Multidisciplinary Approach for Mood Disorders & Alzheimer's

1. Day 1 1-2:30pm - MDWP1 • Introduction (10 min): What we said we would do in the application • Why the two paradigms (LPS and BCG) were chosen • What we hope the behavioural experiments will show • Process for choosing molecules • What we have actually done and what next? • LPS model – Diana • BCG model – Jason/Niels • Additional info (Jon)?

2. Workplansand timeline (from interview slides April 2014) Complementary expertise and assets - academic strengths in translational research and industrial strengths in drug development – combined in a multidisciplinary consortium Part 2 - Mood Disorders Therapeutic PoC trial of an anti-inflammatory in immuno-stratified TRD patients. Part 1 - Mood Disorders MD WP1 – Inflammatory preclinical models MD WP2 – “Immunomarkers” in Treatment Resistant Depression (TRD) Milestone Review Part 2 - Alzheimer’s Disease Experimental Medicine study of an anti-inflammatory in early AD. Part 1 - Alzheimer’s Disease AD WP1 – Clinical Informatics AD WP2 – Animal models AD WP3 – Hypothesis-driven immune system biomarkers • Technical Platforms: • TP1 - Peripheral cytometry, cell sorting, transcriptomics • TP2 – Proteomics in blood and CSF • TP3 – PET imaging of brain inflammation • TP4 – MRI of brain structure and function 0 1 2 3 4 5 Years

3. Mood Disorder Workplans(from interview slides April 2014) MD WP2 Peripheral Immunomarker validation in TRD MD WP1 Inflammatory models in rodents • Biomarkers • immunophenotyping • Gene expression • Behaviour • CSF • sMRI, fMRI • Microglia studies • Autoradiography • Histology • Site of drug action • central vsperipheral • Selection of drugs • Effects on mood/inflammation • Primary Cohort • 80 TRD patients and 80 controls • Clinical and cognitive profile • Immunophenoptype • sMRI and fMRI • Secondary Cohort • 20 TRD patients and 20 controls • TSPO PET imaging • CSF sampling Immunopsychiatry May 2014-16 • Output to enable Part 2 clinical therapeutic PoC trial: • Understand the links between peripheral and CNS inflammation, brain function, and behaviour • Identify appropriate biomarkers to choose the right patients • Determine most appropriate drug s for clinical evaluation

4. Objectives of MD Workpackages • Understand the links between peripheral and CNS inflammation, brain function, and behaviour • Identify appropriate biomarkers to choose the right patients • Determine most appropriate drugs for clinical evaluation

5. MDWP1 – Why these 2 preclinical models? From the application (technical appendix): • We will use two inflammatory models of depression-like behaviour in animals …….These studies will be designed to achieve three objectives: • To improve understanding of mechanistic links between peripheral inflammatory challenges, central inflammatory states, brain function, and depressive behaviours. • To test a range of pharmacological tool compounds, with different MOA & different levels of brain penetration, for efficacy on behavioural endpoints and candidate biomarkers • To cross-validate neuroimaging markers of central inflammatory states by immunohistological and transcriptomic measures of brain tissue (LPS challenge studies only).

6. MDWP1 – from application • Assumptions: • LPS was the “work horse” to translate/bridge inflammatory biomarkers, more rapidly screen drugs for “pharmacodynamic” effects in CNS etc • CNS vs systemic site of action? • BCG model – evaluate link between drug effects upon inflammation and “mood”

7. MDWP1 - Process for choosing molecules • A lot of calls!! • Will tally up for SAB etc • Discussions followed these criteria: • Likelihood of a clinical asset for Stage 2? • Appropriate tool molecule for rodent studies (and data package to allow exp design). • Not necessarily the clinical asset • Appropriate scientific rationale • Publishable

8. Compounds from Janssen & Lundbeck# (from interview slides April 2014) *existing clinical stage assets, other reviewed as they progress to clinical stage; #Subject to prior internal company diligence

9. Targeting P2X7 and inflammation in mood (from interview slides April 2014) Anhedonia ? ATP Proinflammatory Cytokines Chronic Stress Despair Clinical Depression P2X7 Neurogenesis • P2X7 is an ATP gated ion channel that mediates IL-1brelease • Located on monocytic-lineage cells, including microglia and astrocyes • Plasma IL-1b is elevated in in animal stress models and in MDD and Bipolar patients (Jones & Thomsen, 2013) • P2X7 antagonists may have an antidepressant effect • “Gain of function” polymorphism assoc’d with mood disorders (Halmai et al., 2013) • P2X7 KO mice are stress resilient • P2X7 antagonist have antidepressant activity in preclinical models • Opportunity to fully characterise P2X7 antagonist in preclinical models of CNS inflammation and potentially in depressed patients

10. Backups

11. MDWP1 – detailed questions (Application Appendix) • Do anti-inflammatory drugs alleviate depressive-like behaviours in rodent models indicative of potential antidepressant efficacy in a sub-group of depressed patients with peripheral inflammation? • Can we attenuate depressive symptoms by administration of anti-cytokine antibodies that reduce peripheral concentrations of pro-inflammatory cytokines but do not cross the BBB? • Is there additional anti-depressant benefit from administration of molecules that can cross the BBB and engage targets in the brain? This question could be tested, for example, by a comparison of drugs that engage the same target but have different degrees of brain penetrance (e.g., peripheral vs nasal anti-TNF centyrin).. • Do drugs that putatively target microglial activation (e.g., P2X7 inhibitors such as JNJ-47965567 or Lu AF27139; or a KCNN4 antagonist such as Lu AF55813) have effects on imaging and CSF markers that are compatible with that mechanism of action? Do microglial-targeting drugs confer additional anti-depressant benefits? • Can we demonstrate that drugs such as chemokine antagonists that block extravasation of activated immune cells across the BBB from the periphery have anti-depressant effects, perhaps especially in the later stages of depressive response to BCG challenge?

12. MDWP1 – from application