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Executive Summary Information

Compound Methotrexate
Toxicities Fibrosis
Mechanisms Toxicity is ascribed to the antifolate activity of the parent compound.
Comments This compound was selected because the fibrotic activity is induced by a relatively limited biochemical activity without accompanying chemical reactivity.
Feedback Contact Gold Compound Working Group (GCWG)
Leadscope Id LS-249
CAS 59-05-2
DrugBank DB00563
ChemSpider 112728
ChEBI 44185
Pathway DBs
KEGG D00142
Assay DBs
PubChem CID 126941
ChEMBL 34259
Omics DBs
ToxCast Accepted yes
ToxBank Accepted yes
Target Dihydrofolate reductase (DHFR) inhibition
Toxicities Fibrosis

In Vivo Data ? Compound Assessment
Adverse Events ? Hepatic fatty infiltration, fibrosis with a potential to progress to cirrhosis, is one of the most severe adverse effects of long-term methotrexate (MTX) treatment.

Other hepatic side effects include acute hepatitis, decrease in serum albumin and liver enzyme elevations. Chronic hepatotoxicity typically develops only after chronic use of higher doses (2 years or more of total doses of 1.5 grams or more) and is more likely in patients who ingest ethanol, who are aged, who are obese, who have chronic renal insufficiency, or who have diabetes.

Reports from case studies having routine serial liver biopsies for psoriasis treated with long-term MTX have an incidence of hepatic fibrosis of 13–34% and cirrhosis of 0–20% Among rheumatoid arthritis (RA) patients taking long-term MTX, the prevalence of significant fibrosis and cirrhosis is rare. Increases in aminotransferases occurred among 23.7% of patients who received ≥7.5mg MTX per week.

Drug-related pathogenic factors:
Both the dose and frequency of MTX administration influence the risk of liver disease. The frequency of advanced fibrosis (bridging fibrosis) increases from 0% at a cumulative dose of 1,500 mg to 2.6% at 3,000 mg and 8.2% at 6,000 mg . Daily or alternate-day MTX administration is associated with a fourfold increase in the incidence of fibrosis or cirrhosis compared with once-weekly dosing. A weekly MTX dose of ≤20 mg has been associated with lower rates of cirrhosis (0-4%) compared with weekly doses of >20 mg (3-26%).

Host-related pathogenetic factors:
An individual's susceptibility to hepatoxicity could be related to the genetic determinants of drug uptake, metabolism and elimination. Several genetic association studies have investigated the role of single nucleotide polymorphisms in the MTHFR gene as a determinant of MTX toxicity. A 2009 meta-analysis of eight studies concluded that the C677T polymorphism of MTHFR was associated with an increased risk of MTX-related toxicity (OR 1.71, 95% CI 1.32-2.21). Single nucleotide polymorphisms in the genes encoding proteins involved in cellular MTX uptake (folate transporter) and export (ABC transporters) could also influence susceptibility to adverse reactions. Studies have reported associations between MTX toxicity and RFC1 A80G (which may alter the affinity of the carrier to folate), ABCB1 C3435T (which may decrease the activity of the efflux transporter) and ABCC2 G1058A.[59-61]

Cardiovascular side effects including pericarditis, pericardial effusion, myocardial ischemia, hypotension and ventricular arrhythmias have rarely been associated with MTX. Chemical pleuritis secondary to MTX in some patients after high-dose therapy.

-K. Lindsay et al. " Liver fibrosis in patients with psoriasis and psoriatic arthritis on long-term, high cumulative dose methotrexate therapy", Rheumatology (2009) 48 (5): 569-572.
-R Sotoudehmanesh et al. "Methotrexate Hepatotoxicity in Patients with Rheumatoid Arthritis" Middle East Journal of Digestive Diseases/ Vol.2 / No.2/ Sept. 2010
Toxicity Mechanisms ? The exact mechanisms underlying MTX hepatotoxicity are unclear, although they are assumed to be related to the cellular pathway. MTX is a folate analog that enters the cell bound to folate trasporter and is pumped out by the ATP-binding cassette (ABC) family of transporters.

