NGR-Based Radiopharmaceuticals for Angiogenesis Imaging: A Preclinical Review.

Angiogenesis plays a crucial role in tumour progression and metastatic spread; therefore, the development of specific vectors targeting angiogenesis has attracted the attention of several researchers. Since angiogenesis-associated aminopeptidase N (APN/CD13) is highly expressed on the surface of activated endothelial cells of new blood vessels and a wide range of tumour cells, it holds great promise for imaging and therapy in the field of cancer medicine. The selective binding capability of asparagine-glycine-arginine (NGR) motif containing molecules to APN/CD13 makes radiolabelled NGR peptides promising radiopharmaceuticals for the non-invasive, real-time imaging of APN/CD13 overexpressing malignancies at the molecular level. Preclinical small animal model systems are major keystones for the evaluation of the in vivo imaging behaviour of radiolabelled NGR derivatives. Based on existing literature data, several positron emission tomography (PET) and single-photon emission computed tomography (SPECT) radioisotopes have been applied so far for the labelling of tumour vasculature homing NGR sequences such as Gallium-68 (68Ga), Copper-64 (64Cu), Technetium-99m (99mTc), Lutetium-177 (177Lu), Rhenium-188 (188Re), or Bismuth-213 (213Bi). Herein, a comprehensive overview is provided of the recent preclinical experiences with radiolabelled imaging probes targeting angiogenesis.

Scandium-44: Diagnostic Feasibility in Tumor-Related Angiogenesis.

Angiogenesis-related cell-surface molecules, including integrins, aminopeptidase N, vascular endothelial growth factor, and gastrin-releasing peptide receptor (GRPR), play a crucial role in tumour formation. Radiolabelled imaging probes targeting angiogenic biomarkers serve as valuable vectors in tumour identification. Nowadays, there is a growing interest in novel radionuclides other than gallium-68 (68Ga) or copper-64 (64Cu) to establish selective radiotracers for the imaging of tumour-associated neo-angiogenesis. Given its ideal decay characteristics (Eß+average: 632 KeV) and a half-life (T1/2 = 3.97 h) that is well matched to the pharmacokinetic profile of small molecules targeting angiogenesis, scandium-44 (44Sc) has gained meaningful attention as a promising radiometal for positron emission tomography (PET) imaging. More recently, intensive research has been centered around the investigation of 44Sc-labelled angiogenesis-directed radiopharmaceuticals. Previous studies dealt with the evaluation of 44Sc-appended avb3 integrin-affine Arg-Gly-Asp (RGD) tripeptides, GRPR-selective aminobenzoyl-bombesin analogue (AMBA), and hypoxia-associated nitroimidazole derivatives in the identification of various cancers using experimental tumour models. Given the tumour-related hypoxia- and angiogenesis-targeting capability of these PET probes, 44Sc seems to be a strong competitor of the currently used positron emitters in radiotracer development. In this review, we summarize the preliminary preclinical achievements with 44Sc-labelled angiogenesis-specific molecular probes.

In Vivo Imaging of Acute Hindlimb Ischaemia in Rat Model: A Pre-Clinical PET Study.

BACKGROUND: To better understand ischaemia-related molecular alterations, temporal changes in angiogenic Aminopeptidase N (APN/CD13) expression and glucose metabolism were assessed with PET using a rat model of peripheral arterial disease (PAD). METHODS: The mechanical occlusion of the base of the left hindlimb triggered using a tourniquet was applied to establish the ischaemia/reperfusion injury model in Fischer-344 rats. 2-[18F]FDG and [68Ga]Ga-NOTA-c(NGR) PET imaging performed 1, 3, 5, 7, and 10 days post-ischaemia induction was followed by Western blotting and immunohistochemical staining for APN/CD13 in ischaemic and control muscle tissue extracts. RESULTS: Due to a cellular adaptation to hypoxia, a gradual increase in [68Ga]Ga-NOTA-c(NGR) and 2-[18F]FDG uptake was observed from post-intervention day 1 to 7 in the ischaemic hindlimbs, which was followed by a drop on day 10. Conforming pronounced angiogenic recovery, the NGR accretion of the ischaemic extremities differed significantly from the controls 5, 7, and 10 days after ischaemia induction (p ≤ 0.05), which correlated with the Western blot and immunohistochemical results. No remarkable radioactivity was depicted between the normally perfused hindlimbs of either the ischaemic or the control groups. CONCLUSIONS: The PET-based longitudinal assessment of angiogenesis-associated APN/CD13 expression and glucose metabolism during ischaemia may continue to broaden our knowledge on the pathophysiology of PAD.

Radiolabeled NGR-Based Heterodimers for Angiogenesis Imaging: A Review of Preclinical Studies.

