PET Imaging of the P2X7 Ion Channel with a Novel Tracer [18F]JNJ-64413739 in a Rat Model of Neuroinflammation
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The P2X7 receptor, an adenosine triphosphate (ATP)-gated purinoreceptor, has emerged as one of the key players in neuroinflammatory processes. Therefore, developing a positron emission tomography (PET) tracer for imaging of P2X7 receptors in vivo presents a promising approach to diagnose, monitor, and study neuroinflammation in a variety of brain disorders. To fulfill the goal of developing a P2X7 PET ligand as a biomarker of neuroinflammation, [18F]JNJ-64413739 has been recently disclosed.
We evaluated [18F]JNJ-64413739 in a rat model of neuroinflammation induced by an intracerebral injection of lipopolysaccharide (LPS). In vivo brain uptake was determined by PET imaging. Upregulation of neuroinflammatory biomarkers was determined by quantitative polymerase chain reaction (qPCR). Distribution of the tracer in the brain was determined by ex vivo autoradiography (ARG). The specificity of [18F]JNJ-64413739 was confirmed by performing blocking experiments with the P2X7 antagonist JNJ-54175446.
Brain regions of rats injected with LPS had a significantly increased uptake (34 % ± 3 % s.e.m., p = 0.036, t test, standardized uptake value measured over the entire scanning period) of [18F]JNJ-64413739 relative to the corresponding brain regions of control animals injected with phosphate-buffered saline (PBS). The uptake in the contralateral regions and cerebellum was not significantly different between the groups of animals. The increase in uptake of [18F]JNJ-64413739 at the LPS-injected site observed by PET imaging was concordant with ex vivo ARG, upregulation of neuroinflammatory biomarkers, and elevated P2X7 expression levels.
While further work is needed to study [18F]JNJ-64413739 in other types of neuroinflammation, the current results favorably characterize [18F]JNJ-64413739 as a potential PET tracer of central neuroinflammation.
Key wordsP2X7 receptor Neuroinflammation Positron emission tomography (PET) PET imaging [18F]JNJ-64413739 Lipopolysaccharide LPS Rats
Neuroinflammation is thought to play a role in many neuropsychiatric and neurological conditions [1, 2, 3, 4]; however, its exact contribution remains poorly understood [5, 6, 7]. This is partially due to limited diagnostic tools to measure inflammation in the central nervous system (CNS) . Positron emission tomography (PET) enables non-invasive visualization and quantification of molecular targets within CNS . Development of specific PET ligands for key neuroinflammatory targets would allow assessing the role of these targets in different brain diseases and aid selection of appropriate anti-neuroinflammatory treatment .
Recently, several targets have been investigated for PET imaging of neuroinflammation [11, 12, 13, 14, 15, 16, 17, 18]. Specifically, multiple PET tracers binding the 18-kDa translocator protein (TSPO) have been evaluated in psychiatric  and neurodegenerative disorders [2, 19, 20]. There was limited success [21, 22, 23, 24], but also failures [25, 26, 27, 28, 29, 30]. Some failures were due to existing genetic polymorphisms affecting TSPO binding in humans [31, 32, 33]. While this limitation can be corrected [34, 35, 36, 37], the non-specific role of TSPO in neuroinflammation are more difficult to address [9, 38, 39], as TSPO is involved in many cellular functions not specific to central inflammation [40, 41, 42, 43, 44].
In contrast, P2X7 receptor activation lies upstream of a cascade of events leading to microglial activation [45, 46, 47]. P2X7 receptor activation is causally linked to the production of proinflammatory cytokines within the brain [48, 49, 50]. Therefore, P2X7 receptor PET tracers is a promising tool to study central neuroinflammation.
Currently, a few P2X7 receptor PET tracers have been reported [46, 51, 52, 53, 54, 55]. We previously demonstrated that [18F]JNJ-64413739 is suitable to quantify central target engagement of P2X7 therapeutic compounds . Here, we evaluate [18F]JNJ-64413739 as a tool to measure neuroinflammation.
