99mTc-labeled TSPO ligand CB86 targeting macrophages for rheumatoid arthritis SPECT imaging and preliminary evaluation of anti-inflammatory effect

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. 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. background monitor of anti-inflammatory therapy, suggesting its potential as a novel promising molecular probe targeting TSPO for arthritic SPECT


Introduction
Rheumatoid arthritis (RA) is a chronic autoimmune disease that primarily manifests as synovial inflammation. Persistent synovitis leads to severe progressive joint damage, functional disability, morbidity, and increased mortality (1). Moreover, RA is also a systemic autoimmune disease that can affect many tissues and organs and is associated with other diseases, including infections, malignancies and cardiovascular diseases (2). Today, approximately 1-2% of the world population gets injured with RA, most frequently found in developed countries, such as Europe and North America (3). Women predominantly more often get affected than men with ages ranging from 40 to 60 years (4). The etiology of RA is very complex and has not yet been fully elucidated. It has a wide spectrum of clinical characteristics, variability in disease severity, progression and differences in therapeutic response (5). These heterogeneous phenotypes may indicate that variety of factors can contribute in developing RA, including genetic and environmental factors. Among environmental factors, smoking has by far the strongest association with RA due to smoking cigarette smoke inducing pro-inflammatory immune responses via vimentin (6).
Clinical studies have shown that immunological and inflammatory processes resulting in joint destruction have already been set off at the very beginning of RA (7). Thus, it seems reasonable that therapeutic intervention should start as soon as the diagnostic has been established, with the aim of stopping inflammation before irreversible damage is caused (1). Currently, clinical diagnosis of RA is based on the 2010 classification criteria proposed by the American College of Rheumatology (ACR) / European League Against Rheumatism (EULAR) (8). These criteria include characteristics appearing early in the RA course, level of inflammatory markers (Creactive protein, CRP and Erythrocyte Sedimentation Rate, ESR) and autoantibodies in serum (Rheumatoid Factor, RF and Anti-citrullinated protein antibodies, ACCP).
However, they still are not very sensitive and specific to RA (9). Conventional imaging techniques, including plain radiography, ultrasonography, CT, MRI, has poor 6 sensitivity and specificity in the detection of the inflammatory process that happens in the initial stages of RA because these imaging models are anatomical contexts and are rather limited in identifying a pathological condition in early and very early disease stages (10). Therefore, the development of non-invasive, highly sensitive and specific tests/ imaging techniques is essential for very early detection of RA.
Nuclear medicine imaging provides a large scale opportunity in the diagnosis of diseases. These techniques are based on the detecting of gamma rays emitted from biologically active molecules labeled with radioactive isotopes and allow noninvasive in vivo detection of different physiologic and pathologic processes with high sensitivity and specificity. Thus, nuclear medicine imaging has significant potential for timely diagnosis and adequate follow-up of diseases (11). In clinical practice, 99m Tc-MDP bone SPECT (Single Photon Emission Computed Tomography) and 18 F-FDG PET (Positron Emission Tomography) are increasingly used to diagnose inflammatory arthritis including RA. They have high sensitivity but low specificity. In many cases, distinction between inflammatory and metastatic bone processes may be difficult (11). Mounting data of evidence have shown that macrophages are key effector cells in the pathogenesis of RA (12), since macrophages are the major source of cytokines that contribute to synovial inflammation in early stages of RA, and then bone erosion (13). The increase in the number of macrophages in the synovium is an early hallmark of active rheumatic disease (14). Therefore a specific tracer of such a process would be more specific and possibly also enable an earlier detection of RA. Recently, specific ligands targeting macrophage receptors such as CD20 receptor, interleukin-1 (IL-1) receptor, etc. have been investigated in the patients with RA using 99m Tc-anti-CD20, 123 I-IL-1ra and 124 I-anti-CD20, illustrating the interest for molecular imaging in this type of pathology (15)(16)(17).The drawbacks of probes with antibodies severely hamper their clinical applications due to their large size resulting in slow tumor accumulation and slow clearance from the circulation (18).
The translocator protein 18 kDa (TSPO), previously known as the peripheral-type benzodiazepine receptor (PBR) (19), is located in the outer mitochondrial membrane, where it has a function in cholesterol transport from the outer to the inner mitochondrial membrane as a rate-limiting step in steroid biosynthesis (20).
Moreover, TSPO is also involved in apoptosis, cell proliferation, anion transport, regulation of mitochondrial functions and immunomodulation (19). Under normal physiological conditions, TSPO levels in macrophages are very low, but a strong increase in TSPO levels occurs in an activated state of macrophages in response to inflammation (21). Hence, TSPO is considered as a promising biomarker for inflammatory diseases (22). Previous studies showed that PET imaging based TSPO ligands, such as 11 C® PK11195, 11 C-DPA-713, and 18 FDPA-714, can visualize RA (2,23,24). Although PET has higher resolution and sensitivity, SPECT held several advantages over PET including lower cost, more widespread availability, favorable physical and imaging characteristics (γ ray = 140 keV, half-life = 6.02 hours). In additional, the preparation of 99m Tc-labeled tracers is efficient, reproducible, and simple, making it easy for clinical use. Therefore, in the present study, we aimed to develop the 99m Tc-labeled TSPO ligand CB86 as a novel SPECT probe for imaging of rheumatoid arthritis and initially evaluating the effectiveness of steroid antiinflammatory therapy. Wistar rats, aged 6-8 weeks (200 to 300 g), were purchased from the Experimental Animal Center of Xiamen University (Xiamen, China). SPECT imaging studies were performed using a nanoScan-SPECT/CT scanner (Mediso, Budapest, Hungary).

