Drivers of Methotrexate Polyglutamate Concentration in Erythrocytes: Insights from Immune-Mediated Inflammatory Diseases and Pediatric Acute Lymphoblastic Leukemia

Janani Sundaresan; Wout J Hamelink; Renske C F Hebing; Maartje van de Meeberg; Montse Janssen Bonás; Inge M van der Sluis; Pascal H P de Jong; Martijn Heymans; Robert de Jonge; Maurits C F J de Rotte; Maja Bulatović-Ćalasan

doi:10.3390/ph19020267

Drivers of Methotrexate Polyglutamate Concentration in Erythrocytes: Insights from Immune-Mediated Inflammatory Diseases and Pediatric Acute Lymphoblastic Leukemia

Pharmaceuticals (Basel). 2026 Feb 4;19(2):267. doi: 10.3390/ph19020267.

Affiliations

¹ Laboratory of Specialized Diagnostics & Research, Department of Laboratory Medicine, Amsterdam University Medical Center, 1105 AZ Amsterdam, The Netherlands.
² Department of Pharmacy, Sint Maartenskliniek, 6574 NA Nijmegen, The Netherlands.
³ Department of Gastroenterology and Hepatology, Amsterdam University Medical Center, Amsterdam Gastroenterology Endocrinology Metabolism Research Institute, 1105 AZ Amsterdam, The Netherlands.
⁴ Department of Gastroenterology and Hepatology, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands.
⁵ ILD Center of Excellence (Member of the European Reference Network-Lung), St. Antonius Hospital, 3435 CM Nieuwegein, The Netherlands.
⁶ Princess Máxima Center for Pediatric Oncology, 3584 CS Utrecht, The Netherlands.
⁷ Department of Rheumatology, Erasmus MC, 3015 GD Rotterdam, The Netherlands.
⁸ Department of Epidemiology and Data Science, Amsterdam UMC, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands.
⁹ Department of Rheumatology & Clinical Immunology, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands.

Abstract

Background/Objectives: Methotrexate (MTX) is a cornerstone drug used to treat immune-mediated inflammatory diseases (IMIDs) in low doses (10-30 mg/week), and malignancies in high doses (5000 mg/m²/2 weeks). Its active metabolites, Methotrexate polyglutamates (MTX-PG_2-5), quantified in erythrocytes, are associated with efficacy. This study aimed to compare erythrocyte MTX-PG concentrations in patients with IMIDs and pediatric acute lymphoblastic leukemia (ped-ALL) treated with low-dose or high-dose MTX, respectively, and to identify clinical, demographic, and treatment-related factors influencing their concentration. Methods: A total of 567 patients with rheumatoid arthritis, juvenile idiopathic arthritis, Crohn's disease, sarcoidosis, and ped-ALL were included. Erythrocyte MTX-PG concentration data was collected after 3 months (2.5 months for ped-ALL patients) of MTX-use. Multivariate linear regressing modelling adjusting for age, sex, body mass index (BMI), smoking status, starting MTX dose, route of MTX administration, use of predniso(lo)ne, disease-modifying anti-rheumatic drugs (DMARDs), and folic (or folinic) acid was performed. Results: Intravenous high-dose MTX increased MTX-PG_4&5 accumulation. Despite 50-fold higher doses in ped-ALL, MTX-PG_2-5sum concentrations were similar to those seen with subcutaneous low-dose MTX used in IMIDs. Age positively influenced MTX-PG concentrations, while DMARD use reduced MTX-PG_2-3&5 concentrations. Interestingly, predniso(lo)ne use was associated with higher MTX-PG_4&5 concentrations and folic (or folinic) acid with higher MTX-PG_3-5 concentrations. Conclusions: This is the first study to compare erythrocyte MTX-PG concentration in low-dose and high-dose patients. Intravenous high-dose MTX administration increased long-chain MTX-PG_4&5 concentrations, with MTX-PG_2-5sum concentrations similar compared to low-dose subcutaneous MTX use. This study demonstrated that route of administration, age, and concomitant therapies such as DMARDs, predniso(lo)ne, and folic (or folinic) acid significantly influence MTX-PG concentrations.

Keywords: immune-mediated inflammatory diseases; methotrexate polyglutamate; multivariate linear regression modelling; pediatric acute lymphoblastic leukemia; pharmacokinetics and drug metabolism.