Methotrexate Bis

Author: Andrea Rolfo
Date: 25/02/2010


Umberto Barbero
Andrea Rolfo



Methotrexate (amethopterin) is the first example of enzyme inhibitor used in oncology.
It is a structural analogue of folic acid, from which differ only by the absence of a hydroxyl
on the amino group in C4 of the pteridine ring and a methyl group in N10. The active portion of the molecule is the pteridine ring.


It is part of drugs antimetabolites: structural analogues of important biological compounds for the synthesis and the duplication of genetic material and thus essential for cell replication.



  • Choriocarcinoma, coriadenoma
  • Acute lymphoblastic leukemia (child)
  • Meningeal leukemic infiltrates
  • Osteosarcoma
  • Mycosis fungoides
  • Burkitt’s Lymphoma
  • Non-Hodgkin's Lymphoma
  • Breast, head, neck, ovary and bladder cancer

non neoplastic:

  • Psoriasis
  • Not allogeneic marrow transplantation
  • Organ transplants
  • Dermatomyositis
  • Reumatoid Arthritis
  • Wegener's granulomatosis
  • Crohn's Disease
  • Sarcoidosis
  • Placenta accreta


Administration: may be given orally, by subcutaneous, intravenous, intramuscular or intrathecal way. In order to decrease incidence of side effects is typical use weekly doses.

Absorption: for doses below 25 mg/m2 there is a rapid and complete gastrointestinal absorption, while for higher doses the absorption would not be complete so you have to switch to intravenous administration. May reduce the absorption:

  • Food
  • Antibiotics administered orally but not absorbable. (Eg, vancomycin, neomycin)
  • The more rapid transit through the gastrointestinal tract, such as diarrhea, while the slower transit time in the GI tract by constipation increase absorption.

Following intravenous or intramuscular administration, maximum plasma concentrations are reached between 30 minutes and 2 hours, whereas after oral administration are reached between 1 and 4 hours. Equivalent Plasma concentrations to those obtained intravenously are obtained by intra-arterial way. Following oral administration the concentrations are slightly lower than those achieved by intravenous way.

Distribution: Methotrexate is actively transported across cell membranes: it is rapidly distributed in tissues (highest concentrations in the kidneys, gallbladder, spleen, liver and skin) and more slowly in the cavity (peritoneal and pleural): if those are expanded (eg ascites) may act as a reservoir of drug accumulation by increasing the likelihood of toxic effects. It can also be detained in the form of polyglutamate for long periods, for example from weeks to several months in the kidneys and liver. It can overcome the placental barrier (with teratogenic effect) and reaching the milk. Methotrexate is 50% bound to plasma proteins (especially albumin), and may be displaced by several drugs including sulfonamides, salicylates, tetracycline, chloramphenicol and phenytoin. Is poorly metabolized, only 10% of the absorbed dose, but some metabolites such as 7-hydroxy-methotrexate is nephrotoxic. The concentrations in the LCR are only 3% of the plasma so effective doses for the treatment of malignancies of the CNS can only be achieved with dosing at high doses (> 1.5 g/m2) or intrathecal administration (but this often cause meningism ).

Elimination: is mainly eliminated by the kidneys (90% of the absorbed dose is excreted within 48 hours unchanged) by glomerular filtration and tubular secretion, and consequently the simultaneous use of drugs that reduce the blood supply to the kidney (eg NSAIDs) , nephrotoxic drugs (eg, cisplatin) or drugs that are weak organic acids and therefore compete with the tubular transport (eg aspirin) may increase the half-life of methotrexate and its toxic effects. For the same reason, the accumulation occurs more rapidly in patients with renal dysfunction. Small amounts of the drug are excreted in the feces, probably trough the gall.


Cell’s Penetration:

Methotrexate is very similar folic acid therefore penetrate cells in the same way

The mechanisms are 3:
1. Carrier for folate (mechanism temperature and energy dependent)
2. Reduced folate’s carrier
3. a channel active at low pH (poor studies)


Methotrexate is polyglutamated by an enzyme, folil-poligluttamato synthetase, the one acting on natural folic acid. Glutamation, with 2-5 residues, negatively charges molecule and prevents it from escaping from the cell.

