Introduction

Methotrexate is a cornerstone drug in both anticancer chemotherapy and immunosuppressive therapy. Structurally, it is a folic acid antagonist and functions as an antimetabolite.

It is widely used in:

• Various malignancies like acute lymphoblastic leukemia (ALL)
• Autoimmune diseases such as rheumatoid arthritis and psoriasis
• Ectopic pregnancy management

Due to its dual role in oncology and immunology, Methotrexate is a high-yield topic in exams like GPAT, NIPER, NEET-PG, NCLEX, and essential for PharmD, MBBS, and B.Pharm students.

Stepwise Mechanism of Action of Methotrexate

1. Cellular Uptake:
   Methotrexate enters the cell via the reduced folate carrier (RFC), mimicking folic acid.
2. Inhibition of Dihydrofolate Reductase (DHFR):
   Methotrexate competitively inhibits DHFR, the enzyme that converts dihydrofolate (DHF) to tetrahydrofolate (THF). THF is essential for purine and thymidylate (dTMP) synthesis.
3. ↓ THF → Impaired DNA Synthesis:
   Lack of THF disrupts one-carbon transfer reactions needed for purine (adenine, guanine) and thymidylate synthesis, leading to defective DNA replication.
4. Cell Cycle Arrest in S-phase:
   Rapidly dividing cells are most affected, especially in the S-phase, where DNA synthesis is active. This leads to cytotoxicity in malignant cells and immunosuppression in autoimmune diseases.
5. Polyglutamation & Prolonged Action:
   Inside the cell, methotrexate undergoes polyglutamation, which enhances retention and sustains DHFR inhibition, making its effects more long-lasting.
6. Additional Anti-inflammatory Mechanism:
   Methotrexate also promotes adenosine release, which contributes to its anti-inflammatory action in diseases like rheumatoid arthritis.

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Pharmacokinetic Parameters of Methotrexate

Parameter	Value
Bioavailability	70–90% (oral, low dose); lower at high doses
Onset of action	Weeks (in autoimmune diseases)
Half-life	3–10 hours (low dose), up to 15 hours (high dose)
Protein binding	~50%
Metabolism	Liver (partial), polyglutamation in tissues
Excretion	Primarily renal (glomerular filtration & secretion)

Clinical Uses of Methotrexate

Antineoplastic Indications:

• Acute lymphoblastic leukemia (ALL)
• Choriocarcinoma
• Non-Hodgkin’s lymphoma
• Breast and head & neck cancers

Immunosuppressive/Anti-inflammatory Uses:

• Rheumatoid arthritis (low-dose, weekly)
• Psoriasis
• Systemic lupus erythematosus (off-label)
• Ectopic pregnancy

Adverse Effects of Methotrexate

• Myelosuppression (neutropenia, anemia, thrombocytopenia)
• Hepatotoxicity (elevated transaminases, fibrosis)
• Pulmonary fibrosis and pneumonitis
• Stomatitis and mucositis
• Teratogenicity (Category X in pregnancy)
• GI upset, nausea, diarrhea

Folinic acid (Leucovorin) rescue is used to prevent toxicity in high-dose therapy.

Comparative Analysis: Methotrexate vs Azathioprine

Feature	Methotrexate	Azathioprine
Class	Antimetabolite (Folate antagonist)	Purine analog
Primary Action	Inhibits DHFR → ↓ DNA synthesis	Inhibits purine synthesis (via 6-MP)
Use in RA	First-line DMARD	Second-line agent
Onset of Action	4–6 weeks	6–12 weeks
Monitoring	CBC, LFTs, renal	CBC, LFTs
Common Side Effects	Myelosuppression, hepatotoxicity, lung tox.	Myelosuppression, hepatotoxicity

Practice MCQs: Methotrexate

Q1. Methotrexate belongs to which class of drugs?
A. Alkylating agent
B. Folic acid antagonist ✅
C. Purine analog
D. Topoisomerase inhibitor

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Q2. The primary site of action of methotrexate is:
A. Thymidylate synthase
B. Dihydrofolate reductase ✅
C. Adenylosuccinate synthetase
D. DNA polymerase

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Q3. Which of the following pathways is directly blocked by methotrexate?
A. Pyrimidine methylation
B. One-carbon folate pathway ✅
C. GABA synthesis
D. Cholesterol biosynthesis

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Q4. What is the main result of methotrexate-induced inhibition of tetrahydrofolate synthesis?
A. Enhanced RNA polymerase activity
B. DNA synthesis inhibition ✅
C. Increased histamine release
D. Beta-lactamase activation

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Q5. Which of the following drugs is used as a rescue agent to prevent methotrexate toxicity?
A. Folic acid
B. Vitamin B12
C. Leucovorin (Folinic acid) ✅
D. Prednisone

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Q6. Methotrexate is most effective in which phase of the cell cycle?
A. G1
B. S ✅
C. G2
D. M

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Q7. Which of the following is NOT an approved use of methotrexate?
A. Rheumatoid arthritis
B. Acute lymphoblastic leukemia
C. Psoriasis
D. Hypertension ✅

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Q8. Which toxicity is most closely associated with long-term low-dose methotrexate?
A. Retinal damage
B. Hepatotoxicity ✅
C. Pancreatitis
D. Cardiotoxicity

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Q9. Which statement about methotrexate excretion is true?
A. Excreted through bile
B. Eliminated via sweat
C. Excreted unchanged primarily through the kidneys ✅
D. Exhaled via lungs

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Q10. Methotrexate is absolutely contraindicated in which condition?
A. Osteoarthritis
B. Pregnancy ✅
C. Male infertility
D. Anemia of chronic disease

FAQs

Q1: Can methotrexate be used in pregnancy?
No. It is absolutely contraindicated due to its teratogenic effects (FDA Category X).

Q2: What is the role of folinic acid (leucovorin)?
Folinic acid rescues normal cells by bypassing the DHFR blockade and preventing methotrexate toxicity.

Q3: Is methotrexate used in low or high doses for rheumatoid arthritis?
Low-dose, once-weekly methotrexate is the first-line DMARD for RA.

Q4: What monitoring is required during methotrexate therapy?
Regular CBC, liver function tests (LFTs), and renal function tests are essential to detect toxicity early.

References

• KD Tripathi – Essentials of Medical Pharmacology, 8th Edition
• Sparsh Gupta – Review of Pharmacology
• Goodman & Gilman – The Pharmacological Basis of Therapeutics