The potent and selective adenosine A2AR antagonists P400 and P625 protect against symptoms in autoimmune experimental encephalomyelitis by attenuating neuroinflammation and demyelination

M Morozzi; V Borgonetti; C Sasia; G Videtta; S Calenda; D Catarzi; F Varano; V Colotta; N Galeotti

doi:10.1016/j.neuropharm.2026.110857

The potent and selective adenosine A_2AR antagonists P400 and P625 protect against symptoms in autoimmune experimental encephalomyelitis by attenuating neuroinflammation and demyelination

Neuropharmacology. 2026 May 1:288:110857. doi: 10.1016/j.neuropharm.2026.110857. Epub 2026 Jan 27.

Authors

M Morozzi¹, V Borgonetti², C Sasia³, G Videtta⁴, S Calenda⁵, D Catarzi⁶, F Varano⁷, V Colotta⁸, N Galeotti⁹

Affiliations

¹ Department of Neurosciences, Psychology, Drug Research and Child Health (Neurofarba), Section of Pharmacology and Toxicology, University of Florence, Viale G. Pieraccini 6, 50139, Florence, Italy. Electronic address: martina.morozzi@unifi.it.
² Department of Neurosciences, Psychology, Drug Research and Child Health (Neurofarba), Section of Pharmacology and Toxicology, University of Florence, Viale G. Pieraccini 6, 50139, Florence, Italy. Electronic address: vittoria.borgonetti@unifi.it.
³ Department of Neurosciences, Psychology, Drug Research and Child Health (Neurofarba), Section of Pharmacology and Toxicology, University of Florence, Viale G. Pieraccini 6, 50139, Florence, Italy. Electronic address: chiara.sasia@unifi.it.
⁴ Department of Neurosciences, Psychology, Drug Research and Child Health (Neurofarba), Section of Pharmacology and Toxicology, University of Florence, Viale G. Pieraccini 6, 50139, Florence, Italy. Electronic address: giacomina.videtta@unifi.it.
⁵ Department of Neurosciences, Psychology, Drug Research and Child Health (Neurofarba), Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Via Ugo Schiff, 6, Sesto Fiorentino, 50019, Italy. Electronic address: sara.calenda@unifi.it.
⁶ Department of Neurosciences, Psychology, Drug Research and Child Health (Neurofarba), Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Via Ugo Schiff, 6, Sesto Fiorentino, 50019, Italy. Electronic address: daniela.catarzi@unifi.it.
⁷ Department of Neurosciences, Psychology, Drug Research and Child Health (Neurofarba), Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Via Ugo Schiff, 6, Sesto Fiorentino, 50019, Italy. Electronic address: flavia.varano@unifi.it.
⁸ Department of Neurosciences, Psychology, Drug Research and Child Health (Neurofarba), Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Via Ugo Schiff, 6, Sesto Fiorentino, 50019, Italy. Electronic address: vittoria.colotta@unifi.it.
⁹ Department of Neurosciences, Psychology, Drug Research and Child Health (Neurofarba), Section of Pharmacology and Toxicology, University of Florence, Viale G. Pieraccini 6, 50139, Florence, Italy. Electronic address: nicoletta.galeotti@unifi.it.

PMID: 41610910
DOI: 10.1016/j.neuropharm.2026.110857

Abstract

Multiple sclerosis (MS) is an autoimmune chronic inflammatory disease of the central nervous system characterized by oxidative stress, demyelination, and neuronal damage. Current MS therapies are unsatisfactory and new therapies are encouraged. Adenosine is highly implicated in MS as it regulates, via activation of its A_2A receptor (A_2AR), the inflammatory and immune response. The aim of this study is to investigate the therapeutic potential of new selective A_2AR antagonists, P400 and P625, in the experimental autoimmune encephalomyelitis (EAE), the principal animal model of MS. P400 and P625 administration (10 μg/mouse intranasal) for 14 days reduced motor disability (clinical score, rotarod) and thermal (hot plate) and mechanical (von Frey) hypernociceptive symptoms associated with a chronic MS model. Quantitative analysis of lymphocytes infiltration in the spinal cord sections stained with hematoxylin and eosin (H&E) showed a larger number of inflammatory cells in EAE sections that were markedly reduced by P400 and P625. P400 also reduced the immunostaining of Iba1, marker of microglia. Luxol Fast Blue (LFB) staining and myelin basic protein (MBP) immunostaining of spinal cord sections showed a robust loss of myelin that was partially restored by P400 and P625. Both treatments increased spinal neurofilament H (NfH) and GAP43 protein expression compared to untreated immunize mice. These data illustrate the efficacy of the new selective A_2AR antagonists in ameliorating EAE symptoms by attenuating neuroinflammation and demyelination. These findings further highlight A_2AR blocking as a promising perspective to control neurological disturbances in MS patients.

Keywords: A(2A)R antagonists; Adenosine; Demyelination; EAE symptoms; Multiple sclerosis; Neuroinflammation.

MeSH terms

Adenosine A2 Receptor Antagonists* / pharmacology
Adenosine A2 Receptor Antagonists* / therapeutic use
Animals
Calcium-Binding Proteins
Demyelinating Diseases* / drug therapy
Demyelinating Diseases* / pathology
Encephalomyelitis, Autoimmune, Experimental* / drug therapy
Encephalomyelitis, Autoimmune, Experimental* / pathology
Female
Mice
Mice, Inbred C57BL
Microfilament Proteins / metabolism
Microglia / drug effects
Microglia / pathology
Myelin Basic Protein / metabolism
Neuroinflammatory Diseases* / drug therapy
Neuroinflammatory Diseases* / pathology
Peptide Fragments
Receptor, Adenosine A2A / metabolism
Spinal Cord / drug effects
Spinal Cord / metabolism
Spinal Cord / pathology

Substances

Adenosine A2 Receptor Antagonists
Microfilament Proteins
Aif1 protein, mouse
Receptor, Adenosine A2A
Peptide Fragments
Myelin Basic Protein
Calcium-Binding Proteins