CA IX/carbonic anhydrase (IC50 = 30 nM)

Acetazolamide also inhibits hCA II with an IC50 of 130 nM[1]. As a tiny heteroaromatic sulfonamide, acetazolamide (Ace) binds to several carbonic anhydrases with great affinity and inhibits the activity of carbonic anhydrases (CAs)[2]. The treatments with high Acetazolamide concentration (AceH, 50 nM), Cisplatin (Cis; 1 μg/mL), and Cis in combination with low Acetazolamide concentration (AceL, 10 nM) greatly decreased the viability of Hep-2 cells in comparison to the control group[2]. P53 expression levels are markedly elevated upon treatment with the Acetazolamide/Cis combination, as seen by the significantly higher P53 protein expression levels following both AceL+Cis and AceH+Cis treatments in comparison to the control group. Comparing the Ace/Cis combo treatment to the control group, it reduces the bcl-2/bax expression ratio considerably and enhances the expression of the caspase-3 protein. When compared to the control group, the AceL, AceH, Cis, and AceL+Cis therapies dramatically lower the bcl-2/bax ratio[2]. Hep-2 cell apoptosis is efficiently promoted by combined Ace and Cis treatment[2]. The expression of AQP1 mRNA in Hep-2 cells is significantly reduced by combined Ace/Cis therapy. In Hep-2 cells, the AceH and AceL+Cis treatments both reduce the expression of aquaporin-1 (AQP1) mRNA in comparison to the control group[2].

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Acetazolamide (AZ) , MS-275 and AZ + MS-275 treatments inhibit growth of NB SH-SY5Y cells. AZ, MS-275 and AZ + MS-275 treatments reduced migration capacity in NB SH-SY5Y cells. Results: We evaluated the antitumor potential of the HDAC inhibitor (HDACi), pyridylmethyl-N-{4-[(2-aminophenyl)-carbamoyl]-benzyl}-carbamate (MS-275) in combination with a pan CA inhibitor, acetazolamide (AZ) on NB SH-SY5Y, SK-N-SH and SK-N-BE(2) cells. The key observation was that the combination AZ + MS-275 significantly inhibited growth, induced cell cycle arrest and apoptosis, and reduced migration capacity of NB cell line SH-SY5Y. [2]

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The aim of the present study was to determine whether acetazolamide (Ace) treatment enhances the chemosensitivity of Hep-2 laryngeal cells to cisplatin (Cis). At the logarithmic growth phase, Hep-2 cells were treated with Ace, Cis or both, and cell viability was detected using an MTT assay. The degree of apoptosis was detected using flow cytometry. Expression levels of apoptosis-related proteins, including BCL2 apoptosis regulator (bcl-2), BCL2 associated X (bax) and caspase-3, and of proliferation-related proteins, including proliferating cell nuclear antigen (PCNA) and tumor protein p53 (P53), were detected using western blotting. mRNA expression levels of aquaporin-1 (AQP1) in each group were detected using reverse transcription-polymerase chain reaction. Compared with the drugs used alone, treatment with both Ace and Cis displayed synergistic effects on the growth inhibition and apoptosis induction in Hep-2 cells. The Ace/Cis combination decreased the expression of PCNA but increased the expression of p53. In addition, the combination treatment decreased the ratio of bcl-2/bax and increased the expression of caspase-3, as well as decreased the expression of AQP1. These results demonstrated that the combined use of Ace and Cis enhanced the chemosensitivity of laryngeal carcinoma cells.[3]

In neuroblastoma (NB) SH-SY5Y xenografts, acetazolamide (40 mg/kg) greatly amplifies the inhibitory effect of MS-275 on tumorigenesis[3]. ? In NB SH-SY5Y xenografts, acetazolamide (40 mg/kg) and/or MS-275 therapy decrease HIF1-α and CAIX expression[3]. ? In NB SH-SY5Y xenografts, the expression of mitotic and proliferative markers is decreased by acetazolamide (40 mg/kg), MS-275, and acetazolamide+MS-275[3]. ? The gonococcal load in the vagina of infected mice is dramatically reduced by 90% when acetazolamide (50 mg/kg; PO) is administered for three days[6].

