4-[18F]Fluorobenzoyl-C2A domain of synaptotagmin I-glutathione-S-transferase


Leung K.



In vitro Rodents



Apoptosis (programmed cell death) plays an important role in the pathophysiology of many diseases, such as cancer, neurodegenerative disorders, vascular disorders, atherosclerosis, and chronic hepatitis, as well as in the biology of normal cells, such as epithelial cells and immune cells (1). During apoptosis, there is rapid redistribution of phosphatidylserine (PS) from the inner membrane leaflet to the outer membrane leaflet, exposing the anionic head group of PS. PS is also accessible for annexin V (or annexin A5) binding in necrosis because of disruption of the plasma membrane. Annexin V, a 36-kDa protein, binds to PS with high affinity (Kd = 7 nM). Annexin V has been radiolabeled with 123I and 99mTc for single-photon emission computed tomography imaging and with 124I for positron emission tomography (PET) (2-4). Annexin V has been successfully labeled with 18F (18F-labeled annexin V (4-[18F]FBA)) and is currently being developed as a PET agent for imaging apoptosis as well as necrosis (5, 6). Cy5.5-Annexin V has been evaluated as an optical probe in small animals. Annexin V-CLIO-Cy5.5 (AnxCLIO-Cy5.5) is a multimodal agent that consists of CLIO nanoparticles labeled with annexin V and Cy5.5 (7).

Synaptotagmins are integral membrane proteins of synaptic vesicles with calcium ion (Ca2+) binding domains (8). The C2A domain of synaptotagmin I (Syt1) binds two to three Ca2+ ions and also binds anionic phospholipids including PS. Calcium binding to Syt1 participates in triggering neurotransmitter release at the synapse. Davletov et al. (9) showed that C2A-glutathione-S-transferase (C2A-GST) fusion protein binds specifically Ca2+ and anionic phospholipids. 99mTc-Labeled C2A-GST (99mTc-C2A-GST) has been shown to target apoptotic cells in acute myocardial infarction and tumor models in vivo in animal studies (10-14). Wang et al. (15) prepared 4-[18F]fluorobenzoyl-C2A-GST (4-[18F]FB-C2A-GST) as a PET agent for imaging of apoptotic cells.



Wang et al. (15) prepared 4-[18F]FB-C2A-GST with a two-step synthesis. N-Succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) was synthesized by coupling [18F]SFB to O-(N-succinimidyl)-tetramethyluronium tetrafluoroborate. [18F]SFB and C2A-GST (0.2 mg) in borate buffer (pH 8.4) were incubated for 20 min at room temperature. 4-[18F]FB-C2A-GST was purified with column chromatography with a radiochemical purity of >98%. At the end of synthesis, 30–160 MBq (0.8–4.3 mCi) 4-[18F]FB-C2A-GST (n = 6) was obtained for intravenous injection. The decay-corrected yield was 35%–40% based on [18F]SFB. The total synthesis time was ~120 min from the end of bombardment. The specific activity of 4-[18F]FB-C2A-GST was not reported.

In Vitro Studies: Testing in Cells and Tissues


Wang et al. (15) performed cell-binding assays with 4-[18F]FB-C2A-GST in apoptotic human Jurkat T cells treated with camptothecin. The apoptotic cells showed a 4.6 ± 0.6-fold higher radioactivity uptake than that observed in the untreated control cells.

Animal Studies



Wang et al. (15) performed ex vivo biodistribution studies in normal mice (n = 3/group) at 15, 60, and 120 min after injection of 0.37 MBq (0.01 mCi) 4-[18F]FB-C2A-GST. The initial accumulation expressed as percent injected dose/gram (% ID/g) was high in the lung (~20% ID/g), liver (~15% ID/g), kidney (~12% ID/g), and spleen (~8% ID/g) at 15 min after injection and cleared rapidly afterwards with the exception of the kidney. The kidney accumulation was higher at the later time points (~20%–30% ID/g). The accumulation was low (<2% ID/g) in the heart, intestine, muscle, brain, and stomach. The blood radioactivity level was 0.95% ID/g at 120 min after injection. 4-[18F]FB-C2A-GST was 90% intact in the plasma at 120 min after injection. Dynamic PET scans with 4-[18F]FB-C2A-GST clearly visualized the liver, lungs, and heart during the first 10 min, at which point the radioactivity levels declined. On the other hand, the radioactivity level in the kidneys increased gradually with time. NO blocking studies were reported.

Other Non-Primate Mammals


Wang et al. (15) induced tumor apoptosis with a single dose of paclitaxel in rabbits (n = 6) bearing VX2 lung tumors in the right lung. PET imaging studies were performed with 4-[18F]FB-C2A-GST before paclitaxel treatment and at 72 h after paclitaxel treatment. PET imaging with [18F]FDG was also performed to confirm the presence of tumors before paclitaxel treatment. Little 4-[18F]FB-C2A-GST accumulation was found in the [18F]FDG-avid tumor lesions. However, intense 4-[18F]FB-C2A-GST accumulation was found in the tumor lesion. Standard uptake values were 0.47 ± 0.29 (n = 6) and 0.009 ± 0.001 (n = 2) for the paclitaxel-treated group and the untreated group (P < 0.001), respectively. Histological staining of tumor sections showed a significant increase (P < 0.001) in the number of apoptotic cells in the paclitaxel-treated group (80.0 ± 8.7%) compared with the number of apoptotic cells in the untreated group (5.0 ± 0.8%).

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.


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