Clemastine fumarate accelerates accumulation of disability in progressive multiple sclerosis by enhancing pyroptosis.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS). Clemastine fumarate, the over-the-counter antihistamine and muscarinic receptor blocker, has remyelinating potential in MS. A clemastine arm was added to an ongoing platform clinical trial TRAP-MS (NCT03109288) to identify a cerebrospinal fluid (CSF) remyelination signature and to collect safety data on clemastine in patients progressing independently of relapse activity (PIRA). The clemastine arm was stopped per protocol-defined criteria when 3/9 patients triggered individual safety stopping criteria (χ2 p=0.00015 compared to remaining TRAP-MS treatments). Clemastine treated patients had significantly higher treatment-induced disability progression slopes compared to remaining TRAP-MS participants (p=0.0075). Quantification of ~7000 proteins in CSF samples collected before and after clemastine treatment showed significant increase in purinergic signaling and pyroptosis. Mechanistic studies showed that clemastine with sub-lytic doses of extracellular ATP activates inflammasome and induces pyroptotic cell death in macrophages. Antagonist of the purinergic channel P2RX7 that is strongly expressed in oligodendrocytes and myeloid cells, blocked these toxic effects of clemastine. To further confirm our results, we performed the bulk RNA-seq analysis of primary human macrophages (MDMs) and THP-1 cells to identify signaling pathways affected by clemastine (+ATP) stimulation. Ingenuity Pathway Analysis (IPA) predicted inflammasome activation, pyroptosis, and immunogenic cell death as pathways affected by ATP+CLM treatment.