Nrf2

Dimethyl fumarate (DMF; 20–200 μM; 24 hours) significantly lowers SGC-7901, HT29, HCT116, and CT26 viability [1]. In CT26 cells, dimethyl fumarate (DMF; 100 μM; 3–24 hours) dramatically activates p38, ERK, and JNK[1]. By decreasing inflammatory transduction pathways involving GSH depletion, raising ROS, and stimulating MAPK-mediated signaling, dimethyl fumarate acts [1]. By lowering the expression of MHC class II, CD80, and CD86, as well as the synthesis of inflammatory cytokines (IL-12 and IL-6), dimethyl fumarate prevents dendritic cell (DC) development. Dimethyl fumarate reduces DC maturation and cumulative Th1 and Th17 cell secretion by blocking NF-κB and ERK1/2-MSK1 signaling. Dimethyl fumarate also impairs p65 nuclear translocation and phosphorylation [2]. Dimethyl fumarate (DMF), an immunological antioxidant response cell viability assay [1]

Dimethyl fumarate (DMF; 50 mg/kg; daily; for 7 days) administration was demonstrated to upregulate Nrf2-regulated cytoprotective genes' mRNA and protein levels and to reduce 6-OHDA-induced C57BL striae. Eight-week-old male C57BL/6 mice serve as an animal model for body oxidation [4].

Cell Viability Assay[1]
Cell Types: SGC-7901, HT29, HCT116 and CT26 Cell
Tested Concentrations: 20 μM, 50 μM, 100 μM, 200 μM incubation Modulators and inducers, inhibiting HIV replication and neurotoxin release [3].
Incubation Duration: 24 hrs (hours)
Experimental Results: diminished cell viability in SGC-7901, HT29, HCT116 and CT26 cancer cells.

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Western Blot Analysis [1]
Cell Types: CT26 cancer cells
Tested Concentrations: 100 μM
Incubation Duration: 3 hrs (hours), 6 hrs (hours), 12 hrs (hours), 24 hrs (hours)
Experimental Results: JNK, p38 and ERK were Dramatically activated in CT26 cells after 3 to 24 hrs (hours) of treatment .

Animal/Disease Models: Male C57BL/6 mice (8weeks old)[4]
Doses: 50 mg/kg
Route of Administration: po (oral gavage); daily; for 7 days
Experimental Results:Was shown to upregulate mRNA and protein levels of Nrf2 and Nrf2-regulated cytoprotective genes.

Absorption, Distribution and Excretion
Once ingested, dimethyl fumarate is rapidly hydrolyzed by esterases to form monomethyl fumarate (MMF). Therefore, there is a negligible amount of dimethyl fumarate in the body, and all pharmacokinetic information is quantified with MMF. The time to maximum concentration (tmax) of MMF ranges between 2 and 2.5 hours. In patients with multiple sclerosis given 240 mg of dimethyl fumarate two times a day with food, the Cmax and AUC were 1.87 mg/L and 8.21 mg⋅hr/L, respectively. High-fat, high-calorie meals decrease the Cmax of MMF by 40% and cause a tmax delay from 2 hours to 5.5 hours; however, these changes are not considered clinically significant.
The main route of elimination of dimethyl fumarate is by CO₂ exhalation, which accounts for 60% of the dose. The other minor routes of elimination are through the kidney (16% of the dose) and feces (1% of the dose). Trace amounts of unchanged monomethyl fumarate (the active metabolite of dimethyl fumarate) are present in urine.
In healthy people, monomethyl fumarate (MMF) has a variable volume of distribution of 53 to 73 litres.
Monomethyl fumarate (MMF), the active metabolite of dimethyl fumarate, has a rapid clearance. Its apparent clearance (Cl/F) appears to be dose-independent.
After oral administration of Tecfidera, dimethyl fumarate undergoes rapid presystemic hydrolysis by esterases and is converted to its active metabolite, monomethyl fumarate (MMF). Dimethyl fumarate is not quantifiable in plasma following oral administration of Tecfidera. Therefore all pharmacokinetic analyses related to Tecfidera were performed with plasma MMF concentrations. ... The median Tmax of MMF is 2-2.5 hours. The peak plasma concentration (Cmax) and overall exposure (AUC) increased approximately dose proportionally in the dose range studied (120 mg to 360 mg). Following administration of Tecfidera 240 mg twice a day with food, the mean Cmax of MMF was 1.87 mg/L and AUC was 8.21 mg.hr/L in MS patients.
Exhalation of CO2 is the primary route of elimination, accounting for approximately 60% of the Tecfidera dose. Renal and fecal elimination are minor routes of elimination, accounting for 16% and 1% of the dose respectively. Trace amounts of unchanged monomethyl fumarate (MMF) were present in urine.
The apparent volume of distribution of monomethyl fumarate (MMF) varies between 53 and 73 L in healthy subjects. Human plasma protein binding of MMF is 27-45% and independent of concentration. /Monomethyl fumarate, active metabolite/

