End-to-end capabilities to assess Neuroinflammation

Acute inflammation – induced post injury like traumatic brain injury, spinal cord injury or stroke • Star ts 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 multiple sclerosis (MS)• Long-term inflammation can last for months or years • Symptoms include fatigue, abdominal pain, fever, rash, joint pain

Primary human and rodent cellsMurine glia-neuron co-culturesMurine primary microglial cultures

Rodent models of inflammation LPS inducedDelayed type hypersensitivityExperimental autoimmune uveitisNeuropathic pain

Peripheral nervous systemCentral nervous system

Hippocampal slicesBrain slices and sectionsHuman microglia from post-mortem brains

In vitro assays and models Ex vivo assays In vivo assays

and modelsBiomarker

identification

Assays • Microglia/astroglia activation • Pro- and anti-inflammatory • cytokines expression• Cell phenotyping

Imaging • PET imaging with TSPO ligand• PET imaging with Arterial Input Function• SPECT imaging• MRI• IVIS

Assays • Microdialysis• MSD assays• Luminex• Quanterix Simoa• Erenna Singulex

Assays • Microglia/astroglia activation • Hippocampal slice activation• Macrophage activation • Pro- and anti-inflammatory cytokines expression

Pre-clinical nuclear imaging provides a translational approach to monitor progression of inflammation in vivo.Neuroinflammation is associated with progression of various neurodegenerative diseases, including multiple sclerosis,Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and stroke. Activation of the mitochondrial translocatorprotein (TSPO) and changes in metabolic activity are linked to neuroinflammation and TSPO ligands can be used for in vivoPET imaging

Neuroinflammation was studied in cuprizone mouse model of multiple sclerosis. C57Bl/6 female mice were given cuprizone(0.3% w/w) in their diet or regular powdered diet. Exposure lasted 6 weeks after which the cuprizone supplementation of thediet was discontinued. SPECT/CT imaging was performed on week 7. Significant (p<0.05) increase in TSPO ligand (123I-CLINDE) binding to various brain regions was quantified from induced mice compared to naive (Figure 3).

MULTIPLE SCLEROSISINTRODUCTION

TSPO expression in the brain is associated with activation of microglia, therefore TSPO is potential target to evaluateneuroinflammatory changes in a variety of CNS disorders. However, there is always a baseline expression present whichprohibits the use of reference tissue models in dynamic PET imaging. To reach optimal readouts from the imaging andunderstand PET tracer kinetics, metabolite corrected arterial input function (AIF) has to be collected from the imaged animal.LPS i k t t i ti l t hi h d t i d t l l i fl ti i t LPS i f i

ARTERIAL INPUT FUNCTION FROM RODENTS

PET imaging. A B

LPS is a known potent immunostimulant, which was used to induce acute local neuroinflammation in rats. LPS infusion wasdone to right striatum and rats were scanned with TSPO ligand 18F-FEPPA. To obtain blood input function, the tail artery andvein were cannulated, connected to a coincidence counter and a peristaltic pump to maintain constant flow rate. Animalswere scanned for 90 minutes and samples for plasma and parent fraction were collected during the scan (Figures 1 and 2).

D1

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

Naive Cuprizone

D8

D15

CNeuritis model was used to study perineural inflammation and neuropathic pain both in mice and rats. Induction was doneusing modified Complete Freund's adjuvant in Oxygel band wrapped around sciatic nerve. Animals did not exhibit mutilationof limbs or demonstrate moribund behavior, or behavior that would otherwise indicate severe spontaneous pain (shaking orlicking of paws) during the study.Rats were imaged with FDG-PET 6 days post surgical operation. Rats were scanned using FDG-PET on days 7 and 21 aswell with 123I-CLINDE on day 4

NEUROPATHIC  PAIN

PET images 1, 8 and 15 days post LPS infusion with 18F-FEPPA (A). Metabolite corrected AIF was generated for each scan. Higher traceraccumulation was seen to the injection site. Radioactivity distribution between whole blood and plasma during the PET scan (B). Fraction ofunmetabolized 18F-FEPPA in plasma during the PET scan (C). Plasma and parent fraction curves are required for quantitative PET analysis oftracer without reference region.

VEHICLE MPTPA

BA

VT-values of 18F-FEPPA in nucleus accumbens, striatum and thalamus of both hemispheres. Highest values were seen in right striatum. VT is lowon 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

MURINE MODEL OF NEUROINFLAMMATIONDrug Efficacy

8 EVERY STEP OF THE WAY

NOVEL BIOMARKER IDENTIFICATIONIdentification and validation of biomarkers and the development of quantitative assays in primary cells and disease tissue, 

pharmacodynamic and disease models and as efficacy and translational medicine markers in the clinicpharmacodynamic and disease models, and as efficacy and translational medicine markers in the clinic

MesoScale, Luminex or

Proteomic markers

Genomic markers

MesoScale, Luminex or Quanterix for multiplexed

immunoassysFACS

analysis

Epigenetic markers

Readouts Assay Technologies

Phosphoprotein markers Mass spectrometry

9 EVERY STEP OF THE WAY

NEUROINFLAMMATORY MODELSExperimental autoimmune uveitis (mouse and rat models

7 EVERY STEP OF THE WAY

CVN MOUSE (APPSweDI/NOS2-/-)Example data

Inflammatory Cells and Amyloid PlaquesActivated microglia or monocyte derived cells (CD45‐antibody) colocalize with amyloid plaques (WO‐2 antibody) in 12‐month old CVN mouse brains. 

