Pharmacological inhibition of the NLRP3 inflammasome as a potential target for multiple sclerosis induced central neuropathic pain

Abstract

The NOD-like receptor (NLR) family pyrin domain-containing protein 3 (NLRP3) inflammasome is implicated in the pathogenesis of multiple diseases including neuroinflammation associated with multiple sclerosis (MS). However, the extent to which NLRP3 has a pathobiological role in MS-associated central neuropathic pain (CNP) is unknown. Hence, the present study was designed to address this issue using an optimised relapsing–remitting experimental encephalomyelitis (RR-EAE)-mouse model of MS-associated neuropathic pain. RR-EAE mice with fully developed mechanical allodynia in the bilateral hindpaws (paw withdrawal thresholds (PWTs) ≤ 1 g) at day 16 post-immunisation (p.i.) were administered single oral bolus doses of MCC950, a selective and potent small-molecule inhibitor of NLRP3, once daily for 21 consecutive days. Following administration of the first dose of MCC950 at 50 mg kg−1, the mean (± SEM) peak anti-allodynic effect was observed at ~ 1 h post-dosing with a duration of action of ~ 2 h. Following chronic dosing with MCC950, mechanical allodynia in the bilateral hindpaws was progressively reversed by oral treatment with MCC950 (50 mg kg−1 day−1), but not vehicle. Specifically, by day 25 p.i. and continuing until study completion on day 36 p.i., bilateral hindpaw PWTs of RR-EAE mice treated with MCC950 (50 mg kg−1 day−1) did not differ significantly (P > 0.05) from the corresponding hindpaw PWTs for the sham (control) group. In addition, MCC950 at 50 mg kg−1 day−1 attenuated disease relapses in RR-EAE mice indicated by tail limpness as well as hindlimb weakness. Together, our findings suggest that inhibition of NLRP3 inflammasome activation may be a potential therapeutic approach to alleviate MS-associated CNP and disease relapses in patients with RR-MS.

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Abbreviations

CNP:

Central neuropathic pain

NLRP3:

NOD-like receptor (NLR) family pyrin domain-containing protein 3

PWT:

Paw withdrawal threshold

RR-EAE:

Relapsing–remitting experimental autoimmune encephalomyelitis

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Acknowledgements

This work was supported by an Australian Research Council (ARC) Linkage Grant [LP120200623] and by a National Health and Medical Research Council (NHMRC) Grant [APP1059239]. The authors also acknowledge the Queensland Government Smart State Research Programme for supporting CIPDD research infrastructure. CIPDD is also supported by Therapeutic Innovation Australia (TIA). TIA is supported by the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS) program. We also gratefully acknowledge Avril A.B. Robertson for formulating MCC950 and Ruby Pelingon and Mark Butler for conducting the LC-MS/MS of MCC950 in RR-EAE-mouse plasma samples at the Institute for Molecular Bioscience (IMB), The University of Queensland, Australia.

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Affiliations

Authors

Contributions

NK and AK contributed to the research design and performance of the in vivo experiments. NK performed the data analysis of the in vivo experiments and drafted the manuscript; DB performed in vitro experiments and the associated data analysis. The study was conceived and supervised by MS and MC. All authors edited, read and approved the final manuscript.

Corresponding author

Correspondence to Maree T. Smith.

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Conflict of interest

Professor Matt Cooper is CEO and a shareholder in Inflazome Ltd. This company is developing drugs to address clinical unmet needs in inflammatory disease by targeting inflammasomes. All other authors have no conflicts of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Fig. S1

Optimisation of the anti-allodynic dose of MCC950 in our optimised RR-EAE-mouse model of MS-neuropathic pain. The RR-EAE mice, but not sham mice, developed robust mechanical allodynia in the bilateral hindpaws (mean PWT ≤ 1 g) by day 14 (p.i.) that was persistent in vehicle-treated RR-EAE mice until study completion (day 36 p.i.). By contrast, mechanical allodynia in the bilateral hindpaws of RR-EAE treated with oral MCC950 at 20 mg kg−1 day−1 for days 16–26 p.i. followed by 50 mg kg−1 day−1 for days 27–36 p.i. was significantly attenuated compared with vehicle (PBS)-treated RR-EAE. The higher dose of MCC950 (50 mg kg−1 day−1) produced more robust anti-allodynic effects in RR-EAE mice, such that PWTs in these mice were comparable to sham mice by study completion on day 36 p.i.. The shaded area highlights the chronic MCC950 or vehicle treatment period (days 16–36 p.i.). PWT at or below the horizontal dotted line (PWT ≤ 1 g) indicates fully developed mechanical allodynia. Vertical dotted line indicating day 27, whereby RR-EAE mice receiving MCC950 at 50 mg kg−1 day−1 for 10 days. *P ≤ 0.05 (two-way ANOVA, post hoc: Bonferroni). EAE, experimental autoimmune encephalomyelitis; g, gram; kg, kilogram; mg, milligram; PWT, Paw withdrawal threshold; p.i., post-immunisation (JPEG 216 kb)

