DNMT/DNA methyltransferases
Guadecitabine sodium targets DNA methyltransferases (DNMTs), the family of enzymes responsible for catalyzing the addition of methyl groups to DNA. By inhibiting DNMT activity, the compound induces DNA hypomethylation, leading to the reactivation of silenced tumor suppressor genes and other epigenetically regulated genes. This mechanism is particularly relevant in hematological malignancies such as AML and MDS, where aberrant DNA methylation plays a pathogenic role.

After treating HCT116 colorectal cancer cells for six days, guadecitabine sodium (SGI-110 sodium) was shown to cause a dose-dependent increase in p16 expression. Furthermore, p16 protein levels increased in T24 and HCT116 cells treated with guadecitabine sodium or 5-aza-CdR for three days in a dose-dependent manner, suggesting that guadecitabine sodium suppresses DNA methylation and causes modifications to mRNA and mRNA. capacity to trigger p16. amounts of protein and 5-aza-CdR. Consequently, guadecitabine sodium has a similar effect on p16 expression to 5-aza-CdR in terms of inhibiting DNA methylation in the 5' region and inducing p16 gene expression in T24 and HCT116 cells, with the former having a stronger effect. In both cell lines, induction was linked to demethylation of the gene's 5' region. At test levels up to 1 μM, guadecitabine sodium was marginally less hazardous than 5-aza-CdR, but at 10 μM, it demonstrated equal toxicity [1].

---

In vitro, Guadecitabine sodium causes a dose-dependent increase in p16 expression in HCT116 colorectal cancer cells following six days of treatment. This demonstrates its capacity to reactivate epigenetically silenced genes through DNMT inhibition. The compound effectively reduces DNA methylation in various cancer cell lines, with potency comparable to that of 5-azacitidine (5-Aza-CdR).

The toxicity of guadecitabine sodium (SGI-110 sodium) was comparable to that of 5-Aza-CdR, but it was also effective in lowering DNA methylation and slowing tumor growth at a dose of 10 mg/kg. In vivo p16 gene expression is efficiently restored by guadecitabine sodium. The p16 gene is substantially methylated in parental EJ6 cells. In vivo, guadecitabine sodium has the ability to significantly lower the p16 promoter region's DNA methylation level. Because guadecitabine sodium is more well-tolerated than 5-Aza-CdR in vivo, it may be a more appealing option for future clinical uses [2].

---

Methylation of CpG islands in promoter regions is often associated with gene silencing and aberrant DNA methylation occurs in most cancers, leading to the silencing of some tumor suppressor genes. Reversal of this abnormal hypermethylation by DNA methylation inhibitors is effective in reactivating methylation-silenced tumor suppressor genes both in vitro and in vivo. Several DNA methylation inhibitors have been well studied; the most potent among them is 5-aza-2'-deoxycytidine (5-Aza-CdR), which can induce myelosuppression in patients. guadecitabine (S110) is a dinucleotide consisting of 5-Aza-CdR followed by a deoxyguanosine, which we previously showed to be effective in vitro as a DNA methylation inhibitor while being less prone to deamination by cytidine deaminase, making it a promising alternative to 5-Aza-CdR. Here, we show that guadecitabine (S110) is better tolerated than 5-Aza-CdR in mice and is as effective in vivo in inducing p16 expression, reducing DNA methylation at the p16 promoter region, and retarding tumor growth in human xenograft. We also show that guadecitabine (S110) is effective by both i.p. and s.c. deliveries. guadecitabine (S110) therefore is a promising new agent that acts similarly to 5-Aza-CdR and has better stability and less toxicity[2].

---

In vivo, Guadecitabine sodium at 10 mg/kg is an effective dose for reducing DNA methylation and retarding tumor growth in preclinical mouse models. In a xenograft study, subcutaneous administration of Guadecitabine (1 mg/kg; single daily for 13 consecutive days) significantly decreased mean tumor volume in mice. The compound caused roughly the same level of toxicity as 5-Aza-CdR while maintaining efficacy.

