DNMT1; DNMT3A; DNMT3B

Decitabine therapy significantly decreased cell proliferation of SNU719, NCC24, and KATOIII after 96 h following exposure to Decitabine. Decitabine causes G2/M arrest and death in EBVaGC, decreases invasion ability, and upregulates E-cadherin expression in EBVaGC [1]. Only high dosages (10 μM) of Decitabine (0.1-1 μM; 24-72 hours) produce G2 arrest, accompanied by a reduction in G1 cells [3]. Decitabine upregulates DCTPP1 and dUTPase expression in HeLa cells [4].

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The present study investigated the effect of a DNA demethylating agent, decitabine, against Epstein-Barr virus-associated gastric cancer (EBVaGC). Decitabine inhibited cell growth and induced G2/M arrest and apoptosis in EBVaGC cell lines. The expression of E-cadherin was up-regulated and cell motility was significantly inhibited in the cells treated with decitabine. The promoter regions of p73 and RUNX3 were demethylated, and their expression was up-regulated by decitabine. They enhanced the transcription of p21, which induced G2/M arrest and apoptosis through down-regulation of c-Myc. Decitabine also induced the expression of BZLF1 in SNU719. Induction of EBV lytic infection was an alternative way to cause apoptosis of the host cells. This study is the first report to reveal the effectiveness of a demethylating agent in inhibiting tumor cell proliferation and up-regulation of E-cadherin in EBVaGC. J. Med. Virol. 89:508-517, 2017. © 2016 Wiley Periodicals, Inc. [1]

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The DNA methyltransferase inhibitors azacytidine and decitabine represent archetypal drugs for epigenetic cancer therapy. To characterize the demethylating activity of azacytidine and decitabine we treated colon cancer and leukemic cells with both drugs and used array-based DNA methylation analysis of more than 14,000 gene promoters. Additionally, drug-induced demethylation was compared to methylation patterns of isogenic colon cancer cells lacking both DNA methyltransferase 1 (DNMT1) and DNMT3B. We show that drug-induced demethylation patterns are highly specific, non-random and reproducible, indicating targeted remethylation of specific loci after replication. Correspondingly, we found that CG dinucleotides within CG islands became preferentially remethylated, indicating a role for DNA sequence context. We also identified a subset of genes that were never demethylated by drug treatment, either in colon cancer or in leukemic cell lines. These demethylation-resistant genes were enriched for Polycomb Repressive Complex 2 components in embryonic stem cells and for transcription factor binding motifs not present in demethylated genes. Our results provide detailed insights into the DNA methylation patterns induced by azacytidine and decitabine and suggest the involvement of complex regulatory mechanisms in drug-induced DNA demethylation. [3]

In female CD-1 mice, decitabine (1.0 mg/kg, po) in combination with tetrahydrouridine (THU) results in severe toxicity and increases susceptibility to decitabine toxicity related to decitabine plasma levels [5]. C57BL/6 mice with established EL4 tumors show regression when given decitabine (1.0 mg/kg; i.p.; once daily for 5 days) [7].

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Lack of immunogenicity of cancer cells has been considered a major reason for their failure in induction of a tumor specific T cell response. In this paper, we present evidence that decitabine (DAC), a DNA methylation inhibitor that is currently used for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and other malignant neoplasms, is capable of eliciting an anti-tumor cytotoxic T lymphocyte (CTL) response in mouse EL4 tumor model. C57BL/6 mice with established EL4 tumors were treated with DAC (1.0 mg/kg body weight) once daily for 5 days. We found that DAC treatment resulted in infiltration of IFN-γ producing T lymphocytes into tumors and caused tumor rejection. Depletion of CD8(+), but not CD4(+) T cells resumed tumor growth. DAC-induced CTL response appeared to be elicited by the induction of CD80 expression on tumor cells. Epigenetic evidence suggests that DAC induces CD80 expression in EL4 cells via demethylation of CpG dinucleotide sites in the promoter of CD80 gene. In addition, we also showed that a transient, low-dose DAC treatment can induce CD80 gene expression in a variety of human cancer cells. This study provides the first evidence that epigenetic modulation can induce the expression of a major T cell co-stimulatory molecule on cancer cells, which can overcome immune tolerance, and induce an efficient anti-tumor CTL response. The results have important implications in designing DAC-based cancer immunotherapy. [7]

