[11C]Fentanyl: Radiosynthesis and Preclinical PET Imaging for Its Pharmacokinetics

[11C]Fentanyl: Radiosynthesis and Preclinical PET Imaging for Its Pharmacokinetics | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article [ 11 C]Fentanyl: Radiosynthesis and Preclinical PET Imaging for Its Pharmacokinetics Woochan Kim, Aaron K. Wozniak, Nathaniel J. Burkard, Michael L. Freaney, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7367969/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Oct, 2025 Read the published version in EJNMMI Radiopharmacy and Chemistry → Version 1 posted 5 You are reading this latest preprint version Abstract Background Fentanyl is a potent synthetic opioid widely used for pain management and anesthesia, but the high prevalence of its misuse and its key contribution to overdose fatalities in the United States have made it a major drug of concern. Although fentanyl’s onset, duration, and toxicity depend on its pharmacokinetics and specific tissue distribution, most studies have focused primarily on plasma concentrations, leaving its distribution in critical tissues largely unexplored (this knowledge gap limits our understanding of fentanyl’s clinical effects, tissue accumulation, and the factors influencing its efficacy and safety). Here, we report the radiosynthesis of [ 11 C]fentanyl for PET imaging and present a preliminary whole-body pharmacokinetic study in rodents. Results [ 11 C]Fentanyl was synthesized in 42 mins in a high radiochemical yield (10.4 ± 5.7%, n = 5), radiochemical purity (> 99%), and molar activity (up to 2571.5 GBq/µmol at EOB). N , N -diisopropylethylamine in chloroform was optimal for amidation. PET imaging in rats revealed rapid brain uptake (SUV max 2.71 ± 1.04 g/mL) and fast washout (T 1/2 = 5.06 min), both significantly increased by efflux transporter inhibition or knockout. Peripherally, high and prolonged uptake in adipose tissues was observed (SUV max = 1.73 ± 0.313 g/mL, T 1/2 = 177 min), with > 60% of C-11 remaining as unchanged [ 11 C]fentanyl at 60 min. Conclusions We successfully developed and automated the radiosynthesis of [ 11 C]fentanyl, enabling PET imaging that revealed rapid brain kinetics and a critical role of P-gp/BCRP efflux in fentanyl disposition in brain. Prolonged retention in adipose tissue may delay brain clearance, potentially increasing the risk of re-narcotization (as has been reported in clinical cases after naloxone reversal). These findings advance our ability to quantify fentanyl tissue distribution and pharmacokinetics in the brain and body and provide a valuable tool for further studies in preclinical and clinical settings. Fentanyl Carbon-11 Positron emission tomography Pharmacokinetics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Fentanyl is a potent synthetic mu-opioid receptor (MOR) agonist, widely used in clinical settings not only as an adjunct in anesthesia but also for managing acute post-operative pain and breakthrough cancer pain [ 1 ]. The rise of illicitly manufactured fentanyl has led to widespread misuse, making it the main driver of the devastating overdose crisis in the United States. In 2022, fentanyl and analogues were involved in nearly 74,000 drug overdose deaths [ 2 ]. The severity of fentanyl-related overdoses has been exacerbated by its mixture with other drugs such as heroin, cocaine, and methamphetamine, often consumed unknowingly by users, which has significantly complicated overdose reversal efforts [ 3 ]. Given fentanyl's critical role both in medicine and in the overdose crisis, investigating its pharmacokinetics has been essential for optimizing its therapeutic use [ 4 ] while also enhancing our understanding of overdose mechanisms, ultimately informing more effective management strategies [ 5 , 6 ]. For instance, preclinical and clinical investigations of fentanyl's pharmacokinetics have helped to optimize dosages for various patient populations [ 7 , 8 ]. However, these studies mostly concern fentanyl’s dosages relevant to anesthesia and analgesia rather than patterns reported and observed in individuals who misuse fentanyl outside of medical settings. Another important consideration is the observation among some misusers of a secondary fentanyl peak, with an abrupt increase in fentanyl plasma concentration and associated respiratory depression. A previous study found that over a 240-minute period, healthy volunteers injected with 0.5 mg fentanyl IV showed secondary peaks in plasma concentration between 45 and 90 minutes after administration [ 9 ]. Fentanyl’s pharmacokinetics are also impacted by demographic and clinical characteristics of a user, including age, obesity, metabolic function, among others. These factors, in addition to an individual’s history of fentanyl misuse, may drastically alter its pharmacokinetics and consequently, its physiological effects and overdose risk [ 10 ]. Preclinical studies using animal models have been crucial in exploring the threshold doses for severe respiratory depression associated with fentanyl overdose and addiction. These investigations provide valuable insights into the physiological effects of fentanyl at different plasma concentrations and can inform strategies for intervention. However, translating these findings to human physiology would benefit from in vivo non-invasive methods for direct measurement of fentanyl’s biodistribution and kinetics in the brain and body. Positron emission tomography (PET) is a powerful quantitative imaging technique that allows for the in vivo measurement of drug concentrations and distributions in target tissues using radiolabeled compounds. Unlike conventional pharmacokinetic studies that rely on plasma drug concentrations, PET provides direct visualization and quantification of drug levels in tissues relevant to both therapeutic effects and adverse events, such as the brain [ 11 ]. This non-invasive approach offers significant advantages for translational research, enabling repeated measurements and flexible experimental designs in laboratory animals and in humans, which would be particularly valuable for understanding the rapid onset and duration of fentanyl's effects. While early studies utilized tritium- and carbon-14 labeled fentanyl to investigate its metabolism and biodistribution in preclinical models, these radiotracers are not optimal for dynamic PET imaging as each subject has to be scarified for each time point [ 12 , 13 ]. Therefore, a critical gap exists in our ability to non-invasively quantify fentanyl concentrations and kinetics in brain and other organs with PET that could also be eventually used for studies in humans. To address this limitation, we herein report the radiosynthesis of carbon-11 labeled fentanyl and present preliminary PET studies conducted in rodents, paving the way for translational pharmacokinetic investigations in humans. Material and Methods Materials 4-Anilino-N-phenethyl-piperidine (4-ANPP) was purchased from Cayman Chemical. The aqueous hydrochloric acid solution (2 N, RICCA Chemical Company, TX) was diluted with water for semi-preparative HPLC. Absolute Ethanol and sodium phosphate buffer (45 mM phosphate, 60 mEq sodium) were obtained from Warner-Graham Company and Hospira Inc., respectively. Tetrahydrofuran (THF) was purified by distillation with sodium (dispersion in mineral oil, Strem Chemicals). All the other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and were used without any further purification. Radiosynthesis was fully carried out and optimized with a commercially available module (Synthra MeIPlus Research). Radiochemical purity and molar activity were determined using an Agilent 1100 Series HPLC system (column, Agilent Eclipse XDB C-18 column, 150 x 4.6 mm, 5 µm; mobile phase, isocratic 0.1% trifluoroacetic acid solution/acetonitrile = 70/30; flow rate, 1 mL/min; detection wavelength, 210 nm) and a radiometric detector equipped with a B-FC-4100 BGO High Voltage Detector. Animal use and protocol were approved by the institutional Animal Care & Use Committee (National Institutes of Mental Health; MIB-03, MIB-04). Wistar rats (male; 297 ± 39.9 g, Envigo, Indianapolis, IN) were used for PET studies. P-gp and BCRP KO mice (female, 25.7 ± 2.22 g, bred in-house) and FVB mice (female, 25.3 ± 0.985 g, Charles River Laboratories, Wilmington, MA) were used to examine P-gp and BCRP influence on fentanyl pharmacokinetics. Both rats and mice were housed under a 12-hour light/dark cycle. An LFER 150 PET/CT Scanner (Mediso Ltd., Budapest, Hungary) was used for dynamic PET study. Radiosynthesis of [C]fentanyl Ethylmagnesium bromide solution in diethyl ether (3 M, 500 µL) was diluted with freshly distillated THF (500 µL) in the glove box 10 minutes prior to [ 11 C]CO 2 delivery. The resulting solution was flushed through the polyethylene tube (0.034 inch I.D., 0.060 inch O.D., Scientific commodities, Inc.). After excess volume of solution was removed by flushing with nitrogen gas, the tube related to a 4-port 2-way valve (V-101D, IDEX Health & Science) in a closed position for the loop. This valve was installed as shown in Fig. 1 . C-11 labeled carbon dioxide ([ 11 C]CO 2 ) was produced from the on-site cyclotron (GE PET trace 800, GE Healthcare, OH) by the 14 N (p,α) 11 C nuclear reaction using the nitrogen target containing trace of oxygen (1%) and cryogenically trapped in a stainless coil (Length, 200 mm; OD, 1/16″; ID, 0.7mm) at -185°C. After radioactivity within the cold trap plateaued, the cold trap temperature was increased to 100°C and [ 11 C]CO 2 was released into the tubing using helium flow (3.5 mL/min). The carboxylation occurred for 1 min and then the contents of the tubing was eluted using THF (450 µL) into the first reaction vessel contains phthaloyl dichloride (52 µL), 2,6-di-tert-butylpyridine (62 µL), dimethylformamide (2.64 µL). The crude mixture was distilled to remove excess THF under a stream of helium (8.5 mL/min) by heating to 89°C. After injection of chloroform (200 µL), the second distillate portion (90–130°C) was collected to the second reaction vessel containing 4-ANPP (1 mg, 3.6 µmol), chloroform (50 µL) and DIPEA (8 µL, 46 µmol). The second reaction vessel was heated to 60°C and maintained for 5 min for [ 11 C]fentanyl synthesis. Afterwards, chloroform was removed by heating to 100°C under helium flow (8.5 mL/min), followed by cooling down to 30°C. The crude mixture was diluted (HPLC solvent (900 µL) mixed with 12 M HCl (5 µL)) and purified by semi-preparative HPLC (column, Chromolith RP-18 monolithic HPLC column, 100x10 mm, 5 µm; mobile phase: 0.01 M hydrochloric acid/ethanol = 80/20; flow rate, 5 mL/min; detection wavelength, 210 nm) ( Fig. S1 ). [ 11 C]fentanyl was collected at 11 min and pH was adjusted with 1M NaOH solution and sodium phosphate buffer. Small Animal PET Studies Anesthesia was initially induced using isoflurane (5%) in a stream of oxygen gas (1.25 L/min) for 5 minutes, then maintained at low isoflurane (1–2%) throughout the study. A catheter connected with a tubing (BTPE-10, 48 cm; Instech Laboratories, Inc., PA) was inserted into the tail vein. After a CT scan, a dynamic PET scan was performed in a list mode for 90 min simultaneously from the start of [ 11 C]fentanyl administration in one-minute bolus using a PHD 2000 syringe pump (Harvard Apparatus, Holliston, MA). Vital signs were measured with a Physiosuite or MouseStat (Kent Scientific., Torrington, Connecticut). The acquired dynamic PET data was reconstructed into time 23 frames (6x20s, 5x60s, 4x120s, 3x300s, 3x600s, 2x900s). Elacridar (3 mg/Kg; TargetMol Chemicals Inc., Boston, MA) was prepared and administrated at 15 min prior to [ 11 C]fentanyl injection as shown in the previous literature [ 14 ]. Ex Vivo Biodistribution Studies: Radiometric HPLC analysis [ 11 C]Fentanyl was intravenously injected into anesthetized Wistar rats (n = 12). Blood samples (0.2 to 0.5 mL) were collected from the femoral artery through BTPU-27 tubing (Instech Laboratories, Inc., Plymouth Meeting, PA) at 1, 1.5, 3, 5, 10, 15, 30, 45, and 60 minutes after radiotracer injection (n = 1–6 per time point). Samples were processed for both radiolabeled metabolite analysis and total radioactivity quantification. For metabolite analysis, each blood sample was