To achieve this goal, we used a rat model of local neuroinflammation induced by intracerebral injection of lipopolysaccharide (LPS). We selected this model because (a) priming of Toll-like receptors (TLR) by LPS is one of the hallmarks of P2X7-mediated release of pro-inflammatory cytokines [57, 58, 59] and (b) local LPS injection results in a spatially confined region of neuroinflammation, while other, less affected regions can serve as reference .
We studied [18F]JNJ-64413739 uptake in the LPS injected, control (PBS-injected), and reference brain regions by in vivo PET imaging. We compared PET imaging results to the results from ex vivo ARG, immunohistochemistry (IHC), and gene expression (qPCR) analysis. We confirmed the specificity of binding of [18F]JNJ-64413739 to P2X7 receptors by performing competition studies with the P2X7 antagonist JNJ-54175446.
Materials and Methods
Details are provided in the electronic supplementary material (ESM).
Selection of LPS Dose and Time Points: Longitudinal Pilot PET Imaging Studies with [18F]JNJ-64413739
Verification of Induced Neuroinflammatory Markers at Day 2 Post-LPS (20 μg): a qPCR Study
Concordance Between In vivo [18F]JNJ-64413739 PET Imaging, [18F]JNJ-64413739 Ex vivo ARG, and Immunohistochemistry of Iba1
In vivo PET Imaging Study with [18F]JNJ-64413739 in Naïve, PBS-Injected and LPS-Injected Rats
To evaluate the uptake of the [18F]JNJ-64413739 by in vivo PET scans, we statistically compared normalized uptake (SUVR, cerebellum as a reference area) of [18F]JNJ-64413739 between the injected and control sites and found significantly higher uptake of [18F]JNJ-64413739 in the LPS-injected site (37 % increase in SUVRs measured over entire scan period, n = 5, p = 0.0152, t test). In contrast, there was no statistical difference between the injected and control sites in PBS-injected group (3 % increase in SUVRs in the injected site measured over entire scan period, n = 5, p = 0.79, t test) and corresponding sites in the naïve group (1.4 % difference in SUVRs measured over entire scan period, n = 4, p = 0.22, t test).
Effect of JNJ-54175446 on PET Imaging with [18F]JNJ-64413739
To demonstrate specificity of increased uptake of [18F]JNJ-64413739 at the LPS-injected site, we performed blocking studies with the previously reported P2X7 antagonist JNJ-54175446 . The results of the displacement study are provided in the ESM.
The results of experiments with JNJ-54175446 support the finding that the [18F]JNJ-64413739 signal at the LPS-injected site is likely specific to P2X7.
Availability of Data and Materials
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.
We evaluated the novel P2X7 antagonist PET ligand, [18F]JNJ-64413739, in a preclinical neuroinflammatory rat model. We demonstrated increased uptake of the PET ligand at the neuroinflammatory site. These findings show promise for [18F]JNJ-64413739 as a PET ligand to study P2X7 receptors in a variety of brain disorders involving neuroinflammation [3, 4]. We acknowledge that local LPS-induced changes in neuroinflammation, as described in our paper, are extreme and most likely do not represent neuroinflammatory changes in chronic CNS diseases; detailed discussion is provided in the ESM. Nonetheless, this model was set up to address some key scientific questions described here. We plan to extend these findings in more physiologically relevant models of chronic neuroinflammation.
In recent years, a few PET tracer candidates to image P2X7 receptors have been proposed , and some were evaluated in preclinical models of neuroinflammation [47, 63, 64]. For example, [11C]GSK1482160 showed increased tracer uptake in both systemic LPS (5 mg/kg) model in mice  and in rats with experimental autoimmune encephalomyelitis (EAE) . These and other recent results [55, 56] are a promising demonstration of the potential of a P2X7 receptor tracer. However, short half-life of carbon-11 may restrict the clinical application of C-11-labeled PET tracers; it would be preferable to use F-18-labeled tracers. Prior to our work, only one F-18-labeled P2X7 receptor tracer, [18F]EFB , has been tested in preclinical models of neuroinflammation. The preliminary evaluation of [18F] EFB was promising; however, limited brain penetration and suboptimal imaging quality may limit the use of this tracer.
