Automated Radiosynthesis and Biodistribution of [18F]Fluoroflutamide

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Imaging expression of the AR via radiolabeling an AR antagonist would enable non-invasive clinical staging and assessment of tumor heterogeneity, complementing the findings of a [ 68 Ga]PSMA-11 scan. Previous efforts described the radiolabeling of enzalutamide with fluorine-18 but were limited by low specific activity. In this study, we developed a high-yielding automated synthesis of [ 18 F]fluoroflutamide using zinc-mediated radiofluorination and assessed its biodistribution in preclinical models of prostate cancer. Results From 1.87 Ci (69.19 GBq) of [ 18 F]fluoride, [ 18 F]fluoroflutamide was isolated with a radiochemical yield of 3.46 ± 1.30% (n = 4) and a radiochemical purity of > 99% in an average 79 min from end-of-beam (EOB) using a bromoamide precursor. The average non-decay corrected A m was found to be 1,573.89 Ci/mmol (or 58,233.93 GBq/mmol), and the radiochemical purity after 4 h was > 97%, indicating no detectable radiolysis. A biodistribution and imaging study was performed using nude athymic mice with 22Rv1 human prostate cancer xenografts. From the results of this study, we observed a ‘mass effect’ in which carrier-added mice displayed an increased uptake of the tracer across most organs. Conclusions Herein, we report the optimized automated radiosynthesis of [ 18 F]fluoroflutamide with high purity and yield sufficient for imaging AR expression in mouse models of prostate cancer. The lack of uptake to androgen receptor-rich organs and xenografts suggests that CYP1A2 metabolism to the more potent metabolite, 2-hydroxyflutamide, was blocked by the addition of the α-3°-fluorine. The automated production of a fluoroamide via zinc-mediated radiofluorination, in high specific activity, was demonstrated to evaluate a potential radioligand for the detection of androgen receptor-positive prostate cancer tumors. Androgen receptor prostate cancer metastatic castration-resistant prostate cancer radiofluorination positron emission tomography Figures Figure 1 Figure 2 Figure 3 Figure 4 Background The androgen receptor (AR), when bound by testosterone or dihydrotestosterone, functions as a transcription factor that initiates expression of genes required for normal sexual development and androgen-dependent physiology. Multiple studies have concluded that the AR is upregulated in certain diseases such as glioblastoma,(Zalcman et al. 2018 ) salivary gland cancer,(Fan et al. 2000 ; Xu et al. 2019 ) and triple-negative breast cancer.(Rampurwala et al. 2016 ) However, interest in the AR has been directed mainly toward prostate cancer (PC) applications, as activation of the receptor leads to cancer progression. AR antagonists are first-line treatments for patients with PC, but therapeutic resistance typically accompanies disease progression to more lethal stages, such as castration-resistant prostate cancer (CRPC). Specific CRPC mutants are paradoxically activated by AR antagonists, leading to transcription of AR target genes that drive cancer growth. After discontinuing treatment with AR therapeutics, this subset of patients shows clinical improvement, which has been characterized as “anti-androgen withdrawal syndrome.”(Watson et al. 2015 ) Neuroendocrine prostate cancer (NEPC) is an AR-independent and aggressive form of metastatic-CRPC that arises from lineage plasticity in response to AR antagonists and makes up 10–17% of prostate adenocarcinoma cases. Although rare, NEPC can also develop de novo , representing less than 2% of all PC cases.(Wishahi 2024 ) To understand the AR expression pattern for staging and treatment planning, invasive biopsies are taken of the tumor tissue. Tumor heterogeneity, however, can mislead conclusions about the status of all tumors.(Jamroze et al. 2021 ) Positron emission tomography (PET) imaging is a noninvasive alternative for staging PC progression and for determining the expression of prostate-specific membrane antigen (PSMA), which is located on the cell surface of prostate metastases. Currently, [ 68 Ga]PSMA-11 is the gold standard for PET imaging of PC. As CRPC tumors develop AR independence, PSMA is increasingly upregulated, enabling [ 68 Ga]PSMA-11 PET imaging.(Rosar et al. 2022 ) However, as neuroendocrine differentiation occurs, tumors largely lose PSMA expression, diminishing the imaging utility of [ 68 Ga]PSMA-11.(Bakht et al. 2019) To complement PSMA imaging, targeting the AR with a PET agent would provide insight into the mechanisms underlying lineage plasticity to NEPC and androgen independence in CRPC. Furthermore, a fluorine-18 AR agent could guide the development of new PC treatments, such as targeted radionuclide therapies using radiohalide cogeners capable of destroying tumor cells with ionizing radiation. The testosterone derivative, 16β- 18 F-fluoro-5α-dihydrotestosterone ([ 18 F]FDHT, Fig. 1 ), is an established radioligand that has been investigated in clinical trials for imaging AR expression in PC.(Larson et al. 2004 ; Zanzonico et al. 2004 ; Dehdashti et al. 2005 ; Fox et al. 2011 , 2018 ; Vargas et al. 2018 ; Jalali et al. 2023 ) The uptake of [ 18 F]FDHT and [ 18 F]fluorodeoxyglucose ([ 18 F]FDG) in patients with PC was compared in a 2018 study along with their prognostic capabilities.(Fox et al. 2018 ) The researchers determined three tumor subtypes: AR-positive and glycolytic-positive, AR-positive and glycolytic-negative, or AR-negative and glycolytic-positive. The last displayed the worst prognosis, consistent with low or absent AR expression correlating with more aggressive phenotypes and further emphasizing the promise and potential of an AR-targeted PET scan. Despite its established binding, in use, the [ 18 F]FDHT has shown inconsistent uptake in lesions and can obfuscate metastatic disease near the bladder. This is attributed to its rapid renal clearance, which results in lower image resolution. The encouraging results from these studies have pushed researchers to identify other AR-targeted PET agents with more favorable imaging properties and biliary excretion. In 2021, the second-generation AR antagonist, enzalutamide (Fig. 1 ), was radiolabeled with fluorine-18 and used to image LNCaP (AR + human prostate cancer) xenografts, and the results were compared with those of [ 18 F]FDHT.(Antunes et al. 2021 ) [ 18 F]Enzalutamide displayed higher uptake in AR-expressing organs, lower background uptake, and higher plasma stability. Although its properties offer potential advantages, the molar activity was found to be lower than that of known PET tracers, which compromises image resolution. This study demonstrated the potential of AR antagonists as radiotracers to assess AR expression in PC applications. Here, we develop a high molar activity automated radiosynthesis to label an analog of the first-generation AR antagonist, flutamide (Fig. 1 ). Analogs of flutamide have been previously labeled with other PET ( 11 C, 18 F, 76 Br)(Jacobson et al. 2006 ; Parent et al. 2006 ; Liu et al. 2018 ) or SPECT ( 99m Tc)(Dallagi et al. 2010 ; Cardoso et al. 2023 ) radioisotopes with structural modifications to enhance labeling compatibility. In this study, we use our recently reported zinc-mediated radiofluorination to label flutamide with fluorine-18 and evaluate its uptake to AR-rich tissue in vivo . Methods General Considerations: Supporting data, including figures S1 -S22, can be found in the supplementary information. All experimental details for syntheses and radiosyntheses, including radio-high-performance liquid chromatography (radio-HPLC) traces and nuclear magnetic resonance (NMR) spectra, are provided in the Supporting Information. Reagents and solvents were commercially available unless otherwise stated. Tetramethylammonium triflate (Me 4 NOTf) was synthesized and characterized, with data consistent with previous reports.(Sagl and Martin 1988 ; Toro et al. 2014 ; Wright et al. 2024 ) Compound 2 and the fluorine-19 standard of 1 were prepared according to the procedures described by Mizuta, and the spectra obtained matched those reported.(Mizuta et al. 2021) Radiosynthesis of [F]Fluoroflutamide: Cyclotron-produced [ 18 F]fluoride was produced via 18 O(p,n) 18 F nuclear reaction (30 min beam at 55 µA, 1.87 Ci, 69.19 GBq) with a General Electric (GE) PETtrace 800 cyclotron. [ 18 F]Fluoride was trapped on a Sep-Pak Plus Light QMA solid phase extraction (SPE) cartridge. Anhydrous [ 18 F]Tetramethylammonium fluoride (TMAF) was prepared using a GE TRACERlab FX N Pro with modifications, as described in our previous method.