Automated synthesis and preliminary evaluation of [18F]FDPA for cardiac inflammation imaging in rats after myocardial infarction

A translocator protein 18 kDa targeted radiotracer, N,N-diethyl-2-(2-(4-[18F]fluorophenyl)-5,7-dimethylpyrazolo[1,5-a] pyrimidin-3-yl) acetamide ([18F]FDPA), was automated synthetized and evaluated for cardiac inflammation imaging. Various reaction conditions for an automated synthesis were systematically optimized. MicroPET/CT imaging were performed on normal rats and rats with myocardial infarction (MI). Normalized SUV ratios of [18F]FDPA to [13N]NH3 (NSRs) in different regions were calculated to normalize the uptake of [18F]FDPA to perfusion. The amount of TBAOMs and the volume/proportion of water were crucial for synthesis. After optimization, the total synthesis time was 68 min. The non-decay corrected radiochemical yields (RCYs) and molar activities were 19.9 ± 1.7% and 169.7 ± 46.5 GBq/μmol, respectively. In normal rats, [18F]FDPA showed a high and stable cardiac uptake and fast clearance from other organs. In MI rats, NSRs in the peri-infarct and infarct regions, which were infiltrated with massive inflammatory cells revealed by pathology, were higher than that in the remote region (1.20 ± 0.01 and 1.08 ± 0.10 vs. 0.89 ± 0.05, respectively). [18F]FDPA was automated synthesized with high RCYs and molar activities. It showed a high uptake in inflammation regions and offered a wide time window for cardiac imaging, indicating it could be a potential cardiac inflammation imaging agent.


Scientific Reports
| (2020) 10 Translocator protein 18 kDa (TSPO) is mainly localized in the outer mitochondrial membrane. It is associated with various biological processes such as controlling the translocation of cholesterol, regulating mitochondrial membrane potential, mediating immune response, modulating voltage dependent calcium channels and apoptosis 1 . Since it is overexpressed in the activated microglia, the TSPO targeted imaging was focused on evaluating neuroinflammation in the past, including Alzheimer's disease, Parkinson's disease and dementia [2][3][4] . Mitochondria take up 20-30% of the myocardial intracellular volume 5 , making TSPO an attractive biomarker for the diagnosis and evaluation the treatment effects of cardiac diseases [6][7][8] . Inflammation plays an important role in the healing process after myocardial ischemia 9 . Some clinical trials of anti-inflammatory drugs have been performed on patients with acute myocardial ischemia 10,11 . TSPO is also involved in cardiac inflammation related to macrophage infiltration 12 . Therefore, it might be used to monitor inflammatory response, facilitating physicians to choose the appropriate patients and right time for intervention. Positron emission tomography (PET) is a noninvasive technology to monitor functional and physiological changes in vivo. Currently, several studies reported that TSPO PET imaging could assess cardiac inflammation, such as myocarditis 13,14 and inflammation after ischemia 15 , making it a "hot target" for cardiac inflammation imaging.
In this report, we optimized an automated synthesis of [ 18 F]FDPA based on the previously reported spirocyclic iodonium ylide method 19 , and were first to evaluate [ 18 F]FDPA for myocardial inflammation imaging.
Radionuclide production. [  Final automated synthesis process. The optimal synthesis process was described as follows ( Fig. 1).  4 Ac by volume at a flow rate of 4 mL/min. According to ultraviolet (UV, λ = 254 nm) and radiochemical detectors of the module, the desired product (t R = 16.5-17.5 min) was collected into a pear-shaped bottle preloaded with 50 mL water. The mixture was trapped on a C18 SPE cartridge (Waters Corporation, USA), and washed with 10 mL water. The product was eluted with 1 mL ethanol to product vial subsequently, followed by 10 mL sterile water. The solution was passed through a Cathivex-GV filter, and collected with a 25 mL sterile vial. The radiochemical purity (RCP) and molar activity of the final [ 18 F]FDPA solution were determined by reinjecting the product onto the analytical HPLC column and analyzed with the HPLC method mentioned above. The radioactive fraction was measured by a radio detector (Flow-Count, Bioscan Inc., USA) for molar activity calculation. The mass of the product was calculated by comparing the area under the UV curve at 254 nm with that of standard reference.
Animal model. Male Sprague-Dawley rats (300-350 g) were purchased from SPF (Beijing) Biotechnology Co., Ltd. (China). Rats were maintained in a temperature-controlled room (25 °C) with a natural day/night cycle and fed with a standard rodent diet and water.
