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
Dimethyl fumarate is rapidly hydrolyzed by esterases to monomethyl fumarate (MMF) after ingestion. Therefore, the amount of dimethyl fumarate in the body is extremely low, and all pharmacokinetic information is quantified using MMF as the indicator. The time to peak concentration (tmax) of MMF is 2 to 2.5 hours. In patients with multiple sclerosis, taking 240 mg of dimethyl fumarate twice daily with food resulted in a Cmax of 1.87 mg/L and an AUC of 8.21 mg⋅hr/L. High-fat, high-calorie diets reduced the Cmax of MMF by 40% and delayed tmax from 2 hours to 5.5 hours; however, these changes were not clinically significant. The primary route of elimination of dimethyl fumarate is through exhaled carbon dioxide, accounting for 60% of the dose. Other minor routes of elimination include renal (16% of the dose) and fecal (1% of the dose). Trace amounts of unmetabolized monomethyl fumarate (the active metabolite of dimethyl fumarate) are present in urine. In healthy individuals, the volume of distribution of monomethyl fumarate (MMF) is 53 to 73 liters. MMF is the active metabolite of dimethyl fumarate and is rapidly cleared. Its apparent clearance (Cl/F) appears to be dose-independent. Following oral administration of Tecfidera, dimethyl fumarate undergoes rapid first-pass hydrolysis by esterases, converting to its active metabolite, monomethyl fumarate (MMF). Dimethyl fumarate cannot be quantitatively detected in plasma after oral administration of Tecfidera. Therefore, all pharmacokinetic analyses related to Tecfidera were performed using plasma MMF concentrations. …The median time to peak concentration (Tmax) of MMF is 2–2.5 hours. Within the studied dose range (120 mg to 360 mg), peak plasma concentration (Cmax) and total exposure (AUC) increased approximately proportionally to the dose. In patients with multiple sclerosis (MS), after taking 240 mg Tecfidera twice daily with food, the mean Cmax of MMF was 1.87 mg/L and the AUC was 8.21 mg·hr/L. Exhaled carbon dioxide was the primary clearance route, accounting for approximately 60% of the Tecfidera dose. Renal and fecal clearance were secondary clearance routes, accounting for 16% and 1% of the dose, respectively. Trace amounts of unmetabolized monomethyl fumarate (MMF) were present in urine. The apparent volume of distribution of monomethyl fumarate (MMF) in healthy subjects ranged from 53 to 73 liters. The human plasma protein binding rate of MMF was 27% to 45%, independent of concentration. /Methyl fumarate, active metabolite/

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Metabolism/Metabolite
Dimethyl fumarate is rapidly hydrolyzed by esterases in the gastrointestinal tract, tissues, and blood to produce its active metabolite, monomethyl fumarate (MMF). MMF is then further metabolized via the tricarboxylic acid cycle (TCA cycle). The major metabolites of dimethyl fumarate are MMF, glucose, citrate, and fumarate. Cytochrome P450 (CYP) enzymes are not involved in the metabolism of dimethyl fumarate.
