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Christine Parker GSK, Experimental Medicine Imaging

TSPO Enabling Studies to Assist Technical Platform 3 (TP3). Christine Parker GSK, Experimental Medicine Imaging Federico Turkheimer , Camilla Simmons, Marta Vicente Rodriguez and Diana Cash KCL 1 March 2017. Aim.

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Christine Parker GSK, Experimental Medicine Imaging

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  1. TSPO Enabling Studies to Assist Technical Platform 3 (TP3) Christine Parker GSK, Experimental Medicine Imaging Federico Turkheimer , Camilla Simmons, Marta Vicente Rodriguez and Diana Cash KCL 1 March 2017

  2. Aim To design a series of enabling studies, that allows for interrogation of TSPO PET ligand binding characteristics to different cell types in the neuro-inflammatory pathway, under both basal and diseased conditions, which will aid interpretation of PET data with these radioligands in clinical studies of Alzheimer’s Disease and Mood Disorders

  3. Proposed Initial TSPO Enabling Study Brain In vivo PET [11C]PK11195 and/or 2nd generation TSPO ± Inflammatory stimulus Ex vivo and subsequent in vitro Autoradiography on same sections [11C]/[3H]PK11195 and 2nd generation TSPO In vitro IHC/staining on adjacent tissue sections e.g. for activated microglia, Macrophages and astrocytes Ex vivo Dissection [11C]PK11195 and/or 2nd generation TSPO Correlation with ex vivo autoradiography from identical regions Correlation with in vitro autoradiography from identical regions Correlation with in vivo PET data from identical regions Correlation with ex vivo dissection data from identical regions Correlation with in vivo PET data from identical regions Deeper understanding of cell types bound by TSPO PET ligands

  4. Three-Stage Approach • Stage 1 • Demonstrate utility of the paradigm along with methods capability in WT animals ± inflammatory challenge (e.g. LPS) • Stage 2 • If Stage 1 is successful, repeat enabling study with animal models of disease being utilised in AD WP2 and MD WP1. Potential to demonstrate: • Regional CNS localisation of TSPO signal (and hence comparison between animal models and WT data (Stage 1)) • Involvement of specific cell phenotypes • Stage 3 • Utilise these enabling methods in drug intervention studies with animals in AD WP2 and MD WP1. Potential ability to demonstrate: • Alteration in TSPO signal change following drug treatment (e.g. P2X7 antagonist (JNJ54175446); CSF1R antagonist(JNJ40346527)) • Cellular phenotypic changes associated with drug treatment • Longitudinal effects (over multiple scanning sessions)

  5. Contribution to the Clinical Study PART 1 – TSPO • Increase our understanding of cell types contributing to the TSPO PET signal under both basal and diseased conditions, and how these may be altered with drug intervention • More accurate kinetic analysis and interpretation of TSPO PET data in clinical studies of depression (taking both changes in blood volume/flow and tissue density into account)

  6. Current Status • Study centre = KCL • Embedded post-doctoral researcher aligned to this enabling study now in place (as part of GSK’s in-kind contribution) • Marta Vicente Rodriguez • Stage 1 of the enabling study due to initiate mid/end April 2017 • Once Home office licence is in place • Between now and mid/end April assisting in MDWP1 data capture and analysis / familiarisation on techniques to be utilised in the TSPO enabling study • E.g. MicroPET study, radiation use, in vitro techniques etc...

  7. Resource and Operational Requirements • Scientific input required from a number of different scientific fields -  • e.g. IHC, autoradiography, in vivo, animal models, relevant WTs, relevant inflammatory stimulus etc... • Access to relevant laboratories and specific equipment -  • e.g. Preclinical PET camera, autoradiography, IHC set up etc... • Infra-structure/set-up across study site(s) -  • PET and tracer ex vivo studies at St Thomas’s Hospital, KCL • Long-lived ex vivo and in vitro studies at the James Black Centre, Institute of Psychiatry, Psychology and Neuroscience (IOPPN), KCL • Financial considerations -  • e.g. Consumables etc... Now included as part of GSK’s in-kind contribution • Formation of a working group - 

  8. Estimated Study Timings Nov 2017 May 2018 Dec 2018 Apr 2017

  9. Second Target for Imaging • P2X7 receptor • PDE4 enzyme • SV2A vesicle protein • COX-2 enzyme • CD11b integrin

  10. P2X7 Receptor • Available PET radioligands: [18F]JNJ64413739 and [11C]GSK1482160 (both CNS penetrant) • P2X7 = Widely expressed in microglia and astrocytes • 2-5 fold increased expression in astrocytes and microglia in LPS rat model • 70% increased expression in AD microglia • Increased P2X7 expression drives microglial activation, rather than P2X7 over-expression being a consequence of microglial activation Choi et al 2007; Skaper et al 2010; Y. Yiangou et al 2006; Michel et al 2007; Gao et al 2015

