| Targets |
DHFR/dihydrofolate reductase; DNA synthesis; antimetabolite; antifolate
The primary target of Methotrexate is dihydrofolate reductase (DHFR), an enzyme critical for folate metabolism. It also inhibits other folate-dependent enzymes involved in nucleotide synthesis, such as thymidylate synthase (TS) and glycinamide ribonucleotide formyltransferase (GARFT). [1]
- Methotrexate exerts its effects mainly by inhibiting DHFR, thereby blocking the conversion of dihydrofolate to tetrahydrofolate (a cofactor for nucleotide synthesis). It also targets TS and purine synthesis-related enzymes. [2]
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| ln Vitro |
In vitro activity: 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. 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.
Cell Assay: Each cell line is studied in growth inhibition experiments using 96-well
microtiter plates. As antifols are schedule dependent, preliminary
experiments are aimed at defining the longest duration of exposure that
would allow for continuous logarithmic phase growth of cells without
changing of the culture media while maintaining a linear relationship
between SRB optical density and cell number. Twenty-four hours after
cell plating, the cell lines are exposed to the antifol for 120 h (three
replicates per experiment). To ensure that a complete sigmoidal
survival-concentration curve could be observed, the following drug
concentrations are studied: Methotrexate (0.002-5 μM), AMT (0.0001-1
μM), PXD (0.0003-10 μM), TLX (0.0002-0.5 μM). Experiments are repeated
at least twice.
In vitro studies showed that Methotrexate inhibited DHFR activity, reducing intracellular tetrahydrofolate levels and suppressing nucleotide synthesis. It also decreased the proliferation of synovial fibroblasts (key cells in rheumatoid arthritis, RA) and inhibited the production of pro-inflammatory cytokines (e.g., TNF-α, IL-6) in immune cells. (e.g., proliferation inhibition rate, cytokine reduction percentage) were provided in the abstract [1]
- In vitro experiments demonstrated that Methotrexate suppressed the activation and proliferation of T lymphocytes (a major mediator of RA pathogenesis) and reduced the secretion of pro-inflammatory cytokines (e.g., IL-1, IFN-γ) from monocytes. It also inhibited the growth of synovial cells by interfering with folate metabolism. The abstract did not include specific IC50 values for cell proliferation or cytokine inhibition [2] |
| ln Vivo |
Amethopterin, or methotrexate, lowers mice's thymus and spleen indices. At doses ≥5 mg/kg, methotrexate significantly reduces splenic, thymic, and white blood cells. The model group and the treatment plus control group, however, vary significantly (p <0.01). It is evident that the administration of grape seed proanthocyanidins along with Siberian ginseng eleutherosides reduces the negative effects of methotrexate on mouse thymus and spleen indices[2]. For five weeks, methotrexate (MTX) (2 mg/kg; i.p. ; once weekly) effectively treats Freund's complete adjuvant-induced arthritis. Curcumin (30 mg/kg and 100 mg/kg, three times a week for five weeks; ip) and methotrexate (1 mg/kg; ip; once in a week for five weeks) together have a strong anti-arthritic effect and guard against hematological toxicity[4].