Quantitative DNA Methylation Analysis by Methylation-Specific Single Nucleotide Extension (Ms-SNuPE)[2]
Two µg of each genomic DNA sample was converted with sodium bisulfite as previously described, and each region of interest was amplified by PCR. The PCR conditions for p16 were as follows: 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 1 min, annealing at 62°C for 1 min, and extension at 72°C for 1 min, and a final extension at 72°C for 10min. The bisulfite specific-PCR primer sequences are as follows: p16 sense, 5’- GTA GGT GGG GAG GAG TTT AGT T-3’, p16 antisense, 5’- TCT AAT AAC CAA CCA ACC CCT CCT-3’. The Ms-SNuPE conditions for p16 were as follows: 95°C for 2 min, 50°C for 2 min, and 72°C for 1 min. The p16 SNuPE primers are as follows: 5’-TTT TAG GGG TGT TAT ATT-3’, 5’-TTT TTT TGT TTG GAA AGA TAT-3’, and 5’-TTT GAG GGA TAG GGT-3’. The PCR amplicons were extracted with the Qiagen Gel Extraction Kit, and Ms-SNuPE analysis was performed to examine the methylation level changes as previously described.

---

In vitro enzyme inhibition assays for Guadecitabine sodium typically measure DNMT activity in cell-free systems using DNA substrate and radiolabeled S-adenosylmethionine (SAM) as the methyl donor. The compound's active metabolite, decitabine, is incorporated into DNA and forms covalent adducts with DNMT enzymes, leading to their irreversible inhibition. Activity is quantified by measuring the reduction in DNA methylation levels using methods such as liquid chromatography-mass spectrometry (LC-MS) or ELISA-based methylation assays.

In vitro treatment for tumor cells with guadecitabine (S110)[1]
Cells (3–4 × 105) were seeded in a T75 tissue culture flask and treated 24 h later with guadecitabine (S110), 5-AZA-CdR, or 3′-3′-DpG, by replacing the medium with fresh one containing 1 μM or 10 μM of guadecitabine (S110), 1 μM of 5-AZA-CdR, or 3′-3′-DpG, every 12 h for 2 days (4 pulses) and then with fresh medium without drugs for additional 2 days. Control cultures were treated under similar experimental conditions in the absence of drug.[1]

---

Cytotoxicity assay[3]
Cytolytic activity of HLA-A2-restricted gp100-specific CTL was tested against Mel 275 melanoma cells untreated or treated with 1 μM guadecitabine (S110), using the Colorimetric Cytotoxicity Assay Kit that quantitatively measures LDH release. Cells were used at effector/target (E/T) ratios of 25/1, 12/1, 6/1, and 3/1. The percentage of specific lysis was determined following the manufacturer’s instruction.[3]
The blocking effect of HLA class I antigens and ICAM-1 was studied on guadecitabine (S110)-treated Mel 275 melanoma cells pre-incubated with 20 μg/ml of the anti-HLA class I mAb W6/32 or the anti-ICAM-1 mAb 84H10, for 30 min at 37 °C. Then, cells were washed and used as targets for HLA-A2-restricted gp100-specific CTL in LDH release assay at E/T ratio of 25/1.

---

In vitro cellular assays for Guadecitabine sodium involve treating cancer cell lines such as HCT116, RH30, or RH41 with the compound at various concentrations for 5–6 days. Following treatment, DNA methylation levels are assessed by bisulfite sequencing, methylation-specific PCR, or global methylation ELISA. Gene reactivation is evaluated by measuring expression of methylation-silenced genes such as p16 via qRT-PCR or Western blotting. Cell viability and proliferation are also assessed to determine anti-cancer activity.