We conducted PET scans with [18F]JNJ-64413739 in naïve animals and animals injected either with PBS or LPS (20 μg, 2 days post-LPS). The TACs in cerebellum and contralateral site were the same in all groups of animals (Fig. 4, blue and green colors). PBS-injected sites were indistinguishable from uptake in naïve animals, indicating that the surgery did not lead to increased [18F]JNJ-64413739 uptake. In contrast, we found a statistically significant increase of [18F]JNJ-64413739 uptake in the LPS-injected site relative to the control site (37 % increase in SUVRs measured over entire scan period, n = 5, p = 0.0152, t test). To further confirm target specificity, we showed a significant blocking effect by using P2X7 antagonist JNJ-54175446 . These findings support our proposal that the observed increase of [18F]JNJ-64413739 signal at the LPS-injected site is likely specific to P2X7 receptors. The increased uptake of [18F]JNJ-64413739 is most likely a consequence of LPS-induced activation of microglial cells that express P2X7 receptors, as supported by increased staining with Iba1.
To relate increased uptake of [18F]JNJ-64413739 to neuroinflammation induced by LPS in brain tissue, we measured mRNA levels of neuroinflammatory marker genes Tspo, Aif1, and P2rx7 in both LPS- and PBS-injected brains. As expected, expression levels of all genes were elevated in LPS-injected hemisphere relative to PBS-injected hemisphere (more than 7, 5, 2 times for Tspo, Aif1, and P2rx7, respectively, Fig. 2). Interestingly, we also detected increased mRNA levels of all genes studied, but especially P2rx7, in the contralateral hemisphere of LPS-injected brains. This “spreading of the inflammation” represents a novel observation, at least, for the genes studied. Further studies are needed to determine whether this effect is specific to LPS or is a general neuroinflammatory phenomenon .
To investigate whether increased [18F]JNJ-64413739 uptake corresponds to an increased number of microglial cells post-LPS administration, we compared [18F]JNJ-64413739 uptake and intensity of IHC staining for the microglial marker Iba1. We performed in vivo PET scans, ex vivo ARG, and IHC staining in the same animal and used adjacent brain sections for ex vivo ARG and IHC staining. We found a similar increase in signals at the LPS-injected site relative to the contralateral site: PET scans, ARG, and IHC showed a 29 %, 26 %, and 25 % increase, respectively (Fig. 3, top row). The difference between injected and contralateral site in PBS-injected control rats was smaller: PET scans, ARG, and IHC showed a 4 %, 1 %, and 12 % increase, respectively (Fig. 3, bottom row). Overall, the in vivo PET imaging, ex vivo ARG and Iba1 IHC staining showed concordant results (Fig. 3).
The work presented in this paper demonstrates that (1) the brain regions of rats injected with LPS had a significantly increased uptake of [18F]JNJ-64413739 relative to the PBS-injected controls; (2) the specificity of increased uptake at LPS-injected sites was confirmed by pre-treatment and displacement studies with specific P2X7 antagonist JNJ-54175446; and (3) the observed by in vivo PET increase in uptake of [18F]JNJ-64413739 at the LPS-injected site was concordant with ex vivo autoradiography (ARG), upregulation of neuroinflammatory biomarkers, and elevated P2X7 expression levels. While further work is needed to study [18F]JNJ-64413739 in other models of neuroinflammation, the current results favorably characterize [18F]JNJ-64413739 as a potential imaging biomarker of central neuroinflammation and is an important step toward developing PET tracer to image neuroinflammation in the clinic.
Experimental planning: TB, CX, GC, CH, WZ, HK, AB, AKS. [18F]JNJ-64413739 preparation: GC, CH, WZ. Drugs and formulations: TB, WZ, ML. Animal procedures: TB. Imaging procedures and imaging data analysis: TB. Ex vivo autoradiography and immunohistochemistry: CX. RNA extraction, reverse transcription PCR and quantitative real-time PCR: NT, YH. Figure preparation: TB, CX, YH. Manuscript preparation: TB, AKS, CX, AB, GC, CH, WZ, NT, YH, ML.
Janssen Research and Development, LLC
Compliance with Ethical Standards
Ethics Approval and Consent to Participate
All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (US National Institutes of Health), and the research protocol was approved by the IACUC.
Consent for Publication
The authors declare that they have no conflict of interests
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