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Decitabine (5-aza-2'-deoxycytidine; DAC) in combination with tetrahydrouridine (THU) is a potential oral therapy for sickle cell disease and β-thalassemia. A study was conducted in mice to assess safety of this combination therapy using oral gavage of DAC and THU administered 1 hour prior to DAC on 2 consecutive days/week for up to 9 weeks followed by a 28-day recovery to support its clinical trials up to 9-week duration. Tetrahydrouridine, a competitive inhibitor of cytidine deaminase, was used in the combination to improve oral bioavailability of DAC. Doses were 167 mg/kg THU followed by 0, 0.2, 0.4, or 1.0 mg/kg DAC; THU vehicle followed by 1.0 mg/kg DAC; or vehicle alone. End points evaluated were clinical observations, body weights, food consumption, clinical pathology, gross/histopathology, bone marrow micronuclei, and toxicokinetics. There were no treatment-related effects noticed on body weight, food consumption, serum chemistry, or urinalysis parameters. Dose- and gender-dependent changes in plasma DAC levels were observed with a Cmax within 1 hour. At the 1 mg/kg dose tested, THU increased DAC plasma concentration (∼ 10-fold) as compared to DAC alone. Severe toxicity occurred in females receiving high-dose 1 mg/kg DAC + THU, requiring treatment discontinuation at week 5. Severity and incidence of microscopic findings increased in a dose-dependent fashion; findings included bone marrow hypocellularity (with corresponding hematologic changes and decreases in white blood cells, red blood cells, hemoglobin, hematocrit, reticulocytes, neutrophils, and lymphocytes), thymic/lymphoid depletion, intestinal epithelial apoptosis, and testicular degeneration. Bone marrow micronucleus analysis confirmed bone marrow cytotoxicity, suppression of erythropoiesis, and genotoxicity. Following the recovery period, a complete or trend toward resolution of these effects was observed. In conclusion, the combination therapy resulted in an increased sensitivity to DAC toxicity correlating with DAC plasma levels, and females are more sensitive compared to their male counterparts. [5]

Incorporation assay [8]
Twenty-four hours before treatment, cells were seeded in triplicate in 12-well plates, at a density of 2 × 105 cells per well and then incubated with 100 nM [3H]-decitabine (or other concentrations, if indicated). After incubation for 24 h (or other time periods, if indicated), cells were washed with phosphate buffered saline (PBS). For incorporation measurements, DNA or RNA was purified using DNeasy Blood and Tissue kit or RNeasy kit, respectively, and quantified by a ultraviolet (UV) photometer. The purified samples were mixed with liquid scintillation cocktail and their radioactivity was measured by liquid scintillation counting. Measurements were normalized to the amount of DNA (or RNA). The percentage of substitution of decitabine for cytosine was calculated as the amount of decitabine incorporated as a fraction of total cytosine, as described previously. For competition experiments, cells were either treated with 100 nM [3H]-decitabine in addition with increasing concentrations (100 nM, 500 nM, 1 μM, 2 μM) of [14C]-deoxycytidine or were treated with 100 nM [14C]-deoxycytidine in addition with increasing concentrations (100 nM, 500 nM, 1 μM, 2 μM) of [3H]-decitabine. After 24 h, cells were washed with PBS, DNA was extracted using DNeasy Blood and Tissue kit and was measured using liquid scintillation counting.

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DNA methylation analysis[8]
Genomic DNA was isolated from cells using the DNeasy Blood and Tissue kit. Global DNA methylation levels were determined by capillary electrophoresis, as described previously.