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Metabolism / Metabolites
Dimethyl fumarate is quickly hydrolyzed by esterases in the gastrointestinal tract, tissues, and blood to form monomethyl fumarate (MMF), its active metabolite. MMF then undergoes subsequent metabolism through the tricarboxylic acid (TCA) cycle. The main metabolites of dimethyl fumarate are MMF, glucose, citric, and fumaric acid. Cytochrome P450 (CYP) enzymes do not participate in the metabolism of dimethyl fumarate.
In humans, Tecfidera is extensively metabolized by esterases, which are ubiquitous in the gastrointestinal tract, blood and tissues, before it reaches the systemic circulation. Further metabolism occurs through the tricarboxylic acid (TCA) cycle, with no involvement of the cytochrome P450 (CYP) system. A single 240 mg (14)C-dimethyl fumarate dose study identified monomethyl fumarate, fumaric and citric acid, and glucose as the major metabolites in plasma. The downstream metabolism of fumaric and citric acid occurs through the TCA cycle, with exhalation of CO2 serving as a primary route of elimination. Less than 0.1% of the dose is excreted as unchanged dimethyl fumarate in urine.
In humans, dimethyl fumarate is extensively metabolized by esterases, which are ubiquitous in the gastrointestinal tract, blood, and tissues, before it reaches the systemic circulation. Further metabolism of monomethyl fumarate (MMF) occurs through the tricarboxylic acid (TCA) cycle, with no involvement of the cytochrome P450 (CYP) system. MMF, fumaric and citric acid, and glucose are the major metabolites in plasma.

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Biological Half-Life
The dimethyl fumarate metabolite monomethyl fumarate (MMF) has a short half-life of about 1 hour. MMF does not accumulate after repeated doses of dimethyl fumarate.
The terminal half-life of monomethyl fumarate (MMF) is approximately 1 hour and no circulating MMF is present at 24 hours in the majority of individuals. /Monomethyl fumarate, active metabolite/

