bacteriostatic; folate synthesis

Once 100 μg of protein has been added to cell lysates, dapsone is tested using the DHPS activity assay. In the DHPS activity assay, Dapsone has an IC50 of 3.0 μg/ml for E. Coli C600; however, at 256 μg/ml, Dapsone does not inhibit the growth of E. Coli C600. IC50 and MIC values for Dapsone for the recombinant strain containing M. leprae folP1 (pML101) are 0.06 μg/ml and 1 μg/ml, respectively[2].

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Against Leishmania major promastigotes, Dapsone showed an EC₅₀ of 92 μM. Against L. braziliensis promastigotes, the EC₅₀ was 147 μM. [3]
Against intracellular amastigotes of L. major within infected macrophages, Dapsone exhibited an EC₅₀ of 93.7 μM. Against L. braziliensis amastigotes, the EC₅₀ was 54.5 μM. [3]
Dapsone demonstrated low cytotoxicity against mouse peritoneal macrophages (EC₅₀ = 1490 μM), 3T3 fibroblasts (EC₅₀ = 433 μM), and HaCaT keratinocytes (EC₅₀ = 3186 μM) after 48 hours of treatment. [3]
The selectivity index (ratio of cytotoxicity EC₅₀ against peritoneal macrophages to anti-amastigote activity EC₅₀) was higher than 15 for both L. major and L. braziliensis. [3]
At a concentration of 500 μM, Dapsone reduced the number of L. braziliensis amastigotes by 71% and L. major amastigotes by 45%. At 25 μM, it reduced amastigote numbers by approximately 9.1 μM for L. braziliensis and 20 μM for L. major. [3]

Dapsone (topical treatment; 50 mg/kg; twice daily; 30 days) causes lesions to be about 186 mm2 in size in BALB/c mice infected with L. major as opposed to 125 mm2 in control mice. Moreover, 9.6±8.5 µg of DAP/mg of skin was measured for the lesions treated with DAP cream. But compared to mice that were not given the cream, the amount of parasites discovered in the spleen was noticeably reduced in the mice that received the cream[3].

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In BALB/c mice infected with L. major in the tail base, topical treatment with a Dapsone cream (10% w/w) or a pluronic lecithin emulgel (DAP-PLE) applied twice daily for 30 days not only failed to cure cutaneous leishmaniasis lesions but worsened their size and appearance compared to untreated controls. Lesion sizes increased faster in treated groups. [3]
Despite the lack of efficacy on skin lesions, topical treatment with Dapsone formulations significantly reduced the parasite burden in the spleen of infected mice. [3]
In an ear infection model, treatment with DAP-PLE was stopped after 10 days due to a 2.6-fold increase in lesion volume compared to controls, accompanied by skin irritation, peeling, and hair loss. However, a reduction in spleen parasite burden was also observed. [3]

Anti-promastigote activity (MTT assay): Briefly, 2×10⁵ promastigotes per well were seeded in a 96-well plate with different concentrations of Dapsone and incubated at 26°C for 48 hours. Then, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well and plates were incubated for another 4 hours. After incubation, 100 μL of DMSO was added to solubilize the formazan crystals. Absorbance was measured at 570 nm using a microplate reader to determine parasite viability. EC₅₀ values were calculated by fitting data to a dose-effect sigmoid curve. [3]
Anti-amastigote activity in infected macrophages: Mouse peritoneal macrophages were seeded in Labtek plates and infected with metacyclic promastigotes of Leishmania at a parasite-to-macrophage ratio of 7:1 for L. major or 10:1 for L. braziliensis. After overnight infection, cells were treated with different concentrations of Dapsone (25-1000 μM) for 48 hours. Subsequently, slides were fixed, stained with Giemsa, and the percentage of infected macrophages and the number of amastigotes per 100 macrophages were counted under an optical microscope. EC₅₀ was calculated based on these counts. [3]
Cytotoxicity assay (MTT assay): Mouse peritoneal macrophages, 3T3 fibroblasts, or HaCaT keratinocytes were seeded at 2×10⁵ cells per well in 96-well plates. After 24 hours, cells were treated with different concentrations of Dapsone for 48 hours. Then, MTT solution was added, and after incubation, DMSO was added to solubilize the formazan. Absorbance was measured at 570 nm to determine cell viability. EC₅₀ values were calculated. [3]

