Overview of Pfizer Research in Neuroinflammation

1. Overview of Pfizer Research in Neuroinflammation Zoë Hughes, Tarek Samad, Peter Cox, Jonathan Sporn, Rita Balice-Gordon NIMA-The Neuroimmunology Consortium London, 30th October 2015

2. Overview of Presentation- Mechanisms and Models Acute effects of LPS PDE4 inhibition LPS-induced encephalitis MAGL inhibition Neuroinflammation associated with amyloidosis GFAP-IL-6 overexpressing mouse JAK1 inhibition Impact on circadian rhythm CK1 inhibition

3. PDE4 Inhibition Attenuates LPS-induced Inflammation Joseph Hedde, Ashley Hanks, Radka Graf, Zoë Hughes

4. PDE4 Inhibition Inhibits Production of Cytokines • Activation of TLR4 causes upregulation of PDE4 leading to decreased cAMP • Inhibition of PDE4 increases cAMP leading to • Inhibition of p38 MAPK preventing activation induced pro-inflammatory gene transcription (IL-1b, IL-6, IL-8, TNFa) • Activation of PKA leading to activation of ICER which represses pro-inflammatory gene expression • Decreased cytokine release Modified from Yougbare et al 2013

5. Anti-inflammatory Effects of PDE4 Inhibition PDE4 Inhibitors Decrease LPS-Induced TNFα Release from Human PBMC PDE4 gene encodes proteins that specifically hydrolyze cAMP PDE4 is expressed in immune cells and is upregulated in inflammatory states PDE4-/- mice show reduced responses to inflammatory challenge PDE4 inhibitors are effective in preclinical models of inflammation and approved for severe COPD and psoriatic arthritis PDE4B-/- mice show reduced sensitivity to the behavioral effects of LPS PDE4B in human microglia is upregulated in response to IFNγ

6. Effects of Acute LPS in Mice-Modulation by PDE4 Inhibition Protein (MSD ELISA) Plasma Cytokines (TNFα, IL-6, IL-1β) 1 mRNA Protein (MSD ELISA) Brain Cytokines (TNFα, IL-6, IL-1β) 2 Modulation by PDE4 Inhibitors Ex vivo binding- TSPO ligand Microglial Activation Protein (Western Blot) 3 IHC (Iba1) Motivation (Progressive Ratio) Behaviour 4 Cognition (Touchscreen LD)

7. Co-administration of a PDE4 Inhibitor Attenuated LPS-Induced Increases in Cytokines 1 Plasma Takedown 4 h post LPS (1 mg/kg, ip) ± PDE4i 2 Brain Takedown 4 h post LPS (10 mg/kg, ip) ± PDE4i

8. Sub-chronic LPS Stimulation of Microglial Activation Measured by Ex Vivo Binding 3 IHC for Iba1 Literature report: LPS (1 mg/kg, ip) x 4 days LPS increase in [3H]DAA1106 Binding Blocked by PDE4i Ex vivo TSPO Binding LPS (1 mg/kg, ip) x 5 days ± co-administration of PDE4i * # 28% • Western Blot for TSPO • Antibody not specific

9. LPS Affects Behavior in Tests of Motivation and Cognition Progressive Ratio Responding Decreased by LPS and Restored by PDE4i 4 Motivation LPS (0.32 mg/kg, ip) decreases motivation 4h later ± PDE4i Prolonged Deficits in Cognitive Performance Mice Rats 4 Cognition Time-course of LPS effects on location discrimination touchscreen task Veh LPS

10. MAGL Inhibition Modulates Neuroinflammation Justin Piro & Tarek Samad

11. MAGL Inhibition: A Bi-directional Mechanism of Action CB1/2 Signaling DAG Lipase AEA 2-AG NAPE-PLD DAG NAPE MAGL FAAH X MAGL Glycerol Ethanolamine PLA2 Arachidonic acid P-Lipids CNS Specific Cyt P450 EET’s Cyclooxygenase Prostaglandins/Thromboxanes Lipoxygenase Leuokotrienes/Lipoxins MAGL Inhibition Spatiotemporally Enhances Endocannabinoid Signaling While Simultaneously Reducing Arachidonate Production

12. Inhibition of MAGL Regulates Endocannabinoid and Arachidonate Levels: PK/PD profile PK JZL184 16 mg/kg i.p. PD

13. MAGL Blockade Impairs LPS-induced Neuroinflammatory Response in Mouse Brain Endotoxin Induced Encephalitis Model T=0 Animals IP dosed with saline or LPS (20mg/kg) T=30 min Animals IP dosed with PEG300:Tween80=4:1 or JZL184 (16mg/kg) T=1 hr T=2 hr T=4 hr T=6 hr T=8 hr Animals Sac’d Brain is removed and rapidly frozen <30sec for metabolite and cytokine analysis

