99mTc-labeled TSPO ligand CB86 targeting macrophages for rheumatoid arthritis SPECT imaging and preliminary evaluation of anti-inflammatory effect
Authors: Liu Peng , Yang Rongshui , Su Fu , Hou Zhenyu , Dong Wentao , Guo Zhide , Ma Chao , Li Huaibo , Wang Weixing , su xinhui 
Published: December 2019

Abstract
Background and Objective TSPO (translocator protein, 18 kDa) is up-regulated in activated macrophages, and serves as an attractive target for macrophages molecular imaging. Previous studies showed that TSPO radiotracer can visualize arthritis via positron emission tomography (PET). Compared with PET, single photon emission computed tomography (SPECT) has several advantages, such as lower cost and commercial availability. The aim of the present study is to develop the 99mTc-labeled TSPO ligand CB86 as a novel SPECT probe for imaging of rheumatoid arthritis and preliminary evaluating the effectiveness of steroid anti-inflammatory therapy. Methods A novel TSPO ligand CB86 was linked to DTPAA and then labeled with 99mTc to obtain 99mTc-DTPA-CB86. The labeling efficiency, radiochemical purity, and stability were determined in vitro. In vitro cellular uptake, efflux and binding affinity of 99mTc-DTPA-CB86 to TSPO were performed on RAW264.7 macrophage cells. The distribution and SPECT studies were conducted on Freund’s Adjuvant-Induced Left Arthritis in rats after the injection of 99mTc-DTPA-CB86 with or without co-injection of unlabeled DTPA-CB86. Result The radiosynthesis of 99mTc-DTPA-CB86 was completed successfully with the labeling yields and radiochemical purity of 95.86 ± 2.45 % and 97.45 ± 0.69 %, respectively. 99mTc-DTPA-CB86 displayed good stability, which the radiochemical purity was more than > 90%, in the saline or mouse serum at 4 h. It also exhibited high specific TSPO binging in RAW264.7 macrophage cells in vitro. The highest uptake ratio was (36.45 ± 2.18) % at 3 h after incubation, and decreased significantly after adding excessive unlabeled DTPA-CB86. 99mTc-DTPA-CB86 bound to TSPO with low nanomolar affinity (IC50 =0.49 nM) in RAW264.7 cells. The cell efflux study showed that 99mTc-DTPA-CB86 has good cell retention by RAW264.7 cells, with only about 13.99 % (decreased from (33.31 ± 2.34) % to (19.32 ± 2.01) % of total input radioactivity) of 99mTc-DTPA-CB86 efflux observed during 4.5 h to 8 h incubation. Biodistribution studies showed the left inflammatory ankle uptake was 2.35±0.10 %ID/g, and the inflammatory ankle to muscle ratio was 3.01 ± 0.09 at 180 min after injection. Small animal SPET imaging studies revealed that 99mTc-DTPA-CB86 could clearly identify left inflammatory ankle with good contrast at 30-180 min after injection. Uptake of 99mTc-DTPA-CB86 in the inflammatory ankles could be largely blocked by an excess of unlabled DTPA-CB86. Furthermore, 99mTc-DTPA-CB86 accumulation in the left inflammatory ankles significantly decreased in RA rats treated with dexamethasone. Conclusion 99mTc-DTPA-CB86 can be readily synthesized, clearly visualized arthritis with low background and monitor therapy response of anti-inflammatory therapy, suggesting its potential as a novel promising molecular probe targeting TSPO for arthritic SPECT imaging. Key words TSPO (translocator protein, 18 kDa); Technetium radioisotope; SPECT imaging; Rheumatoid arthritis; Macrophages; Glucocorticoids.