Tail model efficacy study: Female BALB/c mice were infected by subcutaneous inoculation of 10⁵ infective metacyclic promastigotes of L. major at the base of the tail. After 4-6 weeks, when measurable lesions (average surface of 10 mm²) developed, topical treatment was initiated. Mice were divided into groups: untreated, treated with Dapsone cream (10% w/w), or treated with DAP-PLE (10% w/v). Treatments were administered topically twice daily with 50 mg of the formulation for a period of 30 days. Lesion size was measured every 3 days. [3]
Ear model efficacy study: Mice were infected by subcutaneous inoculation of 10³ infective metacyclic promastigotes of L. major in the ear. When lesion volume reached approximately 5 mm³, topical treatment with DAP-PLE was initiated (50 mg, twice daily). Treatment was stopped at day 10 due to severe lesion progression. [3]
Pharmacokinetic and toxicity sampling: During the tail model study, blood samples were drawn at various time points (days 0, 1, 2, 3, 12, 18) prior to the morning application to determine plasma Dapsone concentrations, hemoglobin, and hematocrit levels. At the end of the study, spleen, liver, lymph nodes, and skin lesions were collected for parasite burden quantification (by qPCR) and iron content analysis (in spleen). [3]

Absorption, Distribution and Excretion
Bioavailability after oral administration is 70% to 80%.
Renal
Absorption after oral administration is almost complete… For every 1 kg increase in body weight above 62.3 kg, clearance (CL) increases by 0.03 L/h, and volume of distribution (Vd) increases by 0.7 L. Dapsone undergoes N-acetylation via NAT2. Dapsone is oxidized to hydroxylamine via CYP2E1, with less effect from CYP2C. Hydroxylamine enters erythrocytes, leading to the formation of methemoglobin. Sulfones tend to remain in the skin, muscle, and especially the liver and kidneys for up to 3 weeks. Sulfones excreted in bile are reabsorbed in the intestines, leading to prolonged blood retention; therefore, periodic interruption of treatment is recommended. The concentration ratio of epithelial lining fluid to plasma is 0.76 to 2.91; the concentration ratio of cerebrospinal fluid to plasma is 0.21 to 2.01. Approximately 70-80% of dapsone doses are excreted in the urine as acid-labile mono-N-glucuronide and mono-N-sulfamate. After oral administration, dapsone is rapidly and almost completely absorbed from the gastrointestinal tract, reaching peak serum concentrations within 4-8 hours. The volume of distribution of dapsone in adults is reported to be 1.5-2.5 L/kg. Dapsone is distributed in most body tissues. It has been reported that dapsone can remain in the skin, muscles, kidneys, and liver; trace amounts may persist in these tissues for up to 3 weeks after discontinuation of dapsone treatment. Dapsone is also distributed in sweat, saliva, sputum, and tears, and in bile. Dapsone can cross the placental barrier. Dapsone is also distributed in breast milk. Dapsone binds to plasma proteins at a rate of 50-90%. The major metabolite of dapsone, monoacetyl dapsone, is almost completely bound to plasma proteins. Approximately 20% of each dose of dapsone is excreted unchanged in the urine, 70-85% is excreted as water-soluble metabolites in the urine, and a small amount is excreted in the feces. Dapsone is mainly excreted in the urine as acid-labile mono-N-glucuronide and mono-N-aminosulfonate derivatives, as well as some unidentified metabolites. For more complete data on the absorption, distribution, and excretion of dapsone (8 types in total), please visit the HSDB record page. Metabolites/Metabolites are primarily metabolized in the liver, mediated by CYP2E1. After 10-14 days of cell culture, 0.1 mmol and 1.0 mmol of 4'-amino-4'-hydroxyaminodiphenyl sulfone (dapsone metabolites) significantly inhibited the proliferation of bone marrow leukocyte colony-forming units. Dapsone is acetylated in the liver to produce monoacetyl and diacetyl derivatives. The major metabolite of dapsone is monoacetyl dapsone (MADDS). The acetylation rate of dapsone is genetically determined and varies from person to person, but is generally constant for each individual. The drug is also hydroxylated in the liver to hydroxylamine dapsone (NOH-DDS). NOH-DDS appears to be the cause of methemoglobinemia and hemolysis caused by this drug. Known metabolites of dapsone include N-hydroxydapsone and monoacetyldapsone. Dapsone is slowly and almost completely absorbed in the gastrointestinal tract and distributed throughout the body. It is acetylated in the liver to monoacetyl and diacetyl derivatives. The major metabolite of dapsone is monoacetyldapsone. The acetylation rate of dapsone is genetically determined and varies from person to person, but is generally constant for each individual. The drug is hydroxylated in the liver to hydroxylamine dapsone (NOH-DDS). NOH-DDS appears to be the cause of methemoglobinemia and hemolysis caused by this drug. Metabolites are primarily excreted in the urine. Only a small amount of dapsone is excreted in the feces. (A617, A618)
Elimination route: kidney
Half-life: 28 hours (range 10-50 hours)