14. MAGL Inhibition Ameliorates Neuroinflammation Associated with Amyloidosis in PS1/APP Mice Piro et. al. Cell Reports 2012

15. MAGL Inhibition Reduces Pro-Inflammatory Cytokines in a CB1/CB2 Independent Manner in PS1/APP Mice Piro et. al. Cell Reports 2012

16. MAGL Disruption Reduces Levels of Inflammatory Cytokines in PS1/APP Mouse Brains mRNA Protein Piro et. al. Cell Reports 2012

17. JAK1 Inhibition in Neuroinflammation James Duerr, Peter Cox, Tarek Samad

18. JAK2 JAK1 JAK2 JAK2 STAT 1, 3, 5, 6 STAT 1, 3 STAT 4, 3 STAT 3, 5 JAK/STAT pathways central to cytokine signaling Cytokines signal through JAK/STAT combinations gc cytokines (IL-2, IL-4, IL-7, IL-9, IL-15, IL-21) EPO,TPO GM-CSF Type I IFNsIL-10 IL-6 IFNγ IL-23 IL-12 JAK1 TYK2 JAK3 JAK1 TYK2 JAK2 STAT 1, 2, 3, 4, 5 Six STAT members—regulate transcription Different STAT gene signatures Figure adapted from Riese RJ, et al. Best Pract Clin Res Rheumatol. 2010;24:5113-526.

19. JAK/STAT pathways in CNS inflammatory disorders Inflammatory cytokines Neuronal dysfunction associated w/ CNS inflammation Autoimmune Disorders with CNS component Neurodegenerative Disease associated w/ inflammation JAK1 STAT1,3,5 CNS Microglia astrocytes Gene Transcription (Immune response, Inflammation, Cell survival) Periphery Immune cells; cytokines • Brain-penetrant JAK inhibitors have potential to target multiple cell populations in the CNS: • Brain-resident immune cells producing/responding to pro-inflammatory cytokines (microglia, astrocytes). • Peripheral immune cells that have crossed the BBB (e.g., encephalitogenic T-cells and macrophages).

20. Human astrocyte cell lines responsive to multiple JAK-signaling cytokines and sensitive to JAK inhibitors Cytokine-induced pSTAT response in Human Astrocytoma Line CCF-STTG • JAK inhibitors effective at reducing basal and IL6-induced pSTAT3 levels. • IL6-stimulated astrocyte IC50s of JAK1 inhibitors comparable to those seen in Human whole blood assays (e.g., IFNa-stimulated pSTAT3, IL15-stimulated pSTAT5).

21. JAK1 activity correlates with IL-6 mediated STAT3 signaling in astrocytoma cell line Comparison with JAK1, JAK2, and TYK2 selective tools confirms dominant role of JAK1 in IL6 signaling in astrocytoma cell line. J. Duerr

22. Brain-penetrant inhibitor potently reduces central phospho-STAT3 levels GFAP-IL6 WT Non-BP JAK1i BP JAK1i VEH VEH • Brain-penetrant JAK1 inhibitor potently reduces phospho-STAT3 in a mouse transgenic model of neuroinflammation (central overexpression of IL6). • The effect is not seen with a non-brain penetrant compound with similar PK and peripheral potency.

23. Disrupted Circadian Rhythm and Immune Function- Role for CK1d/e Inhibition Travis Wager, Arthur Simen, Jonathan Sporn

24. Relationship between Dysregulation of Circadian Rhythm and Inflammatory Disorders • Some MDD patients have dysregulated circadian rhythms • Emerging data suggest that immune responses are dependent on time of day • The CK1d/e Inhibitor, PF-670462, entrains animals with desynchronized activity Vipr 2 -/- mice Healthy :Depression Vipr 2 -/- mice show weak to no synchrony in constant dark conditions. This asynchrony is reversed by PF-670462 Constant light Wild type mice kept in constant light conditions show asynchrony. This asynchrony is reversed by PF-670462 1. Proc Natl Acad Sci USA 2010,107, 15240-15245.

25. Evaluation of a Range of Mechanisms in a Range of Models Acute effects of LPS PDE4 inhibition LPS-induced encephalitis MAGL inhibition Neuroinflammation associated with amyloidosis GFAP-IL-6 overexpressing mouse JAK1 inhibition Impact on circadian rhythm CK1 inhibition

26. Questions?