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Methotrexate

For research use only.

Catalog No.S1210 Synonyms: NCI-C04671

41 publications

Methotrexate Chemical Structure

CAS No. 59-05-2

Methotrexate (MTX, NCI-C04671), analog of folic acid, is a nonspecific inhibitor of the dihydrofolate reductase(DHFR) of bacteria and cancerous cells as well as normal cells. It forms an inactive ternary complex with DHFR and NADPH. Methotrexate (MTX) induces apoptosis.

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Selleck's Methotrexate has been cited by 41 publications

Purity & Quality Control

Choose Selective ADC Cytotoxin Inhibitors

Biological Activity

Description Methotrexate (MTX, NCI-C04671), analog of folic acid, is a nonspecific inhibitor of the dihydrofolate reductase(DHFR) of bacteria and cancerous cells as well as normal cells. It forms an inactive ternary complex with DHFR and NADPH. Methotrexate (MTX) induces apoptosis.
Targets
hDHFR [4]
(Activated peripheral T cells)
24 nM
In vitro

Methotrexate (0.1-10 mM) induces apoptosis of in vitro activated T cells from human peripheral blood. Methotrexate achieves clonal deletion of activated T cells in mixed lymphocyte reactions. Methotrexate can selectively delete activated peripheral blood T cells by a CD95-independent pathway. [1] Methotrexate is taken up by cells via the reduced folate carrier and then is converted within the cells to polyglutamates. Methotrexate leads to diminished production of leukotriene B4 by neutrophils stimulated ex vivo. Methotrexate polyglutamates inhibit the enzyme aminoimidazolecarboxamidoadenosineribonucleotide (AICAR) transformylase more potently than the other enzymes involved in purine biosynthesis. Methotrexate is also known to suppress TNF activity by suppressing TNF-induced nuclear factor-κB activation in vitro, in part related to a reduction in the degradation and inactivation of an inhibitor of this factor, IκBα, and probably related to the release of adenosine. Methotrexate suppresses the production of both TNF and IFN-γ by T-cell-receptor-primed T lymphocytes from both healthy human donors and RA patients. Methotrexate treatment is associated with a significant decrease of TNF-α-positive CD4+ T cells, while the number of T cells expressing the anti-inflammatory cytokine IL-10 increased. [2]

