(M1330-02-12) PET/CT Imaging of Bleomycin-Induced Murine Pulmonary Fibrosis Using a Radiolabeled PEGylated Functional Upstream Domain Peptide

Purpose: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease associated with a high mortality rate¹. Early detection and sensitive assessment of fibrotic activity are essential for improving clinical outcomes. Aberrant deposition of extracellular matrix proteins, such as fibronectin (FN) and collagen, in the patients’ lungs, is a hallmark of this disease². Specifically, FN fibrillogenesis serves as a scaffold for deposition of other ECM proteins, making it an attractive target for detecting early fibrotic activity in IPF². Functional Upstream Domain (FUD) peptide is a 6-kDa peptide that binds to the N-terminal 70-kDa domain of FN, a critical site for the FN assembly³. In this study, we investigated the spatiotemporal localization of radiolabeled 20-kDa PEGylated FUD (PEG-FUD)⁴ in the bleomycin-induced murine model of pulmonary fibrosis, determining the capacity of radiolabeled PEG-FUD to target the area of active fibrosis in the fibrotic lungs.

Methods: PEG-FUD peptide was conjugated with p-SCN-Bn-NOTA and radiolabeled with ⁶⁴Cu (⁶⁴Cu-PEG-FUD). The radiolabeled 20-kDa PEGylated mutated control peptide (⁶⁴Cu-PEG-mFUD), which has a compromised binding affinity for FN, served as a control⁵. On day 0, mice were treated with bleomycin (1 U/kg) or vehicle (0.9% normal saline) intratracheally. Eleven days later, 200-300 μCi of ⁶⁴Cu-PEG-FUD or ⁶⁴Cu-PEG-mFUD was intravenously delivered to mice treated with either bleomycin or saline. The positron emission tomography/computed (PET/CT) imaging was acquired at 6 h and one-day post-injection of the probe. After the completion of in vivo imaging studies, the mice were euthanized and the major organs harvested for ex vivo biodistribution. The uptake of the probes was presented as percent injected activity per gram of tissue (%IA/g).

Results: The presence of lung injury 11 days after the bleomycin treatment was demonstrated by increased radiodensity on lung CT images. ⁶⁴Cu-PEG-FUD exhibited significantly higher targeting to the injured lung compared to controls, healthy mice treated with ⁶⁴Cu-PEG-FUD and injured mice treated with ⁶⁴Cu-PEG-mFUD. Both ⁶⁴Cu-PEG-FUD and ⁶⁴Cu-PEG-mFUD were primarily eliminated via kidney. The ex vivo biodistribution data corroborated in vivo imaging data, confirming the specific lung uptake of ⁶⁴Cu-PEG-FUD in the lungs of bleomycin-treated mice (P < 0.001, N=3 per group).

Conclusion: Here, we demonstrate that ⁶⁴Cu-PEG-FUD preferentially localizes to the injured mouse lungs 11 days after bleomycin treatment. Our results show initial evidence that FN-targeting radiolabeled PEG-FUD can be utilized to detect the fibrotic regions in the lung in a sensitive and non-invasive manner.

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2. Upagupta, C.; Shimbori, C.; Alsilmi, R.; Kolb, M., Matrix abnormalities in pulmonary fibrosis. European respiratory review: an official journal of the European Respiratory Society 2018, 27 (148).
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4. Zbyszynski, P.; Tomasini-Johansson, B. R.; Peters, D. M.; Kwon, G. S., Characterization of the PEGylated Functional Upstream Domain Peptide (PEG-FUD): a Potent Fibronectin Assembly Inhibitor with Potential as an Anti-Fibrotic Therapeutic. Pharmaceutical research 2018, 35 (7), 126.
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Acknowledgments: This work was supported, in part, by the University of Wisconsin – Madison, Carbone Cancer Center Support Grant (P30 CA014520), and the National Institutes of Health (R01 CA257837, R01 HL146402, and T32CA009206). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We would like to acknowledge the Small Animal Imaging and Radiotherapy Facility (SAIRF) and Cyclotron group at the University of Wisconsin – Madison for helping with the experiments. We would also like to thank Dr. Deane Mosher and Dr. Donna Peters for their scientific support.

Figure 1. A) Axial PET/CT images of normal saline or bleomycin-treated mouse lungs one-day post injection of 64Cu-PEG-FUD or 64Cu-PEG-mFUD. B) Ex vivo lung uptake data demonstrating the preferential uptake of 64Cu-PEG-FUD in bleomycin-treated mouse lungs (n=3 per group). ***: P< 0.001