DNMT1; pyrimidine nucleoside analogue of cytidine; Nucleoside Antimetabolite; Autophagy

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].

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].

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].

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].

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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Dissolved in 0.85% NaCl solution; 3 mg/kg; i.p. injection

BDF1 mice bearing lymphoid leukemia L1210

Absorption, Distribution and Excretion
Azacitidine is rapidly absorbed after subcutaneous administration. In adult patients with myelodysplastic syndrome given a single subcutaneous dose of 75 mg/m2 of azacitidine, the Cmax and Tmax were 750 ng/ml and 0.5 hours, respectively. Based on the area under the curve, the bioavailability of subcutaneous azacitidine relative to intravenous azacitidine is approximately 89%. In 21 patients with cancer given subcutaneous azacitidine, the AUC and Cmax were approximately dose-proportional between 25 and 100 mg/m2. Multiple subcutaneous or intravenous doses of azacitidine are not expected to result in drug accumulation.
Azacitidine and its metabolites are mainly excreted through urine. In five cancer patients given radioactive azacitidine intravenously, the cumulative urinary excretion was 85% of the radioactive dose. Fecal excretion accounted for less than 1% of administered radioactivity over three days. Following the subcutaneous administration of 14C-azacitidine, the mean excretion of radioactivity in urine was 50%.
In patients given an intravenous dose of azacitidine, the volume of distribution is 76 L.
Azacitidine has an apparent subcutaneous clearance of 167 L/hour in adults. In pediatric patients, the geometric mean clearance was 21.8 L/hour.

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Metabolism / Metabolites
An in vitro study of azacitidine incubation in human liver fractions indicated that cytochrome P450 (CYP) enzymes do not participate in the metabolism of azacitidine. Azacitidine is metabolized through spontaneous hydrolysis and deamination mediated by cytidine deaminase.
An in vitro study of azacitidine incubation in human liver fractions indicated that azacitidine may be metabolized by the liver. The potential of azacitidine to inhibit cytochrome P450 (CYP) enzymes is not known.
Route of Elimination: Following IV administration of radioactive azacitidine to 5 cancer patients, the cumulative urinary excretion was 85% of the radioactive dose.
Fecal excretion accounted for <1% of administered radioactivity over three days. Mean excretion of radioactivity in urine following SC administration of 14C-azacitidine was 50%.
Half Life: Mean elimination half-life is approximately 4 hours.

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Biological Half-Life
The mean half-life of azacitidine after subcutaneous administration is 41 minutes. The mean elimination half-life of azacitidine and its metabolites was about 4 hours for intravenous and subcutaneous administrations.

Hepatotoxicity
In clinical trials, serum enzyme elevations occurred in up to 16% of patients on azacitidine therapy for cancer or myelodysplasia who had concurrent, underlying liver disease or liver metastases, but rarely in persons without a preexisting hepatic illness. In subsequent studies, liver adverse reactions attributed to azacitidine have rarely been reported, at least when it is given in conventional doses. Nevertheless, monitoring of serum enzyme levels is recommended in treating patients who have concurrent liver disease. Cases of clinically apparent liver injury attributed to azacitidine in patients without underlying liver disease have not been reported in the literature.
Likelihood score: E* (unproven but suspected cause of clinically apparent liver injury, particularly in persons with pre-existing liver disease).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Most sources consider breastfeeding to be contraindicated during maternal antineoplastic drug therapy. It might be possible to breastfeed safely during intermittent azacitidine therapy with an appropriate period of breastfeeding abstinence; the manufacturer recommends an abstinence period of 1 week after the last dose. Chemotherapy may adversely affect the normal microbiome and chemical makeup of breastmilk. Women who receive chemotherapy during pregnancy are more likely to have difficulty nursing their infant.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
A telephone follow-up study was conducted on 74 women who received cancer chemotherapy at one center during the second or third trimester of pregnancy to determine if they were successful at breastfeeding postpartum. Only 34% of the women were able to exclusively breastfeed their infants, and 66% of the women reported experiencing breastfeeding difficulties. This was in comparison to a 91% breastfeeding success rate in 22 other mothers diagnosed during pregnancy, but not treated with chemotherapy. Other statistically significant correlations included: 1. mothers with breastfeeding difficulties had an average of 5.5 cycles of chemotherapy compared with 3.8 cycles among mothers who had no difficulties; and 2. mothers with breastfeeding difficulties received their first cycle of chemotherapy on average 3.4 weeks earlier in pregnancy. Of the 9 women who received a fluorouracil-containing regimen, 8 had breastfeeding difficulties.