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Gonococcal infections represent an urgent public health threat worldwide due to the increasing incidence of infections that has been accompanied by an increase in bacterial resistance to most antibiotics. This has resulted in a dwindling number of effective treatment options. Undoubtedly, there is a critical need to develop new, effective anti-gonococcal agents. In an effort to discover new anti-gonococcal therapeutics, we previously identified Acetazolamide, a carbonic anhydrase inhibitor, as a novel inhibitor of Neisseria gonorrhoeae. Acetazolamide exhibited potent anti-gonococcal activity in vitro as it inhibited growth of strains of N. gonorrhoeae at concentrations that ranged from 0.5 to 4 μg/mL. The aim of this study was to investigate the in vivo efficacy of acetazolamide in a mouse model of N. gonorrhoeae genital tract infection. Compared to vehicle-treated mice, acetazolamide significantly reduced the gonococcal burden by 90% in the vagina of infected mice after three days of treatment. These results indicate that acetazolamide warrants further investigation as a promising treatment option to supplement the limited pipeline of anti-gonococcal therapeutics.[6]

AlamarBlue cytotoxicity assay [2]
Standard protocol was performed as describe. Percent survival vs. control (DMSO- 0.2x10−4μM) of cells when treated with Acetazolamide (AZ) , MS-275 and AZ + MS-275 were observed using AlamarBlue agent agent (10% of total volume) was added to each well for 4 h before fluorometric detection. Fluorescence was measured using the SPECTRAmax Gemini Spectrophotometer (excitation 540 nm; emission 590 nm).

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Propidium Iodide cell cycle assay [2]
Briefly, 2 × 106 cells treated with Acetazolamide (AZ) and/or MS-275 were lifted by citrate saline and fixed in 80% ice-cold ethanol for 48 h. Cells were then pelleted and re-suspended in 2 mg/mL RNase A for 5 min. A 0.1 mg/mL propidium iodide solution was added, incubated for 30 min at RT, and cells filtered through a cell-strainer into a 5 mL polystyrene tube. Labeled cells were analyzed on a BD FACSCAN flow cytometer. Data was fitted by the Watson-Pragmatic model on FlowJo Software.

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Wound healing assay [2]
SH-SY5Y cells were seeded in a 48-well plate on glass cover slips and allowed to adhere overnight at a density of 105 cells/well in 500 μl culture medium in triplicate. Wells were marked with a straight black line on the bottom for orientation. At the time of 90% confluence, cell monolayers were scratched with a 200 μl pipette tip using the marker guide. Loosened non-adherent cells were washed off with medium. Fresh medium was added to the cultures with additions of Acetazolamide (AZ) (10 μM, 20 μM, 40 μM) and MS-275 (0.75 μM, 1.5 μM and 3 μM) and cultured for 48 h. After the 48 h period cells were washed with PBS and fixed in 4% paraformaldehyde. After three washes in PBS, cells were stained with 1% Crystal violet in 20% methanol. Phase contrast light microscopic images (10x original magnification) were taken at time points of 0, 48 and 72 h of treatment. Migrated cells were counted manually to quantify numbers of cells migrated to wound area using NIH Image J program. Each experiment was conducted three times in triplicate and one representative assay is shown.

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For the drug treatments, Hep-2 cells were treated with Acetazolamide (ACE) (a low concentration of 1×10−8 mol/l, termed here Acetazolamide (ACE)L; or a high concentration of 5×10−8 mol/l, termed here AceH), Cis (1 µg/ml) alone, or Cis in combination with Ace (AceL+Cis, or AceH+Cis) for 48 h. Cells that were treated with equal volumes of vehicle were used as control. Ace was used at 1×10−8 or 5×10−8 mol/l in all experiments. Cis was used at 1 µg/ml in all experiments. Both Cis and Ace were dissolved in dimethyl sulfoxide (DMSO) and then added to PBS to dilute to the final working concentrations. The final concentration of DMSO in cultures did not exceed 0.5%. HUVECs were treated with AceH alone, Cis alone or in combination (AceH+Cis) or control (vehicle) for 48 h.[3]

