Automated Preparation of [18F]Flutamide for Androgen Expression Monitoring

Introduction: In the USA during 2023, nearly 300,000 men were diagnosed with prostate cancer (PC), which is the 4th most diagnosed cancer worldwide. Androgen deprivation therapy is employed in patients who present with advanced disease. However, resistance mechanisms lead to a decline in treatment response and the onset of castration-resistant PC. This lethal stage of the disease coincides with mutations and overexpression of the androgen receptor (AR), and highly invasive biopsies are required to evaluate AR for treatment. Alternatively, agents such as [¹⁸F]16β-fluoro-5α-dihydrotestosterone ([¹⁸F]FDHT) can assess AR via PET imaging non-invasively.¹ However, the rapid metabolism of [¹⁸F]FDHT by the kidneys can lead to low image contrast and confound lesion detection localized near the bladder. Radioligands based on nonsteroidal antiandrogens that are instead metabolized in the liver, such as ¹⁸F-enzalutamide, can potentially address these limitations.² However, the radiochemistry employed to prepare this agent afforded poor molar activities (A_m), reducing utility. Herein, we describe [¹⁸F]flutamide as an alternative imaging agent prepared with Zn-mediated amide radiofluorination, a powerful tool for installing ¹⁸F into sterically congested systems which offers elevated A_m.³

Methods: Automated amide radiofluorination protocols were conducted in a GE synthesis module. [¹⁸F]Fluoride was produced via the ¹⁸O(p,n)¹⁸F reaction using a GE PETTrace cyclotron. [¹⁸F]Fluoride was trapped on a preconditioned Sep-Pak and eluted into the reactor using Me₄NOTf in H₂O. A solution of K₂₂₂ in MeCN was added to the reaction vessel for azeotropic drying at 100 °C. The reactor was cooled to 60 °C before the sequential addition of a solution of TBD and Zn(OTf)₂ in DMSO (500 μL), followed by a solution of precursor in DMSO. The reactor was heated to 80 °C for 20 min before cooling and dilution. The product was purified via HPLC and reformulated on a C18 cartridge. Preliminary preclinical evaluations, including buffer/rodent serum stability, radiometabolites, and in vitro binding affinity, are currently under investigation.

Results: Previously, we successfully prepared the requisite [¹⁸F]flutamide imaging agent with Zn-mediated radiosynthesis of unprotected α-tertiary amides in 24 ± 3% radiochemical conversion (RCC,n=2) under manual conditions. This was achieved using Zn(OTf)₂ as a mediator, triazabicyclodecene (TBD) as a base, [¹⁸F]TMAF as a fluorine-18 source in DMSO solvent. Enough [¹⁸F]flutamide was obtained under manual conditions using bromoflutamide as a precursor to warrant translation to automated production. Modifications were made to improve the radiochemical yield of [¹⁸F]flutamide. For example, alternative reaction concentrations, temperatures, and times were investigated. Buffer and serum stability of closely related analogs was established, and future studies shall aim to confirm whether [¹⁸F]flutamide exhibits comparable stability.

Conclusions: To our knowledge, a reliable one-step protocol to label flutamide at the α-position other than Zn-mediated radiofluorination does not exist. Installation of fluorine-18 at the hindered, α-tertiary position may confer several pharmacokinetic benefits. First, the fluorine-18 atom is sterically protected, reducing metabolic defluorination. Second, substituting the C-H bond may inhibit metabolism to the corresponding α-hydroxy analog, improving signal-to-noise ratio. Third, molar activity should be enhanced relative to other methodologies that suffer from fluorine-19 or precursor contamination. We hope this analog will exhibit utility for assessing AR density, tumor heterogeneity, and PC disease progression, thereby addressing the limitations of other PET AR imaging agents and improving outcomes for patients with advanced PC.

Acknowledgments: NIH R01EB021155 & K99EB031564.

Refs: 1. Beattie et al, J. Nucl. Med., 2010, 51, 182; 2. Atunes et al, J. Nucl. Med., 2021, 62, 1140; 3. Wright et al, Angew. Chem. Int. Ed., 2023, 63, e202316365

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