Toxicity Summary
IDENTIFICATION AND USE: Dimethyl fumarate is a white to off-white powder formulated into delayed release capsules. It is used for the treatment of patients with relapsing forms of multiple sclerosis. Dimethyl fumarate is also used as a biocide to kill molds that may cause products such as furniture or shoes to deteriorate during storage or transportation in a humid climate. Placed in "Desiccant" sachets inside the furniture or footwear boxes, dimethyl fumarate evaporates and impregnates the product, protecting it from molds. HUMAN EXPOSURE AND TOXICITY: When used as a biocide, dimethyl fumarate has caused painful dermatitis. The fact that in serious cases the dermatitis is particularly difficult to treat adds to the damage. Dimethyl fumarate also has toxicity related to its use as a treatment for multiple sclerosis. A patient with multiple sclerosis who was being treated with dimethyl fumarate developed progressive multifocal leukoencephalopathy (PML), and later died. The patient who died was not taking any other drugs that affect the immune system or drugs that are thought to be associated with PML. Patients taking dimethyl fumarate should be advised to contact their clinician if they develop any symptoms that may be suggestive of PML. Treatment with dimethyl fumarate should not be initiated in patients with signs and symptoms of a serious infection. Dimethyl fumarate was clastogenic in the in vitro chromosomal aberration assay in human peripheral blood lymphocytes in the absence of metabolic activation. ANIMAL STUDIES: Acute toxicity studies were performed in mice and rats using oral and intraperitoneal routes. In mice, reduced motility, ataxia, dyspnea, cyanosis, muscular hypotonia were observed at oral doses as low as 681 mg/kg. Ataxia and hypopnea were observed at i.p. doses as low as 464 mg/kg. In rats, ataxia, muscular hypotonia, inhibited respiratory rate and motility were noted at oral doses as low as 2610 mg/kg. Reduced food intake and decreased body weight gain were seen at 1470 and 2150 mg/kg, respectively. Ataxia, muscular hypotonia, reduced motility and respiratory rate were also observed at intraperitoneal doses as low as 681 mg/kg. Dyspnea (825 mg/kg); tremor, pilo-erection (1000 mg/kg); abdominal positioning (1470 mg/kg) were also noted. In these studies, the kidneys, forestomach and liver were identified as target organs. In mice, oral administration of dimethyl fumarate (25, 75, 200, and 400 mg/kg/day) for up to two years resulted in an increase in nonglandular stomach (forestomach) and kidney tumors: squamous cell carcinomas and papillomas of the forestomach in males and females at 200 and 400 mg/kg/day; leiomyosarcomas of the forestomach at 400 mg/kg/day in males and females; renal tubular adenomas and carcinomas at 200 and 400 mg/kg/day in males; and renal tubule adenomas at 400 mg/kg/day in females. In rats, oral administration of dimethyl fumarate (25, 50, 100, and 150 mg/kg/day) for up to two years resulted in increases in squamous cell carcinomas and papillomas of the forestomach at all doses tested in males and females, and in testicular interstitial (Leydig) cell adenomas at 100 and 150 mg/kg/day. In rats administered dimethyl fumarate orally (25, 100, 250 mg/kg/day) throughout organogenesis, embryo fetal toxicity (reduced fetal body weight and delayed ossification) were observed at the highest dose tested. This dose also produced evidence of maternal toxicity (reduced body weight). Oral administration of dimethyl fumarate (25, 100, and 250 mg/kg/day) to rats throughout organogenesis and lactation resulted in increased lethality, persistent reductions in body weight, delayed sexual maturation (male and female pups), and reduced testicular weight at the highest dose tested. Neurobehavioral impairment was observed at all doses. In rabbits administered dimethyl fumarate orally (25, 75, and 150 mg/kg/day) throughout organogenesis, embryo lethality and decreased maternal body weight were observed at the highest dose tested. In male rats, oral administration of dimethyl fumarate (75, 250, and 375 mg/kg/day) prior to and throughout the mating period had no effect on fertility; however, increases in non-motile sperm were observed at the mid and high doses. In female rats, oral administration of dimethyl fumarate (20, 100, and 250 mg/kg/day) prior to and during mating and continuing to gestation day 7 caused disruption of the estrous cycle and increases in embryo lethality at the highest dose tested. Testicular toxicity (germinal epithelial degeneration, atrophy, hypospermia, and/or hyperplasia) was observed at clinically relevant doses in mice, rats, and dogs in subchronic and chronic oral toxicity studies of dimethyl fumarate. Dimethyl fumarate was not mutagenic in the in vitro bacterial reverse mutation (Ames) assay, and it was not clastogenic in the in vivo micronucleus assay in the rat.