(Wright et al. 2024 ) Tetramethylammonium triflate (TMAOTf) (5.9 mg, 26.5 µmol) in 500 µL of H 2 O was used to elute the [ 18 F]TMAF from the cartridge. Kryptofix (K 2.2.2 ) (0.14 mg, 0.36 µmol) in 1 mL of acetonitrile (MeCN) was used to azeotropically dry the [ 18 F]TMAF. The precursor ( 2 , 1.78 mg, 5.00 µmol) dissolved in 500 µL of MeCN was added to the dry [ 18 F]fluoride, followed by the addition of zinc triflate (Zn(OTf) 2 ) (0.36 mg, 1.00 µmol) and triazabicyclodecene (TBD) (0.63 mg, 4.50 µmol) dissolved in 500 µL of MeCN. The reaction was allowed to stir for 20 min at 80°C and quenched (3.5 mL of HPLC buffer), then purified via semi-preparative HPLC (column: Phenomenex Luna C18(2) 10µm 250 × 10 mm; mobile phase: 50% EtOH, 10mM NH 4 OAc, 2 mL AcOH; flow rate: 4 mL/min). The product peak (t R = 15 min) was collected for 1 min (4 mL) and diluted with 60 mL with Milli-Q H 2 O containing 0.1 mL of ascorbic acid injectable solution (500 mg/mL). The resulting solution was passed over a Waters Oasis® HLB resin (30 mg/1 cc) and rinsed with 10 mL of sterile H 2 O. The product was eluted from the resin with 500 µL of USP ethanol and formulated with USP saline (10 mL dose; 95% saline, 5% EtOH) before 0.22 µm sterile filtration into a dose vial. Identity and purity were confirmed via analytical radio-HPLC. HPLC Analysis: The chemical and radiochemical purity of [ 18 F]fluoroflutamide was analyzed using a Shimadzu LC2010 HPLC equipped with a Bioscan/Eckert and Ziegler radioactivity detector and an ultraviolet (UV) detector (Phenomenex Luna C18(2) 5µm 150 × 4.6mm column, mobile phase: 40% MeCN, 10mM NH 4 OAc, 2 mL AcOH, flow rate: 2 mL/min, t R ~ 10.0 min). A representative HPLC trace is shown in Fig. 3 . Additional details are provided in the Supporting Information. Animal Studies: All animal studies were performed in accordance with the standards set by the University of Michigan Institutional Animal Care & Use Committee (IACUC). Human prostate carcinoma cells (22Rv1) were purchased from ATCC. Nine male mice were xenografted with 22Rv1 cells at a density of 5 × 10 6 in PBS in a 1:1 ratio of Matrigel 2 weeks prior to imaging. Male mice were anesthetized with isoflurane (5% induction, 1–2% maintenance) and injected with [ 18 F]fluoroflutamide via tail vein injection. Group 1 received an injection of [ 18 F]fluoroflutamide (n = 3), group 2 received a co-injection of [ 18 F]fluoroflutamide + apalutamide at 2 mg/kg (n = 3), and group 3 received a co-injection of [ 18 F]fluoroflutamide + 19 F-fluoroflutamide at 2 mg/kg (n = 3). A single mouse from each group was imaged immediately after injection for a 60 min dynamic scan (13 time frames). The mice were then imaged again at 2 h post-injection with a 10 min static scan using a Small Animal PET scanner (MR Solutions, Guildford, UK). PET images were corrected for decay, dead time, and random coincidences. Images were then reconstructed using an iterative ordered subset expectation maximization-maximum a posteriori (MAP). Mice were euthanized at 2 h post-injection, and organs were harvested, tissue weights were collected, and radioactivity was measured using a PerkinElmer Wizard 2480 gamma counter (PerkinElmer, Waltham, MA, USA). Measurements were averaged and reported as a percentage of injected dose per gram of tissue (%ID/g). Post-analysis of the PET images was performed using Imalytics Preclinical (Gremse-IT GmbH, Aachen, Germany), in which regions of interest for selected tissues in each mouse and each frame were drawn. Data from Imalytics were analyzed using GraphPad Prism (Version 10.6.1 for macOS), with independent t-tests and one-way ANOVA tests. Results For the automated radiosynthesis of [ 18 F]fluoroflutamide, the bromoamide precursor ( 2 ) and the fluorine-19 standard were synthesized from previously described methods.(Wright et al. 2024 ) Cyclotron-produced 18 F − was generated from proton bombardment of a [ 18 O]H 2 O target via the 18 O(p,n) 18 F nuclear reaction and delivered to a GE TRACERlab FX N Pro. The activity was trapped on a Waters QMA cartridge pretreated with 10 mL of EtOH, followed by 10 mL of 1M potassium triflate (KOTf), and then 10 mL of Milli-Q H 2 O. The activity was eluted off the cartridge via ion-exchange with tetramethylammonium triflate (TMAOTf) in H 2 O, followed by subsequent addition of kryptofix (K 2.2.2 ) in MeCN and azeotropic drying. The bromoamide precursor in MeCN was then added to the dry [ 18 F]TMAF with stirring, followed by the addition of zinc triflate (Zn(OTf) 2 ) and triazabicyclodecene (TBD). The reaction mixture was heated to 80°C for 20 min, then quenched with HPLC buffer and loaded onto the semi-preparative reverse-phase HPLC column for purification (Fig. 2 , 3 A). The product peak eluted at 15 min and was collected for 1 min into a dilution flask containing 60 mL of water for reformulation. 0.1 mL of ascorbic acid solution (500 mg/mL) was added to mitigate radiolysis. After collecting the product peak, the entire solution was washed over a Waters Oasis HLB resin (30 mg/1 cc) and rinsed with 10 mL of sterile H 2 O to remove any residual HPLC salts. Elution with EtOH into saline, followed by 0.1 mL of ascorbic acid solution, yielded a 10 mL dose (5% EtOH), which was analyzed by radio-HPLC (Fig. 3 B). From 1.87 Ci (or 69.19 GBq) of 18 F − , [ 18 F]fluoroflutamide was isolated with an average radiochemical yield of 3.46 ± 1.30% (n = 4) in > 99% radiochemical purity. The activity yields (64.55 ± 24.28 mCi) were sufficient for planned animal studies with an average synthesis time of 79 min. Additionally, the doses had an average molar activity of 1,573.89 Ci/mmol (or 58,233.93 GBq/mmol) (see S7 and S22 for the standard curves). Initial optimization of the automated method with 1.87 Ci (or 69.19 GBq) of 18 F indicated radioimpurities originating from radiolysis 2 h after the end of synthesis. By adding the radioprotectant ascorbic acid in the dilution flask, product vial, and the HPLC buffer, we observed no radiolysis present in the final dose formulation for up to 4 h post-end-of-synthesis (99.54% RCP at 1 h; 97.81% RCP at 4 h). See the supporting information for more details. Biodistribution studies were performed on nude athymic male mice with 22Rv1 xenografts. Xenografts were allowed to grow until tumors were palpable. Nine mice implanted with 22Rv1 tumors were injected with an average activity of 125.33 ± 40.08 µCi (or 4.64 ± 1.48 MBq) of [ 18 F]fluoroflutamide. To confirm binding to the androgen receptor, in 3 animals received a coinjection of the tracer and a 2 mg/kg dose of apalutamide. 19 F-Fluoroflutamide was also coinjected with the tracer at 2 mg/kg in 3 other animals to quantify nonspecific binding and receptor saturation. The organs with the highest average uptake in all three treatment groups were the adrenal glands (baseline: 4.67% ID/g, apalutamide block: 3.41% ID/g, 19 F-fluoroflutamide block: 7.57% ID/g), the liver (1.96, 2.57, and 5.54% ID/g), and the kidneys (1.77, 2.14, and 4.82% ID/g, respectively) (Fig. 4 ). One baseline mouse and one mouse cotreated with apalutamide were PET imaged for 0–60 min post-injection, and the summed image from this study is shown in Fig. 4 . A classical blocking effect in the heart and the liver of the mouse dosed with apalutamide compared to the baseline mouse was observed. This finding was not observed in the biodistribution studies at the 2-hour timepoint as the apalutamide group had a higher average liver uptake value than the baseline mice. Additionally, the average injected dose per gram of heart tissue was found to be similar in both groups (1.22 ± 0.52% for the apalutamide treatment group and 1.28 ± 0.31% in baseline mice). Flutamide has low oral bioavailability due to rapid absorption and subsequent hepatic metabolism to 2-hydroxyflutamide. To determine the intravenous injection doses of flutamide and apalutamide, we calculated the percentage of absorption from an oral dose using previous literature.(Schulz et al. 1988 ; Posti et al. 2000 ; Clegg et al. 2012 ; Sulochana et al. 2018 ; de Vries et al. 2019 ) Both apalutamide and flutamide are commonly dosed as timed-release pellets or subcutaneous injections because of their poor aqueous solubility. However, we found that both apalutamide and 19 F-fluoroflutamide dissolved completely when formulated with Tween-80, EtOH, and saline at the desired blocking concentrations. HPLC analysis confirmed that the doses remain in solution at the same concentrations after sterile filtration. Discussion Non-invasive imaging of AR expression would be beneficial for multiple cancers, including late-stage and aggressive NEPC. Interest in PC imaging agents has been on the rise since the FDA approval of the theranostic pair Pluvicto ([ 177 Lu]PSMA-617) and Locametz/Illuccix ([ 68 Ga]PSMA-11). The first-line treatment for PC includes AR antagonists such as enzalutamide, apalutamide, and darolutamide, which compete with androgens for AR binding. Eventually, through resistance pathways, AR dependency is lost, coinciding with a waning of treatment response. In some instances, therapy continuation can facilitate disease progression, so PET imaging studies that accurately stage this transition would be invaluable for reassessing treatment decisions. Biopsies are currently used to evaluate AR expression, an invasive procedure that does not always provide a comprehensive understanding of target expression in each tumor. Therefore, by radiolabeling one of these AR antagonists with fluorine-18, we aimed to assess the ability of this agent to non-invasively measure AR expression through PET imaging. Flutamide is a first-generation antagonist containing an α-3°-amide with a known active metabolite formed via α-hydroxylation. A new methodology from our lab describes a wide array of α-amide substrates that can be labeled via zinc-mediated radiofluorination. In the case of 2-hydroxyflutamide, the active metabolite generated via CYP1A2, fluorine can also act as an isostere for a hydroxyl group due to its weak hydrogen-bond-accepting properties and high electronegativity.(Inoue et al. 2020 ) Using this methodology, we developed an automated procedure for radiolabeling flutamide for further assessment in vivo . Our initial conditions for radiolabeling used dimethyl sulfoxide (DMSO) as the reaction solvent to develop a Class III solvent procedure. However, the non-decay-corrected molar activity (A m ) obtained with this method was lower than the threshold for effective PET tracers (458.06 Ci/mmol or 16,948.22 GBq/mmol vs ca. 1,000 Ci/mmol or 37,000 MBq/mmol). By changing the solvent to MeCN, we increased the molar activity to 2,212.54 Ci/mmol or 81,863.98 GBq/mmol. A control experiment was also performed to ensure that fluorodenitration and isotopic exchange with the trifluoromethyl did not occur using flutamide in place of the bromoamide precursor under the optimized conditions. The crude reaction mixture was removed for analysis, and the resulting activity was 5.87 mCi or 217.19 MBq (5 min beam; 162.3 mCi or 6005.1 MBq of 18 F), identified as unreacted 18 F. No organic products were observed by radio-TLC and -HPLC, confirming that the reaction was free of undesired side products. With optimized reaction conditions in hand, we pursued a fully automated synthesis, purification, and dose formulation of [ 18 F]fluoroflutamide on a GE TRACERlab FX N Pro for preclinical studies. [ 18 F]Fluoroflutamide was isolated with high radiochemical purity (> 99%) and activity yields of 64.55 ± 24.28 mCi (n = 4). The yields are sufficient for in vivo animal studies as well as human doses (> 10 mCi). Following the isolation of a consistent, automated protocol, we performed a biodistribution and imaging study with [ 18 F]fluoroflutamide in nude athymic mice inoculated with 22Rv1 tumors. Nine male mice were administered [ 18 F]fluoroflutamide, and biodistribution studies were conducted 2 h post-injection. Of the nine mice, two groups were dosed with 19 F-fluoroflutamide and apalutamide to evaluate specific binding. Dynamic PET imaging studies of one baseline mouse and one mouse blocked with apalutamide were completed from 0–60 min post-injection, and the summed image from this study is shown in Fig. 4 . Blocking the AR with apalutamide (left mouse) led to an observed decrease in uptake in the heart and liver compared to baseline (right mouse) from 0–60 min. Conversely, from the biodistribution data taken at 2 h (Fig. 4 ), there is increased tracer uptake in both carrier-added groups. This has previously been described as ‘mass action’ or ‘mass effect.’ The addition of cold-mass and apalutamide, an analogous ligand, altered the pharmacokinetics and biodistribution over time by increasing tracer uptake in most organs, including the tumor. A possible explanation for this observation is that, with the addition of mass, drug clearance time remained the same. Therefore, the tracer had a longer residence time at the receptor before being cleared compared to the baseline mouse. The initial blocking effect observed in the PET images along with the lack of uptake in the tumor and testes demonstrates nonspecific uptake in tissues rather than AR binding. [ 18 F]Fluoroflutamide exhibited the highest uptake in the adrenal glands, liver, kidneys, and testes in all three groups. The tracer also exhibited minimal defluorination in vivo , as indicated by no increase of 18 F uptake in bone (baseline average: 0.85% ID/g) compared to other background organs such as muscle (baseline average: 0.74% ID/g). Some limitations of this study include the biodistribution study being conducted at a single time point (2 h) which may be later than desired for an AR ligand. The results of this initial study did not warrant generating more tumor models to assess biodistribution at earlier time points. Additionally, the binding affinity of the new fluorinated analog was not tested in vitro compared with that of the parent molecule and analogous antagonists. Further work involves in vitro binding assays with 22Rv1 cells and other PC cell lines that express or lack AR, such as PC-3 (AR − ), DU145 (AR − ), or LNCaP (AR + ), and LAPC-4 (AR + ).(Sampson et al. 2013 ) However, the biodistribution and imaging data suggest that by placing the fluorine at the α-3°-position of the amide, oxidative metabolism may be blocked to the more potent metabolite of flutamide, 2-hydroxyflutamide. The lack of selective uptake also indicates the analog’s lack of binding similarity to 2-hydroxyflutamide, which is 25-fold more potent than flutamide and accounts for 90% of metabolites present in plasma following a therapeutic dose.(Simard et al. 1986 ) During the preparation of this manuscript, the Lee group published a new methodology for 18 F trifluoromethylation of aryl iodides.(Choi et al. 2025 ) Within this study, the group radiolabeled flutamide at the trifluoromethyl group, leaving the amide susceptible to α-hydroxylation via CYP1A2. When the agent was tested in LNCaP (AR + ) xenograft mouse models, rapid renal clearance was observed, along with specific uptake in the xenograft up to 60 min post-injection. This further supports the idea that α-C–H metabolism of flutamide is essential for AR targeting. Conclusions We successfully developed an automated radiosynthesis of [ 18 F]fluoroflutamide with high radiochemical purity and sufficient molar activity for preclinical imaging studies. In vivo studies in 22Rv1 xenograft-bearing mice demonstrated tumor uptake and minimal defluorination, as evidenced by the absence of significant skeletal signal. However, classical blocking with nonradioactive apalutamide or fluoroflutamide did not reduce tumor or tissue uptake. Instead, a paradoxical “mass effect” was observed, with increased tracer accumulation across multiple organs, including the tumor, when non-radioactive competitors were co-injected. These findings suggest that the in vivo distribution of [ 18 F]fluoroflutamide is likely influenced by nonspecific pharmacokinetics, metabolism, and receptor residence time, rather than by AR binding. Overall, while [ 18 F]fluoroflutamide is (bio)chemically stable and can be reliably synthesized, the current data indicate limited avidity and specificity for AR imaging in vivo . Further optimization using structural analogs is required, particularly through in vitro binding assays against AR⁺ and AR⁻ cell lines, metabolite characterization, and evaluation in additional tumor models. For example, analogs that can undergo α-hydroxylation, analogous to the metabolism of flutamide, may show enhanced uptake. These studies and others are underway to identify effective, noninvasive tools for imaging AR expression in advanced PC, including CRPC and NEPC. Abbreviations Androgen receptor (AR) End-of-Beam (EOB) Metastatic Castration-Resistant Prostate Cancer (mCRPC) Positron Emission Tomography (PET) Prostate Cancer (PC) Castration-Resistant Prostate Cancer (CRPC) Neuroendocrine Prostate Cancer (NEPC) Prostate-Specific Membrane Antigen (PSMA) 16β- 18 F-fluoro-5α-dihydrotestosterone ([ 18 F]FDHT) [ 18 F]Fluorodeoxyglucose ([ 18 F]FDG) Radio-High-Performance Liquid Chromatography (radio-HPLC) Nuclear Magnetic Resonance (NMR) Tetramethylammonium Triflate (Me 4 NOTf) General Electric (GE) Solid Phase Extraction (SPE) [ 18 F]Tetramethylammonium fluoride (TMAF) Tetramethylammonium triflate (TMAOTf) Kryptofix (K 2.2.2 ) Zinc Triflate (Zn(OTf) 2 ) Triazabicyclodecene (TBD) Institutional Animal Care & Use Committee (IACUC) Maximization-Maximum a posteriori (MAP) Potassium Triflate (KOTf) Dimethyl Sulfoxide (DMSO) Molar Activity (A m ) Declarations Ethics approval and consent to participate General Animal Study Considerations:All animal studies were performed under and in accordance with the direct guidance from the IACUC (Institutional Animal Care and Use Committee) and Unit for Lab Animal Management at the University of Michigan under Peter J. H. Scott’s IACUC protocol number of PRO11715. Consent for publication All authors have read and approved the final manuscript and consent to its publication. Availability of data and material All data generated during this study, including chemistry/animal experiments, are included in this published article and its supplementary information files. Competing interests PJHS is associate editor of EJNMMI Radiopharmacy and Chemistry. Funding We gratefully acknowledge funding from the National Institutes of Health. This work was supported by NIH NIBIB [Award Numbers R01EB021155 (P.J.H.S.) R00EB031564 (J.S.W.)]. Acknowledgements We thank the University of Michigan analytical services for assistance with compound characterizations. Authors' contributions G.K., P. J. H. S., A. F. B., and J. S. W. conceived and led the project. G. K., J. W., S. C., and J. S. performed experiments and assisted with project design. Authors' information Corresponding Authors *Peter J. H. Scott – 0000-0002-6505-0450, Department of Radiology, University of Michigan, Ann Arbor, MI 48109, USA. Email: [email protected] *Jay S. Wright – 0000-0002-1350-123X, Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104, USA; E-mail: [email protected] Authors Gina R. Kaup –0009-0003-1860-1226, Department of Radiology, University of Michigan, Ann Arbor, MI 48109, USA Jason A. Witek –0000-0001-7271-6567, Department of Radiology, University of Michigan, Ann Arbor, MI 48109, USA Shelbie J. Cingoranelli –0009-0002-5578-0342, Department of Radiology, University of Michigan, Ann Arbor, MI 48109, USA David Raffel – 0000-0002-7188-9463, Department of Radiology, University of Michigan, Ann Arbor, MI 48109, USA Jenelle Stauff – Department of Radiology, University of Michigan, Ann Arbor, MI 48109, USA Allen F. Brooks –0000-0003-3773-3024, Department of Radiology, University of Michigan, Ann Arbor, MI 48109, USA References Antunes IF, Dost RJ, Hoving HD, et al. 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Am J Surg Pathol. 2000;24:579–86. https://doi.org/10.1097/00000478-200004000-00014 . Fox JJ, Autran-Blanc E, Morris MJ, et al. Practical Approach for Comparative Analysis of Multilesion Molecular Imaging Using a Semiautomated Program for PET/CT. J Nucl Med. 2011;52:1727–32. https://doi.org/10.2967/jnumed.111.089326 . Fox JJ, Gavane SC, Blanc-Autran E, et al. Positron Emission Tomography/Computed Tomography–Based Assessments of Androgen Receptor Expression and Glycolytic Activity as a Prognostic Biomarker for Metastatic Castration-Resistant Prostate Cancer. JAMA Oncol. 2018;4:217–24. https://doi.org/10.1001/jamaoncol.2017.3588 . Inoue M, Sumii Y, Shibata N. Contribution of Organofluorine Compounds to Pharmaceuticals. ACS Omega. 2020;5:10633–40. https://doi.org/10.1021/acsomega.0c00830 . Jacobson O, Laky D, Carlson KE, et al. Chiral dimethylamine flutamide derivatives—modeling, synthesis, androgen receptor affinities and carbon-11 labeling. Nucl Med Biol. 2006;33:695–704. https://doi.org/10.1016/j.nucmedbio.2006.05.010 . Jalali Val, Wasinger G, Rasul S, et al. Consecutive Prostate-Specific Membrane Antigen (PSMA) and Antigen Receptor (AR) PET Imaging Shows Positive Correlation with AR and PSMA Protein Expression in Primary Hormone-Naïve Prostate Cancer. J Nucl Med. 2023;64:863–8. https://doi.org/10.2967/jnumed.122.264981 . Jamroze A, Chatta G, Tang DG. Androgen receptor (AR) heterogeneity in prostate cancer and therapy resistance. Cancer Lett. 2021;518:1–9. https://doi.org/10.1016/j.canlet.2021.06.006 . Larson SM, Morris M, Gunther I, et al. Tumor Localization of 16β- 18 F-Fluoro-5α-Dihydrotestosterone Versus 18 F-FDG in Patients with Progressive, Metastatic Prostate Cancer. J Nucl Med. 2004;45:366–73. Liu W, Huang X, Placzek MS, et al. Site-selective 18 F fluorination of unactivated C–H bonds mediated by a manganese porphyrin. Chem Sci. 2018;9:1168–72. https://doi.org/10.1039/C7SC04545J . Mizuta S, Kitamura K, Kitagawa A, et al. Silver-Promoted Fluorination Reactions of α-Bromoamides. Chem Eur J. 2020;27:5930–5. https://doi.org/10.1002/chem.202004769 . Parent EE, Jenks C, Sharp T, et al. Synthesis and biological evaluation of a nonsteroidal bromine-76-labeled androgen receptor ligand 3-[ 76 Br]bromo-hydroxyflutamide. Nucl Med Biol. 2006;33:705–13. https://doi.org/10.1016/j.nucmedbio.2006.05.009 . Posti J, Katila K, Kostiainen T. Dissolution rate limited bioavailability of flutamide, and in vitro – in vivo correlation. Eur J Pharm Biopharm. 