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Biological half-life
28 hours (range 10-50 hours)
Elimination half-life is 20-30 hours.
The plasma half-life of dapsone varies considerably among individuals. The plasma half-life of dapsone is 10-83 hours, with an average of 20-30 hours. Dapsone has been reported to have a plasma protein binding rate of approximately 70%. [3] The plasma concentration of dapsone was measured after topical application of dapsone cream (10% w/w) to mice infected with Leishmania. The mean plasma concentration was 23 μg/mL 8 hours after the first administration and 21 μg/mL 24 hours later. The concentration gradually decreased over time, dropping to 13 μg/mL by day 18 of treatment. [3] The theoretical steady-state flux (Jss) of sulfamic acid from saturated aqueous solution, calculated using the Potts-Guy equation, is 0.102 μg/cm²/h. [3]

Toxicity Summary
Identification and Uses: Dapsone is a white or off-white crystalline powder. Dapsone is approved for the treatment of herpetic dermatitis and leprosy. It is particularly suitable for the treatment of linear immunoglobulin A (IgA) dermatitis, bullous systemic lupus erythematosus, persistent raised erythema, and subkeratotic pustular dermatitis. It can be used as an alternative treatment for Pneumocystis carinii pneumonia (PCP) and for the prevention of Pneumocystis carinii infection. The US FDA has designated dapsone as an orphan drug for the treatment and prevention of PCP. It is also effective in treating endogenous uveitis, chronic conjunctivitis, keratitis, and keratoconjunctivitis caused by a previously unreported pathogenic microorganism, Micromyces intracellularis. Dapsone can also be used as an analytical reagent and an epoxy resin curing agent. Human Exposure and Toxicity: Methemoglobinemia is the main and persistent characteristic of dapsone poisoning. Clinical manifestations may include headache, dizziness, agitation, restlessness, nausea, vomiting, abdominal pain, bluish-gray cyanosis, tachycardia, hyperventilation, coma, convulsions, jaundice, and intravascular hemolysis. Hematologic adverse reactions are most severe in patients with G-6-PD deficiency. The incidence of dapsone allergy syndrome is approximately 0.5% to 3.6%, with a reported mortality rate of 9.9%. Dapsone may induce acute pancreatitis. Animal studies: In a 78-week study, dapsone was added to the diet of rats. Mesenchymal tumors of abdominal organs or peritoneal tissue were observed only in male rats in the low-dose and high-dose groups. The most common tumor types were fibromas, fibrosarcomas, or sarcomas of the spleen or peritoneum. When dapsone was added to the diet of mice at 500 or 1000 ppm for 78 weeks, no carcinogenicity was found in either male or female mice. In rats, intragastric administration of 100 mg/kg dapsone for 104 weeks resulted in splenic sarcoma in male rats and a high incidence of C-cell thyroid cancer in both male and female rats. Tumors developed after lifetime treatment with the maximum tolerated dose. Oral administration to sheep resulted in neurological symptoms. Goats treated with an intramammary preparation containing dapsone, chloramphenicol, and hyaluronidase developed ataxia, blindness, recumbency, abdominal distension, opisthotonus, groaning, and involuntary leg movements. Oral administration of dapsone to mice did not induce significant immunotoxicity. The mechanism of action of dapsone on bacteria and protozoa is the same as that of sulfonamides, namely, inhibiting dihydrofolate synthesis by competing with para-aminobenzoic acid for the active site of dihydropteroate synthase. The anti-inflammatory effect of this drug is unrelated to its antibacterial effect, and its mechanism is not fully elucidated.