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Annexin V apoptosis assay [3]
Quantification of apoptotic cells was performed by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining using a FITC-Annexin V Apoptosis Detection kit. At the logarithmic growth phase, Hep-2 cells were placed in 6-well plates. The cells were treated with Acetazolamide (ACE)L, AceH, Cis, AceL+Cis, AceH+Cis, or vehicle for 48 h. Then, cells were washed in PBS, digested with trypsin, and resuspended in calcium-enriched HEPES buffer. This suspension was stained with Annexin V-FITC and PI for 15 min, as per the manufacturer's instructions. Finally, the cells were analyzed by FlowJo software.

Xenograft studies for determining the in vivo efficacy of Acetazolamide (AZ) , MS-275, and Acetazolamide (AZ)  + MS-275 combination [2]
For the in vivo xenograft study, 4–6 weeks-old female NOD/SCID mice were obtained from the animal facility. Subcutaneous xenograft tumors were developed by injecting SH-SY5Y cells (2 × 106) into the inguinal fat pad of NOD/SCID mice. When tumor diameter reached 0.5 cm, the mice were randomized into four groups (5 mice per group). The control and treatment groups received intraperitoneal injections of vehicle (PBS) or Acetazolamide (AZ) (40 mg/kg), MS-275 (20 mg/kg) or the combination, respectively, every day for 2 weeks. Experiments were terminated when tumor sizes exceeded 2 cm3 in volume or animals showed signs of morbidity. Tumor diameters were measured on a daily basis until termination. The long (D) and short diameters (d) were measured with calipers. Tumor volume (cm3) was calculated as V = 0.5 × D × d2. After euthanizing the mice, tumors were resected, weighed and fixed in 10% neutral-buffered formalin at room temperature and processed for histopathology. For the in vivo serial heterotransplantation analysis, 2x106 untreated and pretreated Acetazolamide (AZ)  + MS-275 cells, manually and enzymatically dissociated from treated tumors, were injected subcutaneously to NOD/SCID mice. Growth rates were measured 2–3 times per week. On the 38th day, the animals were sacrificed, after which tumors were removed and weighed.

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Mice infection and treatment [6]
Two days after pellet implantation (Day 0), the vagina of each mouse was rinsed with 50 mM HEPES (pH = 7.4), and each mouse was inoculated intravaginally with 20 μL of the prepared bacterial suspension of N. gonorrhoeae FA1090 (1.2 × 108 CFU/mL). Two days post-infection (Day +2), mice were randomly allocated into groups (n=10) and administered either Acetazolamide (50 mg/kg) or the vehicle (DMSO-Tween 80-PBS, 1:1:8) orally for three consecutive days. As a positive control, one group of mice was administered a single intraperitoneal dose of ceftriaxone (15 mg/kg in water).

Absorption, Distribution and Excretion
Carbonic anhydrase inhibitors are avidly bound by carbonic anhydrase and, accordingly, tissues rich in this enzyme will have higher concentrations of carbonic anhydrase inhibitors following systemic administration. /Carbonic Anhydrase Inhibitors/
Inhibitors of Carbonic Anhydrase. Drug: acetazolamide; Oral Absorption: nearly complete; Plasma Half-Life: 6-9 hours; and Route of Elimination: renal excretion of intact drug. /From table/
ACETAZOLAMIDE RELATED TO RESPONSE IN RABBIT; KIDNEY RESPONSE, MEASURED BY MONITORING URINE FLOW & NA ELIMINATION, URINE FLOW & NA ELIMINATION OCCUR IMMEDIATELY AFTER INJECTION CORRELATED WITH LOG DOSE.
IV BOLUS INJECTIONS OF (14)C-LABELED, ACETAZOLAMIDE WERE MADE IN RABBITS. PLASMA, URINE, & WASHED RED BLOOD CELL CONCN WERE MEASURED, THE LATTER INDICATING BOUND DRUG.
For more Absorption, Distribution and Excretion (Complete) data for ACETAZOLAMIDE (6 total), please visit the HSDB record page.

