Preclinical characterization of [¹⁸F]D₂-LW223: an improved metabolically stable PET tracer for imaging the translocator protein 18 kDa (TSPO) in neuroinflammatory rodent models and non-human primates

Abstract

Positron emission tomography (PET) targeting translocator protein 18 kDa (TSPO) can be used for the noninvasive detection of neuroinflammation. Improved in vivo stability of a TSPO tracer is beneficial for minimizing the potential confounding effects of radiometabolites. Deuteration represents an important strategy for improving the pharmacokinetics and stability of existing drug molecules in the plasma. This study developed a novel tracer via the deuteration of [¹⁸F]LW223 and evaluated its in vivo stability and specific binding in neuroinflammatory rodent models and nonhuman primate (NHP) brains. Compared with LW223, D₂-LW223 exhibited improved binding affinity to TSPO. Compared with [¹⁸F]LW223, [¹⁸F]D₂-LW223 has superior physicochemical properties and favorable brain kinetics, with enhanced metabolic stability and reduced defluorination. Preclinical investigations in rodent models of LPS-induced neuroinflammation and cerebral ischemia revealed specific [¹⁸F]D₂-LW223 binding to TSPO in regions affected by neuroinflammation. Two-tissue compartment model analyses provided excellent model fits and allowed the quantitative mapping of TSPO across the NHP brain. These results indicate that [¹⁸F]D₂-LW223 holds significant promise for the precise quantification of TSPO expression in neuroinflammatory pathologies of the brain.

Introduction

Neuroinflammation, characterized by an inflammatory response in the nervous system caused by pathological conditions such as infection, trauma, and toxin accumulation, is a critical process in many brain diseases, including stroke, Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis, Huntington’s disease (HD), migraine, epilepsy, multiple sclerosis (MS), spinal cord injury, and depression [1, 2]. This inflammatory response is mediated mainly by cytokines, chemokines, and reactive oxygen species produced by various cells, including microglia, astrocytes, endothelial cells, and peripherally derived immune cells [3–5]. Microglia play a central role in central nervous system (CNS) inflammation. As innate immune cells, microglia play a role in primary immune surveillance and macrophage-like activities within the CNS. They undergo proliferation and accumulation at lesion sites, actively monitoring the CNS microenvironment. Microglia are crucial for maintaining neuronal homeostasis, promoting neuronal growth, pruning excessive synapses for optimal neuronal plasticity, and eliminating cellular debris, protein aggregates, and various pathogens. In response to pathological stimuli, microglia can rapidly transform into reactive microglia, which not only provide neuroprotection but also release proinflammatory cytokines (interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α)), chemokines (CCL2, CCL5, CXCL1), and reactive oxygen species, contributing to neuronal damage. Investigating the behavior and function of microglia in various CNS disorders is essential for obtaining a deeper understanding of neuroinflammatory processes and developing therapeutic interventions [6–8].

The translocator protein 18 kDa (TSPO) is an integral membrane protein composed of 169 amino acids. Its structure encompasses five transmembrane α-helical domains, and it is located predominantly in the contact site between the outer and inner mitochondrial membranes of mitochondria [9]. This protein is expressed across various tissues and is evolutionarily conserved, indicating its critical role in numerous biological processes related to mitochondrial bioenergetics, such as the regulation of steroidogenesis, apoptosis, cellular respiration and oxidative processes, mitochondrial metabolism, immunomodulation, and ion transport [10]. Additionally, TSPO is upregulated in activated microglia in various CNS pathologies and psychiatric diseases. In contrast, TSPO levels remain low in the normal brain. This characteristic indicates that TSPO can serve as a biomarker of activated microglia and as a candidate for new diagnostic and therapeutic targets for CNS diseases [11, 12]. Positron emission tomography (PET), a noninvasive imaging technique that provides diverse biological information for various targets, is pivotal in diagnosis, therapeutic evaluation, and drug development [13–15]. Several TSPO-targeted radioligands have been developed for neuroimaging applications [16]. In 1984, the first-generation radioligand [¹¹C]-PK11195, a class of isoquinoline carboxamide, was radiolabeled with carbon-11 and widely used in PET imaging for various brain diseases, including Parkinson’s disease [17], dementia [18], and cerebral small vessel disease [19]. However, the use of [¹¹C]-PK11195 is limited by its weak signal-to-noise ratio, hampering its quantification owing to its high lipophilicity and the short half-life of carbon-11 [20]. To address these limitations, second-generation TSPO-targeted radioligands were designed to maintain a high affinity for TSPO with lower lipophilicity and utilize fluorine-18, which has a longer half-life (109.7 min) than carbon-11, for radiolabeling [21]. Second-generation TSPO radioligands, such as [¹⁸F]PBR111, [¹⁸F]FDPA, and [¹⁸F]FEMPA, present higher TSPO-specific signals than first-generation probes do. However, a drawback is that these second-generation radioligands are sensitive to the TSPO single nucleotide polymorphism (SNP) rs6971 [22]. This rs6971 SNP can cause individuals with the same TSPO density but different genotypes to exhibit varying PET signals, limiting the consistency of the results. Therefore, third-generation TSPO radioligands have been designed to exhibit low sensitivity to rs6971. [¹⁸F]LW223, an ¹⁸F-labeled analog of PK11195, was initially developed for detecting macrophage-driven inflammation [23]. Unlike its predecessors, [¹⁸F]LW223 is not susceptible to genetic polymorphisms. Previous studies conducted by our research team have demonstrated its potential for accurate and sensitive quantification of TSPO in the monkey brain, with promising initial assessments in the human brain [24]. Notably, during these investigations, we serendipitously observed high nondisplaceable uptake in the axial skeleton of nonhuman primates, which may have been caused by defluorination in vivo.