In Vivo Drug Tolerability Study[2]
Non-tumor-bearing athymic nu/nu mice were divided into six treatment groups with six animals per group. Treatments of S110 and 5-Aza-CdR were prepared in PBS and administered intravenously (IV) through tail vein injections. Doses and dosing schedules were designed so that after seven days each group received molar equivalents of either S110 or 5-Aza-CdR. Animals were treated on the following schedules for three weeks: Group 1 received 36.6 mg/kg S110 once weekly (Mon.) and Group 2 was administered 15 mg/kg 5-Aza-CdR once weekly. Group 3 was dosed with 18.3 mg/kg S110 twice weekly (Tues. and Thurs.) and group 4 received 7.5 mg/kg 5-Aza-CdR twice weekly. Finally, groups 5 and 6 received 12.2 and 5.0 mg/kg of S110 and 5-Aza-CdR, respectively administered three times weekly (Mon., Wed., and Fri.). Tolerability was grossly evaluated by body weight measurements and morbidity. Body weight measurements were recorded twice weekly.[2]

---

In vivo xenograft drug efficacy studies with intraperitoneal delivery[2]
The EJ6 human bladder cancer cell was used for this study, and experiments were done similarly to previously described. EJ6 cells (5 × 105/injection) suspended in PBS were inoculated subcutaneously (SQ) into the right and left back (along the midaxillary lines) of 4- to 6-week-old female BALB/c athymic nude-Foxn1nu mice. Mice were randomly divided into 3 groups. After 2–3 weeks and after macroscopic tumors (50–200 mm3) had formed, treatments were initiated. Tumors were measured with calipers, and tumor volumes (TVs) were calculated with the following formula: TV = LD2/2 (where L is the longest diameter and D is the shortest diameter). The fold differences in tumor growth among the various mice groups were calculated using relative TVs (RTVs), which are calculated as follows: RTV = TVn/TV0, where TVn is the tumor volume in mm3 at a given day n and TV0 is the tumor volume in mm3 at day 0 (initial treatment). Mice were weighed at the beginning and end of treatment to determine toxicity. The percent weight change for each mouse was calculated with the following formula: [(W6−W0)/W0] × 100% (where Wn is the mouse weight on day n).

5-Aza-CdR was used as the positive control and 0.45% PBS was used as the negative control. PBS, 5-Aza-CdR (dose of 5 mg/kg in PBS), and S110 (dose of 10 mg/kg in PBS) were administered daily by intraperitoneal (IP) injection over a period of 6 days.

All mice were sacrificed 24 hours after the last treatment. At this time, tumors were removed and each tumor was divided into two separate portions. One portion was immediately homogenized in TRIzol reagent for RNA extraction, and the other portion was immediately frozen in liquid nitrogen for DNA extraction later. Genomic DNA and RNA would be used for analysis of the methylation status of p16 promoter by Ms-SNuPE and of gene expression by real time RT–PCR, respectively. [2]

---

In vivo xenograft drug efficacy studies with subcutaneous delivery[2]
Athymic nu/nu mice were inoculated subcutaneously in the right hind flank with 107 EJ6 bladder cancer cells. After tumors reached 0.5 cm in diameter, animals were stratified into three groups with eight animals per group to begin treatments. Doses and dosing schedules were designed so that each group received molar equivalents of either S110 or 5-Aza-CdR. The agents were administered SQ once weekly at a dose of 12.2 mg/kg for S110 and 5.0 mg/kg for 5-Aza-CdR for three weeks. The study included an appropriate PBS control group. Tumor sizes by caliper and body weight measurements were taken twice weekly to monitor tumor growth inhibition and tolerability.

---

In vivo animal experiments for Guadecitabine sodium are conducted in mouse xenograft models bearing human tumor cells. A typical protocol involves subcutaneous administration of the compound at doses of 1–10 mg/kg, administered daily for 13 consecutive days. Tumor volume is measured periodically to assess anti-tumor efficacy, while DNA methylation levels in tumor tissues are analyzed to confirm target engagement. Body weight and general health are monitored to evaluate toxicity.

Guadecitabine sodium is designed as a prodrug with improved pharmacokinetic properties compared to decitabine. Its dinucleotide structure protects the active metabolite from rapid degradation by cytidine deaminase, resulting in a longer in vivo half-life and extended exposure. Following subcutaneous administration, the compound is slowly released and metabolized to decitabine, which is then incorporated into DNA to exert its DNMT-inhibitory effects. This prolonged exposure profile enables more sustained DNA hypomethylation and enhanced anti-tumor activity.

