{"title":"Application of copper (I) selective ligands for PET imaging of reactive oxygen species through metabolic trapping","authors":"Tetsuro Tada , Yuki Mizuno , Yuki Shibata , Hironobu Yasui , Yuji Kuge","doi":"10.1016/j.nucmedbio.2024.108914","DOIUrl":null,"url":null,"abstract":"<div><h3>Introduction</h3><p>Reactive oxygen species (ROS) are attractive targets for clinical PET imaging. In this study, we hypothesized that PET imaging of ROS would be possible by using chelating ligands (L) that form stable complexes with copper (I) but not with copper (II), based on metabolic trapping. Namely, when [<sup>64</sup>Cu][Cu<sup>I</sup>(L)<sub>2</sub>]<sup>+</sup> is oxidized by ROS, the oxidized complex will release [<sup>64</sup>Cu]Cu<sup>2+</sup>. Then, the released [<sup>64</sup>Cu]Cu<sup>2+</sup> will be trapped inside the cell, resulting in PET signal depending on the redox potential of ROS. To examine the potential of this novel molecular design for ROS imaging, we synthesized copper (I) complexes with bicinchoninic acid (BCA) disodium salt and bathocuproinedisulfonic acid (BCS) disodium salt and evaluated their reactivity with several kinds of ROS. In addition, the cellular uptake of [<sup>64</sup>Cu][Cu<sup>I</sup>(BCS)<sub>2</sub>]<sup>3−</sup> and the stability of [<sup>64</sup>Cu][Cu<sup>I</sup>(BCS)<sub>2</sub>]<sup>3−</sup> in a biological condition were also evaluated.</p></div><div><h3>Methods</h3><p>[<sup>64</sup>Cu]Cu<sup>2+</sup> was reduced to [<sup>64</sup>Cu]Cu<sup>+</sup> by ascorbic acid and coordinated with BCA and BCS in the acetate buffer to synthesize [<sup>64</sup>Cu][Cu<sup>I</sup>(BCA)<sub>2</sub>]<sup>3−</sup> and [<sup>64</sup>Cu][Cu<sup>I</sup>(BCS)<sub>2</sub>]<sup>3−</sup>. The radiochemical yields were determined by thin-layer chromatography (TLC). After [<sup>64</sup>Cu][Cu<sup>I</sup>(BCS)<sub>2</sub>]<sup>3−</sup> was incubated with hydroxyl radical, lipid peroxide, superoxide, and hydrogen peroxide, the percentage of released [<sup>64</sup>Cu]Cu<sup>2+</sup> from the parent complex was evaluated by TLC. HT-1080 human fibrosarcoma cells were treated with 0.1 % Dimethyl sulfoxide (control), imidazole ketone erastin (IKE), or IKE + ferrostatin-1 (Fer-1). Then, the uptake of [<sup>64</sup>Cu][Cu<sup>I</sup>(BCS)<sub>2</sub>]<sup>3−</sup> to HT-1080 cells in each group was evaluated as %Dose/mg protein. Lastly, [<sup>64</sup>Cu][Cu<sup>I</sup>(BCS)<sub>2</sub>]<sup>3−</sup> was incubated in human plasma, and its intact ratio was determined by TLC.</p></div><div><h3>Results</h3><p>The radiochemical yield of [<sup>64</sup>Cu][Cu<sup>I</sup>(BCS)<sub>2</sub>]<sup>3−</sup> (86 ± 1 %) was higher than that of [<sup>64</sup>Cu][Cu<sup>I</sup>(BCA)<sub>2</sub>]<sup>3−</sup> (44 ± 3 %). [<sup>64</sup>Cu][Cu<sup>I</sup>(BCA)<sub>2</sub>]<sup>3−</sup> was unstable and partially decomposed on TLC. After [<sup>64</sup>Cu][Cu<sup>I</sup>(BCS)<sub>2</sub>]<sup>3−</sup> was reacted with hydroxyl radical, lipid peroxide, and superoxide, 67 ± 2 %, 44 ± 13 %, and 22 ± 3 % of total radioactivity was detected as [<sup>64</sup>Cu]Cu<sup>2+</sup>, respectively. On the other hand, the reaction with hydrogen peroxide did not significantly increase the ratio of [<sup>64</sup>Cu]Cu<sup>2+</sup> (4 ± 1 %). These results suggest that [<sup>64</sup>Cu][Cu<sup>I</sup>(BCS)<sub>2</sub>]<sup>3−</sup> could be used for detecting high-redox-potential ROS such as hydroxyl radical and lipid peroxide with high selectivity. The cellular uptake values of [<sup>64</sup>Cu][Cu<sup>I</sup>(BCS)<sub>2</sub>]<sup>3−</sup> in the control, IKE, and Fer-1 group were 42 ± 2, 54 ± 2, and 47 ± 5 %Dose/mg protein (<em>n</em> = 3), respectively, suggesting the ROS specific uptake of [<sup>64</sup>Cu][Cu<sup>I</sup>(BCS)<sub>2</sub>]<sup>3−</sup>. On the other hand, the intact ratio after the incubation of [<sup>64</sup>Cu][Cu<sup>I</sup>(BCS)<sub>2</sub>]<sup>3−</sup> in human plasma was 9 ± 5 %.</p></div><div><h3>Conclusion</h3><p>PET imaging of ROS would be possible by using a copper (I) selective ligand, based on metabolic trapping. Although improvement of the membrane permeability and the stability of copper (I) complexes is required, the present results pave the way for the development of novel <sup>64</sup>Cu-labeled complexes for PET imaging of ROS.</p></div>","PeriodicalId":19363,"journal":{"name":"Nuclear medicine and biology","volume":"134 ","pages":"Article 108914"},"PeriodicalIF":3.6000,"publicationDate":"2024-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Nuclear medicine and biology","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0969805124000404","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"RADIOLOGY, NUCLEAR MEDICINE & MEDICAL IMAGING","Score":null,"Total":0}
引用次数: 0
Abstract
Introduction
Reactive oxygen species (ROS) are attractive targets for clinical PET imaging. In this study, we hypothesized that PET imaging of ROS would be possible by using chelating ligands (L) that form stable complexes with copper (I) but not with copper (II), based on metabolic trapping. Namely, when [64Cu][CuI(L)2]+ is oxidized by ROS, the oxidized complex will release [64Cu]Cu2+. Then, the released [64Cu]Cu2+ will be trapped inside the cell, resulting in PET signal depending on the redox potential of ROS. To examine the potential of this novel molecular design for ROS imaging, we synthesized copper (I) complexes with bicinchoninic acid (BCA) disodium salt and bathocuproinedisulfonic acid (BCS) disodium salt and evaluated their reactivity with several kinds of ROS. In addition, the cellular uptake of [64Cu][CuI(BCS)2]3− and the stability of [64Cu][CuI(BCS)2]3− in a biological condition were also evaluated.
