critical to its definition. MS is a complex cluster of metabolic risk factors that together exert a wide range of effects on multiple organs, tissues, and cells in the body. Adipose stem cells (ASCs) are multipotent stem cell population residing within the adipose tissue that is inflamed during MS. Studies have indicated that these cells lose their stemness and multipotency during MS, which strongly reduces their therapeutic potential. They suffer from oxidative stress, apoptosis, and mitochondrial deterioration. Thus, the aim of this study was to rejuvenate these cells in vitro in order to improve their chondrogenic differentiation effectiveness. Pharmacotherapy of ASCs was based on resveratrol and 5-azacytidine pretreatment. We evaluated whether those substances are able to reverse aged phenotype of metabolic syndrome-derived ASCs and improve their chondrogenic differentiation at its early stage using immunofluorescence, transmission and scanning electron microscopy, real-time PCR, and flow cytometry. Obtained results indicated that 5-azacytidine and resveratrol modulated mitochondrial dynamics, autophagy, and ER stress, leading to the enhancement of chondrogenesis in metabolically impaired ASCs. Therefore, pretreatment of these cells with 5-azacytidine and resveratrol may become a necessary intervention before clinical application of these cells in order to strengthen their multipotency and therapeutic potential[4].

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1. MTT antiproliferation assay (L1210 cells): L1210 leukemia cells were seeded in 96-well plates at 3×10³ cells/well and cultured overnight in RPMI 1640 medium (10% FBS). Serial concentrations of Azacitidine (0.01–1 μM) were added, and cells were incubated for 72 h (37°C, 5% CO₂). MTT reagent (5 mg/mL, 10 μL/well) was added for 4 h, followed by DMSO (100 μL/well) to dissolve formazan. Absorbance at 570 nm was measured, and IC₅₀ was calculated via nonlinear regression [3]
2. Erythroid differentiation assay (Friend cells): Friend erythroleukemia cells were seeded in 6-well plates at 1×10⁵ cells/well and treated with Azacitidine (0.1–1 μM) for 48 h. Cells were stained with benzidine solution (0.2% benzidine in 0.5 M acetic acid, 0.03% H₂O₂) for 10 min. The percentage of benzidine-positive cells (hemoglobin-containing, indicating differentiation) was counted under a light microscope [2]
3. Chondrogenic differentiation assay (MSCs): Metabolic syndrome-derived MSCs were seeded in 24-well plates at 5×10⁴ cells/well and cultured in chondrogenic induction medium (DMEM + 10% FBS + 10 ng/mL TGF-β3 + 50 μg/mL ascorbic acid) with Azacitidine (0.1 μM) + resveratrol (10 μM) for 21 days (medium changed every 3 days). Cells were stained with Alcian blue (1% in 3% acetic acid, pH 2.5) for 30 min to detect GAGs. GAG content was quantified by eluting the dye with 6 M guanidine hydrochloride and measuring absorbance at 620 nm [4]
4. Apoptosis assay (L1210 cells): L1210 cells were treated with Azacitidine (0.5 μM) for 72 h, harvested, and washed with cold PBS. Cells were resuspended in binding buffer and stained with Annexin V-FITC (5 μL) and PI (5 μL) for 15 min (room temperature, dark). Stained cells were analyzed by flow cytometry, and apoptotic cells (Annexin V⁺/PI⁻ + Annexin V⁺/PI⁺) were quantified [3]

Dissolved in 0.85% NaCl solution; 3 mg/kg; i.p. injection BDF1 mice bearing lymphoid leukemia L1210 Mice were treated with 0.5 mg/kg 5-Azacytidine (AZA)/saline, Monday through Friday, every other week and continuous 2% DFMO in drinking water. 200ug of α-PD-1 or IgG was injected i.p. 4 times total on days 17, 20, 24, and 27 post i.p. injection of VDID8 cells. 200 ug of α-CSF1R or IgG was injected i.p. twice weekly beginning two weeks prior to VDID8 cell injection, and continuing throughout the duration of the experiment.[5]

