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| Targets |
Methotrexate is a potent inhibitor of the enzyme dihydrofolate reductase (DHFR). It acts as an antifolate agent by competitively inhibiting DHFR, thereby preventing the conversion of folic acid to tetrahydrofolate. Tetrahydrofolate is an essential cofactor in the synthesis of thymidylate, purines, and several amino acids. By blocking this pathway, methotrexate inhibits DNA synthesis, repair, and cellular replication, particularly affecting rapidly dividing cells such as cancer cells, activated lymphocytes, and bone marrow progenitors. In addition to DHFR inhibition, methotrexate also inhibits other folate-dependent enzymes including thymidylate synthase (TS) and aminoimidazole carboxamide ribonucleotide (AICAR) transformylase, contributing to its anti-inflammatory and immunomodulatory effects. Methotrexate-d3 diammonium is the deuterated version of this compound; as an internal standard, it is not intended to exert pharmacological activity but serves as a tracer for quantitative bioanalysis.
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| ln Vitro |
Methotrexate demonstrates potent in vitro activity against a wide range of cancer cell lines. The antiproliferative effect is mediated through DHFR inhibition, resulting in depletion of reduced folate pools and disruption of de novo purine and thymidylate biosynthesis. In cell culture systems, methotrexate inhibits the growth of leukemia, lymphoma, breast, lung, colon, and osteosarcoma cell lines with IC50 values typically in the nanomolar to low micromolar range, depending on cell line sensitivity and culture conditions. The cytotoxicity of methotrexate is cell cycle phase-specific, primarily affecting S-phase cells where DNA synthesis is active. Methotrexate also exhibits in vitro immunosuppressive activity by inhibiting T-cell proliferation and inducing T-cell apoptosis, which underlies its efficacy in autoimmune diseases such as rheumatoid arthritis. Methotrexate-d3 diammonium is not intended for direct in vitro activity studies; rather, it is used as an internal standard to accurately quantitate non-labeled methotrexate in cell lysates and culture media.
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| ln Vivo |
Methotrexate demonstrates in vivo antitumor activity in various animal models, including murine leukemia (L1210, P388), solid tumors, and human tumor xenografts in immunodeficient mice. The antitumor efficacy is dose-dependent and schedule-dependent, with optimal activity observed with intermittent high-dose or continuous infusion schedules. In animal models of autoimmune diseases such as adjuvant-induced arthritis and collagen-induced arthritis, methotrexate reduces joint swelling, inflammation, and bone erosion. The anti-inflammatory effects are mediated, in part, by the release of adenosine, a potent anti-inflammatory molecule that reduces neutrophil adhesion and cytokine production. Methotrexate-d3 diammonium, when used as an internal standard, allows precise quantification of methotrexate levels in plasma, tissues, and other biological samples from treated animals, enabling pharmacokinetic and pharmacodynamic studies. The compound enables researchers to track the distribution, metabolism, and elimination of methotrexate in animal models with high accuracy.
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| Enzyme Assay |
For in vitro DHFR enzyme inhibition assays, the standard protocol uses recombinant human DHFR. The reaction mixture (total volume 100 microL) contains 50 mM potassium phosphate buffer (pH 6.8-7.4), 50-100 microM NADPH, 50-100 microM dihydrofolate (DHF), and varying concentrations of methotrexate (0.01 nM to 1 microM). The reaction is initiated by the addition of DHFR enzyme (0.01-0.1 U). The enzymatic activity is measured spectrophotometrically by monitoring the decrease in absorbance at 340 nm (ε340 = 12,300 M-¹ cm-¹) as NADPH is oxidized to NADP+ concomitant with the reduction of DHF to tetrahydrofolate (THF). The reaction is monitored at 25-37degC for 5-10 minutes in a UV-Vis spectrophotometer or microplate reader. The IC50 value is determined by plotting percent inhibition versus logarithm of inhibitor concentration. For the deuterated version, as an analytical internal standard, it is not typically used in enzyme inhibition assays. However, the compound can be used in LC-MS/MS-based assays to quantify methotrexate in reaction mixtures after enzymatic reactions.
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| Cell Assay |
For in vitro cell-based cytotoxicity assays, various cancer cell lines including CCRF-CEM (acute lymphoblastic leukemia), MCF-7 (breast cancer), HeLa (cervical cancer), and A549 (lung cancer) can be used. Cells are seeded in 96-well plates at a density of 5,000-10,000 cells per well in RPMI-1640 or DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. After overnight incubation at 37degC with 5% CO2, cells are treated with increasing concentrations of methotrexate (0.001 microM to 100 microM) for 48-72 hours. At the end of treatment, cell viability is assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) or CCK-8 assay. For MTT, 20 microL of MTT solution (5 mg/mL in PBS) is added to each well and incubated for 4 hours at 37degC. The resulting formazan crystals are dissolved in 150 microL DMSO, and absorbance is measured at 570 nm using a microplate reader. IC50 values are calculated using non-linear regression analysis. For the deuterated version, it is used as an internal standard to quantify methotrexate concentrations in the culture medium by LC-MS/MS, enabling the study of drug uptake and efflux in different cell lines.
