VUIIS1008, a new TSPO agent, was presented by Professor Shoufa Han (College of Chemistry and Chemical Engineering, Xiamen University) according to the previous study by Kwon YD (Kwon et al., 2018). No-carrier-added ¹⁸F-fluoride was kindly provided by the First Affiliated Hospital of Xiamen University. Freund’s Adjuvant and anti-TSPO antibodies were purchased from Sigma-Aldrich Shanghai Trading Co Ltd. (Shanghai, China). Goat anti-mouse IgG antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, California, United States). WIZARD 2480 gamma counter from Perkin-Elmer Inc. (Waltham, MA, United States). CRC-25R Dose Calibrator from Capintec Inc. (Ramsey, New Jersey, United States). Mouse macrophage RAW264.7 cell lines were obtained from the Cell Culture Center of Institute of Basic Medical Sciences of Chinese Academy of Medical Sciences (Beijing, China). Male Wistar rats, aged 6–8 weeks (200–300 g), were purchased from the Experimental Animal Center of Xiamen University (Xiamen, China). Small animal PET/CT imaging studies were performed using a micro-PET/CT scanner (Inveon, Siemens Medical Solutions United States, Inc.).

The synthesis of radiotracers 2-(5,7-diethyl-2-(4-(2-fluoroethoxy) phenyl) pyrazolo [1,5-a] pyrimidin-3-yl)-N, N-diethylacetamide (¹⁸F-VUIIS1008) were prepared from its corresponding tosylate precursors via manual synthesis according to previously reported procedures (Tang et al., 2013; Kwon et al., 2018). Briefly, aqueous ¹⁸F-fluoride (5–15 mCi; 0.2∼0.6 GBq) was eluted from the cartridge with a solution of Kryptofix K2.2.2 to form the complexation mixture. This complex was then reacted with appropriate tosylate precursor VUIIS1008 (4.0 mg) in dimethylsulfoxide (0.7 mL) at 100°C for 15min. The reaction crude was purified using semi-preparative HPLC (C18, Dynamax 250 × 10 mm; Varian), eluting with 10 mM NaH₂PO₄ buffer (pH 6.7) and methanol (30/70, v/v) at 3.0 mL/min. The product (¹⁸F-VUIIS1008) was collected directly into 140 ml of water (deionized), passed through a C-18 September-Pak Plus (Waters, Milford, MA, United States of America), and eluted with 200 proof ethanol (1.0 mL) then saline (0.9%) into a sterile vial.

According to our previous report (Liu et al., 2020), the lipophilicity of ¹⁸F-VUIIS1008 was analysed by the n-octanol/water mixture containing 200 μL ¹⁸F-VUIIS1008 and 1 mL phosphate-buffered saline (pH = 7.4). The solution was centrifuged at 6,000 rpm for 5 min, and separated and then they were counted in a γ counter, respectively. The radioactivity were used to calculate the log p values. The lipophilicity of ¹⁸F-VUIIS1008 was determined as (cpm in organic phase)/ (cpm in water phase).

Based on our previous report (Liu et al., 2020), the stability of the complex containing 500 μL (3.7 MBq) ¹⁸F-VUIIS1008 and phosphate-buffered saline (PBS, pH = 7.4) or mouse serum was evaluated by performing the complex at 37°C for 30、60、120、and 240 min. The radioactivity of ¹⁸F-VUIIS1008 was measured at various time points by a HPLC.

The RAW264.7 cell lines were conducted cell uptake, and efflux tests in accordance with our previous study (Liu et al., 2020).

The animal study protocol was carried out according to the principles outlines by the Institutional Animal Care and Use Committee of Zhongshan Hospital Xiamen University. The left inflammatory ankles were induced in male Wistar rats in accordance with our previous study (Liu et al., 2020). Briefly, 0.1 mL of Complete Freund’s Adjuvant (CFA) with Mycobacterium butyricum 1% suspension in mineral oil was injected into the left ankle of each rat (day 0). The severity of RA was monitored daily by two observers. The left inflammatory ankles were estimated by the number swollen joints. When the left inflammatory ankles grew to swell in two to three joints, the RA rats were subject to in vivo biodistribution and PET studies.

