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%, though this decreases at oral doses above 25mg due to saturation of the carrier mediated transport of methotrexate.. Methotrexate has a Tmax of 1 to 2 hours. oral doses of 10-15µg reach serum levels of 0.01-0.1µM.
Methotrexate is >80% excreted as the unchanged drug and approximately 3% as the 7-hydroxylated metabolite. Methotrexate is primarily excreted in the urine with 8.7-26% of an intravenous dose appearing in the bile.
The volume of distribution of methotrexate at steady state is approximately 1L/kg.
Methotrexate clearance varies widely between patients and decreases with increasing doses. Currently, predicting clearance of methotrexate is difficult and exceedingly high serum levels of methotrexate can still occur when all precautions are taken.
In adults, oral absorption of methotrexate appears to be dose dependent. Peak serum levels are reached within one to two hours. At doses of 30 mg/sq m or less, methotrexate is generally well absorbed with a mean bioavailability of about 60%. The absorption of doses greater than 80 mg/sq m is significantly less, possibly due to a saturation effect.
After intravenous administration, the initial volume of distribution is approximately 0.18 L/kg (18% of body weight) and 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 to albumin.
At serum methotrexate concentrations exceeding 0.1 umol/mL passive diffusion becomes a major means of intracellular transport of the drug. The drug is widely distributed into body tissues with highest concn in the kidneys, gallbladder, spleen, liver, and skin.
For more Absorption, Distribution and Excretion (Complete) data for METHOTREXATE (10 total), please visit the HSDB record page.

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Metabolism / Metabolites
Methotrexate is metabolized by folylpolyglutamate synthase to methotrexate polyglutamate in the liver as well as in tissues. Gamma-glutamyl hydrolase hydrolyzes the glutamyl chains of methotrexate polyglutamates converting them back to methotrexate. A small amount of methotrexate is also converted to 7-hydroxymethotrexate.
After absorption, methotrexate undergoes hepatic and intracellular metabolism to form methotrexate polyglutamate, metabolites which by hydrolysis may be converted back to methotrexate. Methotrexate polyglutamates inhibit dihydrofolate reductase and thymidylate synthetase. Small amounts of these polyglutamate metabolites may remain in tissues for extended periods; the retention and prolonged action of these active metabolites vary among different cells, tissues, and tumors. In addition, small amounts of methotrexate polyglutamate may be converted to 7-hydroxymethotrexate; accumulation of this metabolite may become substantial following administration of high doses of methotrexate, since the aqueous solubility of 7-hydroxymethotrexate is threefold to fivefold lower than that of the parent compound. Following oral administration of methotrexate, the drug also is partially metabolized by the intestinal flora.
After absorption, methotrexate undergoes hepatic and intracellular metabolism to form methotrexate polyglutamate, metabolites which by hydrolysis may be converted back to methotrexate. Methotrexate polyglutamates inhibit dihydrofolate reductase and thymidylate synthetase. Small amounts of these polyglutamate metabolites may remain in tissues for extended periods; the retention and prolonged action of these active metabolites vary among different cells, tissues, and tumors. In addition, small amounts of methotrexate polyglutamate may be converted to 7-hydroxymethotrexate; accumulation of this metabolite may become substantial following administration of high doses of methotrexate, since the aqueous solubility of 7-hydroxymethotrexate is threefold to fivefold lower than that of the parent compound. Following oral administration of methotrexate, the drug also is partially metabolized by the intestinal flora. Renal excretion is the primary route of elimination, and is dependent upon dosage and route of administration (A620).
Route of Elimination: Renal excretion is the primary route of elimination and is dependent upon dosage and route of administration. IV administration, 80% to 90% of the administered dose is excreted unchanged in the urine within 24 hours. There is limited biliary excretion amounting to 10% or less of the administered dose.
Half Life: Low doses (less than 30 mg/m^2): 3 to 10 hours; High doses: 8 to 15 hours.

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Biological Half-Life
The half life of low dose methotrexate is 3 to 10 hours in adults. The half life for high dose methotrexate is 8 to 15 hours. Pediatric patients taking methotrexate for acute lymphoblastic anemia experience a terminal half life of 0.7 to 5.8 hours. Pediatric patients taking methotrexate for juvenile idiopathic arthritis experience a half life of 0.9 to 2.3 hours.
Terminal: Low doses: 3 to 10 hours. High doses: 8 to 15 hours. Note: There is wide interindividual variation in clearance rates. Small amounts of methotrexate and its metabolites are protein-bound and may remain in tissues (kidneys, liver) for weeks to months; the presence of fluid loads, such as ascites or pleural effusion, and renal function impairment will also delay clearance.

