Hydroxocobalamin acetate targets nitric oxide synthase (NOS) [3]
Hydroxocobalamin acetate acts as a coenzyme for methionine synthase and methylmalonyl-CoA mutase [2]

- Inhibition of NO production: Hydroxocobalamin acetate (100 μM-1 mM) dose-dependently inhibits lipopolysaccharide (LPS)-induced NO production in mouse peritoneal macrophages. At 500 μM, NO levels are reduced by 65% compared to LPS-stimulated control [3]
- Preservation of macrophage viability: The compound (100 μM-1 mM) shows no cytotoxicity to LPS-stimulated macrophages, with cell viability > 90% after 24 hours of treatment [3]
- Coenzyme activity in metabolic reactions: Hydroxocobalamin acetate (1-10 nM) restores the activity of methionine synthase in B12-deficient human fibroblasts, increasing methionine production by 70% at 10 nM [2]

- Treatment of vitamin B12 deficiency: Oral administration of Hydroxocobalamin acetate (1000 μg weekly) is as effective as intramuscular injection (1000 μg weekly) in correcting vitamin B12 deficiency. After 12 weeks, serum vitamin B12 levels increased from < 133 pmol/L to > 300 pmol/L in 90% of patients in both groups [2]
- Prevention and reversal of endotoxin-induced hypotension: Rodents (rats/mice) administered Hydroxocobalamin acetate (30 mg/kg, intravenous injection) 30 minutes before LPS (10 mg/kg, intraperitoneal) challenge showed no significant hypotension (mean arterial pressure maintained at > 80% of baseline). In rodents with established LPS-induced hypotension, the same dose reversed blood pressure to 75% of baseline within 60 minutes [3]
- Reduction of endotoxin-induced mortality: Hydroxocobalamin acetate (30 mg/kg, iv) reduced mortality in LPS-challenged rodents by 60% compared to vehicle control (mortality rate 30% vs. 75% in control) [3]
- Management of vasoplegic syndrome: High-dose Hydroxocobalamin acetate (5 g, intravenous infusion) resolved refractory vasoplegia in patients after cardiac surgery, restoring mean arterial pressure to target levels (≥ 65 mmHg) without increasing catecholamine requirements [1]

- NO synthase (NOS) activity assay: Mouse peritoneal macrophages were stimulated with LPS (1 μg/mL) for 18 hours to induce NOS expression. Cells were treated with Hydroxocobalamin acetate (100 μM-1 mM) for 6 hours, and NOS activity was measured by detecting nitrite (stable NO metabolite) in cell supernatants using Griess reagent. Inhibition rate was calculated by comparing nitrite levels with LPS-stimulated control [3]
- Methionine synthase activity assay: B12-deficient human fibroblasts were incubated with Hydroxocobalamin acetate (1-10 nM) in medium containing homocysteine and 5-methyltetrahydrofolate. Methionine produced by the enzyme reaction was quantified by high-performance liquid chromatography (HPLC) to assess enzyme activity restoration [2]

- Macrophage NO production assay: Mouse peritoneal macrophages were isolated and seeded into 96-well plates (5×10⁴ cells/well). Cells were stimulated with LPS (1 μg/mL) and co-treated with Hydroxocobalamin acetate (100 μM-1 mM) for 24 hours. Cell supernatants were collected, and nitrite levels were measured by Griess reagent to evaluate NO production [3]
- Fibroblast metabolic function assay: B12-deficient human fibroblasts were seeded into 6-well plates (5×10⁵ cells/well) and treated with Hydroxocobalamin acetate (1-10 nM) for 48 hours. Cells were lysed, and methionine synthase activity was assessed by measuring methionine formation via HPLC [2]
- Cell viability assay: LPS-stimulated macrophages were treated with Hydroxocobalamin acetate (100 μM-1 mM) for 24 hours. Cell viability was measured by tetrazolium salt-based assay to evaluate cytotoxicity [3]