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Hepatotoxicity
Like other sulfonamides, dapsone causes characteristic specific liver damage, manifesting as drug allergy or hypersensitivity reactions.
The typical onset is a sudden onset of fever and rash within days or weeks after drug administration, followed by jaundice. Eosinophilia or lymphocytosis is also common. Its clinical presentation may resemble acute mononucleosis and is often referred to as "sulfone syndrome," a variant of drug eruption syndrome with eosinophilia and systemic symptoms (DRESS). The pattern of liver injury is usually cholestatic or mixed and can be complex and prolonged. In rare cases, dapsone-induced liver injury can lead to acute liver failure. However, most cases resolve rapidly, usually within 2 to 4 weeks after discontinuation of dapsone, unless cholestasis is severe. Probability score: A (Known cause of clinically significant liver injury). Pregnancy and lactation effects: Dapsone can be used during lactation; however, hemolytic anemia may occur, especially in newborns and patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. The period of highest risk of hemolysis in full-term newborns (without G6PD deficiency) can be as short as 8 days after birth. Data suggests that dapsone is advantageous for treating leprosy because it kills the leprosy pathogen in breast milk. Infants should be closely monitored for signs of hemolysis, especially newborns or premature breastfed infants. Topical dapsone gel for treating acne has not been studied during breastfeeding. According to the manufacturer, the blood concentration of topical dapsone gel is only 1% of the oral 100 mg dose. Topical gel is unlikely to affect breastfed infants, but the manufacturer notes that it should not be used during breastfeeding. Until more data are available, other topical medications may be preferred. ◉ Effects on breastfed infants: A 41-day-old breastfed infant developed mild hemolytic anemia; the mother was taking 50 mg of dapsone daily. The hemolysis was likely caused by dapsone in breast milk. A woman with leprosy took dapsone, clofazimine, and rifampin during pregnancy and breastfeeding. Her infant developed skin discoloration due to clofazimine administration, which resolved after 3 months of discontinuation of breastfeeding.
◉ Effects on lactation and breast milk
No relevant published information found as of the revision date.

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Protein binding
70% to 90%

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Toxicity data
LD50: 496 mg/kg (oral, mouse)

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Interactions
……When dapsone is used in combination with trimethoprim, the plasma concentrations of both drugs are higher than when either drug is used alone. In a comparative trial of TMP/dapsone versus TMP-SMX, the observed efficacy rates were 93% and 90%, respectively.
Because these drugs have similar hematological adverse reactions, the risk of these adverse reactions may increase with the concomitant use of folic acid antagonists (such as pyrimethamine) and dapsone. Patients receiving weekly combination therapy with pyrimethamine and dapsone developed agranulocytosis during the second and third months of treatment. If pyrimethamine is used in combination with dapsone, patients should be monitored for hematological adverse reactions more frequently than usual. Because drug effects may be additive, dapsone should be used with caution in patients with G-6-PD deficiency who are receiving or exposed to other drugs or preparations that may induce hemolysis (e.g., nitrites, aniline, phenylhydrazine, naphthalene, metronidazole, nitrofurantoin, primaquine). Multiple studies have shown that concomitant administration of clofazimine does not affect the pharmacokinetics of dapsone, although a few patients receiving both drugs concurrently have reported transient increases in urinary dapsone excretion. In a study of patients with lepromatous leprosy treated with dapsone (100 mg daily) and rifampin (600 mg daily), concomitant administration of clofazimine (100 mg daily) did not affect plasma dapsone concentrations, plasma half-life, or urinary dapsone excretion. There is evidence that dapsone may reduce or counteract some of the anti-inflammatory effects of clofazimine.
It has been reported that approximately 40% of HIV-infected patients receiving didanoxin failed to prevent Pneumocystis carinii pneumonia (PCP) while taking dapsone. This failure rate is significantly higher than the failure rates observed in other studies where dapsone was not used in combination with didanoxin, or in patients receiving sulfamethoxazole-trimethoprim or pentamidine spray for PCP prophylaxis. Although the potential for pharmacokinetic interactions has not been assessed in these patients, studies suggest that buffers present in didanoxin formulations (which maintain a pH of 7–8 to promote gastrointestinal absorption of antiretroviral drugs) may interfere with the gastrointestinal absorption of dapsone.
More complete data on interactions with dapsone (9 in total) can be found on the HSDB record page.

