Hofstetter Michael, Moon Euy Sung, D'Angelo Fabio, Geissbühler Lucien, Alberts Ian, Afshar-Oromieh Ali, Rösch Frank, Rominger Axel, Gourni Eleni
Department of Nuclear Medicine, Inselspital, Bern University Hospital, Freiburgstr. 18, 3010, Bern, Switzerland.
Department of Chemistry - TRIGA site, Johannes Gutenberg - University Mainz, Mainz, Germany.
EJNMMI Radiopharm Chem. 2020 Nov 30;5(1):29. doi: 10.1186/s41181-020-00115-8.
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 AAZTA 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-NH was functionalized with the chelator 6-[Bis (carboxymethyl)amino]-1,4-bis (carboyxmethyl)-6-methyl-1,4-diazepane (AAZTA) through the spacer 4-amino-1-carboxymethyl-piperidine (Pip) to obtain AAZTA-Pip-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH (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 and B 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.
LF1 was labelled with gallium-68, indium-111 and lutetium-177 within 5 min at room temperature (RT). The apparent molar activities (A) were ranging between 50 and 60 GBq/μmol for the Ga-labelled LF1, 10-20 GBq/μmol for the In- and Lu-labelled LF1. The radiotracers were stable for a period of 4 h post labeling exhibiting a hydrophilic profile with an average of a LogD of - 3, while the bound activity to the human serum protein was approximately 10%. Ga-LF1, Lu-LF1 and In-LF1 exhibited high affinity for the PC3 cells, with K 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) was between 0.5 and 0.8 nM which corresponds to approximately 4 × 10 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 Lu-labelled LF1 with rapamycin is applied compared to Lu-laballed LF1 alone.
Encouraged by these promising in vitro data, preclinical evaluation of the LF1 precursor are planned in tumour models in vivo.
基于胃泌素释放肽受体（GRPr）的放射性配体在GRPr阳性癌症（如前列腺癌和乳腺癌）的诊断成像方面显示出巨大潜力。本研究旨在开发和评估一种用于PET/SPECT成像以及术中及治疗应用的通用型基于GRPr的探针。深入研究了通用螯合剂AAZTA对放射性金属标记特性以及所生成放射性示踪剂体外性能的影响。基于GRPr的拮抗剂D - Phe - Gln - Trp - Ala - Val - Gly - His - Sta - Leu - NH通过间隔物4 - 氨基 - 1 - 羧甲基 - 哌啶（Pip）与螯合剂6 - [双（羧甲基）氨基] - 1,4 - 双（羧甲基）- 6 - 甲基 - 1,4 - 二氮杂环庚烷（AAZTA）进行功能化，以获得AAZTA - Pip - D - Phe - Gln - Trp - Ala - Val - Gly - His - Sta - Leu - NH（LF1）。LF1用镓 - 68（PET）、铟 - 111（SPECT，术中应用）和镥 - 177（治疗，SPECT）进行放射性标记。体外评估包括稳定性研究、亲脂性测定、蛋白质结合研究、K和B值的测定以及使用上皮性人前列腺癌细胞系PC3进行的内化研究。进一步进行体外单药治疗以及联合治疗研究，以评估其作为治疗诊断化合物的适用性。
LF1在室温（RT）下5分钟内用镓 - 68、铟 - 111和镥 - 177进行标记。镓标记的LF1的表观摩尔活度（A）在50至60 GBq/μmol之间，铟和镥标记的LF1的表观摩尔活度在10 - 20 GBq/μmol之间。放射性示踪剂在标记后4小时内稳定，呈现亲水性特征，平均LogD为 - 3，而与人血清蛋白的结合活性约为10%。镓 - LF1、镥 - LF1和铟 - LF1对PC3细胞表现出高亲和力，K值分别为16.3±2.4 nM、10.3±2.73 nM和5.2±1.9 nM，使受体饱和所需的放射性示踪剂浓度（B）在0.5至0.8 nM之间，这相当于每个细胞约4×10个受体。在细胞培养中发现低特异性内化率，而总的特异性细胞表面结合摄取总是超过内化活性。体外治疗研究表明，与单独使用镥标记的LF1相比，将镥标记的LF1与雷帕霉素联合应用时，对PC3细胞生长的抑制作用稍强。
受这些有前景的体外数据鼓舞，计划在体内肿瘤模型中对LF1前体进行临床前评估。
