Ankylosing Spondylitis PET Imaging and Quantifications via P2X7 Receptor-Targeting Radioligand [18F]GSK14260

doi:10.21203/rs.3.rs-2212995/v1

Purpose

Ankylosing spondylitis (AS) is a chronic inflammatory disease of the axial spine that manifests with various clinical signs and symptoms; however, the quantitative detection of inflammation in AS remains a drawback in clinical settings. We aimed to investigate the feasibility of using a specific P2X7R-targeting ¹⁸F-labeled tracer [¹⁸F]GSK1482160 for positron emission tomography (PET) imaging and the quantification of AS.

Methods

The radioligand [¹⁸F]GSK1482160 was obtained based on nucleophilic aromatic radiofluorination with [¹⁸F]fluoride. Dynamic [¹⁸F]GSK1482160 and [¹⁸F]FDG micro-PET/CT imaging were performed on AS mouse models and age-matched controls. Tracer kinetics modeling was performed using Logan graphical arterial input function analysis and Patlak models to quantify the in vivo expression of P2X7R and the influx rate of [¹⁸F]FDG, respectively. The post-PET tissues were collected for hematoxylin-eosin, immunohistochemical (IHC), and immunofluorescence (IF) staining.

Results

The decay-corrected radiochemical yield (RCY) of [¹⁸F]GSK1482160 was 20–30%; radiochemical purity, ≥ 98%; and molar activity, 55–85 GBq/µmol. [¹⁸F]GSK1482160 PET/CT imaging revealed that the specific binding in the ankle joint and sacroiliac joint (SIJ) of the AS group (BP_ND^ankle = 13.75 ± 2.20, BP_ND ^SIJ = 15.87 ± 3.90) were significantly higher than that of the control group (BP_ND^ankle = 0.14 ± 0.08, BP_ND^SIJ = 0.75 ± 0.48). In contrast, in [¹⁸F]FDG imaging, there was no significant difference in the uptake in the ankle joint and SIJ between the two groups. IHC and IF staining revealed that the overexpression of P2X7R was colocalized with activated macrophages from the ankle synovium and spinal endplate in mice with AS, indicating that quantification of P2X7R may contribute to the pathogenesis of inflammation in human AS.

Conclusion

This study developed a novel P2X7R-targeting PET tracer [¹⁸F]GSK1482160 to detect the expression of P2X7R in AS mouse models and provided a powerful non-invasive PET imaging and quantification for AS.

Micro-PET/CT

Ankylosing spondylitis (AS)

P2X7R

macrophage

Ankylosing spondylitis (AS) is an insidiously progressive and debilitating form of arthritis involving the axial skeleton, particularly the sacroiliac joints (SIJ) [1]. AS is a disease common in young adults, with a peak incidence between the ages of 20 and 30. Approximately 23,000,000 people in the world are affected by AS and the trend continues to increase [2]. SIJ are most often involved in the early stage of AS, and axial bone fusion can occur in the late stage, resulting in disability and deformity, thereby causing a heavy economic burden on the family and society [3]. Although the presence of the major histocompatibility complex class I allele human leukocyte antigen B27 (HLA-B27) accounts for the majority of genetic risk, evidence from recent studies show that it accounts for only 20–25% of total heritability and 40% of genetic risk [4]. Less than 5% of HLA-B27 carriers in the general population suffer from AS [5].

The accurate diagnosis of AS remains a global problem. At present, in clinical practice, the diagnosis of AS is based on evidence from multiple experimental setups, including blood laboratory testing (HLA-B27 and erythrocyte sedimentation rate) and detection of anatomic abnormalities through X-rays, computed tomography (CT), and magnetic resonance imaging (MRI). These need to be combined with a series of severe clinical symptoms of the patient, such as back pain, spinal deformity, and disability for a comprehensive diagnosis [6]. Existing diagnostic methods have limitations, such as a long diagnostic cycle, low accuracy, and inability to quantify. Timely and accurate diagnosis is of great significance to guide the clinical treatment of AS and determine whether the disease process can be delayed with drugs such as non-steroidal anti-inflammatory drugs, disease-modifying anti-rheumatic drugs, and targeted biological agents [7]. Accurate localization and quantitative detection of AS lesions will help to determine whether AS is in the active or stationary phase and whether spinal osteotomy and orthopedic surgery are suitable. Therefore, a new, sensitive, and non-invasive examination method for AS diagnosis is urgently needed. Accurate localization and quantitative detection of AS are of great significance to evaluate the severity and prognosis of the disease.

