WMIC 2015: Biconjugate Chemistry Award Winners

Targeted imaging of GRP receptor-expressing prostate cancer with 68Ga/IRdye-650 conjugated bombesin antagonist

Hanwen Zhang from Memorial Sloan Kettering Cancer Center

Objectives: Gastrin releasing peptide receptors (GRPr) are frequently overexpressed by human prostate cancer cells, and radiolabeled GRPr-affinity ligands have shown significant promise for in vivo imaging of prostate cancer with PET. The goal of this study was to develop a dual-modality imaging probe that can be used for pre-operative PET imaging and intra-operative optical imaging of prostate cancer.
Methods: We designed and synthesized an IRDye-650 and DOTA conjugated GRPr antagonist, HZ220 (DOTA-Lys(IRDye650)-PEG4-[D-Phe6, Sta13]-BN(6-14)NH2), and studied its uptake at prostate cancer cells in vitro and in vivo. The control GRPr antagonists, DOTA-AR06 (DOTA-PEG4-[D-Phe6, Sta13]-BN(6-14)NH2) and HZ219 (DOTA-Lys-PEG4-[D-Phe6, Sta13]-BN(6-14)NH2) were also synthesized on solid phase by using Fmoc-strategy. 67Ga/68Ga/natGa-labeled ligands were characterized with HPLC or LC-MS. Receptor specific binding of these ligands was compared for prostate cancer cells (PC-3) and tumor uptake in vivo was imaged with PET/CT (Inveon PET/CT) and fluorescence imaging (IVIS spectrum).
Results: HZ220, DOTA-AR06 and HZ219 were synthesized with yield of 20-40%. After HLB cartridge purification, the 68Ga-labeling yields (decay-corrected) for HZ220 and DOTA-AR06 were 56±8% and 84±1%, respectively. Their radiochemical purities were greater than 95%. Ga-HZ220 displayed a lower affinity to GRPr (IC50 value: 21±8 nM) than Ga-DOTA-AR06 or Ga-HZ219 (0.48±0.18 or 0.69±0.18 nM). However, in vivo 68Ga-HZ220 displayed a similar in vivo tumor accumulation as 68Ga-DOTA-AR06 (4.5±0.8 vs 4.1±0.4 %ID/cc at 1 h p.i.); 2/3 of the tumor uptake could be blocked with an excess amount of HZ220 or Ga-DOTA-AR06. IVIS spectrum imaging also visualized PC-3 xenografts in vivo and ex vivo. In addition, both 68Ga-HZ220 and 68Ga-DOTA-AR06 displayed a similar accumulation in all other non-target organs, except for a significantly higher kidney uptake of 68Ga-HZ220 (7.1±1.1 vs 2.3±0.6 %ID/g).
Conclusion: 68Ga-HZ220 is a promising bimodal ligand for non-invasive preoperative PET imaging and intraoperative fluorescent imaging of GRPr-expressing malignancies. Thus, 68Ga-HZ220 PET/fluorescent imaging may improve cancer staging and guide surgical resections.
Acknowledgements: Supported by NIH grant P50-CA84638. MSKCC Small Animal Imaging Core Facility was supported by NIH Small-Animal Imaging Research Program grant R24 CA83084 and NIH Center grant P30 CA08748).


Synthesis and evaluation of Förster Resonance Energy Transfer (FRET) probes detecting changes in cellular redox state

Kaolina Jankowska from The University of Sydney

Oxidative stress is causally linked to many diseases such as obesity [1], cancer [2] and neurodegeneration. [3] It is therefore pertinent to monitor the oxidative capacity of human tissues on cellular and subcellular levels. The only modality able to provide such spatiotemporal resolution is optical imaging enhanced by the use of fluorescent probes.
An ideal optical contrast agent should address certain criteria, including rapid localisation within cells, reversibility and responsiveness to biological changes. Our group has developed FRET agents that, unlike existing probes, satisfy these requirements. These agents contain two fluorophores chemically linked via carbon chains: one containing a redox-sensitive group, and the other a FRET donor or acceptor.
Using combinations of flavin, a widely used redox sensor, with a range of fluorophores, we have synthesised some effective redox probes. The first generation of compounds contained coumarin as a FRET donor. Fluorimetry studies have shown an emission shift to a longer wavelength in increasingly oxidative conditions, while the emission peak of coumarin is unchanged in reducing conditions. Biological studies performed in HeLa cells using confocal microscopy, fluorescence lifetime imaging microscopy and flow cytometry confirm utility of our agents. More recently developed agents employ rhodamine as a FRET acceptor and targeting vector simultaneously.

[1] Rupérez, A. I.; Gil, A.; Aguilera, C. M. Int. J. Mol. Sci. 2014, 15, 3118.
[2] Wang, X.; Wang, W.; Li, L.; Perry, G.; Lee, H.; Zhu, X. Biochim. Biophys. Acta 2014, 1842, 1240.
[3] Sosa, V.; Moliné, T.; Somoza, R.; Paciucci, R.; Kondoh, H.; LLeonart, M. E. Ageing Res. Rev. 2013, 12, 376.\n

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