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Talk Summary:
Invasive fungal infections (IFIs) are a growing global health concern, particularly among immunocompromised individuals. Among the causative pathogens, Aspergillus fumigatus is one of the most common and life-threatening opportunistic fungi. Early diagnosis is critical because delayed treatment is associated with significantly increased morbidity and mortality. However, currently available diagnostic methods, including computed tomography (CT), magnetic resonance imaging (MRI), and microbiological culture, have important limitations. Anatomical imaging lacks pathogen specificity, while culture-based techniques are often invasive, time-consuming, and may yield false-negative results. Consequently, there is a growing need for pathogen-specific molecular imaging agents capable of detecting fungal infections rapidly and noninvasively.
Positron emission tomography (PET) is a highly sensitive imaging modality that can detect disease processes before structural changes become apparent. Although 2-deoxy-2-[¹⁸F]fluoroglucose ([¹⁸F]FDG) is the most widely used PET radiotracer, it accumulates in malignant tissues, sterile inflammation, bacterial infections, and fungal infections alike, making it unsuitable for distinguishing invasive fungal infections from other pathological conditions. To overcome this limitation, our group developed 2-deoxy-2-[¹⁸F]fluorocellobiose ([¹⁸F]FCB), a fluorine-18 labeled analogue of cellobiose that is selectively metabolized by cellulolytic fungi expressing β-glucosidase enzymes.
Although the enzymatic synthesis of [¹⁸F]FCB produced good radiochemical yields and was successfully automated on the Trasis AllInOne synthesizer, the final product consistently contained less than 2% unreacted [¹⁸F]FDG. Despite this seemingly small impurity, residual [¹⁸F]FDG resulted in detectable uptake in highly metabolically active organs such as the heart and brain, thereby reducing image contrast and potentially compromising the interpretation of PET images. This issue is particularly important when imaging fungal infections involving the lungs, myocardium, or central nervous system, where physiological [¹⁸F]FDG uptake is naturally high. Therefore, complete removal of residual [¹⁸F]FDG became essential for improving the diagnostic accuracy of [¹⁸F]FCB.
The objective of the present study was to develop a rapid, fully automated, cassette-based synthesis of [¹⁸F]FCB that eliminates residual [¹⁸F]FDG while maintaining high radiochemical yield and compatibility with current Good Manufacturing Practice (cGMP) production. To achieve this, a novel enzymatic purification strategy was introduced. Following the initial conversion of [¹⁸F]FDG to [¹⁸F]FCB using cellobiose phosphorylase and glucose-1-phosphate, hexokinase and adenosine triphosphate (ATP) were added to the reaction mixture. Hexokinase catalyzed the phosphorylation of any remaining [¹⁸F]FDG to form [¹⁸F]FDG-6-phosphate. Because the phosphorylated product carries a negatively charged phosphate group, it is strongly retained on the FDG purification cartridge, allowing selective recovery of highly purified [¹⁸F]FCB without requiring additional high-performance liquid chromatography purification.
Following optimization of the reaction conditions, the procedure was successfully transferred to a disposable cassette-based Trasis AllInOne automated synthesis module. The automated process included a 20-minute enzymatic conversion, a 5-minute phosphorylation step, cartridge purification, sterile filtration, and final product collection. The total synthesis time was reduced from 75 minutes to 40 minutes, representing a substantial improvement in production efficiency. The automated synthesis consistently produced [¹⁸F]FCB with a decay-corrected radiochemical yield of 45–50% and a radiochemical purity of 100%. In addition, the tracer demonstrated excellent stability in human serum at 37°C, indicating its suitability for in vivo imaging applications.
The biological impact of removing residual [¹⁸F]FDG was evaluated in healthy mice using PET/CT imaging. Animals receiving the improved formulation exhibited significantly lower uptake in the myocardium and brain compared with animals injected with a tracer prepared by the previous method, which contained residual [¹⁸F]FDG. In contrast, tracer uptake in the liver and skeletal muscle remained unchanged, demonstrating that the improved purification specifically reduced nonspecific background activity in highly glucose-dependent tissues. These findings indicate that eliminating residual [¹⁸F]FDG enhances image contrast and may improve the detection of fungal infections in organs with high physiological glucose metabolism.
In conclusion, this study presents a simple and effective enzymatic strategy for the complete removal of residual [¹⁸F]FDG during the synthesis of [¹⁸F]FCB. The method significantly shortens synthesis time, maintains excellent radiochemical yield, achieves 100% radiochemical purity, and is readily implemented on a fully automated cassette-based synthesis platform. The resulting reduction in nonspecific PET signal improves the specificity of [¹⁸F]FCB and supports its future clinical translation as a cGMP-compliant PET radiopharmaceutical for the diagnosis and management of invasive fungal infections.
Presenter Biography:
Dr. Falguni Basuli (Bhattacharyya) earned her Ph.D. in synthetic chemistry from Jadavpur University in Kolkata, India, before immigrating to the United States for advanced postdoctoral research. At Indiana University Bloomington, she focused on organometallic chemistry, catalysis, and mechanistic chemistry. Dr. Basuli subsequently held research appointments at the Indiana University School of Medicine and Brigham and Women’s Hospital/Harvard Medical School, where she expanded her expertise into radiochemistry and positron emission tomography (PET) tracer development. In 2009, Dr. Basuli joined the Chemistry Synthesis Center at the National Institutes of Health (NIH). She is a Senior Associate Scientist with more than 25 years of experience spanning organic, organometallic, and radiopharmaceutical chemistry. Her current research focuses on the design and development of PET radiopharmaceuticals labeled with fluorine-18, copper-64, gallium-68, yttrium-86, and zirconium-89 for preclinical and translational biomedical imaging. Dr. Basuli has authored more than 125 peer-reviewed publications and numerous conference abstracts and is an inventor on four patent applications. Through her extensive expertise in synthetic chemistry, innovative work in radiopharmaceutical development, and strong commitment to collaborative science, she has made significant contributions to the discovery and translation of PET radiotracers.
Author
Falguni Basuli
National Institutes of Health