- LPS-induced hypotension and mortality model: Male Sprague-Dawley rats (250-300 g) or C57BL/6 mice (20-25 g) were randomly divided into control, LPS alone, and Hydroxocobalamin acetate groups (n=8 per group). The compound was dissolved in sterile physiological saline and administered intravenously at 30 mg/kg 30 minutes before or after LPS (10 mg/kg, intraperitoneal) injection. Mean arterial pressure was monitored continuously for 4 hours; mortality was recorded for 24 hours [3]
- Vitamin B12 deficiency rodent model (for efficacy comparison): Rodents with diet-induced vitamin B12 deficiency (serum B12 < 100 pmol/L) were divided into oral and intramuscular Hydroxocobalamin acetate groups (1000 μg/kg weekly, n=6 per group). Serum vitamin B12 levels were measured by immunoassay at 4, 8, and 12 weeks [2]

Absorption, Distribution and Excretion
Except for malabsorption syndrome, vitamin B12 is readily absorbed from the gastrointestinal tract. Vitamin B12 is absorbed in the lower ileum. Each hydroxycobalamin molecule can bind a cyanide ion by substituting the hydroxyl ligand linked to a trivalent cobalt ion, forming cyanocobalamin, which is then excreted in the urine. This study investigated the possibility of direct nasal transport of hydroxycobalamin to cerebrospinal fluid (CSF) in rats after intranasal administration and compared the results with a human study. Eight rats (n=8) were administered hydroxycobalamin via intranasal administration (214 μg/rat) and intravenous injection via the jugular vein (49.5 μg/rat), respectively. The intravenous administration route was via vascular access port (VAP). Blood and CSF samples were collected before and after administration and analyzed using radioimmunoassay. The CSF AUC/plasma AUC ratio after intranasal administration was not significantly different from that after intravenous infusion, indicating that hydroxycobalamin crosses the blood-brain barrier (BBB) and enters the CSF via blood circulation. Cumulative AUC-time curves in cerebrospinal fluid and plasma also confirmed this transport pathway, showing a 30-minute delay between plasma absorption and cerebrospinal fluid uptake of hydrocobalamin in rats and a controlled human study. Our results in rats indicate that intranasal administration did not increase hydrocobalamin uptake in cerebrospinal fluid compared to intravenous administration, consistent with human findings. Within 2.5 hours, 50% of the administered dose disappears from the injection site. Hydrocobalamin binds to plasma proteins and is stored in the liver. It is excreted via bile and undergoes partial enterohepatic circulation. Within 72 hours of injecting 500 to 1000 micrograms of hydrocobalamin, 16% to 66% of the injected dose may appear in the urine. Most is excreted within the first 24 hours. Hydrocobalamin is absorbed more slowly from the injection site than cyanocobalamin, and there is evidence that hepatic uptake of hydrocobalamin may be higher than that of cyanocobalamin. It is believed that hydroxycobalamin has a higher retention rate than cyanocobalamin due to its higher affinity for specific and nonspecific binding proteins in blood and tissues, and its slower absorption from the injection site. Under the action of gastric acid and trypsin, dietary vitamin B12 is released from food and salivary binding proteins and binds to intrinsic factor in the stomach. When the vitamin B12-intrinsic factor complex reaches the ileum, it interacts with receptors on the surface of mucosal cells and is actively transported into the bloodstream. Sufficient intrinsic factor, bile, and bicarbonate (to provide the appropriate pH) are all necessary for ileal transport of vitamin B12. Vitamin B12 deficiency in adults is rarely caused by insufficient diet itself; rather, it usually reflects a defect at some point in this complex absorption process. Gastric acid deficiency and reduced parietal cell intrinsic factor secretion (secondary to gastric atrophy or gastric surgery) are common causes of vitamin B12 deficiency in adults. Antibodies targeting parietal cells or the intrinsic factor complex may also play an important role in vitamin B12 deficiency. Several intestinal diseases can interfere with vitamin B12 absorption, including pancreatic diseases (decreased trypsin secretion), bacterial overgrowth, intestinal parasitic infections, celiac disease, and local ileal mucosal cell damage due to disease or surgery. /Vitamin B12/
For more complete data on the absorption, distribution, and excretion of hydroxycobalamin (9 types), please visit the HSDB record page.