For myocardial infarction (MI) modeling, four rats were anesthetized with an air flow containing 4.0% isoflurane. A left thoracotomy was performed between the third and fourth ribs of the rat. The left pericardium was opened. The left anterior descending branch (LAD) was permanently ligated 1-2 mm below the left atrial appendage, using a 7-0 polypropylene suture with a small curved needle. Successful coronary occlusion was verified by observing the myocardium turned grey after LAD ligation.
All animal experiments were performed according to the laboratory animal management regulations of Beijing, and approved by the Animal Care Committee of Capital Medical University.
MicroPET/CT imaging protocol. PET/CT imaging studies were performed with a dedicated microPET/ CT scanner (Inveon PET/CT, Siemens, Germany). The imaging protocols were shown in Fig. 2.
On the 6th day after surgery, MI rats (350-400 g, n = 3) and normal rats (350-400 g, n = 3) were performed [ 18 F]FDPA imaging study. The rat was placed into a chamber connected to an isoflurane anesthesia unit. Anesthesia was induced using an air flow rate of 2.0 L/min containing 4.0% isoflurane. Then, the animal was immediately placed in a prone position on the scanning bed. The air flow rate was then reduced to 0.8-1.5 L/min with 1-1.5% isoflurane.[ 18 F]FDPA (25-37 MBq) was injected via the tail vein. A 60 min dynamic PET scan (6 frames: 6 × 600 s) was started immediately at the beginning of injection, followed by 10 min CT scan using 'magnification low' acquisition settings (projection: 180; binning: 4 × 4; transaxial field of view: 53.9 mm; axial scanning length: 134 mm; voltage: 80 kV; current: 500 μA). For the blocking study, a MI rat was injected with  Histology. When imaging studies on the 7th Day after surgery were completed, the rats were sacrificed.
The hearts were harvested, and fixed with formalin solution. Each sample was embedded in paraffin, cut into serial sections and mounted on glass slides. Sections were stained with hematoxylin and eosin (H&E) 13 . Tissue sections were examined with a light microscope (Leica DM 3000, Leica, Germany) and processed using Leica Application Suite V4.2 microscope software platform. Statistical analysis. IBM SPSS 19.0 was used for statistical analysis. The data was expressed as mean ± standard deviation (SD). SUVs in the heart at different time points were compared using one-way analysis of variance (ANOVA). NSRs in the peri-infarct, infarct and remote regions at 35 min p.i. were analyzed using Kruskal-Wallis H test. A P value < 0.05 was considered significant.

Results
Optimization of final automated radiosynthesis. As  Optimal automated synthesis process. The optimal synthesis process was described above. Starting from [ 18 F]fluoride trapped on a QMA cartridge, the total synthesis time was 68 min, including HPLC purification and formulation. The RCYs were 19.9 ± 1.7% (n = 3) without correction. The radio-HPLC retention time of [ 18 F]FDPA (t R = 13.3 min) was consistent with the corresponding nonradioactive reference (t R = 12.7 min) ( Supplementary Fig. 1). The RCPs ≥ 99%. The molar activities were 169.7 ± 46.5 GBq/μmol (4.6 ± 1.2 Ci/μmol) at the end of synthesis.
MicroPET/CT imaging. In normal rats, TACs indicated that [ 18 F]FDPA mainly accumulated in the heart, lungs, spleen, kidneys and intestines, in which TSPO were highly expressed (Fig. 3). The initial uptake in lungs were too high that the observation of the heart was interfered. Fortunately, the clearance of [ 18 F]FDPA from lungs was fast. At 20-55 min p.i., the heart showed stable and prominent uptake of [ 18 F]FDPA. The SUVs in the heart were 7.15 ± 0.66 at 20 min and 6.99 ± 0.49 at 55 min p.i. respectively, higher than other organs nearby (Fig. 3). Other tissues in which TSPO were barely expressed, such as muscle (SUVs were 0.63 ± 0.11 at 20 min and 0.62 ± 0.01 at 55 min p.i., respectively), exhibited low uptake of [ 18 F]FDPA.
In the [ 13 N]NH 3 images of MI rats (Fig. 4), a severe perfusion defect with significantly reduced [ 13 N]NH 3 activity was observed in the apex and anterior wall. In [ 18 F]FDG images, the extent of reduced [ 18 F]FDG uptake was smaller than that in [ 13 N]NH 3 images. As shown in Fig. 5, [ 18 F]FDPA was accumulated obviously in periinfarct region, compared with that in remote region or infarct region at 5-55 min p.i. From 25-55 min p.i., the SUVs in peri-infarct regions were maintained consistently, without significant difference (F = 0.064, P = 0.977), as well as the SUVs in remote regions (F = 0.184, P = 0.904) or infarct regions (F = 0.220, P = 0.880) (Fig. 5). At 35 min p.i., the NSRs in the peri-infarct, infarct and remote regions were 1.20 ± 0.01, 1.08 ± 0.10 and 0.89 ± 0.05, respectively (P = 0.027). The NSRs in the peri-infarct, infarct regions were higher than that in remote regions (P = 0.022 and 0.539, respectively). In the blocking study, the uptake of [ 18 F]FDPA was obviously inhibited by PK11195 ( Supplementary Fig. 2), which confirmed the affinity of [ 18 F]FDPA to TSPO. Fig. 6, abundant dark purple stains accumulated in the infarct and peri-infarct regions, indicating these regions were infiltrated by inflammatory cells. In contrast, there was few dark purple stains in the remote myocardium.