Positron emission tomography (PET) is an emerging detection technology, and it is now widely used in the early diagnosis, staging, and restaging of various diseases [8]. At present, [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) PET is the most commonly used PET/CT imaging agent in clinics. However, [¹⁸F]FDG lacks specificity because it can be taken up by metabolically active cells [9]. Therefore, it is important to develop more specific molecular PET/CT imaging agents, which not only better understand the biological characteristics of AS, but also have a potential impact on evaluating new clinical drugs against AS, noninvasively and longitudinally. The P2X7 receptor (P2X7R) is an ATP-gated ion channel that can be expressed by various immune cells, including monocyte-derived cell lines, T and B lymphocytes, and macrophages [10]. When the extracellular ATP concentration is high, the P2X7R is activated, which in turn activates the downstream NLRP3 inflammasome, which activates caspase-1, promotes the maturation and secretion of IL-1β, and produces corresponding mature cytokines [11]. Studies have revealed an association between genetic variations in the P2X7R gene and AS susceptibility [12], and that overexpression of the P2X7R may be involved in the progression of AS. In this study, we successfully synthesized a P2X7R-targeting PET probe [¹⁸F]GSK1482160 by nucleophilic aromatic (SNAr) radiofluorination, with significantly improved radiochemical yield (RCY) and radiometabolism stability compared with previous radiosynthesis methods [13]. A mouse spinal arthritis model was constructed with a proteoglycan and dimethyldioctadecylammonium (DDA) adjuvant, and [¹⁸F]GSK1482160 microPET/CT imaging was performed to investigate the feasibility of the P2X7R for AS.

Radiochemistry

The radiosynthesis of [¹⁸F]GSK1482160 was accomplished by a one-pot ¹⁸F-labeling strategy starting with precursor 10 as shown in Fig. 1a. Et4NHCO3 was first reacted with [¹⁸F]KF to form [¹⁸F]Et4NF. Next, SNAr radiofluorination of [¹⁸F]Et4NF with precursor 10 (2.5 mg) in CH3CN (0.3 mL) was transferred to the reaction vial. After the vial was sealed and heated at 120°C for 15 min, the mixture was diluted with 4 mL mobile phase (CH3CN / water containing 0.1% TFA, 35/65, V/V) and injected into a semipreparative HPLC column (Agilent Technologies, Inc. C18, 5 µm, 9.4 × 250 mm) for further purification. The product was detected with Gabi nova radiation detector and was diluted with 30 mL of water, and then the mixture was passed through a Sep-pak C18 plus cartridge to trap the product. After being washed with 3 times 10 mL of water, the product was eluted with 1 mL of ethanol. The solvent of the collected product was evaporated and dissolved in saline for quality control and other experiments. The radiochemical purity (RCP), radiochemical yield (RCY) and identity of [¹⁸F]GSK1482160 was authenticated by a coinjection with the nonradiolabeled standard GSK1482160 on an analytical HPLC system (Shim-Pack, Inc. C18, 5 µm, 4.6 × 250 mm), UV (254 nm), mobile phase: 35% CH3CN: 65% H2O (0.1% TFA), flow rate: 1 mL/min (Fig. 1b).

In vitro stability

In vitro stability of [¹⁸F]GSK1482160 was assessed in both saline and fatal bovine serum (FBS). [¹⁸F]GSK1482160 (1.34 − 3.35 MBq) was incubated in 0.5 mL of saline and FBS at 37°C separately for different time points (0.5, 1, 1.5, 2 and 2.5 h). After incubation, 50 µL aliquots of the saline samples were

measured by radio-HPLC directly. Correspondingly, 50 µL aliquots of the FBS sample were removed and mixed with 100 µL of ethanol to precipitate the serum protein at each time point and centrifuged at 10 000g for 2 min. After centrifugation, the supernatant was filtrated through 0.22 µm filters (Jinteng Co., Ltd., China). Then, the filtrated samples were analyzed using radio-HPLC (Fig. 1c, d).