Molecolar Target:

Inhibition of DHFR (dihydrofolate reductase), key enzyme in the reduction of folate (dihydrofolate) to THF in the following way:

1 inhibits protein’s synthesis by inhibiting the conversion of glycine to serine and homocysteine to methionine.
2 inhibits the transmethylation of DNA, RNA, phospholipid and some protein.
3 inhibits the synthesis of purines lowering the amount of N5-N10-methylene-tetrahydrofolate.
4 inhibits thymidylate synthetase cofactor for reduction (N5-N10-methylene-tetrahydrofolate)

All this actions leads to apoptosis of tumor cells.

Furthermore, the accumulation of intracellular ribonucleotides’ metabolites stimulates the release of adenosine, a molecule with immunomodulatory and anti-inflammatory action: this could probably be the molecular mechanism underlying the immunosuppressive action methotrexate.


1 The C677T substitution in the methylenetetrahydrofolate reductase. reduces the activity of this enzyme that generates 5-methyl-THF, a cofactor for Metionina synthase, and thus increases the toxicity of methotrexate (Pharmacogenetics of methotrexate: toxicity among marrow transplantation patients varies with the methylenetetrahydrofolate reductase C677T polymorphism.Cornelia M. Ulrich, Yutaka Yasui, Rainer Storb, Mark M. Schubert, John L. Wagner, Jeannette Bigler, Kiley S. Ariail, Cassie L. Keener, Sue Li, Hao Liu, Federico M. Farin, and John D.Potter.) The presence of this polymorphism in leukemic cells thus provides an increased sensitivity to methotrexate. The Italian prevalence is about 15-25% of homozygosity in North-South gradient and it is transmitted as an autosomal recessive trait.

2 The importance of transport in determining the susceptibility is illustrated by the discovery that the reduced folate transporter is highly expressed in iperploide subtype of acute lymphoblastic leukemia due to the presence of multiple pairs of chromosome 21 on which the gene resides. These cells have an increased sensitivity to methotrexate (Pui et al.Germline Genetic Variation in an Organic Anion Transporter polypeptide Associated With Methotrexate Pharmacokinetics and Clinical Effects. J. Clin. Oncol., December 10, 2009; 27.)


  • Myelo suppression
  • Gastrointestinal epithelial suffering
  • Alopecia
  • Dermatitis
  • Interstitial Pneumonia
  • Nephrotoxicity (hydration and alkalinization)
  • Abortion
  • Teratogeneicità
  • Oogenesi and faulty spermatogenesis
  • Hepato

With intermediate and high doses (100-1000 mg / m 2,> 1000 mg/m2) methotrexate is highly toxic and requires the use of an antidote treatment with folinic acid (leucovorin) which is rapidly converted to tetrahydrofolate by carrying out an effect "rescue" on the bone marrow and on the gastrointestinal mucosa (not on the renal toxicity)


1 Reduced Transport within cells (antifolate resistance associated with loss of MRP1 expression and function in Chinese hamster ovary cells with markedly impaired export of folate and Chol. Stark M, Rothem L, Jansen G, Scheffer GL, Goldman ID, Assaraf YG.Department of Biology, The Technion-Israel Institute of Technology, Haifa 32000, Israel. = 4.)
2 Modified binding site for the methotrexate-DHF Reductase (Srimatkandada S, Schweitzer BI, Moroson BA, Dube S, Bertino JR. Amplification of a polymorphic dihydrofolate reductase gene Expressing an enzyme with decreased binding to methotrexate in a human colon carcinoma cell line, HCT-8R4, resistant to this drug. J Biol Chem. 1989 Feb 25; 264 (6) :3524-8 . PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum & ordinalpos = 3.)
3 DHFR increase because of gene’s amplification .Levels of DHFR in the leukemia cells increase within 24 h of treatment of patients, an effect that probably reflects the induction of synthesis of new enzyme. (Leuk Lymphoma. 1996 May; 21 (5-6) :359-68. Methotrexate pharmacology and resistance in childhood acute lymphoblastic leukemia.Matherly LH, Taub JW..)
4 Reduced ability to synthesize the Polyglutamated mtx. (Cancer Res 1992 Mar 15; 52 (6) :1434-8. Mechanisms of natural resistance to antifolates in human soft tissue sarcomas.Li WW, Lin JT, Tong WP, Trippetta TM, Brennan MF, Bertino JR..)
5 Increased expression of drug efflux transporter trough multidrug resistance proteins, MRP (Stashenko et al., 1980)

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