centrifuged at 14500 RPM for 2 minutes (Eppendorf MiniSpin Centrifuge, Enfield, CT). The resulting supernatants were mixed and vortexed with equal volume of acetonitrile and centrifuged again to precipitate plasma proteins prior to radiometric HPLC analysis. In parallel, an aliquot of each blood sample was weighed and measured using the 2480 Wizard gamma counter (Perkin Elmer, Waltham, MA) to determine total radioactivity for SUV calculations. Radiometabolite analysis and total radioactivity quantification were also done in the brain and interscapular brown adipose tissue (BAT). Rats were sacrificed at 15, 30, 45, and 60 minutes after tracer administration (n = 2–4 per time point) and the brain and BAT samples were dissected, weighed, and counted with the gamma counter. Samples were then treated with acetonitrile (500 µL) and homogenized at 3000 RPM for 4 minutes with a homogenizer (099C K54, Glas-Col LLC, Terra Haute, IN). The mixture was centrifuged at 14500 RPM for 2 minutes and supernatants were filtered through polypropylene syringe filters (Tisch Scientific, Cleves, OH) for radiometabolite analysis. Radiometabolite analysis of plasma, brain, and BAT samples were performed with a radiometric HPLC (column, Chromolith Semi-Prep RP-18e endcapped column, 100 x 10 mm, 2 µm; mobile phase, isocratic 0.01M HCl/EtOH = 77/23; flow rate, 5 mL/min; detection wavelength, 210 nm) equipped with a G1367C autosampler (Agilent, Wilmington, DE), two Azura P 4.1S pumps (Knauer, Berlin, Germany), a BlueShadow detector 10D at 210nm (Knauer, Berlin, Germany), and a radiodetector (B-FC-4100 BGO High Voltage Detector) paired with a Colibrick AD converter (DataApex, Prague, Czechia) ( Fig. S2 ). Pharmacokinetic analysis in plasma Pharmacokinetic parameters were generated using a 2-compartment model in a Microsoft Excel Add-in, PKSolver (PMID: 20176408). A weighting of 1/C p 2 was utilized for fitting to the biexponential function, where C p was the plasma concentration. PET Image Processing and Statistical Analysis Reconstructed PET data was co-registered to the rat brain atlas [ 15 ] in PMOD (v2.8 PMOD Technologies, Zurich, Switzerland) and the resulting parameters applied into the corresponding dynamic PET data. Time-activity curves were generated using a regions of interest (ROIs) template and expressed as standard uptake values (SUV). Volumes of interest (VOIs) of peripheral organs were manually identified based on CT and PET images. Results are reported as mean ± standard deviation and analyzed in Microsoft Excel. To compare brain regions within rats a repeated measures one-way ANOVA was performed. Unpaired t-tests were performed for comparison between controls mice and the efflux transporter knockout mice and elacridar pretreatment animals. Results Radiosynthesis of [ 11 C]fentanyl All the steps for [ 11 C]fentanyl radiosynthesis were applied to the commercially available radiochemistry module equipped with minor modifications as shown in Fig. 1 . The averaged total synthesis time was about 42 min (n = 5), providing moderate radiochemical yield (10.4 ± 5.7%, decay collected, n = 5) and high radiochemical purity (> 99%). Sufficient [ 11 C]fentanyl (13.2 ± 7.0 GBq, n = 5) at the end of synthesis was routinely produced from ~ 129.5 GBq (~ 3.5 Ci) of [ 11 C]CO 2 . Molar activity ranged from 384.8 to 2571.5 Gbq/µmol (10.4 to 69.5 Ci/µmol) at the end of bombardment. Co-injection of the nonradioactive fentanyl with [ 11 C]fentanyl in analytical HPLC system showed identity of the product was well established (retention time, 8.9 min) ( Fig. S3 ). PET Study: Brain Pharmacokinetics and Influence of Brain Efflux Pumps Whole brain pharmacokinetics of [ 11 C]fentanyl in the rat brain was characterized by fast and high uptake (SUV max = 2.71 ± 0.122 g/mL, T max = 1.72 min) and fast clearance (T 1/2 = 5.06 min) (Fig. 2 A). Brain uptake was largely homogenous across cortical and subcortical brain regions; consistently, the area under the time-activity curves (Fig. 2 B) did not differ significantly in five brain regions (one-way ANOVA, p = 0.1383) as also shown in the averaged PET image from 0 to 15 minutes (Fig. 2 C). The effect of efflux transporters on [ 11 C]fentanyl brain pharmacokinetics were measured in P-gp and BCRP KO mice and compared to wildtype mice. KO mice had higher whole-brain peak uptake (n = 3, SUV max = 3.94 ± 0.629 g/mL), later peak brain uptake (T max = 2.50 min) and slower clearance (T 1/2 = 14.1 min) in comparison to wildtype mice (n = 4, SUV max = 3.17 ± 1.04 g/mL, T max = 1.83 min, T 1/2 = 8.96 min) (Fig. 3 A). This resulted in higher [ 11 C]fentanyl exposure in the KO’s brain, observed by a higher area under the time-activity curve relative to wildtype mice (Fig. 3 B). This area under the curve difference was significant (unpaired t-test, p = 0.0044 ) and can be visualized in the averaged SUV image (0 to 15 minutes) from a KO and a wildtype mouse (Fig. 3 C). For rats, when P-gp and BCRP efflux transporters were blocked by pretreatment with a 3 mg/kg dose of elacridar, the brain uptake of [ 11 C]fentanyl was higher and peaked later (n = 5, brain/blood max = 2.18 ± 0.367, T max = 1.83 min) and its clearance was slower (T 1/2 = 7.27 min) than the animals pretreated with vehicle (Fig. 4 A). The elacridar pretreated group showed a significantly higher AUC value in comparison to vehicle (Fig. 4 B), also seen in the averaged SUV image (0 to 15 minutes) (Fig. 4 C). PET Study: Whole-body Pharmacokinetics and Ex vivo Radiometric HPLC Analysis Whole body imaging in rats showed rapid [ 11 C]fentanyl uptake in lungs (T max = 1.17 min) and kidneys (T max = 1.83 min) (Fig. 5 A, 5 B) while C-11 uptake in the liver was slower (T max = 10 min) (Fig. 5 A, 5 C), but was the highest of all organs. Clearance of [ 11 C]fentanyl was slow in the kidney (T 1/2 = 11.2 min) and slower in liver (T 1/2 = 59.1 min) (Fig. 5 A, 5 D). The interscapular adipose tissue showed slow peak uptake (SUV max = 1.73 ± 0.313 g/mL, T max = 8 min) and had the slowest clearance of all organs/tissues (T 1/2 = 177 min) (Fig. 5 A). Ex vivo analysis demonstrated that, 30 minutes after [ 11 C]fentanyl injection, less than 50% of total activity in the plasma remained as parent radioactivity. In contrast, a significantly greater proportion of radioactivity remained as unmetabolized parent tracer in the brain (83%) and brown adipose tissue (BAT) (87%) (Fig. 6 ). The plasma pharmacokinetics of [ 11 C]fentanyl plotted as time versus plasma concentration (ng/cc) showed a bi-phasic decline (Fig. 7 A). Parameter estimation of [ 11 C]fentanyl’s pharmacokinetics in plasma revealed short half-lives for the distribution (5.67 ± 3.38 min) and elimination (51.9 ± 1.39 min) phases and a high volume of distribution (2.6 L/kg) at steady state (Fig. 7 B). Discussion Radiosynthesis In this study, the radiolabeling of fentanyl with carbon-11 was achieved through a three-step, two-pot process: (1) [ 11 C]carboxylation of ethylmagnesium bromide, (2) generation and distillation of [ 11 C]propionyl chloride, and (3) [ 11 C]propionylation of 4-ANPP (Scheme 1 ). Initial attempts to use a previously reported one-pot “in-loop” carboxylation and amidation protocol with triethylamine (TEA) in THF resulted in low and inconsistent radiochemical yields, and a requirement for excess precursor (> 7 µmol), which complicating purification process [ 16 ]. These results are likely due to the use of excess thionyl chloride and the low nucleophilicity of the anilinic amine group of 4-ANPP. Since Pike et al. [ 17 ] introduced the two-pot [ 11 C]acylation approach, a key radioactive precursor, [ 11 C]acyl chloride has been utilized in the synthesis of various radiotracers including [ 11 C]diprenorphine [ 17 ], [ 11 C]buprenorphine [ 18 ], [ 11 C]ohmefentanyl [ 19 ], [ 11 C]pyrazosin [ 20 ], [ 11 C]-(+)-PHNO [ 21 ], [ 11 C]WAY-100635 [ 22 ], [ 11 C]cyclophan [ 23 ], [ 11 C]melatonin derivatives [ 24 ], and [ 11 C]physostigmine [ 25 ]. We selected the in-loop carboxylation and distillation of [ 11 C]acyl chloride to improve molar activity and reduce interference from excess chlorinating reagent, thereby minimizing the amount of amine precursor required for amidation. Additionally, this two-pot strategy allowed for the optimization and monitoring of each step via radiometric analysis. For [ 11 C]carboxylation, a loop containing the Grignard reagent (37 µL) was prepared in a glove box using a 4-port-2-way valve, and installed to the radiochemistry module. This procedure strictly excluded ambient carbon dioxide and water, which likely contributed into achieving exceptionally high molar activity (up to 70 Ci/µmol); in other words, most of C-12 mass came from other than commercial ethylmagnesium bromide solution. While trapping efficiency of C-11 radioactivity was > 99% in the loop, the concentration of the Grignard reagent (GR) was critical for production of [ 11 C]propionate. As reported in prior studies [ 16 , 19 , 22 ], the concentration of the GR is critical; low concentrations led to poor [ 11 C]CO₂ conversion, while high concentrations resulted in overreaction. Radiometric HPLC analysis confirmed the formation of [ 11 C]diethyl ketone as a byproduct and the presence of unreacted [ 11 C]CO₂ ( Fig. S4 ). 1.5 M of GR concentration showed 46% of [¹¹C]propionic acid and 10% of [¹¹C]diethyl ketone in total 56% of [ 11 C]CO 2 conversion. The crude [ 11 C]propionyl chloride, generated from [ 11 C]propionate using phthaloyl dichloride, was distilled by heating under a stream of helium. The radioactivity in the initial distillate fraction (20–90°C) accounted for only 1.4–10.5% (n = 5) of the total. Thus, the second fraction (90–130°C) was used for subsequent amidation, dramatically reducing the solvent volume in the second reaction vessel. The distilled [ 11 C]propionyl chloride represented 21 ± 8% (n = 5) of the total radioactivity in the first reaction vessel. The remaining activity (27.6 ± 8%, n = 5) could not be distilled, even at temperatures up to 180°C. As previously mentioned, the low radiochemical yield observed during the acylation of 4-ANPP is likely due to the poor nucleophilicity of its anilinic amine [ 26 ]. In contrast, more nucleophilic amines such as 1-(4-methoxyphenyl)piperazine exhibited high [ 11 C]propionylation yield (> 30%, data not shown). To improve yields with 4-ANPP, various solvent and base combinations were systematically screened using non-radioactive ("cold") propionyl chloride and 4-ANPP under short reaction times ( Fig. S5, S6 ). Among the organic bases tested, DIPEA and 1,2,2,6,6-pentamethylpiperidine (PMP) showed highest yields in both chloroform and THF. Solvent screening with DIPEA revealed that polar chlorinated solvents such as chloroform and dichloromethane were most effective. Based on these results, [ 11 C]propionylation conditions were directly compared to TEA/THF condition (Table 1). While DIPEA/THF gave moderate yield (56.9 ± 10.2%, n = 5), DIPEA/chloroform gave slightly higher yield (62.5 ± 11.3%, n = 3). However, TEA provided very poor yield (7.7%) regardless of solvents, which is consistent with the nonradioactive version of test results. Preclinical PET Studies Understanding fentanyl’s pharmacokinetics throughout the various organs/tissues is invaluable, as its clinical effects, toxicity, and duration of action are determined by its concentrations at specific target sites rather than by plasma levels alone. Organ/tissue-specific data may reveal how fentanyl’s rapid distribution to the brain underlies its fast-acting analgesic and respiratory depressant effects as well as its almost immediate rewarding effects, while its subsequent redistribution to peripheral organs can influence residence time and the pattern of elimination. Knowledge of tissue-level pharmacokinetics thus informs the clinical management of fentanyl toxicity and enhances our understanding of its biodistribution, especially with chronic or high-dose use, ultimately supporting improved therapeutic strategies such as opioid overdose reversal interventions. Our PET imaging results demonstrate rapid and high brain penetration of [ 11 C]fentanyl, consistent with its fast onset of analgesia and the risk of acute respiratory depression when misused; the estimated brain AUC was approximately three