P2X7R cell binding assay

The in vitro fluorescence assay based on ethidium bromide measurement was used to determine the P2X7R inhibitory activities of GSK1482160 following a previous protocol [14]. Briefly, approximately 2 × 10⁵ human HEK293-hP2X7R cells per well were placed in a 96-well poly-lysine-coated black microplate (Corning Inc., Kennebunk, ME) overnight. The HEK293-P2X7R cells were washed three times with cold phosphate-buffered saline (PBS) before performing the assay. Each well was replaced with 90 µL of assay solution containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (10 mM), N-methyl-D-glucamine (5 mM), KCl (5.6 mM), D-glucose (10 mM), and CaCl₂ (0.5 mM) (pH 7.4) and supplemented with 280 mM sucrose. Next, 10 µL of 60 µM BzATP (final 6 µM) and 10 µL of serially diluted compounds (final concentration 0.3–3 µM) were added to each well. After incubation for 15 min, ethidium bromide uptake was detected by measuring the fluorescence with a BioTek Synergy HTX microplate reader (BioTek Instruments Inc., Winooski, VT), using an excitation wavelength of 540 nm and emission wavelength of 615 nm. The results were expressed relative to the maximum accumulation of ethidium bromide when stimulated with BzATP only, and the IC₅₀ values were determined using nonlinear regression by 3-parameter inhibitor-effect equation in Prism (GraphPad Software, Inc., San Diego, CA).

Animal model of AS

All animal studies were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Mice were randomly divided into two groups: a control group containing healthy mice (n = 12) and an AS model group (n = 12). The AS mouse model was constructed as described in previous studies [15, 16]. Briefly, 100 µg proteoglycan lyophilized powder (Sigma, St. Louis, MO, USA) and 20 mg DDA adjuvant (Sigma) were mixed with 200 µL of normal saline to obtain an emulsion. Next, group of AS mice, each received 200 µL intraperitoneal injections of this emulsion at 0, 21, and 42 days. The group of control mice each received 200 µL intraperitoneal injections saline. The mice from both groups were sacrificed at 0, 8, and 14 weeks, respectively, and spinal and ankle tissues were collected for hematoxylin-eosin (H&E) staining.

Micro-PET/CT procedures for AS

After 14 weeks of modeling, PET/CT imaging studies were performed on the AS group (n = 4) and control group (n = 4). The interval between [¹⁸F]FDG and [¹⁸F]GSK1482160 imaging was one day, and the mice were fasted for more than 6 h before [¹⁸F]FDG imaging. The mice were anesthetized with 1.5–2% isoflurane inhalation, and intravenously injected with 200 µL 3.2 ± 0.32 MBq/mouse [¹⁸F]FDG or [¹⁸F]GSK1482160. A dynamic 0–30 min micro-PET/CT (Mediso nanoScan PET/CT imaging system, Mediso Inc, Hungary) scan protocol was performed immediately after [¹⁸F]FDG or [¹⁸F]GSK1482160 injection. Dynamic PET images consisting of 31 frames (10 × 3, 3 × 10, 4 × 60, 6 × 150, and 8 × 300) were reconstructed with CT attenuation correction and isotope decay correction.

PET data analysis

Original [¹⁸F]GSK1482160 and [¹⁸F]FDG PET/CT images were analyzed using the Carimas 2.10 software (Turku PET center, Finland) to obtain the time activity curve (TAC) for regions of interest (ROIs) based on both PET and CT images. The ankle joints, spine, and muscle regions were hand-drawn using the combined PET/CT image at 30 min. PET data in target regions were expressed as standardized uptake values (SUVs). The volume of interest (VOI) consisting of at least 3 ROIs on the target area was obtained. The VOI was plotted on the target lesion and scanned for 30 min for static PET/CT to obtain SUV_max values, and over 30 min for dynamic PET/CT to obtain the TAC. Logan graphical arterial input function analysis (LoganAIF) [17, 18] was used to estimate the binding potential (BP) of [¹⁸F]GSK1482160 across the ankle and spine. TACs of the left ventricle and muscle were obtained from the original micro-PET data and used for arterial or reference input function. Three-dimensional ROIs and corresponding TACs of the ankle joints and spine were obtained and used for output function. Patlak modeling [19] was used to estimate the influx rate of [¹⁸F]FDG in the ankle and spine. The TAC of the left ventricle was obtained from the original micro-PET data and used for arterial input function. The TAC of the left ventricle, ankle, and spine were used to fit standard Patlak modeling to assess FDG tissue kinetics via the least square regression method using Matlab (MathWorks.Inc) program as we previously reported [20]. The goodness-of-fit was evaluated with R-squared. SUV_max was calculated as the activity concentration within the VOI (Bq/mL) divided by injected activity (Bq) and multiplied by body weight (g).

