Background/Purpose: Rheumatoid arthritis (RA) is a chronic autoimmune disease where fibroblast-like synoviocytes (FLS) play a central role in joint inflammation and destruction. Methotrexate (MTX), a standard treatment, has limitations due to systemic toxicity and variable efficacy. RA-FLS exhibit altered glucose metabolism, notably increased glycolysis and upregulation of glucose transporter 1 (GLUT1), presenting a potential therapeutic target. We developed a glucose-methotrexate conjugate (Glc-MTX) designed to selectively target RA-FLS via GLUT transporters, potentially increasing efficacy and reducing toxicity. This study aimed to evaluate for the first time the in vitro effects of the Glc-MTX conjugate compared to free MTX on proliferation, inflammatory mediator production, and GLUT1-dependent uptake in human RA-FLS.

Methods: Cell proliferation of commercially available primary human FLSs, isolated from the synovial tissues of patients with RA and OA, was assessed using the MTT assay after treatment with Glc-MTX or MTX (doses ranging from 0.01 µM to 10 µM) for 24-48h. For inflammatory response, RA-FLS were stimulated with TNF-α (10 ng/ml) for 48h and treated with Glc-MTX or MTX (0.01 µM to 10 µM) for 24-48h. Levels of key inflammatory mediators (IL-6, IL-8) and matrix-degrading enzymes (MMP-1, MMP-3) in RA-FLS supernatants were quantified by ELISA. To assess GLUT1 dependence, RA-FLS were pre-treated with the selective GLUT1 inhibitor BAY-876 (1 mM) for 60 min before treatment.

Results: MTT assays showed that Glc-MTX inhibited RA-FLS proliferation with an IC50 of 2.87 µM, comparable to free MTX (IC50: 2.55 µM). Glc-MTX exhibited greater selectivity for RA-FLS over OA-FLS (IC50: 7.34 µM; Selectivity Index: 2.56) compared to free MTX, which showed higher potency against OA-FLS (IC50: 0.25 µM; Selectivity Index: 0.098). Furthermore, Glc-MTX displayed lower cytotoxic activity against RA-FLS not stimulated with TNF-α compared to those that were stimulated (p=0.013). In TNF-α-stimulated RA-FLS, ELISA results demonstrated that both MTX and Glc-MTX inhibited the secretion of IL-6, IL-8, MMP-1, and MMP-3 compared to stimulated controls, with MTX generally showing stronger inhibition than Glc-MTX at the tested concentrations. Pre-treatment with the GLUT1 inhibitor BAY-876 significantly attenuated the anti-proliferative effect of Glc-MTX on RA-FLS, increasing its IC50 from 2.87 µM to 12.4 µM (p < 0.01), while having minimal impact on the IC50 of free MTX (IC50: 2.55 µM without BAY-876 vs. 2.24 µM with BAY-876, p>0.05).

Conclusion: The Glc-MTX conjugate inhibits RA-FLS proliferation in vitro with potency similar to MTX but demonstrates significantly improved selectivity over OA-FLS. Both Glc-MTX and MTX inhibited inflammatory and matrix-degrading mediator production. The significant reduction in Glc-MTX efficacy following GLUT1 blockade with BAY-876 confirms its reliance on GLUT1-mediated uptake. These findings show the promising potential of Glc-MTX as a novel, targeted therapeutic strategy for RA. Further in vivo studies are required to fully understand the therapeutic potential of Glc-MTX.

« Back to ACR Convergence 2025

ACR Meeting Abstracts - https://acrabstracts.org/abstract/glut1-dependent-targeting-and-enhanced-selectivity-of-a-glucose-methotrexate-conjugate-in-rheumatoid-arthritis-fibroblast-like-synoviocytes/