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

Abstract

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/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.

Fig. 1.. Clemastine-induced changes in disability, metabolism, and inflammatory markers.

(A) Clinical safety was monitored by a continuous, machine-learning derived CombiWISE scale (range 0–100) that correlates strongly with EDSS (range 0–10). (B) A minimum of 4 visits on stable therapy spanning at least 18 months is required to measure baseline (blue) CombiWISE slope. On-therapy CombiWISE slope (orange) is calculated based on 6-months follow-up data collected after theory initiation. (C) 3 out of 9 patients on clemastine therapy triggered safety criteria, in contrast to none of the 63 patient-specific treatments of 6 other TRAP-MS therapies; p-value of probability of this occurrence was based on χ² test. (D) Progression CombiWISE slopes at baseline (B) and therapy (T) were compared between clemastine arm and all other 6 TRAP-MS therapies. Black horizontal line represents median value of the group. Red color indicates 3 patients that triggered safety stopping criteria treatment slope exceeding 5x baseline slope. Displayed p-value was generated from two-sided unaired Wilcoxon rank test comparing therapy-induced change in CombiWISE slope between the clemastine arm and all other therapies. Clemastine induced increase of weight (E), total cholesterol (F), and LDL cholesterol (G) levels between baseline and treatment and these changes showed strong association as seen on example of weight change versus LDL cholesterol change (H). Furthemore, clemastine induced increase of inflammatory biomarker CRP (I). Lipid panel was an optional laboratory test and the results are missing for one patient. EDSS – Expanded Disability Status Scale, CombiWISE – Combinatorial Weigh-adjusted Disability Scale, LDL – low density lipoprotein, CRP – C-reactive protein, Blue line in H represents linear regression line, gray dashed line in D-G and I represents 0 change. The lower and upper hinges of the boxplots correspond to the first and third quartiles (the 25th and 75th percentiles). The upper and lower whiskers extend from the hinge to the largest and smallest value, respectively.

Fig. 2.. Clemastine (CLM) potentiation effect on ATP-induced inflammasome activation and GSDMD-driven pyroptotic cell death in THP-1 cells.

The THP-1 cells were primed with 200 ng/mL LPS overnight and then treated either with medium + DMSO (negative control, Ctrl), 10 μg/mL CLM, 2 mM ATP +/− 10 μg/mL CLM or 2.5 μM Nigericin (positive control) for 3h, 6h, and 18 h. Release of the pro-inflammatory cytokine IL-1β into the culture medium of THP-1 cells (A) and THP-1 Gsdmd ^−/− cells (B), determined by ELISA assay. Activity of Caspase-1 in the culture medium of THP-1 cells (C) and THP-1 Gsdmd ^−/− cells (D), determined using bioluminescent assay. Lytic cell death of THP-1 cells (E) and THP-1 Gsdmd ^−/− cells (F), determined by LDH activity released into the culture medium. Cell viability of THP-1 cells (G) and THP-1 Gsdmd ^−/− cells (H) evaluated by the MTS assay. Data are presented as mean ± SEM of three independent experiments, each performed in duplicate (n=6). One way ANOVA followed by Dunnett’s multiple comparisons test was used to compare the testing groups with control group (Ctrl). One-way ANOVA test followed by Holm-Sidak’s multiple comparison was used to compare ATP and ATP+CLM group (to assess the potentiation effect of CLM). *, P ≤ 0.05. **, P ≤ 0.01. ***, P ≤ 0.001. ****, P ≤ 0.0001. (I) Flow cytometry analysis of cell uptake of SYTOX green in THP-1 cells primed with 200 ng/mL LPS overnight and then treated either with medium + DMSO (control), 10 μg/mL CLM or 2 mM ATP +/− 10 μg/mL for 90 min. Representative plot of two independent experiments.

Fig. 3.. The potentiation effect of Clemastine (CLM) on ATP-induced lytic cell death in primary monocyte-derived macrophages and iPSC-derived oligodendrocytes.

The primary monocyte-derived macrophages (A-D) were primed with 200 ng/mL LPS overnight, then pre-treated with 30 nM JNJ-54175446 (a selective purine P2X7 receptor antagonist) for 1 h, followed by treatment either with medium + DMSO (negative control, Ctrl), 10 μg/mL CLM, 2 mM ATP +/− 10 μg/mL CLM or 10 μM Nigericin (positive control) for 3h, 6h, and 18 h. Levels of the pro-inflammatory cytokine IL-1β (A) and Caspase-1 (B) activity in the culture medium determined by ELISA and bioluminescence assay, respectively. Lytic cell death (C) determined by LDH activity released into the medium and cell viability (D) evaluated by the MTS assay. Data are presented as mean ± SEM of three independent experiments, each performed in duplicate (n=6). Representative fluorescence microscopy images depicting SYTOX Green-stained MDMs treated with drugs for 6 h (E). iPSC-derived oligodendrocytes (F) were primed with 200 ng/mL LPS overnight followed by treatment either with medium + DMSO (negative control, Ctrl), 10 μg/mL CLM, 2mM ATP +/− 10 μg/mL CLM or 10 μM Nigericin (positive control). LDH release after the treatments for 18 h (F). Data are presented as mean ± SEM of four independent samples. One way ANOVA followed by Dunnett’s multiple comparisons test was used to compare the testing groups with control group (Ctrl). One-way ANOVA test followed by Holm-Sidak’s multiple comparison was used to compare ATP and ATP+CLM group (to assess the potentiation effect of CLM). *, P ≤ 0.05. **, P ≤ 0.01. ***, P ≤ 0.001. ****, P ≤ 0.0001.

Fig. 4.. Gene expression associated with pyroptosis signaling pathway in CNS tissue.

Panels (A) and (C) illustrate the profiles of P2RX7 and GSDM family across the lesion types. Meanwhile, panels (B) and (D) show gene expressions based on cell types. P2RX7: purinergic receptor P2X7; GSDM: gasdermin; PJVK: pejvakin; NAWM: normal-appearing white matter; OPC: oligodendrocyte precursor cells.

Fig. 5.. MS severity associated Pyroptosis signature in CSF increases with Clemastine treatment.

CSF biomarker-derived Pyroptosis activation score is significantly elevated in MS patients compared to HD (A) and in progressive MS (prog-MS) patients compared to relapsing-remitting MS (RR-MS) patients (B). The gray area represents HD mean +/− standard deviation. (C) Longitudinal pre-clemastine CSF samples of patients that triggered safety criteria show elevated Pyroptosis activation score. CSF samples collected just prior starting clemastine are highlighted in red. (D) Clemastine-induced yearly change in Pyroptosis activation score is significantly elevated compared to untreated MS patients. The lower and upper hinges of the boxplots correspond to the first and third quartiles (the 25th and 75th percentiles). The upper and lower whiskers extend from the hinge to the largest and smallest value, respectively. Pyroptosis activation scores is strongly associated with the rate of accumulation of cognitive and physical disability, represented by Brain damage severity (E) and MS-DSS (F), respectively. The green line depicts linear regression line, and the gray shaded area shows 95% confidence interval. The gray dashed line depicts 0.