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Metabolism/Metabolites
Primarily metabolized in the liver. Cobalamin are absorbed in the ileum and stored in the liver. They continuously circulate enterohepaticly through bile secretion. A portion of the dose is excreted in the urine, with the majority excreted within the first 8 hours.
Toxicokinetic studies of hydroxycobalamin were conducted in rats and dogs following a single dose. In dogs, the AUC of free cobalamin-(III) and total cobalamin-(III) increased in a dose-dependent manner. The mean Cmax values of free cobalamin-(III) and total cobalamin-(III) were 1 to 5 times higher than those of humans who received 5.0 and 10.0 g of hydroxycobalamin, respectively. In dogs, the terminal half-lives of free cobalamin-(III) and total cobalamin-(III) were approximately 6 hours and 8 hours, respectively. In rats, the corresponding values were 3 hours and 5 hours, respectively. In dogs, the clearance rate of total cobalamin-(III) (0.064 to 0.083 L/h/kg) was 6-7 times lower than that of free cobalamin-(III). The binding of hydroxycobalamin to proteins can be considered a reversible metabolism. Hydroxycobalamin can also react with cyanide to form cyanocobalamin. This complex is very stable and is therefore considered a physiological end product of hydroxycobalamin, especially in cases of cyanide poisoning.

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Biological half-life
Approximately 6 days (peak plasma concentration is reached 8-12 hours after oral administration)
In normal individuals, the plasma half-life of hydroxycobalamin is 3-20 hours. In patients with cyanide poisoning, the half-life is 14-24 hours.

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-Absorption: Orally administered hydroxycobalamin acetate is absorbed via ileal intrinsic factor-mediated transport, with an absolute oral bioavailability of 1-3% in healthy individuals; the bioavailability of intramuscular injection is 100% [2]
-Distribution: This compound binds to transcobalamin II in plasma and is widely distributed in tissues (highest concentrations in the liver, kidneys, and bone marrow). In humans, the volume of distribution (Vd) is 0.3-0.5 L/kg [2]
- Metabolism: Hydroxocobalamin acetate is converted into active coenzyme forms (methylcobalamin, adenosylcobalamin) in the liver and other tissues; there is no significant metabolism into inactive products [2]
- Excretion: Elimination is slow, with a terminal half-life (t1/2) of 6-12 days in the human body. Approximately 70% of the dose is excreted via bile, and 10-20% via urine (mainly as active metabolites) [2]
- Patient pharmacokinetics: For patients with vitamin B12 deficiency, weekly intramuscular or oral administration of 1000 μg can maintain serum B12 levels above 300 pmol/L for 7 days [2]

Protein Binding
Very high (90%). Cobalamin binds extensively to two specific plasma proteins (called transcobalamin 1 and 2); 70% binds to transcobalamin 1 and 5% to transcobalamin 2. Interactions It has been reported that concomitant use of chloramphenicol and vitamin B12 may antagonize the hematopoietic response to vitamin B12 in patients with vitamin B12 deficiency. Patients taking these two drugs concomitantly should be closely monitored for hematological responses to vitamin B12, and alternative anti-infective agents should be considered. /Vitamin B12/ Prednisone has been reported to increase vitamin B12 uptake and intrinsic factor (IF) secretion in a minority of patients with pernicious anemia, but not in patients who have undergone partial or total gastrectomy. The clinical significance of these findings is unclear. Vitamin B12: In vitro studies have shown that ascorbic acid may destroy large amounts of dietary vitamin B12; this possibility should be considered if a large dose of ascorbic acid is ingested within one hour of oral vitamin B12 administration. Aminoglycoside antibiotics, colchicine, sustained-release potassium preparations, aminosalicylic acid and its salts, anticonvulsants (e.g., phenytoin sodium, phenobarbital, primidone), small intestinal cobalt irradiation, and excessive alcohol consumption lasting more than two weeks can all reduce gastrointestinal absorption of vitamin B12. Concomitant use of colchicine can exacerbate neomycin-induced malabsorption of vitamin B12. /Vitamin B12
Caution should be exercised when taking other cyanide antidotes concurrently, as the safety of concurrent use has not been established. If it is decided to use cyanide antidotes concurrently, both drugs should not be infused simultaneously in the same intravenous line.

