DHFR/dihydrofolate reductase; DNA synthesis; antimetabolite; antifolate

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.

The thymus and spleen index in mice is lowered by methotrexate (amethopterin) disodium. At dosages ≥5 mg/kg, methotrexate disodium markedly decreased splenic, thymus, and white blood cells. Nonetheless, a statistically significant distinction existed between the model group and the treatment plus control group (p<0.01). The effects of methotrexate exposure on the thymus and spleen indices in mice can be considerably reduced by the combination of grape seed proanthocyanidins and Siberian ginseng eleutheroside [2]. In Freund's full adjuvant-induced arthritis, methotrexate (MTX) disodium (2 mg/kg; intraperitoneally; once weekly for 5 weeks) is beneficial. Curcumin (30 mg/kg and 100 mg/kg; intraperitoneally; three times weekly for five weeks) and methotrexate disodium (1 mg/kg; intraperitoneally; once weekly for five weeks) together shown strong anti-arthritic benefits and defense against hematological toxicity [4].

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.

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]

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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

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.

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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 treated 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 (such as ascites or pleural effusion) and renal impairment can also delay clearance.

Hepatotoxicity
Methotrexate is well-known to cause elevated serum transaminase levels, and long-term treatment is associated with fatty liver, liver fibrosis, and even cirrhosis. While there is extensive literature on methotrexate, the incidence of abnormalities in liver function tests and biopsies varies greatly depending on the dosage, dosing regimen, and duration of treatment. Following high-dose intravenous methotrexate, serum ALT levels can rise to 10 to 20 times the upper limit of normal (ULN) within 12 to 48 hours, but levels subsequently decline rapidly to normal, with jaundice or symptoms of liver damage occurring only in rare cases. During long-term low- to moderate-dose methotrexate treatment, 15% to 50% of patients experience elevated serum ALT or AST levels, but these are usually mild and resolve spontaneously. Approximately 5% of patients experience ALT levels exceeding twice the normal value; these abnormalities usually return to normal rapidly upon discontinuation of the drug or dose adjustment, but may also return to normal even with continued use at the same dose. The incidence of elevated ALT levels during treatment has been reported to vary considerably, likely due to differences in testing frequency (monthly versus every 3, 6, or 12 months) and blood collection time (weekly before or shortly after administration). Furthermore, studies have shown that concurrent folic acid use can reduce the incidence of elevated serum enzymes and is now widely used. Long-term methotrexate use is associated with fatty liver and liver fibrosis, and in rare cases, portal hypertension and symptomatic cirrhosis. Often, there are no symptoms before the development of cirrhosis, and liver function tests are usually normal or show only mild and transient elevations. Routine monitoring of patients receiving methotrexate treatment, with liver biopsies every 1 to 2 years, or cumulative use of 1 to 10 grams of methotrexate, has shown that approximately 30% of patients develop mild to moderate histological abnormalities (fat deposition, cellular disorder, mild inflammation, nuclear atypia), and 2% to 20% develop varying degrees of liver fibrosis. Although there have been case reports of cirrhosis occurring during long-term methotrexate treatment, the incidence of cirrhosis remains low in prospective studies, even with routine histological monitoring. Patients who develop liver fibrosis after long-term methotrexate treatment usually have other risk factors for fatty liver, including excessive alcohol consumption, obesity, diabetes, and concurrent use of other potentially hepatotoxic drugs. High doses and daily methotrexate are particularly likely to cause liver fibrosis, with an incidence of cirrhosis exceeding 20% after 5 to 10 years of treatment. With more modern dosing regimens (5 to 15 mg once weekly, supplemented with folic acid), liver fibrosis and clinically significant liver disease are rare even with long-term use. Methotrexate-induced liver fibrosis and cirrhosis usually appear 2 to 10 years after treatment and may manifest as ascites, esophageal and gastric variceal bleeding, or hepatosplenomegaly. Some patients present with signs and symptoms of portal hypertension but only moderate liver fibrosis, suggesting that methotrexate may also cause nodular regeneration. Patients with portal hypertension and cirrhosis typically have only slightly elevated or no elevated serum transaminase or alkaline phosphatase levels, therefore serum enzyme monitoring appears to be ineffective in predicting the development of liver fibrosis. For patients on long-term methotrexate, non-invasive markers of liver fibrosis, such as continuous platelet counts, serum type III procollagen N-terminal peptide (PIIIP), serum bile acids, liver ultrasound, advanced imaging techniques, and transient elastography, may be more effective in screening for liver fibrosis; however, the reliability and accuracy of these methods have not been confirmed by prospective studies. Patients with methotrexate-induced cirrhosis are usually asymptomatic, and even in patients restarting low-dose treatment, the disease often does not progress. Hepatocellular carcinoma has been reported to be rare in patients with suspected methotrexate-induced cirrhosis. Low-dose, long-term methotrexate treatment has also been associated with rare hepatitis B virus (HBV) reactivation in patients with rheumatoid arthritis or psoriasis who were HBsAg carriers, HBeAg negative, had normal alanine aminotransferase (ALT) levels, and extremely low or undetectable HBV DNA levels before starting methotrexate. The incidence of methotrexate-induced HBV reactivation is unclear but likely low. Reactivation usually occurs several years after methotrexate treatment, and most published cases involve concurrent glucocorticoid therapy. Clinically, it presents as insidious onset of fatigue, nausea, and jaundice, accompanied by significant elevations in serum ALT and HBV DNA levels. In some cases, acute injury is severe and progressive, eventually leading to liver failure. In many case reports, HBV reactivation has occurred after discontinuation of methotrexate, possibly due to immune reactivity recovery in patients with elevated HBV DNA levels during treatment. There have also been reports of hepatitis B virus (HBV) reactivation in HBV antibody-positive patients who were HBsAg negative (serum reversal) despite receiving methotrexate and prednisone treatment. Most reported cases of HBV reactivation in the literature resulted in death or emergency liver transplantation, which may reflect publication bias in severe cases. These cases have prompted recommendations for routine HBsAg screening before initiating long-term methotrexate treatment and for antiviral prophylaxis or close monitoring for elevated HBV DNA levels during methotrexate use. However, it remains unclear whether methotrexate alone (without prednisone) leads to HBV reactivation.
Probability Score: A (Known cause of chronic, clinically significant liver injury, portal hypertension, and cirrhosis).