Arthritis score evaluation and histology

AS severity was evaluated as described in previous studies [21] using a classical scoring system: grade 0 = normal, 1 = slight redness or swelling in the paw, 2 = moderate redness or swelling involving more than one paw, 3 = obvious erythema or swelling in paws and ankles, and 4 = severe swelling of the entire paw and movement disorder. From the third intraperitoneal injection to tissue harvest, two independent researchers measured the double lower limb arthritis scores weekly and accumulated total scores for each mouse (maximum score = 8) to assess the severity of AS, and micro-PET/CT studies were carried out on the 14th week post DDA treatment.

Tissue histology, H&E immunohistochemistry, immunofluorescence staining

The animals were euthanized using the cervical spine dislocation method. Tissues surrounding the spine and ankle joints were carefully dissected to preserve their integrity. Blocks of spine, ankle joint tissues and human posterior longitudinal ligament tissues were sectioned; samples were dehydrated in a graded ethanol series, embedded in paraffin, serially sectioned to generate 5 µm slices, and stained for H&E, immunohistochemistry (IHC) and immunofluorescence (IF). Images of IHC were acquired and edited using a pathological section scanner (3DHISTECH, Budapest, Hungary). Double IF labeling was performed using a rabbit primary antibody for the murine P2X7R (1:100-fold dilution, ALOMONE LABS Inc., Israel) and rat anti-mCD68 (1:50-fold dilution, Abcam Inc., Waltham, MA). Alexa Fluor 488-conjugated goat antirabbit (1:400) and cyanine dye 3-conjugated goat anti-rat secondary antibodies (1:300) were used to visualize these primary antibodies. Images were acquired using a fluorescence confocal system equipped with a Nikon Eclipse E600 microscope and multiple laser diode modules.

Human specimen collection

Specimens from an AS patient who underwent osteotomy and orthopedic surgery and another patient with intervertebral disc herniation were obtained from the Fifth Affiliated Hospital of Sun Yat-Sen University (FAHSYSU). All subjects were recruited and samples were collected under Institutional Review Board (IRB)-approved protocols (ZDWY.JZWK.002) at FAHSYSU. We complied with all applicable requirements of the local and national regulations and obtained informed consent from each subject before the study began. These protocols were reviewed by the IRB of FAHSYSU before transferring and analyzing the samples. The posterior longitudinal ligament tissue was collected aseptically post-operation. After carefully removing the surrounding adipose tissue, bone, blood vessels, etc., the posterior longitudinal ligament tissue was placed in a clean and sterile 50 mL centrifuge tube on ice, washed with PBS 3 times, and frozen in a liquid nitrogen tank for subsequent experiments.

Statistical analysis

Statistical analysis was performed using SPSS 22.0 software (IBM, USA) and GraphPad Prism 8.0 software (GraphPad Software, Inc., USA). Data are presented as mean ± SD or mean ± SEM. Normally distributed data were analyzed by Student t-test, one-way ANOVA, or Mann–Whitney U parameter analysis. p < 0.05 was considered statistically significant.

Radiochemistry

The one-pot radiation synthesis of [¹⁸F]GSK1482160 lasted approximately 1.5 h, the total RCY was 20–30% (decay corrected to end of synthesis, EOS, n > 20), with high radiochemical purity (> 98%, n > 20) and molar activity (> 68 GBq/µmol, corrected for end of synthesis (EOS) decay). It is noteworthy that the total RCY and stability of [¹⁸F]GSK1482160 were superior to those of [¹⁸F]IUR-1601 and [¹⁸F]IUR-1602 as reported by Zheng et al [22, 23]. It is synthesized a novel precursor from ArCF2SO2CF3 to directly obtain fluorine-18 radiosynthesis of GSK1482160 without any changing structure of this ligand, which promises a broad clinical application.

AS mouse model and behavioral scoring

The AS mouse model was evaluated by joint swelling, arthritis score, and histopathology. Eight weeks after constructing the AS mouse model, obvious symptoms of lower extremity joint swelling began to appear, and with the extension of the modeling time, the degree of lower extremity joint swelling gradually increased and peaked at the 14th week (Fig. 2b). Lower extremity joint inflammation was also scored with a similar protocol (Fig. 2c). The control group did not show the aforementioned changes. We used H&E staining to evaluate the histological features of the ankle, spine, and sacroiliac joints in each group. Pathological changes were recorded in the AS group, comprising synovial hyperplasia and inflammatory infiltration of the ankle joint (Fig. 2d), inflammatory infiltration, and damage to the cartilage endplate of the intervertebral disc (Fig. 2e). However, no obvious signs of inflammation were observed in the control group.