Methods
[64Cu]Cu2+ was reduced to [64Cu]Cu+ by ascorbic acid and coordinated with BCA and BCS in the acetate buffer to synthesize [64Cu][CuI(BCA)2]3− and [64Cu][CuI(BCS)2]3−. The radiochemical yields were determined by thin-layer chromatography (TLC). After [64Cu][CuI(BCS)2]3− was incubated with hydroxyl radical, lipid peroxide, superoxide, and hydrogen peroxide, the percentage of released [64Cu]Cu2+ from the parent complex was evaluated by TLC. HT-1080 human fibrosarcoma cells were treated with 0.1 % Dimethyl sulfoxide (control), imidazole ketone erastin (IKE), or IKE + ferrostatin-1 (Fer-1). Then, the uptake of [64Cu][CuI(BCS)2]3− to HT-1080 cells in each group was evaluated as %Dose/mg protein. Lastly, [64Cu][CuI(BCS)2]3− was incubated in human plasma, and its intact ratio was determined by TLC.
Results
The radiochemical yield of [64Cu][CuI(BCS)2]3− (86 ± 1 %) was higher than that of [64Cu][CuI(BCA)2]3− (44 ± 3 %). [64Cu][CuI(BCA)2]3− was unstable and partially decomposed on TLC. After [64Cu][CuI(BCS)2]3− was reacted with hydroxyl radical, lipid peroxide, and superoxide, 67 ± 2 %, 44 ± 13 %, and 22 ± 3 % of total radioactivity was detected as [64Cu]Cu2+, respectively. On the other hand, the reaction with hydrogen peroxide did not significantly increase the ratio of [64Cu]Cu2+ (4 ± 1 %). These results suggest that [64Cu][CuI(BCS)2]3− could be used for detecting high-redox-potential ROS such as hydroxyl radical and lipid peroxide with high selectivity. The cellular uptake values of [64Cu][CuI(BCS)2]3− in the control, IKE, and Fer-1 group were 42 ± 2, 54 ± 2, and 47 ± 5 %Dose/mg protein (n = 3), respectively, suggesting the ROS specific uptake of [64Cu][CuI(BCS)2]3−. On the other hand, the intact ratio after the incubation of [64Cu][CuI(BCS)2]3− in human plasma was 9 ± 5 %.
Conclusion
PET imaging of ROS would be possible by using a copper (I) selective ligand, based on metabolic trapping. Although improvement of the membrane permeability and the stability of copper (I) complexes is required, the present results pave the way for the development of novel 64Cu-labeled complexes for PET imaging of ROS.
期刊介绍：
Nuclear Medicine and Biology publishes original research addressing all aspects of radiopharmaceutical science: synthesis, in vitro and ex vivo studies, in vivo biodistribution by dissection or imaging, radiopharmacology, radiopharmacy, and translational clinical studies of new targeted radiotracers. The importance of the target to an unmet clinical need should be the first consideration. If the synthesis of a new radiopharmaceutical is submitted without in vitro or in vivo data, then the uniqueness of the chemistry must be emphasized.
These multidisciplinary studies should validate the mechanism of localization whether the probe is based on binding to a receptor, enzyme, tumor antigen, or another well-defined target. The studies should be aimed at evaluating how the chemical and radiopharmaceutical properties affect pharmacokinetics, pharmacodynamics, or therapeutic efficacy. Ideally, the study would address the sensitivity of the probe to changes in disease or treatment, although studies validating mechanism alone are acceptable. Radiopharmacy practice, addressing the issues of preparation, automation, quality control, dispensing, and regulations applicable to qualification and administration of radiopharmaceuticals to humans, is an important aspect of the developmental process, but only if the study has a significant impact on the field.
Contributions on the subject of therapeutic radiopharmaceuticals also are appropriate provided that the specificity of labeled compound localization and therapeutic effect have been addressed.
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