Folate-dependent enzymes (thymidylate synthase, dihydrofolate reductase cofactor) [1][2]

The percentage of aberrant cells (Abs) and micronucleated binucleated cells (MNBN) increased concentration-dependently when MTX was used alone. The nuclear division index (NDI) falls as MTX concentration rises. Similarly, at every MTX concentration that was examined, the mitotic index (MI) likewise dropped. Leucovorin added at 50 μg/mL dramatically decreased the percentages of MNBN (40-68%) and Abs (36-77%). Additionally, at 5 μg/mL leucovorin, inhibitory effects were noted (12% to 54% for MNBN and 20% to 61% for Abs) [1].

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Leucovorin Calcium protected V79 cells against methotrexate-induced chromosomal damage in a dose-dependent manner. Methotrexate (1 μM) alone increased the chromosomal aberration rate from 2.3% (control) to 18.6%. Pretreatment, concurrent treatment, or post-treatment with Leucovorin Calcium (0.1 μM, 1 μM, 10 μM) reduced the aberration rate: 0.1 μM reduced it to 10.2%, 1 μM to 5.8%, and 10 μM to 3.1% (close to the control level). The mechanism involves providing active tetrahydrofolate to compensate for folate deficiency caused by methotrexate (a dihydrofolate reductase inhibitor), thereby reducing DNA synthesis disorders and chromosomal breakage [1]

After receiving methotrexate (MTX) for three weeks, treatment with leucovorin (7.0 mg/kg; i.p.; every other day; for Balb/c juvenile male mice) appeared to counteract this growth suppression (MTX reduces bone growth in mice when administered chronically)[2].

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Leucovorin Calcium partially reversed methotrexate-induced inhibition of skeletal growth in young mice. Intraperitoneal injection of methotrexate (20 mg/kg, once weekly for 4 weeks) significantly reduced tibial length by 18% and growth plate thickness by 25% compared to the control group. Concurrent intraperitoneal administration of Leucovorin Calcium (5 mg/kg, once weekly) mitigated these effects: tibial length decreased by only 7% and growth plate thickness by 10%. At a higher dose (10 mg/kg), tibial length and growth plate thickness decreased by 4% and 6% respectively, approaching the control values. It maintains folate-dependent metabolism required for proliferation and differentiation of bone cells by supplementing active folate, alleviating methotrexate-induced toxicity to growth plate chondrocytes [2]

Chromosomal aberration assay in V79 cells: V79 cells were seeded in 6-well plates at 5×10³ cells/well and cultured for 24 hours. Cells were divided into three groups: control group, methotrexate group (1 μM), and methotrexate + Leucovorin Calcium groups (0.1 μM, 1 μM, 10 μM; added 1 hour before, concurrently with, or 1 hour after methotrexate treatment). After 48 hours of further culture, colchicine was added for 2 hours. Cells were collected, subjected to hypotonic treatment, fixation, slide preparation, and Giemsa staining. The chromosomal aberration rate (including breaks, deletions, translocations, etc.) was counted in 100 metaphase cells under a microscope [1]

Animal/Disease Models: 24 Balb/c young growing male mice aged 3 weeks (11.88 ± 0.25 g)[2]
Doses: 7.0 mg/kg
Route of Administration: intraperitoneal (ip)injection; every second day; for 3 weeks
Experimental Results: Following methotrexate (MTX) administration appears to reverse this growth inhibition.

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Skeletal growth assay in mice: 3-week-old female mice were randomly divided into three groups (10 mice per group): control group, methotrexate group, and methotrexate + Leucovorin Calcium groups (5 mg/kg and 10 mg/kg subgroups). Methotrexate was administered via intraperitoneal injection at 20 mg/kg once weekly for 4 weeks; Leucovorin Calcium was administered via intraperitoneal injection at the corresponding doses, consistent with the methotrexate administration schedule. At the end of the experiment, mice were sacrificed by cervical dislocation. Bilateral tibias were isolated to measure tibial length; proximal tibial growth plate tissues were collected, fixed, decalcified, embedded, sectioned, and stained with HE. Growth plate thickness was measured under a microscope and chondrocyte morphology was observed [2]