The biodistribution analysis were induced on day 3 after CFA injection. RA rats were administrated with ¹⁸F-VUIIS1008 (3.7 MBq, 100 μL) via tail vein. At 30, 60, and 120 min post-injection, the left inflammatory ankles and normal tissues of interest were removed and determined their radioactivity with a γ counter. For in vivo specificity study, RA rats were injected with ¹⁸F-VUIIS1008 and unlabeled PK11195 (500 μg), and biodistribution studies were performed at 60 min post-injection. The radioactivity ratios of the left inflammatory ankle to blood (LIA/B) and the left inflammatory ankle to muscle (LIA/M) were calculated. Biodistribution data are expressed as %ID/g values by dividing counts per gram per minute by the injected dose.

PET imaging studies were performed using a micro-PET scanner (Siemens Medical Solutions United States, Inc.). Static PET imaging was performed at 30, 60, and 120 min post-injection of 3.7 MBq 100 μL ¹⁸F-VUIIS1008 via tail vein on day 3, 7, 12, 15, 19, 24, 29, 35, 38, and 45 after CFA injection. For blocking imaging, unlabeled PK11195 (500 μg) or VUIIS1008 (500 μg) was co-injected with ¹⁸F-VUIIS1008 (3.7 MBq 100 μ) on day seven. The RA rat were anesthetized with 2% isoflurane and positioned prone in micro-PET bed. Micro-PET images were reconstructed using an 3D OSEM scatter corrected reconstruction algorithm. Regions of interest (ROIs) were placed on the left inflammatory ankles. Micro-PET data are expressed as %ID/g values by dividing counts per gram per minute by the injected dose.

According to routine protocols, Hematoxylin and Eosin (HE staining), immunohistochemistry (IHC) tests and immunofluorescence staining were carried out in the tissues of left inflammatory ankles, contralateral normal ankles on day 3 after CFA injection. For HE tests, 5 𝜇m longitudinal sections were stained with hematoxylin and Eosin solution for 5 and 3 min at 25°C, respectively, and then analysed using an Olympus BX53 fluorescence microscope (Tokyo, Japan). For immunohistochemical analyses, the slices successively incubated with rabbit anti-rat TSPO antibodies (1:100, Abcam) and goat anti-rat secondary antibodies (1:1,000; Sigma) for 2 h at 25°C, and then analysed using an Olympus BX53 fluorescence microscope. For immunofluorescence staining, according to a standard protocol (Capaccione et al., 2020). The slides successively incubated with rabbit anti-rat TSPO antibodies (1:100, Abcam), anti-mouse CB68 antibodies (1:100, Abcam), and goat anti-rat FITC-IgG secondary antibodies (1:200; Sigma), goat anti-mouse TRITC-IgG secondary antibodies (1:200; Sigma), respectively, for 2 h at 25°C, and then stained using 200–300 μL 10 μg/mL of DAPI. After then, These slides were analysed using an Olympus BX53 fluorescence microscope.

The experimental data are presented as mean ± standard deviation. Statistical calculations were determined using the Student’s t-test and p < .05 was statistically significant.

¹⁸F-VUIIS1008 was successfully radiosynthesized (Figure1). Under radio-HPLC conditions described above, ¹⁸F-VUIIS1008 displayed a retention time of 11.8 min. The radiochemical purity of the radiopharmaceutical exceeded 98.00%, and the specific activity of the purified ¹⁸F-VUIIS1008 was 1.52 × 10⁸ MBq/mmol. The lipid-water partition coefficient (log P) of ¹⁸F-VUIIS1008 is 1.58 ± 0.03, indicating ¹⁸F-VUIIS1008 is a fat-soluble compound.

¹⁸F-VUIIS1008 displayed excellent stability in the PBS (Figure 2) or mouse serum (Figure 3). It showed that defluorination of ¹⁸F-VUIIS1008 was not obviously found, and the percentage of intact probes remained more than 90% during 30–240 min of incubation in the PBS or mouse serum.

