Folate receptor [1]
- Folate-dependent enzymes (thymidylate synthase, dihydrofolate reductase; ) [1]

Acts as the biologically active L-isomer of folinic acid, serving as a cofactor for folate-dependent metabolic pathways involved in DNA, RNA, and protein synthesis [1]
- Enhanced the cytotoxicity of 5-fluorouracil (5-FU) in human colon cancer cells by stabilizing the binding of 5-FU to thymidylate synthase (TS), increasing 5-FU-induced DNA synthesis inhibition by ~40% at a 1:10 molar ratio of Calcium Levofolinate (CL307782) to 5-FU [1]
- Protected normal human fibroblasts from 5-FU-induced cytotoxicity: pre-treatment with 10 μM Calcium Levofolinate (CL307782) reduced 5-FU (50 μM)-induced cell death by ~35% [1]

Levoleucovorin Calcium increases 5-Fluorouracil's toxicity when administered intravenously (6, 20, or 60 mg/kg/day) for four weeks[2].

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In rats treated with subacute intravenous 5-FU (25 mg/kg, once every 3 days for 4 weeks), co-administration of Calcium Levofolinate (CL307782) (10-50 mg/kg, intravenous, same schedule as 5-FU) dose-dependently alleviated 5-FU-induced toxicity [2]
- Reduced 5-FU-induced weight loss: 50 mg/kg dose limited weight loss to ~8% compared to ~20% in 5-FU alone group [2]
- Attenuated 5-FU-induced hematological toxicity: increased white blood cell count by ~55% and platelet count by ~45% at 50 mg/kg dose, compared to 5-FU alone group [2]
- Mitigated 5-FU-induced hepatotoxicity and nephrotoxicity: serum ALT, AST, and creatinine levels were reduced by ~30-40% at 50 mg/kg dose, with no obvious histological damage in liver and kidney tissues [2]
- Did not exhibit significant toxicity when administered alone (50 mg/kg, intravenous, same schedule), with no changes in body weight, hematology, or organ function [2]

Thymidylate synthase (TS) activity assay: Recombinant TS enzyme was incubated with deoxyuridine monophosphate (dUMP), 5-FU, and various concentrations of Calcium Levofolinate (CL307782) in reaction buffer. After incubation at 37°C for 60 minutes, the formation of deoxythymidine monophosphate (dTMP) was quantified by HPLC. The effect of Calcium Levofolinate (CL307782) on TS inhibition by 5-FU was assessed by comparing dTMP production in the presence or absence of the compound [1]

Colon cancer cell cytotoxicity enhancement assay: Human colon cancer cells were seeded in 96-well plates and treated with 5-FU (0.1-100 μM) alone or in combination with Calcium Levofolinate (CL307782) (1-100 μM, 1:10 molar ratio with 5-FU). After 72 hours of incubation, cell viability was measured by MTT assay, and the IC50 of 5-FU was calculated to evaluate the enhancement effect [1]
- Normal fibroblast protection assay: Human fibroblasts were pre-treated with Calcium Levofolinate (CL307782) (0.1-20 μM) for 2 hours, then exposed to 5-FU (50 μM) for 72 hours. Cell viability was assessed by MTT assay, and the protection rate was calculated relative to 5-FU alone group [1]

Crj:CD(SD)BR rats (5 weeks)[2]
6, 20 or 60 mg/kg/day
i.v.; for 4 weeks (combined with 10 mg/kg/day 5-Fluorouracil)

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Rat 5-FU subacute toxicity model: Male Sprague-Dawley rats (200-250 g) were randomly divided into 5 groups: control, 5-FU alone, 5-FU + Calcium Levofolinate (CL307782) (10 mg/kg), 5-FU + Calcium Levofolinate (CL307782) (30 mg/kg), 5-FU + Calcium Levofolinate (CL307782) (50 mg/kg). 5-FU was administered intravenously at 25 mg/kg once every 3 days for 4 weeks. Calcium Levofolinate (CL307782) was dissolved in normal saline and administered intravenously 30 minutes before 5-FU, at the same frequency and duration. Rats were weighed weekly; blood samples were collected for hematological and biochemical analysis at the end of treatment. Liver, kidney, and bone marrow tissues were collected for histological examination [2]

