Quantitative dual-isotope preclinical SPECT/CT imaging and biodistribution of the mercury-197m/g theranostic pair with [197m/gHg]HgCl2 and a superior [197m/gHg]Hg-tetrathiol complex as a platform for radiopharmaceutical development

Abstract Background Mercury-197m (197mHg, t1/2 = 23.8 h) and mercury-197g (197gHg, t1/2 = 64.14 h) possess favorable nuclear properties for imaging and targeted therapy, but the development of suitable chelators for mercury-based radiopharmaceuticals remains underexplored. Additionally, accurate imaging and quantification of mercury isotopes, particularly in dual-isotope formats, require tools that account for their complex decay schemes. Phantom imaging studies are essential for validating spatial resolution, quantitative accuracy, and isotope-specific calibration prior to in vivo application. In this study, we investigated the commercially available ligand H₄Tetrathiol for chelation of [197m/gHg]Hg²⁺ and developed a robust imaging and quantification pipeline to support the use of these nuclear isomers in preclinical imaging. Results Radiolabeling of H₄Tetrathiol yielded exceptionally efficient complexation, achieving the lowest ligand-to-metal ratio reported for radio-mercury. The resulting [197m/gHg]Hg²⁺-complex demonstrated high in vitro stability in the presence of serum proteins, glutathione, and competing biologically relevant metal ions, though it exhibited kinetic lability when challenged with excess HgCl₂. In vivo biodistribution studies in mice showed a distinct pharmacokinetic profile from unchelated [197m/gHg]HgCl₂, suggesting in vivo complex stability. Phantom imaging studies with a high sensitivity collimator demonstrated submillimeter resolution (≥1.1 mm) for both 197gHg and 197mHg, with decay behavior consistent with known half-lives. To facilitate accurate quantification, we developed HgQuant, a Python-based tool for isotope-specific calibration, Bateman decay correction, and automated dual-isotope analysis. This tool enabled reproducible, time-resolved quantification in both phantom and in vivo settings. Conclusions These results establish Tetrathiol as a promising scaffold for mercury-based theranostics, offering efficient radiolabeling and in vivo stability. The integration of high-resolution imaging and HgQuant-based quantification of each isomer establishes a comprehensive framework for advancing [197m/gHg]Hg radiopharmaceutical development.