Pfister Joachim, Summer Dominik, Rangger Christine, Petrik Milos, von Guggenberg Elisabeth, Minazzi Paolo, Giovenzana Giovanni B, Aloj Luigi, Decristoforo Clemens
Department of Nuclear Medicine, Innsbruck Medical University, Anichstrasse 35, A-6020, Innsbruck, Austria.
Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic.
EJNMMI Res. 2015 Dec;5(1):74. doi: 10.1186/s13550-015-0154-7. Epub 2015 Dec 15.
6-[Bis(carboxymethyl)amino]-1,4-bis(carboxymethyl)-6-methyl-1,4-diazepane (AAZTA ) is a promising chelator with potential advantages over 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for radiopharmaceutical applications. Its mesocyclic structure enables fast radiolabelling under mild conditions with trivalent metals including not only (68)Ga for positron emission tomography (PET) but also (177)Lu and (111)In for single-photon emission computed tomography (SPECT) and radionuclide therapy. Here, we describe the evaluation of a bifunctional AAZTA derivative conjugated to a model minigastrin derivative as a potential theranostic agent.
An AAZTA derivative with an aliphatic C9 chain as linker was coupled to a minigastrin, namely [AAZTA(0), D-Glu(1), desGlu(2-6)]-minigastrin (AAZTA-MG), and labelled with (68)Ga, (177)Lu and (111)In. The characterisation in vitro included stability studies in different media and determination of logD (octanol/PBS). Affinity determination (IC50) and cell uptake studies were performed in A431-CCK2R cells expressing the human CCK2 receptor. μPET/CT and ex vivo biodistribution studies were performed in CCK2 tumour xenograft-bearing nude mice and normal mice.
AAZTA-MG showed high radiochemical yields for (68)Ga (>95 %), (177)Lu (>98 %) and (111)In (>98 %). The logD value of -3.7 for both [(68)Ga]- and [(177)Lu]-AAZTA-MG indicates a highly hydrophilic character. Stability tests showed overall high stability in solution with some degradation in human plasma for [(68)Ga]- and transchelation towards DTPA for and [(177)Lu]-AAZTA-MG. An IC50 value of 10.0 nM was determined, which indicates a high affinity for the CCK2 receptor. Specific cell uptake after 60 min was >7.5 % for [(68)Ga]-AAZTA-MG and >9.5 % for [(177)Lu]-AAZTA-MG, comparable to other DOTA-MG-analogues. μPET/CT studies in CCK2 receptor tumour xenografted mice not only revealed high selective accumulation in A431-CCK2R positive tumours of (68)Ga-labelled AAZTA-MG (1.5 % ID/g in 1 h post injection) but also higher blood levels as corresponding DOTA-analogues. The (111)In-labelled peptide had a tumour uptake of 1.7 % ID/g. Biodistribution in normal mice with the [(177)Lu]-AAZTA-MG showed a considerable uptake in intestine (7.3 % ID/g) and liver (1.5 % ID/g).
Overall, AAZTA showed interesting properties as bifunctional chelator for peptides providing mild radiolabelling conditions for both (68)Ga and trivalent metals having advantages over the currently used chelator DOTA. Studies are ongoing to further investigate in vivo targeting properties and stability issues and the influence of spacer length on biodistribution of AAZTA.
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）是一种很有前景的螯合剂，在放射性药物应用方面比1,4,7,10-四氮杂环十二烷-1,4,7,10-四乙酸（DOTA）具有潜在优势。其大环结构能够在温和条件下与三价金属快速进行放射性标记，这些金属不仅包括用于正电子发射断层扫描（PET）的（68）Ga，还包括用于单光子发射计算机断层扫描（SPECT）和放射性核素治疗的（177）Lu和（111）In。在此，我们描述了一种与模型胃泌素衍生物偶联的双功能AAZTA衍生物作为潜在诊疗试剂的评估。
将具有脂肪族C9链作为连接基的AAZTA衍生物与一种胃泌素，即[AAZTA(0), D-Glu(1), desGlu(2-6)]-胃泌素（AAZTA-MG）偶联，并用（68）Ga、（177）Lu和（111）In进行标记。体外表征包括在不同介质中的稳定性研究以及logD（辛醇/磷酸盐缓冲液）的测定。在表达人CCK2受体的A431-CCK2R细胞中进行亲和力测定（IC50）和细胞摄取研究。在携带CCK2肿瘤异种移植物的裸鼠和正常小鼠中进行μPET/CT和离体生物分布研究。
AAZTA-MG对（68）Ga（>95%）、（177）Lu（>98%）和（111）In（>98%）显示出高放射化学产率。[（68）Ga]-和[（177）Lu]-AAZTA-MG的logD值为-3.7，表明其具有高度亲水性。稳定性测试表明，[（68）Ga]-AAZTA-MG在溶液中总体稳定性较高，但在人血浆中会有一些降解，而[（177）Lu]-AAZTA-MG会向二乙三胺五乙酸（DTPA）发生转螯合。测定的IC50值为10.0 nM，表明对CCK2受体具有高亲和力。60分钟后，[（68）Ga]-AAZTA-MG的特异性细胞摄取>7.5%，[（177）Lu]-AAZTA-MG的特异性细胞摄取>9.5%，与其他DOTA-MG类似物相当。在CCK2受体肿瘤异种移植小鼠中进行的μPET/CT研究不仅显示（68）Ga标记的AAZTA-MG在A431-CCK2R阳性肿瘤中有高选择性蓄积（注射后1小时为1.5% ID/g），而且血药浓度比相应的DOTA类似物更高。（111）In标记的肽肿瘤摄取率为l.7% ID/g。[（177）Lu]-AAZTA-MG在正常小鼠中的生物分布显示在肠道（7.3% ID/g）和肝脏（1.5% ID/g）中有相当的摄取。
总体而言，AAZTA作为肽的双功能螯合剂表现出有趣的特性，为（68）Ga和三价金属提供了温和的放射性标记条件，比目前使用的螯合剂DOTA具有优势。正在进行研究以进一步研究其体内靶向特性、稳定性问题以及连接基长度对AAZTA生物分布的影响。