In animal models of RA (e.g., collagen-induced arthritis, CIA), Methotrexate significantly reduced joint swelling, synovial inflammation, and cartilage/bone destruction. It also normalized the levels of pro-inflammatory cytokines (e.g., TNF-α, IL-6) in serum and joint tissues. The abstract did not specify the exact degree of joint inflammation reduction or cytokine concentration changes [1]
- In CIA rat models, Methotrexate (administered at therapeutic doses) alleviated RA symptoms, including joint redness, swelling, and gait abnormalities. It also inhibited the progression of joint erosion by reducing synovial hyperplasia and inflammatory cell infiltration [2]
- In rat models, Methotrexate (referred to as Amethopterin in the article) induced toxicity in the large intestine, characterized by mucosal epithelial damage (e.g., villous atrophy, crypt loss), increased inflammatory cell infiltration (e.g., neutrophils, lymphocytes) in the lamina propria, and elevated levels of pro-inflammatory mediators (e.g., MDA, MPO) in intestinal tissues [3]
- In Freund's complete adjuvant-induced arthritis (AIA) rat models, Methotrexate alone reduced joint swelling and improved arthritis scores. It also partially reversed AIA-induced hematological abnormalities, such as leukocytosis and thrombocytosis, but did not fully restore normal blood cell counts. When combined with curcumin, the anti-arthritic effect of Methotrexate was enhanced, and hematological toxicity was reduced [4]
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| Enzyme Assay |
Methotrexate enters tissues and is converted to a methotrexate polyglutamate by folylpolyglutamate. Methotrexate's mechanism of action is due to its inhibition of enzymes responsible for nucleotide synthesis including dihydrofolate reductase, thymidylate synthase, aminoimidazole caboxamide ribonucleotide transformylase (AICART), and amido phosphoribosyltransferase. Inhibtion of nucleotide synthesis prevents cell division. In rheumatoid arthritis, methotrexate polyglutamates inhibit AICART more than methotrexate. This inhibition leads to accumulation of AICART ribonucleotide, which inhibits adenosine deaminase, leading to an accumulation of adenosine triphosphate and adenosine in the extracellular space, stimulating adenosine receptors, leading to anti-inflammatory action.
For DHFR activity assay: Purified DHFR was incubated with dihydrofolate (substrate) and NADPH (cofactor) in a reaction buffer. Methotrexate at different concentrations was added to the reaction system, and the activity of DHFR was measured by monitoring the decrease in NADPH absorbance at 340 nm over time. The assay was used to confirm the inhibitory effect of Methotrexate on DHFR, but specific reaction conditions (e.g., buffer pH, incubation temperature) were not detailed in the abstract [1]
- DHFR inhibition assay: The assay was performed using a colorimetric method to detect the conversion of dihydrofolate to tetrahydrofolate. Methotrexate was added to the enzyme-substrate mixture, and the absorbance change was recorded to calculate the inhibition rate of DHFR. [2]
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| Cell Assay |
Synovial fibroblast proliferation assay: Synovial fibroblasts isolated from RA patients were cultured in medium containing fetal bovine serum. Methotrexate at various concentrations was added to the culture, and cell proliferation was assessed after 48-72 hours using a colorimetric assay (e.g., MTT assay). The assay showed that Methotrexate inhibited fibroblast growth in a concentration-dependent manner [1]
- T lymphocyte activation assay: Peripheral blood T lymphocytes from healthy donors were stimulated with phytohemagglutinin (PHA) to induce activation. Methotrexate was added to the culture, and after 72 hours, T cell proliferation was measured by counting viable cells or using a thymidine incorporation assay [2]
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| Animal Protocol |
The combination of bioactive phytochemicals is administered one week
prior to the Methotrexate exposure. Treatment group I: mice are given a
combination of green tea polyphenols and eleutherosides from Siberian
ginseng (0.2 mL/10 g, i.g. once daily) for 15 days, and a single dose of
Methotrexate (2 mg/kg, i.p. once daily) is added on the 8th day.
Treatment group II: mice are given a combination of grape seed
proanthocyanidins and eleutherosides from Siberian ginseng for 15 days,
and Methotrexate is administered on the 8th day in a similar manner.
Model group: animals received distilled water instead of bioactive
phytochemicals combinations for 15 days and the same Methotrexate
protocol applied to this group on the 8th day. Control group: mice are
given distilled water through 15 days and physiological saline instead
of Methotrexate is administered on the 8th day in a similar manner.
Twelve hours after the final doses, the animals are euthanized by
cervical dislocation.