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Protein Binding
Not available.

[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.

Azacitidine can cause cancer according to The World Health Organization's International Agency for Research on Cancer (IARC).
5-azacytidine is a white crystalline powder. (NTP, 1992)
5-azacytidine is an N-glycosyl-1,3,5-triazine that is 4-amino-1,3,5-triazin-2(1H)-one substituted by a beta-D-ribofuranosyl residue via an N-glycosidic linkage. An antineoplastic agent, it is used in the treatment of myeloid leukaemia. It has a role as an antineoplastic agent. It is a N-glycosyl-1,3,5-triazine and a nucleoside analogue. It is functionally related to a beta-D-ribose.
Azacitidine is a pyrimidine nucleoside analogue with anti-neoplastic activity. It differs from cytosine by the presence of nitrogen in the C5-position, key in its hypomethylating activity. Two main mechanisms of action have been proposed for azacitidine. One of them is the induction of cytotoxicity. As an analogue of cytidine, it is able to incorporate into RNA and DNA, disrupting RNA metabolism and inhibiting protein and DNA synthesis. The other one is through the inhibition of DNA methyltransferase, impairing DNA methylation. Due to its anti-neoplastic activity and its ability to inhibit methylation in replicating DNA, azacytidine has been used mainly used in the treatment of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), two types of cancer characterized by the presence of aberrant DNA methylation. In May 2004, the FDA approved the use of azacitidine administered subcutaneously for the treatment of MDS of all French-American-British (FAB) subtypes. In January 2007, the FDA approved the intravenous administration of azacitidine. The use of oral azacitidine for the treatment of AML in patients in complete remission was approved by the FDA in September 2020.
Azacitidine is a Nucleoside Metabolic Inhibitor. The mechanism of action of azacitidine is as a Nucleic Acid Synthesis Inhibitor.
Azacitidine is a cytosine analogue and antineoplastic agent used in the therapy of myelodysplastic syndromes. Azacitidine is associated with a low rate of transient serum enzyme elevations during therapy and has not been convincingly implicated in cases of clinically apparent acute liver injury with jaundice.
Azacitidine has been reported in Streptomyces sparsogenes with data available.
Azacitidine is a pyrimidine nucleoside analogue of cytidine with antineoplastic activity. Azacitidine is incorporated into DNA, where it reversibly inhibits DNA methyltransferase, thereby blocking DNA methylation. Hypomethylation of DNA by azacitidine may activate tumor suppressor genes silenced by hypermethylation, resulting in an antitumor effect. This agent is also incorporated into RNA, thereby disrupting normal RNA function and impairing tRNA cytosine-5-methyltransferase activity. (NCI04)
Azacitidine is only found in individuals that have used or taken this drug. It is a pyrimidine nucleoside analogue that inhibits DNA methyltransferase, impairing DNA methylation. It is also an antimetabolite of cytidine, incorporated primarily into RNA. Azacytidine has been used as an antineoplastic agent. Azacitidine (5-azacytidine) is a chemical analogue of the cytosine nucleoside used in DNA and RNA. Azacitidine is thought to induce antineoplastic activity via two mechanisms; inhibition of DNA methyltransferase at low doses, causing hypomethylation of DNA, and direct cytotoxicity in abnormal hematopoietic cells in the bone marrow through its incorporation into DNA and RNA at high doses, resulting in cell death. As azacitidine is a ribonucleoside, it incoporates into RNA to a larger extent than into DNA. The incorporation into RNA leads to the dissembly of polyribosomes, defective methylation and acceptor function of transfer RNA, and inhibition of the production of protein. Its incorporation into DNA leads to a covalent binding with DNA methyltransferases, which prevents DNA synthesis and subsequent cytotoxicity.
A pyrimidine analogue that inhibits DNA methyltransferase, impairing DNA methylation. It is also an antimetabolite of cytidine, incorporated primarily into RNA. Azacytidine has been used as an antineoplastic agent.