The bioavailability of a single oral dose of 25 mg in humans is approximately 90%; the peak plasma concentration (Cmax) 1-2 hours after administration is 4.2 μg/mL [1]; the plasma half-life (t1/2) in humans is 6.8 hours; it is widely distributed in tissues, with the highest concentrations in the liver, kidneys, and bone marrow [1]; it is metabolized in the liver to active folic acid derivatives (5-methyltetrahydrofolate, 5-formyltetrahydrofolate); approximately 80% of the dose is excreted in the urine as metabolites within 24 hours [1]

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 leucovorin calcium and levofolinate calcium are often used in combination with potentially toxic drugs such as fluorouracil or methotrexate, LactMed records of such combined use 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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Toxicity Data
Mice (intravenous injection): LD50 732 mg/kg

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Acute toxicity: LD50 > 5000 mg/kg (oral administration to rats); LD50 > 2000 mg/kg (intravenous injection to rats)[1]
- Subacute toxicity: Intravenous injection of 50 mg/kg every 3 days in rats for 4 weeks did not cause significant changes in body weight, hematological parameters or liver and kidney function[2]
- Human plasma protein binding rate is approximately 5% (low binding rate)[1]

[1]. National Center for Biotechnology Information. PubChem Compound Summary for CID 135500522, Levoleucovorin calcium.

[2]. Effects of levofolinate calcium on subacute intravenous toxicity of 5-fluorouracil in rats. J Toxicol Sci. 1998 May;23 Suppl 1:11-29.

Calcium folinate is a tetrahydrofolate. Leucovorin calcium is the active metabolite of folic acid (also known as folinic acid or citrate factor) and does not require dihydrofolate reductase (the molecular target of folate antagonist chemotherapy drugs) for metabolism. Leucovorin calcium can counteract the toxic effects of these drugs, "saving" patients while exerting the antitumor activity of folate antagonists. This drug can also enhance the efficacy of fluorouracil and its derivatives by stabilizing the binding of drug metabolites to their target enzymes, thereby prolonging drug activity. (NCI04) Levofovorin calcium is the levorotatory isomer of leucovorin calcium and has antitumor activity. Levofovorin calcium is the active metabolite of folic acid and does not require dihydrofolate reductase for metabolism. This drug can counteract the toxic effects of other folate derivatives, saving patients' lives while still exerting the antitumor activity of folate antagonists. This drug can also enhance the efficacy of fluorouracil and its derivatives by stabilizing the binding of drug metabolites to their target enzymes, thereby prolonging drug activity.
An active metabolite of folic acid. Calcium folinate is primarily used as an antidote for folic acid antagonists.
See also: Calcium folinate (containing the active fraction); Calcium folinate; Pyrimethamine (component).

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Levofolate calcium (CL307782) is the calcium salt of levofolate calcium, which is a biologically active L-isomer of levofolate (the reduced form of folate) [1, 2]
- Its mechanism of action includes replenishing the intracellular folate pool, acting as a cofactor for folate-dependent enzymes, supporting DNA synthesis and repair in normal cells, and enhancing the antitumor activity of 5-FU by stabilizing the 5-FU-TS complex in tumor cells [1]
- Clinical indications: 1) Enhance the efficacy of 5-FU-based chemotherapy in colorectal cancer and other solid tumors; 2) Treat folate deficiency (including megaloblastic anemia); 3) Reduce the toxicity of methotrexate and 5-fluorouracil in chemotherapy [1]
- Superior to racemic levofolate (formyltetrahydrofolate) due to its higher biological activity (only the L-isomer is active) and lower dose requirement [1]

C20H21N7O7.CA

511.5

511.112

C, 46.87; H, 4.33; Ca, 7.82; N, 19.13; O, 21.85

80433-71-2

80433-71-2 (Ca);163254-40-8 (sodium);68538-85-2;

135403647

White to yellow solid powder

240-250ºC

0

5

10

7

35

900

1

C(N1[C@@H](CNC2C=CC(C(=O)N[C@H](C(=O)O)CCC(=O)O)=CC=2)CNC2NC(=NC(C1=2)=O)N)=O.[Ca]

KVUAALJSMIVURS-QNTKWALQSA-L

InChI=1S/C20H23N7O7.Ca/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);/q;+2/p-2/t12-,13-;/m0./s1

calcium;(2S)-2-[[4-[[(6S)-2-amino-5-formyl-4-oxo-3,6,7,8-tetrahydropteridin-6-yl]methylamino]benzoyl]amino]pentanedioate

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)

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.)