1. 68Ga-labeled cyclic NGR peptide for microPET imaging of CD13 receptor expression
Yahui Shao, Wansheng Liang, Fei Kang, Weidong Yang, Xiaowei Ma, Guiyu Li, Shu Zong, Kai Chen, Jing Wang Molecules. 2014 Aug 5;19(8):11600-12. doi: 10.3390/molecules190811600.
Peptides containing the asparagines-glycine-arginine (NGR) motif have been identified as specific ligands binding to CD13/aminopeptidase N (APN) receptor, a tumor neovascular biomarker. In this study, we synthesized a novel NGR-containing peptide (NOTA-G3-NGR), and labeled NOTA-G3-NGR with (68)Ga (t1/2 = 67.7 min). The resulting (68)Ga-NOTA-G3-NGR peptide was subject to in vitro and in vivo characterization. The microPET imaging results revealed that the (68)Ga-NOTA-G3-NGR peptide exhibits rapid and specific tumor uptake, and high tumor-to-background contrast in a subcutaneous HT-1080 fibrosarcoma mouse model. We concluded that the (68)Ga-NOTA-G3-NGR peptide has potential in the diagnosis of CD13-targeted tumor angiogenesis.
2. A comparison of the monomeric [68Ga]NODAGA-NGR and dimeric [68Ga]NOTA-(NGR)2 as aminopeptidase N ligand for positron emission tomography imaging in tumor-bearing mice
Ina Israel, Konstantin Elflein, Andreas Schirbel, Kai Chen, Samuel Samnick Eur J Pharm Sci. 2021 Nov 1;166:105964. doi: 10.1016/j.ejps.2021.105964. Epub 2021 Aug 8.
The aminopeptidase N (APN/CD13) is a key protein specifically expressed on activated endothelial cells and by various tumors, representing a promising target for molecular imaging and therapy of malignant diseases. It is known that the tripeptide NGR is a specific ligand for CD13, therefore radiolabeled NGR peptides are auspicious radiotracers for non-invasive imaging of CD13-positive tumors. From previous studies, it is known that the target affinity could be improved by molecules with multiple ligand sequences. Therefore, the aim of this study was to compare two NGR radioligands [68Ga]NODAGA-NGR (NGR monomer) and [68Ga]NOTA-(NGR)2 (NGR dimer), the latter with two NGR ligand motifs, in vitro and in vivo. CD13 expression was determined by FACS in the human tumor cells A549, SKHep-1, and MDA-MB-231, followed by the investigation of the cell uptake of [68Ga]NODAGA-NGR and [68Ga]NOTA-(NGR)2. For in vivo evaluation of [68Ga]NODAGA-NGR and [68Ga]NOTA-(NGR)2, microPET and biodistribution were carried out in A549- and SKHep-1-bearing mice. After the final examination, tumors were cryo-conserved, cut, and stained against CD13 and CD31. A549 and SKHep-1 cells were identified as CD13 positive, whereas no CD13 expression was detected in MDA-MB-231 cells. The cell uptake study showed relatively low accumulation of both the NGR monomer and dimer in all tumor cell lines examined, with consistently higher cell uptake observed for the dimer than for the monomer. In vivo, [68Ga]NODAGA-NGR and [68Ga]NOTA-(NGR)2 accumulated in the tumors, with slightly higher tumor-to-muscle ratio for the NGR dimer in A549 and SKHep-1. The tumor-to-liver ratio of the NGR dimer was diminished in comparison to the NGR monomer. This finding was confirmed by biodistribution, which revealed higher accumulation in liver and spleen for the NGR dimer. Immunohistochemical staining confirmed the CD13 expression in the tumors and tumor-associated vessels. In conclusion, both the [68Ga]NODAGA-NGR and the [68Ga]NOTA-(NGR)2 were found to be suitable for PET imaging of CD13-positive tumors. Despite slight differences in tumor-to-background ratio and organ accumulation, both radiotracers can be considered comparable.
3. NGR-peptide-drug conjugates with dual targeting properties
Kata Nóra Enyedi, Szilárd Tóth, Gergely Szakács, Gábor Mező PLoS One. 2017 Jun 2;12(6):e0178632. doi: 10.1371/journal.pone.0178632. eCollection 2017.
Peptides containing the asparagine-glycine-arginine (NGR) motif are recognized by CD13/aminopeptidase N (APN) receptor isoforms that are selectively overexpressed in tumor neovasculature. Spontaneous decomposition of NGR peptides can result in isoAsp derivatives, which are recognized by RGD-binding integrins that are essential for tumor metastasis. Peptides binding to CD13 and RGD-binding integrins provide tumor-homing, which can be exploited for dual targeted delivery of anticancer drugs. We synthesized small cyclic NGR peptide-daunomycin conjugates using NGR peptides of varying stability (c[KNGRE]-NH2, Ac-c[CNGRC]-NH2 and the thioether bond containing c[CH2-CO-NGRC]-NH2, c[CH2-CO-KNGRC]-NH2). The cytotoxic effect of the novel cyclic NGR peptide-Dau conjugates were examined in vitro on CD13 positive HT-1080 (human fibrosarcoma) and CD13 negative HT-29 (human colon adenocarcinoma) cell lines. Our results confirm the influence of structure on the antitumor activity and dual acting properties of the conjugates. Attachment of the drug through an enzyme-labile spacer to the C-terminus of cyclic NGR peptide resulted in higher antitumor activity on both CD13 positive and negative cells as compared to the branching versions.