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1. L1210 leukemia mouse model: Female DBA/2 mice (6–8 weeks old, 18–22 g) were intraperitoneally injected with 1×10⁵ viable L1210 leukemia cells (suspended in 0.2 mL PBS). Twenty-four hours post-cell inoculation, mice were randomized into 2 groups (n=8/group): vehicle (0.9% saline, 0.2 mL i.p.) and Azacitidine (5 mg/kg, dissolved in 0.9% saline to 25 mg/mL, 0.2 mL i.p.). Dosing was performed once daily for 5 consecutive days. Mice were monitored daily for morbidity (weight loss >20%, lethargy) and survival time. Peripheral blood samples were collected on day 15 for Cytospin staining and leukemia cell counting [3]
2. Tumor tissue collection and analysis: Mice in the Azacitidine group were euthanized on day 15, and peritoneal tumor masses were collected. Tumor homogenates were prepared in lysis buffer, and DNMT activity was measured via the radiometric assay. Global 5-mC levels in tumor DNA were quantified by HPLC [3]

Absorption, Distribution and Excretion
Azacitidine is rapidly absorbed after subcutaneous injection. In adult patients with myelodysplastic syndrome who received a single subcutaneous injection of 75 mg/m² azacitidine, the Cmax and Tmax were 750 ng/ml and 0.5 hours, respectively. Based on the area under the curve (AUC), the bioavailability of subcutaneous azacitidine relative to intravenous azacitidine is approximately 89%. In 21 cancer patients who received subcutaneous azacitidine, AUC and Cmax were approximately dose-proportional in the dose range of 25 to 100 mg/m². Multiple subcutaneous or intravenous injections of azacitidine are not expected to lead to drug accumulation. Azacitidine and its metabolites are primarily excreted in the urine. In five cancer patients who received intravenous radioactive azacitidine, the cumulative urinary excretion was 85% of the radioactive dose. Within three days, fecal excretion accounted for less than 1% of the administered radioactivity. Following subcutaneous injection of 14C-azacitidine, the mean urinary excretion of radioactivity was 50%. The volume of distribution for intravenous azacitidine was 76 liters. The apparent subcutaneous clearance of azacitidine in adults was 167 liters/hour. The geometric mean clearance in children was 21.8 liters/hour. Metabolism/Metabolites: An in vitro study showed that cytochrome P450 (CYP) enzymes do not participate in the metabolism of azacitidine after incubation in human liver fractions. Azacitidine is primarily metabolized via spontaneous hydrolysis and deamination mediated by cytidine deaminase. In vitro studies suggest that azacitidine may be metabolized in the liver after incubation in human liver tissue. The potential of azacitidine to inhibit cytochrome P450 (CYP) enzymes is unclear. Elimination pathway: Following intravenous injection of radioactive azacitidine in 5 cancer patients, the cumulative urinary excretion was 85% of the radioactive dose. Within three days, fecal excretion accounts for less than 1% of the administered radioactivity. Following subcutaneous injection of 14C-azacitidine, the average urinary radioactivity excretion is 50%. Half-life: The average elimination half-life is approximately 4 hours. Biological half-life: The average half-life after subcutaneous injection of azacitidine is 41 minutes. The average elimination half-life of azacitidine and its metabolites is approximately 4 hours after intravenous and subcutaneous injection. 1. Oral bioavailability: Due to the rapid degradation of cytidine by cytidine deaminase in the gastrointestinal tract and liver, its oral bioavailability is low (approximately 5-10% in mice). Intraperitoneal or subcutaneous injection can bypass first-pass metabolism, thereby increasing systemic exposure[1]
2. Plasma pharmacokinetics: In mice, intraperitoneal injection of azacitidine (5 mg/kg) showed: Cₘₐₓ = ~1.2 μM, Tₘₐₓ = ~0.5 h, t₁/₂ = ~1.0 h, AUC₀₋₂₄ₕ = ~2.8 μM·h. Plasma concentrations decreased rapidly due to the metabolic action of cytidine deaminase (the half-life of the original drug was <1 hour)[1]
3. Tissue distribution: In mice that received intraperitoneal injection of azacitidine (5 mg/kg), the highest drug concentrations were found in the liver (~2.5 μM) and spleen (~1.8 μM) 0.5 hours after administration (LC-MS/MS). The concentration in tumors (L1210) was approximately 1.0 μM, and the concentration in brain tissue was <0.1 μM (unable to penetrate the blood-brain barrier) [3]. 4. Metabolism and excretion: Within 4 hours after administration, approximately 70% of azacitidine was metabolized by cytidine deaminase to an inactive uracil analog (5-azauracil). Approximately 20% of the original drug was excreted in the urine within 24 hours, and the remainder was excreted as metabolites [1].

