Methotrexate update 2014: 70 years in autoimmunity and cancer treatment

Summary

Immunosupressive and antineoplastic activity brought methotrexate to its clinical use many years ago. Although modern antitumour and antiinflammatory drugs and new antifolates have challenged methotrexate, its use in rheumatoid arthritis and in some types of pediatric and adult malignancies remains a gold standard. The principal main mechanism of action of methotrexate is competitive inhibition of dihydrofolate reductase. Recently, other pharmacodynamic effects have been described. Potent inflammation inducers such as leukotriene-B₄ and polyamine synthesis were found to be reduced by methotrexate treatment. Jun N-terminal kinase and B cell lymphoma 2 gene are downregulated by methotrexate. Its metabolite, 7-hydroxymethotrexate and also its intracellular polyglutamated forms show a different spectrum of inhibitory activity as well as potency towards dihydrofolate reductase and other enzymes. In this review, we summarize recent information data on methotrexate in both fields of its use, oncology and rheumatology. We also discuss new biomarkers of toxicity for further improvement of therapeutic protocols.

Keywords:
methotrexate, autoimmunity, neoplasms, biological markers, toxicity, adverse effects

Introduction

Methotrexate (MTX) is an antineoplastic agent first reported by Sidney Farber in 1948 in children with leukaemia [1]. Antiinflammatory activity was observed in patient with rheumatoid arthritis (RA) 3 years later. Since then methotrexate established its sound clinical role among cytotoxic antitumour drugs [2]. In adults, MTX is currently used as an anticancer drug in acute lymphoblastic leukemia (ALL) and lymphomas, leukemic and lymphoma CNS involvement, choriocarcinoma, trophoblastic diseases and osteosarcoma. Acute lymphoblastic leukemia (ALL), lymphomas and osteosarcoma are main indications for MTX in pediatric oncological patients. Methotrexate is used as a conventional immunosuppressive drug in rheumatoid arthritis (adult and juvenile) and in psoriasis patients [3].

New discoveries in mechanisms of action

Methotrexate is a competitive inhibitor of dihydrofolatereductase (DHFR), a key enzyme of the folic acid cycle which helps to maintain the intracellular pool of reduced bioactive folates, i.e. the cofactors in one-carbon metabolic pathways [4].

In autoimmune disorders, other mechanisms which suppress immunocompetent and inflammatory cells may predominate including inhibition of enzymes involved in purine synthesis and an augmented release of adenosine as extensively reviewed elsewhere [5, 6]. Antigen-stimulated proliferation of T cells is decreased by methotrexate in vivo by various mechanisms [7, 8]. In polymorfonuclear leukocytes, methotrexate inhibits activity of 5-lipoxygenase and production of leukotriene B₄ which acts as a potent inflammation inducer [9–11]. Decreased rate of polyamine synthesis resulting from diminished methyl transfer in cells is another possible cellular mechanism of action [12, 13].

Recently, research has focused on other „non-DHFR related“ mechanisms of action. MTX induced activation of Jun N-terminal kinase (JNK) results in expression of JNK-dependent genes that participate in apoptosis [14, 15]. Downregulation of B cell CLL/lymphoma 2 gene (BCL-2) was observed in leukemic and stomach cancer cell lines treated with methotrexate [16, 17].