The toxicity of Guadecitabine sodium is comparable to that of 5-azacitidine (5-Aza-CdR). In preclinical studies, the compound demonstrated myelosuppressive effects consistent with its mechanism as a DNMT inhibitor. At the effective dose of 10 mg/kg, it reduced DNA methylation and slowed tumor growth while maintaining a manageable toxicity profile. Formal toxicology studies have been conducted to support its clinical development for AML and MDS.

[1]. Delivery of 5-aza-2'-deoxycytidine to cells using oligodeoxynucleotides. Cancer Res. 2007 Jul 1;67(13):6400-8.

[2]. S-110, a 5-Aza-2'-deoxycytidine-containing dinucleotide, is an effective DNA methylation inhibitor in vivo and can reduce tumor growth. Mol Cancer Ther. 2010 May;9(5):1443-50.

Guadecitabine sodium is the sodium salt of Guadecitabine, a dinucleotide antimetabolite composed of decitabine linked to deoxyguanosine via a phosphodiester bond, possessing potential antitumor activity. Following phosphodiester bond cleavage and metabolic activation, decitabine is partially incorporated into DNA, inhibiting DNA methyltransferases, leading to nonspecific genome-wide hypomethylation and inducing cell cycle arrest in the S phase. This drug is resistant to cytidine deaminase, which may result in the gradual release of decitabine both inside and outside the cell, thus prolonging decitabine exposure time.

---

Guadecitabine sodium is a second-generation hypomethylating agent that has been investigated in clinical trials for the treatment of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). It is the dinucleotide prodrug of decitabine, designed to overcome the rapid degradation and short half-life of intravenous decitabine. The compound is supplied as a white powder with purity >98% and should be stored at -20°C. It is classified as a research compound and is not approved for clinical use.

C₁₈H₂₃N₉NAO₁₀P

579.39

579.12

C, 37.31; H, 4.00; N, 21.76; Na, 3.97; O, 27.61; P, 5.35

929904-85-8

Guadecitabine;929901-49-5

135564654

White to off-white solid powder

5

12

8

39

1110

6

C1[C@@H]([C@H](O[C@H]1N2C=NC3=C2N=C(NC3=O)N)COP(=O)([O-])O[C@H]4C[C@@H](O[C@@H]4CO)N5C=NC(=NC5=O)N)O.[Na+]

XLHBNJPXFOZFNJ-BYKQGDNKSA-M

InChI=1S/C18H24N9O10P.Na/c19-16-22-6-27(18(31)25-16)12-2-8(9(3-28)35-12)37-38(32,33)34-4-10-7(29)1-11(36-10)26-5-21-13-14(26)23-17(20)24-15(13)30;/h5-12,28-29H,1-4H2,(H,32,33)(H2,19,25,31)(H3,20,23,24,30);/q;+1/p-1/t7-,8-,9+,10+,11+,12+;/m0./s1

sodium;[(2R,3S,5R)-5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methyl [(2R,3S,5R)-5-(4-amino-2-oxo-1,3,5-triazin-1-yl)-2-(hydroxymethyl)oxolan-3-yl] phosphate

SGI-110 sodium; S-110 sodium; SGI110 sodium; Guadecitabine sodium [USAN]; S110 sodium salt; sodium;[(2R,3S,5R)-5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methyl [(2R,3S,5R)-5-(4-amino-2-oxo-1,3,5-triazin-1-yl)-2-(hydroxymethyl)oxolan-3-yl] phosphate; S-110; SGI-110 sodium salt; 0RB89YH367; S110 sodium; SGI 110 sodium; S 110 sodium

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~86.30 mM)
H2O : ~50 mg/mL (~86.30 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.31 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 25.0 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (4.31 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 25.0 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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (4.31 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 33.33 mg/mL (57.53 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

---

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