99mTcO-N,N-Dimethylglycyl-l-lysinyl-l-cysteinylamide-8-amino-3,6-dioxaoctanoic-probestin

99mTcO-N3S-PEG2-probestin

Leung K.

Publication Details

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In vitro Rodents

Background

[PubMed]

Extracellular matrix (ECM) adhesion molecules consist of a complex network of fibronectins, collagens, chondroitins, laminins, glycoproteins, heparin sulfate, tenascins, and proteoglycans that surround connective tissue cells, and they are mainly secreted by fibroblasts, chondroblasts, and osteoblasts (1). Cell substrate adhesion molecules are considered essential regulators of cell migration, differentiation, and tissue integrity and remodeling. These molecules play a role in inflammation and atherogenesis, but they also participate in the process of invasion and metastasis of malignant cells in the host tissue (2). Invasive tumor cells adhere to the ECM, which provides a matrix environment for permeation of tumor cells through the basal lamina and underlying interstitial stroma of the connective tissue. Overexpression of matrix metalloproteinases (MMPs) and other proteases by tumor cells allows intravasation of tumor cells into the circulatory system after degrading the basement membrane and ECM (3).

Tumor angiogenesis represents a continuous and important process in tumor development in which the tumor attempts to gain an independent blood supply (4). This process is driven by the tumor's overproduction of angiogenic factors, which bind to receptors on nearby vessel endothelial cells. Angiogenesis is essential for the growth of solid tumors and their metastases. Imaging angiogenesis may be useful for monitoring angiogenic treatments of tumors and cardiovascular diseases (5-7). Aminopeptidase N (APN, CD13) is a membrane-bound glycoprotein with MMP activity that cleaves unsubstituted N-terminal amino acids with neutral side chains from peptides (8). APN has been shown to play a role in tumor angiogenesis, invasion, and metastasis (9). In addition to endothelial cells of angiogenic vessels, most cells of myeloid origin, epithelial cells, fibroblasts, and smooth muscle cells also express CD13 (10, 11). High levels of CD13 expression were found in solid tumors and tumor vasculatures. Probestin ((2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-l-Leu-l-Pro-l-Pro) is a competitive inhibitor of APN with an inhibition constant (Ki) value of 20 nM. Pathuri et al. (12) prepared 99mTcO-N,N-dimethylglycyl-l-lysinyl-l-cysteinylamide-8-amino-3,6-dioxaoctanoic-probestin (99mTcO-N3S-PEG2-probestin) as a single-photon emission computed tomography (SPECT) for imaging APN expression in tumors in mice.

Synthesis

[PubMed]

N3S-PEG2-Probestin was prepared with solid-phase peptide synthesis (12). 99mTc as pertechnetate (370 MBq (10 mCi)) was added to a solution of N3S-PEG2-probestin (100 µg) containing sodium gluconate and SnCl2. The mixture was incubated for 30 min at 25°C. The labeling efficiency of 99mTc incorporation was 50%–60% with >98% radiochemical purity after high-performance liquid chromatography. The specific activity of 99mTcO-N3S-PEG2-probestin was not reported.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Histoimmunostaining of CD13 was performed on HT-1080 and MCF-7 cancer cell lines (13). The APN enzyme was detected on the cell surface of HT-1080 tumor cells, whereas expression in MCF-7 tumor cells was only at the level of background staining. The APN activity in HT-1080 cells could be blocked with anti-CD13 antibodies.

Pathuri et al. (12) performed in vitro APN inhibition cell assays with HT-1080 cells and Ala-p-nitroanilide as the APN substrate for 30 min at 25°C. The 50% inhibition values were 797, 39.6, and 23.6 µM for probestin, ReO-PEG2-probestin, and ReO-N3S-PEG2-probestin, respectively.

Animal Studies

Rodents

[PubMed]

Pathuri et al. (12) performed ex vivo biodistribution studies of 1.28 MBq (35 μCi) 99mTcO-N3S-PEG2-probestin at 1 h after injection in nude mice (n = 4) bearing HT-1080 fibrosarcoma tumors. The tumor accumulation of radioactivity was 2.88 ± 0.64% injected dose per gram (ID/g). The tumor/blood and tumor/muscle ratios were 4.8 and 5.3, respectively. The radioactivity levels in the kidney (48% ID/g), liver (12% ID/g), and intestine (5% ID/g) were higher than the radioactivity in the spleen (2.4% ID/g), stomach (1.3% ID/g), heart (1.2% ID/g), and lung (0.9% ID/g). No target specificity studies were performed.

SPECT imaging analysis was performed in nude mice bearing the HT-1080 tumors after injection of 25.2 MBq (0.68 mCi) 99mTcO-N3S-PEG2-probestin (12). The tumor was clearly visualized at 0.5 h, along with the kidneys and urinary bladder. At 3 h, the tumor and intestine were still visualized along with a lower radioactivity in the kidneys and urinary bladder. Planar images showed radioactivity in the tumor, thymus, kidneys, liver, and intestine at 1 h after tracer injection. Co-injection of excess ReO-N3S-PEG2-probestin blocked radioactivity in the tumor, thymus, and kidneys, whereas there was an increase in radioactivity in the liver and intestine.

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

NIH Support

S10 RR025652

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