Hepatotoxicity
Methotrexate is well known to cause serum aminotransferase elevations and long term therapy has been linked to development of fatty liver disease, fibrosis and even cirrhosis. The literature on methotrexate is extensive, but with great variability in rates of liver test and biopsy abnormalities at different doses, dose regimens and durations of therapy.
With 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 then fall rapidly to normal with only rare instances of jaundice or symptoms of liver injury. With long term, low-to-moderate dose methotrexate therapy, elevations in serum ALT or AST values occur in 15% to 50% of patients, but are usually mild and self-limiting. Approximately 5% of patients have elevations greater than twice normal and these abnormalities resolve rapidly with discontinuation or dose modification, but can resolve even with continuation at the same dose level. The reported rate of ALT elevations during therapy has varied considerably, perhaps because of differences in frequency of determinations (every month vs every 3, 6 or 12 months) and due to the timing of the blood sampling (whether just before or soon after the once weekly dose). Finally, coadministration of folic acid has been shown to decrease the frequency of serum enzyme elevations and now is commonly used.
Long term therapy with methotrexate has been associated with development of fatty liver and hepatic fibrosis and, in rare instances, portal hypertension and symptomatic cirrhosis. Symptoms are usually absent until cirrhosis is present, and liver tests are typically normal or minimally and transiently elevated. Routine monitoring of patients with regular liver biopsies done at 1 to 2 year intervals or with cumulative methotrexate doses of 1 to 10 grams demonstrates that approximately 30% of patients develop mild-to-moderate histological abnormalities (fat, cellular unrest, mild inflammation, nuclear atypical) and 2 to 20% of patients develop some degree of hepatic fibrosis. Well documented cases of cirrhosis arising during long term methotrexate therapy have been reported, but cirrhosis is rare in prospective series, even with routine histological monitoring. Patients who develop fibrosis on long term methotrexate therapy often have other risk factors for fatty liver disease, including excessive alcohol use, obesity, diabetes and concurrent administration of other potentially hepatotoxic agents. Use of high doses and daily methotrexate dosing is particularly associated with development of hepatic fibrosis and rates of cirrhosis of greater than 20% after 5 to 10 years of treatment. With more modern dose regimens (5 to 15 mg in one dose weekly with folate supplementation), fibrosis and clinically apparent liver disease are rare even with long term use. The hepatic fibrosis and cirrhosis due to methotrexate typically arise after 2 to 10 years of treatment and can present with ascites, variceal hemorrhage or hepatosplenomegaly. Some patients present with signs and symptoms of portal hypertension, yet have only moderate degrees of fibrosis, suggesting that methotrexate may also cause nodular regeneration. Patients who develop portal hypertension and cirrhosis usually have had minimal or no elevations in serum aminotransferase or alkaline phosphatase levels, and monitoring using serum enzymes appears to be poorly predictive of fibrosis development. Noninvasive markers of hepatic fibrosis, such as serial platelet counts, serum procollagen III aminoterminal peptide (PIIIP), serum bile acids, hepatic ultrasound, advanced imaging techniques and transient elastography may be more efficient in screening for fibrosis in patients on long term methotrexate, but the reliability and accuracy of these approaches has not been documented prospectively. Patients with cirrhosis due to methotrexate are often asymptomatic and the condition tends to be non-progressive, even in those who restart low dose therapy. Rare instances of hepatocellular carcinoma have been reported in patients with suspected methotrexate induced cirrhosis.
