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][1]) 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/ATP signaling and pyroptosis cell death. Mechanistic studies showed that clemastine with sub-lytic doses of extracellular ATP activates inflammasome and induces pyroptotic cell death in macrophages. Clemastine with ATP also caused pyroptosis of induced pluripotent stem cell-derived human oligodendrocytes. Antagonist of the purinergic channel P2RX7 that is strongly expressed in oligodendrocytes and myeloid cells, blocked these toxic effects of clemastine. Finally, re-analyses of published snRNAseq studies revealed increased P2RX7 expression and pyroptosis transcriptional signature in microglia and oligodendrocytes in MS brain, especially in chronic active lesions. CSF proteomic pyroptosis score was increased in untreated MS patients, was higher in patients with progressive than relapsing-remitting disease and correlated significantly with rates of MS progression. Thus, pyroptosis is likely first well-characterized mechanism of CNS injury underlying PIRA even outside of clemastine toxicity. One Sentence Summary Clemastine enhanced disability accumulation in patients progressing by non-lesional MS activity by potentiating intrathecal P2RX7 signaling and pyroptosis. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Trial NCT00794352, [NCT03109288][1] ### Funding Statement The study was funded by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by and carried out in accordance with the recommendations of the Central Institutional Review Board (IRB) of the National Institutes of Health (NIH). All subjects provided written informed consent. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as [ClinicalTrials.gov][2]. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT03109288&atom=%2Fmedrxiv%2Fearly%2F2024%2F04%2F10%2F2024.04.09.24305506.atom [2]: http://ClinicalTrials.gov