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Non-human toxicity values
Oral LD50 in rats: 1000 mg/kg
Intraperitoneal LD50 in rats: 196 mg/kg
Oral LD50 in mice: 375 mg/kg
Intraperitoneal LD50 in mice: 313 mg/kg
Subcutaneous LD50 in mice: 329 mg/kg

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In BALB/c mice infected with Leishmania, topical application of dapsone cream (10% w/w) for 18 days resulted in systemic toxicity manifested as significant anemia. The hemoglobin level (12.4 g/dL vs. 17.1 g/dL in the control group) and hematocrit (38.9% vs. 51.3%) in the treated mice were significantly reduced. [3]
As anemia was observed, a significant increase in iron accumulation was observed in the spleen of mice treated with dapsone (3.54 mg/g vs. 2.89 mg/g in the control group). [3] Topical application of DAP-PLE can cause local skin adverse reactions, including irritation, desquamation, and hair loss at the application site. [3] Dapsone hydroxylamine (DHA), the main metabolite that causes dapsone side effects, cannot be produced in mice, suggesting that mice are not an ideal model for studying the systemic toxicity profile of dapsone. [3] Oral administration of dapsone is limited by serious toxic reactions, including hemolytic anemia and methemoglobinemia. [3]

[1]. Dapsone and sulfones in dermatology: overview and update. J Am Acad Dermatol

[2]. Interaction of Pneumocystis carinii dihydropteroate synthase with sulfonamides and diaminodiphenyl sulfone (dapsone).J Infect Dis. 1994 Feb;169(2):456-9.

[3]. Evaluation of Skin Permeation and Retention of Topical Dapsone in Murine Cutaneous Leishmaniasis Lesions.Pharmaceutics. 2019 Nov 13;11(11):607.

Therapeutic Uses
Anti-infective drugs; antimalarial drugs; folic acid antagonists; leprosy treatment drugs. 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 being studied); 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). Dapsone is included in the database. Dapsone is an oral medication. Dapsone is often used in combination with chlorpromazine to treat malaria. Dapsone is also used for the treatment and prevention of Pneumocystis carinii infection, as well as the prevention of Toxoplasma gondii infection… Its anti-inflammatory effect forms the basis for the treatment of pemphigoid, herpetic dermatitis, linear IgA bullous diseases, relapsing chondritis, and ulcers caused by brown spider spiders. Dapsone is approved for the treatment of herpetic dermatitis and leprosy. It is particularly suitable for the treatment of linear immunoglobulin (IgA) dermatitis, bullous systemic lupus erythematosus, persistent raised erythema, and subkeratotic pustular dermatitis. For more complete data on the therapeutic uses of dapsone (14 items in total), please visit the HSDB record page.

