GMP-compliant fully automated radiosynthesis of [18F]FEPPA for PET/MRI imaging of regional brain TSPO expression

Abstract

Background Expression of translocator protein (TSPO) on the outer mitochondrial membrane of activated microglia is strongly associated with neuroinflammation. The second-generation PET ligand [18F]FEPPA specifically binds TSPO to enable in vivo visualization and quantification of neuroinflammation. We optimized a fully automated radiosynthesis method and evaluated the utility of [18F]FEPPA, the second-generation PET ligand specifically binds TSPO, in a mouse model of systemic LPS challenge to detect TSPO-associated signals of central and peripheral inflammation. In vivo dynamic PET/MR imaging was performed in LPS-induced and control mice after [18F]FEPPA administration. The relationship between the [18F]FEPPA signal and the dose of LPS was assessed. The cytokine levels (i.e., TNF-α, Il-1β, Il-6) in LPS-induced mice were measured by RT-PCR. Standard uptake value (SUV), total volume of distribution (VT) and area under the curve (AUC) were determined based on the metabolite-uncorrected plasma input function. Western blotting and immunostaining were used to measure TSPO expression in the brain. Results The fully automated [18F]FEPPA radiosynthesis produced an uncorrected radiochemical yield of 30 ± 2% within 80 min, with a radiochemical purity greater than 99% and specific activity of 148.9‒216.8 GBq/µmol. Significant differences were observed in the brain after [18F]FEPPA administration: SUV, VT and AUC were 1.61 ± 0.1, 1.25 ± 0.12 and 1.58 ± 0.09-fold higher in LPS-injected mice than controls. TNF-α, Il-1β and Il-6 mRNA levels were also elevated in the brains of LPS-injected mice. Western blotting revealed TSPO (p < 0.05) and Iba-1 (p < 0.01) were upregulated in the brain after LPS administration. In LPS-injected mice, TSPO immunoactivity colocalized with Iba-1 in the cerebrum and TSPO was significantly overexpressed in the hippocampus and cerebellum. The peripheral organs (heart, lung) of LPS-injected mice had higher [18F]FEPPA signal-to-noise ratios than control mice. Conclusions Based on the current data on ligand specificity and selectivity in central tissues using 7 T PET/MR imaging, we demonstrate that [18F]FEPPA accumulations significant increased in the specific brain regions of systemic LPS-induced neuroinflammation (5 mg/kg). Future investigations are needed to determine the sensitivity of [18F]FEPPA as a biomarker of neuroinflammation as well as the correlation between the PET signal intensity and the expression levels of TSPO.

Microliter-scale reaction arrays for economical high-throughput experimentation in radiochemistry

“…The highest crude RCY (77 ± 2%, n = 4) was observed at a temperature of 110 °C for 2.0 min, 30 mM precursor in 8 µL of thexyl alcohol: MeCN (1:1 v/v) solvent and 240 nmol of base (TBAHCO 3 ). Compared to literature methods (Supplementary Table S22 ), the reaction time is shorter (2 min vs 10 min 56 – 58 , 61 ), the droplet reaction consumes 40–50 × less precursor, and the overall synthesis is shorter and has higher yield 56 – 58 , 61 . A batch was purified by analytical-scale HPLC (Supplementary Sect.…”

“…As a final example, we performed a very limited optimization of the synthesis of [ 18 F]FEPPA, a radiopharmaceutical that has been used in several pre-clinical and clinical settings in recent years 56 – 60 to look at the overexpression of TSPO, which is known to be associated with a variety of neurodegenerative disorders. With the aid of the high-throughput platform to explore the influence of temperature (Supplementary Sect.…”

“…We started with conditions similar to past droplet studies for other tracers using tosylate precursors. Since literature reports include a range of 9 to 45 mM precursor concentration 56 – 58 , 61 , we chose an initial value of 30 mM. As a function of increasing temperature (Supplementary Figure S44 ), the fluorination efficiency was ~ 10% at 60 °C and sharply increased to reach a plateau after 90 °C.…”

FKBP51 mediates resilience to inflammation-induced anxiety through regulation of glutamic acid decarboxylase 65 expression in mouse hippocampus

“…To overcome the limitations of biochemical and histological examination in identifying Fkbp5 -KO-affected brain regions in mice after inflammation, we performed in vivo 7-T whole-brain PET/MR neuroimaging by using [ 18 F]-FEPPA, a radioligand of 18-kDa TSPO expressed on microglia, to measure the neuroinflammation in mouse brains collected on LPS-D7 [ 43 , 47 , 48 ]. An increase in the expression of Iba-1 and TSPO both used as biomarkers for microglial activation.…”

“…For quantification of neuroinflammation, we used [ 18 F]- N -(2-(2-fluoroethoxy)benzyl)- N -(4-phenoxypyridin-3-yl) acetamide ([ 18 F]-FEPPA) positron emission tomography (PET)/magnetic resonance imaging (MRI) as previously described [ 43 ]. [ 18 F]-FEPPA is a high affinity radioligand for the 18-kDa translocator protein (TSPO) expressed in the outer membrane of the mitochondria, used for detecting glial activation and neuroinflammation in PET [ 44 , 45 ].…”