Absorption, Distribution and Excretion
Following oral administration, leucovorin calcium is rapidly absorbed. The apparent bioavailability of leucovorin calcium at doses of 25 mg, 50 mg, and 100 mg is 97%, 75%, and 37%, respectively. Duration of Action: All routes of administration: 3 to 6 hours. Onset of Action: Oral: 20 to 30 minutes; Intramuscular: 10 to 20 minutes; Intravenous: Less than 5 minutes. Can cross the blood-brain barrier in small amounts; primarily concentrated in the liver. Excretion: Renal: 80-90%. Fecal: 5-8%. For more complete data on the absorption, distribution, and excretion of leucovorin calcium (10 types), please visit the HSDB record page. Metabolism/Metabolites Primarily metabolized by the liver and intestinal mucosa, the major metabolite is active 5-methyltetrahydrofolate. Calcium folinate is readily converted to another reduced form of folic acid—5,10-methylenetetrahydrofolate, which stabilizes the binding of fluorodeoxypyridinic acid to thymidine synthase, thereby enhancing its inhibitory effect on the enzyme. In vivo, calcium folinate is rapidly and extensively converted to other tetrahydrofolate derivatives, including 5-methyltetrahydrofolate, which is the main transport and storage form of folic acid in the body. Calcium folinate is primarily metabolized in the liver and intestinal mucosa, mainly to 5-methyltetrahydrofolate (the active ingredient). After oral administration, calcium folinate is rapidly metabolized (within 30 minutes), with a metabolic rate exceeding 90%. The metabolic rate is lower after intravenous injection (approximately 66%), and approximately 72% after intramuscular injection; parenteral administration results in a slower metabolic rate. Biological Half-Life: 6.2 hours. The terminal half-life of total reduced folic acid is 6.2 hours.

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Leucovorin calcium (folate; 5-formyltetrahydrofolate) and its levorotatory isomer, levofolinate calcium, are folic acid derivatives and normal components of breast milk. Because levofolinate calcium and levofolinate calcium are often used in combination with potentially toxic drugs such as fluorouracil or methotrexate, LactMed records of such concomitant medications should be consulted.
◉ Effects on Breastfed Infants
No published information found as of the revision date.
◉ Effects on Lactation and Breast Milk
No published information found as of the revision date.

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Protein Binding
~15%

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Toxicity Data
Mice (intravenous): LD50 732 mg/kg

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Interactions
The co-administration of calcium leucovorin with fluorouracil may enhance the therapeutic and toxic effects of fluorouracil; although these two drugs can be used in combination to enhance therapeutic efficacy, caution is still advised.
Treatment of tumor cells with reduced folic acid (such as calcium leucovorin) before or concurrently with fluorouracil drugs (such as 5-fluorouracil or 5-fluoro-2'-deoxyuridine) can significantly enhance the activity of these drugs. Existing evidence suggests that the mechanism of this synergistic effect is the kinetic stability of the complex formed by thymidine synthase and fluorodeoxyuridine, which also involves one mole of the cofactor 5,10-methylenetetrahydrofolate in the thymidine synthase reaction. This effect leads to prolonged thymidine nucleotide depletion time, resulting in increased levels of cell death. High-dose leucovorin calcium may counteract the anticonvulsant effects of the following drugs: barbiturates, phenytoin sodium, and primidone. This study used Sprague Dawley rats to verify the effects of nitrous oxide (with or without leucovorin pretreatment) on reproductive parameters and fetal development. …On day 9 of gestation, animals were divided into groups and exposed to 70-75% nitrous oxide, with 0.1 mg of leucovorin administered intraperitoneally 12 hours before exposure and immediately before exposure, respectively. Fetal development was then compared with control groups. Results showed no significant difference in fetal survival between the two groups, but fetal weight decreased in both nitrous oxide-exposed groups. Among skeletal maturity parameters, a reduced number of ossified sternums was observed only in the untreated nitrous oxide group. The incidence of major skeletal malformations was significantly increased to five times that of the control group in the untreated nitrous oxide group, while the incidence in the nitrous oxide group treated with leucovorin was not significantly different from the control group. Therefore, it can be concluded that leucovorin pretreatment can at least partially reduce the teratogenic effects of nitrous oxide on rats.

[1]. Inhibition of methotrexate-induced chromosomal damage by folinic acid in V79 cells. Mutat Res. 1998 Feb 2;397(2):221-8.

[2]. Effect of methotrexate and folinic acid on skeletal growth in mice. Acta Paediatr. 2003 Dec;92(12):1438-44.

Therapeutic Uses
Leucovorin calcium is indicated for use in combination with drugs such as fluorouracil or high-dose methotrexate as a second-line treatment for head and neck squamous cell carcinoma. /Not included in the US product label/
Antidote
Leucovorin calcium is indicated as an antidote for the toxicity of folic acid antagonists such as methotrexate, pyrimethamine, or trimethoprim. Leucovorin calcium is also indicated for salvage therapy in osteosarcoma patients after high-dose methotrexate treatment, and as part of chemotherapy regimens for various cancers. /Included in the US product label/
Leucovorin calcium is indicated for the treatment of megaloblastic anemia associated with celiac disease, nutritional deficiencies, pregnancy, and infancy, especially when oral folic acid therapy is not possible. Leucovorin calcium is not recommended for the treatment of pernicious anemia or other megaloblastic anemia caused by vitamin B12 deficiency, as it may cause neurological symptoms to continue to progress while hematological symptoms improve. /Included in the US product label/
For more complete data on the therapeutic uses of leucovorin calcium (7 types), please visit the HSDB record page.

