Osteoporosis is primarily a chronic degenerative skeletal disease characterized by reduced bone mass, destruction of bone microstructure, and increased bone fragility, leading to a high susceptibility to fractures among patients (Author anonymous, 1993). The gradual decline in bone mass with age, coupled with fractures stemming from age-related bone loss, represents a main cause of disability and mortality in elderly individuals, thereby exacerbating the global economic burden of osteoporosis (Clynes et al., 2020). Consequently, aging-related osteoporosis, a pressing public health issue, is becoming increasingly prevalent with the aging global population.

Osteoporosis is a complex disease that results from the interaction between genetic and environmental factors (Schultz and Wolf, 2019). Increasing evidence from clinical studies has demonstrated that inflammatory diseases are frequently accompanied by significant local or systemic bone loss, highlighting a strong connection between inflammation and osteoporosis (Chotiyarnwong and McCloskey, 2020; Raterman et al., 2020; Kanazawa et al., 2010; Mazzaferro et al., 2018; Baldini et al., 2005). Notably, early research has shown that women with postmenopausal osteoporosis exhibit elevated levels of serum inflammatory markers and P2X7 receptor (P2X7R) (Xiao et al., 2019; Wang et al., 2018). Further investigations into the role of adenosine triphosphate (ATP) and its associated receptors have indicated their involvement in inflammatory response. P2X7R, an ATP-gated cation-selective channel, plays a crucial role in both inflammation and host defense, and is widely distributed in osteocytes, osteoclasts, and osteoblasts, where it is pivotal to bone metabolism. Activation of P2X7R may alleviate osteoporosis by regulating the balance between osteoblasts and osteoclasts (Dong et al., 2020; Ma et al., 2021). Additionally, functional polymorphisms in the P2X7R gene have been shown to significantly influence osteoporosis (Husted et al., 2013), identifying P2X7R as a promising therapeutic target for inflammatory osteoporosis (Huang et al., 2023). Moreover, literature also suggests that P2X7R in microglia within the central nervous system plays a role in affect the regulation of bone metabolism (Nakazato et al., 2014), indicating a potential regulatory pathway between the brain and bone.

P2X7R is widely distributed throughout the central nervous system, particularly in microglia and astrocytes, where it is activated by ATP and plays a critical role in neuroinflammation (Jimenez-Mateos et al., 2019). Given the importance of P2X7R in these processes, it has become an attractive target for molecular imaging using Positron Emission Tomography (PET). PET imaging, an extensively used technique, is capable of non-invasive and real-time visualization of molecular changes in vivo through the use of highly selective probes without inducing pharmacological effects. Among the PET probes targeting P2X7R, the most promising tracer is ¹⁸F-JNJ64413739, reported by Kolb et al. (2019), which showed the upregulation of P2X7R in a rat model of epilepsy. In 2019, Michel Koole et al. further investigated its radiation dosimetry and target occupancy in three healthy individuals, indicating its potential for clinical imaging of P2X7R (Koole et al., 2019). However, the probe’s remarkably low synthetic yield (3.1% ± 2.0%) (Kolb et al., 2019), likely due to the use of chloropyridine as a labeling precursor, constrains its further clinical applications.

To date, no studies have reported on the use of P2X7R probes for brain imaging in a rat model of osteoporosis. In this study, we aim to employ a more electron-deficient nitropyridine as a labeling precursor to improve the labeling yield through an aromatic nucleophilic substitution reaction. We will also perform small-animal PET/CT imaging on a rat model of osteoporosis to explore the change of P2X7R in this condition.

¹⁸F-JNJ-64413739 is a promising PET tracer for P2X7R, but its relatively low synthesis yield (3.1% ± 2.0%) limit its broader application and clinical translation. Our group is dedicated to developing novel PET tracers and advancing their preclinical studies. Considering the chemical structure of ¹⁸F-JNJ-64413739 as well as the reaction mechanism of nucleophilic aromatic substitution used for radio fluorination, we hypothesized that using a more electron-deficient nitropyridine as the labeling precursor could enhance the labeling efficiency. This motivated us to choose compound 1 for further exploration. Through optimizing the labeling condition, including the screening of different bases and reaction times, we successfully achieved automated synthesis of ¹⁸F-JNJ-64413739 using the Trasis AllinOne module. The radiochemical purity exceeded 97%, with the highest n. d. c. RCY reached 12%, 3-fold higher than the results obtained with chloropyridine as the precursor using the TRACERlab FX-FN synthesis module (Kolb et al., 2019). However, the robustness of this radiosynthesis remains a challenge, possibly due to the sensitivity of the labeling reaction to environmental humidity. Our team will continue to optimize the synthesis process to enhance the yield stability of the automated production.

