Synthesis, preliminary evaluation, and comparison of 18F-CNBI and 18F-CNPIFE as PET probes for glycogen synthase kinase-3 imaging in normal mice.

Introduction: Glycogen synthase kinase-3 (GSK-3) is an important biological enzyme involved in phosphorylation of various biological targets. GSK-3 has been involved in numerous biological functions including metabolism, cell signaling, apoptosis, proliferation and many more. Hyperphosphorylation of biological targets catalyzed by GSK-3 led to cause pathological conditions. Therefore, noninvasive imaging of GSK-3 expression and function is of a great importance to understand the disease pathophysiology

Methods: The GSK-3 PET probes were designed by taking a lead from isonicotinamide based GSK-3 inhibitors^1-2. Precursors and reference compounds were synthesized and fully characterized by NMR and HRMS. The ¹⁸F labeling of ¹⁸F-CNBI was performed at 165 ⁰C, 30 min in anhydrous DMSO using 5 mg of the nitro precursor and 4 mg of 18-crown-6 as a catalyst. Whereas ¹⁸F labeling of ¹⁸F-CNPIFE was performed at 125 ⁰C, 12 min in anhydrous DMSO using 3.5 mg of the mesylate precursor. Final products were purified using, Oasis HLB Sep-Pak, followed by reverse phase HPLC and products were concentrated through a standard C-18 plus solid phase extraction cartridge via trap and release. In-vitro kinase assay (IC₅₀ value) were performed using “ADP-Glo^TM” assay (Promega Corporation), selectivity of the PET probes was performed by comparing rad i-TLC with other biological targets. The in vitro blood brain barrier permeability evaluation was performed using parallel artificial membrane permeability assay (PMPA) at pH 7.4. Animal micro-PET imaging was performed as a 30 min dynamic scan after intravenous administration of the PET probes. Biodistribution of PET probes were performed by drawing the region of interest (ROI) as standardized uptake value (SUV) in major organs using image analysis software.

Results: Both ¹⁸F-CNPIFE (A_m= 0.25-1.7GBq/μmoles) and ¹⁸F-CNBI (A_m= 0.002 GBq/μmoles) were synthesized in >97% radiochemical purity. The ¹⁸F-CNPIFE showed 15-17% of uncorrected radiolabeling yield at end of synthesis (EOS). However, ¹⁸F-CNBI showed 5-11 % uncorrected yield at EOS. IC₅₀ values of 19.4±2.5 nM and 19.4±3.8 nM for ¹⁹F-CNPIFE, and 107.6±25.9 nM and 105.3±18.2 nM for ¹⁹F-CNBI, were observed against GSK-3α and GSK-3β, respectively.

In-vivo evaluation of ¹⁸F-CNPIFE and ¹⁸F-CNBI, showed uptake in normal mouse brain. ¹⁸F-CNPIFE showed SUV of 0.74±0.16 at 30 min in mice brain but ¹⁸F-CNBI showed significantly low SUV of 0.08±0.16 at 30 min in mice brain. Additionally, we noticed improvement in brain uptake of both the PET probes after coadministration of nonradioactive reference compound. In case of ¹⁸F-CNPIFE, we noticed SUV of 0.74±0.16 changed to 0.88±0.15 (p=0.10) at 30 min post injection and in case of ¹⁸F-CNBI, SUV of 0.08±0.005 improved to 0.20±0.01 (p=<0.05) and 0.24±0.01 (p=<0.05) at 30 min in mice brain with coadministration of 200 μg and 300 μg of reference compound, respectively (Figure 1). Other than the brain the major uptake of both PET probes was present in liver and gut.

Conclusions: Both the PET probes ¹⁸F-CNPIFE and ¹⁸F-CNBI were successfully synthesized and evaluated in normal mouse model. The PET probe ¹⁸F-CNPIFE showed nanomolar affinity for both GSK-3α and GSK-3β. The ¹⁸F-CNPIFE showed >9-fold higher brain uptake than ¹⁸F-CNBI without coadministration of reference compound and > 4-fold higher uptake than ¹⁸F-CNBI when coadministered with reference compound possibly due to lower sequestration outside the brain. The PET probe ¹⁸F-CNPIFE is promising candidide for GSK-3 imaging and warrants further evaluation in AD mouse model

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