In the human body, Tecfidera is primarily metabolized by esterases that are widely distributed in the gastrointestinal tract, blood, and tissues before entering systemic circulation. Further metabolism occurs via the tricarboxylic acid cycle (TCA cycle) and does not involve the cytochrome P450 (CYP) system. A single 240 mg (14)C-dimethyl fumarate dose study found that the major metabolites in plasma were monomethyl fumarate, fumarate, citrate, and glucose. Downstream metabolism of fumarate and citrate occurs via the TCA cycle, with exhaled carbon dioxide being the primary clearance pathway. Less than 0.1% of the dose is excreted in the urine as unmetabolized dimethyl fumarate. In the human body, dimethyl fumarate is metabolized by esterases widely distributed in the gastrointestinal tract, blood, and tissues before entering systemic circulation. Further metabolism of monomethyl fumarate (MMF) occurs via the tricarboxylic acid cycle (TCA cycle) and does not involve the cytochrome P450 (CYP) system. MMF, fumarate, citrate, and glucose are the major metabolites in plasma. The half-life of monomethyl fumarate (MMF), a metabolite of dimethyl fumarate, is very short, approximately 1 hour. MMF does not accumulate after repeated administration of dimethyl fumarate. The terminal half-life of monomethyl fumarate (MMF) is approximately 1 hour, and it is undetectable in the circulation of most individuals after 24 hours. /Monomethyl fumarate, active metabolite/

Toxicity Summary
Identification and Uses: Dimethyl fumarate is a white to off-white powder formulated as a sustained-release capsule. It is used to treat patients with relapsing-remitting multiple sclerosis (MS). Dimethyl fumarate can also be used as a fungicide to kill mold that can cause products such as furniture or shoes to deteriorate during storage or transport in humid climates. Placing dimethyl fumarate in a desiccant bag inside furniture or shoe boxes allows the fumarate to evaporate and permeate the product, protecting it from mold. Human Exposure and Toxicity: When used as a fungicide, dimethyl fumarate can cause painful dermatitis. In severe cases, this dermatitis is particularly difficult to treat, which exacerbates the damage. Dimethyl fumarate is also toxic as a treatment for MS. One MS patient receiving dimethyl fumarate treatment developed progressive multifocal leukoencephalopathy (PML) and subsequently died. The deceased patient had not taken any other medications that affect the immune system or were considered to be associated with PML. Patients taking dimethyl fumarate should be advised to contact their clinician immediately if any symptoms suggestive of PML occur. Dimethyl fumarate treatment should not be initiated in patients with severe signs and symptoms of infection. In in vitro human peripheral blood lymphocyte chromosome aberration assays, dimethyl fumarate has a chromosome breakage-inducing effect without metabolic activation. Animal studies: Acute toxicity studies were conducted in mice and rats via oral and intraperitoneal routes. In mice, decreased activity, ataxia, dyspnea, cyanosis, and hypotonia were observed at oral doses as low as 681 mg/kg. Ataxia and bradyspnea were observed at intraperitoneal doses as low as 464 mg/kg. In rats, ataxia, hypotonia, decreased respiratory rate, and decreased motor function were observed at oral doses as low as 2610 mg/kg. Decreased food intake and reduced weight gain were observed at doses of 1470 mg/kg and 2150 mg/kg, respectively. At intraperitoneal injections at doses as low as 681 mg/kg also showed ataxia, hypotonia, decreased motor function, and decreased respiratory rate. Additionally, dyspnea (825 mg/kg), tremor, piloerection (1000 mg/kg), and altered abdominal posture (1470 mg/kg) were observed. 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 non-glandular gastric (forestomach) and renal tumors: squamous cell carcinoma and papilloma of the forestomach appeared in male and female mice at doses of 200 and 400 mg/kg/day; leiomyosarcoma of the forestomach appeared in male and female mice at a dose of 400 mg/kg/day; renal tubular adenoma and carcinoma appeared in male mice at doses of 200 and 400 mg/kg/day; and renal tubular adenoma appeared in female mice at a dose of 400 mg/kg/day. In rats, consecutive two-year oral administration of dimethyl fumarate (25, 50, 100, and 150 mg/kg/day) increased the incidence of forestomach squamous cell carcinoma and papilloma in both male and female rats at all tested doses, and also increased the incidence of Leydig cell adenoma at doses of 100 and 150 mg/kg/day. During organogenesis, oral administration of dimethyl fumarate (25, 100, and 250 mg/kg/day) to rats resulted in embryo-fetal toxicity (fetal weight loss and delayed ossification) at the highest tested dose. This dose also showed maternal toxicity (weight loss). During organogenesis and lactation, oral administration of dimethyl fumarate (25, 100, and 250 mg/kg/day) to rats led to increased mortality, persistent weight loss, delayed sexual maturation (in both male and female pups), and decreased testicular weight (highest dose group). Neurobehavioral disorders were observed in all dose groups. During organogenesis, oral administration of dimethyl fumarate (25, 75, and 150 mg/kg/day) to rabbits resulted in embryonic death, with decreased maternal weight observed in the highest dose group. In male rats, oral administration of dimethyl fumarate (75, 250, and 375 mg/kg/day) before and throughout the mating period had no effect on fertility; however, an increase in inactive sperm count was observed in the medium and high dose groups. In female rats, oral administration of dimethyl fumarate (20, 100, and 250 mg/kg/day) before, during, and on day 7 of gestation resulted in estrous cycle disturbances and increased embryonic mortality at the highest tested dose. In subchronic and chronic oral toxicity studies in mice, rats, and dogs, clinically relevant doses of dimethyl fumarate all showed testicular toxicity (germinal epithelial degeneration, atrophy, decreased sperm count, and/or hyperplasia). Dimethyl fumarate did not show mutagenicity in the in vitro bacterial reverse mutation (Ames) assay, nor did it show chromosome breakage in the in vivo rat micronucleus assay.

