Background/Purpose: Arhalofenate is an investigational drug with dual uricosuric and anti-inflammatory properties. Arhalofenate reduced the risk of acute flare while lowering serum urate levels in gout patients in a phase II trial. Previous studies demonstrated that arhalofenate (or its active acid form) suppresses the production of IL-1β in response to urate crystals in macrophages in vitro and in a mouse air pouch model in vivo. In this study, we characterized the molecular mechanism of action of arhalofenate acid, studying urate crystal-induced inflammatory responses in macrophages in vitro.

Methods: Bone marrow derived macrophages (BMDMs) were stimulated with urate crystals in the presence or absence of arhalofenate acid. IL-1b release was measured from conditioned media by ELISA analysis. Western blot was carried out to examine expression of NLRP3, pro-caspase-1 and cleaved caspase-1 (for NLRP3 inflammasome activation); phosphorylation and expression of the nutritional biosensor AMPKa (AMP-activated protein kinase), the NAD⁺-dependent protein deacetylase SIRT1, the PPARg co-activator and master regulator of mitochondrial biogenesis PGC-1a, the nuclear-encoded mitochondrial transcription factor A TFAM, cytosolic thioredoxin 1 (TXN1), nuclear thioredoxin 2 (TXN2), thioredoxin-interacting protein (TXNIP) involved in NLRP3 inflammasome activation; microtubule-associated protein 1 light-chain 3 (LC3) and p62 (an adaptor contributing to autophagic degradation of ubiquitinated substrates and damaged organelle such as damaged mitochondria) for autophagy.

Results: Arhalofenate acid attenuated urate crystal-induced IL-1b production in BMDMs via inhibition of NLRP3 inflamamsome activation, evidenced by inhibition of protein expression of NLRP3 and cleaved caspase-1 (p10). In addition, arhalofenate acid dose-dependently increased phosphorylation of AMPKa, as well as expression of SIRT1 that was dependent on AMPK, since arhalofenate acid failed to increase SIRT1 expression in AMPKa1 knockout (KO) BMDMs. Arhalofenate acid was also unable to significantly inhibit urate crystal-induced IL-1b production in AMPKa1 KO, compared to WT BMDMs, indicating that AMPK mediated inhibitory effect of arhalofenate acid. Moreover, urate crystals concurrently reduced phosphorylation of AMPKa, expression of SIRT1, PGC-1a, TFAM, TXN1, TXN2, but induced expression of TXNIP, all of which were reversed by arhalofenate acid in BMDMs, implicating a role for arhalofenate acid in maintaining mitochondrial function, limiting oxidative stress and preventing TXNIP-mediated inflammasome activation. Furthermore, arhalofenate acid was able to not only promote induction of autophagy but also accelerate autophagic flux.

Conclusion: Arhalofenate acid, the active form of arhalofenate, is anti-inflammatory via activation of AMPK and downstream signaling in macrophages, thereby increasing cellular resistance to stresses induced by urate crystals, mediated by maintaining mitochondrial integrity and cellular quality control through autophagy. These effects likely contribute to gout flare risk reduction by arhalofenate.

« Back to 2017 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/arhalofenate-acid-inhibits-urate-crystal-induced-inflammatory-responses-through-activation-of-amp-activated-protein-kinase-ampk-signaling/