The aryl-18 F bond of [ 18 F]FDPA successfully improved the metabolic stability 18,23 , but also brought a challenge for fluorine-18 labeling. Compared with the manual method 19 , the current study optimized a series of parameters and developed an automated synthesis of [ 18 F]FDPA with better RCC (74.6% vs. 63%), higher molar activities (169.7 ± 46.5 vs. 96 ± 22 GBq/μmol), shorter synthesis time (68 min vs 80 min) and comparable RCYs and RCPs 19 . Compared with the automated method reported recently 24 , the process resulted in better RCYs (19.9 ± 1.7% vs. 15.6 ± 4.2%). www.nature.com/scientificreports/ In this study, several key points affected the final radiochemical yield from the automated synthesis using this method. Firstly, the amount of TBAOMs and the volume/proportion of water for [ 18 F]fluoride elution was crucial to achieving higher RCC and molar activity. Though both the increase of volume or proportion of water can rise elution efficiency, the incremental water may also affect the drying efficiency in the next step. Therefore, we optimized the formulation of stock solution as 20 mg TBAOMs in 0.4 mL CH 3 CN and 0.4 mL H 2 O, instead of the previous method (12 mg TBAOMs in 0.5 mL CH 3 CN and 0.5 mL H 2 O), and resulted in a satisfied elution efficiency. This formulation of stock solution could be a reference for other automated fluorine-18 labeling with TBAOMs. Secondly, the reaction temperature and time also affected the RCC as reported before 19 . In CFN-MPS200 module, the best reaction temperature and time was 100 °C for 15 min. If the reaction time was prolonged or the temperature was increased, RCC decreased accordingly. This may due to the complete evaporation of CH 3 CN under those harsher conditions, which could be observed from the camera of CFN-MPS200 module, leading to no liquid medium available for the reaction. Thirdly, some sterile filters can adversely affect    26 . A similar phenomenon is observed in this study. Since TSPO was highly expressed in normal cardiomyocytes 12 , [ 18 F]FDPA showed certain uptake in normal myocardium, which might complicate the results of cardiac inflammation imaging. Besides that, in the infarct region, due to the dysfunction of mitochondria in cardiomyocyte 15 , the SUVs of [ 18 F]FDPA were not as high as that in remote regions, which affected the visual analysis of inflammation. Therefore, the images of TSPO targeted tracers need an appropriate normalization method for cardiac inflammation imaging. Frank M. Bengel et al. used the polar map of 99m Tc-sestamibi as a reference, successfully assessed the elevated signal of a TSPO tracer 18 F-GE180 in the infarct region of mice at 1 week post MI 15 . In this study, we used the activity of [ 13 N]NH 3 as a reference, calculated the NSRs in the peri-infarct, infarct and remote regions.
TSPO is associated with various cardiac diseases, such as arrhythmia 27 , large vessel vasculitis 28 , cardiac hypertrophy 29 , atherosclerosis 30 and myocarditis 14 . It is worth mentioning that, TSPO is not only a diagnostic marker, but also a therapeutic target for cardiovascular diseases 12 . TSPO ligands have been studied as therapeutic drugs for cardiovascular diseases, including arrhythmia 27 and MI 31 . Therefore, the quantitative determination of TSPO level by TSPO targeted tracers may offer more valuable information for individual treatment strategy and curative effects. Besides that, since TSPO targeted tracers are prominent for neuroinflammatory imaging, whole-body TSPO imaging may be used to evaluate the systemic inflammatory response in certain diseases. TSPO targeted tracers are worth further investigations. At the meantime, follow-up studies, especially with suitable methods for analysis, are warranted in the future.

Conclusion
The synthesis of [ 18 F]FDPA was successfully optimized and automated with good RCYs, high molar activities, and short synthesis time. The fast clearance of [ 18 F]FDPA from non-target organs and the stable uptake in the heart offered a wider time window for cardiac imaging. Higher NSRs from PET imaging and H&E staining showing the presence of inflammatory cells in the peri-infarct and infarct regions suggest that [ 18 F]FDPA could be a potential imaging agent for cardiac inflammation. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.