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

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Interactions
The bioreducing antitumor drug mitomycin C (MMC) requires activation by reductases such as NAD(P)H:quinone oxidoreductase 1 (NQO1)). …We employed a novel approach to enhance the efficacy of MMC by selectively inducing NQO1 in tumor cells in vivo. HCT116 cells were implanted into CD-1 nude mice and fed either a control diet or a diet containing 0.3% dimethyl fumarate (DMF), an NQO1 inducer. Subsequently, mice were treated with saline, 2.0 mg/kg, 3.5 mg/kg, or 2.0 mg/kg mitomycin C (MMC), and the NQO1 inhibitor dicumarol, respectively. The DMF diet increased NQO1 activity in tumors by 2.5-fold, but had no effect on bone marrow cells. Tumor volume in mice treated with the control diet/2.0 mg/kg MMC was the same as in the control group; however, tumor volume in mice treated with the DMF diet/2.0 mg/kg MMC was significantly reduced. Tumor volume in mice treated with DMF/2.0 mg/kg MMC was similar to that in mice treated with the control diet/3.5 mg/kg MMC. Tumor suppression was partially reversed in mice treated with DMF/2.0 mg/kg MMC and dicumarol. Treatment with the DMF diet/2.0 mg/kg MMC did not increase bone marrow suppression or produce any organ toxicity. These results strongly demonstrate that dietary NQO1 inducers can enhance the antitumor activity of bioreducing agents such as mitomycin C (MMC) without increasing toxicity. NQO1 is a reductase crucial for the activation of many bioreducing agents and is also a target for enzyme-directed cancer therapy. Various compounds, including dimethyl fumarate, can selectively induce NQO1 expression in multiple tumor types…RH1 (2,5-diazacyclopropane-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone) is a novel bioreducing agent currently undergoing clinical trials. …HCT116 human colon cancer cells and T47D human breast cancer cells were incubated with dimethyl fumarate or sulforaphane, or without incubation, followed by treatment with mitomycin C or RH1, and cytotoxic activity was determined by colony formation assay (HCT116) or MTT assay (T47D). Treatment with dimethyl fumarate and sulforaphane increased NQO1 activity by 1.4 to 2.8 times and significantly enhanced the antitumor activity of mitomycin C, but had no effect on the antitumor activity of RH1. This appears to be due to the presence of sufficient levels of NQO1 activity in tumor cells to fully activate RH1. HL60 human promyelocytic leukemia cells with low NQO1 activity were implanted into mice. Mice were fed either a control diet or a diet containing dimethyl fumarate and treated with RH1. NQO1 activity in tumors increased, but RH1 did not produce antitumor activity in mice fed either the control diet or the diet containing dimethyl fumarate. This is consistent with a narrow window of NQO1 activity, between RH1 inactivation and maximal RH1 activation. This study suggests that selective induction of NQO1 in tumor cells is unlikely to be an effective strategy for enhancing the antitumor activity of RH1…