Intracellular metabolism

Methotrexate enters the cells through a reduced folate carrier and folate transporters. Other transporters influence the pharmacokinetics of MTX such as the efflux pumps of the ABC superfamily transporters and organic anion transporting peptides [18, 19]. Similarly to folates, MTX undergoes polyglutamation by folylpolyglutamate synthetase (FPGS) [20]. MTX polyglutamates (MTX-Glu_2-7) can be viewed as a depot active form of the drug. They are retained intracellularly and slowly leave the cell after enzymatic hydrolysis to parent drug by folylpolyglutamate hydrolase. MTX polyglutamates have much longer half-life (2–3 weeks) as compared to MTX (4–8 hours). Thus, polyglutamation helps to maintain cytotoxic or immunosuppresive concentrations and makes possible intermittent dosing of MTX once a week in the maintenance therapy of ALL or immunosuppressive treatments. There have been several reports that polyglutamation increases variability in methotrexate efficacy [21, 22]. In breast cancer cells, MTX-Glu_4-5 showed more prolonged action on DHFR than shorter forms [21]. In addition MTX-Glu₂ has been recently proposed as a potential biomarker for clinical response in patients with rheumatoid arthritis treated with methotrexate [23]. Having newly developed methods available that are able to quantitate levels of methotrexate intracellular forms, we expect further elucidation on this topic in the near future [24, 25].

Dosing regimens and effects of extracellular metabolism

MTX dosing vastly varies within clinical protocols for specific diagnoses. Low-dose methotrexate is used to treat RA, psoriasis, and as a maintenance therapy of ALL. According to recommendations of the Czech Society for Rheumatology, low-dose MTX treatment of RA should be initiated at 10–15 mg (oral) weekly [26]. If the clinical response is insufficient, oral administration should be switched for subcutaneous application or oral doses should be increased to 25–30 mg weekly [26]. Initial therapeutic dosage in psoriasis therapy is 7.5 mg MTX weekly that can further be escalated to 30 mg weekly [27]. Acute lymphoblastic leukemia maintenance therapy dose can be 20 mg/m² oral MTX weekly [28, 29]. High-dose MTX is required in cancer treatment. Pediatric ALL protocols include 5000 mg/m² IV MTX with leucovorine (LV) rescue (usual dose 15 mg/m²) [30]. Osteosarcoma high-dose IV MTX therapeutic dose is 10 000–12 500 mg/m² with LV rescue [31]. NHL-BFM 95 protocol contains 1000 mg/m² IV MTX with LV rescue [32]. Intrathecal MTX is administered at 12 mg/m² in prophylaxis of leukemic CNS disease in ALL and NHL. Target intrathecal concentration of the drug is 1 µmol/l [30, 32]. Liver function tests and complete blood count with white blood cell count are main markers of MTX clinical toxicity [26, 27]. High-dose MTX protocols require therapeutic drug monitoring (TDM).

Aldehyde oxidase (E.C. 1.2.3.1) converts MTX into its metabolite 7-hydroxymethotrexate (7-OH MTX) [33, 34]. This metabolite, compared to MTX, shows 200 fold less efficacy in blocking DHFR [33–35]. Furthermore, concentrations of 7-OH MTX inversely correlate with clinical toxicity [36]. Decreased levels of 7-OH MTX were reported to show higher therapeutic efficacy [33].

Methotrexate and its clinical safety

Adverse effects of MTX mostly depend on a dosing regimen. The advantage of low-dose MTX lies in its favourable risk benefit ratio. Main adverse effects in low-dose MTX RA treatment are gastrointestinal intolerance and myelosuppression [37]. Psoriasis patients receiving low-dose MTX appear to have increased risk of non-alcoholic fatty liver disease and liver fibrosis [38–40].

Cross-sectional study of 140 patients with RA treated with MTX (10 mg weekly) found 27% with adverse effects, mainly with hepatotoxicity (8.6%, elevated ALT) and anemia (5.7%) [41]. Most common side effect in retrospective study of 71 Arabic patients with RA (7.5 mg MTX weekly with folic acid 2.5–5 mg weekly) were gastrointestinal disturbances (31%) followed by CNS symptoms (18%) and hepatotoxicity (14%, minor to major elevation of liver function tests) [42]. Study of 673 rheumatic patients (between 1986 and 1999) from Staffordshire Rheumatology Centre identified gastrointestinal symptoms (10.8%), hematological abnormalities (5.5%) and abnormal liver function tests (5.5%) as the most common reasons for cessation of MTX treatment [43]. Type II diabetes mellitus and obesity seem to augment adverse effects of MTX treatment in psoriasis [38]. New markers of MTX induced hepatotoxicity in psoriasis and psoriatic arthritis were recently tested and reviewed [39, 40]. In addition, our data from outpatient blood pressure monitoring indicate, that patients with RA and hypertension treated with MTX preserve more normal diurnal pattern of systolic blood pressure values (dippers) than patients treated with NSAIDs and/or corticosteroids [44].