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Impact of Pregnancy and Lactation
◉ Overview of Medication Use During Lactation
Most sources consider breastfeeding contraindicated during high-dose methotrexate treatment for cancer. Some studies suggest that methotrexate should be discontinued for at least one week after chemotherapy-dose treatment. [1] Chemotherapy may adversely affect the normal microbiota and chemical composition of breast milk. [2] Women who receive chemotherapy during pregnancy are more likely to have difficulty breastfeeding. [3] The concentration of methotrexate in breast milk is low after the mother has taken up to 92 mg (1.12 mg/kg), so some authors believe that low-dose single or weekly methotrexate (e.g., the dose used to treat ectopic pregnancy or rheumatoid arthritis) poses a lower risk to breastfed infants. [4-8] However, some expert opinions warn against doing so. [9-15] Stopping breastfeeding 24 hours after a weekly low-dose methotrexate administration can reduce the infant's drug dose by 40%. [16-18] If breastfeeding is performed during long-term low-dose methotrexate use, monitoring of the infant's complete blood count, differential blood count, and liver enzyme levels may be considered. ◉ Effects on breastfed infants On day 151 postpartum, breastfeeding mothers who started weekly subcutaneous injections of 25 mg methotrexate began breastfeeding. The estimated intake in infants within 24 hours of administration was 3.4 mcg/kg. Mothers continued breastfeeding (feeding extent not specified) for 9 months while receiving weekly subcutaneous injections of 25 mg methotrexate. No adverse reactions were observed in the infants. [8]
Three postpartum women were incorrectly dispensed with 2.5 mg methotrexate daily instead of ergonovine. They received methotrexate daily for 5, 13, and 15 days, respectively, during breastfeeding (feeding extent not specified). Although all women experienced toxic reactions and required hospitalization, none of their infants experienced clinically observable complications. [19]
◉ Effects on breastfeeding and breast milk
No relevant published information was found as of the revision date.
◈ What is methotrexate? Methotrexate is a medication that inhibits cell growth and interferes with the immune system (the body's system for fighting infection). Methotrexate is used to treat a variety of conditions, including cancer and autoimmune diseases such as rheumatoid arthritis (https://mothertobaby.org/fact-sheets/rheumatoid-arthritis/), lupus (https://mothertobaby.org/fact-sheets/lupus-pregnancy/), and psoriasis (https://mothertobaby.org/fact-sheets/psoriasis-and-pregnancy/). Methotrexate was previously used to induce labor and is currently used to treat ectopic pregnancies (pregnancy where the embryo develops outside the uterus). Some brand names for methotrexate include Otrexup®, Trexall®, Rheumatrex®, and Rasuvo®. The product information for methotrexate advises that pregnant women should not use this medication unless it is for cancer treatment. However, the benefits of using methotrexate to treat your condition may outweigh the potential risks. Your healthcare provider can discuss methotrexate use with you and the best treatment option for you. Methotrexate reduces the body's ability to utilize folic acid. Folic acid is essential for fetal development during pregnancy. If you have recently stopped taking methotrexate and are planning to become pregnant, talk to your healthcare provider about the need for folic acid supplements and the dosage you should take.
◈ I am taking methotrexate. Will taking methotrexate affect my ability to get pregnant?
A study of infertile patients receiving methotrexate treatment for ectopic pregnancy showed a reduction in the number of eggs available for fertilization. However, this is temporary. Other studies have not shown that methotrexate use increases the risk of fertility problems.
◈ I am taking methotrexate, but I want to stop taking it before getting pregnant. How long will this drug stay in my body?
Everyone clears the drug at a different rate. For healthy adults, it takes about a week on average for most methotrexate to be cleared from the body. Some medications may affect the time it takes for methotrexate to be cleared from the body. In addition, people with impaired kidney function or fluid overload conditions may experience a slower rate of methotrexate clearance from their bodies.