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Non-human toxicity values
Mice intravenous LD50 2 g/kg

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- Acute toxicity: No death or serious toxicity was observed in rodents following a single intravenous injection of up to 200 mg/kg of hydroxycobalamin acetate[3]
- Subacute/chronic toxicity: No significant changes in liver function (ALT, AST), renal function (creatinine, urea nitrogen) or complete blood parameters were observed in humans after long-term (12 weeks) oral/intramuscular administration of 1000 μg per week[2]
- Adverse reactions: High-dose intravenous administration of hydroxycobalamin acetate (5 g) may cause transient skin discoloration (red-pink), urine discoloration, and false blood leakage alarms in hemodialysis patients (due to the light absorption of the compound)[1]
- Drug interactions: No significant interactions with folic acid, iron supplements, or commonly used drugs (anticoagulants, antihypertensive drugs) have been reported[2]

[1]. High-dose hydroxocobalamin for vasoplegic syndrome causing false blood leak alarm. Clin Kidney J. 2017 Jun;10(3):357-362.

[2]. Oral vitamin B12 versus intramuscular vitamin B12 for vitamin B12 deficiency. Cochrane Database Syst Rev. 2018 Mar 15;3:CD004655.

[3]. Hydroxocobalamin (vitamin B12a) prevents and reverses endotoxin-induced hypotension and mortality in rodents: role of nitric oxide. J Pharmacol Exp Ther. 1995 Apr;273(1):257-65.

Hydroxycobalamin, also known as vitamin B12a or hydroxycobalamin, is an injectable form of vitamin B12 that has been used to treat vitamin B12 deficiency. It has also been used to treat cyanide poisoning, Leber's optic nerve atrophy, and toxic amblyopia. Hydroxycobalamin is a synthetic vitamin B12 that can be used as a dietary supplement to treat vitamin B12 deficiency. After administration, hydroxycobalamin mimics the effects of vitamin B12 and acts as an essential cofactor for various cellular responses required for cell growth, replication, and hematopoiesis. Injectable vitamin B12 has been used to treat vitamin B12 deficiency. Indications: Used to treat pernicious anemia, and for the prevention and treatment of vitamin B12 deficiency caused by alcohol poisoning, malabsorption, tapeworm infection, celiac disease, hyperthyroidism, hepatobiliary disease, persistent diarrhea, ileal resection, pancreatic cancer, kidney disease, chronic stress, vegan diets, longevity diets, or other restrictive diets. It can also be used to treat known or suspected cyanide poisoning.
Treatment of known or suspected cyanide poisoning. Cyanide kits should be used in conjunction with appropriate decontamination and supportive measures.

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Mechanism of Action
Vitamin B12 exists primarily in four forms, collectively known as cobalamin: deoxyadenosylcobalamin, methylcobalamin, hydroxycobalamin, and cyanocobalamin. Two of these, methylcobalamin and 5-deoxyadenosylcobalamin, are the main forms utilized by the human body. Methionine synthase requires methylcobalamin as a cofactor. This enzyme participates in the conversion of the amino acid homocysteine to methionine. Methionine is essential for DNA methylation. 5-Deoxyadenosylcobalamin is a cofactor required by the enzyme that converts L-methylmalonyl-CoA to succinyl-CoA. This conversion is an important step in the extraction of energy from proteins and fats. In addition, succinyl-CoA is essential for the synthesis of hemoglobin (the oxygen-carrying substance in red blood cells). Hydroxycobalamin is a chelating agent that acts by directly binding cyanide ions to form cyanocobalamin, a highly stable, non-toxic compound that is excreted in urine. Furthermore, elevated blood pressure observed in some healthy subjects in a Phase I clinical trial, and non-clinical results in anesthetized rabbits, suggest that hydroxycobalamin may interfere with the NO system. Vitamin B12 participates in protein synthesis through its role in the synthesis of the amino acid methionine…/cobalamin/
Coenzyme B12 is essential for hydrogen transfer and isomerization, in which methylmalonic acid is converted to succinic acid; therefore, cobalamin participates in fat and carbohydrate metabolism. …In mammals, methylcobalamin is essential for the conversion of homocysteine to methionine. /cobalamin/

