| Size | Price | Stock | Qty |
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| 500mg |
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| 5g |
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| Targets |
Leucovorin targets the dihydrofolate reductase (DHFR) pathway as a reduced folate cofactor that bypasses the need for DHFR-mediated reduction, allowing folate-dependent reactions to proceed even when DHFR is inhibited by methotrexate. Leucovorin administration increases the level of reduced folates in tissues, which promotes the inhibition of thymidylate synthase in murine colon tumors. The selectivity of leucovorin rescue may be attributed to direct inhibition by MTX polyglutamyl forms, as well as FH2 polyglutamates that accumulate in their presence, at the levels of thymidylate synthase and transformylation during purine nucleotide biosynthesis. High-dose methotrexate/leucovorin rescue therapy is based on the assumption of differences in the transport system for folate compounds between normal and malignant proliferating cells. After high-dose MTX treatment, the cytotoxic effect of the folate antagonist is compensated for by rescue with leucovorin in low doses only in the normal cell system. Leucovorin ([6R,S]-5-formyl-THF) has been used for many years as rescue treatment to prevent toxicity following high-dose methotrexate therapy, and to modulate the cytotoxicity of 5-FU. It is the most widely used folate in the clinical setting.
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| ln Vitro |
The percentage of aberrant cells (Abs) and micronucleated binucleated cells (MNBN) increased in a concentration-related manner when MTX was used alone. The nuclear division index (NDI) falls as MTX concentration rises. In a similar vein, the mitotic index (MI) dropped for every MTX concentration that was examined. Leucovorin added at 50 μg/mL dramatically decreased the Abs percentage (36–77%) and MNBN percentage (40–68%). Inhibitory effects are likewise produced by leucovorin at 5 μg/mL (12% to 54% for MNBN and 20% to 61% for Abs) [1].
In vitro, leucovorin demonstrates significant activity in various cellular models. Leucovorin (50 μg/mL) dramatically decreases the percentage of aberrant cells (36-77%) and micronucleated binucleated cells (40-68%) induced by methotrexate. In CCRF-CEM cells, leucovorin enhances the cytotoxicity of trimetrexate/fluorouracil but does not increase methotrexate/fluorouracil toxicity. Exposure to 20 mM leucovorin in human colon cancer cell lines results in increased cytotoxicity. The augmentation of leucovorin slows the reduction of folate levels in tissues, thus enhancing the inhibitory effect of thymidylate synthase on murine colonic tumors. Leucovorin (20 mM) increases the cytotoxicity in all three replicate experiments in 10 of the 11 human colorectal carcinoma cell lines. In the MCF-7 human breast cancer cell line, cells exposed to various concentrations of MTX (0.5 to 10 μM) for 24 hours followed by rescue with labeled leucovorin (0.5 to 50 μM) have been used to investigate the mechanism of leucovorin reversal of methotrexate cytotoxicity. |
| ln Vivo |
This growth suppression (chronic administration of the drug MTX reduces bone growth in mice) appears to be reversed by treatment with leucovorin (7.0 mg/kg; i.p.; every other day; for 3 weeks; Balb/c young developing male mice) [2].