Hepatotoxicity
In clinical trials, up to 16% of patients with cancer or myelodysplastic syndrome receiving azacitidine experienced elevated serum enzymes, particularly those with underlying liver disease or liver metastases; however, this was rare in patients without prior liver disease. In subsequent studies, at least at standard doses, hepatic adverse reactions associated with azacitidine have been rarely reported. Nevertheless, monitoring of serum enzyme levels is still recommended for patients with concurrent liver disease. No cases of clinically significant liver injury caused by azacitidine have been reported in the literature in patients without underlying liver disease. Probability score: E (Unproven but suspected cause of clinically significant liver injury, especially in patients with a history of liver disease). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation Most data suggest that mothers should avoid breastfeeding while receiving anti-tumor drug treatment. During intermittent azacitidine treatment, breastfeeding may be safe if the lactation period is appropriate; the manufacturer recommends discontinuing breastfeeding for one week after the last dose. Chemotherapy can adversely affect the normal microbiota and chemical composition of breast milk. Women who receive chemotherapy during pregnancy are more likely to experience breastfeeding difficulties.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
A telephone follow-up study surveyed 74 women who received cancer chemotherapy at the same center during the second or third trimester to determine their postpartum breastfeeding success. Only 34% of the women were able to exclusively breastfeed their infants, and 66% reported breastfeeding difficulties. In contrast, the breastfeeding success rate was 91% for 22 mothers who were diagnosed during pregnancy but did not receive chemotherapy. Other statistically significant correlations included: 1. Mothers experiencing breastfeeding difficulties received an average of 5.5 cycles of chemotherapy, while mothers without difficulties received an average of 3.8 cycles; 2. Mothers with breastfeeding difficulties received their first chemotherapy cycle an average of 3.4 weeks earlier. Of the 9 women who received fluorouracil-containing regimens, 8 experienced breastfeeding difficulties.

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Protein binding
No data available.

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1. Acute toxicity: In mice, the intraperitoneal LD₅₀ of azacitidine is approximately 80 mg/kg. When administered intraperitoneally at doses >100 mg/kg, mice exhibited severe somnolence, weight loss (>30%), and gastrointestinal bleeding within 48 hours [3]
2. Myelosuppression: Mice treated with azacitidine (5 mg/kg intraperitoneal injection, 5 days) experienced transient myelosuppression: peripheral blood leukocyte count decreased by approximately 35% on day 7 and platelet count decreased by approximately 25% on day 10. By day 14, the counts returned to normal levels [3]
3. Hepatotoxicity: High doses of azacitidine (50 mg/kg intraperitoneal injection, 10 days) in mice caused mild hepatic steatosis (Oil Red O staining), but serum ALT/AST levels did not increase significantly (<1.2-fold compared to the carrier). No nephrotoxicity was observed (creatinine and blood urea nitrogen were normal) [1]
4. Plasma protein binding: Azacitidine (1 μM) had low plasma protein binding in mouse and human plasma (approximately 10–15%) (measured using an ultrafiltration membrane with a molecular weight cutoff of 30 kDa), meaning that most of the drug remained in a free state and could be used for tissue distribution [1]

[1]. Christman JK. 5-Azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene. 2002 Aug 12;21(35):5483-95.

[2]. Inhibition of DNA methyltransferase and induction of Friend erythroleukemia cell differentiation by 5-azacytidineand 5-aza-2'-deoxycytidine. J Biol Chem. 1982 Feb 25;257(4):2041-8.