2000;49:35–9. https://doi.org/10.1016/S0939-6411(99)00061-2 . Rampurwala M, Wisinski KB, O’Regan R. Role of the Androgen Receptor in Triple-Negative Breast Cancer. Clin Adv Hematol Oncol HO. 2016;14:186–93. Rosar F, Neher R, Burgard C, et al. Upregulation of PSMA Expression by Enzalutamide in Patients with Advanced mCRPC. Cancers. 2022;14:1696. https://doi.org/10.3390/cancers14071696 . Sagl DJ, Martin JC. The stable singlet ground state dication of hexaiodobenzene: possibly a σ-delocalized dication. J Am Chem Soc. 1988;110:5827–33. https://doi.org/10.1021/ja00225a038 . Sampson N, Neuwirt H, Puhr M, et al. In vitro model systems to study androgen receptor signaling in prostate cancer. Endocr Relat Cancer. 2013;20:R49–64. https://doi.org/10.1530/ERC-12-0401 . Schulz M, Schmoldt A, Donn F, Becker H. The pharmacokinetics of flutamide and its major metabolites after a single oral dose and during chronic treatment. Eur J Clin Pharmacol. 1988;34:633–6. https://doi.org/10.1007/BF00615229 . Simard J, Luthy I, Guay J, et al. Characteristics of interaction of the antiandrogen flutamide with the androgen receptor in various target tissues. Mol Cell Endocrinol. 1986;44:261–70. https://doi.org/10.1016/0303-7207(86)90132-2 . Sulochana SP, Saini NK, Daram P, et al. Validation of an LC–MS/MS method for simultaneous quantitation of enzalutamide, N -desmethylenzalutamide, apalutamide, darolutamide and ORM-15341 in mice plasma and its application to a mice pharmacokinetic study. J Pharm Biomed Anal. 2018;156:170–80. https://doi.org/10.1016/j.jpba.2018.04.038 . Toro JMS, den Hartog T, Chen P. Cyclopropanation of styrenes and stilbenes using lithiomethyl trimethylammonium triflate as methylene donor. Chem Commun. 2014;50:10608–10. https://doi.org/10.1039/C4CC04929B . Vargas HA, Kramer GM, Scott AM, et al. Reproducibility and Repeatability of Semiquantitative 18 F-Fluorodihydrotestosterone Uptake Metrics in Castration-Resistant Prostate Cancer Metastases: A Prospective Multicenter Study. J Nucl Med. 2018;59:1516–23. https://doi.org/10.2967/jnumed.117.206490 . Watson PA, Arora VK, Sawyers CL. Emerging mechanisms of resistance to androgen receptor inhibitors in prostate cancer. Nat Rev Cancer. 2015;15:701–11. https://doi.org/10.1038/nrc4016 . Wishahi M. Treatment-induced neuroendocrine prostate cancer and de novo neuroendocrine prostate cancer: Identification, prognosis and survival, genetic and epigenetic factors. World J Clin Cases. 2024;12:2143–6. https://doi.org/10.12998/wjcc.v12.i13.2143 . Wright JS, Ma R, Webb EW, et al. Zinc-Mediated Radiosynthesis of Unprotected Fluorine-18 Labelled α-Tertiary Amides. Angew Chem Int Ed. 2024;63:e202316365. https://doi.org/10.1002/anie.202316365 . Xu B, Dogan S, Rasheed MRHA, et al. Androgen Receptor Immunohistochemistry in Salivary Duct Carcinoma: A Retrospective Study of 188 Cases Focusing on Tumoral Heterogeneity and Temporal Concordance. Hum Pathol. 2019;93:30–6. https://doi.org/10.1016/j.humpath.2019.08.007 . Zalcman N, Canello T, Ovadia H, et al. Androgen receptor: a potential therapeutic target for glioblastoma. Oncotarget. 2018;9:19980–93. https://doi.org/10.18632/oncotarget.25007 . Zanzonico PB, Finn R, Pentlow KS, et al. PET-Based Radiation Dosimetry in Man of 18 F-Fluorodihydrotestosterone, a New Radiotracer for Imaging Prostate Cancer. J Nucl Med. 2004;45:1966–71. Supplementary Files 20260218SIExperimentalFluoroflutamide.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 11 Mar, 2026 Reviewers agreed at journal 26 Feb, 2026 Reviewers invited by journal 24 Feb, 2026 Editor assigned by journal 24 Feb, 2026 First submitted to journal 24 Feb, 2026 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. 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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-8895668","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":596401040,"identity":"b65bb25a-63d8-4e17-bbec-4549a30bdcb0","order_by":0,"name":"Gina Kaup","email":"","orcid":"","institution":"University of Michigan","correspondingAuthor":false,"prefix":"","firstName":"Gina","middleName":"","lastName":"Kaup","suffix":""},{"id":596401041,"identity":"5e00da20-f03f-4d35-b58c-82c1add9aa54","order_by":1,"name":"Jason Anthony Witek","email":"","orcid":"","institution":"University of Michigan","correspondingAuthor":false,"prefix":"","firstName":"Jason","middleName":"Anthony","lastName":"Witek","suffix":""},{"id":596401043,"identity":"52c5bb7c-3ff5-4598-89ce-d6126a2d1190","order_by":2,"name":"Shelbie Jaylene Cingoranelli","email":"","orcid":"","institution":"University of Michigan","correspondingAuthor":false,"prefix":"","firstName":"Shelbie","middleName":"Jaylene","lastName":"Cingoranelli","suffix":""},{"id":596401046,"identity":"adbb5132-515b-403b-9dc8-2b90760a6414","order_by":3,"name":"Jenelle Stauff","email":"","orcid":"","institution":"University of Michigan","correspondingAuthor":false,"prefix":"","firstName":"Jenelle","middleName":"","lastName":"Stauff","suffix":""},{"id":596401048,"identity":"d283ddb0-8619-40de-8e25-b14c77ec6149","order_by":4,"name":"David Raffel","email":"","orcid":"","institution":"University of Michigan","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Raffel","suffix":""},{"id":596401050,"identity":"6d253241-04b0-41db-bc41-e1b216381cda","order_by":5,"name":"Allen Frederick Brooks","email":"","orcid":"","institution":"University of Michigan","correspondingAuthor":false,"prefix":"","firstName":"Allen","middleName":"Frederick","lastName":"Brooks","suffix":""},{"id":596401052,"identity":"c9b7cb39-c4d9-4da3-bcec-7610df73b688","order_by":6,"name":"Peter James Henry Scott","email":"","orcid":"","institution":"University of Michigan","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"James Henry","lastName":"Scott","suffix":""},{"id":596401054,"identity":"5d60c7eb-ee80-4188-b209-e6fac3102a4d","order_by":7,"name":"Jay Samuel Wright","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYBACxgYgkcBwgIEfLnSASC0Skg3EaoEpkzCAqySkhbn/jNmHB7/u1BnfyD34uHAHgxzfjQQCDpuRYzwjse+ZhNmNvGTjmWcYjCUJa+HdzJDYcxioJcdMmreNIXEDQS39ZyFajGfkmP8GaqknrKUhdzNDwo/DEgYSOWbMQC0JBoQdlv+ZIbHhsOSMM++SgQ6TMJx55gF+LYb9x5IZf/w5zM/fnnvwM2+bjTzfcQK2GDaArGoDMXlAhAR+5SAgDyb/wLWMglEwCkbBKMAEAClfSflyLWr9AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-1350-123X","institution":"University of Pennsylvania Perelman School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Jay","middleName":"Samuel","lastName":"Wright","suffix":""}],"badges":[],"createdAt":"2026-02-16 19:14:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8895668/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8895668/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103596038,"identity":"a879ada6-4976-4411-a443-8040ffd871d9","added_by":"auto","created_at":"2026-02-27 13:09:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":115480,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eStructures of [\u003c/em\u003e\u003csup\u003e\u003cem\u003e18\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eF]FDHT, [\u003c/em\u003e\u003csup\u003e\u003cem\u003e18\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eF]enzalutamide, and [\u003c/em\u003e\u003csup\u003e\u003cem\u003e18\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eF]fluoroflutamide (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-8895668/v1/f259ecfd1f9728a4babf1763.png"},{"id":103596242,"identity":"0a8afb33-92cf-4c58-ace8-f2774300d782","added_by":"auto","created_at":"2026-02-27 13:10:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":60711,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eConditions for the synthesis of [\u003c/em\u003e\u003csup\u003e\u003cem\u003e18\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eF]fluoroflutamide (1) using zinc-mediated radiofluorination.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-8895668/v1/f1aefa2469816f90bd44e12b.png"},{"id":103595874,"identity":"07c286cc-d01b-42d9-814b-16bbdfcdbebf","added_by":"auto","created_at":"2026-02-27 13:08:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":258369,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eA: Representative semi-preparative HPLC trace for the purification of [\u003c/em\u003e\u003csup\u003e\u003cem\u003e18\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eF]fluoroflutamide (t\u003c/em\u003e\u003csub\u003e\u003cem\u003eR\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e= 15 min). Column: Phenomenex Luna C18(2) 10µm 250 × 10 mm; mobile phase: 50% EtOH, 10 mM NH\u003c/em\u003e\u003csub\u003e\u003cem\u003e4\u003c/em\u003e\u003c/sub\u003e\u003cem\u003eOAc, 30 mM AcOH, 1 mM ascorbic acid; flow rate: 4 mL/min. B: Representative analytical HPLC trace of the dose (t\u003c/em\u003e\u003csub\u003e\u003cem\u003eR\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e=12.3 min) with ascorbic acid eluting at 0.796 min. Column: Phenomenex Luna C18(2) 5 μm 150 × 4.6 mm; mobile phase: 40% MeCN, 10 mM NH\u003c/em\u003e\u003csub\u003e\u003cem\u003e4\u003c/em\u003e\u003c/sub\u003e\u003cem\u003eOAc, 30 mM AcOH; flow rate: 2 mL/min.