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Drug Warnings
Rarely, peripheral neuropathy with loss of motor function has been reported in patients receiving high-dose dapsone (200-500 mg daily). Insomnia, headache, nervousness, dizziness, and psychosis have also been reported after taking dapsone.
Resistance to dapsone in Plasmodium falciparum, Plasmodium karyotae, and Mycobacterium leprae primarily stems from gene mutations encoding dihydropteroate synthase.
Hemolysis occurs in almost all patients taking 200-300 mg of dapsone daily. …Methemoglobinemia is also common. A gene defect in NADH-dependent methemoglobin reductase can lead to severe methemoglobinemia after dapsone administration. In addition, isolated cases have been reported with headache, tension, insomnia, blurred vision, paresthesia, reversible peripheral neuropathy (thought to be caused by axonal degeneration), drug fever, hematuria, pruritus, and various rashes. A potentially fatal infectious mononucleosis-like syndrome occurs occasionally.
Glucose-6-phosphate dehydrogenase (G6PD) protects red blood cells from oxidative damage. However, nearly 500 million people worldwide suffer from G6PD deficiency, the most common being G6PD-A-, one of 100 variants. Dapsone is an oxidizing agent that can cause severe hemolysis in patients with G6PD deficiency. Therefore, G6PD deficiency testing should be performed whenever possible before using dapsone. For more complete data on drug warnings for dapsone (11 in total), please visit the HSDB records page. Pharmacodynamics Dapsone is a sulfone drug with anti-inflammatory, immunosuppressive, antibacterial, and antibiotic properties. Dapsone is a leading drug in multidrug therapy recommended by the World Health Organization for the treatment of leprosy. As an anti-infective, dapsone is also used to treat malaria and, in recent years, to treat Pneumocystis carinii pneumonia in AIDS patients. Dapsone is rapidly and almost completely absorbed from the gastrointestinal tract. It is distributed throughout the body's water and is present in all tissues. However, it tends to remain in the skin and muscles, especially in the liver and kidneys: traces of the drug can be detected in these organs for up to 3 weeks after discontinuation. Dapsone (4,4′-diaminodiphenyl sulfone) is a sulfone derivative with antibacterial and anti-inflammatory properties. It was first used in the 1940s to treat leprosy, and later to treat skin diseases such as acne and herpetic dermatitis. [3] Some clinical studies have reported that oral dapsone can treat cutaneous leishmaniasis (CL) with efficacy, but its use is limited due to its poor water solubility, low bioavailability and hemotoxicity. [3] Some countries have approved topical dapsone preparations (e.g., 5% gel) for the treatment of acne vulgaris and for the treatment of other inflammatory skin diseases. [3] The physicochemical properties of dapsone include a molecular weight of 248.3, a LogP value of 0.94 and a melting point of 175-176°C. [3] The study concluded that, among the topical preparations tested, dapoxetine at a dose of 500 mg/kg/day did not have sufficient safety due to its poor skin retention and systemic toxicity (anemia) and was not suitable for the topical treatment of cutaneous leishmaniasis. [3]

C12H12N2O2S

248.3009

248.061

C, 58.05; H, 4.87; N, 11.28; O, 12.89; S, 12.91

80-08-0

Dapsone-d8;557794-38-4;Dapsone-d4;1346602-12-7;Dapsone-13C12;1632119-29-9

2955

Crystals from 95% ethanol
White or creamy white crystalline powder

1.4±0.1 g/cm3

511.7±35.0 °C at 760 mmHg

175-177 °C(lit.)

263.2±25.9 °C

0.0±1.3 mmHg at 25°C

1.662

0.94

2

4

2

17

306

0

O=S(C1C=CC(N)=CC=1)(C1C=CC(N)=CC=1)=O

MQJKPEGWNLWLTK-UHFFFAOYSA-N

InChI=1S/C12H12N2O2S/c13-9-1-5-11(6-2-9)17(15,16)12-7-3-10(14)4-8-12/h1-8H,13-14H2

4-(4-aminophenyl)sulfonylaniline

Diaphenylsulfone; 4,4'-Sulfonyldianiline; 4,4'-Diaminodiphenyl sulfone; Sulfona; 4,4′-Diaminodiphenyl sulfone; DDS

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 : 49~250 mg/mL ( 197.34~1006.85 mM )
Ethanol : ~10 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.07 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 (10.07 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 (10.07 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: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.5 mg/mL (10.07 mM)

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