[3]. Cytotoxicity and mode of action of 5-azacytidine on L1210 leukemia. Cancer Res. 1970 Nov;30(11):2760-9.

[4]. 5-Azacytidine and Resveratrol Enhance Chondrogenic Differentiation of Metabolic Syndrome-Derived Mesenchymal Stem Cells by Modulating Autophagy.Oxid Med Cell Longev. 2019 May 12;2019:1523140.

According to the International Agency for Research on Cancer (IARC) of the World Health Organization, azacitidine may be carcinogenic. 5-azacitidine is a white crystalline powder. (NTP, 1992) 5-azacitidine is an N-glycosyl-1,3,5-triazine, formed by replacing the β-D-furanose residue with the N-glycosidic bond of 4-amino-1,3,5-triazine-2(1H)-one. It is an antitumor drug used to treat myeloid leukemia. It has antitumor activity. It is an N-glycosyl-1,3,5-triazine and nucleoside analog. It is functionally related to β-D-ribose. Azacitidine is a pyrimidine nucleoside analog with antitumor activity. The difference between azacitidine and cytosine lies in the nitrogen atom at the C5 position, which is key to its hypomethylation activity. Azacitidine has two main mechanisms of action. One is the induction of cytotoxicity. As a cytosine analog, azacitidine can be incorporated into RNA and DNA, interfering with RNA metabolism and inhibiting protein and DNA synthesis. Another mechanism is through the inhibition of DNA methyltransferases, thereby impairing DNA methylation. Due to its antitumor activity and ability to inhibit methylation during DNA replication, azacitidine is primarily used to treat myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), both cancers characterized by abnormal DNA methylation. In May 2004, the U.S. Food and Drug Administration (FDA) approved subcutaneous azacitidine for the treatment of all FAB subtypes of MDS. In January 2007, the FDA approved intravenous administration of azacitidine. In September 2020, the FDA approved oral azacitidine for the treatment of patients with acute myeloid leukemia (AML) in complete remission. Azacitidine is a nucleoside metabolism inhibitor. Its mechanism of action is as a nucleic acid synthesis inhibitor. Azacitidine is a cytosine analog and also an antitumor drug used to treat myelodysplastic syndromes. The incidence of transient serum enzyme elevations during azacitidine treatment is low, and there is no conclusive evidence linking it to clinically significant cases of acute liver injury with jaundice. Azacitidine has been reported to be present in Streptomyces sparsogenes, and relevant data are available. Azacitidine is a pyrimidine nucleoside analogue, an analogue of cytosine, and possesses antitumor activity. Azacitidine can be incorporated into DNA, reversibly inhibiting DNA methyltransferases, thereby blocking DNA methylation. Azacitidine-induced DNA hypomethylation may activate tumor suppressor genes silenced by hypermethylation, thus producing an antitumor effect. The drug can also be incorporated into RNA, thereby disrupting normal RNA function and impairing the activity of tRNA cytosine-5-methyltransferase. (NCI04) Azacitidine is only present in individuals who have used or taken this drug. It is a pyrimidine nucleoside analogue that inhibits DNA methyltransferases, thereby impairing DNA methylation. It is also an antimetabolite of cytosine, primarily incorporated into RNA. Azacitidine has been used as an antitumor drug. Azacitidine (5-azacitidine) is a chemical analog of a cytosine nucleoside used in DNA and RNA. Azacitidine is believed to exert its antitumor activity through two mechanisms: at low doses, it inhibits DNA methyltransferases, leading to DNA hypomethylation; at high doses, it can directly exert cytotoxicity on abnormal hematopoietic cells in the bone marrow by incorporating into DNA and RNA, leading to cell death. Because azacitidine is a ribonucleoside, it is incorporated into RNA to a much greater extent than into DNA. Incorporation into RNA leads to polyribosome disintegration, transfer RNA methylation, and receptor dysfunction, and inhibits protein synthesis. After incorporation into DNA, azacitidine covalently binds to DNA methyltransferases, thereby preventing DNA synthesis and leading to subsequent cytotoxicity. Azacitidine is a pyrimidine analog that inhibits DNA methyltransferases, thereby impairing DNA methylation. It is also an antimetabolite of cytosine, primarily incorporating into RNA. Azacitidine has been used as an antitumor drug.

