Abstract View
Development of Radiolabeled Amonafide Derivatives as A GSH-Responsive PET ligand
Category: Radiopharmaceuticals for Oncology
Authors:
Na Yeong Hwang¹·², Meiying Jin², Ji Young Choi², Hyung-Jun Im¹·³·⁴, Byung Chul Lee²·³
¹Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul, Republic of Korea ²Department of Nuclear Medicine, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Republic of Korea ³Department of Applied Bioengineering, Graduate School of Convergence Science and Technology, Seoul National University, Seoul, Republic of Korea ⁴Cancer Research Institute, Seoul National University, Seoul, Republic of Korea
Objective: Tumor microenvironments are characterized by elevated intracellular glutathione (GSH) levels, which are closely associated with antioxidant defense and drug resistance. We developed GSH-responsive radiolabeled Amonafide derivatives ([⁶⁸Ga]- and [18F]-labeled Amonafides) incorporating a disulfide (DS) linker, and evaluated their tumor selectivity, intracellular retention, and PET imaging capability.
Methods: The precursor DOTA-Amonafide-DS was synthesized through a five-step procedure introducing a DS linker into Amonafide and conjugating DOTA to the imide group, and confirmed by NMR and LC-MS. Radiolabeling with ⁶⁸Ga was carried out in HEPES buffer (pH 4.5) at 90 °C. GSH-responsive activation was evaluated using fluorescence analysis. Intracellular DNA intercalation and nuclear retention were assessed in GSH-positive U87-MG cells with confocal microscopy for 24 h. In vivo PET imaging and biodistribution studies were performed in U87-MG and LNCaP xenografts, respectively.
Results: [⁶⁸Ga]Ga-DOTA-Amonafide-DS was synthesized with a radiochemical yield (RCY) of 35.4% (decay-corrected, isolated by radio-HPLC). Radiochemical identity was confirmed by HPLC co-injection analysis with [natGa]Ga-DOTA-Amonafide-DS (TR = 14.6 min). Amonafide-DS showed no fluorescence, whereas a rapid fluorescence enhancement at 545 nm was observed in the presence of GSH. Confocal microscopy revealed strong nuclear fluorescence signals in U87-MG cells that persisted for 24 h, confirming prolonged nuclear retention through DNA intercalation. In vivo PET imaging showed significant tumor uptake (5.06 %ID/g at 60 min post-injection) in U87-MG xenografts. Substantial high uptake was also observed in the intestine, likely attributable to endogenous GSH levels in intestinal epithelial cells. In contrast, LNCaP tumors with relatively low GSH expression showed limited uptake. Notably, radiolabeled Amonafide lacking the DS linker accumulated in tumors, underscoring the functional specificity of the DS linker in vivo.
Conclusion: [⁶⁸Ga]Ga-DOTA-Amonafide-DS exhibited GSH-responsive activation, prolonged nuclear retention, and significant tumor uptake in the U87-MG xenografts. The limited uptake observed in the low-GSH LNCaP xenograft supports the tumor-selective mechanism of the DS linker. These findings suggest the radiolabeled Amonafide represents a promising PET radioligand for imaging redox-associated tumor microenvironments. Al¹⁸F-labeling on the same precursor is currently underway to evaluate extended pharmacokinetics in vivo.
Reference:
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