Background

[PubMed]

Alexa Fluor 680-glycylglycylglycine-bombesin[7-14]NH₂ peptide (Alexa Fluor 680-G-G-G-BN[7-14]NH₂), a peptide analog of human gastrin-releasing peptide (GRP) conjugated with Alexa Fluor 680, was developed for optical imaging of tumors with overexpressed GRP receptors (1). Alexa Fluor 680 is a fluorescence dye with an absorption maximum of 679 nm, an emission maximum of 720 nm, and an extinction coefficient of 180,000 cm−¹M−¹ (2).

The amphibian bombesin (BBN or BN), a peptide of 14 amino acids, is an analog of human GRP (a peptide of 27 amino acids) that binds to GRP receptors (GRP-R) with high affinity and specificity (1, 3). Both GRP and BN share an amidated C-terminus sequence homology of 7 amino acids, -Trp-Ala-Val-Gly-His-Leu-Met-NH₂. BN-Like peptides have been shown to induce various biological responses in diverse tissues, including the central nervous system (CNS) and the gastrointestinal (GI) system (4, 5). They also act as potential growth factors for both normal and neoplastic tissues. Specific BN receptors (BN-R) have been identified on CNS and GI tissues and a number of tumor cell lines. The BN-R superfamily includes at least 4 different subtypes, namely the GRP-R subtype (BB2), the neuromedin B (NMB) receptor subtype (BB1), the BB3 subtype, and the BB4 subtype (6). The findings of GRP-R overexpression in various human tumors, such as breast, prostate, lung, colon, ovarian, and pancreatic cancers, provide opportunities for tumor imaging by designing specific molecular imaging agents to target the GRP-R (1, 7-10).

A review by Smith et al. (10) indicated varying degrees of success in the current development of GRP-R–targeted radiopharmaceuticals for diagnostic or therapeutic applications. Various BN analogs have been labeled with ^99mTc and ¹¹¹In for single-photon emission computed tomography imaging. BN analogs labeled with ⁶⁸Ga-, ¹⁸F-, or ⁶⁴Cu have been studied for positron emission tomography imaging (8, 11, 12). Optical imaging is a method that utilizes light photons emitted from bioluminescence and fluorescence probes (13). Depth penetration is a major limiting factor for in vivo optical imaging. Currently, in vivo optical imaging has wide applications in small animal imaging but only limited applications in large animal and human studies (14). Near-infrared (NIR) fluorescence imaging (light range, 650−900 nm) has the advantages of relatively higher tissue penetration and lower autofluorescence from nontarget tissue. Alexa fluorochromes are spectrally unique fluorescent probes with relatively high quantum yields in their excitation and emission wavelength ranges (2, 15). These fluorochromes can be used in succinimidyl ester form for conjugation with the primary amines of biomolecules. Ma et al. (1) prepared and evaluated the fluorescence probe Alexa Fluor 680-G-G-G-BN[7-14]NH₂ for optical imaging of breast cancer in mice.

Synthesis

[PubMed]

Ma et al. (1) reported the preparation of Alexa Fluor 680-G-G-G-BN[7-14]NH₂. The BN peptide has the sequence of H₂N-Gly-Gly-Gly-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (H₂N-G-G-G-Q-W-A-V-G-H-L-M-(NH₂)) and was synthesized by the standard 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry with use of an automated peptide synthesizer (16). In this solid-phase peptide synthesis, the reaction of O-benzotriazolyl-tetramethyluronium hexafluorophosphate activated carboxyl groups on the reactant with the N-terminal amino group on the growing peptide provided for stepwise amino acid addition. Rink amide O-benzotriazolyl-tetramethyluronium hexafluorophosphate resin and the Fmoc-protected amino acids with side-chain protections were used to synthesize the NH₂-G-G-G-BN[7-14]NH₂ peptide. The final product was cleaved by a cocktail of trifluoroacetic acid, thioanisol, ethanedithiol, and water. The yield of the crude peptide was ~80%, which was purified by high-performance liquid chromatography (HPLC). The purified peptide was prepared as a 10-mg/ml solution with sodium bicarbonate (pH 8.5) for conjugation with the fluorescence label. The Alexa Fluor 680 succinimidyl ester in dimethylformamide was added to the NH₂-G-G-G-BN[7-14]NH₂ solution, and the reaction mixture was allowed to incubate at room temperature for 5 h in darkness. The resulting Alexa Fluor 680-G-G-G-BN[7-14]NH₂ conjugate was purified by HPLC. The yield of the peptide conjugate was ~60%. Coupling to the less reactive amidated C-terminus was not observed. Electrospray ionization mass spectrometry analysis showed a molecular ion of 1953.2, which was consistent with the calculated value of 1952.8.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Ma et al. (1) conducted in vitro competitive binding assays of Alexa Fluor 680-G-G-G-BBN[7-14]NH₂ for the GRP-R in human T-47D breast cancer cell line, which is known to express the GRP-R in very high numbers. Ma et al. used ¹²⁵I-Tyr⁴-BN as the competitive ligand in these assays. The 50% inhibitory concentration (IC₅₀) of Alexa Fluor 680-G-G-G-BBN[7-14]NH₂ was determined to be 7.7 ± 1.4 nM. The efficiency of in vitro cell internalization and residualization of Alexa Fluor 680-G-G-G-BN[7-14]NH₂ was also studied in the T-47D cells (1). Cell washing and confocal fluorescence microscopy image analysis after 40 min of incubation showed that Alexa Fluor 680-G-G-G-BN[7-14]NH₂ was effectively internalized and retained within the cells. The study did not attempt to quantify the data. When 5 μg of wild-type BN[1-14] was incubated with the cells before incubation with Alexa Fluor 680-G-G-G-BN[7-14]NH₂, the binding and internalization of Alexa Fluor 680-G-G-G-BN[7-14]NH₂ were completely or significantly blocked.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

References

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This MICAD chapter is not included in the Open Access Subset, because it was authored / co-authored by one or more investigators who was not a member of the MICAD staff.