Low dose, long term methotrexate therapy has also been implicated in rare instances of reactivation of hepatitis B in patients with rheumatoid arthritis or psoriasis who were HBsAg carriers, without HBeAg and with normal ALT levels and no detectable or low levels of HBV DNA before starting methotrexate. The frequency of reactivation with methotrexate is unknown, but is probably low. Reactivation typically presents after years of therapy with methotrexate and most published cases were also receiving corticosteroids. The clinical presentation is characterized by insidious onset of fatigue, nausea and jaundice accompanied by marked elevations in serum ALT and HBV DNA levels. In some instances, the acute injury is severe and progressive resulting in liver failure. In many case reports, reactivation occurred when methotrexate was withdrawn, perhaps as a result of restoration of immune reactivity in those in whom HBV DNA levels have risen during treatment. Reactivation has also been described in patients with antibodies to HBV without HBsAg (reverse seroconversion) treated with methotrexate and prednisone. The cases of reactivation of hepatitis B published in the literature have mostly resulted in death or emergency liver transplantation, perhaps reflecting publication bias for more severe cases. These cases have led to recommendations for routine screening for HBsAg before starting long term methotrexate therapy and prophylaxis with antiviral agents or careful monitoring for rises in HBV DNA levels if methotrexate is used. However, whether methotrexate on its own, without prednisone, can cause reactivation of hepatitis B is not clear.
Likelihood score: A (well known cause of chronic, clinically significant liver injury, portal hypertension and cirrhosis).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Most sources consider breastfeeding to be contraindicated during maternal high-dose antineoplastic drug therapy with methotrexate. An abstinence period of at least 1 week after chemotherapy doses of methotrexate has been suggested.[1] Chemotherapy may adversely affect the normal microbiome and chemical makeup of breastmilk.[2] Women who receive chemotherapy during pregnancy are more likely to have difficulty nursing their infant.[3]
Maternal doses of methotrexate up to 92 mg (1.12 mg/kg) produce low levels in milk, leading some authors to state that low single or weekly doses, such as those used for ectopic pregnancy or rheumatoid arthritis, are of low risk to the breastfed infant,[4-8] although some expert opinion warns against this use.[9-15] Withholding breastfeeding for 24 hours after a weekly low dose of methotrexate may decrease the infant's dose by 40%.[16-18] If breastfeeding during long-term, low-dose methotrexate use is undertaken, monitoring of the infant's complete blood count and differential and liver enzymes could be considered.
◉ Effects in Breastfed Infants
On day 151 postpartum, weekly methotrexate 25 mg subcutaneously begun a nursing mother. The estimated intake of the infant at that time was 3.4 mcg/kg in the first 24 hours after administration. The mother continued to breastfeed (extent not stated) for an additional 9 months while receiving subcutaneous methotrexate 25 mg weekly. No adverse effects were noted in the infant.[8]
Three postpartum women were erroneously dispensed methotrexate 2.5 mg daily instead of methylergonovine. They took methotrexate daily for 5, 13 and 15 days, respectively, while they were breastfeeding (extent not stated). Although all of the women developed toxicity and required hospitalization, none of their infants had clinically observable complications.[19]
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.
◈ What is methotrexate?
Methotrexate is a medication that can stop the growth of cells and can interfere with the immune system (the system the body uses to fight off infections).Methotrexate is prescribed to treat many conditions, including cancer and autoimmune conditions like 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 used to induce abortions and is currently used to treat ectopic pregnancies (pregnancies that grow outside the uterus). Some brand names for methotrexate are: Otrexup®, Trexall®, Rheumatrex®, and Rasuvo®.The product label for methotrexate recommends people who are pregnant not use this medication unless it is being used for cancer treatment. But, the benefit of using methotrexate to treat your medical condition may outweigh possible risks. Your healthcare providers can talk with you about using methotrexate and what treatment is best for you.Methotrexate lowers the body’s ability to use folic acid. During pregnancy, folic acid is important for development of the baby. If you have recently stopped taking methotrexate and are planning to get pregnant, talk with your healthcare provider about taking a folic acid supplement and what dose you should take.
◈ I take methotrexate. Can it make it harder for me to get pregnant?
One study on infertility patients treated with methotrexate for ectopic pregnancy suggested a lower number of eggs available for fertilization. This finding was temporary. Other studies have not shown an increased chance of problems with fertility with the use of methotrexate.
◈ I am taking methotrexate, but I would like to stop taking it before becoming pregnant. How long does the drug stay in my body?