MTX is retained within the cell as a polyglutamate that inhibits dihydrofolate reductase, thymidylate synthase and AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) transformylase, leading to impaired pyrimidine and purine synthesis. In addition, MTX indirectly affects MTHFR (methylenetetrahydrofolate reductase) and hence the generation of methionine from homocysteine.

MTX therapy in patients with RA has been shown to raise plasma homocysteine levels, although this effect varies depending on concurrent administration of folate. Excess homocysteine can generate oxidative stress or sensitize the cell to its cytotoxic effects. Homocysteine has been shown to induce endoplasmic reticulum (ER) stress, which, when unresolved, leads to fatty infiltration of the liver. Homocysteine, in addition, can also activate pro-inflammatory cytokines. The combination of these insults could contribute to the activation of hepatic stellate cells, which leads to liver fibrosis.



In specimens with moderate or severe fibrosis, fibrous septa sometimes extend along the canals of Hering (coH). These findings suggest that scarring of the coH might be a consequence of the toxic effects of methotrexate.


-P. Hytiroglou et al. "The Canals of Hering Might Represent a Target of Methotrexate Hepatic Toxicity" Am J Clin Pathol 2004;121:324-329
Therapeutic Target ? MTX is an antineoplastic anti-metabolite with immunosuppressant properties. MTX anti-tumor activity is a result of the inhibition of folic acid reductase, leading to inhibition of DNA synthesis and inhibition of cellular replication. MTX selectively affects the most rapidly dividing cells (neoplastic and psoriatic cells). MTX is also indicated for severe rheumatoid arthritis.


-Kremer, J. M. "Toward a better understanding of methotrexate". Arthritis Rheum. 50, 1370-1382 (2004).

FDA and Label Information

The following link will display all of the currently approved FDA drug products on the market. The web page will contain a table listing all current products by their respective Tradenames and primary active ingredients. The list is navigable by simply clicking on the product of interest, which will in turn list all of the NDA's and ANDA's associated with that product. From here users can click on a specific NDA or ANDA and see documents that have been submitted for the product that the FDA has made available via their website. The types of documents include approval history, letters, reviews and labels.
FDA Approved Products

This next url will perform a search in the FDA's system for all labels for products that contain Methotrexate as an active ingredient.
FDA Label Search

Since every unique drug product receives a separate label, we have included a link to another resource for a specific Acetaminophen product (Methotrexate sodium tables - by Rebel Distributors Corp.) which displays the drug label information.

PubMed references

The following resource link will perform a PubMed query for the terms " Methotrexate " and "liver toxicity".
Methotrexate Search

The following resource link will perform a PubMed query for the terms " Methotrexate " and "cardio toxicity".
Methotrexate Search

PK-ADME ? Compound Assessment
PK parameters ? Bioavailability: about 60%. (oral 35%, i.m. 75%)

Volume of distribution: 0.4 to 0.8 L/kg Protein binding: 50%, primarily to albumin

Elimination: with i.v. 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: 3 to 10 hours; high doses: 8 to 15 hours.



Mean C(max) of MTX at 1-2 h after ingestion of 2mg capsule was 0.215 and 0.252 microM, respectively. The mean C(max) of 7-OH MTX was 0.0334 and 0.0289 microM


-Shiozawa K et al. "Serum levels and pharmacodynamics of methotrexate and its metabolite 7-hydroxy methotrexate in Japanese patients with rheumatoid arthritis treated with 2-mg capsule of methotrexate three times per week" Mod Rheumatol. 2005;15(6):405-9.

A twenty fold difference between highest and lowest peak levels (Cmax: 0.11 to 2.3 micromolar after a 20 mg/m2 dose) has been reported. Significant interindividual variability has also been noted in time to peak concentration (Tmax: 0.67 to 4 hrs after a 15 mg/m2 dose).

It is generally accepted that serum concentrations of MTX that exceed 0.05 micromolar for greater than 24 hours result in cytotoxic effects.


