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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.

Purpose Peptide receptor radionuclide therapy (PRRT) is commonly performed in the treatment of neuroendocrine tumors (NET), where somatostatin analogs (DOTATATE) are radiolabeled with 90 Y, 68 Ga or 111 In for pre-therapeutic and therapeutic purposes. Quantitative evaluation of the biokinetic data can be performed by using physiologically based pharmacokinetic (PBPK) models. Knowledge about the biodistribution in a pre-clinical setting would allow optimizing the translation from bench to bedside. The aim of this study was to develop a PBPK model to describe the biodistribution of a novel sst2-targeting radiotracer. Methods Biokinetic data of six mice after injection of 18 F-SiFAlin-Asp 3 -PEG 1 -TATE were investigated using two PBPK models. The PBPK models describe the biodistribution of the tracer in the tumor, kidneys, liver, remainder and whole body via blood flow to these organs via absorption, distribution, metabolism and excretion. A recently published sst2 PBPK model for humans (model 1) was used to describe the data. Physiological information in this model was adapted to that of a mouse. Model 1 was further modified by implementing receptor-mediated endocytosis (model 2). Model parameters were fitted to the biokinetic data of each mouse. Model selection was performed by calculating Akaike weights w i using the corrected Akaike Information Criterion (AICc). Results The implementation of receptor-mediated endocytosis considerably improved the description of the biodistribution (Akaike weights w 1 = 0% and w 2 = 100% for model 1 and 2, respectively). The resulting time-integrated activity coefficients determined by model 2 were for tumor (0.05 ± 0.02) h, kidneys (0.11 ± 0.01) h and liver (0.02 ± 0.01) h. Conclusion Simply downscaling a human PBPK model does not allow for an accurate description of 18 F-SiFAlin-Asp 3 -PEG 1 -TATE in mice. Biokinetics of this tracer can be accurately and adequately described using a physiologically based pharmacokinetic model including receptor-mediated endocytosis. Thus, an optimized translation from bench to bedside is possible.

Heterobivalent peptidic ligands (HBPLs), designed to address two different receptors independently, are highly promising tumor imaging agents. For example, breast cancer has been shown to concomitantly and complementarily overexpress the neuropeptide Y receptor subtype 1 (NPY(Y₁)R) as well as the gastrin-releasing peptide receptor (GRPR). Thus, radiolabeled HBPLs being able to bind these two receptors should exhibit an improved tumor targeting efficiency compared to monospecific ligands. We developed here such bispecific HBPLs and radiolabeled them with 68Ga, achieving high radiochemical yields, purities, and molar activities. We evaluated the HBPLs and their monospecific reference peptides in vitro regarding stability and uptake into different breast cancer cell lines and found that the 68Ga-HBPLs were efficiently taken up via the GRPR. We also performed in vivo PET/CT imaging and ex vivo biodistribution studies in T-47D tumor-bearing mice for the most promising 68Ga-HBPL and compared the results to those obtained for its scrambled analogs. The tumors could easily be visualized by the newly developed 68Ga-HBPL and considerably higher tumor uptakes and tumor-to-background ratios were obtained compared to the scrambled analogs in and ex vivo. These results demonstrate the general feasibility of the approach to use bispecific radioligands for in vivo imaging of breast cancer.

The Silicon-Fluoride-Acceptor (SiFA)-(18)F-labeling strategy has been shown before to enable the straightforward and efficient (18)F-labeling of complex biologically active substances such as proteins and peptides. Especially in the case of peptides, the radiolabeling proceeds kit-like in short reaction times and without the need of complex product workup. SiFA-derivatized, (18)F-labeled Tyr(3)-octreotate (TATE) derivatives demonstrated, besides strong somatostatin receptor (SSTR) binding, favorable in vivo pharmacokinetics as well as excellent tumor visualization by PET imaging. In this study, we intended to determine the influence of the underlying molecular design and used molecular scaffolds of SiFAlin-TATE derivatives on SSTR binding as well as on the in vivo pharmacokinetics of the resulting (18)F-labeled peptides. For this purpose, new SiFAlin-(Asp)n-PEG1-TATE analogs (where n = 1-4) were synthesized, efficiently radiolabeled with (18)F in a kit-like manner and obtained in radiochemical yields of 70-80%, radiochemical purities of ≥97%, and nonoptimized specific activities of 20.1-45.2 GBq/μmol within 20-25 min starting from 0.7-1.5 GBq of (18)F. In the following, the radiotracer's lipophilicities and stabilities in human serum were determined. Furthermore, the SSTR-specific binding affinities were evaluated by a competitive displacement assay on SSTR-positive AR42J cells. The obtained in vitro results support the assumption that aspartic acids are able to considerably increase the radiotracer's hydrophilicity and that their number does not affect the SSTR binding potential of the TATE derivatives. The most promising tracer (18)F-SiFAlin-Asp3-PEG1-TATE [(18)F]6 (LogD = -1.23 ± 0.03, IC50 = 20.7 ± 2.5 nM) was further evaluated in vivo in AR42J tumor-bearing nude mice via PET/CT imaging against the clinical gold standard (68)Ga-DOTATATE as well as the previously developed SiFAlin-TATE derivative [(18)F]3. The results of these evaluations showed that [(18)F]6-although showing very similar chemical and in vitro properties to [(18)F]3-exhibits not only a slowed renal clearance compared to [(18)F]3, but also a higher absolute tumor uptake compared to (68)Ga-DOTATATE, and furthermore enables excellent tumor visualization with high image resolution. These results emphasize the importance of systematic study of the influence of molecular design and applied structure elements of peptidic radiotracers, as these may considerably influence in vivo pharmacokinetics while not affecting other parameters such as radiochemistry, lipophilicity, serum stability, or receptor binding potential.

