Effect of the versatile bifunctional chelator AAZTA5 on the radiometal labelling properties and the in vitro performance of a gastrin releasing peptide receptor antagonist

Michael Hofstetter; Euy Sung Moon; Fabio D'Angelo; Lucien Geissbühler; Ian Alberts; Ali Afshar-Oromieh; Frank Rösch; Axel Rominger; Eleni Gourni

doi:10.1186/s41181-020-00115-8

Effect of the versatile bifunctional chelator AAZTA⁵ on the radiometal labelling properties and the in vitro performance of a gastrin releasing peptide receptor antagonist

EJNMMI Radiopharm Chem. 2020 Nov 30;5(1):29. doi: 10.1186/s41181-020-00115-8.

Authors

Michael Hofstetter¹, Euy Sung Moon², Fabio D'Angelo¹, Lucien Geissbühler¹, Ian Alberts¹, Ali Afshar-Oromieh¹, Frank Rösch², Axel Rominger¹, Eleni Gourni³

Affiliations

¹ Department of Nuclear Medicine, Inselspital, Bern University Hospital, Freiburgstr. 18, 3010, Bern, Switzerland.
² Department of Chemistry - TRIGA site, Johannes Gutenberg - University Mainz, Mainz, Germany.
³ Department of Nuclear Medicine, Inselspital, Bern University Hospital, Freiburgstr. 18, 3010, Bern, Switzerland. eleni.gourni@insel.ch.

Abstract

Background: Gastrin Releasing Peptide receptor (GRPr)-based radioligands have shown great promise for diagnostic imaging of GRPr-positive cancers, such as prostate and breast. The present study aims at developing and evaluating a versatile GRPr-based probe for both PET/SPECT imaging as well as intraoperative and therapeutic applications. The influence of the versatile chelator AAZTA⁵ on the radiometal labelling properties and the in vitro performance of the generated radiotracers were thoroughly investigated. The GRPr-based antagonist D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH₂ was functionalized with the chelator 6-[Bis (carboxymethyl)amino]-1,4-bis (carboyxmethyl)-6-methyl-1,4-diazepane (AAZTA⁵) through the spacer 4-amino-1-carboxymethyl-piperidine (Pip) to obtain AAZTA⁵-Pip-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH₂ (LF1). LF1 was radiolabelled with gallium-68 (PET), indium-111 (SPECT, intraoperative applications) and lutetium-177 (therapy, SPECT). In vitro evaluation included stability studies, determination of lipophilicity, protein-binding studies, determination of K_d and B_max as well as internalization studies using the epithelial human prostate cancer cell line PC3. In vitro monotherapy as well as combination therapy studies were further performed to assess its applicability as a theranostic compound.

Results: LF1 was labelled with gallium-68, indium-111 and lutetium-177 within 5 min at room temperature (RT). The apparent molar activities (A_m) were ranging between 50 and 60 GBq/μmol for the ⁶⁸Ga-labelled LF1, 10-20 GBq/μmol for the ¹¹¹In- and ¹⁷⁷Lu-labelled LF1. The radiotracers were stable for a period of 4 h post labeling exhibiting a hydrophilic profile with an average of a LogD_octanol/PBS of - 3, while the bound activity to the human serum protein was approximately 10%. ^68/natGa-LF1, ^177/natLu-LF1 and ^111/natIn-LF1 exhibited high affinity for the PC3 cells, with K_d values of 16.3 ± 2.4 nM, 10.3 ± 2.73 nM and 5.2 ± 1.9 nM, respectively, and the required concentration of the radiotracers to saturate the receptors (B_max) was between 0.5 and 0.8 nM which corresponds to approximately 4 × 10⁵ receptors per cell. Low specific internalization rate was found in cell culture, while the total specific cell surface bound uptake always exceeded the internalized activity. In vitro therapy studies showed that inhibition of PC3 cells growth is somewhat more efficient when combination of ¹⁷⁷Lu-labelled LF1 with rapamycin is applied compared to ¹⁷⁷Lu-laballed LF1 alone.

Conclusion: Encouraged by these promising in vitro data, preclinical evaluation of the LF1 precursor are planned in tumour models in vivo.

Keywords: AAZTA; Gastrin releasing peptide receptor (GRPr); Imaging; Peptide radionuclide therapy; Prostate cancer.