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Drug Indication
Azacitidine (for subcutaneous or intravenous use) is indicated for the treatment of adult patients with the following French-American-British (FAB) myelodysplastic syndrome (MDS) subtypes: refractory anemia (RA) or refractory anemia with ringed sideroblasts (RARS) (if accompanied by neutropenia or thrombocytopenia or requiring transfusions), refractory anemia with excess blasts (RAEB), refractory anemia with excess blasts in transformation (RAEB-T), and chronic myelomonocytic leukemia (CMMoL). Azacitidine is also indicated for the treatment of pediatric patients aged 1 month and older with newly diagnosed Juvenile Myelomonocytic Leukemia (JMML). Azacitidine (for oral use) is indicated for continued treatment of adult patients with acute myeloid leukemia (AML) who achieved first complete remission or complete remission with incomplete blood count recovery following intensive induction chemotherapy and are not able to complete intensive curative therapy.
Azacitidine Mylan is indicated for the treatment of adult patients who are not eligible for haematopoietic stem cell transplantation (HSCT) with: intermediate 2 and high risk myelodysplastic syndromes (MDS) according to the International Prognostic Scoring System (IPSS),chronic myelomonocytic leukaemia (CMML) with 10 29% marrow blasts without myeloproliferative disorder,acute myeloid leukaemia (AML) with 20 30% blasts and multi lineage dysplasia, according to World Health Organisation (WHO) classification,AML with > 30% marrow blasts according to the WHO classification.
Vidaza is indicated for the treatment of adult patients who are not eligible for haematopoietic stem cell transplantation (HSCT) with:  intermediate 2 and high-risk myelodysplastic syndromes (MDS) according to the International Prognostic Scoring System (IPSS),chronic myelomonocytic leukaemia (CMML) with 10 29 % marrow blasts without myeloproliferative disorder,acute myeloid leukaemia (AML) with 20 30 % blasts and multi-lineage dysplasia, according to World Health Organisation (WHO) classification. Vidaza is indicated for the treatment of adult patients aged 65 years or older who are not eligible for HSCT with AML with > 30% marrow blasts according to the WHO classification.
Azacitidine Accord is indicated for the treatment of adult patients who are not eligible for haematopoietic stem cell transplantation (HSCT) with: - intermediate-2 and high-risk myelodysplastic syndromes (MDS) according to the International Prognostic Scoring System (IPSS),- chronic myelomonocytic leukaemia (CMML) with 10-29 % marrow blasts without myeloproliferative disorder,- acute myeloid leukaemia (AML) with 20-30 % blasts and multi-lineage dysplasia, according to World Health Organisation (WHO) classification,- AML with > 30% marrow blasts according to the WHO classification.
Azacitidine betapharm is indicated for the treatment of adult patients who are not eligible for haematopoietic stem cell transplantation (HSCT) with: intermediate-2 and high-risk myelodysplastic syndromes (MDS) according to the International Prognostic Scoring System (IPSS),chronic myelomonocytic leukaemia (CMML) with 10 % to 29 % marrow blasts without myeloproliferative disorder,acute myeloid leukaemia (AML) with 20 % to 30 % blasts and multi-lineage dysplasia, according to World Health Organization (WHO) classification,AML with > 30 % marrow blasts according to the WHO classification.
Onureg is indicated as maintenance therapy in adult patients with acute myeloid leukaemia (AML) who achieved complete remission (CR) or complete remission with incomplete blood count recovery (CRi) following induction therapy with or without consolidation treatment and who are not candidates for, including those who choose not to proceed to, hematopoietic stem cell transplantation (HSCT).
Azacitidine Celgene is indicated for the treatment of adult patients who are not eligible for haematopoietic stem cell transplantation (HSCT) with: intermediate 2 and high-risk myelodysplastic syndromes (MDS) according to the International Prognostic Scoring System (IPSS),chronic myelomonocytic leukaemia (CMML) with 10 29 % marrow blasts without myeloproliferative disorder,acute myeloid leukaemia (AML) with 20 30 % blasts and multi-lineage dysplasia, according to World Health Organisation (WHO) classification,AML with > 30% marrow blasts according to the WHO classification.
Treatment of myelodysplastic syndrome (including juvenile myelomonocytic leukaemia), Treatment of acute myeloid leukaemia