Micro-PET/CT imaging ankle joints of the AS

Compared with the control group, significant inflammatory uptake of a developer was observed in the ankle joints in the AS group after [¹⁸F]GSK1482160 injection; however, [¹⁸F]FDG uptake was not significantly different between these two groups (Fig. 3a). The dynamic uptake activity curve of ankles in the two groups was also consistent with the observation in the image (Fig. 3b, c). The SUV_max values (Fig. 3d) and BP_ND^ankle value (Fig. 3f) of [¹⁸F]GSK1482160 in ankle joints in the AS group were significantly higher than that of the control group, the SUV_max values (Fig. 3e) and the influx rates (K_i^ankle) of (Fig. 3g) [¹⁸F]FDG were not significantly different between the two groups. Quantitative analysis revealed a high correlation between [¹⁸F]GSK1482160 uptake in the ankle joints and arthritis scores; however, [¹⁸F]FDG uptake was not associated with arthritis scores (Fig. 3h, i).

Micro-PET/CT imaging spinal joints of the AS

We compared the uptake of [¹⁸F]GSK1482160 and [¹⁸F]FDG in the spine in both groups. A strong focal [¹⁸F]GSK1482160 uptake was observed from coronal, sagittal, and cross-sections 30 min after injection, in the coccygeal vertebra (CV), SIJ, lumbar 6 (L6), and lumbar 5 (L5) of the spine in the AS group with a relatively clear adjacent background; however, there was no significant uptake in the control group. Uptake of [¹⁸F]FDG was approximately the same in both groups (Fig. 4a). The dynamic uptake activity curve of the spine between the two groups was also consistent with the observation in the image (Fig. 4b, c). The SUV_max values of [¹⁸F]GSK1482160 in SIJ of the AS group at each time point were significantly higher than those of the control group (Fig. 4d); however, the SUV_max values of [¹⁸F]FDG were not significantly different between the two groups (Fig. 4e). By comparing the uptake of [¹⁸F]GSK1482160 in various parts of the spine, we observed significant differences in the caudate vertebra, SIJ, L6, and L5 between the AS group and the control group, whereas [¹⁸F]GSK1482160 uptake in the L4 was roughly the same in both groups (Fig. 4f, h). The uptake of [¹⁸F]FDG was not significantly different between the two groups (Fig. 4g, i).

Tracer kinetics

To further quantify the in vivo PET results, 0–30 min kinetic data were characterized for the tracer kinetics of [¹⁸F]GSK1482160 in the ankle and SIJ by LoganAIF modeling (Table. 1). In the AS group, BP_ND^ankle = 13.75 ± 2.20 and BP_ND^SIJ = 15.87 ± 3.09 were significantly higher than that in the control group (Fig. 5). The 0–30 min kinetic data for [¹⁸F]FDG were characterized by Patlak modeling (Table. 2). There was no significant difference in the influx rates (K_i) of [¹⁸F]FDG between these two groups (p ＞0.05).

Histologic analysis from AS mice

Inflammatory infiltration of the ankle joint (green arrows) was the pathological feature of the ankle joints of mice in the AS group; however, there was no corresponding change in the control group. The colocalizations of P2X7R (green), CD68 (macrophages, red), and DAPI (blue) were indicated only in AS group. Histological analysis of mouse ankle joints in [¹⁸F]GSK1482160-positive regions showed an enrichment of immune cells in the ankle and metacarpophalangeal joint regions, and P2X7R (green) was expressed on the surface of CD68-positive (red) macrophages in immune cell enrichment regions (Fig. 6).

Histologic analysis from human specimen

We collected supraspinous ligament specimens from the patient with AS and that with disc herniation for histological examination. Inflammatory infiltration of the supraspinous ligaments (green arrows) was the pathological feature of the AS group; however, there was no corresponding change in the control group. Consistent with what was observed in animal models of AS, colocalizations of P2X7R (green), CD68 (macrophages, red), and DAPI (blue) were indicated only in the patient with AS, whereas there was no corresponding change in the control group. (Fig. 7).

