Inosine-Triphosphate-Pyrophosphatase Activity as a Potential Predictor of Methotrexate Remission in Juvenile Idiopathic Arthritis

Objective: Methotrexate (MTX) is the first-line therapy for juvenile idiopathic arthritis (JIA), but up to 40% of patients do not respond to it. Low inosine triphosphate pyrophosphatase (ITPA) activity has been associated with reduced clinical remission. We investigated the role and underlying mechanisms of ITPA in vitro. Methods: ITPA enzymatic activity was measured by high-performance liquid chromatography with ultraviolet detection in erythrocyte lysates from patients with JIA (enrolled in Trieste, Italy; Florence, Italy; and Kansas City, Missouri) receiving weekly MTX (10-15 mg/m2) for at least 12 months. Remission was evaluated by Wallace et al criteria. ITPA rs1127354, which reduces enzyme activity, was genotyped. Immortalized human hepatocytes were transfected with either an ITPA-containing or empty plasmid to evaluate cytotoxicity (MTT), ADORA2A expression (quantitative polymerase chain reaction), intracellular cyclic AMP (cAMP) (enzyme-linked immunosorbent assay), PKA-Cα expression (immunoblot), and extracellular adenosine metabolites (liquid chromatography-tandem mass spectrometry). Results: A total of 195 patients with JIA (mean age: 8 years, range: 0-22 years, 74% female participants) were enrolled. Patients reaching remission (48%) had higher ITPA activity than nonresponders during therapy and at treatment onset (meta-analysis: P = 0.0004 and P = 0.007, respectively). Predictive cutoffs for overall and onset measurements were identified: 181.9 and 177.8 nmol of inosine monophosphate per hour. None of the patients with variant ITPA rs1127354 achieved remission (Fisher's exact test P = 0.0063); however, 48% of wild-type patients failed to respond. ITPA-overexpressing cells showed elevated MTX sensitivity, cAMP levels, PKA-Cα expression, and hypoxanthine (all P < 0.05, two-way analysis of variance). Conclusion: ITPA activity is a promising predictive biomarker for MTX response in JIA, outperforming ITPA rs1127354 genotyping. Its predictive value is evident at treatment initiation, supporting its use for personalized treatment. Mechanistically, higher enzyme activity may enhance MTX efficacy through the adenosine/inosine-ADORA2A pathway.