Deuterium (D), a stable hydrogen isotope (H), is nontoxic and nonradioactive. Deuterated drugs contain deuterium atoms, representing an important strategy for improving the pharmacokinetics of existing drug molecules due to the closer junction of the carbon‒deuterium bond (C‒D) compared with the conventional carbon‒hydrogen bond (C‒H). This proximity may impart greater metabolic stability to deuterated drugs because greater energy is required when the drug is transferred to reach the transition state or dissociation limit [25]. In nuclear molecular imaging, radiometabolites can confound imaging results and diminish imaging quality due to off-target effects. Deuterating pharmaceuticals might be a useful strategy for improving metabolic resistance and minimizing the formation of radiometabolites [26–28]. Given that deuterated drugs may be beneficial to the original drug, we hypothesized that deuterated [¹⁸F]LW223 could decrease undesirable uptake in the axial skeleton, serving as a metabolically more stable analog (Fig. 1a). Our objective was to synthesize and evaluate the pharmacokinetics of [¹⁸F]D₂-LW223, the deuterated form of [¹⁸F]LW223. Moreover, we plan to conduct preliminary evaluations to assess the feasibility of [¹⁸F]D₂-LW223-PET for detecting inflammation in rodent models of brain disease and for brain quantification in nonhuman primates.

Fig. 1. Chemical structures and radiosynthesis of TSPO ligands.

(A) LW223 (compound 1) and D₂-LW223 (compound 2). b Radiosynthesis of [¹⁸F]D₂-LW223, data were shown as mean ± SD.

Materials and methods

Chemistry

All commercial compounds were available from Sigma-Aldrich and used without further purification. D₂-LW223 and LW223 standard compounds and their labeling precursor were synthesized in house. Sep-Pak® Light Accell™ QMA and Sep-Pak® Light C18 cartridges were purchased from Waters. Cathivex GV 0.22 µm sterile filters were supplied from Millipore.

[¹⁸F]D₂-LW223 and [¹⁸F]LW223 were prepared using the TRACERlab® FX2 N module (GE, USA) or in an AllInOne® synthesis module (Trasis, Belgium). ¹⁸F-Fluoride, obtained by irradiating H₂¹⁸O with a proton beam generated from a Minitracer Qilin 10.0 MeV cyclotron (GE, USA), was transformed to the modules and trapped on the preconditioned QMA solid-phase extraction cartridge. The cartridge was washed with a solution composed of TEAB (6 mg in 0.7 ml of MeCN and 0.3 ml of ultrapure water) in the reactor and was azeotropically firstly dried at 65 °C, and then dried at 100 °C with portions of anhydrous MeCN (1.0 ml) added during the process. The precursor solution (4 mg in 1 ml of MeCN) was added to the reactor which was sealed and heated for 10 min at 100 °C. Then after adding MeCN (1.5 ml) and water (1 ml) to the reactor for diluting, the solution was purified by a high-performance liquid chromatography (HPLC) equipped with CAPCELL PAK C18 UG80 5 μm, 10 × 250 mm (Osaka soda, Japan) with the mobile phase of MeCN/water (70:30 v/v) and the flow rate of 3 ml/min. The fraction needed was collected at the retention time of 14 min, diluted with about 100 ml water, and passed through a preconditioned C18 solid-phase extraction cartridge, which was then washed with water (10 ml). Then the product was eluted with ethanol (1.0 ml), and diluted with saline containing ascorbic acid (Vc saline, 1% w/w, 11.5 ml). Finally, this solution was passed through a GV sterile filter (0.22 µm) into a sterile vacuum bottle to yield a sterile [¹⁸F]D₂-LW223 or [¹⁸F]LW223 solution ready for injection.

Animals

LPS-induced neuroinflammatory models

The animal experiments were carried out following ethical rules of Emory university under approved IACUC protocols. A mouse model involving intracerebroventricular administration of LPS (i.c.v. LPS model) has been extensively utilized for evaluating neurotracers. This model is recognized for its ability to induce a robust focal inflammatory lesion. CD-1 Mice (n = 4) were treated with unilateral stereotaxic (coordinates: −2.6 mm dorsal/ventral, −1.5 mm lateral, and −0.2 mm anterior/posterior from bregma) injection of 10 µg of LPS (in 2 μL saline) and saline (sham) by using a 2 μL Neuros™ microsyringe (Hamilton, USA) and injection rate of approx. 0.5 μL/min [29]. All procedures were approved by the Animal Care and Use Committee (No. PROC00021845).

Ischemic rat models

Six adult Sprague Dawley rats (Male, over eight-week-old, weighting 250–300 g) were housed at a constant temperature of 22 ± 2 °C maintained on a 12–12 h light-dark cycle (7:00 am–7:00 pm) and provided with food and water ad libitum. Animals were anesthetized with isoflurane (1.5%–2% in air). Temperature was maintained at 37 °C throughout the surgery using a self-regulating heating blanket.

Endothelin-1 (ET-1, GL biochem, Shanghai) was dissolved in sterile saline to 0.2 μg/μL were delivered near the rat middle cerebral artery (MCA) by stereotaxic injection as previously described with minor modification [30]. Briefly, to cover the MCA as much as possible, a total of 5 μL of ET-1 was injected at two depths (AP: +0.9 mm, ML: +5.2 mm, DV: −8.1/−8.3 mm) at the speed of 0.5 μL/min by a micro infusion pump. The needle was left in situ for 8 min after the injection before being slowly removed. After the surgery, the animals were resuscitated on a heated mat at 37 °C and returned to a clean cage after full recovery. All animal procedures were approved by the Animal Care and Use Committee at Jinan University with approval No. IACUC-20220905-11.

Cynomolgus monkeys

Experiments were implemented with three cynomolgus monkeys (6.3 ± 1.2 years, 6.12 ± 0.86 kg, male). The procedures of using these animals were in accordance with ethical guidelines of the Animal Care and Use Committee of Guangdong Landau Biotechnology Co. Ltd., which has been approved by the institutional Animal Care and Use Committee (IACUC) with approval No. LDACU 20200323-01. Previously confirming regular quarantine and health status of experimental cynomolgus monkey and fasting for 12–15 h, the animals were continuously conducted to a physiological monitor and vital signs (heart rate, blood pressure, respirations, SPO2, EKG, ETCO2, and body temperature) during each imaging scan. Monkeys #1, #2, and #3 each underwent 2 baseline dynamic PET scans and 1–2 blocking (PK11195 at a dose of 10 mg/kg) dynamic PET scans with simultaneous arterial blood sampling (Table S1).

Radioligand binding assay of LW223 and D₂-LW223

The affinity of LW223 and D₂-LW223 for the TSPO was evaluated by use of a membrane-binding assay with [³H]PK11195 as the radioligand and rat kidney tissue similar to a literature procedure [31]. Compounds were dissolved in DMSO (10 mM) and diluted in Tris assay buffer to create 11 half-log dilutions from 10 µM to 0.1 nM. [³H]PK11195 was diluted with Tris buffer. Typically, the assay concentration of [³H]PK11195 is a value between one half the K_D and the K_D of a particular radioligand at its target. Reactions were incubated at room temperature for 1.5 h, then harvested by rapid filtration onto Whatman GF/B glass fiber filters. After washing with chilled Tris buffer, filters were dried overnight, and EcoScint scintillation cocktail was added for liquid scintillation courting. Raw data (dpm) representing total [³H]PK11195 binding (i.e., specific and nonspecific binding) were plotted against the logarithm of the molar concentration of the competitor (i.e., LW223, D2-LW223 and Ro5-4864 as control) using GraphPad Prism and fitted using one-site K_i model.

In vitro stability

[¹⁸F]D₂-LW223 solution obtained by automatic synthesis was placed at room temperature for 30 min, 2 h, and 4 h, and radiochemical purity was confirmed by the analytical HPLC system.

Measurement of LogD_7.4

LogD_7.4 of [¹⁸F]D₂-LW223 was measured by partition between n-octanol and 1 × phosphate-buffered saline (1 × PBS, pH 7.4) at room temperature. Briefly, an aliquot of 15 µCi of [¹⁸F]D₂-LW223 was added to a 15 mL microtube containing 1 × PBS (3.00 ml, 3 g, pH 7.4) and n-octanol (3.65 ml, 3 g). The mixture was vortexed for 30 s and then centrifuged at 3500 rpm for 5 min. A Subsample of the 1-octanol (0.6 ml × 3) and 1 × PBS (0.6 ml × 3) layers were transformed into a pre-weighed tube for being evaluated by gamma counter after weighing. This process was repeated until consistent LogD_7.4 values were obtained, with three consecutive equilibration procedures being performed for each LogD_7.4 measurement. The count per minute (CPM) of each sample was measured with an automatic gamma counter (WIZARD2 2480, PerkinElmer, USA). The LogD_7.4 was calculated as follows:

$${\mathrm{Log}D}_{7.4}=\log \left[\mathrm{cpm}/\mathrm{g}\left(\mathrm{n}-\mathrm{octanol}\right)÷\mathrm{cpm}/\mathrm{g}\left(\mathrm{PBS}\right)\right]$$

Measurement of unbound fraction ($f_{\mathrm{u}}$) in plasma

Arterial blood (2 ml) from cynomolgus monkeys was collected, then centrifuged for 3 min at 4 °C (14,000 rpm) (SCILOGEX, USA) to obtain plasma, which then was mixed with 150 µL [¹⁸F]D₂-LW223 solution. After being incubated for 5 min at ambient temperature, the sample was vortexed 30 with Cold PBS (300 µL, pH 7.4, 5 °C). The sample (ca. 400 µL) was then loaded onto the reservoir of an Amicon® Ultra-0.5 ml device (Merck Millipore Ltd. Tullagreen, Carrigtwohill, Co. Cork, IRELAND) in quadruplicate and centrifuged at 14,000 × g for 20 min at 4 °C. After that, the filter was inverted and centrifuged again at 1000 × g for 2 min at 4 °C in a new centrifuge tube. Each aliquot was collected and weighed, and counts were determined with an automatic gamma counter (WIZARD2 2480, PerkinElmer, USA). The f_u value was calculated as follows:

$$f_{\mathrm{u}}=1-\left(\mathrm{CPM}\mathrm{of}\mathrm{filter}\mathrm{membrane}\right)/\left(\left(\mathrm{CPM}\mathrm{of}\mathrm{filter}\mathrm{solution}\right)+\left(\mathrm{CPM}\mathrm{of}\mathrm{concentrated}\mathrm{solution}\right)+\left(\mathrm{CPM}\mathrm{of}\mathrm{filter}\mathrm{membrane}\right)\right)\times 100\%$$

MicroPET imaging in rodent models

LPS-induced neuroinflammatory mice model imaging study

The microPET scans were carried out with a Genisys 8 PET scanner (Sofie Biosciences, Culver, CA, USA). The model group (n = 4) and sham group (n = 4) were scanned under anesthesia with 1%–2% (v/v) isoflurane. The radioligand (ca. 45 µCi/100 µL) was injected into the tail vein via a preinstalled catheter. The PET dynamic scan was acquired for 60 min and reconstructed using G8 software (Analysis Tools and System Setup/Diagnostics Tool, Sofie Biosciences). The PET images were analyzed using PMOD 4.3 (PMOD technology, Switzerland, License ID: 2595). The V_T values for each brain region was determined.

Ischemic rat model imaging study

The microPET study was performed using an IRIS small animal PET/CT imaging system (inviscan SAS, Strasbourg, France) 10 days (baseline) and 11 days (blocking with 5 mg/kg PK11195) after ischemic surgery for ischemic rats (n = 3). PET data were reconstructed with a three-dimensional ordered-subset expectation-maximization (3D-OSEM) algorithm with a Monte-Carlo based accurate detector model, which provides an 80 mm transaxial field of view (FOV), and a 94 mm axial FOV for imaging. Rats were anesthetized with 2%–3% (v/v) isoflurane during the scans, and their body temperatures were maintained at 37.5 °C (Temperature regulation unit, Minerve equipment veterinaire, Marne, France). [¹⁸F]D₂-LW223 (15–20 MBq) was intravenously injected via the tail vein, and a 60-min list-mode emission scan was carried out immediately. The time frame reconstruction was as follows: 20 s × 6 frames, 30 s × 8 frames, 1 min × 4 frames, 2 min × 5 frames, 4 min × 5 frames and 5 min × 4 frames. For blocking experiments, PK11195 (5 mg/kg), solved in 1 ml of water containing 5% DMSO and 60% PEG 400, was pre-injected 1 min before [¹⁸F]D₂-LW223 administration. Data modeling for PET scans was performed on sinograms, which were using three-dimensional changed into two-dimensional sinograms by Fourier rebinning. Dynamic image reconstruction was done by filtered back-projection using Hanning’s filter with a Nyquist cut-off frequency of 0.5 cycles/pixel. PET images were analyzed using PMOD 4.0 (PMOD technology, Switzerland, License ID: 5645) with a high-resolution magnetic resonance imaging (MRI) template. The regions of interest (ROIs) were manually placed on the infarcted areas on the ipsilateral side defined on a summation static image for each experiment. The ipsilateral ROI was copied and symmetrically pasted into the contralateral side on the same slice to yield a contralateral ROI of identical volume and shape. A time–activity curve for each brain region was determined.

Immunofluorescence staining

LPS-induced neuroinflammatory mice model

The mice were perfused with saline and fixed by 4% paraformaldehyde in sodium phosphate buffer (PBS). The brain tissues were removed from the skull and stored in a 30% sucrose solution. The brains were embedded with Optimal Cutting Temperature compound (OCT) and cut into 10 μm thick sections with a cryostat microtome (RWD FS800; RWD Life Science Co., Ltd, China). The slices were permeated with Triton X-100 (in TBST) and blocked with 5% bovine serum albumin at room temperature for 1 h. The brains sections were incubated with TSPO (ab109497, Abcam, 1:1000), Iba-1(PA5-143572, Thermo Fisher Scientific, 1:1000), and GFAP (PA1-10004, Thermo Fisher Scientific, 1:50) overnight at 4 °C. The brain slices were washed three times with PBS after incubation with the primary antibodies. Subsequently, the sections were incubated with secondary antibody Alexa Fluor^® 488 (ab150113, Abcam, 1:500) and Alexa Fluor^® 647 (ab150175, Abcam, 1:500) for 1 h at room temperature and then washed with PBS three times. The sections were then stained with DAPI staining solution (Thermo Fisher Scientific, P36935). Finally, fluorescence images were obtained using fluorescence microscopy (Nikon SoRa SD, USA), and the results were analyzed using ImageJ 1.50 software (National Institutes of Health, Bethesda, MD, USA).

Ischemic rat models

Brain cryosections with a thickness of 8 μm were obtained 10 days after ischemic surgery for ischemic rats (n = 3). During the fixation process, the tissue is first allowed to equilibrate at room temperature for 10–20 min, then fixed with 4% formaldehyde for 15 min, and washed three times with phosphate-buffered saline (PBS), each for 5 min. Then permeabilized with 1% Triton X-100 at room temperature for 20 min, and again washed three times with PBS. For blocking, after briefly drying the slices, a barrier pen is used to draw around the tissue to prevent antibody leakage, then dried off PBS, and blocked with 2% BSA for 30 min, followed by two PBS washed. For primary antibody incubation, a pre-diluted primary antibody solution (Iba-1 1:1000, Wako019-19741; 1:1000 TSPO, Abcam ab109497) is added to the slices to completely cover the samples, and then incubated overnight at 4 °C in a humidified chamber. The next day, after briefly drying the slices, a pre-diluted fluorescent secondary antibody, goat anti-mouse/goat anti-rabbit (1:1000, ThermoFisher), was added to the brain tissue and incubated in the dark at 37 °C for 60 min. All brain tissue slices were mounted with medium containing 4′,6-diamidino-2-phenylindole (DAPI) and then observed with a confocal microscope (Olympus FV3000) and TissueFAXS (TissueGnostics, Vienna, AUT).

NHP imaging data acquisition

MRI scan

All cynomolgus monkeys have received a brain MRI scan before PET imaging. After anesthetized with ketamine (10 mg/kg, i.m.), the cynomolgus monkeys were conducted a 3.0 T discovery MR 750 (GE Discovery 750, Milwaukee, USA) scan to get anatomical images via brain coil (8 HRBRAIN). The parameters for 3D Bravo T1 sequence include: repetition time = 8.4 ms, echo time = 3.5 ms, slice thickness = 0.5 mm, matrix size = 300 × 300, FOV = 15× 15 cm.

Brain dynamic PET scan and processing

Baseline NHP brain images were acquired using a PET/CT system (GE Discovery Elite 690, USA). After previous CT images were acquired, 60 min dynamic PET scans were acquired alongside intravenous bolus injection of [¹⁸F]D₂-LW223. For the blocking study, a 60 min dynamic scan of 10 mg/kg PK11195 administered via an intravenous catheter 1 min before tracer injection was performed. The parameters of CT and PET data collecting include a slice thickness of 3.27 mm, slice interval of 3.75 mm, and matrix size of 256 × 256. The cynomolgus monkeys were anesthesia-inducted with ketamine (i.m., 0.1–0.2 ml/kg) and with atropine (i.m., 0.1 ml/kg), and then maintained with 2% isoflurane and 98% oxygen. The femoral artery of each cynomolgus monkey was cannulated for blood sample collecting. For each scan, radioactivity of 0.3–0.5 mCi/kg was injected as an i.v. bolus and PET data were collected for 100 min in 3-D mode. The raw data were reconstructed to the sequence setting to 12 × 5 s, 6 × 10 s, 8 × 30 s, 4 × 1 min, 5 × 2 min, 5 × 4 min, 10 × 6 min.

Normalized with the open template of NHP by SPM12 [32, 33], the individual brain MRI template and the corresponding PET images were co-registered by PMOD 4.0 (PMOD technology, Switzerland, License ID: 5645) for generating the volumes of interest (VOIs) and time-activity curves (TACs), including the frontal cortex, temporal cortex, parietal cortex, occipital cortex, insula cortex, striatum, hippocampus, thalamus, amygdala, cingulate, globus pallidus, corpus callosum, and cerebellum. Standard uptake values (SUVs) were calculated as SUV = [(VOI activity) ×(body weight)]/injected dose.

Bone uptakes derived from static NHP whole-body PET scan

Whole-body PET images, generally from the top of the skull to mid-thigh, were acquired 60 to 70 min after intravenous injection of [¹⁸F]D₂-LW223 and [¹⁸F]LW223 at a dose of 0.03–0.05 mCi/kg body weight. The head position was fixed with a stereotactic frame. The body of the subject was in the supine position. CT and PET data were collected with a slice thickness of 3.27 mm, slice interval of 3.75 mm, matrix size of 256 × 256 and scan FOV of 70 cm in 3D time-of-flight (TOF) mode. CT data acquired for attenuation corrections were reconstructed in standard mode with DFOV of 30 cm and window width/window level 100/45, with advanced statistical iterative reconstruction of 40%. The PET data were attenuation-corrected by integrated CTAC technology. The PET/CT data were analyzed with the PMOD 4.0 (PMOD technology, Switzerland, License ID: 5645). Using the aortic arch as the reference region, the average SUVr of the lumbar vertebra 3–5 and humerus head, which was decay corrected back to the radioligand injection time point, was quantitatively extracted based on individual NHP scans.

Blood sampling and processing

Arterial blood samples were collected at a series of time points (10, 20, 30, 40, 50, 60, 75, 90, 105, 120, 150, 180, 210 s, followed by 5, 10, 15, 20, 30, 45, 60, 75, 90 min) during the PET scans consistently with previous study [24]. Among them, a total of 6 blood samples at the time points of 2, 5, 10, 30, 60, and 90 min were drawn in 2 ml whole blood respectively for plasma metabolite analysis, while the rest were 0.5 ml. The solution of Heparin and saline (100 IU/ ml) was used to prevent arterial blood coagulation during sampling. 100 µL of whole-blood and plasma were counted with a correction of radioactivity decay from the injection time using a gamma counter (WIZARD2 2480, PerkinElmer, USA) to obtain the whole-blood and plasma time-radioactivity curves. Whole-blood samples in centrifuge tubes were centrifuged at 6000 r/min and 4 °C for 5 min in a KDC-140HR centrifuge (Zhongke Zhongjia, Co., Ltd., China) to separate the plasma.

As for plasma metabolite analysis, each plasma sample (collected at 2, 5, 10, 30, 60, 90 min) was transferred into a new centrifuge tube mixed with 250 µL of MeCN, and centrifuged at 14,000 r/min and 4 °C for 3 min. Repeating until no visible precipitate, which is a symbol of completed deproteinization, the final supernatant was filtered and co-injected with D₂-LW223 standard into a semipreparative HPLC system (column: CAPCELL PAK C18 UG80 5 μm, 10 × 250 mm; mobile phase: 90% MeCN and 10% water; flow rate: 3 ml/min). The eluent fractions were collected with an automated fraction collector (30 s for each section) and counted with the automatic γ-counters. The percentage of [¹⁸F]D₂-LW223 (standard UV peak as an indicator on HPLC) to total radioactivity was calculated as follows: [CPM for UV peak area/(total CPM)] × 100%.

Kinetic modeling

Kinetic modeling of [¹⁸F]D₂-LW223 was conducted using a one-tissue compartment model (1TCM), two-tissue compartment model (2TCM), and Logan graphical analysis (LGA) with the arterial plasma input function corrected by radio-metabolites to calculate the kinetic rate constants (K) and the total volume of distribution (V_T) in different brain tissues [34]. The Akaike information criterion (AIC) [35] was used to compare the goodness of fit using the 1TCM and 2TCM which lower AIC was indicative of a better model. The non-displaceable volume of distribution (V_ND) and TSPO occupancy by 10 mg/kg PK11195 were calculated using the Lassen plot. The average baseline (n = 6) and blocking V_T (n = 4) estimated from 2TCM were used for the Lassen plot [36].

Statistical analysis

All results data were analyzed using GraphPad Prism (version 8.0.1). Continuous variables were presented as mean ± standard deviation (SD). The t-test was used for calculating differences between [¹⁸F]LW223 and [¹⁸F]D₂-LW223. The strength and direction of associations between SUV and V_T were assessed by linear regression. In addition, Pearson correlation was used to evaluate the associations between V_T estimated by 2TCM and Logan Plot. A P value smaller than 0.05 was considered statistically significant.

Results

Pharmacological properties and radiochemistry

Substitution of the two hydrogen atoms on the carbon atom adjacent to the fluorine ion in LW223 (1) with deuterium yielded D₂-LW223 (2), the structure of which is depicted in Fig. 1a. An in vitro competition-binding assay demonstrated that this deuterated TSPO ligand 2 exhibited enhanced binding affinity, with a K_i of 2.14 nM, compared with that of LW223 (K_i = 5.40 nM), as determined via [³H]PK11195 in our radioligand competition binding assay (Fig. S1a and S1b). The off-target pharmacological profile of ligand 2 is presented in Figure S1c. Following these findings, [¹⁸F]D₂-LW223 was synthesized as illustrated in Fig. 1b via the GE TRACERlab® FX2N synthesizer or the Trasis AllinOne® synthesis module. The radiosynthesis was completed in 70–80 min, achieving an average nondecay-corrected radiochemical yield (n.d.c. RCY) of 33% ± 3.5%, with radiochemical purity exceeding 99% and a molar activity of 3.9 ± 1.5 Ci/μmol (n = 15). The identity of the radiolabeled compound was verified by coinjecting it with the corresponding standard Compound 2 via analytical HPLC.

As shown in Fig. S2, no unexpected radioactive peaks were detected over at least two half-lives, confirming the excellent stability of the formulated [¹⁸F]D₂-LW223. Lipophilicity, an empirical indicator of a ligand’s ability to permeate the blood‒brain barrier (BBB) via passive diffusion, was assessed. The LogD_7.4 of [¹⁸F]D₂-LW223 was determined to be 2.00 ± 0.004 (n = 3), which is lower than that of [¹⁸F]LW223 (2.31 ± 0.13) and falls within the optimal range for CNS-targeted PET imaging (1 < LogD < 3) [37]. Additionally, when the ultrafiltration method was used, the unbound fraction (f_u) of [¹⁸F]D₂-LW223 was found to be 3.70% ± 0.52% (n = 3), which was lower than that of [¹⁸F]LW223 (5.80% ± 1.42%).

Plasma metabolite study and bone uptake analysis

At 60 min postadministration, [¹⁸F]D₂-LW223 exhibited a parent fraction of 55.24% ± 10.6% in the plasma, significantly higher than that of [¹⁸F]LW223, which was 30.05% ± 4.77% (P < 0.05, Fig. 2a). A representative statistical PET scan of the whole-body cynomolgus monkey is shown in Fig. 2b. A comparison of the averaged SUVr between lumbar vertebrae 3–5 and the humerus head, with the aortic arch as the reference region, revealed a lower uptake of [¹⁸F]D₂-LW223 relative to [¹⁸F]LW223 (Supplementary Table S2).

Fig. 2. Ex vivo and in vivo metabolic stability assessment.

Higher intact tracer fraction ratio in plasma over 60 min of [¹⁸F]D₂-LW223 than that of [¹⁸F]LW223 (n = 3, *P < 0.05) b Lower radioactivity accumulation observed in vertebras (red arrows) and humerus head (yellow arrow) with [¹⁸F]D₂-LW223 compared to [¹⁸F]LW223.

MicroPET imaging and immunofluorescence in LPS mouse models

To investigate inflammatory changes, microPET imaging of [¹⁸F]D₂-LW223 was performed in an LPS-induced mouse model and a sham control group. As shown in Fig. 3a, the uptake of [¹⁸F]D₂-LW223 in the whole brains of the LPS-induced mice was greater than that in the control group, demonstrating that LPS can lead to the occurrence of inflammation and the upregulation of TSPO. To characterize the regioselectivity by which LPS induces the expression of TSPO, the distribution volume in different brain regions was calculated. The V_T analysis generated from dynamic PET scans confirmed notably elevated uptake of [¹⁸F]D₂-LW223 in the LPS-injected mouse brain regions, particularly in the cortex (28% increase), hippocampus (33%), and striatum (30%) (Fig. 3b). To further explore the relationship between TSPO expression and the LPS-induced activation of microglia, immunofluorescence imaging of TSPO and Iba-1 in the cortex is shown in Fig. 3c. The results revealed that the robust inflammatory response induced by LPS led to the activation of microglia (Iba1) in the cortex accompanied by the upregulation of TSPO. The quantitative assessment of TSPO and Iba1 expression via immunofluorescence staining revealed that the 38% increase in the protein level of TSPO in the LPS model was consistent with the trends observed via PET imaging (Fig. 3d, e).

Fig. 3. PET imaging study and biochemical analysis in LPS inflammation mouse models.

a, b Representative PET images and regional V_T values analysis by Logan Graphic Analysis. c Images (× 20) of double immunofluorescence staining of TSPO (green) and Iba-1 (red) in cortex tissue sections. d, e Quantitative assessment of TSPO and Iba1 expression. Each group consisted of four mice, with data expressed as mean mean ± SD (***P < 0.001, ****P < 0.0001).

MicroPET imaging and immunofluorescence in the focal cerebral ischemic rat models

As depicted in Fig. 4a, brain PET imaging was conducted to evaluate [¹⁸F]D₂-LW223 signals in ischemic animals at 10 days (baseline) and 11 days (preblock) postsurgery. TACs for [¹⁸F]D₂-LW223 revealed that a stable SUV (peak ca. 1.20) was reached approximately 5 min postinjection (Fig. S3a). When blocked with PK11195 (5 mg/kg), uptake on the ipsilateral side was notably reduced compared to the baseline during a 60-min dynamic scan, with a reduction of area under curve (AUC) exceeding 50% (Fig. S3b). Conversely, on the contralateral side, [¹⁸F]D₂-LW223 exhibited limited peak brain uptake with only marginal changes observed between baseline and blocking conditions (Fig. S3). Quantitative analysis of TACs within a 40 to 60 min imaging window revealed that the total uptake of [¹⁸F]D₂-LW223, as derived from AUC on the ipsilateral side, was significantly higher than that on the contralateral side. This uptake was reduced by more than 50% following blockade with PK11195 (Fig. 4b). Additionally, TSPO expression was evaluated in brain slices harvested 10 days postsurgery, also using Iba1 as a marker for microglia activation. Immunofluorescence results demonstrated an increased intensity of TSPO-positive cells on the ipsilateral side (Fig. 4c−e), which further confirmed excellent in vivo specificity of [¹⁸F]D₂-LW223 for PET imaging of TSPO.

Fig. 4. PET imaging study and biochemical analysis in MCAO rat models.

a, b Representative 40–60 min averaged PET-MRI images and area under curve analysis in the baseline and 5 mg/kg of PK11195 blocking conditions. c Immunofluorescence images (100×) of microglia cells marked with iba1 and TSPO taken 10 days post-infarction. d, e Quantitative analyses of TSPO and Iba1 expression. Each group consisted of three rats, with data expressed as mean ± SD (*P < 0.05, **P < 0.01, ****P < 0.0001).

PET imaging and pharmacokinetic studies in cynomolgus monkey brains

A representative PET-MRI image of a cynomolgus monkey brain is presented in Fig. 5a. TACs showing the uptake of [¹⁸F]D₂-LW223 in various brain regions during baseline studies demonstrated rapid traversal of the BBB, as depicted in Fig. S4, with a mean time to peak of approximately 2 min. Blocking experiments were conducted via the administration of 10 mg/kg PK11195. The results of the blocking studies revealed a higher peak SUV of approximately 2.5 than that reported in the baseline study, which was approximately 1.7. Notably, the TACs from the blocking scans exhibited a significantly accelerated washout rate, likely attributable to the inhibition of uptake in both the CNS and peripheral tissues.

Fig. 5. PET imaging and kinetic modeling of [¹⁸F]D₂-LW223 conducted twice in three cynomolgus monkey brains under baseline conditions.

a MRI and PET averaged images 60 to 90 min post-injection. b Metabolite-corrected plasma time-activity curve. c Plasma parent fraction over time (n = 6). d Tissue-compartment modeling analysis. 2TCM (full line) showed better fitting with the representative striatal TAC (circles) compared to 1TCM (dashed line). e Representative Logan graphical linear fit of striatal TAC (circles). f Averaged V_T values estimated from 2TCM and LGA from 6 baseline scans. g Correlation between the baseline V_T obtained from LGA and 2TCM in 10 brain regions. n = 6 (two-tailed Pearson correlation analysis: correlation coefficient = 0.9687, P < 0.0001, y = 1.047x - 0.028).

Figure 5b illustrates the metabolite-corrected plasma TACs of [¹⁸F]D₂-LW223. Analysis revealed that the radiometabolite fraction in the plasma exhibited greater hydrophilicity and a shorter retention time than did the parent radioligand, as indicated in Fig. S5a, suggesting a more limited ability to penetrate the BBB. The intact radiotracer present in the plasma under baseline conditions was shown in Fig. 5c. Additionally, the stability of [¹⁸F]D₂-LW223 in plasma postadministration was higher than that of [¹⁸F]LW223, as shown in Fig. S6, respectively, due to the slower metabolism rate. Pharmacokinetic analysis of [¹⁸F]D₂-LW223 was conducted with the 1-tissue compartment model (1TCM), 2-tissue compartment model (2TCM), and LGA. The tissue-compartment modeling analysis demonstrated that the 2TCM closely approximated the actual PET imaging data, validating its efficacy in accurately estimating the dynamic processes of [¹⁸F]D₂-LW223 uptake and distribution within the brain (Fig. 5d). A representative Logan plot for the striatum is depicted in Fig. 5e, while Fig. 5f illustrates the V_T values derived from both the 2TCM and LGA, demonstrating a high correlation (Pearson’s r = 0.9687, P < 0.0001; Fig. 5g). The kinetic parameters are summarized in Table 1, with [¹⁸F]D₂-LW223 V_T values ranging from 1.03 to 1.24 ml/cm³ across all brain regions, which are consistently lower than those of [¹⁸F]LW223 (Table S3).

Table 1.

Pharmacokinetic parameters of [¹⁸F]D₂-LW223 (n = 6) estimated from different fitting models.

Region	K1 (mL·cm-3·min-1)	k2 (/min)	k3 (/min)	k4 (/min)	VT (mL/cm3)
2-TCM
Striatum	0.08 ± 0.06	0.23 ± 0.28	0.11 ± 0.10	0.05 ± 0.02	1.15 ± 0.13
Hippocampus	0.09 ± 0.09	0.28 ± 0.41	0.11 ± 0.09	0.05 ± 0.02	1.19 ± 0.13
Thalamus	0.08 ± 0.07	0.24 ± 0.36	0.10 ± 0.10	0.06 ± 0.03	1.14 ± 0.13
Frontal cortex	0.08 ± 0.07	0.21 ± 0.28	0.09 ± 0.08	0.04 ± 0.02	1.24 ± 0.11
Cerebellum	0.10 ± 0.08	0.51 ± 0.65	0.20 ± 0.09	0.06 ± 0.02	1.03 ± 0.19
1-TCM
Striatum	0.05 ± 0.03	0.06 ± 0.04			0.99 ± 0.11
Hippocampus	0.06 ± 0.04	0.06 ± 0.04			1.00 ± 0.09
Thalamus	0.06 ± 0.03	0.06 ± 0.04			0.99 ± 0.13
Frontal cortex	0.05 ± 0.04	0.05 ± 0.04			1.02 ± 0.11
Cerebellum	0.05 ± 0.02	0.06 ± 0.01			0.85 ± 0.08
Logan
Striatum					1.15 ± 0.11
Hippocampus					1.20 ± 0.12
Thalamus					1.13 ± 0.12
Frontal cortex					1.23 ± 0.10
Cerebellum					1.10 ± 0.24

V_T images from the baseline and blocking studies (10 mg/kg PK11195, i.v.) were generated via LGA (Fig. 6a). As depicted in Fig. 6b, preadministration of 10 mg/kg PK11195 significantly reduced the baseline Logan V_T, indicating a high in vivo binding specificity of [¹⁸F]D₂-LW223. In vivo binding specificity was further assessed via Lassen plot analysis, revealing a V_ND of 0.30 ml/cm³ for [¹⁸F]D₂-LW223, which is lower than that of [¹⁸F]LW223 (0.63 ml/cm³, Fig. S7). The correlation between the SUV (60–70 min), SUV (70–80 min), and SUV (80–90 min) with V_T, as shown in Figure S8, suggested that a 10-min static scan conducted 60 min postinjection may serve as a simplified protocol for the dynamic acquisition of [¹⁸F]D₂-LW223 due to its robust relationship (R² = 0.8826, P = 0.0239).

Fig. 6. PET-MR images and quantitative analysis in monkey brain.

a Parametric V_T-based images of baseline (left) and blocking (right) with 10 mg/kg of PK11195, estimated from LGA. (PET-MR images and quantitative analysis in monkey brain. b V_T in different brain regions under baseline (n = 6) and blocking (n = 4) conditions.

Discussion

The development of a quantitative and noninvasive tool for visualizing TSPO expression provides a promising approach for investigating neuroinflammation in various disease states. This study presents an in vivo assessment of a novel deuterated ¹⁸F-labeled TSPO-targeted PET radiotracer, [¹⁸F]D₂-LW223, in monkeys, along with a comparison with [¹⁸F]LW223. The synthesis of [¹⁸F]D₂-LW223 was automated using the GE TRACERlab® FX2N module or the Trasis AllinOne® synthesis module, resulting in high radiochemical yields and purities, as well as molar activities. Furthermore, [¹⁸F]D₂-LW223 exhibited excellent stability up to 4 h after formulation, allowing for a suitable transportation and administration time window. Consistent with its favorable in vitro LogD_7.4, [¹⁸F]D₂-LW223 demonstrated robust tracer uptake into the CNS. We anticipated that [¹⁸F]D₂-LW223 possessed lower lipophilicity compared to [¹⁸F]LW223, potentially leading to reduced nonspecific binding in the brain. The lower f_u of [¹⁸F]D₂-LW223 relative to [¹⁸F]LW223 may contribute to a decreased rate of compound metabolism or drug excretion. Notably, [¹⁸F]D₂-LW223 exhibited a higher plasma parent fraction at 60 min postadministration (55.24% ± 10.6%, n = 3) compared to [¹⁸F]LW223 (30.05% ± 4.77%). This enhanced metabolic stability of the deuterated compound may be attributed to its kinetic isotope effect difference in cleaving the C-D and C-H bonds. Furthermore, the major radioactive metabolite detected in the plasma appears to be more polar than the parent radioligand, indicating a reduced likelihood of crossing the blood‒brain barrier and potentially simplifying the quantitative analysis of PET imaging data. It has been reported that the cleavage rate of the C-H bond exceeds that of the C-D bond by a factor of 6.7 [38]. Therefore, our investigation explored whether deuterium substitution can attenuate the defluorination rate of [¹⁸F]LW223. As anticipated, our findings from whole-body PET scans revealed a modest reduction in tracer bone uptake (Fig. 2b), attributable to deuterium substitution, thereby enhancing the in vivo metabolic stability. This approach of deuteration to modulate PET ligands and mitigate defluorination rates has demonstrated success in numerous instances as documented in prior studies [14, 15, 39–41].

To assess its potential for visualizing neuroinflammation, [¹⁸F]D₂-LW223 was subsequently investigated in neuroinflammatory rodent models, including mice induced by LPS injection and rats suffering from cerebral ischemia. Intrastriatal administration of LPS provoked an inflammatory response characterized by heightened TSPO expression, with vehicle injection serving as a sham control. Remarkably, [¹⁸F]D₂-LW223 PET imaging effectively distinguished the LPS-injected group from the control, manifesting elevated tracer uptake in the former. These findings are consistent with prior studies utilizing [¹¹C]PK11195, [¹⁸F]GE-180, [¹⁸F]CB251, and [¹⁸F]DPA714 in comparable models [42, 43]. Subsequent assessment of [¹⁸F]D₂-LW223 in a preclinical cerebral ischemic rat model revealed rapid and pronounced uptake kinetics within ischemic regions, peaking within 10 min postadministration and persisting at a plateau for 50 min thereafter. Notably, preblocking studies demonstrated rapid displacement from the ipsilateral area by PK11195, affirming the specific binding of [¹⁸F]D₂-LW223 to TSPO in vivo, paralleling prior investigations in cerebral ischemic rats [44, 45]. These observations underscore the specificity of [¹⁸F]D₂-LW223 binding to TSPO and its increased binding within inflammatory brain regions due to TSPO overexpression (Figs. 3d and 4d).

Regional TACs indicated that the peak uptake was reached within 5 min after injection in all brain regions, possibly revealing the fast and reversible brain uptake kinetics (Fig. S4). Scans of healthy cynomolgus monkeys show relatively low uptakes in all brain regions, where there is a low expression level of TSPO in normal brain tissue [46]. In the studies of kinetic models for PET data analysis, similar to other TSPO PET tracers [47], 2TCM of [¹⁸F]D₂-LW223 showed a better fit to region time–activity curves than 1TCM. Moreover, the V_T generated by 2TCM has a statistical relationship with that generated by Logan analysis. A further comparison of microparameters (K₁(ml·cm^-3·min^-1)), the transport rate of ligand from plasma to tissue; k₂(min⁻¹), the transport rate of ligand from tissue to plasma, k₃ (min⁻¹) and k₄ (min⁻¹), the transport rates between free and bound fraction in tissue and V_T of 2TCM between [¹⁸F]D₂-LW223 and [¹⁸F]LW223 demonstrated that [¹⁸F]D₂-LW223 was calculated to have lower V_T attributes with lower K₁ and k₃, which means that there was a smaller portion in the brain region and a lower bound fraction in the brain. These results may be attributed to complex factors, including higher metabolic stability in plasma, a lower LogD_7.4, meaning low extraction across the blood‒brain barrier, and less unbound free fractions. The V_T of [¹⁸F]D₂-LW223 calculated from 2TCM was also lower than that of [¹¹C]PK11195 (2.6–4.56 ml/cm³) [48], [¹⁸F]SF51 (11.86–16.19 ml/cm³) [49], [¹¹C]ER716 (Cerebellum, 11.3 ml/cm³) [50] and so on. However, low uptake in healthy brain might reduce the signal-to-noise ratio for this tracer and make the tracer more susceptible to showing increased uptake when increasing TSPO expression. Due to the ubiquitous expression of TSPO across all brain regions, identifying areas devoid of uptake for use as reference regions presents a significant challenge. Consequently, the V_ND serves as the sole metric for calculating the binding potential. The V_ND of [¹⁸F]D₂-LW223 (0.30 ml/cm³), as determined through the Lassen plot method, was lower than [¹⁸F]LW223 (0.63 ml/cm³) and other TSPO-PET tracer such as ¹⁸F-SF51 (1.73 ml/cm³) [49], which means less nondisplaceable distribution volume. Moreover, the summed 10-min static average SUV values derived from dynamic scans after 60 min postadministration revealed the best correlation with V_T across analyzed brain regions. Therefore, it is reasonable that a static scan performed 60 min postinjection can partially replace long-time dynamic scans and arterial blood collection.

In this study, we discovered a novel PET radioligand, [¹⁸F]D₂-LW223, designed to target the translocator protein 18 kDa (TSPO). Our investigations reveal that [¹⁸F]D₂-LW223 exhibited superior physicochemical and pharmacological characteristics and demonstrated promising in vivo brain kinetics. Compared to [¹⁸F]LW223, this novel deuterated radioligand showed enhanced metabolic stability, reduced defluorination, and lower binding and fast clearance in healthy brain tissue. Preliminary clinical evaluations in rodent models of neuroinflammation indicated specific binding of [¹⁸F]D₂-LW223 to TSPO in regions exhibiting inflammatory responses. Furthermore, pharmacokinetic analysis using 2TCM yielded excellent fits, facilitating the quantitative mapping of TSPO distribution in cynomolgus monkeys. The V_T values across different brain regions were strongly correlated with the average SUV within the 60–70 min timeframe. These findings suggest that [¹⁸F]D₂-LW223 may have significant potential for advanced TSPO quantification in neurological research.

Author contributions

Study conceptualization: HX, SHL, and LW Probe development and characterization: KL, JHC, JM, CCD, CYB, YBG, YFJ, HYW, LH, JQH, JJW, and CYZ. Data acquisition, analyses, and quality control: KL, JHC, HX, SHL, and LW. KL, JHC, TW, YLL, and SY established animal models and performed immunofluorescence staining experiments. Manuscript drafting, editing, and reviewing: KL, JHC, HX, SHL, and LW. All authors reviewed and approved the final version of this manuscript.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41401-024-01375-9.