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Toxicity Data
LC (mouse) > 3,100 mg/m3/10min

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Interactions
The bioreductive antitumor agent, mitomycin C (MMC), requires activation by reductive enzymes like NAD(P)H:quinone oxidoreductase 1 (NQO1). ...A novel approach /was used/ to increase MMC efficacy by selectively inducing NQO1 in tumor cells in vivo. CD-1 nude mice were implanted with HCT116 cells, and fed control diet or diet containing 0.3% of the NQO1 inducer, dimethyl fumarate (DMF). The mice were then treated with saline, 2.0, 3.5 or 2.0 mg/kg MMC and dicoumarol, an NQO1 inhibitor. The DMF diet increased NQO1 activity by 2.5-fold in the tumors, but had no effect in marrow cells. Mice given control diet/2.0 mg/kg MMC had tumors with the same volume as control mice; however, mice given DMF diet/2.0 mg/kg MMC had significantly smaller tumors. Tumor volumes in mice given DMF/2.0 mg/kg MMC were similar to those in mice given control diet/3.5 mg/kg MMC. Tumor inhibition was partially reversed in mice given DMF/2.0 mg/kg MMC and dicoumarol. DMF diet/2.0 mg/kg MMC treatment did not increase myelosuppression and did not produce any organ toxicity. These results provide strong evidence that dietary inducers of NQO1 can increase the antitumor activity of bioreductive agents like MMC without increasing toxicity.
NQO1 is a reductive enzyme that is important for the activation of many bioreductive agents and is a target for an enzyme-directed approach to cancer therapy. It can be selectively induced in many tumor types by a number of compounds including dimethyl fumarate... . RH1 (2,5-diaziridinyl-3-(hydroxymethyl)- 6-methyl-1,4-benzoquinone) is a new bioreductive agent currently in clinical trials. ... HCT116 human colon cancer cells and T47D human breast cancer cells were incubated with or without dimethyl fumarate or sulforaphane followed by mitomycin C or RH1 treatment, and cytotoxic activity was measured by a clonogenic (HCT116) or MTT assay (T47D). Dimethyl fumarate and sulforaphane treatment increased NQO1 activity by 1.4- to 2.8-fold and resulted in a significant enhancement of the antitumor activity of mitomycin C, but not of RH1. This appeared to be due to the presence of a sufficient constitutive level of NQO1 activity in the tumor cells to fully activate the RH1. Mice were implanted with HL60 human promyelocytic leukemia cells, which have low levels of NQO1 activity. The mice were fed control or dimethyl fumarate-containing diet and were treated with RH1. NQO1 activity in the tumors increased but RH1 produced no antitumor activity in mice fed control or dimethyl fumarate diet. This is consistent with a narrow window of NQO1 activity between no RH1 activation and maximum RH1 activation. This study suggests that selective induction of NQO1 in tumor cells is not likely to be an effective strategy for enhancing the antitumor activity of RH1...
... The effects of butylated hydroxyanisole (BHA) /were/ compared with those of other inducers of DT-diaphorase. Rats were dosed with BHA, butylated hydroxytoluene (BHT), ethoxyquin (EQ), dimethyl fumarate (DMF) or disulfiram (DIS) and then challenged with a toxic dose of the naphthoquinones. All the inducers protected against the hemolytic anemia induced by 2-methyl-1,4-naphthoquinone in rats, with BHA, BHT and EQ being somewhat more effective than DMF and DIS. A similar order of activity was recorded in the relative ability of these substances to increase hepatic activities of DT-diaphorase, consistent with a role for this enzyme in facilitating conjugation and excretion of this naphthoquinone. In contrast, all the compounds increased the hemolytic activity of 2-hydroxy-1,4-naphthoquinone. DMF and DIS were significantly more effective in this regard than BHA, BHT and EQ. DMF and DIS also caused a much greater increase in levels of DT-diaphorase in the intestine, suggesting that 2-hydroxy-1,4-naphthoquinone is activated by this enzyme in the gut. BHA, BHT and EQ had no effect on the nephrotoxicity of 2-hydroxy-1,4-naphthoquinone, but the severity of the renal lesions was decreased in rats pre-treated with DMF and DIS. The results of the present experiments show that modulation of tissue levels of DT-diaphorase may not only alter the severity of naphthoquinone toxicity in vivo, but may also change the relative toxicity of these substances to different target organs.
DT-diaphorase is a two-electron reducing enzyme that activates the bioreductive anti-tumour agent, mitomycin C (MMC). Cell lines having elevated levels of DT-diaphorase are generally more sensitive to MMC. ... DT-diaphorase can be induced in human tumor cells by a number of compounds, including 1,2-dithiole-3-thione. ... This study ... investigated whether induction of DT-diaphorase could enhance the cytotoxic activity of MMC in six human tumor cell lines representing four tumor types. DT-diaphorase was induced by many dietary inducers, including ... dimethyl fumarate ... .The cytotoxicity of MMC was significantly increased in four tumor lines with the increase ranging from 1.4- to threefold. In contrast, MMC activity was not increased in SK-MEL-28 human melanoma cells and AGS human gastric cancer cells, cell lines that have high base levels of DT-diaphorase activity. Toxicity to normal human marrow cells was increased by 50% when MMC was combined with 1,2-dithiole-3-thione, but this increase was small in comparison with the threefold increase in cytotoxicity to tumor cells. ...
For more Interactions (Complete) data for DIMETHYL FUMARATE (9 total), please visit the HSDB record page.