times of plasma AUC. Fentanyl distribution in the brain was widespread and did not preferentially accumulate in opioid receptor-rich regions, suggesting largely non-specific signals. This was further supported by naloxone pretreatment studies, which showed no significant change in brain uptake or regional distribution (data not shown). Both brain permeability and clearance were significantly altered by inhibition or genetic knockout of two major efflux pumps, resulting in increases of up to 34% in rats and 81% in mice. These findings are consistent with previous reports indicating that fentanyl is a substrate for P-gp and BCRP, and that efflux pump inhibition is associated with enhanced central effects and respiratory depression [ 27 ]. This is particularly relevant given that chronic exposure to opioid drugs alters the expression efflux transporters of cerebral blood vessels [ 28 ]. Peripherally, [ 11 C]fentanyl PET showed initial high uptake in the lung, heart, liver, and kidneys. Notably, uptake in brown adipose tissue (BAT) increased gradually and remained elevated throughout the 90-minute scan, with BAT concentrations exceeding those in the brain. The prolonged retention and higher concentration of fentanyl in adipose tissues suggest that fat may serve as a reservoir, delaying fentanyl clearance from the brain and potentially contributing to re-narcotization following opioid reversal. This mechanism may be particularly important in chronic fentanyl users, where delayed elimination could complicate overdose management. Indeed, the re-narcotization observed after fentanyl overdose reversal with naloxone in some fentanyl misusers is believed to reflect fat accumulation from repeated exposures consistent with the presence of fentanyl in urine for up to 1 week in fentanyl misusers [ 10 ]. Experimental data on fentanyl concentrations in human brain and peripheral tissues are extremely limited, primarily derived from postmortem forensic studies and a small number of intraoperative CSF measurements. The use of [ 11 C]fentanyl for PET imaging presents a valuable tool to noninvasively quantify fentanyl pharmacokinetics in various human populations and clinical scenarios, providing better understanding in our understanding of fentanyl disposition and its clinical implications. Conclusion [¹¹C]Fentanyl was reliably synthesized in high molar activity, effectively minimizing isotopic dilution through an automated two-pot synthesis. Rodent PET imaging demonstrated rapid and high brain penetration, with evidence of interaction with brain efflux transporters in vivo. Furthermore, our findings revealed prolonged accumulation of [¹¹C]fentanyl in adipose tissues, suggesting a significant peripheral reservoir. These data indicate that [¹¹C]fentanyl will serve as a valuable tool for elucidating fentanyl's brain and whole-body pharmacokinetics across diverse patient populations, particularly chronic fentanyl misusers. This advancement is anticipated to contribute to the development of improved therapeutic strategies. Declarations Author contributions W.K, A.K.W, N.J.B., N.D.V., and S.W.K. mainly wrote and revised the manuscript. Radiosynthesis development was done by W.K, A.K.W, N.J.B., M.L.F, G.B, W.Z, S.M.E and S.W.K. PET and metabolite analysis were performed and analyzed by N.J.B, A.C, M.L.F, J.L, K.A.O, S.W.K. Full experimental design and supervision was from S.W.K and N.D.V. All authors read and approved the final manuscript. Acknowledgements This study was conducted with support from the Intramural Research Program of the National Institute on Alcohol Abuse and Alcoholism (Y1AA-3009, Volkow). The authors thank the NIH Clinical Center PET Department (Dr. Peter Herscovitch, Mr. Kris Kim, Linwood Tucker, and George Elliott) for their assistance with cyclotron operations. We also acknowledge the NIMH Molecular Imaging Branch, particularly Drs. Robert Innis and Victor Pike, for providing PET imaging infrastructure and valuable scientific comments. Availability of data and material The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. Funding Open access funding was provided by the National Institute on Alcohol Abuse and Alcoholism. This work was supported by the Intramural Research Program of the National Institute on Alcohol Abuse and Alcoholism (ZIA-AA000550, Volkow). Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests Not applicable. References Stanley TH, Fentanyl. J Pain Symptom Manage. 2005;29:S67–71. http://doi:10.1016/j.jpainsymman.2005.01.009 . CDC overdose prevention. About overdose prevention. In: overdose statistics. U.S. Department of Health and Human Services. https://www.cdc.gov/overdose-prevention/about/index.html#:~:text=Drug%20overdoses%20dramatically%20increased%20over,made%20fentanyl%20and%20fentanyl%20analogs . Accessed 7 July 2025. Volkow ND, Califf RM, Sokolowska M, Tabak LA, Compton WM. Testing for fentanyl — urgent need for practice-relevant and public health research. N Engl J Med. 2023;388:2214–7. http://doi:10.1056/NEJMp2302857 . Peng PWH, Sandler AN. A review of the use of fentanyl analgesia in the management of acute pain in adults. Anesthesiology. 1999;90:576–99. https://doi.org/10.1097/00000542-199902000-00034 . Baird A, White SA, Das R, Tatum N, Bisgaard EK. Whole body physiology model to simulate respiratory depression of fentanyl and associated naloxone reversal. Commun Med (Lond). 2024;4:114. https://doi:10.1038/s43856-024-00536-5 . Chaudun F, Python L, Liu Y, Hiver A, Cand J, Kieffer BL, Valjent E, Lüscher C. Distinct micro-opioid ensembles trigger positive and negative fentanyl reinforcement. Nature. 2024;630:141–8. https://doi:10.1038/s41586-024-07440-x . Okada CR, Henthorn TK, Zuk J, Sempio C, Roosevelt G, Ruiz AG, et al. Population pharmacokinetics of single bolus dose fentanyl in obese children. Anesth Analg. 2024;138:99–107. https://doi:10.1213/ANE.0000000000006554 . Shibutani K, Inchiosa MA Jr., Sawada K, Bairamian M. Pharmacokinetic mass of fentanyl for postoperative analgesia in lean and obese patients. Br J Anaesth. 2005;95:377–83. https://doi:10.1093/bja/aei195 . Stoeckel H, Schuttler J, Magnussen H, Hengstmann JH. Plasma fentanyl concentrations and the occurrence of respiratory depression in volunteers. Br J Anaesth. 1982;54:1087–95. https://doi:10.1093/bja/54.10.1087 . Bird HE, Huhn AS, Dunn KE. Fentanyl absorption, distribution, metabolism, and excretion: narrative review and clinical significance related to illicitly manufactured fentanyl. J Addict Med. 2023;17:503–8. https://doi:10.1097/ADM.0000000000001185 . Ghosh KK, Padmanabhan P, Yang CT, Ng DCE, Palanivel M, Mishra S, Christer H, Balázs G. Positron emission tomographic imaging in drug discovery. Drug Discov Today. 2022;27:280–91. 10.1016/j.drudis.2021.07.025 . https://doi . Schneider E, Brune K. Distribution of fentanyl in rats: an autoradiographic study. Naunyn-Schmiedeberg's Arch Pharmacol. 1985;331:359–63. https://doi.org/10.1007/BF00500820 . Nami M, Dabiri M, Shirvani G, Ahmadi Faghih MA, Javaheri M. Preparation of Fentanyl Labeled with Carbon-14. Radiochemistry. 2018;60:42–4. 10.1134/s1066362218010071 . Tang S, Kim SW, Olsen-Dufour A, Pearson T, Freaney M, Singley E, Jenkins M, Burkard NJ, Wozniak A, Parcon P, Wu S, Morse CL, Jana S, Liow J, Zoghbi SS, Vendruscolo JCM, Vendruscolo LF, Pike VW, Koob GF, Volkow ND, Innis RB. PET imaging in rat brain shows opposite effects of acute and chronic alcohol exposure on phosphodiesterase-4B, an indirect biomarker of cAMP activity. Neuropsychopharmacology. 2024;50:444–51. https://doi:10.1038/s41386-024-01988-y . Schiffer WK, Mirrione MM, Biegon A, Alexoff DL, Patel V, Dewey SL. Serial microPET measures of the metabolic reaction to a microdialysis probe implant. J Neurosci Methods. 2006;155:272–84. 10.1016/j.jneumeth.2006.01.027 . https://doi . Rami-Mark C, Ungersboeck J, Haeusler D, Nics L, Philippe C, Mitterhauser M, Willeit M, Lanzenberger R, Karanikas G, Wadsak W. Reliable set-up for in-loop 11 C-carboxylations using Grignard reactions for the preparation of [carbonyl- 11 C]WAY-100635 and [ 11 C]-(+)-PHNO. Appl Radiat Isot. 2013;82:75–80. https://doi:10.1016/j.apradiso.2013.07.023 . Luthra SK, Pike VW, Brady F. The preparation of carbon-11 labelled diprenorphine: a new radioligand for the study of the opiate receptor system in vivo . J Chem Soc Chem Commun. 1985;1423–25. https://doi:10.1039/C39850001423 . Luthra SK, Pike VW, Brady F, Horlock PL, Prenant C, Crouzel C. Preparation of [ 11 C]Buprenorphine-A potential radioligand for the study of the opiate receptor system in vivo . Appl Radiat Isot. 1987;38:65–6. https://doi.org/10.1016/0883-2889(87)90239-5 . Zhu YC, Prenant C, Crouzel C, Comar D, Chi ZQ. Synthesis of [ 11 C]-ohmefentanyl, a novel, highly potent and selective agonist for opiate µ-receptors. J Label Comp Radiopharm. 1992;31:853–60. https://doi.org/10.1002/jlcr.2580311103 . Ehrin E, Luthra SK, Crouzel C, Pike VW. Preparation of carbon-11 labelled prazosin, a potent and selective α1-adrenoreceptor antagonist. J Label Comp Radiopharm. 1988;25:177–83. https://doi.org/10.1002/jlcr.2580250209 . Pfaff S, Philippe C, Nics L, Berroteràn-Infante N, Pallitsch K, Rami-Mark C, Weidenauer A, Sauerzopf U, Willeit M, Mitterhauser M, Hacker M, Wadsak W, Pichler V. Toward the optimization of (+)-[ 11 C]PHNO synthesis: time reduction and process validation. Contrast Media Mol Imaging, 2019; 2019(1):4292596. https://doi.org/10.1155/2019/4292596 McCarron JA, Turton DR, Pike VW, Poole KG. Remotely-controlled production of the 5-HT1A receptor radioligand, [carbonyl- 11 C]WAY-100635, via 11 C-carboxylation of an immobilized Grignard reagent. J Label Comp Radiopharm. 1996. 10.1002/(sici)1099-1344(199610)38:103.0.Co;2-y . https:// . 38:941 – 53. McPherson DW, Hwang D, Fowler JS, Wolf AP, MacCregor RM, Arnett CD. A simple one-pot synthesis of cyclopropane [ 11 C]carbony1 chloride. synthesis and biodistribution of [ 11 C]cyclorphan. J Label Comp Radiopharm. 1986;3:505–14. https://doi.org/10.1002/jlcr.2580230507 . Bars DL, Luthra SK, Pike VW, Duc CL. Preparation of a carbon-11 labelled neurohormone-[ 11 C]melatonin. Appl Radiat Isot. 1987;38:1073–7. https://doi:10.1016/0883-2889(87)90073-6 . Bonnot-Lours S, Crouzel C, Prenant C, Hinnen F. Carbon-11 labelling of an inhibitor of Acetylchoiinesterase: [ 11 C]Physostigmine. J Label Comp Pharm. 1992;33:277–84. https://doi.org/10.1002/jlcr.2580330405 . Cai L, Xu R, Guo X, Pike VW. Rapid room-temperature 11 C-methylation of arylamines with [ 11 C]methyl iodide promoted by solid inorganic-bases in DMF. Eur J Org Chem. 2012;2012(7):1303–10. https://doi.org/10.1002/ejoc.201101499 . Yu C, Yuan M, Yang H, Zhuang X, Li H. P-glycoprotein on blood-brain barrier plays a vital role in fentanyl brain exposure and respiratory toxicity in rats. Toxicol Sci. 2018;164:353–62. https://doi.org/10.1093/toxsci/kfy093 . Schaefer CP, Arkwright NB, Jacobs LM, Jarvis CK, Hunn KC, Largent-Milnes TM, Tome ME, Davis TP. Chronic morphine exposure potentiates pglycoprotein trafficking from nuclear reservoirs in cortical rat brain microvessels. PLoS ONE. 2018;13(2):e0192340. https://doi.org/10.1371/journal.pone.0192340 . Scheme Scheme 1 is available in the Supplementary Files section. Supplementary Files FentanylSIEJNMMIRadiopharmacyandChemistry.docx floatimage1.png Cite Share Download PDF Status: Published Journal Publication published 28 Oct, 2025 Read the published version in EJNMMI Radiopharmacy and Chemistry → Version 1 posted Editorial decision: Minor revision 07 Sep, 2025 Reviewers agreed at journal 18 Aug, 2025 Reviewers invited by journal 18 Aug, 2025 Editor assigned by journal 15 Aug, 2025 First submitted to journal 14 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7367969","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":501733556,"identity":"ab6b2ec7-e498-4276-b1bc-89a84feba5f8","order_by":0,"name":"Woochan Kim","email":"","orcid":"","institution":"National Institute on Alcohol Abuse and Alcoholism","correspondingAuthor":false,"prefix":"","firstName":"Woochan","middleName":"","lastName":"Kim","suffix":""},{"id":501733557,"identity":"122eda31-082f-4484-9a7a-5c9ae07ef70a","order_by":1,"name":"Aaron K. Wozniak","email":"","orcid":"","institution":"National Institute on Alcohol Abuse and