End-to-end capabilities to assess Neuroinflammation

Acute – induced post injury like traumatic injury, injury or stroke • Starts rapidly • Becomes severe in a short timeframe • Symptoms include pain, redness, immobility, swelling, heat

Chronic inflammation – neurodegenerative diseases including Alzheimer’s (AD) , Parkinson’s (PD), Huntington’s (HD) and (MS) • Long-term inflammation can last for months or years • Symptoms include , abdominal pain, fever, rash, joint pain

Rodent models of inflammation LPS induced Primary human and rodent cells Hippocampal slices Delayed type hypersensitivity Murine glia-neuron co-cultures Brain slices and sections Experimental autoimmune uveitis Peripheral nervous system Murine primary microglial cultures Human from post-mortem Neuropathic pain

INTRODUCTION MULTIPLE SCLEROSIS

Pre-clinical nuclear imaging provides a translational approach to monitor progression of inflammation in vivo. Neuroinflammation was studied in cuprizone mouse model of multiple sclerosis. C57Bl/6 female mice were given cuprizone Neuroinflammation is associated with progression of various neurodegenerative diseases, including multiple sclerosis, (0.3% w/w) in their diet or regular powdered diet. Exposure lasted 6 weeks after which the cuprizone supplementation of the Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and stroke. Activation of the mitochondrial translocator diet was discontinued. SPECT/CT imaging was performed on week 7. Significant (p<0.05) increase in TSPO ligand (123I- protein (TSPO) and changes in metabolic activity are linked to neuroinflammation and TSPO ligands can be used for in vivo CLINDE) binding to various brain regions was quantified from induced mice compared to naive (Figure 3). PET imaging. A B

ARTERIAL INPUT FUNCTION FROM RODENTS MURINE MODEL OF NEUROINFLAMMATION In vitro assays TSPO expressionIn in the brain isvivo associated with activation assays of microglia, therefore TSPO is potential target to evaluate BiomarkerDrug Efficacy neuroinflammatory changes in a variety of CNS disorders. However, there is always a baseline expression present which prohibits the use of reference tissue models in dynamic PET imaging. To reach optimal readouts from the imaging and ExCVN vivo MOUSE (APPSweDI/NOS2-/-) assays understand PET tracer kinetics, metabolite corrected arterial input function (AIF) has to be collected from the imaged animal. Example data MICROGLIAL ACTIVATION LPS isaknown potttent immunos timu lan t,whic h was used to idinduce acute lllocal neuroin flamma tion in rats. LPS ifinfus ion was and models done to right striatum andand rats were scanned with TSPOmodels ligand 18F-FEPPA. To obtain blood input function, the tail artery and identification Example Data vein were cannulated, connected to a coincidence counter and a peristaltic pump to maintain constant flow rate. Animals were scanned for 90 minutes and samples for plasma and parent fraction were collected during the scan (Figures 1 and 2). Naive Cuprizone

A B Example SPECT/CT images in naive and cuprizone induced mouse in coronal, horizontal and sagittal view (A). Significant (p<0.05) increase in accumulation of 123I-CLINDE was quantified in all studied brain regions. Image analysis results shown as mean +SD. Quantification was done D1 using PMOD.

NEUROPATHIC PAIN

Neuritis model was used to study perineural inflammation and neuropathic pain both in mice and rats. Induction was done Inflammatory Cells and Amyloid PlaquesHIPPOCAMPAL ACTIVATION D8 C Activated microglia or monocyte derived cells (CD45‐) colocalize with amyloid plaques (WO‐2 using modified Complete Freund's adjuvant in Oxygel band wrapped around sciatic nerve. Animals did not exhibit mutilation Activatedantibody) in microglia 12‐month oldor monocyteCVN mouse brains.derived cells (CD45-antibody) colocalize with amyloid of limbs or demonstrate moribund behavior, or behavior that wouldNOVEL otherwise indicate severe spontaneousBIOMARKER pain (shaking or IDENTIFICATION plaques (WO-2 antibody) in 12-month CVN mouse brains licking of paws) during the study. Rats were imaged withIdentification FDG-PET 6 days post surgicaland operation.validation Rats were of scanned biomarkers using FDG-PET and on days the 7 and development 21 as of quantitative assays in primary cells and disease tissue, 123 D15 well with I-CLINDE on day 4 pharmacodynamic and disease models, and as efficacy and translational medicine markers in the clinic

3 EVERY STEP OF THE WAY

NEUROINFLAMMATORYA MODELS VEHICLE MPTP Multiplex analysis of decreased TNF-alpha Measurement of release after LPS activation using multiplex assays 8 EVERY STEP OF THE WAY levels with dexamethasone treatment in a LPS 18 1 MURINEEVERY STEP OF THE WAYPRIMARY MICROGLIAL CELL CULTURES ExperimentalPET images 1, 8 and autoimmune 15 days post LPS infusion with F-FEPPA uveitis (A). Metabolite (mouse corrected AIF was and generated forrat each scan.models Higher tracer induced neuroinflammation mouse model accumulationPET images was seen post to the LPS injection infusion site. Radioactivity with distribution 18F-FEPPA between whole (A). blood Metabolite and plasma during corrected the PET scan AIF (B). for Fraction of unmetabolizedeach scan.18F-FEPPA Higher in plasma tracer during accumulation the PET scan (C). Plasma was and parentseen fraction to the curves injection are required site. for quantitative PET analysis of MesoScale, Luminex or tracer(B) without Fraction reference of region. unmetabolized 18F-FEPPA in plasma during the PET scan (C) Plasma and parent fraction curves for quantitative PET analysis without Quanterix for multiplexed reference region A B Genomic immunoassys Proteomic markers FACS markers analysis