People eliminate medication at different rates. In healthy adults, it takes up to 1 week, on average, for most of the methotrexate to be gone from the body. Certain medications might affect how long methotrexate clears from the body. In addition, people who have reduced kidney function or a condition that leads to extra body fluid might clear methotrexate more slowly from their body.
◈ How long do I need to wait to become pregnant after I stop taking methotrexate?
Some healthcare providers have suggested waiting 1 to 3 months after stopping methotrexate to ensure the medication has been cleared from the body. The drug label recommends waiting 3 to 6 months after stopping the medication. However, there are no reports of babies being born with methotrexate-related birth defects when a female stops taking this medication before conception.
◈ Does taking methotrexate increase the chance for miscarriage?
Miscarriage is common and can occur in any pregnancy for many different reasons. Small studies have reported an increased chance for miscarriage. Since methotrexate can be used to abort pregnancies or treat ectopic pregnancies, it seems likely that methotrexate use in early pregnancy would increase the chance of miscarriage.
◈ Does taking methotrexate increase the chance of birth defects?
Every pregnancy starts out with a 3-5% chance of having a birth defect. This is called the background risk. Taking methotrexate in the first trimester could increase the chance for a specific pattern of birth defects. This includes malformations of the infant's head, face, limbs, and bones. For other birth defects, like heart defects and oral clefts, the evidence is not strong enough to show that methotrexate is the cause.Limited evidence suggests that methotrexate-related birth defects can occur if a pregnancy is exposed to 10 mg or more of methotrexate per week between 6 and 8 weeks after conception (8 to 10 weeks after the first day of the last menstrual period).One published review of studies reported no increase in miscarriage or birth defects in 101 people with rheumatoid arthritis who were exposed to 5 to 25mg a week of methotrexate in the first trimester. While reassuring, this does not mean there is no increased chance of miscarriage or birth defects when using low-dose methotrexate in the first trimester.
◈ Does taking methotrexate in pregnancy increase the chance of other pregnancy-related problems?
Based on the studies reviewed, poor growth of the developing baby may be associated with methotrexate use during pregnancy.
◈ Does taking methotrexate in pregnancy affect future behavior or learning for the child?
Based on the studies reviewed, developmental delay, learning problems, and intellectual disability have been described in children who were exposed to methotrexate during pregnancy.
◈ Breastfeeding while taking methotrexate:
Methotrexate passes into breast milk in small amounts. The drug label and some healthcare providers do not recommend using methotrexate while breastfeeding and to not breastfeed for 1 week after taking the last dose of methotrexate.Testing of breastmilk in people exposed to methotrexate doses up to 92 mg found low levels of methotrexate in the milk. As a result, some experts suggest that weekly low-dose methotrexate has a low chance of causing problems for the breastfed infant. If a person uses low-dose methotrexate treatment while breastfeeding, monitoring of the baby’s blood count is suggested. Be sure to talk to your healthcare provider about all of your breastfeeding questions.
◈ If a male takes methotrexate, could it affect fertility (ability to get partner pregnant) or increase the chance of birth defects?
The drug label states that males should use effective contraception while on methotrexate and for 3 months after taking the final dose. Methotrexate might cause an increased chance of infertility. Low sperm count has been seen in some males using methotrexate. Most of these males were using high doses of the medication, as well as other medications to treat cancer. Sperm levels returned to normal after the medication was stopped. Males who need to take methotrexate as part of cancer treatment may want to consider banking sperm before treatment.There are no reports suggesting that males who use methotrexate at the time of conception are more likely to conceive a baby with a birth defect. Four studies noted no increase in birth defects in the children of 65 males who had taken methotrexate around the time of conception. In general, exposures that fathers or sperm donors have are unlikely to increase the risks to a pregnancy. For more information, please see the MotherToBaby fact sheet Paternal Exposures at https://mothertobaby.org/fact-sheets/paternal-exposures-pregnancy/.