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Metabolism / Metabolites
ACETAZOLAMIDE DOSE NOT UNDERGO METABOLIC ALTERATION.

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Biological Half-Life
3 to 9 hours
Plasma half-life: 6-9 hours /From table/

Toxicity Summary
IDENTIFICATION: Acetazolamide is of synthetic origin. Acetazolamide exists as a white to faintly yellowish-white, odorless crystalline powder. Acetazolamide is very slightly soluble in water and only slightly soluble in ethanol (approx 750 g/l); it is practically insoluble in ether and chloroform. Uses: Preoperative management of closed-angle glaucoma, or as an adjunct in the treatment of open-angle glaucoma. Abnormal retention of fluid: drug-induced edema, obesity, and congestive cardiac failure. Epilepsy Metabolic alkalemia. Periodic paralysis HUMAN EXPOSURE: Patients with either acute or chronic overdosage with acetazolamide may show signs of dehydration with thirst, lethargy, confusion, poor skin turgor, and prolonged capillary refill time, but may have a paradoxical continued diuresis. Electrolyte abnormalities include hyponatremia, hypokalemia, and a non-anion gap hyperchloremic metabolic acidosis in the more than mild ingestion which may lead to further deterioration in mental status, production of seizures, electrocardiographic abnormalities, and arrhythmias. Prior renal insufficiency will lead to increased toxicity at a given dose. There are idiosyncratic reactions producing bone marrow suppression with hepatic and renal insufficiency. Acetazolamide may also precipitate in the renal tubules producing calculi with renal colic. Hypokalemia may lead to muscular weakness, hyporeflexia, and hypochloremic metabolic alkalosis. In geriatric patients, a chronic metabolic acidosis may lead to a chronic compensatory hyperventilation which increases pulmonary vascular resistance and decreases left ventricular function. This can be especially significant in patients on concurrent beta-blocker or calcium channel blocker therapy. The ventricular fibrillation threshold may then be reduced. Cardiac arrhythmias may occur due to potassium deficiency. Abuse or overdose may result in pancreatitis. Hyperglycemia, hyperuricemia, and hyperlipidemia may occur with acute overdose or in chronic use or abuse. Hypersensitivity reactions such as rash, photosensitivity, thrombocytopenia, and pancreatitis are rare. Contraindications: Renal hyperchloremic acidosis. Addison's disease and all types of suprarenal gland failure. Conditions where there is known depletion of sodium and potassium (at least until this is treated). Long-term administration is contraindicated in patients with chronic closed angle-closure glaucoma. Known sensitivity to sulfonamides. Acetazolamide should not be used to alkalinize urine following salicylate overdose since it may worsen metabolic acidosis. Acetazolamide is well absorbed from the gastrointestinal tract. Acetazolamide is distributed throughout body tissues; it concentrates principally in erythrocytes, plasma and kidneys and to a lesser extent in liver, muscles, eyes and the central nervous system. Acetazolamide does not accumulate in tissues. The drug crosses the placenta in unknown quantities. Acetazolamide is tightly bound to carbonic anhydrase and high concentrations are present in tissues containing this enzyme such as erythrocytes and the renal cortex. There is a small amount of irreversible binding to red cells. It is 70 to 90% bound to plasma protein. Acetazolamide is not metabolized. Acetazolamide is excreted unchanged by the kidneys via tubular secretion and passive reabsorption. There is no evidence of enterohepatic circulation although small amounts of unchanged drug are eliminated in the bile. Acetazolamide is a carbonic anhydrase inhibitor. Acetazolamide reduces the formation of hydrogen and bicarbonate ions from carbon dioxide and water by noncompetitive, reversible inhibition of the enzyme carbonic anhydrase, thereby reducing the availability of these ions for active transport into secretions. One patient died of cholestatic jaundice after taking 13 g of acetazolamide in 26 days. In one patient, fatal bone marrow depression with leukopenia, thrombocytopenia, and anemia occurred after therapy with 500 mg of acetazolamide twice daily for 14 weeks. One case of renal failure (anuria) occurred in a patient after taking 500 mg of acetazolamide twice daily for 2 weeks. There have been no reports of congenital defects despite past widespread use though one women on 750 mg per day for glaucoma during the 1st and 2nd trimester had a baby with a sacrococcygeal teratoma but no causal link could be made. Teratogenicity tests in rats and mice showed the absence of fourth and fifth digits from the right forelimb in the offspring of rats and mice. There were no apparent lessions in the newborn of rabbits and monkeys. The drug crosses the placenta in unknown quantities. Potentially hazardous interactions: The effects of folic acid antagonists, oral hypoglycaemic agents and oral anticoagulants may be increased by acetazolamide. The urinary antiseptic effect of methenamine may be prevented by acetazolamide by keeping the urine alkaline. The alkalinization of the urine by acetazolamide can reduce the urinary excretion of many weak bases (including amphetamine, quinine, quinidine, and diethylcarbamazine) and thus enhance their pharmacological effects. In one patient taking phenytoin and acetazolamide drug-induced osteomalacia was reported. The more serious effects include blood disorders, skin toxicity and renal stone formation. Stevens-Johnson syndrome has not been reported. Symptomatic adverse effects: Flushing, thirst, headache, drowsiness, dizziness, fatigue, irritability, excitement, paresthesias, ataxia, hyperpnoa and gastrointestinal disturbances have all been reported (Dollery). Oral ingestion is the usual means of exposure. There is no appreciable dermal absorption. There is no significant absorption or local irritation. ANIMAL/PLANT STUDIES: Numerous animal studies have demonstrated that the toxicity of acetazolamide was very low in the species studied (mouse, dog, rat, monkey).