Alcoholism","correspondingAuthor":false,"prefix":"","firstName":"Aaron","middleName":"K.","lastName":"Wozniak","suffix":""},{"id":501733558,"identity":"08017081-9496-4820-b86f-0bacbeb3119a","order_by":2,"name":"Nathaniel J. Burkard","email":"","orcid":"","institution":"National Institute on Alcohol Abuse and Alcoholism","correspondingAuthor":false,"prefix":"","firstName":"Nathaniel","middleName":"J.","lastName":"Burkard","suffix":""},{"id":501733559,"identity":"b49c6a7c-1861-4e0f-8c71-22e965da87c0","order_by":3,"name":"Michael L. Freaney","email":"","orcid":"","institution":"National Institute on Alcohol Abuse and Alcoholism","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"L.","lastName":"Freaney","suffix":""},{"id":501733560,"identity":"4fcb38bf-48ae-4b51-9a7d-31f82f4d5550","order_by":4,"name":"Ailen Costamagna-Soto","email":"","orcid":"","institution":"National Institute on Alcohol Abuse and Alcoholism","correspondingAuthor":false,"prefix":"","firstName":"Ailen","middleName":"","lastName":"Costamagna-Soto","suffix":""},{"id":501733561,"identity":"fef1cd42-019e-4f89-a2be-e03b5af1829f","order_by":5,"name":"Kelly O’Conor","email":"","orcid":"","institution":"National Institute on Alcohol Abuse and Alcoholism","correspondingAuthor":false,"prefix":"","firstName":"Kelly","middleName":"","lastName":"O’Conor","suffix":""},{"id":501733562,"identity":"44a4ced3-2837-42e9-a5bc-8e393ce74a25","order_by":6,"name":"Abolgasem Bakhoda","email":"","orcid":"","institution":"National Institute on Alcohol Abuse and Alcoholism","correspondingAuthor":false,"prefix":"","firstName":"Abolgasem","middleName":"","lastName":"Bakhoda","suffix":""},{"id":501733563,"identity":"4d8c2d90-5a11-4bf3-b93f-7a6e85756813","order_by":7,"name":"Seth M. Eisenberg","email":"","orcid":"","institution":"National Institute on Alcohol Abuse and Alcoholism","correspondingAuthor":false,"prefix":"","firstName":"Seth","middleName":"M.","lastName":"Eisenberg","suffix":""},{"id":501733564,"identity":"8cbf440b-d218-4894-a022-217cd5386fd0","order_by":8,"name":"Wenjing Zhao","email":"","orcid":"","institution":"National Institute on Alcohol Abuse and Alcoholism","correspondingAuthor":false,"prefix":"","firstName":"Wenjing","middleName":"","lastName":"Zhao","suffix":""},{"id":501733565,"identity":"ee84fdca-3ceb-4b11-b0c6-543897bfdb36","order_by":9,"name":"Jeih-San Liow","email":"","orcid":"","institution":"National Institute of Mental Health","correspondingAuthor":false,"prefix":"","firstName":"Jeih-San","middleName":"","lastName":"Liow","suffix":""},{"id":501733566,"identity":"a5625380-982c-4c17-b33a-0ca326cd3275","order_by":10,"name":"Nora D. Volkow","email":"","orcid":"","institution":"National Institute on Alcohol Abuse and Alcoholism","correspondingAuthor":false,"prefix":"","firstName":"Nora","middleName":"D.","lastName":"Volkow","suffix":""},{"id":501733567,"identity":"f86fb2b5-0256-402e-8847-a782805383ca","order_by":11,"name":"Sung Won Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYPCCBB4G9gaGwyRq4TlAohYGBokEBmai1MrPyD32mIchTUZ35tuDhwsYauUMDhDQYnAjL92YhyGHx+x2XsLhGQzHjQlrkcgxk+ZhqABqyTE4zMNwLHFmA0GHwbTcPEOkFoYbYC1Ah93gAWmpSewnpMPgzBszyTkGaTxmZ0AOMzhgzE9Ii3x7jpnEm4pke7PjZ4w/81TUybER0gICTDwGcEuJjE3GHwh2HXFaRsEoGAWjYEQBAFFvORbj5HCKAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-4647-4899","institution":"National Institute on Alcohol Abuse and Alcoholism","correspondingAuthor":true,"prefix":"","firstName":"Sung","middleName":"Won","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2025-08-13 20:30:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7367969/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7367969/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s41181-025-00394-z","type":"published","date":"2025-10-28T15:57:42+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89982583,"identity":"5e922c2f-9317-44ce-ab91-7d6fa3863f19","added_by":"auto","created_at":"2025-08-27 06:29:50","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":35344,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the fully automated system used for [\u003csup\u003e11\u003c/sup\u003eC]fentanyl production\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7367969/v1/8014cb390aeb3b7205e8eb73.jpg"},{"id":89984483,"identity":"e16d7996-87fb-4047-86e5-650303148ce8","added_by":"auto","created_at":"2025-08-27 06:37:50","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":20595,"visible":true,"origin":"","legend":"\u003cp\u003eBaseline pharmacokinetics of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl in the brain. (\u003cstrong\u003eA\u003c/strong\u003e) Averaged time-activity curves of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl in standard uptake value (SUV, g/mL) and (\u003cstrong\u003eB\u003c/strong\u003e) The area under the time activity curve (AUC) for the nucleus accumbens (Nac), caudate/putamen (Cd/Pu), hypothalamus (HPTH), thalamus (TH), and cerebellum (CB). (\u003cstrong\u003eC\u003c/strong\u003e) A group-averaged standard uptake value (SUV) image (averaged from 0 to 15 minutes).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7367969/v1/edecee890f13e9dd6b500c57.jpg"},{"id":89984482,"identity":"6c60b04c-584b-46ca-b4ea-4281e50b9a03","added_by":"auto","created_at":"2025-08-27 06:37:50","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":21327,"visible":true,"origin":"","legend":"\u003cp\u003eWhole brain uptake of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl in P-gp and BCRP KO mice versus wildtype mice. (\u003cstrong\u003eA\u003c/strong\u003e) Averaged time-activity curve of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl in the brain of KO mice and wildtype mice. (\u003cstrong\u003eB\u003c/strong\u003e) Area under the time-activity curve for brain uptake of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl in KO mice and wildtype mice. (\u003cstrong\u003eC\u003c/strong\u003e) Representative brain uptake difference between KO and wildtype mice.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7367969/v1/86fce5c4c4afad0ba79b7eed.jpg"},{"id":89982589,"identity":"65c8941a-4983-4eb9-bce1-549b50c42c49","added_by":"auto","created_at":"2025-08-27 06:29:50","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":20388,"visible":true,"origin":"","legend":"\u003cp\u003eWhole brain uptake of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl in Wistar rats pretreated with 3 mg/kg elacridar or vehicle. (\u003cstrong\u003eA\u003c/strong\u003e) Averaged time-activity curve of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl in whole brain of elacridar and vehicle pretreated rats. (\u003cstrong\u003eB\u003c/strong\u003e) Area under the time-activity curve for brain uptake of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl in elacridar and vehicle pretreated rats.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7367969/v1/98a5c04318f162e14d367c89.jpg"},{"id":89982591,"identity":"75d46d1c-47ab-4577-b731-0f325cde1065","added_by":"auto","created_at":"2025-08-27 06:29:50","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":49854,"visible":true,"origin":"","legend":"\u003cp\u003eWhole-body pharmacokinetics of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl in Wistar rats. (\u003cstrong\u003eA\u003c/strong\u003e) Averaged time-activity curves of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl in the brain, liver, lung, kidney and interscapular adipose tissue (n = 3). Standard uptake value (SUV) images of a representative rat time-averaged at (\u003cstrong\u003eB\u003c/strong\u003e) 0-2 minutes, (\u003cstrong\u003eC\u003c/strong\u003e) 2-9 minutes, and (\u003cstrong\u003eD\u003c/strong\u003e) 9-90 minutes.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7367969/v1/3dfdb351c008cc8916c8de2f.jpg"},{"id":89982588,"identity":"0edf7a52-8105-47af-84b4-8381b3051e60","added_by":"auto","created_at":"2025-08-27 06:29:50","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":19000,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolism of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl in the plasma, brain, and interscapular brown adipose tissue (iBAT) of Wistar rats represented as percentage of unchanged [\u003csup\u003e11\u003c/sup\u003eC]fentanyl over time.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7367969/v1/adedb0011302421de4ecd9ee.jpg"},{"id":89982597,"identity":"2a838857-8f88-4c11-bd26-8ad58c021cb0","added_by":"auto","created_at":"2025-08-27 06:29:51","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":35694,"visible":true,"origin":"","legend":"\u003cp\u003ePlasma pharmacokinetics of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl in Wistar rats. (\u003cstrong\u003eA\u003c/strong\u003e) Metabolism corrected plasma concentration (ng/cc) of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl in a representative rat over time. (\u003cstrong\u003eB\u003c/strong\u003e) [\u003csup\u003e11\u003c/sup\u003eC]fentanyl pharmacokinetics parameters calculated from plasma (n = 2).\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7367969/v1/96ce602a3a17704017788a61.jpg"},{"id":95040063,"identity":"90efcdf7-9ffd-46b6-a5eb-7fc64dfc3bf3","added_by":"auto","created_at":"2025-11-03 16:08:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1006571,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7367969/v1/fe5d195c-8404-486d-bb52-4ff93c4bb11b.pdf"},{"id":89982586,"identity":"0f9c5ec4-f9b1-4c3b-8b3e-e51e219e6d27","added_by":"auto","created_at":"2025-08-27 06:29:50","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":486126,"visible":true,"origin":"","legend":"","description":"","filename":"FentanylSIEJNMMIRadiopharmacyandChemistry.docx","url":"https://assets-eu.researchsquare.com/files/rs-7367969/v1/0cfbe8becd5edf1d6def1c05.docx"},{"id":89985394,"identity":"5f633d35-581a-498f-9aa6-1d8653e1f339","added_by":"auto","created_at":"2025-08-27 06:45:50","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":53800,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7367969/v1/8245f19b11eeb3a505084375.png"}],"financialInterests":"","formattedTitle":"\u003cp\u003e[\u003csup\u003e11\u003c/sup\u003eC]Fentanyl: Radiosynthesis and Preclinical PET Imaging for Its Pharmacokinetics\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFentanyl is a potent synthetic mu-opioid receptor (MOR) agonist, widely used in clinical settings not only as an adjunct in anesthesia but also for managing acute post-operative pain and breakthrough cancer pain [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The rise of illicitly manufactured fentanyl has led to widespread misuse, making it the main driver of the devastating overdose crisis in the United States. In 2022, fentanyl and analogues were involved in nearly 74,000 drug overdose deaths [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The severity of fentanyl-related overdoses has been exacerbated by its mixture with other drugs such as heroin, cocaine, and methamphetamine, often consumed unknowingly by users, which has significantly complicated overdose reversal efforts [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGiven fentanyl's critical role both in medicine and in the overdose crisis, investigating its pharmacokinetics has been essential for optimizing its therapeutic use [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] while also enhancing our understanding of overdose mechanisms, ultimately informing more effective management strategies [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. For instance, preclinical and clinical investigations of fentanyl's pharmacokinetics have helped to optimize dosages for various patient populations [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, these studies mostly concern fentanyl\u0026rsquo;s dosages relevant to anesthesia and analgesia rather than patterns reported and observed in individuals who misuse fentanyl outside of medical settings. Another important consideration is the observation among some misusers of a secondary fentanyl peak, with an abrupt increase in fentanyl plasma concentration and associated respiratory depression. A previous study found that over a 240-minute period, healthy volunteers injected with 0.5 mg fentanyl IV showed secondary peaks in plasma concentration between 45 and 90 minutes after administration [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Fentanyl\u0026rsquo;s pharmacokinetics are also impacted by demographic and clinical characteristics of a user, including age, obesity, metabolic function, among others. These factors, in addition to an individual\u0026rsquo;s history of fentanyl misuse, may drastically alter its pharmacokinetics and consequently, its physiological effects and overdose risk [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePreclinical studies using animal models have been crucial in exploring the threshold doses for severe respiratory depression associated with fentanyl overdose and addiction. These investigations provide valuable insights into the physiological effects of fentanyl at different plasma concentrations and can inform strategies for intervention. However, translating these findings to human physiology would benefit from \u003cem\u003ein vivo\u003c/em\u003e non-invasive methods for direct measurement of fentanyl\u0026rsquo;s biodistribution and kinetics in the brain and body.