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The synthesis of 64Cu-chelated porphyrin photosensitizers and their tumor-targeting peptide conjugates for the evaluation of target cell uptake and PET image-based pharmacokinetics of targeted photodynamic therapy agents

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Abstract

Objective

Targeted photodynamic therapy (PDT) is necessary for preventing the side effects associated with PDT, such as photosensitivity caused by the distribution of photosensitizers into normal tissues. In the development of targeted PDT agents, a simple evaluation system of in vivo pharmacokinetics, as well as target cell uptake, is absolutely imperative. We hypothesized that 64Cu chelation with porphyrin photosensitizer-biomacromolecule conjugates may become a simple and versatile labeling strategy for this purpose.

Methods

Protoporphyrin IX (PPIX) and a bombesin (BBN) analog, that interacts with the gastrin-released peptide (GRP) receptor, were used as a photosensitizer and tumor-targeting peptide, respectively. Then, a conjugate of PPIX and BBN analog linked via short polyethylene glycol (PPIX-PEG6-BBN analog) was synthesized and used as a targeted PDT agent. In addition, a 64Cu-chelated PPIX-PEG6-BBN analog was synthesized under optimized reaction conditions. Lastly, cell uptake study and PET image-based pharmacokinetic analyses of the PPIX-PEG6-BBN analog were carried out in a human prostate cancer cell line, PC-3, which highly expresses the GRP receptor, and PC-3 tumor-bearing mice.

Results

It was confirmed that degradation (thought to be based on radiolysis) occurs, and large amounts of 64Cu-labeling compounds are wasted in the reaction mixture. Interestingly, the addition of ethanol into the reaction mixture provides an effective solution for this problem. As for cell uptake study, the [64Cu]PPIX-PEG6-BBN analog demonstrated significantly higher uptake for PC-3 cells than [64Cu]PPIX and, in addition, the uptake of [64Cu]PPIX-PEG6-BBN analog was significantly inhibited by adding excess cold BBN analog peptide. PET image-based pharmacokinetic evaluation revealed that [64Cu]PPIX-PEG6-BBN analog and [64Cu]PPIX rapidly accumulate into the liver and kidney, circulate in blood for a long time compared with normal peptides, and distribute at a low level in the tumor. This result suggested that in vivo biodistribution of PPIX-PEG6-BBN analog is mainly dependent on the lipophilicity of PPIX. Ex vivo measurements of radioactivity distribution after PET studies showed that although there was no remarkable difference in the tumor/skin ratio of radioactivity between [64Cu]PPIX-PEG6-BBN analog and [64Cu]PPIX, the pancreas (an organ that also expresses GRP receptors)/skin ratio was significantly higher in the case of [64Cu]PPIX-PEG6-BBN analog.

Conclusion

We report on the successful synthesis of 64Cu-chelated porphyrin photosensitizers and their tumor-targeting peptide conjugates under conditions in which radiolysis is suppressed. This labeling strategy with porphyrin photosensitizers may be of value for the rapid development of ideal targeted PDT agents.

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Acknowledgments

This work was supported, in part, by a consignment expense for the Molecular Imaging Program entitled “Research Base for Exploring New Drugs” from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. We would like to thank Mr. M. Kurahashi (Sumitomo Heavy Industry Accelerator Service Ltd.) for operating the cyclotron, Dr. K. Takahashi, Dr. Y. Kanayama and Ms. R. Zochi (RIKEN) for supporting 64Cu production, Ms. E. Hayashinaka (RIKEN) for supporting the reconstruction of PET images, and the RIKEN Biomolecular Characterization Team for supporting the MALDI-TOF–MS analysis. The authors declare that they have no financial interests in this study.

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  1. Molecular Probe Dynamics Laboratory, Center for Molecular Imaging Science (CMIS), RIKEN, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan

    Hidefumi Mukai, Yasuhiro Wada & Yasuyoshi Watanabe

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  1. Hidefumi Mukai
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  2. Yasuhiro Wada
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Correspondence to Yasuyoshi Watanabe.

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Mukai, H., Wada, Y. & Watanabe, Y. The synthesis of 64Cu-chelated porphyrin photosensitizers and their tumor-targeting peptide conjugates for the evaluation of target cell uptake and PET image-based pharmacokinetics of targeted photodynamic therapy agents. Ann Nucl Med 27, 625–639 (2013). https://doi.org/10.1007/s12149-013-0728-2

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