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Protein Binding
Methotrexate is 46.5-54% bound to plasma proteins.

[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.

Methotrexate Sodium can cause developmental toxicity according to state or federal government labeling requirements.
Methotrexate disodium is an organic sodium salt that is the disodium salt of methotrexate. It has a role as an EC 1.5.1.3 (dihydrofolate reductase) inhibitor. It contains a methotrexate(2-).
Methotrexate Sodium is the sodium salt of methotrexate, an antimetabolite with antineoplastic and immunomodulating properties. Methotrexate binds to and inhibits the enzyme dihydrofolate reductase, resulting in inhibition of purine nucleotide and thymidylate synthesis and, subsequently, inhibition of DNA and RNA syntheses. Methotrexate also exhibits potent immunosuppressant properties. (NCI04)
An antineoplastic antimetabolite with immunosuppressant properties. It is an inhibitor of TETRAHYDROFOLATE DEHYDROGENASE and prevents the formation of tetrahydrofolate, necessary for synthesis of thymidylate, an essential component of DNA.
See also: Methotrexate (has active moiety).

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Methotrexate can cause developmental toxicity according to an independent committee of scientific and health experts.
Methotrexate is an odorless yellow to orange-brown crystalline powder. (NTP, 1992) It is a chemotherapy drug that interferes with DNA and RNA synthesis.
Methotrexate is a member of pteridines, a monocarboxylic acid amide and a dicarboxylic acid. It has a role as an antineoplastic agent, an antirheumatic drug, an EC 1.5.1.3 (dihydrofolate reductase) inhibitor, a DNA synthesis inhibitor, an abortifacient, a dermatologic drug, an antimetabolite and an immunosuppressive agent. It is functionally related to a L-glutamic acid. It is a conjugate acid of a methotrexate(1-).
Methotrexate is a folate derivative that inhibits several enzymes responsible for nucleotide synthesis. This inhibition leads to suppression of inflammation as well as prevention of cell division. Because of 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. Due to the toxic effects of methotrexate, it is indicated for treatment of some forms of arthritis and severe psoriasis only if first line treatment has failed or patients are intolerant of those treatments. Methotrexate was granted FDA approval on 7 December 1953.
Methotrexate is a Folate Analog Metabolic Inhibitor. The mechanism of action of methotrexate is as a Folic Acid Metabolism Inhibitor.
Methotrexate is an antineoplastic and immunosuppressive agent widely used in the therapy of leukemia, lymphoma, solid tumors, psoriasis and rheumatoid arthritis. When given in high intravenous doses, methotrexate can cause acute elevations in serum enzymes, and long term methotrexate therapy has been associated with frequent but mild elevations in serum liver enzymes and, more importantly, with development of chronic liver injury, progressive fibrosis, cirrhosis and portal hypertension.
Methotrexate has been reported in Quambalaria cyanescens, Gambierdiscus, and Asimina triloba with data available.
Methotrexate is an antimetabolite and antifolate agent with antineoplastic and immunosuppressant activities. Methotrexate binds to and inhibits the enzyme dihydrofolate reductase, resulting in inhibition of purine nucleotide and thymidylate synthesis and, subsequently, inhibition of DNA and RNA syntheses. Methotrexate also exhibits potent immunosuppressant activity although the mechanism(s) of actions is unclear.
Methotrexate is only found in individuals that have used or taken this drug. It is an antineoplastic antimetabolite with immunosuppressant properties. It is an inhibitor of tetrahydrofolate dehydrogenase and prevents the formation of tetrahydrofolate, necessary for synthesis of thymidylate, an essential component of DNA. [PubChem]Methotrexate anti-tumor activity is a result of the inhibition of folic acid reductase, leading to inhibition of DNA synthesis and inhibition of cellular replication. The mechanism involved in its activity against rheumatoid arthritis is not known.
An antineoplastic antimetabolite with immunosuppressant properties. It is an inhibitor of TETRAHYDROFOLATE DEHYDROGENASE and prevents the formation of tetrahydrofolate, necessary for synthesis of thymidylate, an essential component of DNA.
See also: Methotrexate Sodium (has salt form).