3 EVERY STEP OF THE WAY

HIPPOCAMPAL ACTIVATION

4 EVERY STEP OF THE WAY

MICROGLIAL ACTIVATIONExample Data

1 EVERY STEP OF THE WAYMURINE PRIMARY MICROGLIAL CELL CULTURES

2 EVERY STEP OF THE WAY

Measurement of cytokine release after LPS activation using multiplex assays

Flow cytometric characterization of cell populations and surface markers

Activated microglia or monocyte derived cells (CD45-antibody) colocalize with amyloid plaques (WO-2 antibody) in 12-month CVN mouse brains

Increased neuronal damage in hippocampus caused by exposure to amyloid b

PET images post LPS infusion with 18F-FEPPA (A). Metabolite corrected AIF foreach scan. Higher tracer accumulation was seen to the injection site. (B) Fraction of unmetabolized 18F-FEPPA in plasma during the PET scan(C) Plasma and parent fraction curves for quantitative PET analysis withoutreference region

Ocular imaging of uveitis modelPre-clinical nuclear imaging provides a translational approach to monitor progression of inflammation in vivo.Neuroinflammation is associated with progression of various neurodegenerative diseases, including multiple sclerosis,Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and stroke. Activation of the mitochondrial translocatorprotein (TSPO) and changes in metabolic activity are linked to neuroinflammation and TSPO ligands can be used for in vivoPET imaging

Neuroinflammation was studied in cuprizone mouse model of multiple sclerosis. C57Bl/6 female mice were given cuprizone(0.3% w/w) in their diet or regular powdered diet. Exposure lasted 6 weeks after which the cuprizone supplementation of thediet was discontinued. SPECT/CT imaging was performed on week 7. Significant (p<0.05) increase in TSPO ligand (123I-CLINDE) binding to various brain regions was quantified from induced mice compared to naive (Figure 3).

MULTIPLE SCLEROSISINTRODUCTION

TSPO expression in the brain is associated with activation of microglia, therefore TSPO is potential target to evaluateneuroinflammatory changes in a variety of CNS disorders. However, there is always a baseline expression present whichprohibits the use of reference tissue models in dynamic PET imaging. To reach optimal readouts from the imaging andunderstand PET tracer kinetics, metabolite corrected arterial input function (AIF) has to be collected from the imaged animal.LPS i k t t i ti l t hi h d t i d t l l i fl ti i t LPS i f i

ARTERIAL INPUT FUNCTION FROM RODENTS

PET imaging. A B

LPS is a known potent immunostimulant, which was used to induce acute local neuroinflammation in rats. LPS infusion wasdone to right striatum and rats were scanned with TSPO ligand 18F-FEPPA. To obtain blood input function, the tail artery andvein were cannulated, connected to a coincidence counter and a peristaltic pump to maintain constant flow rate. Animalswere scanned for 90 minutes and samples for plasma and parent fraction were collected during the scan (Figures 1 and 2).

D1

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

Naive Cuprizone

D8

D15

CNeuritis model was used to study perineural inflammation and neuropathic pain both in mice and rats. Induction was doneusing modified Complete Freund's adjuvant in Oxygel band wrapped around sciatic nerve. Animals did not exhibit mutilationof limbs or demonstrate moribund behavior, or behavior that would otherwise indicate severe spontaneous pain (shaking orlicking of paws) during the study.Rats were imaged with FDG-PET 6 days post surgical operation. Rats were scanned using FDG-PET on days 7 and 21 aswell with 123I-CLINDE on day 4

NEUROPATHIC  PAIN

PET images 1, 8 and 15 days post LPS infusion with 18F-FEPPA (A). Metabolite corrected AIF was generated for each scan. Higher traceraccumulation was seen to the injection site. Radioactivity distribution between whole blood and plasma during the PET scan (B). Fraction ofunmetabolized 18F-FEPPA in plasma during the PET scan (C). Plasma and parent fraction curves are required for quantitative PET analysis oftracer without reference region.

VEHICLE MPTPA

BA

VT-values of 18F-FEPPA in nucleus accumbens, striatum and thalamus of both hemispheres. Highest values were seen in right striatum. VT is lowon 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

SPECT/CT images in naive andcuprizone induced mouse incoronal, horizontal and sagittalview

Available biomarker readouts

Multiplex analysis of decreased TNF-alphalevels with dexamethasone treatment in a LPSinduced neuroinflammation mouse model