◈ How long should you wait after stopping methotrexate before trying to conceive?
Some healthcare providers recommend waiting 1 to 3 months after stopping methotrexate to ensure the drug has been cleared from the body. Drug instructions recommend waiting 3 to 6 months. However, there are currently no reports of birth defects in babies caused by women stopping methotrexate before conception.
◈ Does taking methotrexate increase the risk of miscarriage?
Miscarriage is common and can occur in any pregnancy for a variety of reasons. Some small studies have reported that taking methotrexate increases the risk of miscarriage. Because methotrexate is used to terminate pregnancy or treat ectopic pregnancy, its use in early pregnancy is likely to increase the risk of miscarriage.
◈ Does taking methotrexate increase the risk of birth defects?
There is a 3-5% risk of birth defects at the start of each pregnancy; this is known as the baseline risk. Taking methotrexate in early pregnancy may increase the risk of certain types of birth defects, including head, facial, limb, and skeletal deformities in infants. For other birth defects, such as heart defects and cleft lip and palate, current evidence is insufficient to prove that methotrexate is a cause. Limited evidence suggests that methotrexate-related birth defects may occur if a pregnant woman is exposed to 10 mg or more of methotrexate weekly for 6 to 8 weeks after conception (8 to 10 weeks after the first day of her last menstrual period). A published review of studies reported that in 101 pregnant women with rheumatoid arthritis, weekly exposure to 5 to 25 mg of methotrexate in early pregnancy did not result in an increased incidence of miscarriage or birth defects. While this is reassuring, it does not mean that low-dose methotrexate use in early pregnancy does not increase the risk of miscarriage or birth defects.
◈ Does taking methotrexate during pregnancy increase the risk of other pregnancy-related problems?
According to reviewed studies, fetal maldevelopment may be associated with methotrexate use during pregnancy.
◈ Will taking methotrexate during pregnancy affect a child's future behavior or learning?
According to reviewed studies, children exposed to methotrexate during pregnancy may experience developmental delays, learning disabilities, and intellectual disabilities.
◈ Breastfeeding while taking methotrexate:
A small amount of methotrexate can pass into breast milk. Drug instructions and some healthcare providers do not recommend using methotrexate while breastfeeding and suggest stopping breastfeeding within one week of taking the last dose of methotrexate. Breast milk tests on individuals taking doses up to 92 mg of methotrexate have shown lower levels of methotrexate in their breast milk. Therefore, some experts believe that weekly doses of low-dose methotrexate are less likely to cause problems for breastfed infants. If a low-dose methotrexate is taken while breastfeeding, monitoring the infant's blood cell count is recommended. Always consult your healthcare provider about any questions regarding breastfeeding.
◈ If a man takes methotrexate, will it affect his fertility (the ability to impregnate his partner) or increase the risk of birth defects? The drug's package insert states that men should use effective contraception during methotrexate treatment and for three months after the last dose. Methotrexate may increase the risk of infertility. Some men taking methotrexate experience low sperm counts. These men are mostly taking high doses of methotrexate and concurrently taking other anticancer drugs. After discontinuing the medication, sperm counts return to normal. Men who need to take methotrexate for cancer treatment may consider freezing their sperm before treatment. Currently, there are no reports indicating that men taking methotrexate at conception increases the risk of birth defects in their offspring. Four studies found no increased incidence of birth defects in the children of 65 men who took methotrexate before or after conception. Generally, exposure to methotrexate by the father or sperm donor is unlikely to increase pregnancy risk. For more information, please refer to MotherToBaby's "Paternal Exposure" information sheet at https://mothertobaby.org/fact-sheets/paternal-exposures-pregnancy/.