Aging is a key risk factors for brain diseases, characterized by a progressive degenerative process often accompanied by chronic low-level inflammation. The ATP-gated P2X7R plays an important role in inflammation and has been implicated in the pathogenesis of several neurodegenerative diseases, including multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, making it a potential therapeutic target (Zheng et al., 2024). Beyond its involvement in neurological conditions, P2X7R is also involved in age-related disease such as cancer, osteoporosis, diabetes and arthritis (Tao et al., 2013; Kvist et al., 2014; Novak and Solini, 2018; Adinolfi et al., 2018), further positioning it as a therapeutic potential in osteoporosis. Studies reported by Juana Maria Sanz and others suggest that targeting P2X7R could help alleviate inflammation-related brain disorders and other age-associated diseases. In elderly Caucasian populations from high-income countries, hypofunctional P2X7R variants might offer protection against prevalent chronic inflammatory diseases, with hypomorphic P2RX7 alleles potentially under positive selection with age (Sanz et al., 2020). The findings indicate that P2X7R is related to both neurological diseases and osteoporosis, suggesting an important role in bridging these conditions, although research in this area remains limited. This study also conducted ¹⁸F-JNJ64413739 PET/CT imaging of osteoporotic rats to provide a molecular imaging foundation for further investigations into the role of P2X7R in the brain-bone axis.

This article analyzed the changes of P2X7R in the brains of osteoporosis rats, and found that compared to normal rats, the radioactivity in the thalamus were significantly increased. Previous studies have emphasized the important role of the thalamus in osteoporosis. Yeh CB et al. and Ito A et al. report that calcitonin can partially reverse ovariectomy-induced hypersensitivity in a rat models and alleviate lower back pain in patients with osteoporosis or neuropathic pain by the modulating receptor or channel expression in thalamus (Yeh et al., 2016; Ito and Yoshimura, 2017). Additionally, other literature has shown that oxidative stress can not only contributes to hyperalgesia in OVX mice but also increase osteoporotic pain by affecting the thalamus (Tu et al., 2020). Within the thalamus, the thalamic reticular nucleus (TRN), a thin shell-like cell band located between the outer medullary lamina and the internal capsule, serves as a crucial pathway for almost all axons connecting the thalamus and cortex. Functioning as a gateway between the thalamus and cortex, the TRN facilitates information exchange within the thalamocortical circuit, owing to its unique neural structure and vital anatomical position (Wang et al., 2021). About the striatum, Liping Zhou et al. showed that icariin, a flavonoid phytoestrogen derived from Herba epimedii, regulated the transcriptional events of estrogen-responsive genes related to bone remodeling and prevented dopaminergic neurons against OVX-induced changes by rescuing expression of estrogen-regulated tyrosine hydroxylase and dopamine transporter in the striatum (Zhou et al., 2020) showed that considerable P2X7R colocalized with microglia after injection of Abeta (1–42) into rat hippocampus in Alzheimer disease (McLarnon et al., 2006). Literature reports that there is a relationship between hippocampus and bone. Hippocampus participates in the balance of the bone-brain endocrine axis (Patricia da Silva et al., 2023) and significant upregulation of doublecortin protein expression was observed in the hippocampus of both calcium- and vitamin D-treated rats (Lapmanee et al., 2023). Microglia, the immune cells in the central nervous system, are widely distributed throughout these regions. Almost all pathological processes in the brain trigger microglia activation. Therefore, when osteoporosis occurs, the upregulation of P2X7R on microglia in the thalamus becomes significant, though this mechanism has been rarely explored in osteoporosis research.

In summary, this study employed the AllinOne module to complete the automated synthesis of the P2X7R imaging agent ¹⁸F-JNJ64413739, with non-corrected synthesis yield up to 12%. This synthetic method is simple and reliable, producing a product meets the standards for clinical injection. Imaging results in osteoporotic rats suggest that ¹⁸F-JNJ64413739 is safe and feasible for use, and the its application holds significant scientific value for exploring the occurrence and progression mechanism of P2X7R-related osteoporosis.