Cell Data
Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
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OMC-1 NILER2NIem:5dHigbY5pcWKrdHnvckBie3OjeR?= M3vRSmlvcGmkaYTpc44hd2ZiaIXtZY4hV02FLUGgZ4VtdCCpcn;3eIghcW5iYTDj[YxtKH[rYXLpcIl1gSCjc4PhfUwhUUN3MDC9JFI1NjZ{MUKg{txONg>? NVy0cYkzRGFidHHy[4V1RSehYnzhcosoKGi{ZX[9K4h1fHC|Oj:ve5d4NmWkaT7hZ{52cy:laHXtZoww[2:vcH;1coRgemWyb4L0Y4NiemRxQ1jFUWJNOzR{NUmvK|5USU6JRWK8M4E,
Hs-578-T NHHhbGNIem:5dHigbY5pcWKrdHnvckBie3OjeR?= M{nRRWlvcGmkaYTpc44hd2ZiaIXtZY4hUHNvNUe4MXQh[2WubDDndo94fGhiaX6gZUBk\WyuII\pZYJqdGm2eTDhd5NigSxiSVO1NEA:KDJ2LkewPVch|ryPLh?= MlW5QIEhfGG{Z3X0QUdg[myjbnunJIhz\WZ;J3j0eJB{Qi9xd4f3MoVjcS6jYz71b{9kcGWvYnyvZ49ueG:3bnTfdoVxd3K2X3PhdoQwS0iHTVLMN|QzPTlxJ{7TRW5ITVJ:L3G+
SNB75 M13Qe2dzd3e2aDDpcohq[mm2aX;uJIF{e2G7 NEDHdJRKdmirYnn0bY9vKG:oIHj1cYFvKFOQQke1JINmdGxiZ4Lve5RpKGmwIHGgZ4VtdCC4aXHibYxqfHliYYPzZZktKEmFNUCgQUAzPC55N{WxJO69VS5? MUC8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:5d4eu[YJqNmGlLoXrM4Np\W2kbD;jc41xd3WwZG;y[ZBwenShY3Hy[E9EUEWPQlyzOFI2QS9pPmPBUmdGWjxxYU6=
T47D NEjiTHJIem:5dHigbY5pcWKrdHnvckBie3OjeR?= MXrJcohq[mm2aX;uJI9nKGi3bXHuJHQ1P0RiY3XscEBoem:5dHigbY4h[SClZXzsJJZq[WKrbHn0fUBie3OjeTygTWM2OCB;IEK0Mlg4QDNizszNMi=> MW[8ZUB1[XKpZYS9K39jdGGwazegbJJm\j1paIT0dJM7Ny:5d4eu[YJqNmGlLoXrM4Np\W2kbD;jc41xd3WwZG;y[ZBwenShY3Hy[E9EUEWPQlyzOFI2QS9pPmPBUmdGWjxxYU6=
NCI-H2029 MV7Hdo94fGhiaX7obYJqfGmxbjDhd5NigQ>? NUH4[5dXUW6qaXLpeIlwdiCxZjDoeY1idiCQQ1mtTFIxOjliY3XscEBoem:5dHigbY4h[SClZXzsJJZq[WKrbHn0fUBie3OjeTygTWM2OCB;IEK1Mlc6PjdizszNMi=> NETsT4Q9[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;3e5cv\WKrLnHjMpVsN2OqZX3icE9kd22yb4Xu[H9z\XCxcoTfZ4Fz\C:FSFXNRmw{PDJ3OT:nQnNCVkeHUkyvZV4>
NB6 MXvHdo94fGhiaX7obYJqfGmxbjDhd5NigQ>? M{PnVmlvcGmkaYTpc44hd2ZiaIXtZY4hVkJ4IHPlcIwh\3Kxd4ToJIlvKGFiY3XscEB3cWGkaXzpeJkh[XO|YYmsJGlEPTBiPTCyOU45PzZ5IN88UU4> MkLlQIEhfGG{Z3X0QUdg[myjbnunJIhz\WZ;J3j0eJB{Qi9xd4f3MoVjcS6jYz71b{9kcGWvYnyvZ49ueG:3bnTfdoVxd3K2X3PhdoQwS0iHTVLMN|QzPTlxJ{7TRW5ITVJ:L3G+
BHT-101 NH3zc|dIem:5dHigbY5pcWKrdHnvckBie3OjeR?= NYL0blNCUW6qaXLpeIlwdiCxZjDoeY1idiCESGStNVAyKGOnbHyg[5Jwf3SqIHnuJIEh[2WubDD2bYFjcWyrdImgZZN{[XluIFnDOVAhRSB{Nj62OFQ{KM7:TT6= NGq0V409[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;3e5cv\WKrLnHjMpVsN2OqZX3icE9kd22yb4Xu[H9z\XCxcoTfZ4Fz\C:FSFXNRmw{PDJ3OT:nQnNCVkeHUkyvZV4>
TK10 MnvES5Jwf3SqIHnubIljcXSrb36gZZN{[Xl? NXvuR3V7UW6qaXLpeIlwdiCxZjDoeY1idiCWS{GwJINmdGxiZ4Lve5RpKGmwIHGgZ4VtdCC4aXHibYxqfHliYYPzZZktKEmFNUCgQUAzPi56NEm1JO69VS5? NV;YVW5uRGFidHHy[4V1RSehYnzhcosoKGi{ZX[9K4h1fHC|Oj:ve5d4NmWkaT7hZ{52cy:laHXtZoww[2:vcH;1coRgemWyb4L0Y4NiemRxQ1jFUWJNOzR{NUmvK|5USU6JRWK8M4E,
NCI-H2405 MkO4S5Jwf3SqIHnubIljcXSrb36gZZN{[Xl? MX3Jcohq[mm2aX;uJI9nKGi3bXHuJG5EUS2KMkSwOUBk\WyuIHfyc5d1cCCrbjDhJINmdGxidnnhZoltcXS7IHHzd4F6NCCLQ{WwJF0hOjlwMEi5OUDPxE1w NF7hb249[SC2YYLn[ZQ:L1:kbHHub{chcHKnZk2nbJR1eHN8Lz;3e5cv\WKrLnHjMpVsN2OqZX3icE9kd22yb4Xu[H9z\XCxcoTfZ4Fz\C:FSFXNRmw{PDJ3OT:nQnNCVkeHUkyvZV4>

... Click to View More Cell Line Experimental Data

Assay
Methods Test Index PMID
Western blot
ppJAK1 / ppJAK2 / JAK1 / JAK2 ; 

PubMed: 26131691     


HDLM-2 cells treated with the indicated concentrations of methotrexate and aminopterin and blotted to show levels of total JAK1 (tJAK1), dual phosphorylated JAK1 (ppJAK1), total JAK2 (tJAK2), dual phosphorylated JAK2 (ppJAK2) and ß-actin shown as a loading control. Levels of ppJAK1 and ppJAK2 are reduced at high drug concentrations.

pSTAT1 / pSTAT3 / pSTAT5 / STAT1 / STAT3 / STAT5; 

PubMed: 26131691     


D) HDLM-2 cells treated with the indicated concentrations of methotrexate and aminopterin and blotted to show levels of total STAT1 (tSTAT1), phosphorylated STAT1 (pSTAT1), total STAT3 (tSTAT3), phosphorylated STAT3 (pSTAT3), total STAT5 (tSTAT5), phosphorylated STAT5 (pSTAT5) and β-actin shown as a loading control. Levels of pSTAT1 and pSTAT5 are reduced at most drug concentrations while levels of pSTAT3 appear to be unaffected.