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Decitabine (5-aza-2'-deoxycytidine, aza-dCyd) is an anti-cancer drug used clinically for the treatment of myelodysplastic syndromes and acute myeloid leukaemia that can act as a DNA-demethylating or genotoxic agent in a dose-dependent manner. On the other hand, DCTPP1 (dCTP pyrophosphatase 1) and dUTPase are two 'house-cleaning' nucleotidohydrolases involved in the elimination of non-canonical nucleotides. In the present study, we show that exposure of HeLa cells to decitabine up-regulates the expression of several pyrimidine metabolic enzymes including DCTPP1, dUTPase, dCMP deaminase and thymidylate synthase, thus suggesting their contribution to the cellular response to this anti-cancer nucleoside. We present several lines of evidence supporting that, in addition to the formation of aza-dCTP (5-aza-2'-deoxycytidine-5'-triphosphate), an alternative cytotoxic mechanism for decitabine may involve the formation of aza-dUMP, a potential thymidylate synthase inhibitor. Indeed, dUTPase or DCTPP1 down-regulation enhanced the cytotoxic effect of decitabine producing an accumulation of nucleoside triphosphates containing uracil as well as uracil misincorporation and double-strand breaks in genomic DNA. Moreover, DCTPP1 hydrolyses the triphosphate form of decitabine with similar kinetic efficiency to its natural substrate dCTP and prevents decitabine-induced global DNA demethylation. The data suggest that the nucleotidohydrolases DCTPP1 and dUTPase are factors involved in the mode of action of decitabine with potential value as enzymatic targets to improve decitabine-based chemotherapy. [3]

Cell Cycle Analysis[1]
Cell Types: HCT116 cells
Tested Concentrations: 0.1, 1, 10 µM
Incubation Duration: 24, 48, 72 hrs (hours)
Experimental Results: Only high drug concentrations (10 µM) resulted in a G2 phase arrest, which was accompanied by a reduction of cells in G1 phase.

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Transport assay [8]
Transport assays were conducted as described previously. Twenty-four hours before treatment, cells were seeded in triplicate in 12-well plates, at a density of 2 × 105 cells per well and then incubated with 100 nM [3H]-decitabine. After incubation for the indicated time periods, cells were washed with PBS and lysed with 0.2% sodium dodecyl sulphate (SDS). The isolated samples were mixed with liquid scintillation cocktail and radioactivity was measured by scintillation counting. In parallel, protein concentration was measured using a bicinchoninic acid (BCA) protein assay to normalize the total cellular uptake to total protein concentrations.

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Cell cycle analysis [8]
Approximately 1 × 106 cells were treated with 100 nM decitabine. Cells were collected at time points indicated and fixed with ice-cold absolute ethanol. After fixation, cells were washed with PBS, centrifuged and resuspended in staining solution (0.1% Triton X-100, 0.2 mg/ml RNase A and 20 μg/ml propidium iodide) for 15 min at 37°C in the dark. For the flow cytometric analyses 10 000 cells were measured with a FACS Canto II (BD Biosciences) and data were analyzed using FlowJo software.

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Whole-genome sequencing [8]
Cells were seeded 24 h prior to the experiment. Cells were treated with 100 nM decitabine for 24 h and genomic DNA was extracted using the DNeasy Blood and Tissue kit. DNA was sheared into fragments of ∼300 bp. Adapters were then ligated and fragments were size selected and purified. Cluster generation was performed on the Illumina cBot. The generated clusters from eight samples (four control, four treatment) were sequenced simultaneously on one lane in an Illumina HiSeq 2000 platform using 101 bp paired-end reads. Quality control of the generated sequences was performed using FastQC. Mapping was done by using Bowtie2 and hg19 as reference. For the analysis of point mutations SAMtools was used. The amount of genomic rearrangements was estimated by determining the proportion of discordantly aligning read pairs. All experiments were repeated with independent biological replicates. Mapping efficiencies and coverages are given in Supplementary Table S2. Sequencing data have been deposited in the SRA database under the accession number SRP040672.

Animal/Disease Models: C57BL/6 mice (bearing EL4 cells)[6]
Doses: 1.0 mg/kg
Route of Administration: intraperitoneal (ip)injection; one time/day for 5 days consecutive
Experimental Results: Caused continuous tumor regression even after Decitabine treatment was stopped.

