Design, Synthesis, In Vitro, and Initial In Vivo Evaluation of Heterobivalent Peptidic Ligands Targeting Both NPY(Y1)- and GRP-Receptors-An Improvement for Breast Cancer Imaging?

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.

Effects of Visible-Light Irradiation of Protoporphyrin†IX on the Self-Assembly of Tubulin Heterodimers.

The formation and the effects of laser irradiation of the complex formed by protoporphyrin IX (PPIX) and tubulin was investigated. We have used tubulin as a model protein to investigate whether docked photoactive ligands can affect the structure and function of polypeptides upon exposure to visible light. We observed that laser irradiation in the Soret band prompts bleaching of the PPIX, which is accompanied by a sharp decrease in the intensity and average fluorescence lifetime of the protein (dominated by the four tryptophan residues of the tubulin monomer). The kinetics indicate non-trivial effects and suggest that the photosensitization of the PPIX bound to tubulin prompts structural alterations of the protein. These modifications were also observed through changes in the energy transfer between Trp residues and PPIX. The results suggest that laser irradiation produces localized partial unfolding of tubulin and that the changes prompt modification of the formation of microtubules in vitro. Measurements of singlet oxygen formation were inconclusive in determining whether the changes are prompted by reactive oxygen species or other excited state mechanisms.

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.