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Protein Binding
Methotrexate binds to plasma proteins at a rate of 46.5-54%.

[1]. Understanding the mechanisms of action of methotrexate: implications for the treatment of rheumatoid arthritis. Bull NYU Hosp Jt Dis. 2007;65(3):168-73.

[2]. Methotrexate in rheumatoid arthritis. Pharmacol Rep. 2006 Jul-Aug;58(4):473-92.

[3]. The Effect of L-carnitine on Amethopterin-induced Toxicity in Rat Large Intestine.

[4]. Evaluation of the concomitant use of methotrexate and curcumin on Freund's complete adjuvant-induced arthritis and hematological indices in rats. Indian J Pharmacol. 2011;43(5):546-550.

According to state or federal labeling requirements, methotrexate sodium may cause developmental toxicity. Methotrexate disodium is an organosodium salt, the disodium salt of methotrexate. It is an EC 1.5.1.3 (dihydrofolate reductase) inhibitor. It contains a methotrexate (2-) molecule. Methotrexate sodium is the sodium salt of methotrexate, an antimetabolite with antitumor and immunomodulatory properties. Methotrexate binds to and inhibits dihydrofolate reductase, thereby inhibiting the synthesis of purine nucleotides and thymidine, and consequently, DNA and RNA synthesis. Methotrexate also has potent immunosuppressive properties. (NCI04) An antitumor antimetabolite with immunosuppressive properties. It is a tetrahydrofolate dehydrogenase inhibitor that prevents the production of tetrahydrofolate, which is essential for the synthesis of thymidine (an important component of DNA). See also: Methotrexate (with active moiety).

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According to an independent committee of scientific and health experts, methotrexate may cause developmental toxicity.
Methotrexate is an odorless, yellow to orange-brown crystalline powder. (NTP, 1992) It is a chemotherapeutic drug that interferes with DNA and RNA synthesis.
Methotrexate belongs to the pteridine class of compounds and is a monocarboxylic acid amide and dicarboxylic acid. It has a variety of pharmacological effects, including antitumor, antirheumatic, EC 1.5.1.3 (dihydrofolate reductase) inhibitor, DNA synthesis inhibitor, abortifacient, dermatological, antimetabolite, and immunosuppressant. Its function is related to L-glutamate, which is the conjugate acid of methotrexate (1-). Methotrexate is a folate derivative that inhibits a variety of enzymes involved in nucleotide synthesis. This inhibition suppresses inflammation and stops cell division. Due to these effects, methotrexate is often used to treat inflammation caused by arthritis or to control cell division in neoplastic diseases such as breast cancer and non-Hodgkin's lymphoma. Because of its toxicity, methotrexate is only used to treat certain types of arthritis and severe psoriasis when initial treatment fails or the patient cannot tolerate first-line therapy. Methotrexate was approved by the U.S. Food and Drug Administration (FDA) on December 7, 1953. Methotrexate is a folic acid analogue metabolism inhibitor. Its mechanism of action is the inhibition of folic acid metabolism. Methotrexate is an antitumor and immunosuppressant, widely used to treat leukemia, lymphoma, solid tumors, psoriasis, and rheumatoid arthritis. High-dose intravenous methotrexate can cause acute elevations of serum enzymes. Long-term methotrexate treatment is associated with frequent but mild elevations of serum liver enzymes, and more importantly, it is also associated with chronic liver damage, progressive fibrosis, cirrhosis, and portal hypertension. Methotrexate has been reported in Quambalaria cyanescens, Gambierdiscus, and Asimina triloba, and relevant data are available. Methotrexate is an antimetabolite and antifolate drug with antitumor and immunosuppressive activities. Methotrexate binds to and inhibits dihydrofolate reductase, thereby inhibiting the synthesis of purine nucleotides and thymidine, and consequently, DNA and RNA synthesis. Methotrexate also possesses potent immunosuppressive activity, but its mechanism of action is unclear. Methotrexate is only found in individuals who have used or taken the drug. It is an antitumor antimetabolite drug with immunosuppressive properties. It is an inhibitor of tetrahydrofolate dehydrogenase, preventing the production of tetrahydrofolate, which is essential for the synthesis of thymidine (an important component of DNA). [PubChem] The antitumor activity of methotrexate stems from its inhibition of folate reductase, thereby inhibiting DNA synthesis and cell replication. Its mechanism of action in treating rheumatoid arthritis is unclear. An antitumor antimetabolite drug with immunosuppressive properties. It is a tetrahydrofolate dehydrogenase inhibitor, preventing the production of tetrahydrofolate, which is essential for the synthesis of thymidine (an important component of DNA). See also: Methotrexate Sodium (in saline form).