Fig. S2

Impact of repeated oral dosing of MCC950 (20–50 mg kg−1 day−1) on the mild relapsing–remitting (RR) disease course in our optimised RR-EAE-mouse model of MS-neuropathic pain. In RR-EAE mice, but not sham mice, the onset of clinical disease (mean score ≥ 0.5 a.u.) was evident by day 4 p.i. followed by consecutive episodes of relapse (mean score ≥ 1 a.u.) and remission (mean score < 0.5 a.u.). This RR-EAE disease course was not changed significantly (P > 0.05) by oral MCC950 at 20 mg kg−1 day−1 for days 16–26 (p.i.) followed by 50 mg kg−1 day−1 for days 27–36 (p.i.) in a manner similar to vehicle-treated RR-EAE mice. The shaded area highlights the chronic treatment (MCC950 or vehicle) period (days 16–36 p.i.). *P ≤ 0.05 (two-way ANOVA, post hoc: Bonferroni). a.u., arbitrary units; EAE, experimental autoimmune encephalomyelitis; kg, kilogram; mg, milligram; PWT, Paw withdrawal threshold; p.i., post-immunisation (JPEG 294 kb)

Fig. S3

Mean (± SEM) plasma concentrations of TNF-α, IL-5, and CXCL1 in RR-EAE mice treated with chronic oral dosing of MCC950 at 50 mg kg−1 day−1 for 21 consecutive days (days 16–36 p.i.). Treatment of RR-EAE mice with MCC950 did not significantly alter the plasma cytokine concentrations at the four timepoints investigated, i.e., day 0, 15, 25, and 36 post-immunisation compared with vehicle-treated RR-EAE mice. The only exception was that the IL-5 concentration was significantly lower in the RR-EAE mice assigned to MCC950 treatment group compared with the vehicle group at day 0. *P ≤ 0.05 (one-way ANOVA with Tukey’s multiple comparison test). pg, picogram; mL, millilitre (JPEG 273 kb)

Table S1

Individual plasma concentrations of IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-10, and IL-12p70 on Day 0 in RR-EAE mice treated chronically with MCC950 at 50 mg.kg−1.day−1 from day 16 to day 36 p.i.; †, plasma pooled from multiple mice; ND, not determinable as value was below the standard curve range (DOCX 16 kb)

Table S2

Individual plasma concentrations of IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-10, and IL-12p70 on Day 15 p.i. in RR-EAE mice treated chronically with MCC950 at 50 mg.kg−1.day−1 from day 16 to day 36 p.i.; †, plasma pooled from multiple mice; ND, not determinable as value was below the standard curve range (DOCX 16 kb)

Table S3

Individual plasma concentrations of IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-10, and IL-12p70 on Day 25 p.i. in RR-EAE mice treated chronically with MCC950 at 50 mg.kg−1.day−1 from day 16 to day 36 p.i.; †, plasma pooled from multiple mice; ND, not determinable as value was below the standard curve range (DOCX 16 kb)

Table S4

Individual plasma concentrations IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-10, and IL-12p70 on Day 36 p.i. in RR-EAE mice treated with chronically with MCC950 at 50 mg.kg−1.day−1 from day 16 to day 36 p.i.; †, plasma pooled from multiple mice; ND, not determinable as value was below the standard curve range (DOCX 17 kb)

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Khan, N., Kuo, A., Brockman, D.A. et al. Pharmacological inhibition of the NLRP3 inflammasome as a potential target for multiple sclerosis induced central neuropathic pain. Inflammopharmacol 26, 77–86 (2018). https://doi.org/10.1007/s10787-017-0401-9

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Keywords

  • Analgesia
  • Experimental autoimmune encephalomyelitis
  • Neuropathic pain
  • Multiple sclerosis
  • Paw withdrawal threshold (PWT)
  • Relapsing–remitting experimental autoimmune encephalomyelitis (RR-EAE)