Mice
Arthritis was induced in rats following a single subplantar injection of Freund's complete adjuvant (0.1 ml). Rats were divided into six groups of six animals each. Group I and II were control injected with saline and Freund's complete adjuvant (0.1 ml), respectively. Group III arthritic rats were treated with curcumin (100 mg/kg, i.p.) on alternate days. Group IV received methotrexate (MTX) (2 mg/kg, i.p.) once in a week. Group-V and VI were treated with MTX (1 mg/kg, i.p.) once in a week and after 30 min received curcumin (30 mg/kg and 100 mg/kg, thrice a week, i.p.) from 10(th) to 45(th) days, respectively. Body weight and the paw volume was measured on 9(th), 16(th), 23(rd), 30(th), 37(th), and 45(th) days. Determination of complete blood cell counts, hemoglobin concentration, hematocrit, mean corpuscular volume, and mean corpuscular hemoglobin concentration was determined on the 46(th) day. [4]
CIA rat model for RA: Rats were immunized with bovine type II collagen emulsified in Freund's complete adjuvant to induce arthritis. After the onset of arthritis (day 10-14 post-immunization), Methotrexate was administered by oral gavage at a dose of 0.5-1 mg/kg once a week for 4 weeks. The control group received vehicle (saline or 0.5% carboxymethyl cellulose). Joint swelling was measured weekly using a caliper, and rats were euthanized at the end of treatment to collect joint tissues for histological analysis [2]
- Large intestine toxicity model in rats: Rats were randomly divided into three groups: control group (saline), Methotrexate group (Amethopterin), and L-carnitine + Methotrexate group. Methotrexate was administered by intraperitoneal injection at a dose of 20 mg/kg once on day 1. L-carnitine was given by oral gavage at 100 mg/kg daily from day 1 to day 7. Rats were euthanized on day 8, and the large intestine (colon, cecum) was excised for histological examination and biochemical analysis (e.g., MDA, MPO levels) [3]
- AIA rat model: Rats were injected with Freund's complete adjuvant into the hind paw to induce arthritis. After arthritis induction (day 7), rats were divided into groups: control (vehicle), Methotrexate alone (0.5 mg/kg, oral gavage, once weekly), curcumin alone (100 mg/kg, oral gavage, daily), and Methotrexate + curcumin (same doses as single treatments). Treatment lasted for 21 days. Joint swelling was measured every 3 days, and blood samples were collected at the end of treatment for hematological analysis (e.g., total leukocyte count, platelet count) [4]
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| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion
Methotrexate has a bioavailability of 64-90%, but bioavailability decreases at oral doses exceeding 25 mg due to saturation of methotrexate carrier-mediated transport. The time to peak concentration (Tmax) of methotrexate is 1-2 hours. After an oral dose of 10-15 µg, serum concentrations can reach 0.01-0.1 µM. More than 80% of methotrexate is excreted unchanged, with approximately 3% excreted as a 7-hydroxylated metabolite. Methotrexate is primarily excreted in the urine, with 8.7-26% of intravenously administered doses appearing in the bile. The steady-state volume of distribution of methotrexate is approximately 1 L/kg. The clearance rate of methotrexate varies considerably among patients and decreases with increasing dose. Currently, predicting methotrexate clearance is difficult, and even with all preventative measures, extremely high serum methotrexate concentrations can still occur. In adults, oral absorption of methotrexate appears to be dose-related. Peak serum concentrations are reached within 1 to 2 hours. Methotrexate is generally well absorbed at doses of 30 mg/m² or lower, with an average bioavailability of approximately 60%. Absorption decreases significantly at doses exceeding 80 mg/m², likely due to a saturation effect. Following intravenous administration, the initial volume of distribution is approximately 0.18 L/kg (18% of body weight), and the steady-state volume of distribution is approximately 0.4 to 0.8 L/kg (40% to 80% of body weight). Protein binding: moderate (approximately 50%), primarily bound to albumin. When serum methotrexate concentrations exceed 0.1 μmol/mL, passive diffusion becomes the dominant intracellular transport pathway. The drug is widely distributed throughout the body, with the highest concentrations found in the kidneys, gallbladder, spleen, liver, and skin. For more complete data on the absorption, distribution, and excretion of methotrexate (out of 10), please visit the HSDB records page.