\u003c/p\u003e\u003cp\u003ePositron emission tomography (PET) is a powerful quantitative imaging technique that allows for the \u003cem\u003ein vivo\u003c/em\u003e measurement of drug concentrations and distributions in target tissues using radiolabeled compounds. Unlike conventional pharmacokinetic studies that rely on plasma drug concentrations, PET provides direct visualization and quantification of drug levels in tissues relevant to both therapeutic effects and adverse events, such as the brain [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This non-invasive approach offers significant advantages for translational research, enabling repeated measurements and flexible experimental designs in laboratory animals and in humans, which would be particularly valuable for understanding the rapid onset and duration of fentanyl's effects.\u003c/p\u003e\u003cp\u003eWhile early studies utilized tritium- and carbon-14 labeled fentanyl to investigate its metabolism and biodistribution in preclinical models, these radiotracers are not optimal for dynamic PET imaging as each subject has to be scarified for each time point [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Therefore, a critical gap exists in our ability to non-invasively quantify fentanyl concentrations and kinetics in brain and other organs with PET that could also be eventually used for studies in humans. To address this limitation, we herein report the radiosynthesis of carbon-11 labeled fentanyl and present preliminary PET studies conducted in rodents, paving the way for translational pharmacokinetic investigations in humans.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMaterials\u003c/h2\u003e\u003cp\u003e4-Anilino-N-phenethyl-piperidine (4-ANPP) was purchased from Cayman Chemical. The aqueous hydrochloric acid solution (2 N, RICCA Chemical Company, TX) was diluted with water for semi-preparative HPLC. Absolute Ethanol and sodium phosphate buffer (45 mM phosphate, 60 mEq sodium) were obtained from Warner-Graham Company and Hospira Inc., respectively. Tetrahydrofuran (THF) was purified by distillation with sodium (dispersion in mineral oil, Strem Chemicals). All the other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and were used without any further purification.\u003c/p\u003e\u003cp\u003eRadiosynthesis was fully carried out and optimized with a commercially available module (Synthra MeIPlus Research). Radiochemical purity and molar activity were determined using an Agilent 1100 Series HPLC system (column, Agilent Eclipse XDB C-18 column, 150 x 4.6 mm, 5 \u0026micro;m; mobile phase, isocratic 0.1% trifluoroacetic acid solution/acetonitrile\u0026thinsp;=\u0026thinsp;70/30; flow rate, 1 mL/min; detection wavelength, 210 nm) and a radiometric detector equipped with a B-FC-4100 BGO High Voltage Detector.\u003c/p\u003e\u003cp\u003eAnimal use and protocol were approved by the institutional Animal Care \u0026amp; Use Committee (National Institutes of Mental Health; MIB-03, MIB-04). Wistar rats (male; 297\u0026thinsp;\u0026plusmn;\u0026thinsp;39.9 g, Envigo, Indianapolis, IN) were used for PET studies. P-gp and BCRP KO mice (female, 25.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.22 g, bred in-house) and FVB mice (female, 25.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.985 g, Charles River Laboratories, Wilmington, MA) were used to examine P-gp and BCRP influence on fentanyl pharmacokinetics. Both rats and mice were housed under a 12-hour light/dark cycle. An LFER 150 PET/CT Scanner (Mediso Ltd., Budapest, Hungary) was used for dynamic PET study.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eRadiosynthesis of [C]fentanyl\u003c/h3\u003e\n\u003cp\u003eEthylmagnesium bromide solution in diethyl ether (3 M, 500 \u0026micro;L) was diluted with freshly distillated THF (500 \u0026micro;L) in the glove box 10 minutes prior to [\u003csup\u003e11\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e delivery. The resulting solution was flushed through the polyethylene tube (0.034 inch I.D., 0.060 inch O.D., Scientific commodities, Inc.). After excess volume of solution was removed by flushing with nitrogen gas, the tube related to a 4-port 2-way valve (V-101D, IDEX Health \u0026amp; Science) in a closed position for the loop. This valve was installed as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. C-11 labeled carbon dioxide ([\u003csup\u003e11\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e) was produced from the on-site cyclotron (GE PET trace 800, GE Healthcare, OH) by the \u003csup\u003e14\u003c/sup\u003eN (p,α)\u003csup\u003e11\u003c/sup\u003eC nuclear reaction using the nitrogen target containing trace of oxygen (1%) and cryogenically trapped in a stainless coil (Length, 200 mm; OD, 1/16\u0026Prime;; ID, 0.7mm) at -185\u0026deg;C. After radioactivity within the cold trap plateaued, the cold trap temperature was increased to 100\u0026deg;C and [\u003csup\u003e11\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e was released into the tubing using helium flow (3.5 mL/min). The carboxylation occurred for 1 min and then the contents of the tubing was eluted using THF (450 \u0026micro;L) into the first reaction vessel contains phthaloyl dichloride (52 \u0026micro;L), 2,6-di-tert-butylpyridine (62 \u0026micro;L), dimethylformamide (2.64 \u0026micro;L). The crude mixture was distilled to remove excess THF under a stream of helium (8.5 mL/min) by heating to 89\u0026deg;C. After injection of chloroform (200 \u0026micro;L), the second distillate portion (90\u0026ndash;130\u0026deg;C) was collected to the second reaction vessel containing 4-ANPP (1 mg, 3.6 \u0026micro;mol), chloroform (50 \u0026micro;L) and DIPEA (8 \u0026micro;L, 46 \u0026micro;mol). The second reaction vessel was heated to 60\u0026deg;C and maintained for 5 min for [\u003csup\u003e11\u003c/sup\u003eC]fentanyl synthesis. Afterwards, chloroform was removed by heating to 100\u0026deg;C under helium flow (8.5 mL/min), followed by cooling down to 30\u0026deg;C. The crude mixture was diluted (HPLC solvent (900 \u0026micro;L) mixed with 12 M HCl (5 \u0026micro;L)) and purified by semi-preparative HPLC (column, Chromolith RP-18 monolithic HPLC column, 100x10 mm, 5 \u0026micro;m; mobile phase: 0.01 M hydrochloric acid/ethanol\u0026thinsp;=\u0026thinsp;80/20; flow rate, 5 mL/min; detection wavelength, 210 nm) (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). [\u003csup\u003e11\u003c/sup\u003eC]fentanyl was collected at 11 min and pH was adjusted with 1M NaOH solution and sodium phosphate buffer.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eSmall Animal PET Studies\u003c/h3\u003e\n\u003cp\u003eAnesthesia was initially induced using isoflurane (5%) in a stream of oxygen gas (1.25 L/min) for 5 minutes, then maintained at low isoflurane (1\u0026ndash;2%) throughout the study. A catheter connected with a tubing (BTPE-10, 48 cm; Instech Laboratories, Inc., PA) was inserted into the tail vein. After a CT scan, a dynamic PET scan was performed in a list mode for 90 min simultaneously from the start of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl administration in one-minute bolus using a PHD 2000 syringe pump (Harvard Apparatus, Holliston, MA). Vital signs were measured with a Physiosuite or MouseStat (Kent Scientific., Torrington, Connecticut). The acquired dynamic PET data was reconstructed into time 23 frames (6x20s, 5x60s, 4x120s, 3x300s, 3x600s, 2x900s). Elacridar (3 mg/Kg; TargetMol Chemicals Inc., Boston, MA) was prepared and administrated at 15 min prior to [\u003csup\u003e11\u003c/sup\u003eC]fentanyl injection as shown in the previous literature [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eEx Vivo Biodistribution Studies: Radiometric HPLC analysis\u003c/h3\u003e\n\u003cp\u003e[\u003csup\u003e11\u003c/sup\u003eC]Fentanyl was intravenously injected into anesthetized Wistar rats (n\u0026thinsp;=\u0026thinsp;12). Blood samples (0.2 to 0.5 mL) were collected from the femoral artery through BTPU-27 tubing (Instech Laboratories, Inc., Plymouth Meeting, PA) at 1, 1.5, 3, 5, 10, 15, 30, 45, and 60 minutes after radiotracer injection (n\u0026thinsp;=\u0026thinsp;1\u0026ndash;6 per time point). Samples were processed for both radiolabeled metabolite analysis and total radioactivity quantification. For metabolite analysis, each blood sample was centrifuged at 14500 RPM for 2 minutes (Eppendorf MiniSpin Centrifuge, Enfield, CT). The resulting supernatants were mixed and vortexed with equal volume of acetonitrile and centrifuged again to precipitate plasma proteins prior to radiometric HPLC analysis. In parallel, an aliquot of each blood sample was weighed and measured using the 2480 Wizard gamma counter (Perkin Elmer, Waltham, MA) to determine total radioactivity for SUV calculations.\u003c/p\u003e\u003cp\u003eRadiometabolite analysis and total radioactivity quantification were also done in the brain and interscapular brown adipose tissue (BAT). Rats were sacrificed at 15, 30, 45, and 60 minutes after tracer administration (n\u0026thinsp;=\u0026thinsp;2\u0026ndash;4 per time point) and the brain and BAT samples were dissected, weighed, and counted with the gamma counter. Samples were then treated with acetonitrile (500 \u0026micro;L) and homogenized at 3000 RPM for 4 minutes with a homogenizer (099C K54, Glas-Col LLC, Terra Haute, IN). The mixture was centrifuged at 14500 RPM for 2 minutes and supernatants were filtered through polypropylene syringe filters (Tisch Scientific, Cleves, OH) for radiometabolite analysis.\u003c/p\u003e\u003cp\u003eRadiometabolite analysis of plasma, brain, and BAT samples were performed with a radiometric HPLC (column, Chromolith Semi-Prep RP-18e endcapped column, 100 x 10 mm, 2 \u0026micro;m; mobile phase, isocratic 0.01M HCl/EtOH\u0026thinsp;=\u0026thinsp;77/23; flow rate, 5 mL/min; detection wavelength, 210 nm) equipped with a G1367C autosampler (Agilent, Wilmington, DE), two Azura P 4.1S pumps (Knauer, Berlin, Germany), a BlueShadow detector 10D at 210nm (Knauer, Berlin, Germany), and a radiodetector (B-FC-4100 BGO High Voltage Detector) paired with a Colibrick AD converter (DataApex, Prague, Czechia) (\u003cb\u003eFig. S2\u003c/b\u003e).\u003c/p\u003e\n\u003ch3\u003ePharmacokinetic analysis in plasma\u003c/h3\u003e\n\u003cp\u003ePharmacokinetic parameters were generated using a 2-compartment model in a Microsoft Excel Add-in, PKSolver (PMID: 20176408). A weighting of 1/C\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e was utilized for fitting to the biexponential function, where C\u003csub\u003ep\u003c/sub\u003e was the plasma concentration.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePET Image Processing and Statistical Analysis\u003c/h2\u003e\u003cp\u003eReconstructed PET data was co-registered to the rat brain atlas [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] in PMOD (v2.8 PMOD Technologies, Zurich, Switzerland) and the resulting parameters applied into the corresponding dynamic PET data. Time-activity curves were generated using a regions of interest (ROIs) template and expressed as standard uptake values (SUV). Volumes of interest (VOIs) of peripheral organs were manually identified based on CT and PET images. Results are reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation and analyzed in Microsoft Excel. To compare brain regions within rats a repeated measures one-way ANOVA was performed. Unpaired t-tests were performed for comparison between controls mice and the efflux transporter knockout mice and elacridar pretreatment animals.