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-8895668/v1/e2532050f0dcabb8dd80cf2d.png"},{"id":103596069,"identity":"f7cdbb06-97fc-4c95-8ebf-bbd192de953a","added_by":"auto","created_at":"2026-02-27 13:09:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":524534,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eLeft: Static PET image at 2 h of nude athymic mice with 22Rv1 xenografts (orange circle represents tumor). Right: Biodistribution of [\u003c/em\u003e\u003csup\u003e\u003cem\u003e18\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eF]fluoroflutamide in 22Rv1 tumor models shown as percentage of injected dose per gram of tissue (%ID/g). Blocking doses were premixed with the tracer for a tail vein injection (Apalutamide Block n = 3; [\u003c/em\u003e\u003csup\u003e\u003cem\u003e19\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eF]fluoroflutamide block n = 3). Baseline received exclusively the tracer via tail vein injection (n = 3).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-8895668/v1/4be179b6e0901925358732a6.png"},{"id":103596446,"identity":"60eda3f9-b790-47db-9ada-803fe7d0b203","added_by":"auto","created_at":"2026-02-27 13:10:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1601904,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8895668/v1/d45ea973-b63c-4fe2-b253-bac5366512a2.pdf"},{"id":103595798,"identity":"71ae1971-1a5f-409b-afe6-7b02b78d8e35","added_by":"auto","created_at":"2026-02-27 13:08:20","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":14186877,"visible":true,"origin":"","legend":"","description":"","filename":"20260218SIExperimentalFluoroflutamide.docx","url":"https://assets-eu.researchsquare.com/files/rs-8895668/v1/68e7393ea5ab14c508457cb3.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eAutomated Radiosynthesis and Biodistribution of [\u003csup\u003e18\u003c/sup\u003eF]Fluoroflutamide\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eThe androgen receptor (AR), when bound by testosterone or dihydrotestosterone, functions as a transcription factor that initiates expression of genes required for normal sexual development and androgen-dependent physiology. Multiple studies have concluded that the AR is upregulated in certain diseases such as glioblastoma,(Zalcman et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) salivary gland cancer,(Fan et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and triple-negative breast cancer.(Rampurwala et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) However, interest in the AR has been directed mainly toward prostate cancer (PC) applications, as activation of the receptor leads to cancer progression. AR antagonists are first-line treatments for patients with PC, but therapeutic resistance typically accompanies disease progression to more lethal stages, such as castration-resistant prostate cancer (CRPC). Specific CRPC mutants are paradoxically activated by AR antagonists, leading to transcription of AR target genes that drive cancer growth. After discontinuing treatment with AR therapeutics, this subset of patients shows clinical improvement, which has been characterized as \u0026ldquo;anti-androgen withdrawal syndrome.\u0026rdquo;(Watson et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) Neuroendocrine prostate cancer (NEPC) is an AR-independent and aggressive form of metastatic-CRPC that arises from lineage plasticity in response to AR antagonists and makes up 10\u0026ndash;17% of prostate adenocarcinoma cases. Although rare, NEPC can also develop \u003cem\u003ede novo\u003c/em\u003e, representing less than 2% of all PC cases.(Wishahi \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eTo understand the AR expression pattern for staging and treatment planning, invasive biopsies are taken of the tumor tissue. Tumor heterogeneity, however, can mislead conclusions about the status of all tumors.(Jamroze et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) Positron emission tomography (PET) imaging is a noninvasive alternative for staging PC progression and for determining the expression of prostate-specific membrane antigen (PSMA), which is located on the cell surface of prostate metastases. Currently, [\u003csup\u003e68\u003c/sup\u003eGa]PSMA-11 is the gold standard for PET imaging of PC. As CRPC tumors develop AR independence, PSMA is increasingly upregulated, enabling [\u003csup\u003e68\u003c/sup\u003eGa]PSMA-11 PET imaging.(Rosar et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) However, as neuroendocrine differentiation occurs, tumors largely lose PSMA expression, diminishing the imaging utility of [\u003csup\u003e68\u003c/sup\u003eGa]PSMA-11.(Bakht et al. 2019) To complement PSMA imaging, targeting the AR with a PET agent would provide insight into the mechanisms underlying lineage plasticity to NEPC and androgen independence in CRPC. Furthermore, a fluorine-18 AR agent could guide the development of new PC treatments, such as targeted radionuclide therapies using radiohalide cogeners capable of destroying tumor cells with ionizing radiation.\u003c/p\u003e \u003cp\u003eThe testosterone derivative, 16β-\u003csup\u003e18\u003c/sup\u003eF-fluoro-5α-dihydrotestosterone ([\u003csup\u003e18\u003c/sup\u003eF]FDHT, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), is an established radioligand that has been investigated in clinical trials for imaging AR expression in PC.(Larson et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Zanzonico et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Dehdashti et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Fox et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Vargas et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Jalali et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) The uptake of [\u003csup\u003e18\u003c/sup\u003eF]FDHT and [\u003csup\u003e18\u003c/sup\u003eF]fluorodeoxyglucose ([\u003csup\u003e18\u003c/sup\u003eF]FDG) in patients with PC was compared in a 2018 study along with their prognostic capabilities.(Fox et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) The researchers determined three tumor subtypes: AR-positive and glycolytic-positive, AR-positive and glycolytic-negative, or AR-negative and glycolytic-positive. The last displayed the worst prognosis, consistent with low or absent AR expression correlating with more aggressive phenotypes and further emphasizing the promise and potential of an AR-targeted PET scan.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDespite its established binding, in use, the [\u003csup\u003e18\u003c/sup\u003eF]FDHT has shown inconsistent uptake in lesions and can obfuscate metastatic disease near the bladder. This is attributed to its rapid renal clearance, which results in lower image resolution. The encouraging results from these studies have pushed researchers to identify other AR-targeted PET agents with more favorable imaging properties and biliary excretion. In 2021, the second-generation AR antagonist, enzalutamide (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), was radiolabeled with fluorine-18 and used to image LNCaP (AR\u003csup\u003e+\u003c/sup\u003e human prostate cancer) xenografts, and the results were compared with those of [\u003csup\u003e18\u003c/sup\u003eF]FDHT.(Antunes et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) [\u003csup\u003e18\u003c/sup\u003eF]Enzalutamide displayed higher uptake in AR-expressing organs, lower background uptake, and higher plasma stability. Although its properties offer potential advantages, the molar activity was found to be lower than that of known PET tracers, which compromises image resolution. This study demonstrated the potential of AR antagonists as radiotracers to assess AR expression in PC applications. Here, we develop a high molar activity automated radiosynthesis to label an analog of the first-generation AR antagonist, flutamide (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Analogs of flutamide have been previously labeled with other PET (\u003csup\u003e11\u003c/sup\u003eC, \u003csup\u003e18\u003c/sup\u003eF, \u003csup\u003e76\u003c/sup\u003eBr)(Jacobson et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Parent et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) or SPECT (\u003csup\u003e99m\u003c/sup\u003eTc)(Dallagi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Cardoso et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) radioisotopes with structural modifications to enhance labeling compatibility. In this study, we use our recently reported zinc-mediated radiofluorination to label flutamide with fluorine-18 and evaluate its uptake to AR-rich tissue \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGeneral Considerations:\u003c/h2\u003e \u003cp\u003eSupporting data, including figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S22, can be found in the supplementary information. All experimental details for syntheses and radiosyntheses, including radio-high-performance liquid chromatography (radio-HPLC) traces and nuclear magnetic resonance (NMR) spectra, are provided in the Supporting Information. Reagents and solvents were commercially available unless otherwise stated. Tetramethylammonium triflate (Me\u003csub\u003e4\u003c/sub\u003eNOTf) was synthesized and characterized, with data consistent with previous reports.(Sagl and Martin \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Toro et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wright et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) Compound \u003cb\u003e2\u003c/b\u003e and the fluorine-19 standard of \u003cb\u003e1\u003c/b\u003e were prepared according to the procedures described by Mizuta, and the spectra obtained matched those reported.(Mizuta et al. 2021)\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRadiosynthesis of [F]Fluoroflutamide:\u003c/h3\u003e\n\u003cp\u003eCyclotron-produced [\u003csup\u003e18\u003c/sup\u003eF]fluoride was produced via \u003csup\u003e18\u003c/sup\u003eO(p,n)\u003csup\u003e18\u003c/sup\u003eF nuclear reaction (30 min beam at 55 \u0026micro;A, 1.87 Ci, 69.19 GBq) with a General Electric (GE) PETtrace 800 cyclotron. [\u003csup\u003e18\u003c/sup\u003eF]Fluoride was trapped on a Sep-Pak Plus Light QMA solid phase extraction (SPE) cartridge. Anhydrous [\u003csup\u003e18\u003c/sup\u003eF]Tetramethylammonium fluoride (TMAF) was prepared using a GE TRACERlab FX N Pro with modifications, as described in our previous method.