Background

Anthracyclines are widely used in cancer treatments but can cause sublethal cardiotoxicity manifesting in heart failure. Doxorubicin provokes lasting damage to cardiomyocytes with resulting impaired contractile machinery and mitochondrial function. BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) is a multi-effector protein that has been shown to play a critical role in doxorubicin-induced mitochondrial damage. A BNIP3 antagonistic peptide designed by our group preserved human donor cardiomyocytes from doxorubicin-induced calcium-overload. For a successful in vivo transition, reliable information on biodistribution of BNIP3 antagonistic peptide is required. We therefore aimed to design a [⁶⁸Ga] peptide conjugate PET tracer to examine stability, pharmacokinetics, and cardiac tissue availability of the BNIP3 antagonistic peptide after in vivo application.  

Methods

For tracer synthesis, the c-terminus of the BNIP3 antagonistic peptide was conjugated to NODAGA. Following [⁶⁸Ga] chelation, its radiochemical purity, in vitro stability and human ex vivo plasm stability was assessed using radio-HPLC. In vivo tissue distribution in main organs heart, liver and kidney as well as saturation experiments were performed in healthy C57BL/6J mice using dynamic and static small-animal positron emission tomography (PET)/computed tomography (CT) imaging (Molecubes b-Cube and X-Cube) and gamma counter measurements.

Results

Radiochemical purity of the [⁶⁸Ga]-NODAGA peptide conjugate was >99%. Human ex vivo plasm stability slightly decreased to 95.8 ± 4.6% after 60 min. Incubation with proteinase K and whole heart muscle tissue lysate proofed degradability of the [⁶⁸Ga]-NODAGA peptide conjugate. Initial uptake after i.v. administration was highest in liver (21.1 ± 3.2%), kidneys (18.75 ± 4.3%) and followed by heart (8.6 ± 4.6%) one min after application. Renal uptake slightly increased over time. Saturation with unlabeled NODAGA peptide conjugate resulted in decreased uptake in all organs examined but increased in the liver. Gamma counter measurements confirmed PET-based assessment.

Conclusion & outlook

This study shows that BNIP3 antagonistic peptide reaches the cardiac target tissue in vivo. Radiolabeling of NODAGA peptide conjugate can be successfully used for analysis of in vivo biodistribution and tissue availability. The labeling was reproducible and stable. In vivo application demonstrated specific uptake in various organs including the heart in healthy animals. The kidney uptake indicated renal excretion. Our promising results facilitate immediate evaluation in an animal model of doxorubicin-induced cardiotoxicity to analyze biodistribution, target tissue availability and bio metabolization in a pathological model and to prepare for further possible clinical translation.