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Interactions
/ACETAZOLAMIDE/ POTENTIATES MERCURIAL DIURETICS /IN ANIMALS/.
Concurrent use /of glucocorticoid corticosteroids, especially with significant mineralocorticoid activity; mineralocorticoid corticosteroids; parenteral amphotericin; corticotropin, especially prolonged therapeutic use/ with carbonic anhydrase inhibitors may result in severe hypokalemia and should be undertaken with caution; serum potassium concentrations and cardiac function should be monitored during concurrent use. /Carbonic anhydrase inhibitors/
Concurrent use of corticosteroids or corticotropin with acetazolamide sodium may increase the risk of hypernatremia and/or edema because these medications cause sodium and fluid retention; the risk with corticosteroids or corticotropin may depend on the patients's sodium requirement as determined by the condition being treated. /Acetazolamide sodium/
The possibility should be considered that concurrent chronic use of corticosteroids or corticotropin with carbonic anhydrase inhibitors may increase the risk of hypocalcemia and osteoporosis because these medications increase calcium excretion. /Carbonic anhydrase inhibitors/
For more Interactions (Complete) data for ACETAZOLAMIDE (16 total), please visit the HSDB record page.

[1]. Dual-tail approach to discovery of novel carbonic anhydrase IX inhibitors by simultaneously matching the hydrophobic and hydrophilic halves of the active site. Eur J Med Chem. 2017 May 26;132:1-10.

[2]. Acetazolamide potentiates the anti-tumor potential of HDACi, MS-275, in neuroblastoma. BMC Cancer. 2017 Feb 24;17(1):156.

[3]. Combined treatment with acetazolamide and cisplatin enhances chemosensitivity in laryngeal carcinoma Hep-2 cells. Oncol Lett. 2018 Jun;15(6):9299-9306.

[4]. Acetazolamide: a forgotten diuretic agent. Cardiol Rev. 2011 Nov-Dec;19(6):276-8.

[5]. Interaction of antihypertensive acetazolamide with nonsteroidal anti-inflammatory drugs. J Photochem Photobiol B. 2013 Aug 5;125:155-63.