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Experimental Design [5]
Mice were assigned to four dose groups and a vehicle control group as shown in Table 1. Animals were gavaged with Decitabine (5-aza-2'-deoxycytidine; DAC) or its vehicle 1 hour ± 5 minutes after administration of THU or its vehicle at a dose volume of 10 mL/kg. The DAC doses were selected based on the range finding study in which the mice tolerated six oral doses (2x/week) of 0.1, 0.2 and 0.4 mg/kg DAC in combination with a fixed dose of 167 mg/kg THU. A fixed THU dose (500 mg/m2) and the optimal timing between THU and DAC administration (60 min) were selected based on previous studies11. Conversion of milligrams per body surface area dose in mice into milligrams per kilogram body weight dose estimation was based on Michaelis constant (km) values for mice obtained from US Food and Drug Administration published guidelines. In brief, the mouse dose in milligrams per body surface area (500 mg/m2) was divided by the km of 3 to convert the dose to milligrams per kilogram body weight (167 mg/kg). The working body weight range of mice in the guideline is 11-34 gram; the body weight range of mice used in this study was 24-38 gram.

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Toxicokinetics [5]
Sample collection tubes were prepared prior to each collection day by adding 10 μL/tube of a 10 mg/mL THU solution. Blood samples (~0.5 mL) were collected via intra-cardiac puncture from non-fasted, anesthetized toxicokinetic animals on study day 1 (Groups 2 to 5) and 58 (Groups 2 to 5 with the exception of Group 4 females) at 15, 30, 60, 90, 120 and 180 minutes after administration of Decitabine (5-aza-2'-deoxycytidine; DAC) from 3 animals/sex/group at each time point. Due to mortality in the Group 4 females, the first 5 surviving animals were necropsied on study day 38 and blood samples were collected from three females per time point at 15, 30, and 120 minutes following administration of DAC. All samples were collected within 5 minutes of the target time.

Absorption, Distribution and Excretion
Decitabine administered intravenously at 15 mg/m2 for three hours every eight hours over three days resulted in a Cmax of 73.8 ng/mL (66% coefficient of variation, CV), an AUC0-∞ of 163 ng\*h/mL (62% CV), and a cumulative AUC of 1332 ng\*h/mL (95% CI of 1010-1730). Similarly, decitabine at 20 mg/m2 for one hour once daily over five days resulted in a Cmax of 147 ng/mL (49% CV), an AUC0-∞ of 115 ng\*h/mL (43% CV), and a cumulative AUC of 570 ng\*h/mL (95% CI of 470-700).
Less than 1% of administered decitabine is excreted in the urine.
Decitabine as an apparent volume of distribution of 4.59 ± 1.42 L/kg.
Decitabine has a clearance of 125 L/hr/m² (53% CV) when administered intravenously at 15 mg/m² for three hours every eight hours over three days, and a clearance of 210 L/hr/m² (47% CV) at 20 mg/m² for one hour once daily over five days.

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Metabolism / Metabolites
Decitabine is phosphorylated inside cells by the sequential action of deoxycytidine kinase, nucleotide monophosphate kinase, and nucleotide diphosphate kinase, prior to being incorporated into newly synthesized DNA by DNA polymerase. Decitabine not incorporated into cellular DNA undergoes deamination by cytidine deaminase followed by additional degradation prior to excretion.

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Biological Half-Life
Decitabine has a half-life of 0.62 hours (49% CV) when administered intravenously at 15 mg/m² for three hours every eight hours over three days, and a half-life of 0.54 hours (43% CV) at 20 mg/m² for one hour once daily over five days.

Hepatotoxicity
In early clinical trials using high doses of decitabine, serum enzyme elevations occurred in up to 16% of patients with underlying liver disease or liver metastases, but rarely in persons without hepatic illness. In subsequent studies, serum ALT elevations were reported in 5% to 15% of treated patients, but all were self-limited and no clinically apparent liver injury was reported. Recent studies have reported elevations in serum bilirubin levels in 7% to 12% of treated patients, but the elevations resolved rapidly and were not associated with other clinical or laboratory evidence of liver injury. Monitoring of serum enzyme levels during treatment is recommended only in patients with concurrent liver disease.
The oral combination therapy of decitabine with cedazuridine appears to have a similar frequency and pattern of adverse events as iv decitabine alone. In several prospective clinical trials, single cycles of oral vs iv decitabine had similar rates of aminotransferase elevations and long term, multi-course regimens of the oral fixed dose combination therapy resulted in serum aminotransferase elevations in 20% to 37% of patients, which were above 5 times the upper limit of normal (ULN) in 2% to 3% with no cases of clinically apparent liver injury attributable to the chemotherapeutic agent.
Thus, despite widescale use as therapy of MDS, decitabine has not been convincingly linked to cases of clinically apparent liver injury. Nevertheless, the frequency of serum enzyme elevations with therapy make it difficult to say that decitabine is totally without potential for causing drug induced liver injury.
Likelihood score: E* (unproven but suspected, rare cause of clinically apparent liver injury).