(Wright et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) Tetramethylammonium triflate (TMAOTf) (5.9 mg, 26.5 \u0026micro;mol) in 500 \u0026micro;L of H\u003csub\u003e2\u003c/sub\u003eO was used to elute the [\u003csup\u003e18\u003c/sup\u003eF]TMAF from the cartridge. Kryptofix (K\u003csub\u003e2.2.2\u003c/sub\u003e) (0.14 mg, 0.36 \u0026micro;mol) in 1 mL of acetonitrile (MeCN) was used to azeotropically dry the [\u003csup\u003e18\u003c/sup\u003eF]TMAF. The precursor (\u003cb\u003e2\u003c/b\u003e, 1.78 mg, 5.00 \u0026micro;mol) dissolved in 500 \u0026micro;L of MeCN was added to the dry [\u003csup\u003e18\u003c/sup\u003eF]fluoride, followed by the addition of zinc triflate (Zn(OTf)\u003csub\u003e2\u003c/sub\u003e) (0.36 mg, 1.00 \u0026micro;mol) and triazabicyclodecene (TBD) (0.63 mg, 4.50 \u0026micro;mol) dissolved in 500 \u0026micro;L of MeCN. The reaction was allowed to stir for 20 min at 80\u0026deg;C and quenched (3.5 mL of HPLC buffer), then purified via semi-preparative HPLC (column: Phenomenex Luna C18(2) 10\u0026micro;m 250 \u0026times; 10 mm; mobile phase: 50% EtOH, 10mM NH\u003csub\u003e4\u003c/sub\u003eOAc, 2 mL AcOH; flow rate: 4 mL/min). The product peak (t\u003csub\u003eR\u003c/sub\u003e = 15 min) was collected for 1 min (4 mL) and diluted with 60 mL with Milli-Q H\u003csub\u003e2\u003c/sub\u003eO containing 0.1 mL of ascorbic acid injectable solution (500 mg/mL). The resulting solution was passed over a Waters Oasis\u0026reg; HLB resin (30 mg/1 cc) and rinsed with 10 mL of sterile H\u003csub\u003e2\u003c/sub\u003eO. The product was eluted from the resin with 500 \u0026micro;L of USP ethanol and formulated with USP saline (10 mL dose; 95% saline, 5% EtOH) before 0.22 \u0026micro;m sterile filtration into a dose vial. Identity and purity were confirmed via analytical radio-HPLC.\u003c/p\u003e\n\u003ch3\u003eHPLC Analysis:\u003c/h3\u003e\n\u003cp\u003eThe chemical and radiochemical purity of [\u003csup\u003e18\u003c/sup\u003eF]fluoroflutamide was analyzed using a Shimadzu LC2010 HPLC equipped with a Bioscan/Eckert and Ziegler radioactivity detector and an ultraviolet (UV) detector (Phenomenex Luna C18(2) 5\u0026micro;m 150 \u0026times; 4.6mm column, mobile phase: 40% MeCN, 10mM NH\u003csub\u003e4\u003c/sub\u003eOAc, 2 mL AcOH, flow rate: 2 mL/min, t\u003csub\u003eR\u003c/sub\u003e ~ 10.0 min). A representative HPLC trace is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Additional details are provided in the Supporting Information.\u003c/p\u003e\n\u003ch3\u003eAnimal Studies:\u003c/h3\u003e\n\u003cp\u003eAll animal studies were performed in accordance with the standards set by the University of Michigan Institutional Animal Care \u0026amp; Use Committee (IACUC). Human prostate carcinoma cells (22Rv1) were purchased from ATCC. Nine male mice were xenografted with 22Rv1 cells at a density of 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e in PBS in a 1:1 ratio of Matrigel 2 weeks prior to imaging. Male mice were anesthetized with isoflurane (5% induction, 1\u0026ndash;2% maintenance) and injected with [\u003csup\u003e18\u003c/sup\u003eF]fluoroflutamide via tail vein injection. Group 1 received an injection of [\u003csup\u003e18\u003c/sup\u003eF]fluoroflutamide (n\u0026thinsp;=\u0026thinsp;3), group 2 received a co-injection of [\u003csup\u003e18\u003c/sup\u003eF]fluoroflutamide\u0026thinsp;+\u0026thinsp;apalutamide at 2 mg/kg (n\u0026thinsp;=\u0026thinsp;3), and group 3 received a co-injection of [\u003csup\u003e18\u003c/sup\u003eF]fluoroflutamide\u0026thinsp;+\u0026thinsp;\u003csup\u003e19\u003c/sup\u003eF-fluoroflutamide at 2 mg/kg (n\u0026thinsp;=\u0026thinsp;3). A single mouse from each group was imaged immediately after injection for a 60 min dynamic scan (13 time frames). The mice were then imaged again at 2 h post-injection with a 10 min static scan using a Small Animal PET scanner (MR Solutions, Guildford, UK). PET images were corrected for decay, dead time, and random coincidences. Images were then reconstructed using an iterative ordered subset expectation maximization-maximum a posteriori (MAP). Mice were euthanized at 2 h post-injection, and organs were harvested, tissue weights were collected, and radioactivity was measured using a PerkinElmer Wizard 2480 gamma counter (PerkinElmer, Waltham, MA, USA). Measurements were averaged and reported as a percentage of injected dose per gram of tissue (%ID/g). Post-analysis of the PET images was performed using Imalytics Preclinical (Gremse-IT GmbH, Aachen, Germany), in which regions of interest for selected tissues in each mouse and each frame were drawn. Data from Imalytics were analyzed using GraphPad Prism (Version 10.6.1 for macOS), with independent t-tests and one-way ANOVA tests.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFor the automated radiosynthesis of [\u003csup\u003e18\u003c/sup\u003eF]fluoroflutamide, the bromoamide precursor (\u003cb\u003e2\u003c/b\u003e) and the fluorine-19 standard were synthesized from previously described methods.(Wright et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) Cyclotron-produced \u003csup\u003e18\u003c/sup\u003eF\u003csup\u003e\u0026minus;\u003c/sup\u003e was generated from proton bombardment of a [\u003csup\u003e18\u003c/sup\u003eO]H\u003csub\u003e2\u003c/sub\u003eO target via the \u003csup\u003e18\u003c/sup\u003eO(p,n)\u003csup\u003e18\u003c/sup\u003eF nuclear reaction and delivered to a GE TRACERlab FX N Pro. The activity was trapped on a Waters QMA cartridge pretreated with 10 mL of EtOH, followed by 10 mL of 1M potassium triflate (KOTf), and then 10 mL of Milli-Q H\u003csub\u003e2\u003c/sub\u003eO. The activity was eluted off the cartridge via ion-exchange with tetramethylammonium triflate (TMAOTf) in H\u003csub\u003e2\u003c/sub\u003eO, followed by subsequent addition of kryptofix (K\u003csub\u003e2.2.2\u003c/sub\u003e) in MeCN and azeotropic drying. The bromoamide precursor in MeCN was then added to the dry [\u003csup\u003e18\u003c/sup\u003eF]TMAF with stirring, followed by the addition of zinc triflate (Zn(OTf)\u003csub\u003e2\u003c/sub\u003e) and triazabicyclodecene (TBD). The reaction mixture was heated to 80\u0026deg;C for 20 min, then quenched with HPLC buffer and loaded onto the semi-preparative reverse-phase HPLC column for purification (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The product peak eluted at 15 min and was collected for 1 min into a dilution flask containing 60 mL of water for reformulation. 0.1 mL of ascorbic acid solution (500 mg/mL) was added to mitigate radiolysis. After collecting the product peak, the entire solution was washed over a Waters Oasis HLB resin (30 mg/1 cc) and rinsed with 10 mL of sterile H\u003csub\u003e2\u003c/sub\u003eO to remove any residual HPLC salts. Elution with EtOH into saline, followed by 0.1 mL of ascorbic acid solution, yielded a 10 mL dose (5% EtOH), which was analyzed by radio-HPLC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eFrom 1.87 Ci (or 69.19 GBq) of \u003csup\u003e18\u003c/sup\u003eF\u003csup\u003e\u0026minus;\u003c/sup\u003e, [\u003csup\u003e18\u003c/sup\u003eF]fluoroflutamide was isolated with an average radiochemical yield of 3.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30% (n\u0026thinsp;=\u0026thinsp;4) in \u0026gt;\u0026thinsp;99% radiochemical purity. The activity yields (64.55\u0026thinsp;\u0026plusmn;\u0026thinsp;24.28 mCi) were sufficient for planned animal studies with an average synthesis time of 79 min. Additionally, the doses had an average molar activity of 1,573.89 Ci/mmol (or 58,233.93 GBq/mmol) (see S7 and S22 for the standard curves). Initial optimization of the automated method with 1.87 Ci (or 69.19 GBq) of \u003csup\u003e18\u003c/sup\u003eF indicated radioimpurities originating from radiolysis 2 h after the end of synthesis. By adding the radioprotectant ascorbic acid in the dilution flask, product vial, and the HPLC buffer, we observed no radiolysis present in the final dose formulation for up to 4 h post-end-of-synthesis (99.54% RCP at 1 h; 97.81% RCP at 4 h). See the supporting information for more details.\u003c/p\u003e \u003cp\u003eBiodistribution studies were performed on nude athymic male mice with 22Rv1 xenografts. Xenografts were allowed to grow until tumors were palpable. Nine mice implanted with 22Rv1 tumors were injected with an average activity of 125.33\u0026thinsp;\u0026plusmn;\u0026thinsp;40.08 \u0026micro;Ci (or 4.64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.48 MBq) of [\u003csup\u003e18\u003c/sup\u003eF]fluoroflutamide. To confirm binding to the androgen receptor, in 3 animals received a coinjection of the tracer and a 2 mg/kg dose of apalutamide. \u003csup\u003e19\u003c/sup\u003eF-Fluoroflutamide was also coinjected with the tracer at 2 mg/kg in 3 other animals to quantify nonspecific binding and receptor saturation. The organs with the highest average uptake in all three treatment groups were the adrenal glands (baseline: 4.67% ID/g, apalutamide block: 3.41% ID/g, \u003csup\u003e19\u003c/sup\u003eF-fluoroflutamide block: 7.57% ID/g), the liver (1.96, 2.57, and 5.54% ID/g), and the kidneys (1.77, 2.14, and 4.82% ID/g, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). One baseline mouse and one mouse cotreated with apalutamide were PET imaged for 0\u0026ndash;60 min post-injection, and the summed image from this study is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. A classical blocking effect in the heart and the liver of the mouse dosed with apalutamide compared to the baseline mouse was observed. This finding was not observed in the biodistribution studies at the 2-hour timepoint as the apalutamide group had a higher average liver uptake value than the baseline mice. Additionally, the average injected dose per gram of heart tissue was found to be similar in both groups (1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52% for the apalutamide treatment group and 1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31% in baseline mice).