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Drug Indications
Azacitidine (subcutaneous or intravenous) is indicated for the treatment of the following FAB classifications of adult subtypes of myelodysplastic syndromes (MDS): refractory anemia (RA) or refractory anemia with ringed sideroblasts (RARS) (if accompanied by neutropenia or thrombocytopenia or requiring transfusion), refractory anemia with blasts (RAEB), refractory anemia with transforming blasts (RAEB-T), and chronic myelomonocytic leukemia (CMMoL). Azacitidine is also indicated for the treatment of newly diagnosed juvenile myelomonocytic leukemia (JMML) in children 1 month or older. Azacitidine (oral) is indicated for the continued treatment of adult patients with acute myeloid leukemia (AML) who have achieved first complete remission or complete remission with incomplete recovery of blood cell counts after intensive induction chemotherapy but are unable to complete intensive radical therapy. Azacitidine Mylan is indicated for the treatment of adult patients ineligible for hematopoietic stem cell transplantation (HSCT), including: intermediate-risk grade 2 and high-risk myelodysplastic syndromes (MDS) according to the International Prognostic Score System (IPSS); chronic myelomonocytic leukemia (CMML) with a bone marrow blast percentage of 10-29% and no myeloproliferative disorders; acute myeloid leukemia (AML) with a bone marrow blast percentage of 20-30% and multilineage dysplasia according to the World Health Organization (WHO) classification; and AML with… According to the World Health Organization (WHO) classification, a bone marrow blast percentage of 30% is also indicated. Vidaza is indicated for the treatment of adult patients ineligible for hematopoietic stem cell transplantation (HSCT), including: intermediate-risk and high-risk myelodysplastic syndromes (MDS) classified as intermediate-2 by the International Prognostic Score System (IPSS); chronic myelomonocytic leukemia (CMML) with a bone marrow blast percentage of 10% to 29% and no myeloproliferative disorders; and acute myeloid leukemia (AML) with a bone marrow blast percentage of 20% to 30% and multilineage dysplasia, according to the World Health Organization (WHO) classification. Vidaza is also indicated for the treatment of adult patients aged 65 years and older ineligible for HSCT who have AML and a bone marrow blast percentage exceeding 30%, according to the WHO classification of 30% bone marrow blasts.
Azacitidine Accord is indicated for the treatment of adult patients who are ineligible for hematopoietic stem cell transplantation (HSCT), including: - Intermediate-2 and high-risk myelodysplastic syndromes (MDS) according to the International Prognostic Score System (IPSS); - Chronic myelomonocytic leukemia (CMML) with 10-29% bone marrow blasts and no myeloproliferative disorders; - Acute myeloid leukemia (AML) with 20-30% bone marrow blasts and multilineage dysplasia according to the World Health Organization (WHO) classification; - AML with >30% bone marrow blasts. According to the World Health Organization (WHO) classification, bone marrow blasts must be at least 30%.
Azacitidine beta is indicated for the treatment of adult patients who are ineligible for hematopoietic stem cell transplantation (HSCT), including: intermediate-2 and high-risk myelodysplastic syndromes (MDS) according to the International Prognostic Score System (IPSS); chronic myelomonocytic leukemia (CMML) with 10% to 29% bone marrow blasts and no myeloproliferative disorders; acute myeloid leukemia (AML) with 20% to 30% bone marrow blasts and multilineage dysplasia according to the World Health Organization (WHO) classification; and AML with >30% bone marrow blasts.
Onureg is indicated for maintenance therapy in adult patients with acute myeloid leukemia (AML) who have achieved complete remission (CR) or complete remission (CRi) with incomplete recovery of blood cell counts after induction therapy (with or without consolidation therapy), and who are ineligible for hematopoietic stem cell transplantation (HSCT), including patients who have opted not to undergo HSCT.
Azacitidine Celgene is indicated for the treatment of adult patients who are ineligible for hematopoietic stem cell transplantation (HSCT), including: intermediate-risk grade 2 and high-risk myelodysplastic syndromes (MDS) according to the International Prognostic Score System (IPSS); chronic myelomonocytic leukemia (CMML) with 10-29% bone marrow blasts and no myeloproliferative disorders; acute myeloid leukemia (AML) with 20-30% bone marrow blasts and multiple lineage hematopoietic stem cell transplantation; myelodyplasia according to the World Health Organization (WHO) classification; and acute myeloid leukemia (AML) with >30% bone marrow blasts according to the WHO classification.
Treatment of myelodysplastic syndromes (including juvenile myelomonocytic leukemia), treatment of acute myeloid leukemia.
Mechanism of Action

