DNMT/DNA methyltransferases

In CTA-negative cancer cells, exposure to guadecitabine increased the expression of the examined cancer/testis antigen (CTA). The findings demonstrate that in tumor cells across all tissue types examined, guadecitabine causes and/or significantly upregulates constitutive levels of MAGE-A3 and NY-ESO-1-specific mRNA expression. On Mel 275 melanoma cells, exposure to guadecitabine increased the constitutive expression levels of HLA class I antigens, HLA-A2 allospecific, and the costimulatory molecule ICAM-1 considerably (p<0.05). The findings demonstrated that constitutive methylation of the CTA promoter in the cancer cells under investigation was significantly reduced (p<0.01) as a consequence of guadecitabine treatment. In Mel 195 and MZ-1257 RCC cells, the average percentage of guadecitabine-induced demethylation in the MAGE-A1 and NY-ESO-1 promoters was 57% and 30%, respectively[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].

---

Tumor growth can be effectively delayed by guadecitabine (S110). Tumors treated with PBS only showed notable growth, while those treated with guadecitabine (S110) did not diminish in size and only expanded extremely slowly. Furthermore, as demonstrated by changes in mouse body weight, guadecitabine given subcutaneously (SQ) generated much less toxicity than when injected intraperitoneally [3].

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 treatment for tumor cells with guadecitabine (S110)[3]
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.[3]

---

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 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.

Guadecitabine (S110) is better tolerated than 5-Aza-CdR in vivo in tumor-free nude mice[2]
While 5-Aza-CdR is an effective DNA methylation inhibitor both in vitro and in vivo, it suffers from the drawback of having toxicity such as myelosuppression in patients. Guadecitabine (S110) is a dinucleotide consisting of 5-Aza-CdR followed by a deoxyguanosine (Figure 1), and has been previously shown by Yoo et al. to be effective in vitro as a DNA methylation inhibitor and comparable to 5-Aza-CdR in decreasing DNA methylation, reactivating p16 expression, depleting DNA methyltransferases and reducing cancer cell growth in vitro. Moreover, it has the advantage of being less prone to deamination by cytidine deaminase, making it a promising alternative to 5-Aza-CdR.

To compare the in vivo tolerability of guadecitabine (S110) to that of 5-Aza-CdR, six groups of six tumor-free female nude mice each were randomly assigned to varying schedules according to Table 1. The groups were paired up so that S-110 was administered at molar equivalents of 5-Aza-CdR at the same frequencies in groups 1 and 2, groups 3 and 4, and groups 5 and 6. We first compared the tolerability by examining the relative body weights throughout the study (Figure 2A). Animals in groups 1 and 2 maintained or steadily gained body weight throughout the course of the study and were euthanized after 36 days. In groups 3 and 4, animals receiving guadecitabine (S110) also maintained or steadily gained weight, whereas the average body weight of those receiving 5-Aza-CdR began to decline after the first week of dosing. While the average body weight of both groups 5 and 6 rapidly decreased after the first week of dosing, eventually leading to death, those receiving S-110 maintained a healthy body weight slightly longer than animals receiving 5-Aza-CdR. It is of interest that despite the same total weekly dosage of 5-Aza-CdR or S110, both drugs cause more toxicity when given in smaller doses and higher frequency. In all three paired comparisons, S110 was better tolerated than 5-Aza-CdR in terms of weight change.

[1]. Epigenetic drug discovery: targeting DNA methyltransferases. J Biomol Screen. 2012 Jan;17(1):2-17.

[2]. S110, 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.

[3]. Immunomodulatory activity of SGI-110, a 5-aza-2'-deoxycytidine-containing demethylating dinucleotide. Cancer Immunol Immunother. 2013 Mar;62(3):605-14.

Guadecitabine is under investigation for the treatment of Previously Treated Metastatic Colorectal Cancer.
Guadecitabine is a dinucleotide antimetabolite composed of a decitabine linked via phosphodiester bond to a deoxyguanosine, with potential antineoplastic activity. Following metabolic activation via cleavage of the phosphodiester bond and incorporation of the decitabine moiety into DNA, guadecitabine inhibits DNA methyltransferase, thereby causing non-specific, genome-wide hypomethylation, and induction of cell cycle arrest at S-phase. This agent is resistant to cytidine deaminase, which may result in gradual release of decitabine both extra- and intra-cellularly, leading to prolonged exposure to decitabine.

---

Drug Indication
Treatment of acute myeloid leukaemia

C18H24N9O10P

557.41122

557.138

C, 38.79; H, 4.34; N, 22.62; O, 28.70; P, 5.56

929901-49-5

Guadecitabine sodium;929904-85-8

135564655

White to off-white solid powder

2.2±0.1 g/cm3

956.4±75.0 °C at 760 mmHg

532.2±37.1 °C

0.0±0.3 mmHg at 25°C

1.922

-4.38

6

12

8

38

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

GUWXKKAWLCENJA-ZNSALQAWSA-N

InChI=1S/C18H24N9O10P/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)/t7-,8+,9-,10+,11-,12-/m1/s1

(2R,3S,5R)-5-(4-amino-2-oxo-1,3,5-triazin-1(2H)-yl)-2-(hydroxymethyl)tetrahydrofuran-3-yl (((2S,3R,5R)-5-(2-amino-6-oxo-1H-purin-9(6H)-yl)-3-hydroxytetrahydrofuran-2-yl)methyl) hydrogen phosphate

SGI 110; S110; SGI-110; S-110; SGI-110 free acid; 2KT4YN1DP7; SGI 110; [(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] hydrogen phosphate; Guanosine, 2'-deoxy-5-azacytidylyl-(3'-5')-2'-deoxy-; SGI110

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product is not stable in solution, please use freshly prepared working solution for optimal results.

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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