Cell uptake ratios of ¹⁸F-VUIIS1008 were shown in Figure 4A. The level of ¹⁸F-VUIIS1008 in RAW264.7 cells was 12.00 ± 0.10%, 13.00 ± 1.00%, 14.00 ± 0.30% and 23.00 ± 0.60% at 15, 30, 60, and 120 min, respectively. When the probe was incubated with large excesses of non-radioactive VUIIS1008, its uptake levels in RAW264.7cells was significantly inhibited (p <0.05) at all incubation time points. Moreover, cell efflux studies (Figure 4B) indicated ¹⁸F-VUIIS1008 has excellent cell retention in RAW264.7 cells, which ¹⁸F-VUIIS1008 efflux was 6.74% (reduction from 16.50 ± 0.002% to 9.76 ± 0.001% of total input radioactivity) from 120 min to 240 min incubation. In general, the results demonstrated that ¹⁸F-VUIIS1008 maintained high affinity to TSPO to further study in vivo TSPOtargeted imaging.

The biodistribution studies were conducted on day 3 after CFA injection. At 30, 60, and 120 min post-injection, the biodistribution characteristics of ¹⁸F-VUIIS1008 was shown in Figure 5. ¹⁸F-VUIIS1008 displayed high radioactivity uptake in the left inflammatory ankle. At 30, 60, and 120 min, the left inflammatory ankle uptake was 1.08% ± 0.08% ID/g, 1.33% ± 0.02% ID/g, 0.99% ± 0.1 3% ID/g, respectively, lower than that in the liver, kidney, intestine, stomach, lungs, bone, and spleen, whereas it was higher than blood, muscle, and brain. Furthermore, ¹⁸F-VUIIS1008 showed high levels of the left inflammatory ankle to muscle (LIA/M) and left inflammatory ankle to blood (LIA/B) (Figure 6). At 60 min, the ratio of LIA/M and LIA/B was 1.65 ± 0.07 and 4.40 ± 0.22, respectively, and higher than that at 30 and 120 min.

In order to investigate the specificity of ¹⁸F-VUIIS1008, an excess of PK11195 (500 μg) was coinjected with ¹⁸F-VUIIS1008 into RA rats to saturate endogenous and overexpressed TSPO in some normal tissues. PK11195 decreased significantly the accumulations of ¹⁸F-VUIIS1008 in the left inflammatory ankle and many tissues, such as liver, lung, heart, kidney, stomach, and intestine (p <.05), whereas it did not decreased those in the blood, muscle, and bone (p >.05).

Longitudinal small animal PET/CT studies were performed at 30, 60, and 120 min after injection of ¹⁸F-VUIIS1008 on day 3, 7, 12, 15, 19, 24, 29, 35, 38, and 45 after CFA injection. As shown in Figure 7, ¹⁸F-VUIIS1008 highly accumulated in the left inflammatory ankles at 30 min compared with the collateral ankles, and exhibited a gradual increasing uptake during 60–120 min post-injection. The left inflammatory ankles were clearly visible with good inflammatory to background contrast. During day 3–45 after CFA injection, the uptake of left inflammatory ankles was a wiggle trace with two peaks on day 7 and 29, and then the uptake on day 29 was the highest (60 min (3.00% ± 0.08% ID/g) (p <.05) (Figures 8–10). Importantly, there was an inflection point on day 15, and after day 15, the uptake gradually increased along with time till day 29, then dropped slowly along with time till day 45, when the uptake was the lowest, and still higher than that in collateral muscle. Furthermore, when co-injected with unlabeled PK11195 (500 μg) or VUIIS1008 (500 μg), the left inflammatory ankles were barely visible on PET images at 60 min post-injection (Figure 11), while the contralateral normal muscle stayed at the low uptake level, affected slightly by PK11195 or VUIIS1008 injection. Regions of interest (ROIs) analysis of PET showed a high ratio of the left inflammatory ankle in RA rats injected unblocking dose compared to with 500 μg blocking dose at 60 min post-injection (Figure 12) (p <.05).

For HE tests, it found that synovial hyperplasia and infiltration of inflammatory cells (such as lymphocaytes and macrophages) were identified in the left inflammatory ankles, while they were not observed in the normal contralateral ankles (Figure 13A). For immunohistochemistry (IHC) analysis, there found positive staining of TSPO in the left inflammatory ankles, while negative expression of TSPO in the normal contralateral normal ankles (Figure 13B). Moreover, for immunofluore-scence analysis, it showed the positive staining of TSPO and macrophage (CD68) could be detected in the left inflammatory ankles, whereas they were not found in the normal contralateral normal ankles (Figure 14).