iEDDA Conjugation Reaction in Radiometal Labeling of Peptides with 68Ga and 64Cu: Unexpected Findings.

The inverse electron demand Diels-Alder conjugation reaction has gained increasing importance over the past few years for efficient in vivo and ex vivo radiometal labeling of antibodies. However, the application of this very fast reaction type has not been studied for radiolabeling of peptides so far. We show here the synthesis of 3-benzyl-1,2,4,5-tetrazine-comprising ((1,4,7,10-tetraazacyclododecane-4,7,10-triyl)triacetic acid-1-glutaric acid) (DOTA-GA) and ((1,4,7-triazacyclononane-4,7-diyl)diacetic acid-1-glutaric acid) (NODA-GA) chelators and their radiometal labeling with 68Ga3+ and 64Cu2+. The secondary labeling precursors 68Ga-DOTA-GA-Tz, 68Ga-NODA-GA-Tz, and 64Cu-DOTA-GA-Tz were obtained in high radiochemical yields (RCYs) and purities as well as molar activities for further labeling of trans-cyclooctene (TCO)-modified peptides. However, the following reactions of the radiometal-labeled tetrazines with different TCO-comprising model peptide analogs unexpectedly resulted in the formation of a considerable amount of side products (20-55%) which limits the overall achievable RCYs and purities as well as molar activities of the target radiopeptides. Under otherwise identical, nonradioactive reaction conditions, this effect could however not be observed. In contrast, the corresponding one-step radiolabeling protocols provided the target 68Ga-labeled radiopeptides in exceptionally high RCYs and purities of ≥99% and molar activities of 68-72 GBq/µmol starting from activities of 340-358 MBq of 68Ga. Thus, the usefulness of the two-step labeling of TCO-modified peptides with radiometal-labeled chelator-tetrazines seems to be limited.

Synthesis, in vitro and in vivo evaluation of 18F-fluoronorimatinib as radiotracer for Imatinib-sensitive gastrointestinal stromal tumors.

INTRODUCTION: Gastrointestinal stromal tumors (GIST) have a wide range of mutations, but can mostly be treated with Imatinib, until eventually resistance towards this tyrosine kinase inhibitor is acquired. Early and non-invasive determination of the sensitivity of the tumor and its metastases towards Imatinib by positron emission tomography (PET) would be beneficial for therapy planning and monitoring. METHODS: We developed a synthesis strategy towards the precursor molecule, performed the 18F-synthesis and in the following evaluated the radioligand in vitro regarding its lipophilicity, stability and biological activity (KIT binding properties) as well as its in vivo properties in GIST tumor-bearing mice. RESULTS: [18F]fluoronorimatinib could be obtained in an overall radiochemical yield of 22.2±3.3% within 90min. The radioligand showed high GIST cell uptake and was able to distinguish between Imatinib-sensitive and resistant tumor cell lines (GIST-T1, GIST882, GIST430) in vitro. Further biological evaluations of the ligand towards 9 different GIST-relevant KIT mutations showed comparable binding affinities compared to the structural lead Norimatinib (65nM vs. 53nM for wt-KIT). The in vivo evaluation of the newly developed radioligand showed tumor-to-background-ratios comparable to previously described, similar radiotracers. CONCLUSIONS: Thus, [18F]fluoronorimatinib is able to distinguish between Imatinib-resistant and sensitive KIT mutations. Although no improvement of in vivo tumor-to-background ratios could be achieved compared to formerly described radioligands, the hepatic uptake could be considerably reduced, being advantageous for the imaging of GIST. Advances in knowledge and implications for patient care: We were able to show that it is possible to significantly reduce the unfavorably high hepatic uptake of small-molecule radioligands applicable for GIST PET imaging. This work can thus be the basis for further work intending to develop a PET-radioligand for Imatinib-dependent GIST imaging.

Rational Design, Development, and Stability Assessment of a Macrocyclic Four-Hydroxamate-Bearing Bifunctional Chelating Agent for 89 Zr.

