Background

[PubMed]

The amphibian bombesin (BBN or BN, a peptide of 14 amino acids) is an analog of human gastrin-releasing peptide (GRP, a peptide of 27 amino acids) that binds to GRP receptors (GRPR) with high affinity and specificity (1, 2). Both GRP and BBN share an amidated C-terminus sequence homology of seven amino acids, Trp-Ala-Val-Gly-His-Leu-Met-NH₂. BBN-Like peptides have been shown to induce various biological responses in diverse tissues, including the central nervous system (CNS) and the gastrointestinal (GI) system. They also act as potential growth factors for both normal and neoplastic tissues (3). Specific BBN receptors (BBN-Rs) have been identified on CNS and GI tissues and on a number of tumor cell lines (4). The BBN-R superfamily includes at least four different subtypes, namely the GRPR subtype (BB2), the neuromedin B receptor subtype (BB1), the BB3 subtype, and the BB4 subtype. The findings of GRPR 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 GRPR (5, 6).

Integrins are a family of heterodimeric glycoproteins on cell surfaces that mediate diverse biological events involving cell–cell and cell–matrix interactions (7). Integrins consist of an α and a β subunit and are important for cell adhesion and signal transduction. The α_vβ₃ integrin is the most prominent receptor affecting tumor growth, tumor invasiveness, metastasis, tumor-induced angiogenesis, inflammation, osteoporosis, and rheumatoid arthritis (8-13). Expression of the α_vβ₃ integrin is strong on tumor cells and activated endothelial cells, whereas expression is weak on resting endothelial cells and most normal tissues. A peptide sequence consisting of Arg-Gly-Asp (RGD) has been identified as a recognition motif used by extracellular matrix proteins (vitronectin, fibrinogen, laminin, and collagen) to bind to a variety of integrins, including α_vβ₃. Various ligands have been introduced for imaging of tumors and tumor angiogenesis (14).

Because breast and prostate cancers express both GRPR and α_vβ₃, Liu et al. (15) designed an RGD-BBN heterodimer in which BBN[7-14]NH₂ and c(RGDyK) are connected with a glutamate linker (BBN on the Glu side chain γ-carboxylate group and RGD on the Glu side chain α-carboxylate group). 1,4,7-triazacyclononane-1,4-7-triacetic acid (NOTA) was used as a bifunctional chelator for labeling RGD-BBN to form ⁶⁴Cu-NOTA-RGD-BBN for use in positron emission tomography (PET) imaging of α_vβ₃ and GRPR in nude mice bearing human tumors. For single-photon emission computed tomography (SPECT), Liu et al. (16) conjugated Glu-RGD-BBN with 6-hydrazinonicotinyl (HYNIC) and labeled the product with ^99mTc, using tricine and trisodium triphenylphosphine-trisulfonate (TPPTS) as the coligands. ^99mTc-HYNIC-RGD-BBN was evaluated in C57/BL6 mice bearing mouse Lewis lung carcinomas (LLC).

Synthesis

[PubMed]

RGD-BBN was prepared with solid-phase peptide synthesis (16). Addition of a HYNIC group to RGD-BBN was performed by mixing 2 µmol RGD-BBN with 6 µmol HYNIC-NHS in sodium bicarbonate buffer (pH 9) for 5 h at room temperature. HYNIC-RGD-BBN was isolated with high-performance liquid chromatography (HPLC), with 56% yield. Measurement with matrix-assisted laser desorption ionization/time of flight mass spectrometry indicated the molecular mass to be m/z 1,918.60 (calculated molecular weight, 1,919.17). For ^99mTc labeling, a solution of 370 MBq (10 mCi) Na^99mTcO₄, SnCl₂, and 10 nmol HYNIC-RGD-BBN in succinate buffer (pH 5) was incubated for 10 min at 25°C. After addition of TPPTS, the mixture was heated for 30 min at 100°C. ^99mTc-HYNIC-RGD-BBN was isolated with HPLC, with 90% yield and a radiochemical purity of >98%. The specific activity was >30 MBq/nmol (0.81 mCi/nmol). The total preparation time was ~50 min.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Liu et al. (16) performed in vitro inhibition studies of HYNIC-RGD-BBN in cultured U87MG cells with ¹²⁵I-c(RGDyK). The 50% inhibition concentration (IC₅₀) values were 18.8 ± 3.8 nM and 10.8 ± 2.6 nM for HYNIC-RGD-BBN and c(RGDyK), respectively. In vitro inhibition studies of HYNIC-RGD-BBN were also performed in cultured PC-3 cells (high GRPR and moderate α_vβ₃ levels) with ¹²⁵I-[Tyr⁴]-BBN; the IC₅₀ values were 104.7 ± 5.8 nM and 71.6 ± 3.1 nM for HYNIC-RGD-BBN and BBN, respectively. The HYNIC-RGD-BBN heterodimer exhibited slightly lower binding affinities for the GRPR and α_vβ₃ integrin receptor than the corresponding unconjugated RGD and BBN peptides. Cellular accumulation of ^99mTc-HYNIC-RGD-BBN in PC-3 tumor cells showed a gradual increase in radioactivity in PC-3 cells from 30 min to 4 h. The cellular radioactivity reached 4.0% of incubation dose at 4 h. Treatment with excess RGD-BBN (1,000 nM) almost completely inhibited the accumulation of ^99mTc-HYNIC-RGD-BBN in PC-3 tumor cells.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

NIH Support

R01 CA120188, R01 CA119053, R21 CA121842, R21 CA102123, P50 CA114747, U54 CA119367, R24 CA93862

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Liu Z., Huang J., Dong C., Cui L., Jin X., Jia B., Zhu Z., Li F., Wang F. 99mTc-labeled RGD-BBN peptide for small-animal SPECT/CT of lung carcinoma. Mol Pharm. 2012;9(5):1409–17. [PubMed: 22452411]