Cell culture
The mouse macrophage RAW264.7 cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were maintained in a humidified atmosphere of 5% CO 2 at 37 °C, with the medium changed every two days. A 70-80% confluent monolayer was detached by 0.1% 9 trypsin and dissociated into a single cell suspension for further cell culture.

Quantitative Real-Time PCR (qRT-PCR)
The expression of TSPO mRNA in RAW264.7 cell lines with or without LPS treatment is determined by the qRT-PCR based on the method described earlier (25,26

Cellular immunofluorescence staining
Cellular immunofluorescence staining was performed as previously described (25,27). Briefly, the cell-seeded coverslips were washed and fixed. The TSPO mAb  in an ice bath. The byproduct was removed by filtration and then purified by column chromatography (hexane/ethyl acetate = 6:1, v/v) to give a white filtrate containing coumarin-CB86.

Fluorescence imaging of CB86 in living cells
The RAW264.7 cells stimulated with LPS were incubated in the probe coumarin-CB86

Labeling DTPA-CB86 with 99m Tc
The radiolabeling method of DTPA-CB86 was performed as our previously described methods (28).  Figure 1.
After shaking for 5 min at room temperature, The solution was centrifuged at 3000 rpm for 5 min. Afterward, 100 μL of the organic phase and water phase were counted in a gamma counter, respectively. The averaged activities from each phase were used to calculate the log P values. The lipid-water partition coefficien (Po/w) of 99m Tc-DTPA-CB86 was calculated as (cpm in organic phase)/(cpm in water phase).
All the experiments were performed with triplicate samples and reported as mean ± SD.

In vitro stability analysis
In vitro stabilities in saline and mouse serum were determined similarly to the procedures previously described with minor modifications (29,30). 99m Tc-DTPA-CB86

Cell assays
14 Cell uptake, blocking, and efflux assays were performed as previously described with minor modifications (28)(29)(30)(31)  Each joint was graded, so the maximum score was 16 for a rat. Meanwhile, two weeks after injecting, the joint thickness was measured by a vernier caliper (Exploit Technology CO., LTD., Taiwan, China). Rats were allowed to grow to around grade 2 and 3 and then the RA rats were subject to in vivo biodistribution and imaging studies.
Anti-inflammatory therapy. RA rat with grade 4 (n =4 for each group) were administered with 0.5 mg/kg.d dexamethasone or saline by stomach perfusion once a day for 2 weeks, and then were imaged at the 7th day of and 15th day after treatment, respectively. The saline treated group as control group.

Biodistribution study
The animal procedures were performed according to a protocol approved by the

Immunohistochemical staining
The slides were blocked with 5% goat serum in PBS containing 0.1% Triton X-100 for 1 h at room temperature and then incubated with rabbit anti-rat TSPO (1:200, Sigma) antibodies for 2 h at room temperature. After being washed, the slides were then incubated for 2 h at room temperature with secondary antibodies Goat antimouse IgG antibody (1:1,000; Sigma).The slides were then examined and photographed using an Olympus BX53 fluorescence microscope (Tokyo, Japan).

Statistical methods
The experimental data were analyzed by SPSS 18.0 (SPSS Company, Chicago, IL, USA). Statistical analysis was performed using two tailed Student's t-test for unpaired data. Data are expressed as mean ± standard deviation and P < 0.05 was considered to indicate a statisti cally significant difference.

Expression of TSPO in the activated RAW264.7 cells
As shown in Figure 1A, qRT-PCR results indicated that the expression of TSPO mRNA in the activated RAW264.7 cells was significantly higher than that in the resting RAW264.7 cells (P <0.01). Consistent with qRT-PCR, Western blot ( Figure 1B) demonstrated that the activated RAW264.7 cells exhibited higher levels of TSPO protein expression. Immunofluorescence analysis showed ( Figure 2) that TSPO widely over-expressed in activated RAW264.7 cells, and is mainly located in the cytoplasm. These results could be concluded that TSPO is excessively expressed in macrophage RAW264.7 cells induced by LPS.