Epigenetic Readouts Assay Technologies markers Flow cytometric characterization of cell populations and surface markers 18 VT-values of F-FEPPA in nucleus accumbens, striatum and thalamus of both hemispheres. Highest values were seen in right striatum. VT is low Increased neuronal damage in caused by exposure to amyloid b on D1, increased thereafter and remained elevated between D8 and D15. Individual data points are plotted in the graphs with mean ±SD (A). 4 EVERY STEP OF THE WAY Tissue/Input curve ratio from right striatum shows equilibration of activity in tissue and plasma (B). Phosphoprotein markers Mass spectrometry INTRODUCTION MULTIPLE SCLEROSIS

Pre-clinical nuclear imaging provides a translational approach to monitor progression of inflammation in vivo. Neuroinflammation was studied in cuprizone mouse model of multiple sclerosis. C57Bl/6 female mice were given cuprizone Available biomarker readouts Neuroinflammation is associated with progression of various neurodegenerative diseases, including multiple sclerosis, (0.3% w/w) in their diet orOcular regular powdered imaging diet. of Exposure uveitis lasted model 6 weeks after which the cuprizone supplementation of the Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and stroke. Activation of the mitochondrial translocator diet was discontinued. SPECT/CT imaging was performed on week 7. Significant (p<0.05) increase in TSPO ligand (123I- 2 EVERY STEP OF THE WAY protein (TSPO) and changes in metabolic activity are linked to neuroinflammation and TSPO ligands can be used for in vivo CLINDE) binding to various brain regions was quantified from induced mice compared to naive (Figure 3). PET imaging. A B

ARTERIAL INPUT FUNCTION FROM RODENTS 9 EVERY STEP OF THE WAY

TSPO expression in the brain is associated with activation of microglia, therefore TSPO is potential target to evaluate SPECT/CT images in naive and neuroinflammatory changes in a variety of CNS disorders. However, there is always a baseline expression present which cuprizone induced mouse in EVERY STEP OF THE WAY 1 prohibits the use of reference tissue models in dynamic PET imaging. To reach optimal readouts from the imaging and coronal, horizontal and sagittal understand PET tracer kinetics, metabolite corrected arterial input function (AIF) has to be collected from the imaged animal. view LPS isaknown potttent immunos timu lan t,whic h was used to idinduce acute lllocal neuroin flamma tion in rats. LPS ifinfus ion was done to right striatum and rats were scanned with TSPO ligand 18F-FEPPA. To obtain blood input function, the tail artery and vein were cannulated, connected to a coincidence counter7 and a peristaltic pump to maintain constantEVERY flow STEP rate. OF Animals THE WAY were scanned for 90 minutes and samples for plasma and parent fraction were collected during the scan (Figures 1 and 2). Naive Cuprizone

A B Example SPECT/CT images in naive and cuprizone induced mouse in coronal, horizontal and sagittal view (A). Significant (p<0.05) increase in accumulation of 123I-CLINDE was quantified in all studied brain regions. Image analysis results shown as mean +SD. Quantification was done D1 using PMOD.

NEUROPATHIC PAIN

Neuritis model was used to study perineural inflammation and neuropathic pain both in mice and rats. Induction was done D8 Assays Assays C Imagingusing modified Complete Freund's adjuvant in Oxygel band wrapped around sciatic nerve. Animals did not exhibit mutilation Assays of limbs or demonstrate moribund behavior, or behavior that would otherwise indicate severe spontaneous pain (shaking or • Microglia/astroglia activation • Microglia/astroglia activation • PETlicking imaging of paws) during the study. with TSPO ligand • Microdialysis Rats were imaged with FDG-PET 6 days post surgical operation. Rats were scanned using FDG-PET on days 7 and 21 as well with 123I-CLINDE on day 4 • Pro- and anti-inflammatory • HippocampalD15 slice activation • PET imaging with Arterial Input Function • MSD assays

• expression • MacrophageA activation • Luminex VEHICLE MPTP • SPECT imaging

• Cell phenotyping • Pro- andPET images anti-inflammatory 1, 8 and 15 days post LPS infusion with 18F-FEPPA (A).cytokines Metabolite corrected AIF was generated for each scan. Higher tracer • Quanterix Simoa accumulation was seen to the injection site. Radioactivity distribution between whole blood and plasma during the PET scan (B). Fraction of • MRI expressionunmetabolized 18F-FEPPA in plasma during the PET scan (C). Plasma and parent fraction curves are required for quantitative PET analysis of tracer without reference region. • IVIS • Erenna Singulex

A B

18 VT-values of F-FEPPA in nucleus accumbens, striatum and thalamus of both hemispheres. Highest values were seen in right striatum. VT is low on D1, increased thereafter and remained elevated between D8 and D15. Individual data points are plotted in the graphs with mean ±SD (A). Tissue/Input curve ratio from right striatum shows equilibration of activity in tissue and plasma (B).

EVERY STEP OF THE WAY 1