…The effects of butylated hydroxyanisole (BHA) were compared with those of other DT-dihydroflavinase inducers. Rats were administered BHA, butylated hydroxytoluene (BHT), ethoxyquinoline (EQ), dimethyl fumarate (DMF), or disulfiram (DIS), respectively, followed by challenge with toxic doses of naphthoquinones. All inducers protected rats from 2-methyl-1,4-naphthoquinone-induced hemolytic anemia, with BHA, BHT, and EQ showing slightly better protection than DMF and DIS. These substances also exhibited a similar activity sequence in their ability to increase hepatic DT-dihydroflavinase activity, consistent with the enzyme's role in promoting naphthoquinone binding and excretion. Conversely, all compounds enhanced the hemolytic activity of 2-hydroxy-1,4-naphthoquinone. DMF and DIS were significantly more effective than BHA, BHT, and EQ in this regard. DMF and DIS also significantly increased intestinal DT-dihydroflavinase levels, suggesting that 2-hydroxy-1,4-naphthoquinone is activated by this enzyme in the intestine. BHA, BHT, and EQ had no effect on the nephrotoxicity of 2-hydroxy-1,4-naphthoquinone, but DMF and DIS pretreatment reduced the degree of kidney damage in rats. These results indicate that regulating the level of DT-dihydroflavinase in tissues can not only alter the in vivo toxicity of naphthoquinone drugs but also change the relative toxicity of these substances to different target organs. DT-dihydroflavinase is a two-electron reductase that activates the bioreducing antitumor drug mitomycin C (MMC). Cell lines with elevated DT-dihydroflavinase levels are generally more sensitive to MMC. …Multiple compounds, including 1,2-dithiocyclopenten-3-thionone, can induce the expression of DT-dihydroflavinase in human tumor cells. …This study…investigated whether inducing DT-dihydroflavinase could enhance the cytotoxic activity of MMC in six human tumor cell lines representing four tumor types. Multiple dietary inducers, including…dimethyl fumarate…, can induce the expression of DT-dihydroflavinase. In four tumor cell lines, MMC showed significantly enhanced cytotoxicity, increasing by 1.4 to 3-fold. Conversely, MMC activity was not increased in SK-MEL-28 human melanoma cells and AGS human gastric cancer cells (these cell lines have high basal levels of DT-dihydroflavinase activity). When MMC was used in combination with 1,2-dithiacyclopenten-3-thione, cytotoxicity against normal human bone marrow cells increased by 50%, but this increase was smaller compared to the three-fold increase in cytotoxicity against tumor cells. ...
For more complete data on interactions of dimethyl fumarate (9 in total), please visit the HSDB record page.

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Non-human toxicity values
Oral LD50 in rats: 2240 mg/kg
Dermal LD50 in rabbits: 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
Dermatological medication; immunosuppressants; radiosensitizers
/Clinical Trials/ ClinicalTrials.gov is a registry and results database that lists human clinical studies funded by public and private institutions worldwide. The website is maintained by the National Library of Medicine (NLM) and the National Institutes of Health (NIH). Each record on ClinicalTrials.gov includes a summary of the study protocol, including: the disease or condition; the intervention (e.g., the medical product, behavior, or procedure under investigation); the title, description, and design of the study; participation requirements (eligibility criteria); the location of the study; contact information for the study location; and links to relevant information from other health websites, such as the NLM's MedlinePlus (which provides patient health information) and PubMed (which provides citations and abstracts of academic articles in the medical field). Dimethyl fumarate is listed in the database.
