Radiopharmacological Evaluation of Cyclohexanediamine-Triazole-Peptide Conjugates Labeled via the Al18F-Approach

Aim/Introduction: The Al18F-labeling method is as modern technique an alternative to conventional 18F-labeling procedures that allows radiofluorination of biomolecules such as peptides and proteins in a one-step procedure in aqueous solution [1]. In search for versatile applicable chelators, which allow stable binding of both 18F and radiometals such as 68Ga or 111In, a cyclohexanediamine-triazole-chelator was designed. This chelator was conjugated via copper-catalyzed azide-alkyne cycloaddition (CuAAC) to the well-known binding motif (glutamate-urea-lysine) of the prostate-specific membrane antigen (PSMA) [2-5] complemented by 2-azidoacetyl moiety as linker unit (ligand L1). Furthermore, ligand L2 was synthesized bearing a 6-azidohexanoyl moiety as linker to investigate the influence of the linker on the stability of final 18F or radiometal complex.
The aim of this study was to investigate the radiopharmacological potential of L1 and L2 after radiolabeling regarding binding properties, cell internalization, and in vivo behavior in a murine prostate cancer model.
Methods: For the in vitro assays PSMA-positive LNCaP cells were used. The incubation with the respective radiolabeled ligand (RCY>95%) was terminated via a cell harvester. Internalization experiments were carried out by the “acid wash” method. In vivo studies (biodistribution and small animal PET) were performed with mice bearing a prostate tumor.
Results: In competition assays versus [177Lu]Lu-PSMA-617 (“gold standard”), the affinity of non-labeled L1 and L2 was slightly lower than that of PSMA-617. Saturation analysis of [68Ga]Ga-L1, [111In]In-L1, and [18F]F-L1 binding on LNCaP homogenate was comparable to [18F]F-L2 binding. The obtained Kd values were in a range of 20 to 30 nM. Internalization experiments with LNCaP cells revealed a lower uptake of the differently labeled L1 and L2 conjugates compared to [64Cu]Cu-PSMA-617.
Furthermore, in vivo behavior of both [18F]F-L1 and [18F]F-L2 was investigated in prostate carcinoma bearing mice by biodistribution experiments and small animal PET imaging. Thereby, PSMA dependent tumor uptake could be observed.
Conclusion: After successful radiolabeling, the conjugates L1 and L2 showed promising binding properties towards PSMA. The chelator presented here offers a flexible platform for radiolabeling of peptides or proteins for various PET and SPECT applications.
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