In vitro cytotoxicity of CB86 and CB86-DTPA
In vitro cytotoxicity of CB86 and CB86-DTPA was determined using the MTT assay in RAW264.7 and 4T1 cells. The cells were incubated with different concentrations of CB86 and CB86-DTPA for 24 hours, respectively. As shown in Figure 3, the cell survival rates of RAW264.7 and 4T1 cells were not significantly different (P > 0.05) between the groups of CB86 and CB86-DTPA. The cell survival rates were > 90% even in the concentration of 20 μM CB86 and CB86-DTPA, indicating that CB86 and CB86-DTPA were safe to the RAW264.7 and 4T1 cells at the test concentrations.

Fluorescence imaging of CB86 in the activated RAW264.7 cells
As shown in Figure 3, the activated RAW264.7 cells were stained with MitoRed a well-established mitochondrial dye, and subjected to confocal fluorescence microscopy. CB86-coumarin was found to be localized to mitochondria ( Figure 4A), whereas, it was barely observed in the presence of CB86 ( Figure 4B). These results indicated that CB86 could bind well with TSPO receptor on the surface of mitochondrion in the activated RAW264.7cells.

In vitro stability analysis
In vitro stability studies (Figure 6, 7) showed that more than 90% of 99m Tc-DTPA -CB86 remained intact during 1 to 4 h of incubation in the saline or mouse serum, indicating that 99m Tc-DTPA-CB86 maintained excellently stable in the saline or mouse serum.

Cell assays
Cell uptake ratios of 99m Tc-DTPA-CB86 were shown in Figure 8A. The binding affinity of 99m Tc-DTPA-CB86 to TSPO was determined through the receptor saturation assay. As shown in Figure 8B, the IC 50 value of 99m Tc-DTPA-CB86 was 0.49 nM.

Biodistribution study
At 30, 90, and 180 min after administration, the biodistribution profiles of 99m Tc-DTPA-CB86 are presented in Figure 9, 99m Tc-DTPA-CB86 exhibited high levels of radioactivity accumulation in the left inflammatory ankle. At 30 min, the left inflammatory ankle uptake was 1.33±0.16 %ID/g, lower than that in the liver

Evaluating therapy response of steroid anti-inflammatory therapy
In dexamethasone treated RA rats, swelling was reduced during the treatment.
Clinical scores of RA rats were reduced during the drugs treatment. As shown in Figure 12, 99m Tc-DTPA-CB86 accumulation in the left inflammatory ankles at 7th day and 15th day after treatment significantly decreased compared to the control group, especially at 15th day after treatment.

Histological findings
HE staining showed that synovial hyperplasia and infiltration of inflammatory cells (such as lymphocaytes and macrophages) could be detected in the left inflammatory ankles ( Figure 13A), while no inflammation signs were observed in the normal contralateral ankles ( Figure 13B). Immunohistochemistry (IHC) showed that positive staining of TSPO could be detected in the left inflammatory ankles ( Figure 13C), while negative expression of TSPO was observed in the normal contralateral normal ankles ( Figure 13B).