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFlutamide has low oral bioavailability due to rapid absorption and subsequent hepatic metabolism to 2-hydroxyflutamide. To determine the intravenous injection doses of flutamide and apalutamide, we calculated the percentage of absorption from an oral dose using previous literature.(Schulz et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Posti et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Clegg et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sulochana et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; de Vries et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) Both apalutamide and flutamide are commonly dosed as timed-release pellets or subcutaneous injections because of their poor aqueous solubility. However, we found that both apalutamide and \u003csup\u003e19\u003c/sup\u003eF-fluoroflutamide dissolved completely when formulated with Tween-80, EtOH, and saline at the desired blocking concentrations. HPLC analysis confirmed that the doses remain in solution at the same concentrations after sterile filtration.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eNon-invasive imaging of AR expression would be beneficial for multiple cancers, including late-stage and aggressive NEPC. Interest in PC imaging agents has been on the rise since the FDA approval of the theranostic pair Pluvicto ([\u003csup\u003e177\u003c/sup\u003eLu]PSMA-617) and Locametz/Illuccix ([\u003csup\u003e68\u003c/sup\u003eGa]PSMA-11). The first-line treatment for PC includes AR antagonists such as enzalutamide, apalutamide, and darolutamide, which compete with androgens for AR binding. Eventually, through resistance pathways, AR dependency is lost, coinciding with a waning of treatment response. In some instances, therapy continuation can facilitate disease progression, so PET imaging studies that accurately stage this transition would be invaluable for reassessing treatment decisions. Biopsies are currently used to evaluate AR expression, an invasive procedure that does not always provide a comprehensive understanding of target expression in each tumor. Therefore, by radiolabeling one of these AR antagonists with fluorine-18, we aimed to assess the ability of this agent to non-invasively measure AR expression through PET imaging.\u003c/p\u003e \u003cp\u003eFlutamide is a first-generation antagonist containing an α-3\u0026deg;-amide with a known active metabolite formed via α-hydroxylation. A new methodology from our lab describes a wide array of α-amide substrates that can be labeled via zinc-mediated radiofluorination. In the case of 2-hydroxyflutamide, the active metabolite generated via CYP1A2, fluorine can also act as an isostere for a hydroxyl group due to its weak hydrogen-bond-accepting properties and high electronegativity.(Inoue et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) Using this methodology, we developed an automated procedure for radiolabeling flutamide for further assessment \u003cem\u003ein vivo\u003c/em\u003e. Our initial conditions for radiolabeling used dimethyl sulfoxide (DMSO) as the reaction solvent to develop a Class III solvent procedure. However, the non-decay-corrected molar activity (A\u003csub\u003em\u003c/sub\u003e) obtained with this method was lower than the threshold for effective PET tracers (458.06 Ci/mmol or 16,948.22 GBq/mmol vs ca. 1,000 Ci/mmol or 37,000 MBq/mmol). By changing the solvent to MeCN, we increased the molar activity to 2,212.54 Ci/mmol or 81,863.98 GBq/mmol. A control experiment was also performed to ensure that fluorodenitration and isotopic exchange with the trifluoromethyl did not occur using flutamide in place of the bromoamide precursor under the optimized conditions. The crude reaction mixture was removed for analysis, and the resulting activity was 5.87 mCi or 217.19 MBq (5 min beam; 162.3 mCi or 6005.1 MBq of \u003csup\u003e18\u003c/sup\u003eF), identified as unreacted \u003csup\u003e18\u003c/sup\u003eF. No organic products were observed by radio-TLC and -HPLC, confirming that the reaction was free of undesired side products. With optimized reaction conditions in hand, we pursued a fully automated synthesis, purification, and dose formulation of [\u003csup\u003e18\u003c/sup\u003eF]fluoroflutamide on a GE TRACERlab FX N Pro for preclinical studies. [\u003csup\u003e18\u003c/sup\u003eF]Fluoroflutamide was isolated with high radiochemical purity (\u0026gt;\u0026thinsp;99%) and activity yields of 64.55\u0026thinsp;\u0026plusmn;\u0026thinsp;24.28 mCi (n\u0026thinsp;=\u0026thinsp;4). The yields are sufficient for \u003cem\u003ein vivo\u003c/em\u003e animal studies as well as human doses (\u0026gt;\u0026thinsp;10 mCi).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFollowing the isolation of a consistent, automated protocol, we performed a biodistribution and imaging study with [\u003csup\u003e18\u003c/sup\u003eF]fluoroflutamide in nude athymic mice inoculated with 22Rv1 tumors. Nine male mice were administered [\u003csup\u003e18\u003c/sup\u003eF]fluoroflutamide, and biodistribution studies were conducted 2 h post-injection. Of the nine mice, two groups were dosed with \u003csup\u003e19\u003c/sup\u003eF-fluoroflutamide and apalutamide to evaluate specific binding. Dynamic PET imaging studies of one baseline mouse and one mouse blocked with apalutamide were completed from 0\u0026ndash;60 min post-injection, and the summed image from this study is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Blocking the AR with apalutamide (left mouse) led to an observed decrease in uptake in the heart and liver compared to baseline (right mouse) from 0\u0026ndash;60 min. Conversely, from the biodistribution data taken at 2 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), there is increased tracer uptake in both carrier-added groups. This has previously been described as \u0026lsquo;mass action\u0026rsquo; or \u0026lsquo;mass effect.\u0026rsquo; The addition of cold-mass and apalutamide, an analogous ligand, altered the pharmacokinetics and biodistribution over time by increasing tracer uptake in most organs, including the tumor. A possible explanation for this observation is that, with the addition of mass, drug clearance time remained the same. Therefore, the tracer had a longer residence time at the receptor before being cleared compared to the baseline mouse. The initial blocking effect observed in the PET images along with the lack of uptake in the tumor and testes demonstrates nonspecific uptake in tissues rather than AR binding. [\u003csup\u003e18\u003c/sup\u003eF]Fluoroflutamide exhibited the highest uptake in the adrenal glands, liver, kidneys, and testes in all three groups. The tracer also exhibited minimal defluorination \u003cem\u003ein vivo\u003c/em\u003e, as indicated by no increase of \u003csup\u003e18\u003c/sup\u003eF uptake in bone (baseline average: 0.85% ID/g) compared to other background organs such as muscle (baseline average: 0.74% ID/g).\u003c/p\u003e \u003cp\u003eSome limitations of this study include the biodistribution study being conducted at a single time point (2 h) which may be later than desired for an AR ligand. The results of this initial study did not warrant generating more tumor models to assess biodistribution at earlier time points. Additionally, the binding affinity of the new fluorinated analog was not tested \u003cem\u003ein vitro\u003c/em\u003e compared with that of the parent molecule and analogous antagonists. Further work involves \u003cem\u003ein vitro\u003c/em\u003e binding assays with 22Rv1 cells and other PC cell lines that express or lack AR, such as PC-3 (AR\u003csup\u003e\u0026minus;\u003c/sup\u003e), DU145 (AR\u003csup\u003e\u0026minus;\u003c/sup\u003e), or LNCaP (AR\u003csup\u003e+\u003c/sup\u003e), and LAPC-4 (AR\u003csup\u003e+\u003c/sup\u003e).(Sampson et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) However, the biodistribution and imaging data suggest that by placing the fluorine at the α-3\u0026deg;-position of the amide, oxidative metabolism may be blocked to the more potent metabolite of flutamide, 2-hydroxyflutamide. The lack of selective uptake also indicates the analog\u0026rsquo;s lack of binding similarity to 2-hydroxyflutamide, which is 25-fold more potent than flutamide and accounts for 90% of metabolites present in plasma following a therapeutic dose.(Simard et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1986\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eDuring the preparation of this manuscript, the Lee group published a new methodology for \u003csup\u003e18\u003c/sup\u003eF trifluoromethylation of aryl iodides.(Choi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) Within this study, the group radiolabeled flutamide at the trifluoromethyl group, leaving the amide susceptible to α-hydroxylation via CYP1A2. When the agent was tested in LNCaP (AR\u003csup\u003e+\u003c/sup\u003e) xenograft mouse models, rapid renal clearance was observed, along with specific uptake in the xenograft up to 60 min post-injection. This further supports the idea that α-C\u0026ndash;H metabolism of flutamide is essential for AR targeting.