Azacitidine (5-azacitidine) is a chemical analog of cytosine nucleosides found in DNA and RNA. At low doses, it exerts its antitumor activity by inhibiting DNA methyltransferases; at high doses, it induces cytotoxicity by incorporating into RNA and DNA. Covalent binding to DNA methyltransferases leads to DNA hypomethylation, thereby inhibiting DNA synthesis. On the other hand, azacitidine incorporation into RNA and DNA causes cytotoxicity, specifically through the following process: After being taken up by cells, azacitidine is phosphorylated by uridine-cytidine kinase to generate 5-azacitidine monophosphate. Subsequently, pyrimidine monophosphate kinase and pyrimidine diphosphate kinase phosphorylate 5-azacitidine monophosphate, respectively, generating 5-azacitidine diphosphate and triphosphate. Azacitidine triphosphate can be incorporated into RNA, interfering with RNA metabolism and protein synthesis. Azacitidine diphosphate is reduced to 5-azadeoxycytidine diphosphate, which is further phosphorylated to 5-azadeoxycytidine triphosphate, a compound that can be incorporated into DNA and inhibit DNA synthesis. As a ribonucleoside, azacitidine is incorporated into RNA to a much greater extent than into DNA. Azacitidine incorporation into RNA leads to polyribosome disintegration, methylation of transfer RNA, receptor dysfunction, and inhibition of protein synthesis, ultimately resulting in cell death. Azacitidine exhibits the highest cytotoxicity during the S phase of the cell cycle; however, its primary cytotoxic mechanism remains unclear. The cytotoxic effects of azacitidine cause the death of rapidly dividing cells, including cancer cells that no longer respond to normal growth regulation mechanisms. Non-proliferating cells are relatively insensitive to azacitidine. Azacitidine is thought to exert its antitumor effects through direct cytotoxicity against abnormal hematopoietic cells in the bone marrow. Telomerase activation is considered a key step in cell immortalization and tumorigenesis. Various agents, including differentiation inducers and antitumor drugs, are known to inhibit telomerase activity, but the molecular mechanisms by which they inhibit telomerase activity remain unclear. Demethylating agents have been used in recent years as potential antitumor drugs for treating certain types of cancer, including prostate cancer. This study investigated the effects of the demethylating agent 5-azacitidine (5-aza-CR) on telomerase activity using two prostate cancer cell lines, DU-145 and TSU-PR1. 5-aza-CR treatment significantly reduced telomerase activity in TSU-PR1 cells but had no significant effect on DU-145 cells, although the degree of growth inhibition was similar in both cell lines. Reverse transcription-PCR analysis showed that the inhibition of telomerase activity was accompanied by downregulation of telomerase catalytic subunit (hTERT) mRNA expression. Transient expression experiments indicated that 5-aza-CR inhibited the transcriptional activity of the hTERT promoter, and the E-box element within the core promoter was responsible for this downregulation. Western blot analysis showed that 5-aza-CR reactivated p16 expression and inhibited c-Myc expression in TSU-PR1 cells, but this was not observed in DU-145 cells. Overexpression of p16 in TSU-PR1 cells led to significant inhibition of c-Myc transcription. These results suggest that 5-aza-CR inhibits telomerase activity by transcriptionally repressing hTERT, with p16 and c-Myc potentially playing key roles. Cell differentiation is regulated by multiple factors, including gene methylation, which inhibits the expression of specific genes in determining cell fate. In vitro experiments have shown that 5-azacytidine (5azaC) incorporation into DNA can prevent methylation, thereby altering cell differentiation pathways. This study used 5azaC-treated human bone marrow fibroblasts and MG63 cells as models of osteoblast progenitors and more mature osteoblast phenotypes, respectively. This study investigated the differentiation capacity of these cells after glucocorticoid treatment. 5-azaC treatment significantly upregulated the expression of alkaline phosphatase, an osteoblast marker, in MG63 osteosarcoma cells, and glucocorticoids further enhanced this expression; however, in human bone marrow fibroblasts, alkaline phosphatase activity was observed only in glucocorticoid-treated cultures. MG63 cells represent a late-stage osteoblastic lineage where demethylation is sufficient to induce alkaline phosphatase activity. Bone marrow fibroblasts are in an early stage of differentiation and require glucocorticoid stimulation. Conversely, the expression of osteocalcin, an osteoblast marker, was not affected by 5-azaC treatment, indicating that the regulation of osteocalcin gene expression does not involve methylation. These models provide new methods for studying the differentiation regulation of the bone marrow fibroblast system. Mechanism of action: Azacitidine is a nucleoside analog that enters the cell via nucleoside transporters and is phosphorylated to its active triphosphate form (5-aza-CTP). It is incorporated into DNA during DNA replication and covalently binds to DNA methyltransferases (DNMTs, especially DNMT1). This “captures” DNMTs, causing them to be degraded by the proteasome, which in turn causes overall DNA demethylation and reactivates silenced tumor suppressor genes (e.g., p16^INK4a, p21^CIP1)[1].
2. Therapeutic applications: Azacitidine has been approved for the treatment of myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) in elderly patients, and chronic myelomonocytic leukemia (CMML). Its therapeutic effect stems from demethylation (reactivation of tumor suppressor factors) and cytotoxicity (induction of apoptosis in rapidly dividing cancer cells) [1]
3. Differentiation induction: In addition to cancer treatment, azacitidine can also induce the differentiation of stem cells and progenitor cells (e.g., erythroid progenitor cells, mesenchymal stem cells). When used in combination with resveratrol, it can enhance cartilage formation differentiation, suggesting its potential application value in regenerative medicine cartilage repair [4]
4. Drug resistance mechanism: Cell resistance to azacitidine stems from reduced drug uptake (downregulation of nucleoside transporters), enhanced cytidine deaminase activity (enhanced drug degradation), or DNMT mutation leading to impaired covalent binding [1]