AS is an inflammation-related disease with pathological processes that are difficult to quantify. Inflammation, bone erosion, and syndesmophyte formation may be ongoing for decades; therefore, animal models are ideal for experimental studies. We used a proteoglycan and DDA adjuvant to create an inflammation-associated AS model that mimics the pathological process of AS in patients. The condition began with peripheral joint symptoms, including ankle synovitis, synovial hyperplasia, cartilage erosion, and advanced spinal destruction. Several methods, including clinical-grade analysis and histological techniques, were applied to detect the inflammatory status of AS, which involves the ankle, SIJ, and spine. H&E staining confirmed that the pathological changes in the ankle joint and spine of the mouse AS model were inflammatory infiltration. At the same time, micro-PET/CT imaging showed that [¹⁸F]GSK1482160 had specific high uptake in the ankle and spine of the AS model, which meant that the P2X7R was highly expressed. Similarly, immunofluorescence staining indicated that the highly expressed P2X7R colocalized with macrophages. However, the influx rates of [¹⁸F]FDG into the ankle and spine were not significantly different between the AS group and the control group.

A recent study showed that the P2X7R abundantly accumulates in activated macrophages and the aggregation of macrophages is significantly associated with disease progression in AS [24], which is the same as our results. At present, a molecular probe targeting the P2X7R has been used in nerve and blood vessel PET/CT imaging studies [25]; however, there have been no reports regarding the detection of AS. Therefore, based on previous studies by our group, the P2X7R specific radioligand [¹⁸F]GSK1482160 was successfully used for PET/CT imaging of AS. As for the performance of [¹⁸F]GSK1482160 in AS imaging studies, we observed that with the increase in time, the high uptake of the probe by the ankle can be significantly observed within 15–30 min after injection. Compared with [¹⁸F]FDG, [¹⁸F]GSK1482160 has significantly higher specificity for AS detection in AS models. Similarly, we observed that the P2X7R was also highly expressed in the SIJ of the spine with a gradual decline upward along the SIJ, which is highly similar to the pathological process of AS. This proves once again that our AS model highly simulates AS in clinical settings.

We successfully used [¹⁸F]GSK1482160 and PET/CT imaging to accurately locate and quantitatively analyze the systemic lesions of AS, thus providing a new idea for the clinical diagnosis of AS. In addition, we used the Logan graphic modeling method to analyze the tracer dynamics of [¹⁸F]GSK1482160 and [¹⁸F]FDG. The specificity of [¹⁸F]GSK1482160 in AS detection was much higher than that of [¹⁸F]FDG; this provides potent medical imaging and quantification for future clinical applications. All these results suggest that [¹⁸F]GSK1482160 can reflect the degree of inflammation of an AS model by monitoring the level of P2X7R expressed on macrophages in the ankle and spine.

Additional studies have reported the use of F-18-fluoride, [¹⁸F]NaF PET/CT, and other techniques for the diagnosis and treatment of AS [26]. However, these studies are still in their infancy and need further exploration, which may be the future development direction for AS diagnosis. Our study provides a novel potential molecular biomarker for AS diagnosis and assessment, with the potential to achieve precise localization and early diagnosis of AS in the future. However, there are still some limitations in our study, such as the small number of human clinical samples and the lack of human clinical research. Although the clinical samples of patients with AS are very precious and difficult to collect, more specimens may need to be collected in the future to verify our conclusions. We believe that if this study is applied to clinical research, the precise positioning and quantitative evaluation technology of AS will likely greatly promote the clinical diagnosis and staging of AS. It will also provide a reliable theoretical basis for the formulation of a diagnosis and treatment plan for AS. In the future, our research group will continue to actively promote the clinical translation of [¹⁸F]GSK1482160 and strive to further verify our conclusions on patients with AS as soon as possible. If this assumption is established, it will hopefully help solve the diagnostic problem of AS and may provide new ideas for the treatment of AS.

We successfully developed a novel [¹⁸F]GSK1482160 tracer and PET/CT imaging to accurately locate and quantitatively analyze the systemic lesions in mouse AS models, thereby providing a novel clinical diagnostic method for AS. The Logan graphic modeling indicated that the specificity of [¹⁸F]GSK1482160 in detecting AS was much higher than that of [¹⁸F]FDG and that [¹⁸F]GSK1482160 can reflect the degree of inflammation in AS by monitoring the level of P2X7R expressed on macrophages in the ankle and spine. This provides potent medical imaging and quantification for clinical application in AS. Our research is of great clinical importance to further elucidate the pathogenesis of AS and potential molecular biomarkers.

Acknowledgments

We would like to thank Dr. Chunlei Han (Turku PET center, Finland) and Min Yang for technique supports for the tracer kinetics modeling.

Funding

This work was supported by National Natural Science Foundation of China (81871382, 82150610508,12126603),Guangdong-Macao Science and Technology Innovation Joint Funding Project (0056/2021/AGJ), Zhuhai City Innovation and Innovation Team Project (ZH0406190031PWC), Zhuhai City Industry-University-Research Cooperation Project (ZH22017002210017PWC), and grants from the Department of Science and Technology of Guangdong Province to the Guangdong Provincial Key Laboratory of Biomedical Imaging (2018B030322006).