Peptidic radiotracers are highly potent substances for the specific in vivo imaging of various biological targets with Single Photon Emission Computed Tomography and Positron Emission Tomography. However, some radiolabeled peptides such as bombesin analogs were shown to exhibit only a limited stability, hampering a successful target visualization. One option to positively influence the stability of radiolabeled peptides is the introduction of certain artificial molecular scaffolds. In order to comparatively assess the influence of different structure elements on the stability of radiolabeled peptides and to identify those structure elements being most useful for peptide radiotracer stabilization, several monomeric and dimeric bombesin derivatives were synthesized, exhibiting differing molecular designs and the chelator NODAGA for (68) Ga-labeling. The radiolabeled peptides were evaluated regarding their in vitro stability in human serum to determine the influence of the introduced molecular scaffolds on the peptides' serum stabilities. The results of the evaluations showed that the introduction of scaffold structures and the overall molecular design have a substantial impact on the stabilities of the resulting peptidic radiotracers. But besides some general trends found using certain scaffold structures, the obtained results point to the necessity to empirically assess their influence on stability for each susceptible peptidic radiotracer individually.

As the gastrin releasing peptide receptor (GRPR) is overexpressed on several tumor types, it represents a promising target for the specific in vivo imaging of these tumors using positron emission tomography (PET). We were able to show that PESIN-based peptide multimers can result in substantially higher GRPR avidities, highly advantageous in vivo pharmacokinetics and tumor imaging properties compared to the respective monomers. However, the minimal distance between the peptidic binders, resulting in the lowest possible system entropy while enabling a concomitant GRPR binding and thus optimized receptor avidities, has not been determined so far. Thus, we aimed here to identify the minimal distance between two GRPR-binding peptides in order to provide the basis for the development of highly avid GRPR-specific PET imaging agents. We therefore synthesized dimers of the GRPR-binding bombesin analogue BBN(7-14) on a dendritic scaffold, exhibiting different distances between both peptide binders. The homodimers were further modified with the chelator NODAGA, radiolabeled with (68)Ga, and evaluated in vitro regarding their GRPR avidity. We found that the most potent of the newly developed radioligands exhibits GRPR avidity twice as high as the most potent reference compound known so far, and that a minimal distance of 62 bond lengths between both peptidic binders within the homodimer can result in concomitant peptide binding and optimal GRPR avidities. These findings answer the question as to what molecular design should be chosen when aiming at the development of highly avid homobivalent peptidic ligands addressing the GRPR.

Peptidic radiotracers are highly potent substances for the specific in vivo imaging of various biological targets with Single Photon Emission Computed Tomography and Positron Emission Tomography. However, some radiolabeled peptides such as bombesin analogs were shown to exhibit only a limited stability, hampering a successful target visualization. One option to positively influence the stability of radiolabeled peptides is the introduction of certain artificial molecular scaffolds. In order to comparatively assess the influence of different structure elements on the stability of radiolabeled peptides and to identify those structure elements being most useful for peptide radiotracer stabilization, several monomeric and dimeric bombesin derivatives were synthesized, exhibiting differing molecular designs and the chelator NODAGA for (68) Ga-labeling. The radiolabeled peptides were evaluated regarding their in vitro stability in human serum to determine the influence of the introduced molecular scaffolds on the peptides' serum stabilities. The results of the evaluations showed that the introduction of scaffold structures and the overall molecular design have a substantial impact on the stabilities of the resulting peptidic radiotracers. But besides some general trends found using certain scaffold structures, the obtained results point to the necessity to empirically assess their influence on stability for each susceptible peptidic radiotracer individually.

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 β3 -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.