Since angiogenesis/neoangiogenesis has a major role in tumor development, progression and metastatic spread, the establishment of angiogenesis-targeting imaging and therapeutic vectors is of utmost significance. Aminopeptidase N (APN/CD13) is a pivotal biomarker of angiogenic processes abundantly expressed on the cell surface of active vascular endothelial and various neoplastic cells, constituting a valuable target for cancer diagnostics and therapy. Since the asparagine-glycine-arginine (NGR) sequence has been shown to colocalize with APN/CD13, the research interest in NGR-peptide-mediated vascular targeting is steadily growing. Earlier preclinical experiments have already demonstrated the imaging and therapeutic feasibility of NGR-based probes labeled with different positron emission tomography (PET) and single-photon emission computed tomography (SPECT) radionuclides, including Gallium-68 (68Ga), Copper-64 (64Cu), Technetium-99m (99mTc), Lutetium-177 (177Lu), Rhenium-188 (188Re) or Bismuth-213 (213Bi). To improve the tumor binding affinity and the retention time of single-receptor targeting peptides, NGR motifs containing heterodimers have been introduced to identify multi-receptor overexpressing malignancies. Preclinical studies with various tumor-bearing experimental animals provide useful tools for the investigation of the in vivo imaging behavior of NGR-based heterobivalent ligands. Herein, we review the reported preclinical achievements on NGR heterodimers that could be highly relevant for the development of further target-specific multivalent compounds in diagnostic and therapeutic settings.

Aminopeptidase N (CD13) targeted MR and NIRF dual-modal imaging of ovarian tumor xenograft.

The development of tumor-specific imaging nanoprobes with the potential to improve the accuracy of cancer diagnosis has become an area of intense research. Aminopeptidase N (CD13) predominantly expresses on the surface of ovarian tumor cells and can be specifically recognized by Asn-Gly-Arg (NGR) peptide. The applicability of CD13 as a target for specific ovarian tumor imaging, however, remains unexploited so far. In this study, Cy5.5-labeled, NGR-conjugated iron oxide nanoparticles (Cy5.5-NGR-Fe3O4 NPs) were prepared as an ovarian tumor specific bimodal imaging nanoprobe. It is demonstrated that the conjugation of NGR targeting moiety leads to a higher cellular uptake toward ES-2 cells, the human ovarian carcinoma cells that highly express CD13. Moreover, magnetic resonance imaging of ovarian tumor xenograft reveals that the Fe3O4-Cy5.5-NGR NPs results in a significant T2* signal reduction in the tumor. Meanwhile, near infrared fluorescence imaging indicates a higher accumulation of Fe3O4-Cy5.5-NGR NPs in the tumor xenograft. Therefore, CD13 could be applied as a novel and efficient target for constructing ovarian tumor specific nanoprobes with improved accuracy for ovarian tumor diagnosis.

13F-1, a novel 5-fluorouracil prodrug containing an Asn-Gly-Arg (NO2) COOCH3 tripeptide, inhibits human colonic carcinoma growth by targeting Aminopeptidase N (APN/CD13).

13F-1 is a 5-fluorouracil prodrug containing an Asn-Gly-Arg (NO2) COOCH3 tripeptide. 13F-1 might possess the activity against cancer growth by targeting Aminopeptidase N (APN/CD13). Our goal in this study was to evaluate the inhibitory effect of 13F-1 on the growth of human colonic carcinoma by both in vitro and in vivo studies. Experiments were performed in colonic carcinoma Colo205 cells, which highly express APN/CD13 on cell surface. The inhibition of 13F-1 on cancer cell growth was estimated by the colorimetric and clonogenic assays. The assays of Annexin V-FITC/PI and JC-1 fluorescence probe were employed to determine the apoptotic cells. Further experiment was performed in mice bearing Colo205 xenografts. 13F-1 was injected for three consecutive weeks. The specimens of Colo205 xenografts were removed for TUNEL staining and western blotting analysis. The expressions of APN/CD13 were analyzed by immunofluorescent flow cytometry and western blotting assays. 13F-1 significantly inhibited Colo205 cell proliferation. 13F-1 by injection delayed the expansion of Colo205 xenografts without significant toxicity to mice. The inhibitory effect of 13F-1 might arise from its role in apoptotic induction. Further analysis indicated that 13F-1 strongly inhibited APN/CD13 expression on cancer cell surface. In contrast, 5-FU did not affect APN/CD13 expression. These results indicated the mechanism of 13F-1 action that 13F-1׳s effect was associated with its role in suppression of APN/CD13 expression. Conclusion, 13F-1 could be developed as a promising agent for treatment of cancers with high expression of APN/CD13.

Development of NGR peptide-based agents for tumor imaging.

Molecular imaging allows direct visualization of targets and characterization of cellular pathways, as long as a high signal/background ratio can be achieved, which requires a sufficient amount of probes to accumulate in the imaging region. The Asn-Gly-Arg (NGR) tripeptide selected by phage display can specifically target tumor vasculature. Recognizing the aminopeptidase N (APN or CD13) receptor on the membrane of tumor cells, the peptide can be further internalized into cytoplasma by the endosomal pathway. Hence NGR can serve as an ideal candidate for tumor imaging, once it is conjugated with fluorescent or radiolabeled imaging probes. Herein, we highlight some recent developments of NGR peptide based imaging of tumors. Although still in the preliminary stage, some NGR probes have shown potential as promising agents in future clinical applications.