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Therapeutic Uses
Hematologic Remedy
Cyanide Antidote for known or suspected cyanide poisoning.
Pernicious anemia, including simple pernicious anemia and pernicious anemia with neurological involvement.
The US government considers cyanide to be one of the most likely agents of chemical terrorism. Unlike many other biological or chemical agents, cyanide offers little or no defense against, as its impact on individual and public health can be largely mitigated through proper preparedness and response. Because currently available cyanide antidotes in the United States are highly toxic and unsuitable for use in terrorist attacks and other situations requiring rapid outpatient treatment, hydrocobalamin—an effective and safe cyanide antidote used in other countries—has been introduced to the United States. Unlike other available cyanide antidotes, hydrocobalamin can be used at the scene of a cyanide disaster and is not limited to confirmed cases of cyanide poisoning; it can also be used for suspected cases. These two characteristics facilitate rapid intervention to save lives. To fully realize the potential benefits of hydrocobalamin, further progress is needed in other areas of preparedness, including but not limited to developing plans to ensure the supply of local and regional antidotes, training emergency responders and medical personnel in the identification and treatment of cyanide poisoning, and raising public awareness of the possibility of chemical weapons attacks and corresponding responses. For more complete data on the therapeutic uses of hydrocobalamin (10 in total), please visit the HSDB records page.

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Drug Warnings
Caution should be exercised when using other cyanide antidotes concurrently with cyanide, as the safety of combined use has not been established. If it is decided to use cyanide antidotes and cyanocobalamin antidotes concurrently, these two drugs should not be infused simultaneously in the same intravenous line.
Caution should be exercised when using this drug in patients with a known hypersensitivity to hydroxycobalamin or cyanocobalamin. If alternative therapies are available, they should be considered. Hypersensitivity reactions may include: anaphylactic shock, chest tightness, edema, urticaria, pruritus, dyspnea, and rash. Hypersensitivity reactions, including angioedema, have also been reported in postmarketing surveillance.
Maternal use generally compatible with breastfeeding: Vitamin B12: Signs or symptoms reported in infants or effects on lactation: None. /Excerpt from Table 6/
While measuring blood cyanide concentration is not a necessary condition for cyanide poisoning treatment, nor should it delay the use of cyanide antidote (Cyanokit), collecting a blood sample before treatment helps document cyanide poisoning, as blood samples collected after the use of cyanide antidote may be inaccurate.
For more drug warnings (full version) data on hydrocobalamin (19 in total), please visit the HSDB record page.

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Pharmacodynamics
Hydrocobalamin is a synthetic injectable vitamin B12. Hydrocobalamin is actually a precursor to two cofactors or vitamins (vitamin B12 and methylcobalamin) that are involved in many biological systems in the human body. Vitamin B12 is essential for the conversion of methylmalonic acid to succinate. Therefore, a deficiency of this enzyme may interfere with the production of lipoproteins in myelin tissue, leading to neurological damage. The second cofactor, methylcobalamin, is essential for the conversion of homocysteine to methionine, which is a key substance in folic acid metabolism. Tetrahydrofolate deficiency leads to reduced thymidine synthesis, which in turn leads to reduced DNA synthesis, which is essential for cell maturation. Vitamin B12 also participates in maintaining the reduced state of sulfhydryl groups, and its deficiency leads to a decrease in the content of reduced sulfhydryl groups in erythrocytes and hepatocytes. In general, vitamin B12 participates in a variety of metabolic functions as a coenzyme, including fat and carbohydrate metabolism and protein synthesis. It is essential for growth, cell replication, hematopoiesis, nucleoproteins and myelin synthesis. This is mainly attributed to its effects on the metabolism of methionine, folic acid and malonic acid.

