FARMACOGENETICA E FARMACOCINETICA DELLA TERAPIA CON METOTREXATO PER L’ARTRITE IDIOPATICA GIOVANILE

Methotrexate (MTX) is the recommended conventional disease-modifying antirheumatic drug for juvenile idiopathic arthritis (JIA). However, its mechanism of action at low doses remains incompletely understood, and up to 40% of patients do not respond. The project had two main aims: to investigate MTX’s mechanism of action and to evaluate inosine triphosphate pyrophosphate (ITPA) activity as a predictive biomarker of MTX response in JIA. For the first aim, the SW982 fibroblast-like synoviocyte (FLS) line was treated with MTX, with or without an inflammatory stimulus, to assess cytotoxicity (MTT), IL-6, IL-8, MMP-9, MMP-2, long intergenic non-coding RNA-p21 (lincRNA-p21), ADORA expression (qPCR), p53, p65, and PKA-Cα levels (immunoblot), and adenosine metabolites in supernatants (LC-MS/MS). Plasma cell-free RNA (cfRNA) from 13 JIA patients was analyzed to correlate lincRNA-p21 expression with disease activity markers. MTX decreased IL-6 and p65 expression, while increasing lincRNA-p21 and p53. Despite the lack of changes in PKA-Cα levels, adenosine and uric acid were higher in cells treated with the combination of inflammatory stimulus and MTX. A correlation emerged between lincRNA-p21 and both erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). Thus, lincRNA-p21 may contribute to the anti-inflammatory effects of MTX in FLS through a mechanism similar to that described in T cells. Its relevance compared with the adenosine-related pathway and its potential as a biomarker require further investigation. Assessing lincRNA-p21 expression in plasma could offer a minimally invasive biomarker. For the second aim, ITPA enzymatic activity was measured in erythrocyte lysates (HPLC-UV) from 195 JIA patients (Trieste, Florence, Kansas City) on weekly MTX (10-15 mg/m2) for ≥12 months. Remission was assessed by Wallace criteria. ITPA rs1127354 and rs7270101, which reduce enzyme activity, were genotyped. Immortalized human hepatocytes (IHH) were transfected with either an ITPA-containing or empty plasmid and treated with MTX to evaluate cytotoxicity (MTT), ADORA2A expression (qPCR), intracellular cyclic AMP (cAMP) (ELISA), PKA-Cα expression and phosphorylated cAMP Response Element-Binding protein (CREB) (immunoblot), and extracellular adenosine metabolites (LC-MS/MS). Patients reaching remission had higher ITPA activity than non-responders with predictive cutoffs of 181.9 and 177.8 nmol of IMP/h for overall and onset measurements. None of the rs1127354 carriers achieved remission, but 48% of wild-type patients failed to respond. No association emerged for rs7270101. ITPA-overexpressing cells showed higher MTX sensitivity, cAMP levels, PKA-Cα and phosphorylated CREB, whereas MOCK cells had higher phosphorylated p65, adenosine, inosine and uric acid levels. These findings strongly support ITPA activity as a potential tool for personalizing MTX therapy.