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eRadiosynthesis of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl\u003c/h2\u003e\u003cp\u003eAll the steps for [\u003csup\u003e11\u003c/sup\u003eC]fentanyl radiosynthesis were applied to the commercially available radiochemistry module equipped with minor modifications as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The averaged total synthesis time was about 42 min (n\u0026thinsp;=\u0026thinsp;5), providing moderate radiochemical yield (10.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.7%, decay collected, n\u0026thinsp;=\u0026thinsp;5) and high radiochemical purity (\u0026gt;\u0026thinsp;99%). Sufficient [\u003csup\u003e11\u003c/sup\u003eC]fentanyl (13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;7.0 GBq, n\u0026thinsp;=\u0026thinsp;5) at the end of synthesis was routinely produced from ~\u0026thinsp;129.5 GBq (~\u0026thinsp;3.5 Ci) of [\u003csup\u003e11\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e. Molar activity ranged from 384.8 to 2571.5 Gbq/\u0026micro;mol (10.4 to 69.5 Ci/\u0026micro;mol) at the end of bombardment. Co-injection of the nonradioactive fentanyl with [\u003csup\u003e11\u003c/sup\u003eC]fentanyl in analytical HPLC system showed identity of the product was well established (retention time, 8.9 min) (\u003cb\u003eFig. S3\u003c/b\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ePET Study: Brain Pharmacokinetics and Influence of Brain Efflux Pumps\u003c/h2\u003e\u003cp\u003eWhole brain pharmacokinetics of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl in the rat brain was characterized by fast and high uptake (SUV\u003csub\u003emax\u003c/sub\u003e = 2.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.122 g/mL, T\u003csub\u003emax\u003c/sub\u003e = 1.72 min) and fast clearance (T\u003csub\u003e1/2\u003c/sub\u003e = 5.06 min) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Brain uptake was largely homogenous across cortical and subcortical brain regions; consistently, the area under the time-activity curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) did not differ significantly in five brain regions (one-way ANOVA, p\u0026thinsp;=\u0026thinsp;0.1383) as also shown in the averaged PET image from 0 to 15 minutes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe effect of efflux transporters on [\u003csup\u003e11\u003c/sup\u003eC]fentanyl brain pharmacokinetics were measured in P-gp and BCRP KO mice and compared to wildtype mice. KO mice had higher whole-brain peak uptake (n\u0026thinsp;=\u0026thinsp;3, SUV\u003csub\u003emax\u003c/sub\u003e = 3.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.629 g/mL), later peak brain uptake (T\u003csub\u003emax\u003c/sub\u003e = 2.50 min) and slower clearance (T\u003csub\u003e1/2\u003c/sub\u003e = 14.1 min) in comparison to wildtype mice (n\u0026thinsp;=\u0026thinsp;4, SUV\u003csub\u003emax\u003c/sub\u003e = 3.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04 g/mL, T\u003csub\u003emax\u003c/sub\u003e = 1.83 min, T\u003csub\u003e1/2\u003c/sub\u003e = 8.96 min) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). This resulted in higher [\u003csup\u003e11\u003c/sup\u003eC]fentanyl exposure in the KO\u0026rsquo;s brain, observed by a higher area under the time-activity curve relative to wildtype mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). This area under the curve difference was significant (unpaired t-test, p\u0026thinsp;=\u0026thinsp;0.0044 ) and can be visualized in the averaged SUV image (0 to 15 minutes) from a KO and a wildtype mouse (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor rats, when P-gp and BCRP efflux transporters were blocked by pretreatment with a 3 mg/kg dose of elacridar, the brain uptake of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl was higher and peaked later (n\u0026thinsp;=\u0026thinsp;5, brain/blood max\u0026thinsp;=\u0026thinsp;2.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.367, T\u003csub\u003emax\u003c/sub\u003e = 1.83 min) and its clearance was slower (T\u003csub\u003e1/2\u003c/sub\u003e = 7.27 min) than the animals pretreated with vehicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The elacridar pretreated group showed a significantly higher AUC value in comparison to vehicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), also seen in the averaged SUV image (0 to 15 minutes) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePET Study: Whole-body Pharmacokinetics and Ex vivo Radiometric HPLC Analysis\u003c/h2\u003e\u003cp\u003eWhole body imaging in rats showed rapid [\u003csup\u003e11\u003c/sup\u003eC]fentanyl uptake in lungs (T\u003csub\u003emax\u003c/sub\u003e = 1.17 min) and kidneys (T\u003csub\u003emax\u003c/sub\u003e = 1.83 min) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) while C-11 uptake in the liver was slower (T\u003csub\u003emax\u003c/sub\u003e = 10 min) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), but was the highest of all organs. Clearance of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl was slow in the kidney (T\u003csub\u003e1/2\u003c/sub\u003e = 11.2 min) and slower in liver (T\u003csub\u003e1/2\u003c/sub\u003e = 59.1 min) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The interscapular adipose tissue showed slow peak uptake (SUV\u003csub\u003emax\u003c/sub\u003e = 1.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.313 g/mL, T\u003csub\u003emax\u003c/sub\u003e = 8 min) and had the slowest clearance of all organs/tissues (T\u003csub\u003e1/2\u003c/sub\u003e = 177 min) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eEx vivo analysis demonstrated that, 30 minutes after [\u003csup\u003e11\u003c/sup\u003eC]fentanyl injection, less than 50% of total activity in the plasma remained as parent radioactivity. In contrast, a significantly greater proportion of radioactivity remained as unmetabolized parent tracer in the brain (83%) and brown adipose tissue (BAT) (87%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The plasma pharmacokinetics of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl plotted as time versus plasma concentration (ng/cc) showed a bi-phasic decline (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Parameter estimation of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl\u0026rsquo;s pharmacokinetics in plasma revealed short half-lives for the distribution (5.67\u0026thinsp;\u0026plusmn;\u0026thinsp;3.38 min) and elimination (51.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39 min) phases and a high volume of distribution (2.6 L/kg) at steady state (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eRadiosynthesis\u003c/h2\u003e\u003cp\u003eIn this study, the radiolabeling of fentanyl with carbon-11 was achieved through a three-step, two-pot process: (1) [\u003csup\u003e11\u003c/sup\u003eC]carboxylation of ethylmagnesium bromide, (2) generation and distillation of [\u003csup\u003e11\u003c/sup\u003eC]propionyl chloride, and (3) [\u003csup\u003e11\u003c/sup\u003eC]propionylation of 4-ANPP (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Initial attempts to use a previously reported one-pot \u0026ldquo;in-loop\u0026rdquo; carboxylation and amidation protocol with triethylamine (TEA) in THF resulted in low and inconsistent radiochemical yields, and a requirement for excess precursor (\u0026gt;\u0026thinsp;7 \u0026micro;mol), which complicating purification process [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These results are likely due to the use of excess thionyl chloride and the low nucleophilicity of the anilinic amine group of 4-ANPP.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSince Pike et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] introduced the two-pot [\u003csup\u003e11\u003c/sup\u003eC]acylation approach, a key radioactive precursor, [\u003csup\u003e11\u003c/sup\u003eC]acyl chloride has been utilized in the synthesis of various radiotracers including [\u003csup\u003e11\u003c/sup\u003eC]diprenorphine [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], [\u003csup\u003e11\u003c/sup\u003eC]buprenorphine [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], [\u003csup\u003e11\u003c/sup\u003eC]ohmefentanyl [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], [\u003csup\u003e11\u003c/sup\u003eC]pyrazosin [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], [\u003csup\u003e11\u003c/sup\u003eC]-(+)-PHNO [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], [\u003csup\u003e11\u003c/sup\u003eC]WAY-100635 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], [\u003csup\u003e11\u003c/sup\u003eC]cyclophan [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], [\u003csup\u003e11\u003c/sup\u003eC]melatonin derivatives [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and [\u003csup\u003e11\u003c/sup\u003eC]physostigmine [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. We selected the in-loop carboxylation and distillation of [\u003csup\u003e11\u003c/sup\u003eC]acyl chloride to improve molar activity and reduce interference from excess chlorinating reagent, thereby minimizing the amount of amine precursor required for amidation. Additionally, this two-pot strategy allowed for the optimization and monitoring of each step via radiometric analysis.\u003c/p\u003e\u003cp\u003eFor [\u003csup\u003e11\u003c/sup\u003eC]carboxylation, a loop containing the Grignard reagent (37 \u0026micro;L) was prepared in a glove box using a 4-port-2-way valve, and installed to the radiochemistry module. This procedure strictly excluded ambient carbon dioxide and water, which likely contributed into achieving exceptionally high molar activity (up to 70 Ci/\u0026micro;mol); in other words, most of C-12 mass came from other than commercial ethylmagnesium bromide solution. While trapping efficiency of C-11 radioactivity was \u0026gt;\u0026thinsp;99% in the loop, the concentration of the Grignard reagent (GR) was critical for production of [\u003csup\u003e11\u003c/sup\u003eC]propionate. As reported in prior studies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], the concentration of the GR is critical; low concentrations led to poor [\u003csup\u003e11\u003c/sup\u003eC]CO₂ conversion, while high concentrations resulted in overreaction. Radiometric HPLC analysis confirmed the formation of [\u003csup\u003e11\u003c/sup\u003eC]diethyl ketone as a byproduct and the presence of unreacted [\u003csup\u003e11\u003c/sup\u003eC]CO₂ (\u003cb\u003eFig. S4\u003c/b\u003e). 1.5 M of GR concentration showed 46% of [\u0026sup1;\u0026sup1;C]propionic acid and 10% of [\u0026sup1;\u0026sup1;C]diethyl ketone in total 56% of [\u003csup\u003e11\u003c/sup\u003eC]CO\u003csub\u003e2\u003c/sub\u003e conversion.\u003c/p\u003e\u003cp\u003eThe crude [\u003csup\u003e11\u003c/sup\u003eC]propionyl chloride, generated from [\u003csup\u003e11\u003c/sup\u003eC]propionate using phthaloyl dichloride, was distilled by heating under a stream of helium. The radioactivity in the initial distillate fraction (20\u0026ndash;90\u0026deg;C) accounted for only 1.4\u0026ndash;10.5% (n\u0026thinsp;=\u0026thinsp;5) of the total. Thus, the second fraction (90\u0026ndash;130\u0026deg;C) was used for subsequent amidation, dramatically reducing the solvent volume in the second reaction vessel. The distilled [\u003csup\u003e11\u003c/sup\u003eC]propionyl chloride represented 21\u0026thinsp;\u0026plusmn;\u0026thinsp;8% (n\u0026thinsp;=\u0026thinsp;5) of the total radioactivity in the first reaction vessel. The remaining activity (27.6\u0026thinsp;\u0026plusmn;\u0026thinsp;8%, n\u0026thinsp;=\u0026thinsp;5) could not be distilled, even at temperatures up to 180\u0026deg;C.