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Drug Indication
Methotrexate oral solution is indicated for pediatric acute lymphoblastic leukemia and pediatric polyarticular juvenile idiopathic arthritis. Methotrexate injections for subcutaneous use are indicated for severe active rheumatoid arthritis, polyarticular juvenile idiopathic arthritis and severe, recalcitrant, disabling psoriasis. It has also been approved by the EMA for the treatment of adult patients requiring systemic therapy for moderate-to-severe plaque psoriasis. Other formulations are indicated to treat gestational choriocarcinoma, chorioadenoma destruens, hydatiform mole, breast cancer, epidermoid cancer of the head and neck, advanced mycosis fungoides, lung cancer, and advanced non-Hodgkin's lymphoma. It is also used in the maintenance of acute lymphocytic leukemia. Methotrexate is also given before treatment with leucovorin to prolong relapse-free survival following surgical removal of a tumour in non-metastatic osteosarcoma.
FDA Label
Nordimet is indicated for the treatment of: active rheumatoid arthritis in adult patients,polyarthritic forms of severe, active juvenile idiopathic arthritis (JIA), when the response to nonsteroidal anti-inflammatory drugs (NSAIDs) has been inadequate,moderate to severe plaque psoriasis in adults who are candidates for systemic therapy, and severe psoriatic arthritis in adult patients, induction of remission in moderate steroid-dependent Crohn's disease in adult patients, in combination with corticosteroids and for maintenance of remission, as monotherapy, in patients who have responded to methotrexate.
In rheumatological and dermatological diseasesActive rheumatoid arthritis in adult patients. Polyarthritic forms of active, severe juvenile idiopathic arthritis (JIA) in adolescents and children aged 3 years and over when the response to non-steroidal anti-inflammatory drugs (NSAIDs) has been inadequate. Severe, treatment-refractory, disabling psoriasis which does not respond sufficiently to other forms of treatment such as phototherapy, psoralen and ultraviolet A radiation (PUVA) therapy and retinoids, and severe psoriatic arthritis in adult patients. In oncologyMaintenance treatment of acute lymphoblastic leukaemia (ALL) in adults, adolescents and children aged 3 years and over.

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Mechanism of Action
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.
Methotrexate and its polyglutanate metabolites reversibly inhibits dihydrofolate reductase, the enzyme that reduces folic acid to tetrahydrofolic acid. Inhibition of tetrahydrofolate formation limits the availability of one-carbon fragments necessary for synthesis of purines and the conversion of deoxyuridylate to thymidylate in the synthesis of DNA and cell reproduction. The affinity of dihydrofolate reductase for methotrexate is far greater than its affinity for folic acid or dihydrofolic acid. and, therefore, even very large doses of folic acid given simultaneously will not reverse the effects of methotrexate. Leucovorin calcium, a derivative of tetrahydrofolic acid, may block the effects of methotrexate if given shortly after the antineoplastic agent. Results of one study indicate that methotrexate also causes an increase in intracellular deoxyadenosine triphosphate, which is thought to inhibit ribonucleotide reduction, and polynucleotide ligase, an enzyme concerned in DNA synthesis and repair. Tissues with high rates of cellular proliferation such as neoplasms, psoriatic epidermis, bone marrow, the lining of the GI tract, hair matrix, and fetal cells are most sensitive to the effects of methotrexate.
Methotrexate ... has immunosuppressive activity, in part possibly as a result of inhibition of lymphocyte multiplication. The mechanism(s) of action in the management of rheumatoid arthritis of the drug is not known, although suggested mechanisms have included immunosuppressive and/or antiinflammatory effects.

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A variety of disease-modifying antirheumatic drugs (DMARDs) are available to control the clinical activity of rheumatoid arthritis (RA). Methotrexate (MTX), an analogue of folic acid and of aminopterin, is the most commonly used DMARD and is now prescribed worldwide to at least 500,000 patients with RA. The mechanism by which MTX used at a low dose modulates inflammation in RA is still unknown. Monitoring of the therapy in terms of MTX concentration in patients with RA seems not to have a significant influence on the effectiveness of the treatment. Two meta-analyses showed that MTX has one of the best efficacy/toxicity ratios. It should be the first DMARD used in the majority of patients with RA at this time. However, a significant number of patients treated only with MTX fail to achieve optimal disease control, so there are many combinations of DMARD regimes. It is hoped that more aggressive use of conventional DMARDs and biological agents will result in less disability and a higher proportion of patients achieving remission. The therapy of RA is a dynamic process and requires maintaining a delicate balance between benefits and risks. Even with the newer biological agents, MTX continues to serve as a reference point and there is still a role for MTX in the treatment of RA patients.[2]

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Methotrexate has been widely used for the treatment of rheumatoid arthritis (RA). The mechanisms of action of methotrexate are complex. Developed as a folic acid analogue, methotrexate inhibits purine and pyrimidine synthesis, which accounts for its efficacy in the therapy of cancer as well as for some of its toxicities. Recently, many studies have focused on the adenosine-mediated antiinflammatory effects of methotrexate. Certain aspects of methotrexate toxicities are also attributed to adenosine release. A better understanding of the mechanisms of action and toxicities of methotrexate will direct clinicians in their treatment approach and toxicity monitoring. Toward that objective, the latest developments in the pharmacokinetics, mechanism of action, pharmacogenetics, and toxicity of methotrexate are herein discussed.[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.)