Competing Interests

All authors have no relevant financial or non-financial interests to disclose.

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Shiyanjin Zhang, Yifan Qiu, Lihua Huang. The first draft of the manuscript was written by Shiyanjin Zhang, Hongjin Jun, Hai Lu and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of FAHSYSU (No. 2019051701 and No. ZDWY. JZWK. 002).

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Consent to publish

The authors affirm that human research participants provided informed consent for publication of the images in Fig. 7a and b.

Wang R, Ward MM. Epidemiology of axial spondyloarthritis: an update. Curr Opin Rheumatol. 2018;30(2):137–43.
Smith JA. Update on ankylosing spondylitis: current concepts in pathogenesis. Curr Allergy Asthma Rep. 2015;15(1):489.
Hwang MC, Ridley L, Reveille JD. Ankylosing spondylitis risk factors: a systematic literature review. Clin Rheumatol. 2021;40(8):3079–93.
Ebrahimiadib N, Berijani S, Ghahari M. Ankylosing Spondylitis. J Ophthalmic Vis Res. 2021;16(3):462–9.
Wang H, Zheng H, Ma Y. Drug treatment of ankylosing spondylitis and related complications: an overlook review. Ann Palliat Med. 2020;9(4):2279–85.
Poddubnyy D. Classification vs diagnostic criteria: the challenge of diagnosing axial spondyloarthritis. Rheumatology (Oxford). 2020;59(Suppl4):iv6–17.
Ritchlin C, Adamopoulos IE. Axial spondyloarthritis: new advances in diagnosis and management. BMJ. 2021;372:m4447.
Tan H, Gu Y, Yu H, et al. Total-Body PET/CT: Current Applications and Future Perspectives. AJR Am J Roentgenol. 2020;215(2):325–37.
Hadad Z, Afzelius P, Sorensen SM, Jurik AG. Clinical relevance of 18F-FDG-PET/CT incidental findings. Dan Med J. 2020;67(10).
Adinolfi E, Giuliani AL, De Marchi E, et al. The P2X7 receptor: A main player in inflammation. Biochem Pharmacol. 2018;151:234–44.
Yue N, Huang H, Zhu X, et al. Activation of P2X7 receptor and NLRP3 inflammasome assembly in hippocampal glial cells mediates chronic stress-induced depressive-like behaviors. J Neuroinflammation. 2017;14(1):102.
Pan Z, Zhang X, Ma Y, et al. Genetic variation of rs7958311 in P2X7R gene is associated with the susceptibility and disease activity of ankylosing spondylitis. Postgrad Med J. 2019;95(1123):251–7.
Yang RY, Gao X, Gong K, et al. Synthesis of ArCF2X and [(18)F]Ar-CF3 via Cleavage of the Trifluoromethylsulfonyl Group. Org Lett. 2022;24(1):164–8.
Jin H, Han J, Resing D, et al. Synthesis and in vitro characterization of a P2X7 radioligand [(123)I]TZ6019 and its response to neuroinflammation in a mouse model of Alzheimer disease. Eur J Pharmacol. 2018;820:8–17.
Shen J, Yang L, You K, et al. Indole-3-Acetic Acid Alters Intestinal Microbiota and Alleviates Ankylosing Spondylitis in Mice. Front Immunol. 2022;13:762580.
Ishikawa LL, Colavite PM, Da RL, et al. Commercial bovine proteoglycan is highly arthritogenic and can be used as an alternative antigen source for PGIA model. Biomed Res Int. 2014;2014:148594.
Logan J, Volkow ND, Fowler JS, et al. Effects of blood flow on [11C]raclopride binding in the brain: model simulations and kinetic analysis of PET data. J Cereb Blood Flow Metab. 1994;14(6):995–1010.
Jin H, Yue X, Liu H, et al. Kinetic modeling of [(18) F]VAT, a novel radioligand for positron emission tomography imaging vesicular acetylcholine transporter in non-human primate brain. J Neurochem. 2018;144(6):791–804.
Patlak CS, Blasberg RG. Graphical evaluation of blood-to-brain transfer constants from multiple-time uptake data. Generalizations J Cereb Blood Flow Metab. 1985;5(4):584–90.
Yang M, Lin Z, Xu Z, et al. Influx rate constant of 18F-FDG increases in metastatic lymph nodes of non-small cell lung cancer patients. Eur J Nucl Med Mol Imaging. 2020;47(5):1198–208.
Yang L, Liu B, Zheng J, et al. Rifaximin Alters Intestinal Microbiota and Prevents Progression of Ankylosing Spondylitis in Mice. Front Cell Infect Microbiol. 2019;9:44.
Gao M, Wang M, Glick-Wilson BE, et al. Synthesis and preliminary biological evaluation of a novel P2X7R radioligand [(18)F]IUR-1601. Bioorg Med Chem Lett. 2018;28(9):1603–9.
Gao M, Wang M, Glick-Wilson BE, et al. Synthesis and initial in vitro characterization of a new P2X7R radioligand [(18)F]IUR-1602. Appl Radiat Isot. 2019;144:10–8.
Akhtari M, Zargar SJ, Vojdanian M, Jamshidi A, Mahmoudi M. Monocyte-derived and M1 macrophages from ankylosing spondylitis patients released higher TNF-alpha and expressed more IL1B in response to BzATP than macrophages from healthy subjects. Sci Rep. 2021;11(1):17842.
Fu Z, Lin Q, Xu Z, et al. P2X7 receptor-specific radioligand (18)F-FTTM for atherosclerotic plaque PET imaging. Eur J Nucl Med Mol Imaging. 2022;49(8):2595–604.
Raynor WY, Borja AJ, Hancin EC, et al. Novel Musculoskeletal and Orthopedic Applications of (18)F-Sodium Fluoride PET. PET Clin. 2021;16(2):295–311.

