Methotrexate (MTX) continues to be the first-line disease-modifying antirheumatic drug (DMARD) in the treatment of juvenile idiopathic arthritis (JIA). However, response to MTX is often delayed and variable, requiring the initiation of second-line therapies, most notably biologic DMARDs. Recognizing the goal of early initiation of effective therapy in the treatment of JIA, there remains a need to identify predictive biomarkers to guide drug selection in the treatment of JIA. This study uses a plasma metabolomic profiling approach to identify metabolic biomarkers associated with MTX non-response in JIA.

A multi-platform ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) approach was used to obtain a 902 plasma metabolite profile in pretreatment samples from patients with JIA initiating MTX therapy ( n  = 60) in a single-center, prospective observational study. The cohort consisted of 67 % female patients, with a median age of 10.75 years. Patients were classified as responders ( n  = 45) or non-responders ( n  = 15), with non-response defined as the need to add or switch to a biologic DMARD within the first six months of therapy. Unpaired univariate analysis was performed using MetaboAnalyst 5.0 to identify significant metabolites ( p   <  0.05). Enrichment analysis was conducted using Chemical Similarity Enrichment Analysis (ChemRich) and identified chemical clusters that differed between the groups using an FDR-adjusted p -value ( q -value) threshold of 0.05. A metabolic network map was generated using MetaMapp and visualized using Cytoscape 3.9.1. Receiver operator characteristic (ROC) curve analyses were conducted to identify metabolites that effectively discriminate MTX responders and non-responders. Biomarker performance was assessed based on the ROC Area Under the Curve (AUC). Regression modeling was used to investigate the independent association between the identified metabolites, while controlling for clinical covariates, including baseline 71-joint count, Juvenile Arthritis Disease Activity Score (JADAS-71), and Active Joint Count (AJC).

Out of 902 pretreatment plasma metabolites measured, 63 were found to significantly differ based on MTX response. Chemical cluster analysis identified six metabolite clusters as significant, including indoles, sugar alcohols, xanthines, trimethylammonium compounds, glycodeoxycholic acid, and dipeptides. Metabolic pathway analysis reveals that tryptophan, lipids, amino acids, and nucleic acids are key pathways that differ between MTX responders and non-responders. ROC analysis identified eight metabolites with an AUC > 0.75, which included multiple metabolites related to gut microbial metabolism, such as hippurate, indole-2-one, indolepropionate, anthranilate, phenylacetylglutamine, p-cresol sulfate, and several unidentified metabolites. Increased plasma hippurate levels were found to be the most discriminating metabolite (AUC=0.781). Multivariate models that included baseline JADAS-71, AJC, and hippurate levels demonstrated that increased plasma hippurate levels were independently associated with MTX non-response at 6 months.

These findings support plasma metabolomic differences associated with the response to MTX in JIA. The identification of hippurate, indoles, and other gut microbial-derived metabolites continues to draw attention to the potential relationship between the gut microbiome and MTX treatment response in JIA.