DHFR/dihydrofolate reductase; DNA synthesis; antimetabolite; antifolate

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].

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.

Toxicity Summary
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.

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Toxicity Data
Man(iv): TD: 740 mg/kg
Mouse(ip): LD50 mg/kg
Rat(po): LD50 135 mg/kg
Rat(ip): LD50 6 mg/kg
LD50: 43 mg/kg (Oral, Rat) (A308)

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Interactions
Oral neomycin may decreases absorption of oral methotrexate.
Severe, sometimes fatal, toxicity (including hematologic and GI toxicity) has occurred following administration of a non-steroidal anti-inflammatory agent (eg, indomethacin, ketoprofen) concomitantly with methotrexate (particularly with high dose therapy) in patients with various malignant neoplasms, psoriasis, or rheumatoid arthritis.
Concomitant use of penicillins (e.g., amoxicillin, carbenicillin, mezlocillin) may decrease renal clearance of methotrexate, presumably by inhibiting renal tubular secretion of the drug. Increased serum concentrations of methotrexate, resulting in GI or hematologic toxicity, have been reported in patients receiving low- or high-dose methotrexate therapy concomitantly with penicillins, and patients receiving the drugs concomitantly should be carefully monitored.
Concurrent adminstration of intrathecal methotrexate and acyclovir may result in neurological abnormalities; use with caution.
For more Interactions (Complete) data for METHOTREXATE (16 total), please visit the HSDB record page.

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Non-Human Toxicity Values
LD50 Rat oral 180 +/- 45 mg/kg body weight
LD50 Rat ip 6-25 mg/kg body weight
LD50 Mice ip 94 +/- 9 mg/kg body weight

[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.

Therapeutic Uses
Abortifacient Agents, Nonsteroidal; Antimetabolites, Antineoplastic; Antirheumatic Agents; Dermatologic Agents; Enzyme Inhibitors; Folic Acid Antagonists; Immunosuppressive Agents; Nucleic Acid Synthesis Inhibitors
Methotrexate is indicated for treatment of breast carcinoma, head and neck cancers (epidermoid), non-small cell lung carcinoma (especially squamous cell types), small cell lung carcinoma, and gestational trophoblastic tumors (gestational choriocarcinoma, chorioadenoma destruens, hydatidiform mole). /Included in US product labeling/
Methotrexate is indicated for treatment of cervical carcinoma, ovarian carcinoma, bladder carcinoma, colorectal carcinoma, esophageal carcinoma, gastric carcinoma, pancreatic carcinoma, and penile carcinoma. /NOT included in US product labeling/
Methotrexate is indicated for treatment of acute lymphocytic leukemia and prophylaxis and treatment of meningeal leukemia. /Included in US product labeling/
For more Therapeutic Uses (Complete) data for METHOTREXATE (17 total), please visit the HSDB record page.

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Drug Warnings
Methotrexate is a highly toxic drug with a very low therapeutic index and a therapeutic response is not likely to occur without some evidence of toxicity. ... When methotrexate is used in combination with other antineoplastic agents and/or radiation therapy, toxic reactions may be more severe than would occur with methotrexate therapy alone. Although doses of methotrexate used in the management of psoriasis and rheumatoid arthritis are usually lower than those used in antineoplastic chemotherapy, severe toxicity may occur in any patient receiving the drug and deaths have been reported with the use of methotrexate in the management of psoriasis and rheumatoid arthritis.
Methotrexate should be used with extreme caution in patients with infection, peptic ulcer, ulcerative colitis, or debility, and in very young or geriatric patients. Methotrexate should be used with extreme caution, if at all, in patients with malignant disease who have preexisting liver damage or impaired hepatic function, preexisting bone marrow depression, aplasia, leukopenia, thrombocytopenia, or anemia; the drug is usually contraindicated in patients with impaired renal function. In the management of psoriasis, methotrexate is contraindicated in patients with poor nutritional status or severe renal or hepatic disorders, those with overt or laboratory evidence of an immunodeficiency syndrome, and in those with preexisting blood dyscrasias such as bone marrow hypoplasia, leukopenia, thrombocytopenia, or clinically important anemia; relative contraindications also include cirrhosis, active or recent hepatitis, or excessive alcohol consumption. In the management of rheumatoid arthritis, methotrexate is contraindicated in patients with preexisting blood dyscrasias such as bone marrow hypoplasia, leukopenia, thrombocytopenia, or clinically important anemia; those with overt or laboratory evidence of immunodeficiency syndromes; and those with excessive alcohol consumption, alcoholic liver disease, or chronic liver disease.
Elevations in serum uric acid concentrations may occur in patients receiving methotrexate as a result of cell destruction and hepatic and renal damage. In some patients, uric acid nephropathy and acute renal failure may result. Tumor lysis syndrome associated with other cytotoxic drugs (e.g., fludarabine, cladribine), also has been reported in patients with rapidly growing tumors who were receiving methotrexate. Pharmacologic and appropriate supportive treatment may prevent or alleviate this complication. Methotrexate also was reported to precipitate acute gouty arthritis in two patients being treated for psoriasis. Administration of large volumes of fluids, alkalinization of the urine, and/or administration of allopurinol may be useful in preventing acute attacks of hyperuricemia and uric acid nephropathy.
Severe nephropathy manifested by azotemia, hematuria, and renal failure may occur in patients receiving methotrexate; fatalities have been reported. In one study, postmortem examination revealed extensive necrosis of the epithelium of the convoluted tubules. In patients with renal impairment, methotrexate accumulation and increased toxicity or additional renal damage may occur.
For more Drug Warnings (Complete) data for METHOTREXATE (22 total), please visit the HSDB record page.

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Pharmacodynamics
Methotrexate inhibits enzymes responsible for nucleotide synthesis which prevents cell division and leads to anti-inflammatory actions. It has a long duration of action and is generally given to patients once weekly. Methotrexate has a narrow therapeutic index. Do not take methotrexate daily.

C20H22N8O5

454.44

454.171

C, 52.86; H, 4.88; N, 24.66; O, 17.60

59-05-2

Methotrexate disodium;7413-34-5;Methotrexate hydrate;133073-73-1;Methotrexate monohydrate;6745-93-3; Methotrexate-d3; 432545-63-6; 7413-34-5 (disodium); 7532-09-4 (monosodium); 15475-56-6 (sodium); 59-05-2 (free acid)

126941

Light yellow to yellow solid powder

1.4080

561.26°C

195°C

11℃

1.6910

-0.24

5

12

9

33

704

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

FBOZXECLQNJBKD-ZDUSSCGKSA-N

InChI=1S/C20H22N8O5/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)/t13-/m0/s1

(S)-2-(4-(((2,4-diaminopteridin-6-yl)methyl)(methyl)amino)benzamido)pentanedioic acid.

alphamethopterin; amethopterin; methylaminopterin; CL 14377; NCIC04671; WR19039; WR-19039; Rheumatrex; Metatrexan; Hdmtx; Abitrexate; WR 19039; MTX; NCI-C04671; NCI C04671; CL14377; CL-14377; Methotrexate.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.50 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.50 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.50 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (5.50 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.

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Solubility in Formulation 5: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O:5 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)