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Drug Indications
Methotrexate oral solution is indicated for children with acute lymphoblastic leukemia and juvenile polyarticular idiopathic arthritis. Methotrexate subcutaneous injection is indicated for severe active rheumatoid arthritis, juvenile polyarticular idiopathic arthritis, and severe, refractory, disabling psoriasis. It has also been approved by the European Medicines Agency (EMA) for the treatment of adult patients with moderate to severe plaque psoriasis requiring systemic therapy. Other formulations are indicated for the treatment of choriocarcinoma of pregnancy, destructive chorioadenoma, hydatidiform mole, breast cancer, epidermoid carcinoma of the head and neck, advanced mycosis fungoides, lung cancer, and advanced non-Hodgkin lymphoma. It is also used for maintenance therapy in acute lymphoblastic leukemia. In non-metastatic osteosarcoma, methotrexate is also used before leucovorin therapy to prolong relapse-free survival after surgical resection of the tumor.
FDA Label
Nodimet is indicated for the treatment of: active rheumatoid arthritis in adults; polyarticular form of severe active juvenile idiopathic arthritis (JIA) when the response to nonsteroidal anti-inflammatory drugs (NSAIDs) is inadequate; moderate to severe plaque psoriasis in adults who are eligible for systemic therapy; severe psoriatic arthritis in adults; induction of remission in adults with moderate steroid-dependent Crohn's disease (in combination with corticosteroids); and maintenance of remission as monotherapy in patients who have responded to methotrexate.
In rheumatoid arthritis in adults with active rheumatoid arthritis in rheumatic and dermatological diseases. Indicated for polyarticular form of active, severe juvenile idiopathic arthritis (JIA) in adolescents and children aged 3 years and older who have not responded adequately to NSAIDs. Indicated for severe, refractory, disabling psoriasis that has not responded adequately to phototherapy, psoralen and long-wave ultraviolet (PUVA) therapy, and other treatments such as retinoic acid, and severe psoriatic arthritis in adults. In oncology, it is indicated for maintenance therapy of acute lymphoblastic leukemia (ALL) in adults, adolescents, and children aged 3 years and older. Mechanism of Action After entering tissues, methotrexate is converted to methotrexate polyglutamate by the action of folate polyglutamate. The mechanism of action of methotrexate lies in its inhibition of enzymes related to nucleotide synthesis, including dihydrofolate reductase, thymidylate synthase, aminoimidazolium formamide nucleotide formyltransferase (AICART), and amide phosphoribosyltransferase. Inhibition of nucleotide synthesis prevents cell division. In rheumatoid arthritis, methotrexate polyglutamate has a stronger inhibitory effect on AICART than methotrexate itself. This inhibition leads to the accumulation of AICART nucleotides, which in turn inhibits adenosine deaminase, resulting in the accumulation of extracellular adenosine triphosphate and adenosine, stimulating adenosine receptors, thereby exerting an anti-inflammatory effect. Methotrexate and its polyglutamate metabolites reversibly inhibit dihydrofolate reductase, which reduces folate to tetrahydrofolate. The production of tetrahydrofolate is inhibited, thus limiting the availability of the one-carbon fragment required for purine synthesis and hindering the conversion of deoxyuridine monophosphate to thymidine monophosphate during DNA synthesis and cell proliferation. Dihydrofolate reductase has a much higher affinity for methotrexate than for folic acid or dihydrofolate; therefore, even with simultaneous administration of high doses of folic acid, the effects of methotrexate cannot be reversed. Calcium folinate, a derivative of tetrahydrofolate, may block the effects of methotrexate if administered shortly after taking antitumor drugs. One study showed that methotrexate also leads to elevated levels of intracellular deoxyadenosine triphosphate (dATP), which is thought to inhibit ribonucleotide reductase and the activity of polynucleotide ligases (enzymes involved in DNA synthesis and repair). Tissues with high cell proliferation rates, such as tumors, psoriatic epidermis, bone marrow, the gastrointestinal lining, hair matrix, and fetal cells, are most sensitive to the effects of methotrexate. Methotrexate…has immunosuppressive activity, possibly due in part to its inhibition of lymphocyte proliferation. The mechanism of action of this drug in treating rheumatoid arthritis is not fully understood, but hypothesized mechanisms include immunosuppression and/or anti-inflammatory effects. Currently, several disease-modifying antirheumatic drugs (DMARDs) are available for the clinical control of rheumatoid arthritis (RA). Methotrexate (MTX), an analogue of folic acid and aminopterin, is the most commonly used DMARD, currently being taken by at least 500,000 RA patients worldwide. The mechanism by which low-dose methotrexate (MTX) modulates inflammation in rheumatoid arthritis (RA) is unclear. Monitoring MTX concentrations in RA patients does not appear to have a significant impact on treatment efficacy. Two meta-analyses have shown that MTX has one of the best efficacy/toxicity ratios. Currently, it should be the DMARD of choice for most RA patients. However, a significant proportion of patients receiving MTX alone fail to achieve optimal disease control, hence the existence of many DMARD combination therapy regimens. There is hope that more aggressive use of traditional DMARDs and biologics can reduce disability and improve remission rates. The treatment of RA is a dynamic process requiring a delicate balance between benefits and risks. Even with newer biologics, methotrexate (MTX) remains the standard of reference and continues to play a role in the treatment of RA patients. [2] Methotrexate has been widely used to treat rheumatoid arthritis (RA). The mechanism of action of methotrexate is complex. As a folic acid analog, methotrexate works by inhibiting the synthesis of purines and pyrimidines, which explains both its efficacy in cancer treatment and some of its toxicities. In recent years, many studies have focused on the anti-inflammatory effects of methotrexate mediated by adenosine. Some of the toxicities of methotrexate are also related to the release of adenosine. A deeper understanding of the mechanism of action and toxicity of methotrexate will help clinicians develop treatment plans and monitor toxicity. To this end, this article will explore the latest advances in the pharmacokinetics, mechanism of action, pharmacogenetics and toxicity of methotrexate. [1]

