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N-2-(4-[18F]-Fluorobenzamido)ethylmaleimide coupled to cysteine-tagged on the C- or N-terminal of exendin-4

[18F]FBEM-[Cys0]-exendin-4 and [18F]FBEM-[Cys40]-exendin-4
, PhD
National Center for Biotechnology Information, NLM, Bethesda, MD 20894

Created: ; Last Update: March 22, 2012.

Chemical name:N-2-(4-[18F]-Fluorobenzamido)ethylmaleimide coupled to cysteine-tagged on the C- or N-terminal of exendin-4
Abbreviated name:[18F]FBEM-[Cys0]-exendin-4 and [18F]FBEM-[Cys40]-exendin-4
Synonym:
Agent Category:Peptide
Target:Glucogon-like peptide 1 receptor (GLP-1R)
Target Category:Receptor
Method of detection:Positron emission tomography (PET)
Source of signal / contrast:18F
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Structure not available in PubChem.

Background

[PubMed]

Pancreatic neuroendocrine tumors (PNET) are rare and are classified as nonfunctional or functional as discussed in detail by Batcher et al. (1). Nonfunctional PNETs usually grow in size and eventually cause a mass effect in the organ, whereas functional tumors secrete one (or more) active hormones and are subclassified on the basis of the hormone secreted by the cells constituting the tumor (e.g., insulinomas (secrete insulin), glucagonomas (secrete glucagon), somatostatinomas (secrete somatostatin), etc.). Insulinomas are the most common PNETs observed in the clinic, and biochemical tests of these patients show that the individuals have fasting hypoglycemia along with hyperinsulinoma (elevated levels of insulin) (1). Complete surgical resection of the tumor is considered to be the most suitable treatment and cure for the patient (2). Most insulinomas are small and benign, and they exist as single lesions. Imaging techniques such as computed tomography (CT), magnetic resonance imaging (MRI), and endoscopic ultrasound (EUS) are often used to detect and determine the location of these tumors on the pancreas (3). However, the preoperative detection of these lesion with noninvasive imaging is difficult because the techniques are either unable to distinguish the tumor from the normal parenchymal tissue in the pancreas (e.g., CT), are expensive (e.g., MRI), or are invasive and operator-dependent (e.g., EUS) (3, 4).

Investigators have shown that insulinomas characteristically express high levels of glucagon-like peptide 1 (GLP-1) receptors (GLP-1R), and radiolabeled GLP-1–like ligands have been developed and evaluated for the detection of these PNETs (5). Although GLP-1 is the natural ligand for the GLP-1R, a major drawback of using this peptide to detect the lesions is that it is rapidly inactivated (half-life, ~2 min) by proteolytic enzymes while in circulation. As a consequence, investigators identified 111In-labeled exendin-4 or its analogs (exendin is a peptide of 39 amino acids that has a 54% homology with GLP-1, acts as an agonist of the GLP-1R, and is not inactivated by proteolytic enzymes) for the detection of insulinomas in Rip1Tag2 mice that have spontaneous insulinoma (6) and in humans (4). In another study, EM3106B, an analog of GLP-1 that contains lactam bridges, was labeled with 18F by coupling it with N-2-(4-[18F]-fluorobenzamido)ethylmaleimide ([18F]-FBEM), and the labeled compound was successfully used with positron emission tomography (PET) to detect subcutaneous insulinomas in nude mice (7). As an extension of the earlier work, structural analogs of exendin-4 were conjugated with [18F]FBEM for labeling with 18F, and the biodistribution of [18F]FBEM-[Cys0]-exendin-4 and [18F]FBEM-[Cys40]-exendin-4 was studied in mice bearing INS-1 cell xenograft tumors (for structural details of the compounds, see the Synthesis section below) (5). The radiolabeled compounds were also evaluated with PET to detect tumors that overexpressed GLP-1R in these mice.

Synthesis

[PubMed]

Because exendin-4 does not contain a cysteine residue in its native structure, the peptide was modified with a C-terminal cysteine ([Cys0]-exendin-4) or an N-terminal cysteine ([Cys40]-exendin-4) to permit site-specific labeling of the analogs with [18F]FBEM (5). [Cys0]-exendin-4 and [Cys40]-exendin-4 were prepared commercially using solid-phase peptide synthesis procedures. The radiochemical synthesis of [18F]FBEM-[Cys0]-exendin-4 and [18F]FBEM-[Cys40]-exendin-4 has been described by Kiesewetter et al. (5). The radiochemical yield of the labeling reactions for both the tracers was 34.3 ± 3.4%, and the radiochemical purity of the labeled compounds at the time of preparation was >96% as determined with high-performance liquid chromatography (HPLC). The specific activity of the preparations was reported to be 45.51 ± 16.28 GBq/μmol (1.23 ± 0.44 Ci/μmol; n = 7 preparations).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Using [125I]GLP-1 as the ligand in a competition assay with INS-1 cells (Western blot analysis showed that these cells had a high expression of GLP-1R), the binding affinities of unlabeled FBEM-[Cys0]-exendin-4 and FBEM-[Cys40]-exendin-4 were determined to be 1.88 ± 0.075 nM and 1.22 ± 0.049 nM, respectively (5). The 50% inhibition concentration values for GLP-1, FBEM-[Cys0]-exendin-4, and FBEM-[Cys40]-exendin-4 were reported to be 5.33 ± 0.053 nM, 2.99 ± 0.0056 nM, and 1.11 ± 0.057 nM, respectively. This indicated that, among the different ligands of GLP-1R investigated in this study, FBEM-[Cys40]-exendin-4 exhibited the maximum affinity for the receptor.