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Non-Human Toxicity Values
LD50 Rat oral 2240 mg/kg
LD50 Rabbit dermal 1259 mg/kg

[1]. Dimethyl fumarate induces necroptosis in colon cancer cells through GSH depletion/ROS increase/MAPKs activation pathway. Br J Pharmacol. 2015 Aug;172(15):3929-43.

[2]. Dimethyl fumarate inhibits dendritic cell maturation via nuclear factor κB (NF-κB) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) and mitogen stress-activated kinase 1 (MSK1) signaling. J Biol Chem. 2012 Aug 10;287(33):28017-26.

[3]. Dimethyl fumarate, an immune modulator and inducer of the antioxidant response, suppresses HIV replication and macrophage-mediated neurotoxicity: a novel candidate for HIV neuroprotection. J Immunol. 2011 Nov 15;187(10):5015-25.

[4]. Dimethyl fumarate attenuates 6-OHDA-induced neurotoxicity in SH-SY5Y cells and in animal model of Parkinson's disease by enhancing Nrf2 activity. Neuroscience. 2015 Feb 12;286:131-40.

Therapeutic Uses
Dermatologic Agents; Immunosuppressive Agents; Radiation-Sensitizing Agents
/CLINICAL TRIALS/ ClinicalTrials.gov is a registry and results database of publicly and privately supported clinical studies of human participants conducted around the world. The Web site is maintained by the National Library of Medicine (NLM) and the National Institutes of Health(NIH). Each ClinicalTrials.gov record presents summary information about a study protocol and includes the following: Disease or condition; Intervention (for example, the medical product, behavior, or procedure being studied); Title, description, and design of the study; Requirements for participation (eligibility criteria); Locations where the study is being conducted; Contact information for the study locations; and Links to relevant information on other health Web sites, such as NLM's MedlinePlus for patient health information and PubMed for citations and abstracts for scholarly articles in the field of medicine. Dimethyl fumarate is included in the database.
Tecfidera is indicated for the treatment of patients with relapsing forms of multiple sclerosis. /Included in US product label/
EXPL THER Mixtures of fumaric acid esters (FAE) are used as an oral systemic treatment for moderate to severe psoriasis. Large clinical studies with dimethylfumarate (DMF) monotherapy are scarce. The objective of this study is to assess the effectiveness and long-term safety of high-dose DMF monotherapy in moderate to severe psoriasis. A prospective single-blinded follow-up study was performed in a cohort of patients treated with DMF. Patients were followed-up at fixed intervals. Assessment of consecutive photographs was performed by two observers. Primary outcome was a change in static physician global assessment (PGA) score. Safety outcome was defined as incidences of (serious) adverse events. A total of 176 patients with moderate to severe psoriasis were treated with DMF for a median duration of 28 months. The median daily maintenance dosage of 480 mg was reached after a median of 8 months. Psoriasis activity decreased significantly by 1.7 out of five points. A total of 152 patients reported one or more adverse events, such as gastrointestinal complaints and flushing. High-dose DMF monotherapy is an effective and safe treatment option in moderate to severe psoriasis. It can be suggested that 50% of all patients may benefit from high-dose DMF monotherapy. KEYWORDS: Dimethylfumurate; high dose; monotherapy; prospective study; psoriasis