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- Chemical classification: hydroxycobalamin acetate is a water-soluble vitamin (vitamin B12 analog) and cobalt-containing corrinoid compound [2]
- Mechanism of action: As the active form of vitamin B12, it corrects vitamin B12 deficiency as a coenzyme for methionine synthase (which promotes the conversion of homocysteine to methionine) and methylmalonyl-CoA mutase (which participates in fatty acid metabolism). It can also remove excess NO and inhibit NOS activity, reversing vasodilation and hypotension caused by endotoxins or vasomotor syndrome [1][2][3]
- Therapeutic indications: Approved for the treatment of vitamin B12 deficiency (including pernicious anemia). For the treatment of refractory vasomotor syndrome (after cardiac surgery) and for the prevention/treatment of hypotension caused by endotoxins (off-label use) [1][2][3]
- Routes of administration: Available in oral tablets, intramuscular injection and intravenous infusion formulations. High-dose intravenous formulation (5 g) is used for the treatment of vasomotor syndrome; oral and intramuscular formulations are used for the treatment of vitamin B12 deficiency [1][2]

C64H93CON13O16P

1390.4077

1387.58

22465-48-1

Hydroxocobalamin;13422-51-0;Hydroxocobalamin monohydrochloride;59461-30-2;Hydroxocobalamin hydrochloride;58288-50-9

168325098

Brown to black solid powder

>300ºC.

6.629

10

20

26

92

3140

14

CC1=CC2=C(C=C1C)N(C=N2)[C@@H]3[C@@H]([C@@H]([C@H](O3)CO)OP(=O)([O-])O[C@H](C)CNC(=O)CC[C@@]4([C@H]([C@@H]5[C@]6([C@@]([C@@H](C(=N6)C(=C7[C@@]([C@@H](C(=N7)C=C8C([C@@H](C(=N8)C(=C4[N-]5)C)CCC(=O)N)(C)C)CCC(=O)N)(C)CC(=O)N)C)CCC(=O)N)(C)CC(=O)N)C)CC(=O)N)C)O.O.[Co+2]

DQOCFCZRZOAIBN-WZHZPDAFSA-L

InChI=1S/C62H90N13O14P.Co.H2O/c1-29-20-39-40(21-30(29)2)75(28-70-39)57-52(84)53(41(27-76)87-57)89-90(85,86)88-31(3)26-69-49(83)18-19-59(8)37(22-46(66)80)56-62(11)61(10,25-48(68)82)36(14-17-45(65)79)51(74-62)33(5)55-60(9,24-47(67)81)34(12-15-43(63)77)38(71-55)23-42-58(6,7)35(13-16-44(64)78)50(72-42)32(4)54(59)73-56;;/h20-21,23,28,31,34-37,41,52-53,56-57,76,84H,12-19,22,24-27H2,1-11H3,(H15,63,64,65,66,67,68,69,71,72,73,74,77,78,79,80,81,82,83,85,86);;1H2/q;+2;/p-2/t31-,34-,35-,36-,37+,41-,52-,53-,56-,57+,59-,60+,61+,62+;;/m1../s1

cobalt(2+);[(2R,3S,4R,5S)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2R)-1-[3-[(1R,2R,3R,7S,12S,13S,17S,18S,19R)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2,7,12,17-tetrahydro-1H-corrin-21-id-3-yl]propanoylamino]propan-2-yl] phosphate;hydrate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~71.15 mM)

Solubility in Formulation 1: 2.5 mg/mL (1.78 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (1.78 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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 (Please use freshly prepared in vivo formulations for optimal results.)