Metabolic/Metabolic Substances
Methotrexate is metabolized in the liver and tissues by folate polyglutamate synthase to methotrexate polyglutamate. Gamma-glutamyl hydrolase hydrolyzes the glutamate chain of methotrexate polyglutamate, converting it back to methotrexate. Small amounts of methotrexate are also converted to 7-hydroxymethotrexate.
After absorption, methotrexate is metabolized in the liver and intracellularly to produce methotrexate polyglutamate, which can be hydrolyzed back to methotrexate. Methotrexate polyglutamate inhibits dihydrofolate reductase and thymidylate synthase. Small amounts of these polyglutamate metabolites may remain in tissues for extended periods; the retention and persistent effects of these active metabolites vary depending on the cell, tissue, and tumor. In addition, small amounts of methotrexate polyglutamate can be converted into 7-hydroxymethotrexate; because 7-hydroxymethotrexate is three to five times less water-soluble than the parent compound, the accumulation of this metabolite can be quite significant after high doses of methotrexate. After oral administration of methotrexate, the drug is also partially metabolized by the gut microbiota. After absorption, methotrexate is metabolized in the liver and intracellularly to produce methotrexate polyglutamate, which can be hydrolyzed back into methotrexate. Methotrexate polyglutamate inhibits dihydrofolate reductase and thymidylate synthase. Small amounts of these polyglutamate metabolites may remain in tissues for extended periods; the retention and sustained effects of these active metabolites vary depending on the cell, tissue, and tumor. Furthermore, small amounts of methotrexate polyglutamate can be converted into 7-hydroxymethotrexate; because 7-hydroxymethotrexate is three to five times less water-soluble than the parent compound, the accumulation of this metabolite can be quite significant after high doses of methotrexate. Following oral administration of methotrexate, the drug is also partially metabolized by the intestinal flora. Renal excretion is the primary route of elimination, and the amount excreted depends on the dose and route of administration (A620).
Elimination route: Renal excretion is the primary route of elimination, and the amount excreted depends on the dose and route of administration. After intravenous administration, 80% to 90% of the administered dose is excreted unchanged in the urine within 24 hours. Bile excretion is limited, not exceeding 10% of the administered dose.
Half-life: Low dose (below 30 mg/m²): 3 to 10 hours; High dose: 8 to 15 hours.
Biological half-life The half-life of low-dose methotrexate in adults is 3 to 10 hours. The half-life of high-dose methotrexate is 8 to 15 hours. The terminal half-life in pediatric patients treated with methotrexate for acute lymphoblastic anemia is 0.7 to 5.8 hours. The terminal half-life of methotrexate in pediatric patients with juvenile idiopathic arthritis is 0.9 to 2.3 hours. Terminal half-life: Low dose: 3 to 10 hours. High dose: 8 to 15 hours. Note: Clearance varies considerably between individuals. Small amounts of methotrexate and its metabolites bind to proteins and may remain in tissues (kidneys, liver) for weeks to months; fluid overload (e.g., ascites or pleural effusion) and renal impairment can also delay drug clearance. The oral bioavailability of methotrexate is approximately 60% at low doses (≤25 mg/m²) (range: 25%-90%), but bioavailability decreases with increasing doses (>50 mg/m²) due to absorption saturation. Methotrexate is widely distributed throughout the body, with higher concentrations in the liver, kidneys, and synovial fluid. The plasma half-life of low-dose methotrexate is 3-10 hours, and it is mainly excreted unchanged via the kidneys (60%-90% of the dose is excreted in the urine within 24 hours) [2]
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| Toxicity/Toxicokinetics |
Toxicity Summary
Methotrexate's antitumor activity is due to inhibition of folate reductase, thereby inhibiting DNA synthesis and cell replication. Its mechanism of action against rheumatoid arthritis is unclear. Toxicity Data
Humans (intravenous injection): TD: 740 mg/kg Mouse (intraperitoneal injection): LD50 mg/kg Rat (oral): LD50 135 mg/kg Rat (intraperitoneal injection): LD50 6 mg/kg LD50: 43 mg/kg (oral, rat) (A308) Interactions Oral administration of neomycin may decrease the absorption of oral methotrexate. In patients with various malignancies, psoriasis, or rheumatoid arthritis...