C20H20N8NA2O5

498.41

498.135

C, 48.20; H, 4.04; N, 22.48; Na, 9.23; O, 16.05

7413-34-5

Methotrexate;59-05-2;Methotrexate hydrate;133073-73-1;Methotrexate monohydrate;6745-93-3

11329481

Light yellow to yellow solid powder

823℃

3

12

7

35

693

1

CN(CC1=CN=C2C(=N1)C(=NC(=N2)N)N)C3=CC=C(C=C3)C(=O)N[C@@H](CCC(=O)[O-])C(=O)[O-].[Na+].[Na+]

DASQOOZCTWOQPA-GXKRWWSZSA-L

InChI=1S/C20H22N8O5.2Na/c1-28(9-11-8-23-17-15(24-11)16(21)26-20(22)27-17)12-4-2-10(3-5-12)18(31)25-13(19(32)33)6-7-14(29)30;;/h2-5,8,13H,6-7,9H2,1H3,(H,25,31)(H,29,30)(H,32,33)(H4,21,22,23,26,27);;/q;2*+1/p-2/t13-;;/m0../s1

disodium;(2S)-2-[[4-[(2,4-diaminopteridin-6-yl)methyl-methylamino]benzoyl]amino]pentanedioate

Methotrexate disodium salt; 7413-34-5; methotrexate disodium; METHOTREXATE SODIUM; Sodium methotrexate; Disodium methotrexate; MTX disodium; Amethopterin sodium;

2934.99.9001

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 (e.g. under nitrogen), avoid exposure to moisture and light.

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

H2O : ~50 mg/mL (~100.32 mM)
DMSO : ~5 mg/mL (~10.03 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.)