Human Endogenous Metabolite

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 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. Cobalamin is primarily metabolized in the liver. They are absorbed in the ileum and stored in the liver. They continuously circulate in the enterohepatic system via bile secretion. Part of the dose is excreted in the urine, with the majority excreted within the first 8 hours. Cobalt is absorbed through the lungs, gastrointestinal tract, and skin. As it is a component of vitamin B12 (cyanocobalamin), it is distributed throughout most tissues of the body. It is transported via the bloodstream, usually bound to albumin, with the highest concentrations found in the liver and kidneys. Cobalt is primarily excreted in urine and feces. (L29)
Elimination pathway: Each hydroxycobalamin molecule can bind to a cyanide ion by substituting the hydroxyl ligand linked to a trivalent cobalt ion, forming cyanocobalamin, which is then excreted in the urine.
Half-life: Approximately 6 days (peak plasma concentration reached 8-12 hours after oral administration)
Biological half-life
Approximately 6 days (peak plasma concentration 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.

Toxicity Summary
Vitamin B12 exists primarily in four forms, collectively known as cobalamin: deoxyadenosylcobalamin, methylcobalamin, hydroxycobalamin, and cyanocobalamin. Methylcobalamin and 5-deoxyadenosylcobalamin are the two 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 a crucial step in the extraction of energy from proteins and fats. Furthermore, succinyl-CoA is essential for the synthesis of hemoglobin (the oxygen-carrying substance in red blood cells). 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.

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Toxicity Data
LD50: >50 mL/kg (intravenous injection, mice) (L1865)Interactions
It has been reported that co-administration of chloramphenicol and vitamin B12 may antagonize the hematopoietic response to vitamin B12 in patients with vitamin B12 deficiency. Patients taking these two drugs concurrently should be closely monitored for hematologic responses to vitamin B12, and alternative anti-infective agents should be considered.
/Vitamin B12/
Prednisone has been reported to increase the absorption of vitamin B12 and the secretion of intrinsic factor (IF) in a small number of patients with pernicious anemia, but this effect is not observed 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 can destroy large amounts of dietary vitamin B12; this possibility should be considered if a large dose of ascorbic acid is ingested within 1 hour after oral administration of vitamin B12.
/Vitamin B12/
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 2 weeks can all reduce the gastrointestinal absorption of vitamin B12. Neomycin-induced malabsorption of vitamin B12 may be exacerbated by concomitant use of colchicine. /Vitamin B12
Caution should be exercised when using other cyanide antidotes and cyanide neutralizing agents concurrently, as the safety of combined use has not been established. If it is decided to use other cyanide antidotes and cyanide neutralizing agents concurrently, they should not be administered simultaneously via the same intravenous infusion line.

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

[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.

Hydroxocobalamin, also known as vitamin B12a or simply hydroxycobalamin, is an injectable form of vitamin B12 that was previously used to treat vitamin B12 deficiency. It was also used to treat cyanide poisoning, Leber's optic atrophy, and toxic amblyopia. Hydroxocobalamin is an antidote. It is a synthetic vitamin B12 available as a dietary supplement to treat vitamin B12 deficiency. After administration, hydroxycobalamin mimics vitamin B12 and acts as an important cofactor for various cellular responses required for cell growth, replication, and hematopoiesis. Hydroxocobalamin is only present in individuals who have used or taken the drug. It is an injectable form of vitamin B12 that was previously used to treat vitamin B12 deficiency. [PubChem] An injectable vitamin B12 preparation used to treat vitamin B12 deficiency. See also: Hydroxocobalamin acetate (active ingredient); Hydroxocobalamin hydrochloride (active ingredient). Indications For the treatment of 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. Cyanide kits should be used in conjunction with appropriate decontamination and supportive measures. 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 to convert L-methylmalonyl-CoA to succinyl-CoA. This conversion is a crucial step in the extraction of energy from proteins and fats. Furthermore, 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. Additionally, 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 Use
Important for treating known or suspected cyanide poisoning.

Pernicious anemia, including simple pernicious anemia and pernicious anemia with neurological involvement.

The U.S. government considers cyanide one of the most likely chemical terrorist agents. Unlike many other biological or chemical agents, which have little or no defense against cyanide, the effects of cyanide on individual and public health can be largely mitigated through proper preparedness and response. Because currently available cyanide antidotes in the U.S. 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 U.S. 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 hydroxycobalamin, 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 countermeasures. For more complete data on the therapeutic uses of hydroxycobalamin (one of 10), please visit the HSDB records page. Drug Warnings Caution should be exercised when using other cyanide antidotes and cyanocobalamin concurrently, as the safety of combined use has not been established. If a decision is made to use both cyanide antidotes and cyanocobalamin concurrently, both drugs should not be infused simultaneously in the same intravenous line. Caution should be exercised when using hydroxycobalamin or cyanocobalamin in patients with a known hypersensitivity to either. If alternative therapies are available, they should be considered. Hypersensitivity reactions may include: anaphylactic shock, chest tightness, edema, urticaria, itching, difficulty breathing, and rash. Post-marketing surveillance has also reported allergic reactions, including angioedema.
Maternal use generally compatible with breastfeeding: Vitamin B12: Signs or symptoms reported by infants or effects on lactation: None. /Excerpt from Table 6/
While measuring blood cyanide levels is not a necessary condition for treatment of cyanide poisoning, nor should it delay the use of cyanide antidote (Cyanokit), collecting blood samples 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 succinic acid. Therefore, a deficiency of this enzyme may interfere with the production of lipoproteins in myelin tissue, leading to nervous system damage. The second cofactor, methylcobalamin, is essential for the conversion of homocysteine to methionine, a key substance in folate metabolism. Tetrahydrofolate deficiency leads to reduced thymidine synthesis, which in turn reduces DNA synthesis, crucial for cell maturation. Vitamin B12 also participates in maintaining the reduced state of sulfhydryl groups; its deficiency results in decreased levels of reduced sulfhydryl groups in erythrocytes and hepatocytes. In summary, vitamin B12 acts as a coenzyme in various metabolic functions, including fat and carbohydrate metabolism and protein synthesis. It is essential for growth, cell replication, hematopoiesis, nucleoproteins, and myelin synthesis. This is primarily due to its influence on the metabolism of methionine, folate, and malonic acid.

C62H89CON13O15P

1346.36

1345.567

13422-51-0

Hydroxocobalamin monohydrochloride;59461-30-2;Hydroxocobalamin acetate;22465-48-1;Hydroxocobalamin hydrochloride;58288-50-9

44475014

Purple to black solid powder

200ºC (decomposes)

6.438

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(=C\7/[C@@]([C@@H](C(=N7)/C=C\8/C([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.[OH-].[Co+3]

YOZNUFWCRFCGIH-WZHZPDAFSA-K

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;+3;/p-3/t31-,34-,35-,36-,37+,41-,52-,53-,56-,57+,59-,60+,61+,62+;;/m1../s1

cobalt(3+);[(2R,3S,4R,5S)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2R)-1-[3-[(1R,2R,3R,4Z,7S,9Z,12S,13S,14Z,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;hydroxide

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, avoid exposure to moisture.

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

DMSO: 25 mg/mL (18.57 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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