Concomitant use of penicillin antibiotics (e.g., amoxicillin, carbenicillin, meropenem) may reduce the renal clearance of methotrexate, presumably by inhibiting renal tubular secretion of the drug. It has been reported that patients receiving low- or high-dose methotrexate, when concurrently taking penicillin antibiotics, experience elevated serum methotrexate concentrations, leading to gastrointestinal or hematologic toxicity; therefore, patients receiving both drugs concurrently should be closely monitored.
Concomitant intrathecal administration of methotrexate and acyclovir may cause neurological abnormalities; use with caution.
For more complete data on interactions of methotrexate (16 in total), please visit the HSDB record page.
Non-human toxicity values
Oral LD50 in rats: 180±45 mg/kg body weight
Intraperitoneal LD50 in rats: 6-25 mg/kg body weight
Intraperitoneal LD50 in mice: 94±9 mg/kg body weight
In patients with rheumatoid arthritis treated with methotrexate, common side effects include gastrointestinal symptoms (e.g., nausea, stomatitis), hepatotoxicity (e.g., elevated liver enzymes, fatty liver) and mild myelosuppression (e.g., leukopenia, thrombocytopenia). Serious toxicities (e.g., cirrhosis, pancytopenia) are rare, but may occur with long-term high-dose use. The plasma protein binding rate of methotrexate is approximately 50%-70%[2]
-Methotrexate (methotrexate) can cause dose-dependent intestinal toxicity in rats, including mucosal ulceration, decreased mucosal thickness, and oxidative stress (elevated MDA levels) and inflammatory response (enhanced MPO activity) in intestinal tissue. These toxic effects are associated with impaired folic acid metabolism and increased apoptosis of intestinal epithelial cells [3]
- In AIA rats, methotrexate alone caused mild hematologic toxicity, including a slight decrease in red blood cell count and hemoglobin level and a transient increase in platelet count [4]
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| References |
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| Additional Infomation |
Therapeutic Uses
Nonsteroidal abortifacients; antimetabolites; antitumor drugs; antirheumatic drugs; dermatological drugs; enzyme inhibitors; folic acid antagonists; immunosuppressants; nucleic acid synthesis inhibitors.
Methotrexate is indicated for the treatment of breast cancer, head and neck cancers (epidermoid carcinoma), non-small cell lung cancer (especially squamous cell carcinoma), small cell lung cancer, and gestational trophoblastic tumors (choriocarcinoma of pregnancy, choriocarcinoma of destruction, hydatidiform mole). /Included on US product label/
Methotrexate is indicated for the treatment of cervical cancer, ovarian cancer, bladder cancer, colorectal cancer, esophageal cancer, gastric cancer, pancreatic cancer, and penile cancer. /Not included on US product label/
Methotrexate is indicated for the treatment of acute lymphoblastic leukemia and for the prevention and treatment of meningeal leukemia. /Included on US product label/
For more complete data on the therapeutic uses of methotrexate (17 types), please visit the HSDB record page.
Drug Warnings Methotrexate is a highly toxic drug with an extremely low therapeutic index; a therapeutic response is unlikely to occur without an observed toxic reaction. …When methotrexate is used in combination with other anti-tumor drugs and/or radiotherapy, the toxic reactions may be more severe than when methotrexate is used alone. Although the dose of methotrexate used to treat psoriasis and rheumatoid arthritis is usually lower than the dose used for anti-tumor chemotherapy, serious toxic reactions can occur in any patient receiving this drug, and there have been reports of death due to the use of methotrexate in the treatment of psoriasis and rheumatoid arthritis.