AKT / p-AKT / mTOR / p-mTOR / Beclin 1 / HMGB1 ; 

PubMed: 26702616     


After the cells were exposed to 0.1 μM MTX for the indicated times, the cell lysates were subjected to Western blotting with specific antibodies. The results are representative of three independent experiments. β-actin was used as a loading control.

26131691 26702616
In vivo Methotrexate increases splenocyte AICAR content, raised adenosine concentrations in exudates from carrageenan-inflamed air pouches, and markedly inhibits leukocyte accumulation in inflamed air pouches in mice. Methotrexate-mediated reduction in leukocyte accumulation is partially reversed by injection of adenosine deaminase (ADA) into the air pouch, completely reverses by a specific adenosine A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX), but not affected by an adenosine A1 receptor antagonist, 8-cyclopentyl-dipropylxanthine in mice. [3]

Protocol

Solubility (25°C)

In vitro DMSO 90 mg/mL warmed (198.04 mM)
Water Insoluble
Ethanol Insoluble
In vivo Add solvents to the product individually and in order(Data is from Selleck tests instead of citations):
2% DMSO+30% PEG 300+5% Tween 80+ddH2O
For best results, use promptly after mixing.
5mg/mL

* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

Chemical Information

Molecular Weight 454.44
Formula

C20H22N8O5

CAS No. 59-05-2
Storage powder
in solvent
Synonyms NCI-C04671
Smiles CN(CC1=CN=C2C(=N1)C(=NC(=N2)N)N)C3=CC=C(C=C3)C(=O)NC(CCC(=O)O)C(=O)O

In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)
Dosage mg/kg Average weight of animals g Dosing volume per animal ul Number of animals
Step 2: Enter the in vivo formulation ()
% DMSO % % Tween 80 % ddH2O
CalculateReset

Bio Calculators

Molarity Calculator

Molarity Calculator

Calculate the mass, volume or concentration required for a solution. The Selleck molarity calculator is based on the following equation:

Mass (mg) = Concentration (mM) × Volume (mL) × Molecular Weight (g/mol)

  • Mass
    Concentration
    Volume
    Molecular Weight

*When preparing stock solutions, please always use the batch-specific molecular weight of the product found on the via label and MSDS / COA (available on product pages).

Dilution Calculator

Dilution Calculator

Calculate the dilution required to prepare a stock solution. The Selleck dilution calculator is based on the following equation:

Concentration (start) x Volume (start) = Concentration (final) x Volume (final)

This equation is commonly abbreviated as: C1V1 = C2V2 ( Input Output )

  • C1
    V1
    C2
    V2

* When preparing stock solutions always use the batch-specific molecular weight of the product found on the vial label and MSDS / COA (available online).

The Serial Dilution Calculator Equation

  • Serial Dilutions

  • Computed Result

  • C1=C0/X C1: LOG(C1):
    C2=C1/X C2: LOG(C2):
    C3=C2/X C3: LOG(C3):
    C4=C3/X C4: LOG(C4):
    C5=C4/X C5: LOG(C5):
    C6=C5/X C6: LOG(C6):
    C7=C6/X C7: LOG(C7):
    C8=C7/X C8: LOG(C8):
Molecular Weight Calculator

Molecular Weight Calculator

Enter the chemical formula of a compound to calculate its molar mass and elemental composition:

Total Molecular Weight: g/mol

Tip: Chemical formula is case sensitive. C10H16N2O2 c10h16n2o2

Instructions to calculate molar mass (molecular weight) of a chemical compound:

To calculate molar mass of a chemical compound, please enter its chemical formula and click 'Calculate'.

Definitions of molecular mass, molecular weight, molar mass and molar weight:

Molecular mass (molecular weight) is the mass of one molecule of a substance and is expressed in the unified atomic mass units (u). (1 u is equal to 1/12 the mass of one atom of carbon-12)
Molar mass (molar weight) is the mass of one mole of a substance and is expressed in g/mol.

Molarity Calculator

Mass Concentration Volume Molecular Weight

Clinical Trial Information

NCT Number Recruitment interventions Conditions Sponsor/Collaborators Start Date Phases
NCT03642795 Not yet recruiting Other: Questionnaire Rheumatoid Polyarthritis Centre Hospitalier Universitaire de Nīmes October 2021 --
NCT04668768 Not yet recruiting Drug: Glucarpidase Lymphoma Fox Chase Cancer Center February 2021 Phase 2
NCT04205734 Not yet recruiting Diagnostic Test: MTX Assessment IBD Boston Children''s Hospital December 30 2020 --
NCT04622956 Recruiting Drug: Methotrexate Injectable Solution Graft Vs Host Disease|Hematopoietic Neoplasm University of Sao Paulo General Hospital|Libbs Farmacêutica LTDA October 7 2020 Phase 1|Phase 2

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

Tel: +1-832-582-8158 Ext:3

If you have any other enquiries, please leave a message.

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Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID