Evaluation of [18F]MFPBG: a novel automatically radiolabeled probe for NET PET imaging

Abstract

Background

Cardiac sympathetic nerve imaging is critical for sudden death risk stratification in heart failure patients. Conventional SPECT probe [¹²³I]MIBG is limited by inadequate spatial resolution and restricted quantitative ability. While [¹⁸F]-labeled PET probes have shown promise, some suffer from high hepatic uptake that interferes with cardiac signals, and low radiolabeling efficiency hinders clinical translation. Thus, a novel norepinephrine transporter (NET) probe [¹⁸F]MFPBG was developed for cardiac sympathetic nerve imaging, synthesized via an automated radiolabeling module.

Results

The decay-corrected radiochemical yield of [¹⁸F]MFPBG was 15.14±2.57% (n = 5), with a retention time of 11.87 minutes. The probe exhibited a radiochemical purity of >95% and remained stable (purity >95%) for 4 hours at ambient temperature. The biodistribution studies in ICR mice (n=28) showed a cardiac uptake of 9.34±0.73 %ID/g at 5 minutes post-injection, with the heart to liver ratio (3.99±0.15). At 120 minutes, cardiac uptake remained high (4.88±0.85 %ID/g), and the heart to liver ratio was 4.46±0.85. In Bama miniature pigs, imipramine pre-treatment significantly reduced cardiac uptake compared to controls at 10 minutes (SUV-bw: 2.08±0.16 vs. 3.73±0.29; P < 0.001) and 60 minutes (SUV-bw: 1.21±0.14 vs. 2.15±0.21; P < 0.001).

Conclusions

[¹⁸F]MFPBG demonstrates NET-mediated uptake, stable cardiac retention, low background noise, and suitability for automated radiolabeling, establishing it as a promising agent for cardiac sympathetic nerve imaging with favorable characteristics that warrant further preclinical validation, including in vivo metabolic profiling, to fully assess its clinical translatability.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s13550-026-01482-0.

Background

The norepinephrine transporter (NET), a crucial regulatory component of the sympathetic nervous system, precisely regulates cardiac sympathetic tone by reabsorbing norepinephrine (NE) from the synaptic cleft and the functional state directly influences local NE concentration and myocardial electrical stability [1, 2]. In recent years, advancements in nuclear medicine imaging technology have facilitated the transition of NET-targeted molecular probes from the laboratory to clinical practice, serving as a novel instrument for visualizing cardiac sympathetic integrity, clarifying disease mechanisms, and directing personalized therapy [3]. Risk stratification models utilizing NET imaging have potential to address the shortcomings of existing guidelines, which exclusively rely on an ejection fraction ≤35% as the criterion for implanted cardioverter-defibrillator primary prevention. This method may decrease the oversight of 80% of non-beneficiary sudden death incidents and avert one-third of patients from experiencing the psychological and physiological burdens linked to unwarranted shocks.[4–7]. The conventional SPECT probe [¹²³I]MIBG was the inaugural method to illustrate the strong correlation between denervation and the risk of sudden death in heart failure patients [2, 4]; however, its inadequate spatial resolution, restricted quantitative abilities have hindered its extensive utilization [8].

The introduction of ¹⁸F-labeled PET probes has revitalized NET imaging, utilizing enhanced resolution, absolute quantitative capabilities, and the logistical benefit of centralized distribution due to their half-life. [7, 8]. Phenethylguanidine compounds, which exhibit high NET affinity and possess straightforward labeling and consistent cross-species kinetics, are considered promising prospects for next-generation clinical translation.[3, 9]. Simultaneously, probes like [¹⁸F]LMI1195 and [¹⁸F]4F-MHPG have successfully concluded phase I/II trials [10, 11]. These investigations have validated that these probes can precisely measure regional sympathetic denervation in patients with ischemic and non-ischemic cardiomyopathy. Moreover, the denervation volume is not contingent upon the left ventricular ejection fraction, facilitating more accurate forecasting of malignant arrhythmias and sudden cardiac mortality. This study developed a novel imaging agent, [¹⁸F]MFPBG, utilizing the phenethylguanidine molecule. In comparison to agents like [¹⁸F]MFBG, [¹⁸F]LMI1195, and [¹⁸F]AF78, it demonstrates rapid hepatic clearance, straightforward synthesis, and advantageous pharmacokinetics, indicating extensive application potential [10, 12, 13]. Additionally, it is synthesized using a fully automated cartridge reactor, which improves reproducibility, minimizes radiation exposure, and facilitates large-scale clinical application.

Methods

Synthesis and radiolabeling

The fully protected benzylguanidine precursor (Fig. 1) with high chemical purity (>95%) was supplied by Jiangsu Huajing Molecular Imaging and Drug Research Institute Co., Ltd. (Beijing, China). All other reagents and solvents were commercially available and used directly without further purification. Sep-Pak columns for solid-phase extraction were purchased from Waters Corporation (Milford, Massachusetts, USA).

Fig. 1.

Radiochemical synthesis of [¹⁸F]MFPBG

Labeling and deprotection

[¹⁸F]MFPBG was synthesized using an automated, two-step synthesis with intermediate solid-phase extraction: first, a nucleophilic aromatic substitution reaction occurred between the ¹⁸F-fluoride ion and the precursor, followed by removal of the Boc protecting group under acidic conditions (Fig. 1). This synthetic workflow was completed and optimized on Trasis’s AllInOne synthesis module (Fig. 2). Brief steps are as follows: [¹⁸F]fluoride ions generated by the cyclotron are captured by a QMA anion exchange column, then eluted into a reaction flask using a methanol/water solution (1 mL) containing K₂CO₃ (3.94 mg) and Kryptofix 2.2.2 (13.5 mg). Solvent is removed by heating under a nitrogen stream.

Fig. 2.

An automated synthesis of [¹⁸F]MFPBG. Schematic diagram of Trasis AIO module. (A) 1 mL eluent ( K₂CO₃: 3.94 mg; Kryptofix 2.2.2: 13.5 mg; Methanol: 0.72 mL; H₂O: 0.18 mL); (B) 10 mL ethanol; (C) 1.0 mL DMSO (containing 3.0 mg of precursor MFPBG); (D) 2.0 mL Na₂CO₃ aqueous solution (1 M); (E) 2.0 mL HCl aqueous solution (2 M); (F) 500 mL Water for Injection; (G) 60 mL product collection vial; (H) 20 mL ethanol; (I) 15 mL normal saline (containing 75 mg of sodium ascorbate)

A DMSO solution (1.0 mL) containing the precursor MFPBG (3.0 mg) was added to the dried [¹⁸F]fluoride ion/K₂O-Kryptofix 2.2 complex and reacted at 120 °C for 15 minutes. After reaction completion, the mixture was cooled to 65 °C and diluted with 7 mL ultrapure water. The diluted solution was purified on a Sep-Pak C18 column, sequentially washed with 10 mL ultrapure water, and dried under nitrogen. The fluorine-18-labeled intermediate was eluted back into the reaction flask using 2 mL anhydrous ethanol.

Subsequently, add 2 mL of 2 M HCl aqueous solution to the flask and acid-hydrolyze at 110 °C for 8 minutes. Cool the reaction mixture to 45 °C and neutralize with 2 mL of 1 M Na₂CO₃ aqueous solution. Purify the crude product solution by preparative high-performance liquid chromatography (HPLC).

HPLC purification

The preparative column used was an XBridge BEH C18 OBD column. Mobile phase: acetonitrile and ammonium acetate buffer (containing sodium ascorbate, 30:70, v/v). Flow rate: 5 mL/min. The target product peak (retention time approximately 15.5 min) was automatically collected.

The collected product fraction was diluted with 30 mL of injectable water and desalted and concentrated again through another Sep-Pak C18 column. The column was rinsed with 20 mL of water for injection and air-dried. The product was then eluted into a transfer vial using 1.0 mL of anhydrous ethanol. Finally, the product was diluted with 10 mL of physiological saline containing 50 mg of sodium ascorbate and filtered through a sterile filter membrane to obtain sterile [¹⁸F]MFPBG injection solution.

Quality control and stability

Radiochemical purity and identity were assessed by analytical HPLC using an XBridge BEH C18 column (4.6 × 150 mm) with acetonitrile/ammonium acetate buffer (containing sodium ascorbate, 30:70, v/v) at a flow rate of 1 mL/min. The non-radioactive reference standard [¹⁹F]MFPBG (dissolved in ethanol) and the purified [¹⁸F]MFPBG (in saline-based formulation) were injected separately under identical conditions. The retention time of the [¹⁹F]MFPBG standard was 11.15 min, while that of [¹⁸F]MFPBG was 11.87 min. This slight shift (0.72 min) is attributed to the difference in sample matrices and does not affect identity confirmation or purity assessment. For stability testing, the product was re-analyzed after 4 h at ambient temperature; the retention time was 11.75 min with radiochemical purity >95%.

Animal experiments

All animal procedures complied with institutional and national regulations for the care and use of laboratory animals. The experimental protocol was approved by the Animal Ethics Committee of the Affiliated Hospital of Inner Mongolia Medical University and strictly followed the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

Radionuclide distribution experiment

Twenty-eight normal ICR mice (14 males and 14 females) weighing 20-40 g were purchased from the Animal Experiment Center of Inner Mongolia University. They were randomly divided into 7 groups (4 mice per group). Each mice received an intravenous injection of 7.4 MBq (0.2 mL) of [¹⁸F]MFPBG via the tail vein. After being deeply anesthetized with isoflurane, animals were euthanized by carotid artery transection at 5, 15, 30, 45, 60, 90, and 120 minutes post-injection. The heart, lungs, liver, spleen, kidneys, stomach, intestines, brain, muscle, and blood were removed. These organs and tissues were washed with physiological saline, weighed separately, and their radioactivity was measured using a gamma counter. The radioactivity uptake rate of [¹⁸F]MFPBG in blood and various organs at different time points (%ID/g) and the radioactivity ratio between target organs and non-target organs (T/NT) were calculated.

Blocking experiments

Eight 9-month-old Chinese Bama Miniature Pigs weighing 25–35 kg (four males and four females) were randomly divided into two groups. The experimental group (4 pigs) received 8 mg/kg imipramine, a NET inhibitor with additional affinity for the serotonin transporter, via auricular vein injection. After 2.5 hours, animals were anesthetized with Zoletil 50 and injected with the radiotracer [¹⁸F]MFPBG at a dose of 5.55 MBq/kg. Whole-body PET/CT imaging was performed using a GE Discovery IQ PET/CT scanner. The control group received PET/CT scanning without the injection of inhibitors. Images were obtained at 10, 20, 30, and 60 minutes. The alterations in PET/CT radioactivity levels prior to and after to inhibition were evaluated utilizing Carimas Core software (supplied by Turku PET Center, Finland). For PET imaging analysis, regions of interest (ROIs) were manually delineated on the left ventricular free wall to quantify myocardial uptake. Adjacent blood pool and liver areas were excluded to reduce background contamination. All images were attenuation-corrected using co-registered CT data. No partial volume correction was applied. Given the spatial resolution of the PET system, partial volume effects may lead to an underestimation of true myocardial activity.

Statistical analysis

All data are presented as mean ± standard deviation (SD). Statistical analysis was performed using IBM SPSS Version 23 software, and graphical representations were created using GraphPad Prism 9.0 software. A two-way repeated-measures ANOVA was performed to evaluate the effects of treatment (control vs. imipramine), time (10, 20, 30, 60 min), and their interaction on myocardial SUV-bw. When a significant interaction was found, post-hoc comparisons at individual time points were conducted using the Bonferroni correction for multiple comparisons. A p-value < 0.05 was considered statistically significant. A p-value < 0.05 was considered statistically significant.

Results

Synthesis and quality control of [¹⁸F]MFPBG

The precursor eluted at 31.75 min on analytical HPLC (Fig. 3A). Under identical analytical HPLC conditions, the non-radioactive [¹⁹F]MFPBG reference standard showed a retention time of 11.15 min (Fig. 3B), while the purified [¹⁸F]MFPBG exhibited a retention time of 11.87 min (Fig. 3C). The 0.72 min difference is attributable to the different injection solvents (ethanol vs. saline/ascorbate formulation) and is within the acceptable variability of the method. The radiochemical purity of [¹⁸F]MFPBG was >95% with a molar activity of >2 GBq/μmol. After 4 h at ambient temperature, the retention time remained at 11.75 min with purity >95% (Fig. 3D).

Fig. 3.

Analytical HPLC quality control of [¹⁸F]MFPBG. (A) Precursor (RT = 31.75 min). (B) Non-radioactive [¹⁹F]MFPBG reference standard (RT = 11.15 min, dissolved in ethanol). (C) Purified [¹⁸F]MFPBG (RT = 11.87 min, in saline-based formulation). The slight difference in retention time between (B) and (C) (0.72 min) is due to matrix effects. (D) [¹⁸F]MFPBG after 4 h at ambient temperature (RT = 11.75 min), confirming stability (radiochemical purity >95%). All runs were performed on the same analytical HPLC system under identical mobile phase and flow rate conditions

Radionuclide distribution experiment in mice

The biodistribution of [¹⁸F]MFPBG in normal ICR mice is illustrated (Fig. 4a and Suppl. Table 1) . Five minutes post-injection of the probe, radioactivity swiftly disseminated throughout the body, with predominant accumulation in the heart (9.34 ± 0.73% ID/g) and kidneys (13.53 ± 4.27% ID/g). After 30 minutes, renal absorption diminished to 3.72 ± 0.89% ID/g. This signifies that [¹⁸F]MFPBG is predominantly eliminated by the kidneys, in accordance with PET/CT imaging findings. Significantly, [¹⁸F]MFPBG exhibits a low absorption value in the liver, maintaining a consistently high heart-to-liver ratio, hence minimizing interference with cardiac imaging (Fig. 4b and Suppl. Table 1). An apparent myocardial washout was calculated from the mean cardiac %ID/g values at 15 min (n=4) and 120 min (n=4) in separate groups of mice, yielding a 46.3% reduction. The estimated half-clearance time based on these terminal data is approximately 110 minutes.

Fig. 4.

Biological distribution and uptake ratio of [¹⁸F]MFPBG in ICR mice. (a) Pharmacokinetics and (b) heart background ratios of 18F-MFPBG in ICR mice, evaluated by gamma counter. Background organs showed low activity

Blocking experiments in pigs

[¹⁸F]MFPBG demonstrated a consistent radiotracer distribution inside the left ventricular myocardium of the control group, with low uptake in surrounding organs (Fig. 5) . In pigs pretreated with the NET inhibitor imipramine (8 mg/kg), the myocardial distribution of [¹⁸F]MFPBG was markedly diminished compared to control pigs, commencing 10 minutes post-injection (Fig. 5c and Suppl. Table 2). This signifies that the myocardial uptake of [¹⁸F]MFPBG is NET-dependent, and the tracer is transported via the norepinephrine transporter, rendering it an optimal radiotracer for evaluating cardiac sympathetic nerve density.

Fig. 5.

Cardiac PET images of [¹⁸F]MFPBG in bama pigs. (A) control group (n=4), (B) pretreated with imipramine at 8 mg/kg (n=4) to block NET. These images were acquired at 10, 20, 30 and 60 min after injection. (C) Myocardial uptake in control and imipramine treatment pigs. Heart activity is expressed as SUV_bw. ***p<0.001 versus the control group

Discussion

This research concentrated on the advancement of NET targeted radiopharmaceuticals, detailing the optimization of automated synthesis, in vivo performance assessment, and mechanism validation of [¹⁸F]MFPBG. The objective is to provide a novel tracer for PET imaging of the cardiac sympathetic nervous system (SNS). Presently, significant obstacles in NET imaging encompass inadequate radiolabeling efficiency of tracers and inferior pharmacokinetic properties (e.g., high hepatic uptake interfering with cardiac signals) [11, 14, 15, 15, 16]. To mitigate these constraints, we concentrated on enhancing the automated synthesis process of [¹⁸F]MFPBG and methodically assessed its in vivo efficacy and targeting mechanism to facilitate its clinical translation.

In synthesis, the low radiochemical yield of ¹⁸F-labeled phenylguanidine derivatives presents a significant obstacle for clinical application, principally due to interference from unprotected guanidine structures during nucleophilic fluorination reactions [17, 18]. The core structure of NET-targeted pharmaceuticals, NE-like benzylguanidine, is resistant to monoamine oxidase degradation; however, the unprotected regions, such as the free hydrogen of its imine, are susceptible to forming hydrogen bonds with [¹⁸F] fluoride, hence diminishing nucleophilicity.[17, 19]. Referring to recently developed guanidino protection strategies (e.g., tetraethoxymethylsilyl or triazine ring protection [15, 16, 19, 20]), we discontinued the conventional di-N,N’-tert-butoxy-protected precursor and devised and produced a completely protected benzyl guanidino precursor. The efficient synthesis of [¹⁸F]MFPBG was achieved by the " sequential two-step synthesis with intermediate solid-phase extraction " completely automated cassette method in the Trasis AllInOne module.This method elevated the decay-corrected radiochemical yield from approximately 7% (using earlier non-fully protected strategies) to 15%, while maintaining radiochemical purity above 95% as verified by HPLC [17]. This automated workflow not only simplifies operations but also enhances synthesis reproducibility and elevates the yield of radionuclide probes, laying the foundation for subsequent clinical studies. A point that warrants discussion is the moderate molar activity (>2 GBq/μmol) achieved in this study. While this value is lower than the typical clinical-grade standard (>37 GBq/μmol) and represents a limitation, it is not without precedent in the NET-targeting radiotracer literature. [¹²³I]MIBG, the clinical gold standard for cardiac sympathetic imaging, is routinely used with a specific activity of approximately 0.31 GBq/μmol [21]. The successful clinical use of [¹²³I]MIBG demonstrates that a tracer with modest specific activity can still provide biologically meaningful results for cardiac imaging, where the target (NET) is present at relatively high density in sympathetic nerve terminals. The injected mass was ≤3.7 nmol for mice. For a 30 kg pig receiving 5.55 MBq/kg (total 166.5 MBq) at a molar activity of >2 GBq/μmol, the injected mass was approximately 83 nmol. This mass exceeds typical clinical tracer doses and may approach the threshold for partial receptor occupancy in pigs. However, the robust and significant reduction in cardiac uptake following imipramine blockade (P < 0.001) confirms that the NET-mediated specific signal remains detectable. Nonetheless, we acknowledge that achieving higher molar activity (e.g., >37 GBq/μmol) would be desirable for future clinical translation to minimize any potential mass effect.

Based on synthetic optimization, we assessed the in vivo distribution characteristics of [¹⁸F]MFPBG by PET imaging of miniature Bama pigs and biodistribution experiments in ICR mice. The in vivo imaging results of pigs indicated that [¹⁸F]MFPBG exhibits ideal characteristics with high cardiac uptake and extremely low uptake in non-target organs: cardiac radioactivity accumulation gradually decreased over time but remained significantly higher than in the liver and lungs, where radioactivity levels consistently remained low, enabling clear cardiac imaging. This advantageous biodistribution is likely attributable to its molecular design. The presence of a hydrophilic 1,4-dioxaheptane-2,7-diol side chain on the aromatic ring (Fig. 1) may enhance water solubility, thereby facilitating faster hepatic clearance and reduced liver retention, effectively circumventing the issue of “early high liver uptake” associated with some other tracers [13]. Compared to similar tracers, [¹⁸F]MFPBG exhibits outstanding heart-to-liver ratio: [¹⁸F]AF78 showed 1.60±0.38 at 60 minutes [13], while in non-human primates, [¹⁸F]3F-PHPG and [¹⁸F]4F-MHPG showed values of 2.2±0.8 and 2.5±0.3 at 90 minutes [16].In this study, the heart-to-liver ratio of [¹⁸F]MFPBG compares favorably with values reported for other tracers such as [¹⁸F]AF78 and [¹⁸F]LMI1195. However, these cross-study comparisons are qualitative due to differences in experimental conditions, and definitive conclusions require head-to-head studies. Nevertheless, definitive conclusions still require further rigorous head-to-head comparison under consistent experimental conditions. A higher heart-to-liver ratio mitigate radioactive spillover counts from the liver to the inferior myocardial wall, thus enhancing image quality [22].

The horizontal comparison with the NET-targeted tracers frequently used in clinical practice further demonstrated the applicability potential of [¹⁸F]MFPBG. In comparison to [¹²³I]MIBG, which exhibits substantial hepatic uptake that often obscures the inferior wall signal of the left ventricle [20, 23–25], the 30-minute distribution data for ICR mice indicated that the cardiac uptake of [¹⁸F]MFPBG (7.66±0.94 %ID/g) was marginally higher than that of [¹²³I]MIBG (7.50±0.54 %ID/g), whereas the hepatic uptake (2.24±0.44 %ID/g) was markedly lower than that of [¹²³I]MIBG (6.02±0.60 %ID/g) [26]. [¹¹C]hydroxyephedrine ([¹¹C]HED) can evaluate sympathetic nerve function; however, the short physical half-life of carbon-11 (T1/2=20 minutes) restricts its extensive clinical use [27–29] , whereas the half-life of [¹⁸F] (T1/2=109 minutes) is more appropriate for standard PET imaging procedures. In comparison to other ¹⁸F-labeled analogues, [¹⁸F]MFPBG showed favorable characteristics in our experimental setting: relative to [¹⁸F]MFBG, it shows accelerated hepatic clearance in murine models, with a markedly elevated heart-to-liver ratio at 60 minutes (3.9 vs. 2.31) [30]; Compared with [¹⁸F]LMI1195, which is currently in Phase II clinical trials, the heart-to-lung ratios observed for [¹⁸F]MFPBG in mice (15 min: 1.92±0.08, 60 min: 2.48±0.17) appear higher than those reported for [¹⁸F]LMI1195 (15 min: 1.23±0.50, 60 min: 1.82±0.43), suggesting potentially lower pulmonary background, though a direct comparison is lacking. However, it is important to note that [¹⁸F]LMI1195 has been shown to achieve a high heart-to-liver ratio of 3.5±1.9 at 30 minutes in non-human primates [10], which is comparable to the heart-to-liver ratios observed for [¹⁸F]MFPBG at similar time points (e.g., 3.48±0.56 at 30 minutes in mice and 3.91±0.85 at 60 minutes in mice). Thus, both tracers exhibit favorable cardiac-to-background characteristics, and direct head-to-head comparisons under identical experimental conditions will be necessary to determine their relative performance.

The calculated washout rate of 46.3% over 105 minutes provides important insight into the in vivo behavior of [¹⁸F]MFPBG. A slow washout is a critical feature for a cardiac neuronal tracer, as it provides a stable imaging signal over an extended period, allowing for high-quality imaging and robust parametric kinetic analysis. This apparent slow clearance is a favorable characteristic, resembling the behavior reported for [¹⁸F]LMI1195 in dynamic PET studies, and contrasts with the rapid clearance of [¹¹C]HED. A definitive kinetic comparison will require dynamic PET imaging [35]. This pharmacokinetic advantage, makes [¹⁸F]MFPBG a particularly promising candidate for clinical scenarios where sustained imaging is beneficial, such as delayed imaging to assess denervation in a low-flow state.

