Automated Synthesis and Initial Evaluation of (4′-Amino-5′,8′-difluoro-1′H-spiro[piperidine-4,2′-quinazolin]-1-yl)(4-[(18)F]fluorophenyl)methanone for PET/MR Imaging of Inducible Nitric Oxide Synthase

BACKGROUND: Inducible nitric oxide synthase (iNOS) plays a crucial role in neuroinflammation, especially microglial activity, and may potentially represent a useful biomarker of neuroinflammation. In this study, we carefully defined a strategic plan to develop iNOS-targeted molecular PET imaging using (4′-amino-5′,8′-difluoro-1′H-spiro[piperidine-4,2′-quinazolin]-1-yl)(4-fluorophenyl)methanone ([(18)F]FBAT) as a tracer in a mouse model of lipopolysaccharide- (LPS-) induced brain inflammation. METHODS: An in vitro model, murine microglial BV2 cell line, was used to assess the uptake of [(18)F]FBAT in response to iNOS induction at the cellular level. In vivo whole-body dynamic PET/MR imaging was acquired in LPS-treated (5 mg/kg) and control mice. Standard uptake value (SUV), total volume of distribution (V(t)), and area under the curve (AUC) based on the [(18)F]FBAT PET signals were determined. The expression of iNOS was confirmed by immunohistochemistry (IHC) of brain tissues. RESULTS: At the end of synthesis, the yield of [(18)F]FBAT was 2.2–3.1% (EOS), radiochemical purity was >99%, and molar radioactivity was 125–137 GBq/μmol. In vitro, [(18)F]FBAT rapidly and progressively accumulated in murine microglial BV2 cells exposed to LPS; however, [(18)F]FBAT accumulation was inhibited by aminoguanidine, a selective iNOS inhibitor. In vivo biodistribution studies of [(18)F]FBAT showed a significant increase in the liver and kidney on LPS-treated mice. At 3 h postinjection of LPS, in vivo, the [(18)F]FBAT accumulation ratios at 30 min post intravenous (i.v.) radiotracer injection for the whole brain, cortex, cerebellum, and brainstem were 2.16 ± 0.18, 1.53 ± 0.25, 1.41 ± 0.21, and 1.90 ± 0.12, respectively, compared to those of mice not injected with LPS. The mean area under the curve (AUC(0-30min)), total volume of distribution (V(t), mL/cm(3)), and K(i) (influx rate) of [(18)F]FBAT were 1.9 ± 0.21- and 1.4 ± 0.22-fold higher in the 3 h LPS group, respectively, than in the control group. In the pharmacokinetic two-compartment model, the whole brain K(i) of [(18)F]FBAT was significantly higher in mice injected with LPS compared to the control group. Aminoguanidine, selective iNOS inhibitor, pretreatment significantly reduced the AUC(0-30min) and V(t) values in LPS-induced mice. Quantitative analysis of immunohistochemically stained brain sections confirmed iNOS was preferentially upregulated in the cerebellum and cortex of mice injected with LPS. CONCLUSION: An automated robotic method was established for radiosynthesis of [(18)F]FBAT, and the preliminary in vitro and in vivo results demonstrated the feasibility of detecting iNOS activity/expression in LPS-treated neuroinflammation by noninvasive imaging with [(18)F]FBAT PET/MRI.