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Drug Warnings
A patient with multiple sclerosis who was being treated with dimethyl fumarate developed progressive multifocal leukoencephalopathy (PML), and later died. The patient who died was not taking any other drugs that affect the immune system or drugs that are thought to be associated with PML. Patients taking dimethyl fumarate should be advised to contact their clinician if they develop any symptoms that may be suggestive of PML. Symptoms of PML are diverse, progress over days to weeks, and include the following: progressive weakness on one side of the body or clumsiness of limbs; disturbance of vision; and changes in thinking, memory and orientation, leading to confusion and personality changes. The progression of deficits can lead to severe disability or death. Dimethyl fumarate should be discontinued immediately at the first sign or symptom suggestive of PML and an appropriate diagnostic evaluation should be performed. Lymphocyte counts should be monitored in dimethyl fumarate-treated patients according to approved labeling.
Dimethyl fumarate may decrease lymphocyte counts. In placebo-controlled clinical trials, mean lymphocyte counts decreased by approximately 30% during the first year of treatment with the drug and remained stable thereafter. Mean lymphocyte counts improved 4 weeks following discontinuance of the drug, but did not return to baseline values. Dimethyl fumarate has not been studied in patients with preexisting low lymphocyte counts. Prior to initiation of dimethyl fumarate, a recent (i.e., within 6 months) complete blood cell (CBC) count should be available to identify patients with preexisting low lymphocyte counts. A CBC should also be obtained annually during therapy and as clinically indicated. In patients with serious infections, withholding dimethyl fumarate treatment should be considered until the infection has resolved.
During post marketing experience, hypersensitivity reactions have been reported, including rare reports of anaphylaxis and angioedema in patients treated with Tecfidera. Signs and symptoms have included difficulty breathing, urticaria, and swelling of the throat and tongue.
Treatment with Tecfidera should not be initiated in patients with signs and symptoms of a serious infection. Decreases in lymphocyte counts observed in patients treated with Tecfidera in clinical trials were not associated with increased frequencies of infections. However, due to the potential risk of infections in patients who develop sustained lymphopenia, patients should be instructed to report symptoms of infection to their physician. For patients with signs and symptoms of serious infections, interrupting treatment with Tecfidera should be considered, until the infection(s) resolves.
For more Drug Warnings (Complete) data for DIMETHYL FUMARATE (14 total), please visit the HSDB record page.

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Pharmacodynamics
The physiological effects of dimethyl fumarate on the body are not well understood. It has anti-inflammatory and cytoprotective effects, likely involved in its actions in multiple sclerosis (MS) patients. Dimethyl fumarate does not cause clinically significant QT interval prolongation. However, cases of progressive multifocal leukoencephalopathy, serious opportunistic infections, lymphopenia and liver injury have been reported in MS patients treated with this drug. Dimethyl fumarate may also cause anaphylaxis and angioedema.

C6H8O4

144.12

144.042

C, 50.00; H, 5.60; O, 44.40

624-49-7

Dimethyl fumarate-d6;66487-95-4;Dimethyl fumarate-d2;23057-98-9

637568

White to off-white solid

1.1±0.1 g/cm3

193.0±0.0 °C at 760 mmHg

102-106 °C(lit.)

91.1±0.0 °C

0.5±0.3 mmHg at 25°C

1.435

Endogenous Metabolite

0.62

0

4

4

10

141

0

O(C([H])([H])[H])C(/C(/[H])=C(\[H])/C(=O)OC([H])([H])[H])=O

LDCRTTXIJACKKU-ONEGZZNKSA-N

InChI=1S/C6H8O4/c1-9-5(7)3-4-6(8)10-2/h3-4H,1-2H3/b4-3+

But-2-enedioic acid dimethyl ester

DMF Dimethylfumarate Dimethyl Fumarate

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)

DMSO : ~41.67 mg/mL (~289.11 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (14.43 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 (14.43 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 (14.43 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: 2 mg/mL (13.88 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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Solubility in Formulation 5: 7.5 mg/mL (52.04 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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