Zirconium-89 is a positron-emitting radionuclide of high interest for medical imaging applications with positron emission tomography (PET). For the introduction of this radiometal into biologically active targeting vectors, the chelating agent desferrioxamine B (DFO) is commonly applied. However, DFO is known to form 89 Zr complexes of limited in vivo stability. Herein we describe the rational design and chemical development of a new macrocyclic four-hydroxamate-bearing chelating agent-1,10,19,28-tetrahydroxy-1,5,10,14,19,23,28,32-octaazacyclohexatriacontan-2,6,11,15,20,24,29,33-octaone (CTH36)-for the stable complexation of Zr4+ . For this purpose, we first performed computational studies to determine the optimal chelator geometry before we developed different synthesis pathways toward the target structures. The best results were obtained using an efficient solution-phase-based synthesis strategy toward the target chelating agent. To enable efficient and chemoselective conjugation to biomolecules, a tetrazine-modified variant of CTH36 was also developed. The excellent conjugation characteristics of the so-functionalized chelator were demonstrated on the example of the model peptide TCO-c(RGDfK). We determined the optimal 89 Zr radiolabeling parameters for CTH36 as well as its bioconjugate, and found that 89 Zr radiolabeling proceeds efficiently under very mild reaction conditions. Finally, we performed comparative complex stability tests for 89 Zr-CHT36-c(RGDfK) and 89 Zr-DFO-c(RGDfK), showing improved complex stability for the newly developed chelator CTH36.

Comparative Assessment of Complex Stabilities of Radiocopper Chelating Agents by a Combination of Complex Challenge and in vivo Experiments.

For (64) Cu radiolabeling of biomolecules to be used as in vivo positron emission tomography (PET) imaging agents, various chelators are commonly applied. It has not yet been determined which of the most potent chelators--NODA-GA ((1,4,7-triazacyclononane-4,7-diyl)diacetic acid-1-glutaric acid), CB-TE2A (2,2'-(1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diyl)diacetic acid), or CB-TE1A-GA (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diyl-8-acetic acid-1-glutaric acid)--forms the most stable complexes resulting in PET images of highest quality. We determined the (64) Cu complex stabilities for these three chelators by a combination of complex challenge and an in vivo approach. For this purpose, bioconjugates of the chelating agents with the gastrin-releasing peptide receptor (GRPR)-affine peptide PESIN and an integrin αv ß3 -affine c(RGDfC) tetramer were synthesized and radiolabeled with (64) Cu in excellent yields and specific activities. The (64) Cu-labeled biomolecules were evaluated for their complex stabilities in vitro by conducting a challenge experiment with the respective other chelators as challengers. The in vivo stabilities of the complexes were also determined, showing the highest stability for the (64) Cu-CB-TE1A-GA complex in both experimental setups. Therefore, CB-TE1A-GA is the most appropriate chelating agent for *Cu-labeled radiotracers and in vivo imaging applications.

Bimodal imaging probes for combined PET and OI: recent developments and future directions for hybrid agent development.

Molecular imaging--and especially positron emission tomography (PET)--has gained increasing importance for diagnosis of various diseases and thus experiences an increasing dissemination. Therefore, there is also a growing demand for highly affine PET tracers specifically accumulating and visualizing target structures in the human body. Beyond the development of agents suitable for PET alone, recent tendencies aim at the synthesis of bimodal imaging probes applicable in PET as well as optical imaging (OI), as this combination of modalities can provide clinical advantages. PET, due to the high tissue penetration of the γ-radiation emitted by PET nuclides, allows a quantitative imaging able to identify and visualize tumors and metastases in the whole body. OI on the contrary visualizes photons exhibiting only a limited tissue penetration but enables the identification of tumor margins and infected lymph nodes during surgery without bearing a radiation burden for the surgeon. Thus, there is an emerging interest in bimodal agents for PET and OI in order to exploit the potential of both imaging techniques for the imaging and treatment of tumor diseases. This short review summarizes the available hybrid probes developed for dual PET and OI and discusses future directions for hybrid agent development.

(89)Zr, a radiometal nuclide with high potential for molecular imaging with PET: chemistry, applications and remaining challenges.

Molecular imaging-and especially Positron Emission Tomography (PET)-is of increasing importance for the diagnosis of various diseases and thus is experiencing increasing dissemination. Consequently, there is a growing demand for appropriate PET tracers which allow for a specific accumulation in the target structure as well as its visualization and exhibit decay characteristics matching their in vivo pharmacokinetics. To meet this demand, the development of new targeting vectors as well as the use of uncommon radionuclides becomes increasingly important. Uncommon nuclides in this regard enable the utilization of various selectively accumulating bioactive molecules such as peptides, antibodies, their fragments, other proteins and artificial structures for PET imaging in personalized medicine. Among these radionuclides, 89Zr (t1/2 = 3.27 days and mean Eß+ = 0.389 MeV) has attracted increasing attention within the last years due to its favorably long half-life, which enables imaging at late time-points, being especially favorable in case of slowly-accumulating targeting vectors. This review outlines the recent developments in the field of 89Zr-labeled bioactive molecules, their potential and application in PET imaging and beyond, as well as remaining challenges.