\u003c/p\u003e\u003cp\u003eAs previously mentioned, the low radiochemical yield observed during the acylation of 4-ANPP is likely due to the poor nucleophilicity of its anilinic amine [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In contrast, more nucleophilic amines such as 1-(4-methoxyphenyl)piperazine exhibited high [\u003csup\u003e11\u003c/sup\u003eC]propionylation yield (\u0026gt;\u0026thinsp;30%, data not shown). To improve yields with 4-ANPP, various solvent and base combinations were systematically screened using non-radioactive (\"cold\") propionyl chloride and 4-ANPP under short reaction times (\u003cb\u003eFig. S5, S6\u003c/b\u003e). Among the organic bases tested, DIPEA and 1,2,2,6,6-pentamethylpiperidine (PMP) showed highest yields in both chloroform and THF. Solvent screening with DIPEA revealed that polar chlorinated solvents such as chloroform and dichloromethane were most effective. Based on these results, [\u003csup\u003e11\u003c/sup\u003eC]propionylation conditions were directly compared to TEA/THF condition (Table\u0026nbsp;1). While DIPEA/THF gave moderate yield (56.9\u0026thinsp;\u0026plusmn;\u0026thinsp;10.2%, n\u0026thinsp;=\u0026thinsp;5), DIPEA/chloroform gave slightly higher yield (62.5\u0026thinsp;\u0026plusmn;\u0026thinsp;11.3%, n\u0026thinsp;=\u0026thinsp;3). However, TEA provided very poor yield (7.7%) regardless of solvents, which is consistent with the nonradioactive version of test results.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003ePreclinical PET Studies\u003c/h2\u003e\u003cp\u003eUnderstanding fentanyl\u0026rsquo;s pharmacokinetics throughout the various organs/tissues is invaluable, as its clinical effects, toxicity, and duration of action are determined by its concentrations at specific target sites rather than by plasma levels alone. Organ/tissue-specific data may reveal how fentanyl\u0026rsquo;s rapid distribution to the brain underlies its fast-acting analgesic and respiratory depressant effects as well as its almost immediate rewarding effects, while its subsequent redistribution to peripheral organs can influence residence time and the pattern of elimination. Knowledge of tissue-level pharmacokinetics thus informs the clinical management of fentanyl toxicity and enhances our understanding of its biodistribution, especially with chronic or high-dose use, ultimately supporting improved therapeutic strategies such as opioid overdose reversal interventions.\u003c/p\u003e\u003cp\u003eOur PET imaging results demonstrate rapid and high brain penetration of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl, consistent with its fast onset of analgesia and the risk of acute respiratory depression when misused; the estimated brain AUC was approximately three times of plasma AUC. Fentanyl distribution in the brain was widespread and did not preferentially accumulate in opioid receptor-rich regions, suggesting largely non-specific signals. This was further supported by naloxone pretreatment studies, which showed no significant change in brain uptake or regional distribution (data not shown). Both brain permeability and clearance were significantly altered by inhibition or genetic knockout of two major efflux pumps, resulting in increases of up to 34% in rats and 81% in mice. These findings are consistent with previous reports indicating that fentanyl is a substrate for P-gp and BCRP, and that efflux pump inhibition is associated with enhanced central effects and respiratory depression [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This is particularly relevant given that chronic exposure to opioid drugs alters the expression efflux transporters of cerebral blood vessels [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePeripherally, [\u003csup\u003e11\u003c/sup\u003eC]fentanyl PET showed initial high uptake in the lung, heart, liver, and kidneys. Notably, uptake in brown adipose tissue (BAT) increased gradually and remained elevated throughout the 90-minute scan, with BAT concentrations exceeding those in the brain. The prolonged retention and higher concentration of fentanyl in adipose tissues suggest that fat may serve as a reservoir, delaying fentanyl clearance from the brain and potentially contributing to re-narcotization following opioid reversal. This mechanism may be particularly important in chronic fentanyl users, where delayed elimination could complicate overdose management. Indeed, the re-narcotization observed after fentanyl overdose reversal with naloxone in some fentanyl misusers is believed to reflect fat accumulation from repeated exposures consistent with the presence of fentanyl in urine for up to 1 week in fentanyl misusers [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eExperimental data on fentanyl concentrations in human brain and peripheral tissues are extremely limited, primarily derived from postmortem forensic studies and a small number of intraoperative CSF measurements. The use of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl for PET imaging presents a valuable tool to noninvasively quantify fentanyl pharmacokinetics in various human populations and clinical scenarios, providing better understanding in our understanding of fentanyl disposition and its clinical implications.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e[\u0026sup1;\u0026sup1;C]Fentanyl was reliably synthesized in high molar activity, effectively minimizing isotopic dilution through an automated two-pot synthesis. Rodent PET imaging demonstrated rapid and high brain penetration, with evidence of interaction with brain efflux transporters in vivo. Furthermore, our findings revealed prolonged accumulation of [\u0026sup1;\u0026sup1;C]fentanyl in adipose tissues, suggesting a significant peripheral reservoir. These data indicate that [\u0026sup1;\u0026sup1;C]fentanyl will serve as a valuable tool for elucidating fentanyl's brain and whole-body pharmacokinetics across diverse patient populations, particularly chronic fentanyl misusers. This advancement is anticipated to contribute to the development of improved therapeutic strategies.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003eW.K, A.K.W, N.J.B., N.D.V., and S.W.K. mainly wrote and revised the manuscript. Radiosynthesis development was done by W.K, A.K.W, N.J.B., M.L.F, G.B, W.Z, S.M.E and S.W.K. PET and metabolite analysis were performed and analyzed by N.J.B, A.C, M.L.F, J.L, K.A.O, S.W.K. Full experimental design and supervision was from S.W.K and N.D.V. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e This study was conducted with support from the Intramural Research Program of the National Institute on Alcohol Abuse and Alcoholism (Y1AA-3009, Volkow). The authors thank the NIH Clinical Center PET Department (Dr. Peter Herscovitch, Mr. Kris Kim, Linwood Tucker, and George Elliott) for their assistance with cyclotron operations. We also acknowledge the NIMH Molecular Imaging Branch, particularly Drs. Robert Innis and Victor Pike, for providing PET imaging infrastructure and valuable scientific comments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u0026nbsp;\u003c/strong\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eOpen access funding was provided by the National Institute on Alcohol Abuse and Alcoholism. This work was supported by the Intramural Research Program of the National Institute on Alcohol Abuse and Alcoholism (ZIA-AA000550, Volkow).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eStanley TH, Fentanyl. J Pain Symptom Manage. 2005;29:S67\u0026ndash;71. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi:10.1016/j.jpainsymman.2005.01.009\u003c/span\u003e\u003cspan address=\"http://doi:10.1016/j.jpainsymman.2005.01.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCDC overdose prevention. About overdose prevention. In: overdose statistics. U.S. Department of Health and Human Services. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cdc.gov/overdose-prevention/about/index.html#:~:text=Drug%20overdoses%20dramatically%20increased%20over,made%20fentanyl%20and%20fentanyl%20analogs\u003c/span\u003e\u003cspan address=\"https://www.cdc.gov/overdose-prevention/about/index.html#:~:text=Drug%20overdoses%20dramatically%20increased%20over,made%20fentanyl%20and%20fentanyl%20analogs\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Accessed 7 July 2025.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVolkow ND, Califf RM, Sokolowska M, Tabak LA, Compton WM. Testing for fentanyl \u0026mdash; urgent need for practice-relevant and public health research. N Engl J Med. 2023;388:2214\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi:10.1056/NEJMp2302857\u003c/span\u003e\u003cspan address=\"http://doi:10.1056/NEJMp2302857\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePeng PWH, Sandler AN. A review of the use of fentanyl analgesia in the management of acute pain in adults. Anesthesiology. 1999;90:576\u0026ndash;99. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1097/00000542-199902000-00034\u003c/span\u003e\u003cspan address=\"10.1097/00000542-199902000-00034\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBaird A, White SA, Das R, Tatum N, Bisgaard EK. Whole body physiology model to simulate respiratory depression of fentanyl and associated naloxone reversal. Commun Med (Lond). 2024;4:114. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1038/s43856-024-00536-5\u003c/span\u003e\u003cspan address=\"https://doi:10.1038/s43856-024-00536-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChaudun F, Python L, Liu Y, Hiver A, Cand J, Kieffer BL, Valjent E, L\u0026uuml;scher C. Distinct micro-opioid ensembles trigger positive and negative fentanyl reinforcement. Nature. 2024;630:141\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1038/s41586-024-07440-x\u003c/span\u003e\u003cspan address=\"https://doi:10.1038/s41586-024-07440-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOkada CR, Henthorn TK, Zuk J, Sempio C, Roosevelt G, Ruiz AG, et al. Population pharmacokinetics of single bolus dose fentanyl in obese children. Anesth Analg. 2024;138:99\u0026ndash;107. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1213/ANE.0000000000006554\u003c/span\u003e\u003cspan address=\"https://doi:10.1213/ANE.0000000000006554\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShibutani K, Inchiosa MA Jr., Sawada K, Bairamian M. Pharmacokinetic mass of fentanyl for postoperative analgesia in lean and obese patients. Br J Anaesth. 2005;95:377\u0026ndash;83. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1093/bja/aei195\u003c/span\u003e\u003cspan address=\"https://doi:10.1093/bja/aei195\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStoeckel H, Schuttler J, Magnussen H, Hengstmann JH. Plasma fentanyl concentrations and the occurrence of respiratory depression in volunteers. Br J Anaesth. 1982;54:1087\u0026ndash;95. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1093/bja/54.10.1087\u003c/span\u003e\u003cspan address=\"https://doi:10.1093/bja/54.10.1087\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBird HE, Huhn AS, Dunn KE. Fentanyl absorption, distribution, metabolism, and excretion: narrative review and clinical significance related to illicitly manufactured fentanyl. J Addict Med. 2023;17:503\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1097/ADM.0000000000001185\u003c/span\u003e\u003cspan address=\"https://doi:10.1097/ADM.0000000000001185\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGhosh KK, Padmanabhan P, Yang CT, Ng DCE, Palanivel M, Mishra S, Christer H, Bal\u0026aacute;zs G. Positron emission tomographic imaging in drug discovery. Drug Discov Today. 2022;27:280\u0026ndash;91. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.drudis.2021.07.025\u003c/span\u003e\u003cspan address=\"10.1016/j.drudis.2021.07.025\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi\u003c/span\u003e\u003cspan address=\"https://doi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchneider E, Brune K. Distribution of fentanyl in rats: an autoradiographic study. Naunyn-Schmiedeberg's Arch Pharmacol. 1985;331:359\u0026ndash;63. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF00500820\u003c/span\u003e\u003cspan address=\"10.1007/BF00500820\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNami M, Dabiri M, Shirvani G, Ahmadi Faghih MA, Javaheri M. Preparation of Fentanyl Labeled with Carbon-14. Radiochemistry. 2018;60:42\u0026ndash;4. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1134/s1066362218010071\u003c/span\u003e\u003cspan address=\"10.1134/s1066362218010071\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTang S, Kim SW, Olsen-Dufour A, Pearson T, Freaney M, Singley E, Jenkins M, Burkard NJ, Wozniak A, Parcon P, Wu S, Morse CL, Jana S, Liow J, Zoghbi SS, Vendruscolo JCM, Vendruscolo LF, Pike VW, Koob GF, Volkow ND, Innis RB. PET imaging in rat brain shows opposite effects of acute and chronic alcohol exposure on phosphodiesterase-4B, an indirect biomarker of cAMP activity. Neuropsychopharmacology. 2024;50:444\u0026ndash;51. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1038/s41386-024-01988-y\u003c/span\u003e\u003cspan address=\"https://doi:10.1038/s41386-024-01988-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchiffer WK, Mirrione MM, Biegon A, Alexoff DL, Patel V, Dewey SL. Serial microPET measures of the metabolic reaction to a microdialysis probe implant. J Neurosci Methods. 2006;155:272\u0026ndash;84. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jneumeth.2006.01.027\u003c/span\u003e\u003cspan address=\"10.1016/j.jneumeth.2006.01.027\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi\u003c/span\u003e\u003cspan address=\"https://doi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRami-Mark C, Ungersboeck J, Haeusler D, Nics L, Philippe C, Mitterhauser M, Willeit M, Lanzenberger R, Karanikas G, Wadsak W. Reliable set-up for in-loop \u003csup\u003e11\u003c/sup\u003eC-carboxylations using Grignard reactions for the preparation of [carbonyl-\u003csup\u003e11\u003c/sup\u003eC]WAY-100635 and [\u003csup\u003e11\u003c/sup\u003eC]-(+)-PHNO. Appl Radiat Isot. 2013;82:75\u0026ndash;80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1016/j.apradiso.2013.07.023\u003c/span\u003e\u003cspan address=\"https://doi:10.1016/j.apradiso.2013.07.023\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLuthra SK, Pike VW, Brady F. The preparation of carbon-11 labelled diprenorphine: a new radioligand for the study of the opiate receptor system \u003cem\u003ein vivo\u003c/em\u003e. J Chem Soc Chem Commun. 1985;1423\u0026ndash;25. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1039/C39850001423\u003c/span\u003e\u003cspan address=\"https://doi:10.1039/C39850001423\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLuthra SK, Pike VW, Brady F, Horlock PL, Prenant C, Crouzel C. Preparation of [\u003csup\u003e11\u003c/sup\u003eC]Buprenorphine-A potential radioligand for the study of the opiate receptor system \u003cem\u003ein vivo\u003c/em\u003e. Appl Radiat Isot. 1987;38:65\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0883-2889(87)90239-5\u003c/span\u003e\u003cspan address=\"10.1016/0883-2889(87)90239-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu YC, Prenant C, Crouzel C, Comar D, Chi ZQ. Synthesis of [\u003csup\u003e11\u003c/sup\u003eC]-ohmefentanyl, a novel, highly potent and selective agonist for opiate \u0026micro;-receptors. J Label Comp Radiopharm. 1992;31:853\u0026ndash;60. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jlcr.2580311103\u003c/span\u003e\u003cspan address=\"10.1002/jlcr.2580311103\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEhrin E, Luthra SK, Crouzel C, Pike VW. Preparation of carbon-11 labelled prazosin, a potent and selective α1-adrenoreceptor antagonist. J Label Comp Radiopharm. 1988;25:177\u0026ndash;83. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jlcr.2580250209\u003c/span\u003e\u003cspan address=\"10.1002/jlcr.2580250209\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePfaff S, Philippe C, Nics L, Berroter\u0026agrave;n-Infante N, Pallitsch K, Rami-Mark C, Weidenauer A, Sauerzopf U, Willeit M, Mitterhauser M, Hacker M, Wadsak W, Pichler V. Toward the optimization of (+)-[\u003csup\u003e11\u003c/sup\u003eC]PHNO synthesis: time reduction and process validation. Contrast Media Mol Imaging, 2019; 2019(1):4292596. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2019/4292596\u003c/span\u003e\u003cspan address=\"10.1155/2019/4292596\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcCarron JA, Turton DR, Pike VW, Poole KG. Remotely-controlled production of the 5-HT1A receptor radioligand, [carbonyl-\u003csup\u003e11\u003c/sup\u003eC]WAY-100635, via \u003csup\u003e11\u003c/sup\u003eC-carboxylation of an immobilized Grignard reagent. J Label Comp Radiopharm. 1996. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/(sici)1099-1344(199610)38:10\u0026lt;941::Aid-jlcr906\u0026gt;3.0.Co;2-y\u003c/span\u003e\u003cspan address=\"10.1002/(sici)1099-1344(199610)38:10%3C941::Aid-jlcr906%3E3.0.Co;2-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://\u003c/span\u003e\u003cspan address=\"https://\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 38:941\u0026thinsp;\u0026ndash;\u0026thinsp;53.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMcPherson DW, Hwang D, Fowler JS, Wolf AP, MacCregor RM, Arnett CD. A simple one-pot synthesis of cyclopropane [\u003csup\u003e11\u003c/sup\u003eC]carbony1 chloride. synthesis and biodistribution of [\u003csup\u003e11\u003c/sup\u003eC]cyclorphan. J Label Comp Radiopharm. 1986;3:505\u0026ndash;14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jlcr.2580230507\u003c/span\u003e\u003cspan address=\"10.1002/jlcr.2580230507\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBars DL, Luthra SK, Pike VW, Duc CL. Preparation of a carbon-11 labelled neurohormone-[\u003csup\u003e11\u003c/sup\u003eC]melatonin. Appl Radiat Isot. 1987;38:1073\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1016/0883-2889(87)90073-6\u003c/span\u003e\u003cspan address=\"https://doi:10.1016/0883-2889(87)90073-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBonnot-Lours S, Crouzel C, Prenant C, Hinnen F. Carbon-11 labelling of an inhibitor of Acetylchoiinesterase: [\u003csup\u003e11\u003c/sup\u003eC]Physostigmine. J Label Comp Pharm. 1992;33:277\u0026ndash;84. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jlcr.2580330405\u003c/span\u003e\u003cspan address=\"10.1002/jlcr.2580330405\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCai L, Xu R, Guo X, Pike VW. Rapid room-temperature \u003csup\u003e11\u003c/sup\u003eC-methylation of arylamines with [\u003csup\u003e11\u003c/sup\u003eC]methyl iodide promoted by solid inorganic-bases in DMF. Eur J Org Chem. 2012;2012(7):1303\u0026ndash;10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ejoc.201101499\u003c/span\u003e\u003cspan address=\"10.1002/ejoc.201101499\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu C, Yuan M, Yang H, Zhuang X, Li H. P-glycoprotein on blood-brain barrier plays a vital role in fentanyl brain exposure and respiratory toxicity in rats. Toxicol Sci. 2018;164:353\u0026ndash;62. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/toxsci/kfy093\u003c/span\u003e\u003cspan address=\"10.1093/toxsci/kfy093\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchaefer CP, Arkwright NB, Jacobs LM, Jarvis CK, Hunn KC, Largent-Milnes TM, Tome ME, Davis TP. Chronic morphine exposure potentiates pglycoprotein trafficking from nuclear reservoirs in cortical rat brain microvessels. PLoS ONE. 2018;13(2):e0192340. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0192340\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0192340\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme ","content":"\n\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-radiopharmacy-and-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"erpc","sideBox":"Learn more about [EJNMMI Radiopharmacy and Chemistry](http://ejnmmipharmchem.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/erpc/default.aspx","title":"EJNMMI Radiopharmacy and Chemistry","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Fentanyl, Carbon-11, Positron emission tomography, Pharmacokinetics","lastPublishedDoi":"10.21203/rs.3.rs-7367969/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7367969/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eFentanyl is a potent synthetic opioid widely used for pain management and anesthesia, but the high prevalence of its misuse and its key contribution to overdose fatalities in the United States have made it a major drug of concern. Although fentanyl\u0026rsquo;s onset, duration, and toxicity depend on its pharmacokinetics and specific tissue distribution, most studies have focused primarily on plasma concentrations, leaving its distribution in critical tissues largely unexplored (this knowledge gap limits our understanding of fentanyl\u0026rsquo;s clinical effects, tissue accumulation, and the factors influencing its efficacy and safety). Here, we report the radiosynthesis of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl for PET imaging and present a preliminary whole-body pharmacokinetic study in rodents.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003e[\u003csup\u003e11\u003c/sup\u003eC]Fentanyl was synthesized in 42 mins in a high radiochemical yield (10.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.7%, n\u0026thinsp;=\u0026thinsp;5), radiochemical purity (\u0026gt;\u0026thinsp;99%), and molar activity (up to 2571.5 GBq/\u0026micro;mol at EOB). \u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e-diisopropylethylamine in chloroform was optimal for amidation. PET imaging in rats revealed rapid brain uptake (SUV\u003csub\u003emax\u003c/sub\u003e 2.71\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04 g/mL) and fast washout (T\u003csub\u003e1/2\u003c/sub\u003e = 5.06 min), both significantly increased by efflux transporter inhibition or knockout. Peripherally, high and prolonged uptake in adipose tissues was observed (SUV\u003csub\u003emax\u003c/sub\u003e = 1.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.313 g/mL, T\u003csub\u003e1/2\u003c/sub\u003e = 177 min), with \u0026gt;\u0026thinsp;60% of C-11 remaining as unchanged [\u003csup\u003e11\u003c/sup\u003eC]fentanyl at 60 min.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eWe successfully developed and automated the radiosynthesis of [\u003csup\u003e11\u003c/sup\u003eC]fentanyl, enabling PET imaging that revealed rapid brain kinetics and a critical role of P-gp/BCRP efflux in fentanyl disposition in brain. Prolonged retention in adipose tissue may delay brain clearance, potentially increasing the risk of re-narcotization (as has been reported in clinical cases after naloxone reversal). These findings advance our ability to quantify fentanyl tissue distribution and pharmacokinetics in the brain and body and provide a valuable tool for further studies in preclinical and clinical settings.\u003c/p\u003e","manuscriptTitle":"[11C]Fentanyl: Radiosynthesis and Preclinical PET Imaging for Its Pharmacokinetics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-27 06:29:45","doi":"10.21203/rs.3.rs-7367969/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revision","date":"2025-09-07T05:31:40+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-08-18T08:57:25+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-18T06:46:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-15T09:53:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"EJNMMI Radiopharmacy and Chemistry","date":"2025-08-14T17:07:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-radiopharmacy-and-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"erpc","sideBox":"Learn more about [EJNMMI Radiopharmacy and Chemistry](http://ejnmmipharmchem.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/erpc/default.aspx","title":"EJNMMI Radiopharmacy and Chemistry","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"66b2dba6-8aee-49df-8b1d-ee477545cac7","owner":[],"postedDate":"August 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-03T16:04:10+00:00","versionOfRecord":{"articleIdentity":"rs-7367969","link":"https://doi.org/10.1186/s41181-025-00394-z","journal":{"identity":"ejnmmi-radiopharmacy-and-chemistry","isVorOnly":false,"title":"EJNMMI Radiopharmacy and Chemistry"},"publishedOn":"2025-10-28 15:57:42","publishedOnDateReadable":"October 28th, 2025"},"versionCreatedAt":"2025-08-27 06:29:45","video":"","vorDoi":"10.1186/s41181-025-00394-z","vorDoiUrl":"https://doi.org/10.1186/s41181-025-00394-z","workflowStages":[]},"version":"v1","identity":"rs-7367969","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7367969","identity":"rs-7367969","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}