Tecfidera is indicated for the treatment of patients with relapsing-remitting multiple sclerosis. /Listed on US Product Labels/
Fumarate mixtures (FAE) are available as an oral systemic treatment for moderate to severe psoriasis. Currently, there are few large-scale clinical studies on dimethyl fumarate (DMF) monotherapy. This study aimed to evaluate the efficacy and long-term safety of high-dose DMF monotherapy for moderate to severe psoriasis. This prospective, single-blind follow-up study was conducted on a cohort of patients receiving DMF treatment. Patients were followed up at fixed time intervals. Serial photographs were evaluated by two observers. The primary endpoint was the change in static Physician Overall Assessment (PGA) score. The safety endpoint was defined as the incidence of (serious) adverse events. A total of 176 patients with moderate to severe psoriasis received dimethyl fumarate (DMF) treatment, with a median treatment duration of 28 months. The median maintenance dose of 480 mg was reached after 8 months of treatment. Psoriasis activity significantly decreased by 1.7 points (out of 5). One or more adverse events, such as gastrointestinal discomfort and flushing, were reported in 152 patients. High-dose DMF monotherapy is an effective and safe treatment option for moderate to severe psoriasis. It is estimated that 50% of patients may benefit from high-dose DMF monotherapy. Keywords: Dimethyl fumarate; high dose; monotherapy; prospective study; psoriasis
Drug Warning
A patient with multiple sclerosis receiving dimethyl fumarate developed progressive multifocal leukoencephalopathy (PML) and subsequently died. The deceased patient had not taken any other medications affecting the immune system or those considered to be associated with progressive multifocal leukoencephalopathy (PML) prior to death. Patients taking dimethyl fumarate should be advised to contact their doctor immediately if any symptoms that may indicate PML occur. Symptoms of PML are varied and can gradually worsen over days to weeks, including: progressive weakness or clumsiness on one side of the body; visual disturbances; and changes in thinking, memory, and orientation, leading to confusion and personality changes. Disease progression can lead to severe disability or death. Dimethyl fumarate should be discontinued immediately upon the appearance of any signs or symptoms indicative of PML, and appropriate diagnostic evaluation should be performed. Lymphocyte counts in patients receiving dimethyl fumarate should be monitored according to the approved drug label. Dimethyl fumarate may decrease lymphocyte counts. In placebo-controlled clinical trials, patients experienced a mean decrease in lymphocyte count of approximately 30% during the first year of treatment with this drug, after which it remained stable. Four weeks after discontinuation, the mean lymphocyte count improved but did not return to baseline levels. Dimethyl fumarate has not been studied in patients with a history of low lymphocyte counts. A recent complete blood count (CBC) should be performed within the last 6 months before initiating dimethyl fumarate treatment to identify patients with a history of low lymphocyte counts. A CBC should be performed annually during treatment, and follow-up should be conducted as clinically necessary. For patients with severe infections, discontinuation of dimethyl fumarate treatment should be considered until the infection is resolved. Post-marketing experience has reported hypersensitivity reactions, including rare anaphylactic shock and angioedema. Signs and symptoms include dyspnea, urticaria, and swelling of the throat and tongue. Dimethyl fumarate should not be initiated in patients with severe signs and symptoms of infection. In clinical trials, the decrease in lymphocyte count observed in patients treated with Tecfidera was not associated with an increased incidence of infection. However, due to the potential risk of infection in patients with persistent lymphopenia, they should be advised to report any symptoms of infection to their physician. For patients with severe signs and symptoms of infection, Tecfidera treatment should be considered for discontinuation until the infection resolves.
For more complete data on drug warnings for dimethyl fumarate (14 of them), please visit the HSDB record page.

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Pharmacodynamics
The physiological effects of dimethyl fumarate on humans are not fully understood. It has anti-inflammatory and cytoprotective effects, which may be related to its role in patients with multiple sclerosis (MS). Dimethyl fumarate does not cause clinically significant QT interval prolongation. However, there have been reports of multiple sclerosis patients receiving this drug experiencing progressive multifocal leukoencephalopathy, severe opportunistic infections, lymphopenia, and liver damage. Dimethyl fumarate may also cause allergic reactions 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.)