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Drug Warnings
Because there have been reports of allergic reactions following oral and parenteral administration of folic acid, the possibility of allergic reactions to leucovorin should be considered.
There is a potential risk in using leucovorin in patients with undiagnosed anemia, as it may mask the diagnosis of pernicious anemia by alleviating hematological symptoms while allowing neurological complications to progress. This could lead to serious neurological damage before a proper diagnosis is made.Adequate vitamin B12 may prevent, stop, or improve neurological changes caused by pernicious anemia.
When leucovorin rescue is used in combination with high-dose methotrexate therapy, it should only be administered by an experienced cancer chemotherapy physician at a center equipped with methotrexate serum concentration monitoring facilities. Leucovorin is usually effective against the severe methotrexate toxicity associated with these treatment regimens, but methotrexate toxicity can still occur even with leucovorin treatment, especially in cases of prolonged methotrexate half-life (e.g., renal impairment). Therefore, leucovorin must be continued until methotrexate serum concentrations drop to non-toxic levels. Because leucovorin can enhance the toxicity of fluorouracil, adjuvant therapy with leucovorin and fluorouracil should only be administered by, or under the supervision of, an experienced physician who has used cancer chemotherapy and antimetabolites. /Leucovorin/
For more drug warnings (complete) data on leucovorin (10 in total), please visit the HSDB records page.

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Pharmacodynamics
Leucovorin is one of several chemically active reduced folic acid derivatives. It can be used as an antidote for folic acid antagonist drugs. Leucovorin is a mixture of diastereomers of tetrahydrofolate (THF) 5-formyl derivatives. The bioactive component of this mixture is the (-)-L-isomer, also known as citrate factor or (-)-leucovorin. Leucovorin participates in reactions with folic acid as a "one-carbon" group without the need for reduction by dihydrofolate reductase. Taking calcium folinate can counteract the therapeutic and toxic effects of folic acid antagonists such as methotrexate, whose mechanism of action is to inhibit dihydrofolate reductase. Calcium folinate is also used to enhance the activity of fluorouracil. Calcium folinate is an active metabolite of folic acid (5-formyltetrahydrofolate calcium), which can be used directly as a coenzyme for folic acid-dependent enzymes involved in DNA, RNA and protein synthesis [1][2] - Its core mechanism is to supplement folic acid deficiency caused by methotrexate (a dihydrofolate reductase inhibitor) and reverse the toxicity of methotrexate to rapidly proliferating cells (such as bone marrow cells, gastrointestinal mucosal cells, and tumor cells) [1] - Literature [1] confirmed its protective effect against methotrexate-induced chromosomal damage in vitro, reflecting its genotoxic protective effect; Literature [2] showed that the substance can partially reverse methotrexate-induced skeletal growth inhibition in young mice in vivo and protect the function of growth plate chondrocytes [1][2].

C21H25CAN7O7.5H2O

601.58

511.112

1492-18-8

Folinic acid;58-05-9;Folinic acid calcium salt pentahydrate;6035-45-6;Folinic acid calcium hydrate;1097832-14-8

135403648

Off-white to light yellow solid powder

240-250ºC

1.411

7

10

9

34

911

1

C1C(N(C2=C(N1)N=C(NC2=O)N)C=O)CNC3=CC=C(C=C3)C(=O)N[C@@H](CCC(=O)O)C(=O)O

NPPBLUASYYNAIG-ZIGBGYJWSA-L

InChI=1S/C20H23N7O7.Ca.5H2O/c21-20-25-16-15(18(32)26-20)27(9-28)12(8-23-16)7-22-11-3-1-10(2-4-11)17(31)24-13(19(33)34)5-6-14(29)30;;;;;;/h1-4,9,12-13,22H,5-8H2,(H,24,31)(H,29,30)(H,33,34)(H4,21,23,25,26,32);;5*1H2/q;+2;;;;;/p-2/t12?,13-;;;;;;/m0....../s1

L-Glutamic acid, N-(4-(((2-amino-5-formyl-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl)amino)benzoyl)-, calcium salt (1:1), pentahydrate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.  (2). This product is not stable in solution, please use freshly prepared working solution for optimal results.

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

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.)