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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 decitabine 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
Decitabine exhibits negligible (< 1%) plasma protein binding.

[1]. Decitabine inhibits tumor cell proliferation and up-regulates E-cadherin expression in Epstein-Barr virus-associated gastric cancer. J Med Virol. 2017 Mar;89(3):508-517.
[2]. Enzymology of purine and pyrimidine antimetabolites used in the treatment of cancer. Chem Rev. 2009 Jul;109(7):2880-93.
[3]. Azacytidine and decitabine induce gene-specific and non-random DNA demethylation in human cancer cell lines. PLoS One. 2011 Mar 7;6(3):e17388.
[4]. The nucleotidohydrolases DCTPP1 and dUTPase are involved in the cellular response to decitabine. Biochem J. 2016 Sep 1;473(17):2635-43.
[5]. Subchronic oral toxicity study of decitabine in combination with tetrahydrouridine in CD-1 mice. Int J Toxicol. 2014 Mar-Apr;33(2):75-85.
[6]. DNA methyltransferase expression in triple-negative breast cancer predicts sensitivity to decitabine. J Clin Invest. 2018 Jun 1;128(6):2376-2388.
[7]. Low dose decitabine treatment induces CD80 expression in cancer cells and stimulates tumorspecific cytotoxic T lymphocyte responses. PLoS One. 2013 May 9;8(5):e62924.
[8]. Quantitative determination of decitabine incorporation into DNA and its effect on mutation rates in human cancer cells. Nucleic Acids Res. 2014 Oct 29; 42(19): e152.

Pharmacodynamics
Decitabine is a prodrug analogue of the natural nucleotide 2’-deoxycytidine, which, upon being phosphorylated intracellularly, is incorporated into DNA and exerts numerous effects on gene expression. The use of decitabine is associated with neutropenia and thrombocytopenia. In addition, decitabine can cause fetal harm in pregnant women; effective contraception and avoidance of pregnancy are recommended during treatment with decitabine.

C8H12N4O4

228.21

228.085

C, 42.10; H, 5.30; N, 24.55; O, 28.04

2353-33-5

451668

White to off-white solid

1.9±0.1 g/cm3

485.8±55.0 °C at 760 mmHg

~200 °C (dec.)

247.6±31.5 °C

0.0±2.8 mmHg at 25°C

1.780

-1.93

3

4

2

16

356

3

O1[C@]([H])(C([H])([H])[C@@]([H])([C@@]1([H])C([H])([H])O[H])O[H])N1C([H])=NC(N([H])[H])=NC1=O

XAUDJQYHKZQPEU-KVQBGUIXSA-N

InChI=1S/C8H12N4O4/c9-7-10-3-12(8(15)11-7)6-1-4(14)5(2-13)16-6/h3-6,13-14H,1-2H2,(H2,9,11,15)/t4-,5+,6+/m0/s1

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

5-Aza-2'-deoxycytidine; deoxyazacytidine; 2353-33-5; Dacogen; 2'-Deoxy-5-azacytidine; 5-Azadeoxycytidine; AzadC; 5-aza-CdR; 5-aza-dCyd; Deoxycytidine; NSC127716; NSC 127716; NSC-127716; dezocitidine; Brand name: Dacogen. Abbreviations: 5AZA; DAC

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 requires protection from light (avoid light exposure) during transportation and storage.

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: ≥ 2.5 mg/mL (10.95 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (10.95 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (10.95 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.

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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: 10 mg/mL (43.82 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.)