[6]. In vivo efficacy of acetazolamide in a mouse model of Neisseria gonorrhoeae infection. Microb Pathog. 2022 Mar;164:105454.

Therapeutic Uses
Anticonvulsants; Carbonic Anhydrase Inhibitors; Diuretics
MEDICATION (VET): IN LAMINITIS, UDDER EDEMA, ENTEROTOXEMIA, ASCITES, & GLAUCOMA IN VARIOUS SPECIES.
Carbonic anhydrase inhibitors are indicated primarily as adjuncts to other agents in the treatment of open-angle (chronic simple) glaucoma and secondary glaucoma, and to lower intraocular pressure prior to surgery for some types of glaucoma. /Carbonic anhydrase inhibitors; Included in US product labeling/
Acetazolamide is used to lower intraocular pressure in the treatment of malignant (ciliary block) glaucoma, which may occur after inflammation surgery, trauma, or use of miotics. /NOT included in US product labeling/
For more Therapeutic Uses (Complete) data for ACETAZOLAMIDE (13 total), please visit the HSDB record page.

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Drug Warnings
VET: CONTRAINDICATED IN ADRENAL FAILURE OR LOW POTASSIUM AND SODIUM SYNDROMES.
SAFE USE OF THESE AGENTS DURING PREGNANCY HAS NOT BEEN ESTABLISHED. THESE AGENTS ARE CONTRAINDICATED IN PT WITH IDIOPATHIC RENAL HYPERCHLOREMIC ACIDOSIS, RENAL FAILURE, KNOWN DEPLETION OF SODIUM & OF POTASSIUM, ADDISON'S DISEASE, & PT KNOWN TO BE SENSITIVE TO THIS CLASS OF DRUGS. /CARBONIC ANHYDRASE INHIBITORS/
DIURETICS, SUCH AS ACETAZOLAMIDE & THIAZIDES, CAN ALKALINIZE URINE & THUS THEORETICALLY WOULD LIMIT USEFULNESS OF METHENAMINE AS WELL AS ITS MANDELATE & HIPPURATE SALTS AS URINARY TRACT ANTI-INFECTIVE AGENTS.
Maternal Medication usually Compatible with Breast-Feeding: Acetazolamide: Reported Sign or Symptom in Infant or Effect on Lactation: None. /from Table 6/
For more Drug Warnings (Complete) data for ACETAZOLAMIDE (13 total), please visit the HSDB record page.

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Pharmacodynamics
Acetazolamide is a potent carbonic anhydrase inhibitor, effective in the control of fluid secretion, in the treatment of certain convulsive disorders and in the promotion of diuresis in instances of abnormal fluid retention. Acetazolamide is not a mercurial diuretic. Rather, it is a nonbacteriostatic sulfonamide possessing a chemical structure and pharmacological activity distinctly different from the bacteriostatic sulfonamides.

C4H6N4O3S2

222.237

221.988

C, 21.62; H, 2.72; N, 25.21; O, 21.60; S, 28.85

59-66-5

Acetazolamide;59-66-5; 1424-27-7 (sodium)

1986

White to off-white solid powder

1.7±0.1 g/cm3

256-261°C

1.641

-0.26

2

7

2

13

297

0

BZKPWHYZMXOIDC-UHFFFAOYSA-N

InChI=1S/C4H6N4O3S2/c1-2(9)6-3-7-8-4(12-3)13(5,10)11/h1H3,(H2,5,10,11)(H,6,7,9)

N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)acetamide

Acetazolamide; Diluran; Diamox; Defiltran; 59-66-5; Diamox; Acetamox; Nephramide; Glaupax; N-(5-Sulfamoyl-1,3,4-thiadiazol-2-yl)acetamide; Acetazolamid; PIM005; Glaupax

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 : ~50 mg/mL (~224.97 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (11.25 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 (11.25 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 (11.25 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: 1.96 mg/mL (8.82 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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 (Please use freshly prepared in vivo formulations for optimal results.)