  11. P2X7 Receptor – [18F]JNJ64413739Primate Imaging Baseline (vehicle) 0.1 mg/kg JNJ’739 2.5 mg/kg JNJ’739 Janssen information - CONFIDENTIAL

  12. P2X7 Receptor – [18F]JNJ64413739Rodent Imaging ± LPS Challenge Intra-striatal injection of PBS (control 5 µl) or LPS (20µg in 5µl PBS) Janssen information - CONFIDENTIAL

  13. P2X7 Receptor – [18F]JNJ64413739Rodent Imaging ± LPS Challenge Intra-striatal injection of PBS (control 5 µl) or LPS (20µg in 5µl PBS) Janssen information - CONFIDENTIAL

  14. P2X7 Receptor – [11C]GSK1482160Primate Imaging • C57BL6 mice (n=3/group) • LPS challenge (5mg/Kg LPS; IP) • Animals scanned 72 hours post saline / LPS challenge • LPS + homologous block: unlabelled GSK1482160 (1mg/Kg) administered i.v. 10 min prior to tracer • Saline = open circles; LPS challenge = filled circles; LPS+block = grey circles Territo et al (2016) Journal of Nuclear Medicine – jnumed.116.181354 – advanced online publication

  15. PDE4 Enzyme • Clinically available PET radioligand: [11C]R(-)Rolipram (CNS penetrant) • Cyclic adenosine monophosphate (cAMP) is a critical regulator of microglia homeostasis • PDE4 is a predominant negative modulator of cyclic AMP signalling within microglia, and hence represents a promising target for modulating immune function • PDE4 expression is regulated by inflammation, and in turn, PDE4 inhibition may function to alter microglia reactivity Pearse and Hughes, 2016

  16. Synaptic Vesicle Density Marker 2A (SV2A) • Clinically available PET radioligand: [11C]UCB-J (CNS penetrant) • Levels of SV2A via the [11C]UCB-J PET ligand have been bench-marked against the known synaptic density biomarker, synaptophysin, and demonstrated to be closely correlated (Finnema et al, 2016) • Levels of synaptophysin have previously been demonstrated to decrease in the mouse ME7 model of AD (Gray et al, 2009) • How levels of SV2A are affected clinically in depression are currently unknown

  17. COX-2 Enzyme • Available PET radioligands: [11C]Celecoxib and [11C]Rofecoxib (both CNS penetrant) • COX-2 = Expressed in neurons • Markedly up-regulated by inflammation in microglia and possibly other cell types • Both [11C]Celecoxib and [11C]Rofecoxib are unsuitable for in vivo PET assessment due to low affinities Ji et al, 2013

  18. CD11b Integrin • Available PET radioligand: [18F]VHH7 (low CNS penetrancy) • There is a strong induction of CD11b expression in models of cortical impact and LPS • [18F]VHH7 is unsuitable for in vivo PET assessment due to its potentially low brain penetration

  19. Summary

  20. Back-Up

  21. TSPO • Translocator protein (TSPO) is an 18 kDa protein located on the outer mitochondrial membrane • TSPO has many proposed functions depending on the tissue • The most studied of these include roles in the immune response, steroid synthesis and apoptosis • TSPO expressed throughout the brain and body • Located in mast cells and macrophages, indicating its role in the immune system • TSPO exerts many actions on immune cells including modulation of oxidative bursts by neutrophils and macrophages, inhibition of the proliferation of lymphoid cells and secretion of cytokines by macrophages • Expression of TSPO linked to inflammatory responses has been demonstrated after ischaemia-reperfusion injury, brain injury and some neurodegenerative diseases • TSPO up-regulation occurs in activated microglial cells and reactive astrocytes, which represent two major components of the neuroinflammatory process

  22. TSPO • Clinically available PET radioligands for the TSPO, including the first generation agent: [11C]PK11195; and a wealth of second generation agents, including: [11C]PBR28, [18F]PBR111, [18F]GE-180, [11C]DPA713, [18F]DPA714 and [11C]DAA1106 • The proportion of TSPO radioligand bound to each of the inflammatory cell types under both basal and diseased conditions has to date, yet to be determined. • Understanding this division of binding would provide vital information regarding the interpretation of TSPO PET data from neurodegenerative studies, including those investigating Alzheimer’s and Mood Disorders

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