Info
Background:
Alzheimer’s Disease (AD) is a neurodegenerative disorder involving amyloid-beta (Aβ) aggregation, hyperphosphorylation of tau, and neuroinflammation. AD is the sixth‐leading cause of death in the United States and the fifth‐leading cause of death among Americans age 65 and older.1Several treatments are now available for AD, most of which target Aβ, but unfortunately they have not been successful in halting or reversing disease progression2. Thus, there is a critical unmet need for new therapies targeting other major pathological hallmarks of AD, such as restoring healthy immune function and mitigating neuroinflammation. Importantly, the ability to non-invasively track the efficacy of such interventions can expedite clinical trials and support treatment plans3,4. G protein-coupled receptor 84 (GPR84) is a promising biomarker of neuroinflammation that is selectively upregulated on activated microglia and macrophages. Utilizing a novel 18F-labeled GPR84-PET tracer, [18F]MGX-38, we demonstrate the ability to detect small yet significant temporal changes within the early neuroinflammatory stages of murine AD.
Hypothesis/Objective:
To investigate whether GPR84-PET imaging can be used to track subtle alterations in innate immune activation within a murine model of AD using a novel small molecule radiotracer.
Methods:
[18F]MGX-38 was synthesized via copper (II) mediated radiofluorination of the MGX-38-Bpin precursor using azeotropically dried [¹⁸F]fluoride, Cu(OTf)₂, and pyridine in DMA/1-butanol at 130°C for 18 minutes (Fig S2A). The crude product was purified by semi-preparative HPLC, isolated via C18 cartridge trapping, and formulated in ethanol/saline. Product identity and molar activity were confirmed by analytical HPLC. To assess the specificity of [18F]MGX-38, binding studies were performed using hGPR84-expressing HEK293 cells versus parental controls, with and without co-incubation of a structurally distinct GPR84 antagonist, GLPG1205. 3.5 – 4 month-old 5xFAD mice were used to model AD. Age/sex-matched C57BL/6J mice were used as wild-type controls. Static 10-min PET/CT imaging of 4-month-old female 5xFAD mice (n=14) and wild-type (WT) controls (n=14) was conducted at 30-40 min post injection of [18F]MGX-38. Mice were perfused to remove any unbound intravascular tracer and tissues of interest were analyzed via gamma counting and ex-vivo autoradiography.
Results:
Results: [18F]MGX-38 was synthesized in 76 minutes with 23.85 ± 2.58 % radiochemical yield (non-decay corrected, n=3), >99% radiochemical purity, and 5.06 ± 1.59 Ci/mmol (or 187.35 ± 58.88 GBq/mmol) molar activity (Figure S2A). Cell binding studies demonstrated 3.24-fold higher binding of [18F]MGX-38 in hGPR84-expressing HEK293 cells versus parental control cells after 40 minutes incubation (P <0.0001, n=8). Co-incubation with the GPR84 antagonist GLPG1205 (35 μM) reduced tracer binding by 64.87%, confirming high specificity of [18F]MGX-38 for GPR84 (P <0.0001, n=8, Figure S2B). PET/CT images revealed higher PET signal within the brain of 5xFAD mice when compared to controls (Fig 1A). Quantification of PET data using brain atlas mapping revealed significantly higher signal within pathology-associated regions including the cortex (P < 0.001), hippocampus (****P < 0.0001), and thalamus (P < 0.001) in addition to the whole-brain (P < 0.001, Fig 1B).Ex-vivo autoradiography of the same mouse brains verified significantly elevated 18F-MGX-38 binding cortex (P < 0.01), hippocampus (P < 0.0001) and thalamus brain regions P < 0.0001, Fig S2D, E). Ex vivo gamma counting showed significantly higher binding in the whole brain and liver of 5xFAD mice compared to controls. (Fig. S2C).
Conclusion:
[18F]MGX-38 is a new, promising small molecule PET tracer for accurately and sensitively detecting early innate immune activation in a murine AD model. Notably, PET/CT images in addition to ex vivo measures revealed significantly higher binding of [18F]MGX-38 in 5xFAD versus age-sex-matched wild-type mice within pathology-associated regions. Longitudinal GPR84-PET imaging of 5xFAD mice from the same cohort, alongside histological analyses, is ongoing to determine the utility of this PET tracer for tracking neuroinflammation with increased disease severity in addition to assessing its ability to monitor response to novel immunomodulatory therapies.
Image/Figure:
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Image/Figure Caption:
Figure 1. Increased binding of GPR84-PET tracer within 5xFAD pathology-associated regions. (A) Representative coronal PET-CT images exhibit higher [18F]MGX-38 binding when compared to age/sex-matched wild-type control at 4 months of age. (B) PET quantification of pathology-associated regions (cortex, hippocampus, thalamus) and whole-brain show significant binding in 5xFAD mice (n=14) when compared to wild-type counterparts (n=14) at 4 months of age. Statistical analyses were performed unpaired t-tests *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
Author
Jonathan Green
Stanford School of Medicine