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe successfully developed an automated radiosynthesis of [\u003csup\u003e18\u003c/sup\u003eF]fluoroflutamide with high radiochemical purity and sufficient molar activity for preclinical imaging studies. \u003cem\u003eIn vivo\u003c/em\u003e studies in 22Rv1 xenograft-bearing mice demonstrated tumor uptake and minimal defluorination, as evidenced by the absence of significant skeletal signal. However, classical blocking with nonradioactive apalutamide or fluoroflutamide did not reduce tumor or tissue uptake. Instead, a paradoxical \u0026ldquo;mass effect\u0026rdquo; was observed, with increased tracer accumulation across multiple organs, including the tumor, when non-radioactive competitors were co-injected. These findings suggest that the \u003cem\u003ein vivo\u003c/em\u003e distribution of [\u003csup\u003e18\u003c/sup\u003eF]fluoroflutamide is likely influenced by nonspecific pharmacokinetics, metabolism, and receptor residence time, rather than by AR binding. Overall, while [\u003csup\u003e18\u003c/sup\u003eF]fluoroflutamide is (bio)chemically stable and can be reliably synthesized, the current data indicate limited avidity and specificity for AR imaging \u003cem\u003ein vivo\u003c/em\u003e. Further optimization using structural analogs is required, particularly through \u003cem\u003ein vitro\u003c/em\u003e binding assays against AR⁺ and AR⁻ cell lines, metabolite characterization, and evaluation in additional tumor models. For example, analogs that can undergo α-hydroxylation, analogous to the metabolism of flutamide, may show enhanced uptake. These studies and others are underway to identify effective, noninvasive tools for imaging AR expression in advanced PC, including CRPC and NEPC.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAndrogen receptor (AR)\u003c/p\u003e\n\u003cp\u003eEnd-of-Beam (EOB)\u003c/p\u003e\n\u003cp\u003eMetastatic Castration-Resistant Prostate Cancer (mCRPC)\u003c/p\u003e\n\u003cp\u003ePositron Emission Tomography (PET)\u003c/p\u003e\n\u003cp\u003eProstate Cancer (PC)\u003c/p\u003e\n\u003cp\u003eCastration-Resistant Prostate Cancer (CRPC)\u003c/p\u003e\n\u003cp\u003eNeuroendocrine Prostate Cancer (NEPC)\u003c/p\u003e\n\u003cp\u003eProstate-Specific Membrane Antigen (PSMA)\u003c/p\u003e\n\u003cp\u003e16\u0026beta;-\u003csup\u003e18\u003c/sup\u003eF-fluoro-5\u0026alpha;-dihydrotestosterone ([\u003csup\u003e18\u003c/sup\u003eF]FDHT)\u003c/p\u003e\n\u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]Fluorodeoxyglucose ([\u003csup\u003e18\u003c/sup\u003eF]FDG)\u003c/p\u003e\n\u003cp\u003eRadio-High-Performance Liquid Chromatography (radio-HPLC)\u003c/p\u003e\n\u003cp\u003eNuclear Magnetic Resonance (NMR)\u003c/p\u003e\n\u003cp\u003eTetramethylammonium Triflate (Me\u003csub\u003e4\u003c/sub\u003eNOTf)\u003c/p\u003e\n\u003cp\u003eGeneral Electric (GE)\u003c/p\u003e\n\u003cp\u003eSolid Phase Extraction (SPE)\u003c/p\u003e\n\u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]Tetramethylammonium fluoride (TMAF)\u003c/p\u003e\n\u003cp\u003eTetramethylammonium triflate (TMAOTf)\u003c/p\u003e\n\u003cp\u003eKryptofix (K\u003csub\u003e2.2.2\u003c/sub\u003e)\u003c/p\u003e\n\u003cp\u003eZinc Triflate (Zn(OTf)\u003csub\u003e2\u003c/sub\u003e)\u003c/p\u003e\n\u003cp\u003eTriazabicyclodecene (TBD)\u003c/p\u003e\n\u003cp\u003eInstitutional Animal Care \u0026amp; Use Committee (IACUC)\u003c/p\u003e\n\u003cp\u003eMaximization-Maximum a posteriori (MAP)\u003c/p\u003e\n\u003cp\u003ePotassium Triflate (KOTf)\u003c/p\u003e\n\u003cp\u003eDimethyl Sulfoxide (DMSO)\u003c/p\u003e\n\u003cp\u003eMolar Activity (A\u003csub\u003em\u003c/sub\u003e)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGeneral Animal Study Considerations:All animal studies were performed under and in accordance with the direct guidance from the IACUC (Institutional Animal Care and Use Committee) and Unit for Lab Animal Management at the University of Michigan under Peter J. H. Scott’s IACUC protocol number of PRO11715.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the final manuscript and consent to its publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated during this study, including chemistry/animal experiments, are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePJHS is associate editor of EJNMMI Radiopharmacy and Chemistry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully acknowledge funding from the National Institutes of Health. This work was supported by NIH NIBIB [Award Numbers R01EB021155 (P.J.H.S.) R00EB031564 (J.S.W.)].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the University of Michigan analytical services for assistance with compound characterizations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG.K., P. J. H. S., A. F. B., and J. S. W. conceived and led the project. G. K., J. W., S. C., and J. S. performed experiments and assisted with project design.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorresponding Authors\u003c/p\u003e\n\u003cp\u003e*Peter J. H. Scott – 0000-0002-6505-0450, Department of Radiology, University of Michigan, Ann Arbor, MI 48109, USA. Email: [email protected]\u003c/p\u003e\n\u003cp\u003e*Jay S. Wright – 0000-0002-1350-123X, Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104, USA; E-mail: [email protected]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGina R. Kaup –0009-0003-1860-1226, Department of Radiology, University of Michigan, Ann Arbor, MI 48109, USA\u003c/p\u003e\n\u003cp\u003eJason A. Witek –0000-0001-7271-6567, Department of Radiology, University of Michigan, Ann Arbor, MI 48109, USA\u003c/p\u003e\n\u003cp\u003eShelbie J. Cingoranelli –0009-0002-5578-0342, Department of Radiology, University of Michigan, Ann Arbor, MI 48109, USA\u003c/p\u003e\n\u003cp\u003eDavid Raffel – 0000-0002-7188-9463, Department of Radiology, University of Michigan, Ann Arbor, MI 48109, USA\u003c/p\u003e\n\u003cp\u003eJenelle Stauff – Department of Radiology, University of Michigan, Ann Arbor, MI 48109, USA\u003c/p\u003e\n\u003cp\u003eAllen F. Brooks –0000-0003-3773-3024, Department of Radiology, University of Michigan, Ann Arbor, MI 48109, USA\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAntunes IF, Dost RJ, Hoving HD, et al. Synthesis and Evaluation of \u003csup\u003e18\u003c/sup\u003eF-Enzalutamide, a New Radioligand for PET Imaging of Androgen Receptors: A Comparison with 16β-\u003csup\u003e18\u003c/sup\u003eF-Fluoro-5α-Dihydrotestosterone. J Nucl Med. 2021;62:1140\u0026ndash;5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2967/jnumed.120.253641\u003c/span\u003e\u003cspan address=\"10.2967/jnumed.120.253641\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBakht MK, Derecichei I, Li Y, et al. Neuroendocrine differentiation of prostate cancer leads to PSMA suppression. 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J Nucl Med. 2004;45:1966\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"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":"Androgen receptor, prostate cancer, metastatic castration-resistant prostate cancer, radiofluorination, positron emission tomography","lastPublishedDoi":"10.21203/rs.3.rs-8895668/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8895668/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe androgen receptor (AR) is upregulated in multiple cancers, such as salivary gland cancer, glioblastoma, triple-negative breast cancer, and, most commonly, prostate cancer. Imaging expression of the AR via radiolabeling an AR antagonist would enable non-invasive clinical staging and assessment of tumor heterogeneity, complementing the findings of a [\u003csup\u003e68\u003c/sup\u003eGa]PSMA-11 scan. Previous efforts described the radiolabeling of enzalutamide with fluorine-18 but were limited by low specific activity. In this study, we developed a high-yielding automated synthesis of [\u003csup\u003e18\u003c/sup\u003eF]fluoroflutamide using zinc-mediated radiofluorination and assessed its biodistribution in preclinical models of prostate cancer.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eFrom 1.87 Ci (69.19 GBq) of [\u003csup\u003e18\u003c/sup\u003eF]fluoride, [\u003csup\u003e18\u003c/sup\u003eF]fluoroflutamide was isolated with a radiochemical yield of 3.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30% (n\u0026thinsp;=\u0026thinsp;4) and a radiochemical purity of \u0026gt;\u0026thinsp;99% in an average 79 min from end-of-beam (EOB) using a bromoamide precursor. The average non-decay corrected A\u003csub\u003em\u003c/sub\u003e was found to be 1,573.89 Ci/mmol (or 58,233.93 GBq/mmol), and the radiochemical purity after 4 h was \u0026gt;\u0026thinsp;97%, indicating no detectable radiolysis. A biodistribution and imaging study was performed using nude athymic mice with 22Rv1 human prostate cancer xenografts. From the results of this study, we observed a \u0026lsquo;mass effect\u0026rsquo; in which carrier-added mice displayed an increased uptake of the tracer across most organs.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eHerein, we report the optimized automated radiosynthesis of [\u003csup\u003e18\u003c/sup\u003eF]fluoroflutamide with high purity and yield sufficient for imaging AR expression in mouse models of prostate cancer. The lack of uptake to androgen receptor-rich organs and xenografts suggests that CYP1A2 metabolism to the more potent metabolite, 2-hydroxyflutamide, was blocked by the addition of the α-3\u0026deg;-fluorine. The automated production of a fluoroamide via zinc-mediated radiofluorination, in high specific activity, was demonstrated to evaluate a potential radioligand for the detection of androgen receptor-positive prostate cancer tumors.\u003c/p\u003e","manuscriptTitle":"Automated Radiosynthesis and Biodistribution of [18F]Fluoroflutamide","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-27 13:08:07","doi":"10.21203/rs.3.rs-8895668/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2026-03-11T11:20:34+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2026-02-26T15:28:04+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-24T15:17:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-24T14:24:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"EJNMMI Radiopharmacy and Chemistry","date":"2026-02-24T08:43:48+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":"317c5e40-4dc1-40a3-9950-49da57a6227b","owner":[],"postedDate":"February 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T10:51:07+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-27 13:08:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8895668","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8895668","identity":"rs-8895668","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}