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Methotrexate (MTX) is the cornerstone of therapy in the treatment of rheumatoid arthritis (RA). However, its efficacy and toxicity are variable and remain unpredictable. Interindividual variation in the metabolism of MTX by the enzyme folyl polyglutamate synthetase (FPGS) has been associated with response variability in RA. In this work, we propose the development of a FPGS phenotyping assay that can be evaluated as a tool for the prediction of efficacy and toxicity in patients with RA prior to initiating MTX therapy. FPGS activity was measured in erythrocyte lysate by monitoring methotrexate polyglutamate (MTX + Glun) formation using ultra-performance liquid chromatography tandem–mass spectrometry (UPLC/MS/MS). Erythrocyte FPGS activity was measured in newly diagnosed RA (n = 35) and osteoarthritis (n = 7) patients. The enzymatic assay was optimized for measuring FPGS activity in 25 µL of packed erythrocytes over two hours. The coefficient of variation for intra- and inter-day analysis was found to be 5% and 12%, respectively. The method was used to measure FPGS enzyme kinetics, resulting in a mean (SD) Km of 30.3 (4.8) µM and a Vmax of 612 (193) pmol MTX + Glu2/h/mL of packed erythrocytes. Mean (SD) erythrocyte FPGS activity in patients with RA was found to be 445.93 (344.50) pmol MTX + Glu2/h/mL and with a 26-fold difference in the range (range: 83–2179 pmol MTX + Glu2/h/mL) whereas for patients with OA, it was found to be 409.80 (157.66) pmol MTX + Glu2/h/mL with a 3.5-fold difference in the range (range: 200.95–683.93 pmol MTX + Glu2/h/mL). Monitoring erythrocyte FPGS activity may be a feasible strategy of phenotyping for methotrexate efficacy and toxicity in patients with RA.

Methotrexate (MTX) efficacy in the treatment of rheumatoid arthritis (RA) is variable and unpredictable, resulting in a need to identify biomarkers to guide drug therapy. This study evaluates changes in the plasma metabolome associated with response to MTX in RA with the goal of understanding the metabolic basis for MTX efficacy towards the identification of potential metabolic biomarkers of MTX response. Plasma samples were collected from healthy control subjects (n = 20), and RA patients initiating MTX therapy (n = 20, 15 mg/week) before and after 16 weeks of treatment. The samples were analyzed by a semi-targeted metabolomic analysis, and then analyzed by univariate and multivariate methods, as well as an enrichment analysis. An MTX response was defined as a clinically significant reduction in the disease activity score in 28 joints (DAS-28) of greater than 1.2; achievement of clinical remission, defined as a DAS-28 < 2.6, was also utilized as an additional measure of response. In this study, RA is associated with an altered plasma metabolome that is normalized following initiation of MTX therapy. Metabolite classes found to be altered in RA and corrected by MTX therapy were diverse and included triglycerides (p = 1.1 × 10−16), fatty acids (p = 8.0 × 10−12), and ceramides (p = 9.8 × 10−13). Stratification based on responses to MTX identified various metabolites differentially impacted in responders and non-responders including glucosylceramides (GlcCer), phosphatidylcholines (PC), sphingomyelins (SM), phosphatidylethanolamines (PE), choline, inosine, hypoxanthine, guanosine, nicotinamide, and itaconic acid (p < 0.05). In conclusion, RA is associated with significant alterations to the plasma metabolome displaying at least partial normalization following 16 weeks of MTX therapy. Changes in multiple metabolites were found to be associated with MTX efficacy, including metabolites involved in fatty acid/lipid, nucleotide, and energy metabolism.