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Updated: May 9, 2025
Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis
Published on: September 9, 2022
The P2X7R-antagonist AFC-5128 ameliorates chronic experimental autoimmune encephalomyelitis in a preventive and
Robert Hoffrogge1, Anna Karachunskaya1, Neele Heitmann1
1Department of Neurology, St. Josef-Hospital, Bochum, Germany.
Background:
Multiple sclerosis (MS) is characterized by chronic inflammation driven by central nervous system (CNS)-resident immune cells such as microglia, especially during the progressive phase of the disease. The P2X7 receptor (P2X7R), a risk protein for MS, is ubiquitously expressed on immune cells. AFC-5128, a CNS-penetrating small molecule inhibitor of P2X7R, is a promising agent for the treatment of autoimmune diseases such as MS.
Methods:
In vitro, the effects on the calcium influx of primary murine microglia were assessed via Fluo-4 calcium imaging. In vivo, MOG35-55 immunized C57BL/6 mice were treated with AFC-5128, fingolimod (FTY) or vehicle in different treatment paradigms. The mice were scored daily. Microglial marker expression, immune cell phenotyping and serum cytokine analyses were performed via flow cytometry. Immune cell infiltration, demyelination and Iba1+/CD3+ cells were detected in spinal cord cross-sections. The effects of MOG35-55 T-cell restimulation were assessed in vitro.
Results:
In vitro, treatment of primary microglia with 10 µM AFC-5128 reduced the influx of calcium following ATP stimulation (p<0.0001). In vivo, treatment of mice with AFC-5128 led to a reduction in overall EAE scores in acute and chronic EAE, with the best effects using 200 mg/kg body weight AFC-5128 (p<0.0001). Peripheral immune cell subsets (B cells, T cells and macrophages) and serum cytokine levels of chronic EAE mice treated in a therapeutic paradigm were not affected. While the expression of homeostasis markers of microglia in AFC-5128-treated mice was not affected, there was a trend toward lower expression of phagocytosis-associated markers. Late therapeutic treatment with AFC-5128 had only mild effects on chronic EAE.
Conclusion:
The treatment of EAE mice with AFC-5128 improved acute and chronic EAE in different treatment paradigms, with positive effects on histological markers and slight modulation of microglial marker expression. Mechanistically, calcium influx of microglia was reduced following AFC-5128 treatment, which implies the ability of AFC-5128 to stabilize calcium homeostasis. Therefore, therapeutic inhibition of P2X7R via AFC-5128 has the potential for translation into a treatment of both relapsing and progressive forms of multiple sclerosis.
Insights
AFC-5128, a novel P2X7 receptor inhibitor, effectively reduced inflammation and improved symptoms in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS). This suggests AFC-5128
Area of Science:
- Neuroimmunology
- Pharmacology
Background:
- Multiple sclerosis (MS) involves chronic central nervous system (CNS) inflammation driven by microglia.
- The P2X7 receptor (P2X7R) is a risk factor for MS and is present on immune cells.
- AFC-5128 is a CNS-penetrant small molecule inhibitor of P2X7R with potential for treating autoimmune diseases like MS.
Purpose of the Study:
- To evaluate the efficacy of AFC-5128 in a mouse model of multiple sclerosis.
- To investigate the mechanism of action of AFC-5128 on microglial activity and immune responses.
Main Methods:
- In vitro: Assessed calcium influx in primary murine microglia using Fluo-4 imaging.
- In vivo: Treated MOG35-55 immunized mice with AFC-5128 or fingolimod (FTY) in various treatment paradigms.
- Analyzed clinical scores, microglial markers, immune cell phenotyping, serum cytokines, and spinal cord histology.
Main Results:
- AFC-5128 significantly reduced calcium influx in microglia in vitro (p<0.0001).
- AFC-5128 treatment decreased overall EAE scores in acute and chronic models, with optimal effects at 200 mg/kg (p<0.0001).
- Peripheral immune cell populations and serum cytokines were largely unaffected; a trend towards reduced microglial phagocytosis markers was observed.
Conclusions:
- AFC-5128 demonstrates therapeutic potential by improving EAE outcomes and modulating microglial activity.
- The drug's ability to reduce microglial calcium influx suggests a mechanism for stabilizing calcium homeostasis.
- AFC-5128 shows promise for treating both relapsing and progressive forms of multiple sclerosis.
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