Background

[PubMed]

Receptor guanylate cyclase C (GC-C) is a type I transmembrane glycoprotein expressed on intestinal brush border membranes of intestinal epithelial cells and transformed human colon cancer cell lines (e.g., T84) (1). GC-C is expressed in very low levels on the other tissues and is highly expressed on the primary and metastatic colon cancer tissues (2). Peptides such as the Escherichia coli heat-stable enterotoxin (STh) and uroguanylin have been shown to inhibit the growth of colorectal cancer (CRC) cells in animal models (3-5). STh and uroguanylin are agonistic for the GC-C receptor with nanomolar affinity, resulting in activation of the guanylyl cyclase that increases the intracellular concentration of guanosine 3,5-cyclic monophosphate. Uroguanylin and guanylin are endogenous peptides, which are secreted into the lumen of the gut by enterochromaffin cells to regulate ion and fluid homeostasis (6). However, their expression is lost during cancer transformation (5, 7).

CRC is the second most common type of cancer in the United States (8). The United States Food and Drug Administration (FDA) has approved scintigraphic agents that are based on monoclonal antibodies (mAbs) for the imaging of colorectal cancers (9), but these agents have limitations because of a lack of penetration in tumors and prolonged blood circulation as a result of their large molecular size. Furthermore, the pharmacokinetics of intact radiolabeled mAbs, with high liver uptake and slow blood elimination, are generally not ideal for imaging. 1,4,7,10-Tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid (DOTA) derivatives of STh have been evaluated for in vivo imaging in nude mice bearing colorectal xenografts (10-12). Uroguanylin (NDDCELCVNVACTGCL) is a 16 amino acid peptide with two disulfide bonds and is less complex than STh, which contains three disulfide bonds. The N-terminal end of uroguanylin and E³-uroguanylin (NDECELCVNVACTGCL) was conjugated with DOTA for ¹¹¹In-radiolabeling. ¹¹¹In-DOTA-uroguanylin and ¹¹¹In-DOTA-E³-uroguanylin have been evaluated in nude mice bearing human colon adenocarcinoma tumors (13).

Synthesis

[PubMed]

N-Hydroxysuccinimide (NHS) ester of DOTA (NHS-DOTA) was used in a 100-fold excess to conjugate the N-terminal end of uroguanylin and E³-uroguanylin to form DOTA-uroguanylin and DOTA-E³-uroguanylin after being incubated overnight at 4°C (13). DOTA-uroguanylin and DOTA-E³-uroguanylin were isolated with high-performance liquid chromatography (HPLC) and identified with mass spectroscopy. There was one DOTA molecule per peptide. DOTA-uroguanylin or DOTA-E³-uroguanylin (24 nmol) was mixed with 7.5–92.5 MBq (0.2–2.5 mCi) ¹¹¹InCl₃ and incubated for 60 min at 37°C. ¹¹¹In-DOTA-uroguanylin and ¹¹¹In-DOTA-E³-uroguanylin were purified with HPLC. The labeling yield and the radiochemical purity were not reported. The specific activity was not reported.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The binding affinities of uroguanylin, DOTA-uroguanylin, E³-uroguanylin, and DOTA-E³-uroguanylin for GC-C were determined using T84 cells (13). In this assay, ¹²⁵I-F19-STh(1-19) was incubated with 3 × 10⁶ cells in the presence of 0.1–1,000 nM corresponding peptide for 60 min at 37°C. The 50% inhibition concentration (IC₅₀) values were 39.9, 34.5, 5.0, and 9.6 nM for uroguanylin, DOTA-uroguanylin, E³-uroguanylin, and DOTA-E³-uroguanylin, respectively.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

NIH Support

P50 CA103130-01

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