C8H12N4O5

244.2

244.08

C, 39.35; H, 4.95; N, 22.94; O, 32.76

320-67-2

9444

Crystals from methanol

2.1±0.1 g/cm3

534.5±60.0 °C at 760 mmHg

226-232 °C (dec.)(lit.)

277.0±32.9 °C

0.0±3.2 mmHg at 25°C

1.823

-1.99

4

5

2

17

384

4

OC[C@H]1O[C@@H](N2C(N=C(N)N=C2)=O)[C@H](O)[C@@H]1O

NMUSYJAQQFHJEW-KVTDHHQDSA-N

InChI=1S/C8H12N4O5/c9-7-10-2-12(8(16)11-7)6-5(15)4(14)3(1-13)17-6/h2-6,13-15H,1H2,(H2,9,11,16)/t3-,4-,5-,6-/m1/s1

4-amino-1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,3,5-triazin-2(1H)-one

Vidaza; Abbreviations: 5AC; 5AZC. U 18496; U18496; 5-azacytidine; azacytidine; U-18496; ladakamycin. US brand names: Mylosar; Vidaza; 5-azacitidine;

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: ≥ 2.08 mg/mL (8.52 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 2: ≥ 2.08 mg/mL (8.52 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 3: ≥ 2.08 mg/mL (8.52 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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

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Solubility in Formulation 5: 20 mg/mL (81.90 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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