Table 1

Pharmacokinetic results of [¹⁸F]GSK1482160 by LoganAIF modeling (mean ± SD, n = 4)
	DV	Ic	R²	MSE	DVR	BP
AS ^ankle	2.536 ± 1.27	-5.14 ± 1.42	0.985 ± 0.01	1.042 ± 0.18	14.751 ± 2.20	13.751 ± 2.20
Control ^ankle	0.303 ± 0.06	-3.427 ± 1.77	0.988 ± 0.01	0.941 ± 0.43	1.138 ± 0.08	0.138 ± 0.08
P value	0.013	0.183	0.563	0.681	< 0.001	< 0.001
AS ^SIJ	2.825 ± 1.26	-3.619 ± 2.21	0.996 ± 0.005	0.565 ± 0.36	16.87 ± 3.09	15.87 ± 3.09
Control ^SIJ	0.457 ± 0.11	-1.528 ± 1.43	0.995 ± 0.002	0.811 ± 0.23	1.751 ± 0.48	0.751 ± 0.48
P value	0.009	0.163	0.832	0.294	< 0.001	< 0.001

Table 2

Pharmacokinetic results of [¹⁸F]FDG by Patlak modeling (mean ± SD, n = 4)
	K_i (min^− 1)	Ic	R²	MSE
Control ^ankle	0.046 ± 0.01	0.348 ± 0.36	0.942 ± 0.01	0.218 ± 0.03
AS ^ankle	0.048 ± 0.01	0.589 ± 0.16	0.933 ± 0.03	0.21 ± 0.06
P value	0.580	0.253	0.584	0.810
Control ^SIJ	0.049 ± 0.01	0.614 ± 0.22	0.94 ± 0.03	0.234 ± 0.08
AS ^SIJ	0.048 ± 0.01	0.258 ± 0.03	0.979 ± 0.01	0.115 ± 0.03
P value	0.739	0.018	0.048	0.037

supplementalFiles.docx

Ankylosing Spondylitis PET Imaging and Quantifications via P2X7 Receptor-Targeting Radioligand [¹⁸F]GSK14260

Status:

Journal Publication

Version 1

Abstract

Purpose

Methods

Results

Conclusion

Figures

Introduction

Materials And Methods

Radiochemistry

In vitro stability

P2X7R cell binding assay

Animal model of AS

Micro-PET/CT procedures for AS

PET data analysis

Arthritis score evaluation and histology

Tissue histology, H&E immunohistochemistry, immunofluorescence staining

Human specimen collection

Statistical analysis

Results

Radiochemistry

AS mouse model and behavioral scoring

Micro-PET/CT imaging ankle joints of the AS

Micro-PET/CT imaging spinal joints of the AS

Tracer kinetics

Histologic analysis from AS mice

Histologic analysis from human specimen

Discussion

Conclusion

Declarations

References

Tables

Supplementary Files

Status:

Journal Publication

Version 1