Discussion
Rheumatoid arthritis (RA) is a chronic systemic autoimmune inflammatory disease of unknown cause that affects over 1.0% of the population in developed countries. It may begin at any age but usually initiates between 30 and 50 years old and is approximately three-times more common in the female gender (7). RA is characterized by chronic inflammation not only mainly involving the synovium of both small and large joints, but also affecting skin, eyes, lungs, heart and blood vessels, leading to anatomical alteration and functional disability (7). The etiology of RA is very complex and is yet to be explored properly. It has a wide spectrum of clinical manifestations, variability in disease severity, progression and differences in therapeutic response. These heterogeneous phenotypes of RA may suggest that variety of factors can contribute in the development of this complex trait, which includes environmental, hormonal and genetic factors (5). Recently, mounting data of evidence suggest that macrophages play a central role in the pathogenesis of RA, since they generate cytokines that not only initiate inflammation leading to synovitis, but also promote synovitis development contributing to destruction of cartilage and bone. Increased numbers of macrophages in the RA synovial tissue has a significant correlation with the degree of disease activity, including C-reactive protein levels, swollen joint count, synovial lining layer thickness, and joint severity (33). Numerous preclinical and clinical studies indicated that changes in numbers of synovial sublining macrophages and the expression of inflammatory products correlate with clinical improvement (33,34). Therefore, synovial macrophages have great value as a molecular target for disease diagnosis, therapeutic intervention, and a predictive marker of the response to antirheumatic treatment. Accordingly, imaging of lesional macrophages could serve as a biomarker of disease progression and therapeutic intervention.
Imaging of RA using conventional methods such as CT and MRI provides information mainly about structural changes in the involved tissues. Thus, conventional radiography has poor sensitivity in the detection of the inflammatory process that happens in the initial stages of RA. On the other hand, nuclear medicine provides functional assessment of physiological processes using radiolabeled targeting specific elements of the inflammatory process, and therefore has significant potential for timely diagnosis and adequate follow-up of RA. Several single photon emission computed tomography (SPECT) and positron emission tomography (PET) radiopharmaceuticals have been developed and applied in this field (11). Those radiopharmaceuticals mainly include not-targeting specific tracers, such as 99m Tc-MDP and 18 F-FDG, and targeting specific tracers, such as 99m Tc-anti-CD20, 123 I-IL-1ra and 124 I-anti-CD20. Nonetheless, the pitfalls of probes including low specificity, or large size severely hamper their clinical applications (18 of applied activity at 180 min (Fig. 8A). The cell efflux study showed that 99 Tc m -DTPA-CB86 has good cell retention by RAW264.7 cells (Fig. 8A). This accumulation is TSPO specific receptor binding since the rapid cellular uptake of the tracer could be effectively blocked by cold DTPA-CB86 (Fig. 8A), suggesting that labeling has not influenced the ability of CB86 to bind specifically to TSPO. These results warranted the further evaluation of the probe for in vivo TSPO-targeted imaging.
99m Tc-DTPA-CB86 showed good in vivo pharmacokinetics for TSPO targeted SPECT imaging. 99m Tc-DTPA-CB86 exhibited rapid inflammatory ankle accumulation and blood clearance, which are the major advantages of using small molecules as imaging agents compared to large long circulating proteins such as full antibodies or antibody fragments (18). It rapidly localized in the left inflammatory ankles and showed good inflammatory uptake, retention, and inflammatory -to-muscle ratios ( Fig. 9, 10, and 11). The left inflammatory ankles could be clearly visualized with good contrast with good contrast by SPECT at 30-180 min after injection. It is also interesting to find out that the inflammatory uptake of the 99m Tc-DTPA-CB86, and inflammatory to muscle ratio are higher than those of the 18 F-DPA-714, 11 C-DPA-713,and (R)-11 C-PK11195 (2,23). Evaluation of the probe in these RA rats demonstrated that 99m Tc-DTPA-CB86 is a promising agent for TSPO imaging.
In this study, the kidney and liver showed the highest uptake because they are the major organs of metabolism. In agree with previous study (38), radioactivity was found in the lung, heart, intestine, and stomach since these normal organs have moderate TSPO expression. A high expression of the target in normal organ might appreciable influence the imaging results, especially when the target level in the lesion is low. After optimization of spiking doses was administered to saturate the target expression in normal organ, an increase lesion-normal ratio could be achieved (18,29). The in vivo TSPO binding specificity of 99m Tc-DTPA-CB86 was also verified. When 300 µg of unlabeled DTPA-CB86 was co-injected, uptakes in high TSPO expression organs/tissues, such as inflammatory ankle, lung, heart, intestine, and stomach lung, heart, intestine, and stomach, were both significantly reduced (P < 0.05).
According to the European League Against Rheumatism (EULAR), RA should initially be treated with synthetic disease-modifying antirheumatic drugs (DMARDs) in combination with glucocorticoids (39). Moreover, The guides underline the importance of glucocorticoids and reflect convincing evidence for their beneficial effects (39). In this study, RA rats treated with dexamethasone once a day for 2 weeks, their swelling ankles and clinical scores were reduced during the treatment.
99m Tc-DTPA-CB86 accumulation in the left inflammatory ankles at 7th day and 15th day after treatment significantly decreased compared to the control group, especially at 15th day after treatment (Fig. 12). The results indicated that 99m Tc-DTPA-CB86 SPECT may real-time monitor therapy response of anti-inflammatory 28 therapy.

Conclusion
This study demonstrates that 99m Tc-DTPA-CB86 SPECT imaging can identify the activated macrophages in the synovial joints in RA rat models and monitor therapy response of anti-inflammatory therapy. 99m Tc-DTPA-CB86 SPECT may be a useful biomarker as a non-invasive imaging method for clinical management of RA. The immunofluorescence of the activated RAW264.7 cells. Fluorescence imaging of CB86 in the activated RAW264.7 cells. The activated RAW264.7 cells Biodistribution results for 99mTc-DTPA-CB86 in RA rats. Data are expressed as %ID/g at vario Figure 10 SPECT/CT imaging of 99mTc-DTPA-CB86 in RA rat models co-injected with 0 μg dose (unbloc 41 Figure 11 The ratio of the left inflammatory ankle to muscle based on SPECT imaging between 0 μg (un The findings of HE staining (A,B) and immunohistochemistry (C,D) in the left inflammatory an