Comparison of multimodal imaging of antisense oligonucleotides (ASOs): PET, SPECT, cryo-fluorescence tomography (CFT), and ARG of mice co-injected with 89Zr, 125I or IR750-labeled ASO

Purpose: Quantitative whole-body autoradiography (QWBA) is a validated method, but it requires the challenging step of radiolabeling oligonucleotides. This study examined whether imaging modalities could provide alternatives to QWBA by co-administering nuclide- or fluorescence-labeled antisense oligonucleotides (ASOs) to mice and comparing the results with QWBA. Procedures: This study evaluated the biodistribution of an ASO conjugated with deferoxamine-star and labeled with zirconium-89 ([ ⁸⁹ Zr]Zr-DFO*, for PET), labeled with iodine-125 ([ ¹²⁵ I]-ASO, for SPECT and autoradiography (ARG)), or conjugated to IR750 for cryo-fluorescence tomography (CFT). Twelve mice were dosed with [ ⁸⁹ Zr]Zr-DFO*-ASO, [ ¹²⁵ I]-ASO, and ASO-IR750 subcutaneously, and then imaged for up to 10 days. Following imaging, two mice per timepoint were euthanized for CFT, and slices were pulled for ARG. For QWBA, [ ¹⁴ C]-ASO was administered subcutaneously. Results: ⁸⁹ Zr-PET, ¹²⁵ I-SPECT, and ¹²⁵ I-ARG showed similar biodistributions, with concentration rankings in major organs and temporal changes being comparable to those of ¹⁴ C -QWBA. The IR750-CFT data showed all regions having the highest signals at the first timepoint, which was unlike the other modalities; however, the regions with high-intensity signals were consistent. Conclusions: The ASO was successfully radiolabeled with ⁸⁹ Zr and ¹²⁵ I, the radioactive concentrations in main organs were similar, and there was good agreement with ¹⁴ C-QWBA; thus, these labeling and imaging modalities appear useful for assessing ASO biodistribution. Although CFT data were not completely comparable with ¹⁴ C-ARG, the regions with high concentrations were consistent, and high-resolution three-dimensional images were obtained. These images may be useful for exploratory evaluation of the distribution in the early stages of drug discovery.