In vivo, leucovorin demonstrates efficacy in reversing methotrexate-induced toxicity and enhancing anticancer effects. Leucovorin (7.0 mg/kg; intraperitoneal; every other day) reverses methotrexate-induced bone growth suppression in mice. Leucovorin administration increases the level of reduced folates in tissues, which promotes the inhibition of thymidylate synthase in two murine colon tumors. In the clinical setting, leucovorin rescue allows for the use of very large doses of methotrexate and results in a greater response rate at a lower cost in toxicity than methotrexate alone in equivalent dosage. Leucovorin calcium supplies the product of the inhibited enzyme and thus can prevent, and rescue normal cells from, the adverse biologic effects of methotrexate. When combined with 5-FU, leucovorin stabilizes the drug's binding to the enzyme thymidylate synthase, enhancing its cancer-killing effects. Leucovorin is also used to treat certain types of anemia caused by folic acid deficiency. |
| Enzyme Assay |
In vitro enzyme/receptor binding assays for leucovorin typically evaluate its role as a reduced folate cofactor in folate-dependent enzymatic reactions. The compound is not an enzyme inhibitor but rather a substrate or cofactor that bypasses the need for DHFR-mediated reduction. Assays may measure the conversion of leucovorin to other reduced folate species by enzymes such as folylpolyglutamate synthetase. For studies investigating leucovorin's interaction with methotrexate, assays may measure the displacement of MTX from DHFR or the competition for cellular uptake via the reduced folate carrier. The selectivity of leucovorin rescue may be attributed to direct inhibition by MTX polyglutamyl forms, as well as FH2 polyglutamates that accumulate in their presence, at the levels of thymidylate synthase and transformylation during purine nucleotide biosynthesis. Binding studies may use radiolabeled leucovorin or folate analogs to assess uptake and metabolism in cells.
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| Cell Assay |
In vitro cellular assays for leucovorin typically employ folate-dependent cell lines to evaluate its ability to rescue cells from methotrexate-induced toxicity and to enhance the cytotoxicity of fluoropyrimidines. CCRF-CEM leukemia cells or other folate-dependent cell lines are cultured in folate-free media supplemented with leucovorin at various concentrations. Methotrexate is added to induce folate depletion and cell growth inhibition, and leucovorin is added at various concentrations to rescue cells from MTX toxicity. Cell viability is measured via MTT, resazurin reduction, or colony formation assays. For studies evaluating leucovorin's enhancement of 5-FU cytotoxicity, cells are treated with 5-FU and leucovorin in combination, and cell viability is assessed. In MCF-7 breast cancer cells, cells are exposed to MTX for 24 hours followed by rescue with labeled leucovorin (0.5 to 50 μM). Human lymphoid cell lines have been studied for leucovorin requirements to protect from MTX-induced growth suppression.
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| Animal Protocol |
Animal/Disease Models: 24 3weeks old Balb/c young male mice (11.88±0.25g) [2]
Doses: 7.0mg/kg Route of Administration: intraperitoneal (ip) injection; every other day; for 3 weeks Experimental Results: Administration of methamine This growth inhibition appears to be reversed by pterin (MTX). In vivo animal experiments for leucovorin typically employ mouse models to evaluate its rescue effects following methotrexate administration. Mice are administered methotrexate at high doses to induce toxicity, followed by leucovorin rescue at various doses and schedules. Leucovorin (7.0 mg/kg; intraperitoneal; every other day) reverses methotrexate-induced bone growth suppression in mice. For studies evaluating leucovorin's enhancement of anticancer therapy, tumor-bearing mice are treated with 5-FU and leucovorin in combination, and tumor growth is measured. Murine colon tumor models have been used to demonstrate that leucovorin administration increases the level of reduced folates in tissues, promoting the inhibition of thymidylate synthase. In xenograft models, the combination of leucovorin with 5-FU has been shown to enhance antitumor activity. Dosing regimens vary depending on the experimental objectives, with acute studies using single doses and chronic studies using repeated daily administration. |
| ADME/Pharmacokinetics |
The pharmacokinetics of leucovorin are characterized by rapid absorption and distribution following oral or intravenous administration. Leucovorin is a reduced folic acid and an active metabolite of folic acid (also known as folinic acid). It is a biological folic acid that is generally administered along with methotrexate (MTX) as a rescue agent to decrease MTX-induced toxicity. Leucovorin is water-soluble, with a solubility of ≥15.04 mg/mL in water with gentle warming. The compound has a molecular weight of 601.58 and a molecular formula of C₂₁H₂₅CaN₇O₇·5H₂O. Following administration, leucovorin is converted to other reduced folate species that participate in one-carbon transfer reactions. The pharmacokinetics of leucovorin are influenced by the route of administration, with oral administration resulting in lower bioavailability compared to intravenous administration due to first-pass metabolism. The compound is distributed throughout the body, with higher concentrations in the liver and other tissues with high folate requirements. Excretion occurs primarily in the urine.