The uptake, internalization, and efflux of [18F]FBEM-[Cys0]-exendin-4 and [18F]FBEM-[Cys40]-exendin-4 were investigated with INS-1 cells (5). Both tracers had similar uptake values in the cells (~0.5% of added dose), and after 2 h of incubation, 80% of the radioactivity had been internalized by the cells. Both probes were reported to have a gradual and biphasic efflux from the cells.

In another study, unlabeled FBEM-[Cys40]-exendin-4 was incubated with mouse serum and human plasma, respectively, for up to 1 h at 37°C (5). At the end of the incubation, a radio-HPLC analysis of the mixture showed that 20% and 32% of the compound was adsorbed to the serum and the plasma proteins, respectively.

Animal Studies

Rodents

[PubMed]

The biodistribution of [18F]FBEM-[Cys0]-exendin-4 and [18F]FBEM-[Cys40]-exendin-4 was studied in mice bearing INS-1 cell xenograft tumors or MDA-MB-435 cell xenograft tumors (do not express the GLP-1R; negative controls) as described by Kiesewetter et al. (5). The animals (n = 6 mice/group; under anesthesia) were injected with 3.44 ± 0.26 MBq (~100 μCi) of each tracer through the tail vein, and microPET images were acquired at 1 h and 2 h postinjection (p.i.). From the images it was determined that the radioactivity uptake values with the 18F-labeled [Cys0]-and [Cys40] analogs of exendin-4 in the INS-1 cell tumors at 1 h p.i. were 7.20 ± 1.26% injected dose per gram tissue (% ID/g) and 25.25 ± 3.39% ID/g, respectively. With [18F]FBEM-[Cys0]-exendin-4, the accumulation of radioactivity in the MD-MB-435 tumors was 0.67 ± 0.06% ID/g during the same period. At 2 h p.i., the amount of label in the tumors was ~4% ID/g and ~20% ID/g with [18F]FBEM-[Cys0]-exendin-4 and [18F]FBEM-[Cys40]-exendin-4, respectively. Ex vivo quantification of radioactivity in the different tissues of the animals showed that the tumor/kidney and tumor/liver ratios with [18F]FBEM-[Cys0]-exendin-4 were 0.48 and 7.4, respectively, and with [18F]FBEM-[Cys40]-exendin-4 these ratios were 4.8 and 39.9, respectively.

For blocking studies, the mice (n = 4 animals) were injected with 200 μg (~45 nmol) unlabeled [Cys0]-exendin-4 or [Cys40]-exendin-4 10 min before either [18F]FBEM-[Cys0]-exendin-4 or [18F]FBEM-[Cys40]-exendin-4 were administered to the animals (5). An ex vivo analysis of the major tissues from the animals showed that the radioactivity from both the tracers was reduced by >87% (P < 0.05) in the INS-1 tumors, pancreas, and lungs. With the [Cys40] isomer, there was a 52% (P < 0.05) inhibition of uptake in the intestine, but the liver, kidneys, and muscle showed a >49% increase in accumulation of radioactivity. However, the investigators did not explain the increased uptake of label observed in these organs.

From these studies, the investigators concluded that the [Cys40] isomer was a candidate for further development as a GLP-1R imaging agent because, compared with [18F]FBEM-[Cys0]-exendin-4, a higher uptake of radioactivity in the tumor was observed with [18F]FBEM-[Cys40]-exendin-4 and this labeled analog generated better tumor/nontarget tissue ratios in the rodents (5).

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.

Supplemental Information

[Disclaimers]

No information is currently available.

NIH Support

Studies presented in this chapter were supported by the Intramural Research Program of the National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health.

References

1.
Batcher E., Madaj P., Gianoukakis A.G. Pancreatic neuroendocrine tumors. Endocr Res. 2011;36(1):35–43. [PubMed: 21226566]
2.
Mathur A., Gorden P., Libutti S.K. Insulinoma. Surg Clin North Am. 2009;89(5):1105–21. [PMC free article: PMC3470467] [PubMed: 19836487]
3.
McAuley G., Delaney H., Colville J., Lyburn I., Worsley D., Govender P., Torreggiani W.C. Multimodality preoperative imaging of pancreatic insulinomas. Clin Radiol. 2005;60(10):1039–50. [PubMed: 16179163]
4.
Wild D., Macke H., Christ E., Gloor B., Reubi J.C. Glucagon-like peptide 1-receptor scans to localize occult insulinomas. N Engl J Med. 2008;359(7):766–8. [PubMed: 18703486]
5.
Kiesewetter D.O., Gao H., Ma Y., Niu G., Quan Q., Guo N., Chen X. (18)F-radiolabeled analogs of exendin-4 for PET imaging of GLP-1 in insulinoma. Eur J Nucl Med Mol Imaging. 2012;39(3):463–73. [PMC free article: PMC3617488] [PubMed: 22170321]
6.
Wild D., Behe M., Wicki A., Storch D., Waser B., Gotthardt M., Keil B., Christofori G., Reubi J.C., Macke H.R. [Lys40(Ahx-DTPA-111In)NH2]exendin-4, a very promising ligand for glucagon-like peptide-1 (GLP-1) receptor targeting. J Nucl Med. 2006;47(12):2025–33. [PubMed: 17138746]
7.
Gao H., Niu G., Yang M., Quan Q., Ma Y., Murage E.N., Ahn J.M., Kiesewetter D.O., Chen X. PET of insulinoma using 18F-FBEM-EM3106B, a new GLP-1 analogue. Mol Pharm. 2011;8(5):1775–82. [PMC free article: PMC3185201] [PubMed: 21800885]

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