Ministry of Health Oswaldo Cruz Foundation Oswaldo Cruz

Ministry of Health Oswaldo Cruz Foundation Oswaldo Cruz Institute Cellular Comunication Laboratory Immunopharmacology Laboratory The P 2 X 7 R activation and its correlation with the inflammatory and nociceptive signaling: A promising target for the treatment of painful diseases. Rômulo José Soares Bezerra, Ph. D. E-mail: cyentysta@gmail. com

Purinergic Receptors P 1 Receptors (activated by adenosine) A 1, A 2 A, A 2 B, A 3 avelic & Burnstock, 1998. P 2 Receptors (activated by nucleotides) P 2 Y P 2 X G-coupled Ionotropic P 2 Y 1, P 2 Y 2, P 2 Y 4, P 2 Y 6, P 2 Y 11, P 2 Y 12, P 2 Y 13, P 2 Y 14 P 2 X 1, P 2 X 2, P 2 X 3, P 2 X 4, P 2 X 5, P 2 X 6, P 2 X 7

Expression of P 2 X 7 R in mammals Cells from CNS: -Microglia; -Astrocytes; Cells of the hematopoietic lineage: -Oligodendrocytes; -Monocytes; -Some neurons populations. -Macrophages; -Lymphocytes; -Mast cells; -Dendritic cells. Microglia Astrocytes Monocytes Oligodendrocytes Neurons Macrophages Lymphocytes Mast cells Dendritic cells Ravelic & Burnstock, 1998; Lister et al. , 2007; Coddou et al.

Activation of P 2 X 7 Receptor Na+ ATP ATP ATP Na+ Ca++ Na+ BE YP Na+ Ca++ ? ATP LY YP IP BE LY extracellular ATP intracellular IP 668, 39 Da BE 394, 31 Da LY 457, 24 Da YP 629 Da K+ K+ K+

Effects associated to P 2 X 7 R Inflammasome activation P 2 X 7 R activated Cell death ROS generation Caspases activation Transcription factors activation such NF-κB and proinflammatory genes transcription such COX-2 and i. NOS Cytokines secretion: IL-1β, IL-18 e TNF-α Phospholipases secretion: PLA 2 and PLD MAPK activation 1. Proinflammatory profile; 2. Pro-nociceptive. Lister et al. , 2007; Friedle et al. , 2010; Bartlett et al. , 2014; Rein & Torres, 2009; Riteau et al. , 2012; Latz et al. , 2013; Honore et al. , 2006; BarberàCremades et al. , 2012.

Diseases associated with P 2 X 7 R - Neurophatic pain Buchanan et al. , 2006. Important therapeutic target - Neurodegenerative diseases Friedle; Curet & Watters, 2010. - Rheumatoid arthritis - - Chronic inflammation - Burnstock et al. , 2009. Lister et. al. , 2007.

Activity from new natural compounds discovered by our research group • Our aim: Identify new compounds from natural proucts that may be candidates for potential antagonist to P 2 X 7 receptor.

Screening Method

Natural Products Library EXTRACTS Extracts with activity Active extracts P 1 A 2 A P 1 H 11 0 0 P 2 A 2 A P 2 H 11 1 8067 P 3 A 2 A P 3 H 11 0 0 P 4 A 2 A P 4 H 11 0 0 P 5 A 2 A P 5 H 11 2 8549 & 8568 P 6 A 2 A P 6 H 11 0 0 P 7 A 2 A P 7 H 11 0 0 P 8 A 2 A P 8 H 11 0 0 P 9 A 2 A P 9 H 11 0 0 P 10 A 2 A P 10 H 11 0 0 P 11 A 2 A P 11 H 11 0 0 P 12 A 2 A P 12 H 11 0 0 P 13 A 2 A P 13 H 11 0 0 P 14 A 2 A P 14 H 11 0 0 P 15 A 2 A P 15 H 11 0 0 P 16 A 2 A P 16 H 11 0 0 P 17 A 2 A P 17 H 11 0 0 P 18 A 2 A P 18 H 11 0 0 P 19 A 2 A P 19 H 11 0 0

Inhibitory Concentration (IC 50) obtained

Inhibitory Concentration (IC 50) obtained

Inhibitory Concentration (IC 50) obtained

Activity on the IL-1β Release

Activity on the Nitric Oxide Release

Activity on the Reactive Oxygen Species (ROS) release

Cytotoxicity

Pharmacologic Characterization

Nociception

Nociception

Next Steps

Synthetic Compounds Alves, L. A. et al. 2013

Natural Compounds

Thank You!

Inhibition of pain in vivo models using P 2 X 7 antagonists Pain attenuation in vivo models of neurophatic pain Labasi et al. , 2002; Honore et al. , 2006; Mc. Garaughty et Pain attenuation in vivo models of inflammatory pain

Inhibition of pain in vivo models using P 2 X 7 antagonists from natural products Rheedia longifolia Compound: Amentoflavone - P 2 X 7 • Model of writhing induced by acetic acid using the leaves of Rheedia longifolia Triana & Planch Santos, J. A. A et al. , 2010.

Inhibition of pain in vivo models using P 2 X 7 antagonists from natural products Number of indomethacin induced gastric lesions Model of neurogenic nociception induced by capsaicin Inflammatory nociception observed by von Frey model