https://www.targetedonc.com/news/first-patient-enrolled-in-phase-iii-study-of-novel-agent-arfolitixorin-in-mcrc

(6R)-5,10-methylenetetrahydrofolate is the (6R)-stereoisomer of 5,10-methylenetetrahydrofolate. It functions as a metabolite in E. coli, a mouse metabolite, and a cofactor. It is the conjugate acid of (2-)-5,10-methylenetetrahydrofolate. Modufolate is being investigated for the treatment of osteosarcoma. 5,10-methylenetetrahydrofolate is present in or produced by E. coli (K12 strain, MG1655 strain). (6R)-5,10-methylenetetrahydrofolate has been reported in humans, and relevant data are available. Afotesalin is the R-isomer of fotesalin, a reduced folate biomodulator and the active metabolite of the folate drugs leucovorin (LV) and levofolinate (l-LV). It can be used to enhance the efficacy of certain antimetabolites (e.g., the cytotoxic drug 5-fluorouracil (5-FU)) and reduce and prevent certain antimetabolite-related adverse reactions. Its effects are similar to those of high-dose methotrexate (HD) treatment. Following fotesalin administration, 5,10-methylenetetrahydrofolate (MTHF), as a reduced folate substrate for thymidylate synthase (TS), stabilizes the covalent binding of the 5-FU metabolite 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) rather than deoxyuridine monophosphate (dUMP) to the target enzyme TS when co-administered with 5-fluorouracil (5-FU), thereby inhibiting TS activity. This inhibits the synthesis of deoxythymidine monophosphate (dTMP), leading to the depletion of thymidine triphosphate (TTP), an essential component of DNA. This inhibits DNA synthesis, thereby suppressing cell proliferation and inducing tumor cell death. Because MTHF stabilizes and enhances the ternary complex, its combination with 5-FU enhances DNA synthesis inhibition and increases the cytotoxicity of 5-FU. MTHF is the active form of folic acid and an active metabolite of LV and l-LV; therefore, arfolitixorin can be activated without conversion to its active metabolite. During DNA synthesis, a ternary complex is formed between the reduced folic acid substrate MTHF, the TS enzyme, and dUMP, converting dUMP into dTMP, a DNA building block essential for DNA synthesis.

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Drug Indications
Treatment of colorectal cancer

C20H23N7O6

457.4399

457.171

31690-11-6

31690-11-6 (free);149930-93-8 (sulfate);154705-24-5 (sodium);

135398645

Typically exists as solid at room temperature

0.718

6

10

7

33

911

2

O=C1C2=C(N=C(N([H])[H])N1[H])N([H])C([H])([H])[C@]1([H])C([H])([H])N(C3C([H])=C([H])C(C(N([H])[C@]([H])(C(=O)O[H])C([H])([H])C([H])([H])C(=O)O[H])=O)=C([H])C=3[H])C([H])([H])N12

QYNUQALWYRSVHF-OLZOCXBDSA-N

InChI=1S/C20H23N7O6/c21-20-24-16-15(18(31)25-20)27-9-26(8-12(27)7-22-16)11-3-1-10(2-4-11)17(30)23-13(19(32)33)5-6-14(28)29/h1-4,12-13H,5-9H2,(H,23,30)(H,28,29)(H,32,33)(H4,21,22,24,25,31)/t12-,13+/m1/s1

(4-((R)-3-amino-1-oxo-1,2,5,6,6a,7-hexahydroimidazo[1,5-f]pteridin-8(9H)-yl)benzoyl)-L-glutamic acid

(6R)-5,10-Methylene-5,6,7,8-tetrahydrofolic acid; (6R)-5,10-Methylenetetrahydrofolate; 5,10-Methylene-(6R)-tetrahydrofolic acid;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)