What is the mechanism of action of Methotrexate (MTX)?

Mechanism of Action of Methotrexate

Methotrexate primarily works as a competitive inhibitor of dihydrofolate reductase (DHFR), decreasing folate cofactors required for nucleic acid synthesis, while also inhibiting 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (ATIC), which increases endogenous adenosine and provides anti-inflammatory effects. 1

Cellular Transport and Metabolism

Methotrexate enters cells through specific transport mechanisms:

• Transported into cells via the solute carrier family 19, member 1 (SLC19A1) 2
• Can be actively transported out of cells by ATP-binding cassette transporters (ABCC1-4, ABCG2) 2
• Undergoes polyglutamation (activation) under the enzymatic control of folylpolyglutamate synthetase (FPGS) 2, 1
• Polyglutamation is a dynamic process where glutamate residues can be removed by γ-glutamyl hydrolase (GGH) 2
• Polyglutamated forms have increased intracellular retention and enhanced anti-inflammatory activity 2, 1

Primary Mechanisms of Action

1. Inhibition of Folate Pathway

• Competitively inhibits dihydrofolate reductase (DHFR), preventing the reduction of dihydrofolates to tetrahydrofolates 3
• This inhibition interferes with DNA synthesis, repair, and cellular replication 3
• The polyglutamated form also inhibits thymidylate synthase (TYMS), which converts deoxyuridylate (dUMP) to deoxythymidylate (dTMP) in the pyrimidine biosynthetic pathway 2, 4

2. Adenosine-Mediated Anti-inflammatory Effects

• In its polyglutamated form, methotrexate inhibits aminoimidazole-4-carboxamide ribonucleotide transformylase (ATIC) 2, 1
• This inhibition results in increased levels of endogenous adenosine 2, 1
• Adenosine acts on adenosine receptors (ADORA A1 and 2a) producing anti-inflammatory effects 2
• This mechanism is particularly important in rheumatoid arthritis treatment 1, 5

3. JAK/STAT Pathway Inhibition

• Recent evidence suggests methotrexate inhibits the JAK/STAT pathway activity 6
• This pathway is central to both inflammatory and immune systems 6
• This mechanism may explain many of the disease-modifying anti-rheumatic drug (DMARD) effects of methotrexate 6

Differential Effects Based on Dosing

• Low-dose methotrexate (<25 mg per week) primarily decreases proliferation of lymphoid cells and has direct immunosuppressive effects 1
• Higher doses target rapidly dividing malignant cells by inhibiting DNA synthesis 3
• At high doses, methotrexate may overcome resistance caused by:
  • Impaired active transport
  • Decreased affinity of DHFR for methotrexate
  • Increased levels of DHFR from gene amplification
  • Decreased polyglutamation 3

Clinical Implications

• Understanding methotrexate's mechanisms explains why folic acid supplementation reduces side effects without significantly compromising efficacy 1
• Methotrexate's effect on rapidly proliferating tissues explains both its therapeutic effects and common side effects (bone marrow suppression, GI mucosa effects) 3, 4
• In rheumatoid arthritis, effects on articular swelling and tenderness can be seen as early as 3-6 weeks, though it does not induce disease remission 3
• In psoriasis, methotrexate targets the increased rate of epithelial cell production in the skin 3

Pharmacokinetic Considerations

• Oral absorption appears to be dose-dependent with mean bioavailability of about 60% at doses ≤30 mg/m² 3
• Terminal half-life is approximately 3-10 hours for low-dose therapy and 8-15 hours for high-dose therapy 3
• Renal excretion is the primary route of elimination (80-90% excreted unchanged in urine within 24 hours) 3
• Delayed drug clearance is a major factor responsible for methotrexate toxicity 3

Understanding methotrexate's complex mechanisms of action helps explain its efficacy across multiple disease states while also providing insight into its side effect profile and potential drug interactions.