Reagents Glu406, Ile 418, Lys417, and Tyr453 are interacting with levomefolic acid (5-MTHF). Lys 417 and Tyr 453 are physiologically significant in the interaction between S1 and ACE and may function as S1 Potential Inhibitors of: ACE2 interaction with SARS-CoV-2 virus [1]. Levomefolate (5-MTHF) (50 nM, 72 hours) boosts intracellular folate metabolic activity in human lymphoblastoid cell lines (LCL) by 7-fold, which is 2 times greater than folic acid. This makes it an effective folate supplement agent[2].

Absorption, Distribution and Excretion
L-methylfolate is absorbed in the proximal small intestine via active proton-coupled folate transporter (PCFT), which transports both oxidized and reduced folate. Passive diffusion also occurs in the proximal and distal small intestine. The mean peak plasma concentration after a single oral dose of 906 nmol of L-methylfolate in healthy women is 39.4 nmol/L. It is primarily excreted via the kidneys or feces. Since over 99% of tissue folate exists as polyglutamate, only a small amount of excreted L-methylfolate is excreted unchanged. A portion of L-methylfolate is secreted into bile. It circulates in free form or loosely bound to plasma proteins. Metabolism/Metabolites L-methylfolate is further converted to tetrahydrofolate (THF) by the vitamin B12-dependent enzyme methionine synthase, and then efficiently polyglutamates via folate polyglutamate synthase (FPG). Folic acid in its polyglutamic acid form is a more effective substrate for enzymes involved in the folate-dependent response.

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Biological Half-Life
After daily oral administration of 5 mg of L-methylfolate for 7 consecutive days, the average elimination half-life is approximately 3 hours.

Protein Binding

Approximately 56% binds to plasma proteins.

[1]. Virtual screening and molecular dynamics study of approved drugs as inhibitors of spike protein S1 domain and ACE2 interaction in SARS-CoV-2. J Mol Graph Model. 2020 Dec;101:107716.

[2]. Simultaneous quantification of intracellular concentrations of clinically important metabolites of folate-homocysteine cycle by LC-MS/MS. Anal Biochem. 2020 Sep 15;605:113830.

5-Methyltetrahydrofolate is a derivative formed by substituting a methyl group at the 5-position of 5,6,7,8-tetrahydrofolate. It is a human metabolite functionally related to 5,6,7,8-tetrahydrofolate and is the conjugate acid of 5-methyltetrahydrofolate (2-). 5-Methyltetrahydrofolate is a methylated derivative of tetrahydrofolate. L-methylfolate (INN) is produced by methylenetetrahydrofolate reductase using 5,10-methylenetetrahydrofolate as a substrate, and is catalyzed by 5-methyltetrahydrofolate-homocysteine methyltransferase (also known as methionine synthase) to reduce homocysteine to methionine. L-methylfolate is a metabolite of folic acid (vitamin B9) and the main active form of folic acid in food and blood circulation, accounting for 98% of the total folic acid in human plasma. It can cross various cell membranes, including the blood-brain barrier, to enter different tissues and plays an important role in DNA synthesis, the cysteine cycle, and the regulation of homocysteine. In tissues, it methylates homocysteine to produce methionine and tetrahydrofolate (THF). L-methylfolate has been approved as a food additive and is recognized as a GRAS (Generally Recognized As Safe) compound. L-methylfolate is commercially available in its calcium salt crystalline form (Metafolin®), and its stability is sufficient for use as a dietary supplement. Because L-methylfolate is less likely to mask symptoms of vitamin B12 deficiency, it is a more suitable supplement than folic acid. 5-Methyltetrahydrofolate is a metabolite found in or produced by Escherichia coli (K12 strain, MG1655 strain). L-methylfolate is a folic acid analogue. There are reports and data regarding the effects of L-methylfolate in humans. L-methylfolate is a nutritional supplement containing the bioactive form of vitamin B9 folic acid—5-methyltetrahydrofolate (L-methylfolate)—which has potential antitumor activity. After administration, L-methylfolate provides methyl groups, thereby increasing DNA methylation levels in promoter regions of certain pro-tumorigenic genes, reversing hypomethylation of these genes, and inactivating them. This may lead to a reduction in tumor cell proliferation and tumor progression. Furthermore, taking L-methylfolate may enhance the cytotoxic effects of other chemotherapeutic drugs on tumor cells. Unlike folic acid, L-methylfolate can cross the blood-brain barrier, potentially offering benefits for the treatment of brain tumors. Hypomethylation of DNA in certain genes leads to chromosomal instability and promotes tumorigenesis and development.
See also: 5-methyltetrahydrofolate (note moved to).

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Drug Indications
For the treatment and prevention of folic acid deficiency, and as an antidote for folic acid antagonists. L-methylfolate has been added to oral contraceptives to reduce the risk of neural tube defects due to folic acid deficiency in pregnant women who conceive during or shortly after taking the product. Its use as a treatment for cardiovascular disease and as adjunctive therapy to antidepressants is currently under investigation. Mechanism of Action L-methylfolate plays a crucial role in the methylation of homocysteine to methionine, acting as a methyl donor in the reaction catalyzed by vitamin B12-dependent methionine synthase. Homocysteine must be further metabolized via transsulfurization, converting to cysteine, taurine, and glutathione through a vitamin B6-dependent process, or remethylated back to methionine. The methionine generated by L-methylfolate's remethylation of homocysteine is further metabolized into the downstream metabolite S-adenosylmethionine (SAMe). SAMe participates in various biochemical methyl donor reactions, including the synthesis of monoamine neurotransmitters. Studies have shown that high levels of homocysteine in plasma are associated with increased arterial plaque formation. Pharmacodynamics L-methylfolate is the active metabolite of folic acid and also a methyl donor in one-carbon metabolism. It regulates important cellular functions such as DNA biosynthesis, gene expression regulation, amino acid synthesis and metabolism, and myelin synthesis and repair. As the only form of folic acid capable of crossing the blood-brain barrier, it acts as a cofactor in the synthesis of monoamine neurotransmitters such as dopamine, serotonin, and norepinephrine. L-folate also participates in erythrocyte production.

C20H25N7O6

459.46

459.186

31690-09-2

Levomefolic acid-13C,d3;1356019-94-7;Levomefolate calcium;151533-22-1;Levomefolic acid-13C5;2687960-08-1

135398561

Light yellow to yellow solid powder

1.6±0.1 g/cm3

1.733

-2.61

7

10

9

33

865

2

CN1[C@H](CNC2=C1C(=O)NC(=N2)N)CNC3=CC=C(C=C3)C(=O)N[C@@H](CCC(=O)O)C(=O)O

ZNOVTXRBGFNYRX-STQMWFEESA-N

InChI=1S/C20H25N7O6/c1-27-12(9-23-16-15(27)18(31)26-20(21)25-16)8-22-11-4-2-10(3-5-11)17(30)24-13(19(32)33)6-7-14(28)29/h2-5,12-13,22H,6-9H2,1H3,(H,24,30)(H,28,29)(H,32,33)(H4,21,23,25,26,31)/t12-,13-/m0/s1

(2S)-2-[[4-[[(6S)-2-amino-5-methyl-4-oxo-3,6,7,8-tetrahydropteridin-6-yl]methylamino]benzoyl]amino]pentanedioic acid

LMSR Levomefolinic acidLevomefolinic acid Metafolin BodyfolinNutrifolin Levomefolate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

H2O : ~2.08 mg/mL (~4.53 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.)