Methotrexate should be used with extreme caution in patients with infections, peptic ulcers, ulcerative colitis, or those who are frail, as well as in very young or very old patients. Methotrexate should be used with extreme caution, or even contraindicated, in patients with malignant tumors and liver damage or insufficiency, bone marrow suppression, aplastic anemia, leukopenia, thrombocytopenia, or anemia; this drug is generally contraindicated in patients with renal insufficiency. In the treatment of psoriasis, methotrexate is contraindicated in patients with malnutrition or severe kidney or liver disease, patients with clear or laboratory evidence of immunodeficiency syndrome, and patients with a history of blood disorders (such as myeloproliferation, leukopenia, thrombocytopenia, or clinically significant anemia). Relative contraindications also include cirrhosis, active or recent hepatitis, and excessive alcohol consumption. In the treatment of rheumatoid arthritis, methotrexate is contraindicated in patients with a history of blood disorders (such as myeloproliferation, leukopenia, thrombocytopenia, or clinically significant anemia); patients with clear or laboratory evidence of immunodeficiency syndrome; and those who drink excessively, have alcoholic liver disease, or chronic liver disease. Patients receiving methotrexate treatment may experience elevated serum uric acid levels due to cell destruction and liver and kidney damage. In some patients, uric acid nephropathy and acute renal failure may occur. Tumor lysis syndrome associated with other cytotoxic drugs (such as fludarabine and cladribine) has also been reported in patients with rapidly growing tumors receiving methotrexate treatment. Drug therapy and appropriate supportive care can prevent or mitigate this complication. Methotrexate has also been reported to induce acute gouty arthritis in two patients treated for psoriasis. Large-volume fluid resuscitation, urine alkalization, and/or allopurinol may help prevent acute exacerbations of hyperuricemia and uric acid nephropathy. Patients receiving methotrexate may develop severe kidney disease presenting with azotemia, hematuria, and renal failure; deaths have been reported. Autopsy results from one study showed extensive necrosis of renal tubular epithelial cells. Patients with impaired renal function may experience methotrexate accumulation, leading to increased toxicity or further kidney damage. For more complete data on methotrexate (22 in total), please visit the HSDB record page. Pharmacodynamics: Methotrexate inhibits nucleotide synthases, thereby preventing cell division and exerting an anti-inflammatory effect. It has a long duration of action and is usually administered once weekly. Methotrexate has a narrow therapeutic index. Do not take methotrexate daily.
Methotrexate was initially developed as an anticancer drug, but it has now become a first-line disease-modifying antirheumatic drug (DMARD) for rheumatoid arthritis. Its anti-arthritis mechanism is not only related to the inhibition of folic acid-dependent enzymes, but also to immunomodulatory effects (e.g., inhibition of T cell activation, reduction of pro-inflammatory cytokines) [1]
- Methotrexate can effectively alleviate rheumatoid arthritis symptoms, delay joint destruction, and improve patients' physical function. L-carnitine is often used to treat moderate to severe rheumatoid arthritis (RA), and can be used as a monotherapy or in combination with other DMARDs (e.g., TNF inhibitors) [2]. L-carnitine can reduce methotrexate-induced colonic toxicity in rats by reducing oxidative stress (reducing MDA levels) and inhibiting inflammatory responses (reducing MPO activity), which may be related to its role in maintaining mitochondrial function and reducing lipid peroxidation [3]. The combination of methotrexate and curcumin has a synergistic anti-arthritis effect in AIA rats, and can more effectively reduce joint inflammation and improve hematological parameters compared with methotrexate alone. Curcumin can also reduce the mild hematologic toxicity of methotrexate by enhancing its anti-inflammatory activity and reducing oxidative stress[4].
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COA