Therapeutic window ? Highest: i.v. for neoplastic diseases: wide range from 30-40 mg/m2/week to 100-12,000 mg/m2 (with leucovorin rescue)

Lowest: orally for rheumatoid arthritis 7.5 mg weekly

Metabolically activated ? MTX is partially metabolized by intestinal flora after oral administration bacteria to the inactive metabolite 4-amino-4-deoxy-N-methylpteroic acid (DAMPA), which accounts for less than 5% loss of the oral dose.

After absorption, MTX undergoes hepatic and intracellular metabolism to polyglutamated forms which can be converted back to MTX by hydrolase enzymes. These polyglutamates act as inhibitors of dihydrofolate reductase and thymidylate synthetase. Small amounts of MTX polyglutamates may remain in tissues for extended periods. The retention and prolonged drug action of these active metabolites vary among different cells, tissues and tumors.

7-Hydroxy-methotrexate (7-OH-MTX) is the main (much less potent as dihydrofolate reductase inhibitor) metabolite in serum following high-dose MTX; a small amount of metabolism to 7-OH-MTX may occur at low doses.


-M.Joerger et al. "Determinants of the elimination of methotrexate and 7-hydroxy-methotrexate following high-dose infusional therapy to cancer patients" Br J Clin Pharmacol. 2006 July; 62(1): 71–80.

Omics and IC50 Data ? Compound Assessment
Gene expression profiles known. ? References:
-Glenn S. Belinsky et al. "The Contribution of Methotrexate Exposure and Host Factors on Transcriptional Variance in Human Liver" Toxicol. Sci. (2007) 97(2): 582-594.
-Jeffrey F. Waring et al. " Microarray analysis of hepatotoxins in vitro reveals a correlation between gene expression profiles and mechanisms of toxicity" Toxicology Letters, Volume 120, Issues 1-3, 31 March 2001, Pages 359-368.
-MH Lee et al. "Gene expression profiles of murine fatty liver induced by the administration of methotrexate" Toxicology. 2008 Jul 10;249(1):75-84
Proteomics profiles known. ?
Metabonomics profiles known. ?
Fluxomics profiles known. ?
Epigenomics profiles known. ? References:
-S. M. Hanafy et al. "Influence of anticancer drugs on DNA methylation in liver of female mice" American Journal of Molecular Biology, 2011, 1, 62-69
Observed IC50 for in vitro cellular efficacy. ?
Observed IC50 for in vitro cellular toxicity studies. ? Median IC50 78 nM in pediatric leukemia and lymphoma cell lines


-RE. Norris et al. " Clinical potency of methotrexate, aminopterin, talotrexin and pemetrexed in childhood leukemias" Cancer Chemother Pharmacol. 2010 May;65(6):1125-30

Physical Properties ? Compound Assessment
Accepted and listed within the ToxCast/Tox21 program. ? Yes - Included in ToxCast Phase I and II Chemicals List.
Substance stability. ? Light sensitive Methotrexate Sigma Aldrich M8407
Soluble in buffer solution at 30 times the in vitro IC50 for toxicity. ? Water insoluble

estimated intrinsic solubility : 2.8174e-2 mg/ml estimated solubility in pure water at pH 5.04: 5.0672e-3 mg/ml estimated solubility in water at pH 7.4: 43.08 mg/ml (Calculations performed using ACD/PhysChem v 12.0)

Solubility in DMSO 100 times buffer solubility. ? Soluble to 100 mM Tocris Bioscience Product (1230) information
Vessel binding properties. ? The stability of methotrexate was determined in plastic and glass administration containers. It was equally stable over 24 hours in both glass and plastic (polyvinyl chloride) containers.


-J Benvenuto, RW Anderson, K Kerkof, RG Smith, TL Loo. Stability and compatibility of antitumor agents in glass and plastic containers. Am J Hosp Pharm 1981, 88:1914-8
Vapor pressure. (Non-volatile) ? Estimated vapor pressure (25°C): 1.51E-017 mmHg

(Calculation performed using EPI Suite v4.1)

Authors of this ToxBank wiki page

David Bower, Egon Willighagen
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