Notably, the carbon chain length between the benzene ring and the guanidino group markedly influences the pharmacokinetics of NET-targeting tracers. In contrast to tracers featuring a phenylguanidine core structure, phenethylguanidine derivatives such as [¹⁸F]4F-MHPG, [¹⁸F]3F-PHPG, and [¹⁸F]AF78 demonstrate sluggish uptake kinetics in cardiac sympathetic imaging [13, 31], whereas [¹⁸F]MFPBG attains swift cardiac uptake and effective clearance due to optimized carbon chain and PEG modifications, thereby balancing targeting affinity with pharmacokinetic characteristics. In addition, the uptake mechanism of NET-targeted tracers varies among species. For instance, [¹⁸F]LMI1195 binds via the neural uptake mechanism in humans and rabbits, whereas it predominantly utilizes the non-neural method in mice. Additionally, its cardiac uptake is unaffected by uptake-1 blockers, potentially due to the elevated uptake-2 density in the rat heart [32, 33]. To ascertain the targeting mechanism of [¹⁸F]MFPBG, we conducted NET blocking experiments in miniature Bama pigs (an optimal cardiac model for human studies [34]). Following treatment with imipramine, the cardiac uptake of [¹⁸F]MFPBG in miniature Bama pigs was markedly diminished, confirming that its cardiac uptake is predominantly mediated by the norepinephrine transporter (uptake-1 mechanism). Future in vitro assays will be necessary to fully define its binding affinity and selectivity profile across related monoamine transporters. The cardiac uptake pattern resembled that of [¹¹C]HED [35] , further substantiating its potential as a dependable NET-targeted tracer.

Despite [¹⁸F]MFPBG demonstrating excellent performance in synthesis and preliminary evaluation, further exploration of its performance in disease models (such as heart failure or arrhythmia) is needed to validate its clinical applicability. The profound integration of NET imaging with physiology, pathology, and cardiology anticipates that the tracer will facilitate closed-loop management, progressing from "visualized nerves" to "predicting disease events" and ultimately to "guiding precise treatment," thereby offering a novel approach for diagnosing and treating conditions such as heart failure, autonomic neuropathy, and arrhythmia syndrome [23, 27].

It should be noted that the current evidence for tracer specificity relies on in vivo pharmacological blockade. Future in vitro affinity and selectivity assays against related monoamine transporters are warranted to fully characterize its binding profile. A limitation is the absence of in vivo metabolic profiling. The high initial renal uptake we observed cannot be definitively attributed to the parent compound without plasma radio-metabolite analysis, and the stability of the tracer in vivo remains to be confirmed. Furthermore, a limitation of the current biodistribution study is the absence of bone uptake data. Measuring bone radioactivity is critical for assessing potential defluorination of ¹⁸F-labeled tracers. Without this data, the degree of in vivo metabolic defluorination of [¹⁸F]MFPBG cannot be determined. Future studies will prioritize the inclusion of bone tissue analysis and radio-metabolite profiling to comprehensively evaluate in vivo stability.

Conclusion

In this study, an automated radiolabeling method for [¹⁸F]MFPBG was successfully established. Preclinical evaluation in mice and pigs demonstrates that this tracer exhibits NET-mediated cardiac uptake, favorable heart-to-background contrast, and suitable kinetics for PET imaging. These initial results support further preclinical validation, including in vivo metabolic profiling, assessment of defluorination, and studies in disease models, to fully evaluate its clinical translatability.

Abbreviations

[18F]

Fluorine-18

SUV

Standardized uptake value

NET

Norepinephrine transporter

NE

Norepinephrine

SNS

Sympathetic nervous system

Author contributions

Study design: XmW, ZhW, GjZ. Data acquisition: PfX, JL, XaW. Data analysis, writing of manuscript drafts: ZhW,XmW. Preparation of images:ZhW,JL. Interpretation of data:XmW,GjZ. Revising manuscript, approving final content of manuscript: All.

Funding

National Natural Science Foundation of China, U22A6008.

Data availability

The data supporting the findings of this study are accessible from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

This study was approved by the Animal Ethics Committee of the Affiliated Hospital of Inner Mongolia Medical University (NO.DW2025053). All animal procedures in this study were performed in accordance with the Basel Declaration. The committee approved the method of euthanasia (carotid artery transection under deep anesthesia) as it was deemed necessary for rapid tissue collection and to avoid metabolic interference from anesthetic agents in biodistribution studies.

Consent for publication

Not applicable.

Competing interests

Not applicable.