Methotrexate inhibits dihydrofolic acid reductase. Dihydrofolates must be reduced to tetrahydrofolates by this enzyme before they can be utilized as carriers of one-carbon groups in the synthesis of purine nucleotides and thymidylate. Therefore, methotrexate interferes with DNA synthesis, repair, and cellular replication. Actively proliferating tissues such as malignant cells, bone marrow, fetal cells, buccal and intestinal mucosa, and cells of the urinary bladder are in general more sensitive to this effect of methotrexate. When cellular proliferation in malignant tissues is greater than in most normal tissues, methotrexate may impair malignant growth without irreversible damage to normal tissues.

The mechanism of action in rheumatoid arthritis is unknown; it may affect immune function. Two reports describe in vitro methotrexate inhibition of DNA precursor uptake by stimulated mononuclear cells, and another describes in animal polyarthritis partial correction by methotrexate of spleen cell hyporesponsiveness and suppressed IL 2 production. Other laboratories, however, have been unable to demonstrate similar effects. Clarification of methotrexate’s effect on immune activity and its relation to rheumatoid immunopathogenesis await further studies. 

In patients with rheumatoid arthritis, effects of methotrexate on articular swelling and tenderness can be seen as early as 3 to 6 weeks. Although methotrexate clearly ameliorates symptoms of inflammation (pain, swelling, stiffness), there is no evidence that it induces remission of rheumatoid arthritis nor has a beneficial effect been demonstrated on bone erosions and other radiologic changes which result in impaired joint use, functional disability, and deformity. 

Most studies of methotrexate in patients with rheumatoid arthritis are relatively short-term (3 to 6 months). Limited data from long-term studies indicate that an initial clinical improvement is maintained for at least two years with continued therapy.

In psoriasis, the rate of production of epithelial cells in the skin is greatly increased over normal skin. This differential in proliferation rates is the basis for the use of methotrexate to control the psoriatic process. 

Methotrexate in high doses, followed by leucovorin rescue, is used as a part of the treatment of patients with non-metastatic osteosarcoma. The original rationale for high-dose methotrexate therapy was based on the concept of selective rescue of normal tissues by leucovorin. More recent evidence suggests that high-dose methotrexate may also overcome methotrexate resistance caused by impaired active transport, decreased affinity of dihydrofolic acid reductase for methotrexate, increased levels of dihydrofolic acid reductase resulting from gene amplification, or decreased polyglutamation of methotrexate. The actual mechanism of action is unknown.

In a 6-month double-blind, placebo-controlled trial of 127 pediatric patients with juvenile rheumatoid arthritis (JRA) (mean age, 10.1 years; age range, 2.5 to 18 years; mean duration of disease, 5.1 years) on background non-steroidal anti-inflammatory drugs (NSAIDs) and/or prednisone, methotrexate given weekly at an oral dose of 10 mg/m2 provided significant clinical improvement compared to placebo as measured by either the physician’s global assessment, or by a patient composite (25% reduction in the articular-severity score plus improvement in parent and physician global assessments of disease activity). Over two-thirds of the patients in this trial had polyarticular-course JRA, and the numerically greatest response was seen in this subgroup treated with 10 mg/m2/wk methotrexate. The overwhelming majority of the remaining patients had systemic-course JRA. All patients were unresponsive to NSAIDs; approximately one-third were using low-dose corticosteroids. Weekly methotrexate at a dose of 5 mg/m2 was not significantly more effective than placebo in this trial. 

Two Pediatric Oncology Group studies (one randomized and one non-randomized) demonstrated a significant improvement in relapse-free survival in patients with non-metastatic osteosarcoma, when high-dose methotrexate with leucovorin rescue was used in combination with other chemotherapeutic agents following surgical resection of the primary tumor. These studies were not designed to demonstrate the specific contribution of high-dose methotrexate/leucovorin rescue therapy to the efficacy of the combination. However, a contribution can be inferred from the reports of objective responses to this therapy in patients with metastatic osteosarcoma, and from reports of extensive tumor necrosis following preoperative administration of this therapy to patients with non-metastatic osteosarcoma.