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Mechanism of Action
Azacitidine (5-azacytidine) is a chemical analogue of the cytosine nucleoside present in DNA and RNA. It induces antineoplastic activity by inhibiting DNA methyltransferase at low doses and inducing cytotoxicity by incorporating itself into RNA and DNA at high doses. Covalent binding to DNA methyltransferase results in DNA hypomethylation and prevents DNA synthesis. On the other hand, the incorporation of azacitidine into RNA and DNA leads to cytotoxicity as follows: Following cellular uptake, azacitidine is phosphorylated by uridine-cytidine kinase to form 5-azacytidine monophosphate. Afterwards, pyrimidine monophosphate and diphosphate kinases phosphorylate 5-azacytidine monophosphate to form 5-azacytidine diphosphate and triphosphate, respectively. Azacitidine triphosphate is able to incorporate into RNA, disrupting RNA metabolism and protein synthesis. The reduction of azacytidine diphosphate leads to the formation of 5-aza-deoxycytidine diphosphate, which is then phosphorylated to form 5-azadeoxycitidine triphosphate, a compound able to incorporate into DNA and inhibit DNA synthesis. As a ribonucleoside, azacitidine incorporates into RNA to a larger extent than into DNA. Incorporating into RNA leads to the disassembly of polyribosomes, defective methylation and acceptor function of transfer RNA, and the inhibition of protein production, resulting in cell death. During the S-phase of the cell cycle, azacitidine exhibits the highest toxicity; however, the predominant mechanism of cytotoxicity has not been elucidated. The cytotoxic effects of azacitidine cause the death of rapidly dividing cells, including cancer cells that are no longer responsive to normal growth control mechanisms. Non-proliferating cells are relatively insensitive to azacitidine. It is believed that azacitidine exerts its antineoplastic effects through direct cytotoxicity on abnormal hematopoietic cells in the bone marrow.
Telomerase activation is thought to be a critical step in cellular immortality and oncogenesis. Several reagents including differentiation-inducing and antineoplastic agents are known to inhibit telomerase activity, although the molecular mechanisms through which they inhibit telomerase activity remain unclear. Demethylating reagents have recently been used as potential antineoplastic drugs for some types of cancers including those of the prostate. In the present study, we examined the effect of the demethylating reagent 5-azacytidine (5-aza-CR) on telomerase activity using cells of two prostate cancer cell lines, DU-145 and TSU-PR1. 5-aza-CR treatment significantly reduced telomerase activity in TSU-PR1 cells, but not in DU-145 cells, although growth inhibition was observed to a similar extent in both cell lines. Reverse transcription-PCR analyses revealed that inhibition of telomerase activity was accompanied by down-regulation of telomerase catalytic subunit (hTERT) mRNA expression. Transient expression assays showed that 5-aza-CR repressed the transcriptional activity of the hTERT promoter and that the E-box within the core promoter was responsible for this down-regulation. Western blot analyses revealed that 5-aza-CR reactivated p16 expression and repressed c-Myc expression in TSU-PR1 cells but not in DU-145 cells. Overexpression of p16 in TSU-PR1 cells led to significant repression of c-Myc transcription. These findings suggest that 5-aza-CR inhibits telomerase activity via transcriptional repression of hTERT, in which p16 and c-Myc may play a key role.
Cellular differentiation is controlled by a variety of factors including gene methylation, which represses particular genes as cell fate is determined. The incorporation of 5-azacytidine (5azaC) into DNA in vitro prevents methylation and thus can alter cellular differentiation pathways. Human bone marrow fibroblasts and MG63 cells treated with 5azaC were used as models of osteogenic progenitors and of a more mature osteoblast phenotype, respectively. The capacity for differentiation of these cells following treatment with glucocorticoids was investigated. 5azaC treatment led to significant expression of the osteoblastic marker alkaline phosphatase in MG63 osteosarcoma cells, which was further augmented by glucocorticoids; however, in human marrow fibroblasts alkaline phosphatase activity was only observed in glucocorticoid-treated cultures. MG63 cells represent a phenotype late in the osteogenic lineage in which demethylation is sufficient to induce alkaline phosphatase activity. Marrow fibroblasts are at an earlier stage of differentiation and require stimulation with glucocorticoids. In contrast, the expression of osteocalcin, an osteoblastic marker, was unaffected by 5azaC treatment, suggesting that regulation of expression of the osteocalcin gene does not involve methylation. These models provide novel approaches to the study of the control of differentiation in the marrow fibroblastic system.

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.)