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| Animal Protocol |
For in vivo animal experiments, female BALB/c nu/nu mice (6-8 weeks old, 18-22 g) are commonly used for xenograft studies. Tumor cells (e.g., 5 × 10⁶ cells in 100 microL PBS) are implanted subcutaneously in the right flank of each mouse. When tumors reach a volume of approximately 100-200 mm3 (typically 7-14 days post-inoculation), mice are randomized into treatment groups (n=6-10 per group). Methotrexate is administered via intraperitoneal (IP) injection at doses ranging from 5-100 mg/kg, typically once weekly or every 3-4 days, depending on the study design. Vehicle-treated control mice receive saline or other suitable vehicles. Body weight and tumor volumes (measured with calipers using the formula: tumor volume = length × width2 × 0.5) are recorded every 2-3 days. At the end of the experiment (typically 3-4 weeks), mice are euthanized, and tumors, blood samples, and major organs (liver, kidney, spleen, heart) are collected for further analysis. For pharmacokinetic studies, blood samples are collected at predetermined time points (0, 5, 15, 30, 60, 120, 240, 480, 720 minutes post-dose) for LC-MS/MS analysis. Methotrexate-d3 diammonium is used as an internal standard for quantifying non-labeled methotrexate in these samples.
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| ADME/Pharmacokinetics |
Methotrexate exhibits dose-dependent pharmacokinetics. Following intravenous administration, methotrexate shows a tri-exponential plasma concentration-time curve with distribution half-lives (t½alpha) of 0.5-1 hour and (t½beta) of 2-4 hours, and a terminal elimination half-life (t½gamma) of 8-12 hours in humans. In rodents, the elimination half-life is shorter (approximately 1-3 hours). The volume of distribution is approximately 0.4-0.8 L/kg, indicating distribution primarily in extracellular fluid. Methotrexate is approximately 50-60% bound to plasma proteins, primarily albumin. The drug is poorly lipid-soluble and does not cross the blood-brain barrier effectively except at high doses. Methotrexate is excreted primarily unchanged in urine (80-90%) via active tubular secretion and glomerular filtration. Its clearance is reduced in patients with renal impairment. Methotrexate is metabolized in the liver to 7-hydroxymethotrexate (7-OH-MTX) by aldehyde oxidase, which has lower DHFR inhibitory activity. The deuterated version Methotrexate-d3 diammonium, as an internal standard, exhibits identical LC-MS/MS characteristics, with the +3 Da mass shift allowing it to be distinguished from the non-labeled methotrexate during quantitative analysis.
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| Toxicity/Toxicokinetics |
Methotrexate has a well-characterized toxicity profile that includes both acute and chronic adverse effects. The dose-limiting toxicity is myelosuppression, manifested as leukopenia, thrombocytopenia, and anemia. Gastrointestinal toxicities include mucositis, stomatitis, nausea, vomiting, and diarrhea. Hepatotoxicity may occur with chronic use, ranging from transient elevated transaminases to cirrhosis. Nephrotoxicity can result from precipitation of methotrexate and its metabolites in the renal tubules, particularly at high doses. Pulmonary toxicity, including pneumonitis and pulmonary fibrosis, is less common but serious. Neurotoxicity can occur with intrathecal administration or high-dose systemic therapy. Methotrexate is teratogenic and contraindicated during pregnancy. The deuterated compound is not intended for human use but for research use only. As an internal standard, Methotrexate-d3 diammonium is handled in small quantities for analytical purposes; standard precautions for handling cytotoxic compounds should be followed, including the use of appropriate personal protective equipment, handling in designated areas, and proper waste disposal. The compound is for research use only and not for diagnostic or therapeutic applications.
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| References | |
| Additional Infomation |
Methotrexate (Amethopterin) is a first-line antimetabolite and antifolate agent that has been used clinically for over 70 years. It is approved by the FDA and other regulatory agencies worldwide for the treatment of various conditions including acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma, osteosarcoma, breast cancer, lung cancer, head and neck cancer, and gestational trophoblastic neoplasia. In rheumatology, methotrexate is the anchor disease-modifying antirheumatic drug (DMARD) for the treatment of rheumatoid arthritis, as well as for juvenile idiopathic arthritis, psoriasis, and psoriatic arthritis. The mechanism of action involves inhibition of DHFR and subsequent depletion of reduced folate pools, leading to inhibition of DNA synthesis and cell proliferation. Methotrexate-d3 diammonium is a deuterated internal standard specifically designed for LC-MS/MS-based therapeutic drug monitoring (TDM) and pharmacokinetic studies. The compound is formulated as the diammonium salt to enhance aqueous solubility and formulation compatibility. It is available as a research reagent only and is not for human therapeutic use. Storage: powder at -20degC for 3 years or 4degC for 2 years; in solvent at -80degC for 6 months or -20degC for 1 month.
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| Molecular Formula |
C20H25D3N10O5
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| Molecular Weight |
491.52
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| Appearance |
Solid powder
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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: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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) |
DMSO : 50 mg/mL (101.73 mM; with sonication)
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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 | 2.0345 mL | 10.1725 mL | 20.3451 mL | |
| 5 mM | 0.4069 mL | 2.0345 mL | 4.0690 mL | |
| 10 mM | 0.2035 mL | 1.0173 mL | 2.0345 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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