High-dose MTX protocols remain a mainstay in the treatment of childhood malignancies and are clearly reflected in higher and more severe toxicity incidence [45]. Methotrexate high-dose protocols have high risk of nephrotoxicity and tubulopathy that may progress to acute renal damage (ARD) and renal failure [46, 47]. Plasmatic levels of MTX, renal function and hydration must be monitored during high-dose treatment [47]. Appropriate hydration, leucovorine administration and urine alkalization to increase MTX and 7-OH MTX solubility are main preventive steps against crystalluria and ARD [47]. Severe renal impairment during high-dose MTX therapy is managed mainly by high-flux dialysis [47]. Another option is to use carboxypeptidase G2 (glucarpidase), recombinant bacterial enzyme that cleaves carboxyl-terminal glutamate residue from folic acid and its analogues, such as MTX, 7-OH MTX and also LV [48]. Glucarpidase (Voraxaze) was approved by FDA in January 2012. It is IV administered in a single dose of 50 U/kg, however patients should not receive LV within 2 hours before and after administration [47, 49]. Emergency treatment with IT glucarpidase (2000 U reconstituted in 12 ml of normal saline) and systemic LV (4 doses, 100 mg every 6 hours) showed positive outcome in pediatric patients [50]. Therefore IT administration of glucarpidase was proposed as an alternative to standard procedures in intrathecal MTX overdose (drainage by lumbar puncture, ventriculolumbar CSF perfusion, systemic corticosteroids and LV administration) [50].

Allergic reactions to high-dose MTX in children have been reported by various studies although their occurence is relatively rare [51–53]. Another complication in HD MTX treatment is therapy-related neurotoxicity [54]. The authors from the Department of Pediatric Oncology, University Hospital, Brno, described a case of neurotoxicity in 9-year-old boy treated with HD MTX (ALL-BFM 95 protocol). Severe encefalopathy that followed the first HD MTX course in this case correlated with patients five times elevated homocysteine levels. However, these complications, together with laboratory abnormalities, disappeared in the second HD MTX course [55]. Similar cases were observed in other studies [56, 57]. In light of these findings, we may conclude that homocysteine may be a promising pharmacodynamic biomarker of HD MTX-related neurotoxicity in children with cancer.

Conclusion

Methotrexate clinical role within a wide range of modern antineoplastic and immunosuppresive drugs remains untouched. Even though new antifolates are in clinical trials, none of them has shown better risk benefit ratio than the original MTX molecule in terms of pharmacokinetics and pharmacodynamics [58]. High-dose MTX therapeutic protocols went through many years of optimalization and provide safe, efficacious and flexible chemotherapy. We believe that further investigation of MTX effects on cells and finding new but relevant biomarkers in folate metabolism may help consolidate MTX role in current therapeutic protocols and confirm its value in cancer and autoimmunity treatment.

Acknowledgement

All authors listed contributed sufficiently to the manuscript to be included as authors. This work was supported by European Regional Development Fund and the State budget of the Czech Republic for Regional Centre of Applied Molecular Oncology (RECAMO, CZ.1.05/2.1.00/03.0101), by Large Infrastructure Projects of Czech Ministry of Education, Youth and Sports LM2011017 and by IGA Czech Ministry of Health NT14327.

Došlo: 20. 12. 2013

Přijato: 9. 4. 2014

Corresponding author

Bc. Michal Řiháček

Masaryk Memorial Cancer Institute

RECAMO

Žlutý kopec 7

656 53 Brno

Czech Republic

e-mail: michal.rihacek@mou.cz