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| Toxicity/Toxicokinetics |
Effects During Pregnancy and Lactation
◉ Overview of Medication 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, relevant drug records in the LactMed database 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. Leucovorin has a favorable safety profile as a naturally occurring folate derivative. It is generally well-tolerated at therapeutic doses. As a chemoprotectant, leucovorin is used to diminish the hematologic and mucosal toxicity of high-dose methotrexate therapy. Common adverse effects are typically related to the underlying condition being treated or to the concomitant chemotherapy agents. Allergic reactions to leucovorin are rare. The compound is also used to treat certain types of anemia caused by folic acid deficiency. In the context of leucovorin rescue, careful monitoring of methotrexate levels is essential to ensure adequate rescue and to prevent methotrexate toxicity. The compound is not associated with significant organ toxicity at therapeutic doses. However, as with any medication, caution should be exercised in patients with known hypersensitivity to folates or related compounds. The safety of leucovorin in pregnancy and lactation has not been established, and its use should be guided by clinical judgment. |
| References | |
| Additional Infomation |
The active metabolite of folic acid. Calcium folinate is mainly used as an antidote for folic acid antagonists.
See also: Calcium folinate (note moved to). Leucovorin calcium hydrate is a reduced form of folic acid that serves as a vital coenzyme in nucleic acid synthesis. It is FDA-approved for "leucovorin rescue" to reduce methotrexate toxicity in osteosarcoma, for treating folate deficiency megaloblastic anemias, and in combination with 5-fluorouracil for advanced colorectal cancer. Leucovorin calcium is also used to treat certain types of anemia caused by folic acid deficiency. It plays a key role in supportive cancer care as a chemoprotectant, not a chemotherapy drug. When combined with 5-FU, leucovorin stabilizes the drug's binding to the enzyme thymidylate synthase, enhancing its cancer-killing effects. The selectivity of leucovorin rescue may be attributed to direct inhibition by MTX polyglutamyl forms, as well as FH2 polyglutamates that accumulate in their presence. Leucovorin ([6R,S]-5-formyl-THF) has been used for many years as rescue treatment to prevent toxicity following high-dose methotrexate therapy, and to modulate the cytotoxicity of 5-FU, and is the most widely used folate in the clinical setting. Fusilev® (levoleucovorin) is approved by FDA for rescue after high-dose methotrexate therapy in osteosarcoma. |
| Molecular Formula |
C20H31CAN7O12
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|---|---|
| Molecular Weight |
601.58
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| Exact Mass |
601.165
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| CAS # |
6035-45-6
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| Related CAS # |
Folinic acid calcium;1492-18-8;Folinic acid;58-05-9;Folinic acid calcium hydrate;1097832-14-8
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| PubChem CID |
135802074
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| Appearance |
Light yellow to yellow solid powder
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| Melting Point |
240-250ºC
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| Hydrogen Bond Donor Count |
10
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| Hydrogen Bond Acceptor Count |
15
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
40
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| Complexity |
900
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| Defined Atom Stereocenter Count |
1
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| SMILES |
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-].O.O.O.O.O.[Ca+2]
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| InChi Key |
NPPBLUASYYNAIG-ZIGBGYJWSA-L
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| InChi Code |
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
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| Chemical Name |
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
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| Synonyms |
Folinic acid calcium salt pentahydrate Leucovorin calcium hydrate
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| HS Tariff Code |
2934.99.9001
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| Storage |
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. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
H2O : ~10 mg/mL (~16.62 mM)
DMSO : ~1 mg/mL (~1.66 mM) |
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6623 mL | 8.3114 mL | 16.6229 mL | |
| 5 mM | 0.3325 mL | 1.6623 mL | 3.3246 mL | |
| 10 mM | 0.1662 mL | 0.8311 mL | 1.6623 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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