Background

[PubMed]

Bombesin (BBN) is a tetradecapeptide isolated from the European fire-bellied frog (Bombina bombina) (1). BBN possesses a specific C-terminus (Gly-His-Leu-Met), which is necessary for its biological activity (2). Several peptides that are structurally related to BBN have been identified in mammals. Gastrin-releasing peptide (GRP) is a peptide of 27 amino acids from porcine gastric tissues with Gly-His-Leu-Met at its C-terminus. Neuromedin B (NMB) is a peptide of 32 amino acids from porcine spinal cords with Gly-His-Phe-Met at its C-terminus. These peptides are the ligands of a group of receptors called BBN receptors (BB-R). The mammalian BB-R family consists of three subtypes, including the GRP-preferring receptor (GRP-R or BB₂-R (384 amino acids)), the NMB-preferring receptor (NMB-R or BB₁-R (390 amino acids)), and an orphan receptor (BB₃-R (399 amino acids)) (3). These subtypes of BB-R are overexpressed in various diseased tissues. For example, GRP acts as a neurotransmitter and an endocrine cell-growth factor to regulate various functions of gastrointestinal and central nervous systems and to stimulate cell proliferations in lung, colon, stomach, pancreas, breast, and prostate cancers in humans (4). GRP binds to GRP-R as an agonist and is subsequently transported to the perinuclear space via receptor-mediated endocytosis (1), which leads to an accumulation in GRP-R–positive tissues. Thus, tagging GRP with an imaging probe allows for assessment of GRP-R in vivo.

¹⁷⁷Lu-(4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetracetic acid (DOTA))-Gly-4-aminobenzoyl-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (¹⁷⁷Lu-AMBA) is used with single-photon emission computed tomography (SPECT) imaging of GRP-R (5). ¹⁷⁷Lu-AMBA consists of two components: a peptide of eight amino acids that is composed of the seven common amino acids in the C-terminus of BBN/GRP (Trp-Ala-Val-Gly-His-Leu-Met) and a complex of ¹⁷⁷Lu-DOTA attached to the N-terminus of the peptide via a glycyl-4-aminobezoic acid linker. The small peptide accounts for the biological potency and possesses many advantages such as high in vivo stability, high uptake in tumors, low uptake in non-target tissues, and a rapid clearance from blood via the kidney (3). ¹⁷⁷Lu is a radionuclide from the group of rare earth radionuclides, and it is produced by neutron bombardment of purified target material in reactors (6). With a half-life of 6.71 days for β^- emission at 498 keV and 78% branch fraction, ¹⁷⁷Lu has been a very promising radionuclide in radiotherapy for effective destruction of small tumors and metastasis (optimal size 1.2–3.0 mm) while sparing normal tissue (7). ¹⁷⁷Lu also emits low-energy gamma rays at 208 and 113 keV with 10% and 6% abundance, respectively, which allows for direct monitoring of the activity distribution with SPECT and subsequent dosimetry calculations. ¹⁷⁷Lu-AMBA is currently under phase I clinical trials (8).

Synthesis

[PubMed]

Lantry et al. reported the synthesis of ¹⁷⁷Lu-AMBA (5). AMBA was produced with solid-phase peptide synthesis at a 14.5% yield followed by reaction with ~2.2 GBq (59.4 mCi ) ¹⁷⁷LuCl₃ with specific activity of 103.6-151.3 GBq/μmol (2.8–4.09 Ci/μmol) in 0.05 N HCl for 10 min at 100ºC. The produced ¹⁷⁷Lu-AMBA was purified with high-performance liquid chromatography (HPLC).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Lantry et al. examined the specificity of in vitro ¹⁷⁷Lu-AMBA binding to GRP-R in human prostate cancer PC-3 cells (~2.5 × 10⁵ GRP-R per cell) (5). The 50% inhibition concentration (IC₅₀) was measured via competition studies in which six cold metalated ligand (¹⁷⁵Lu-AMBA) solutions ranging from 1.25 × 10^-9 M to 5.0 × 10^-8 M were used to inhibit the binding of ¹²⁵I-[Tyr⁴]-BBN with specific activity of 2.2 Ci/μmol (81.4 GBq/μmol). The binding affinity (K_d) and the maximum binding capacity (B_max) were measured via direct saturation studies, in which 10 ¹⁷⁷Lu-AMBA solutions ranging from 0.0-0.37 MBq/ml (0–0.01 Ci/ml) were used. Both the inhibitory and the saturation studies were performed at 4ºC to eliminate interference from internalization and degradation. The measured IC₅₀, K_d, and B_max values were 2.50 ± 0.50 nmol/L, 1.02 nmol/L, and 414 fmol per 10⁶ PC-3 cells, respectively. The internalization of ¹⁷⁷Lu-AMBA was evaluated in adherent PC-3 cells at 37ºC. After 40 min of incubation, 76.8 ± 1.8% of ¹⁷⁷Lu-AMBA was internalized. Only 2.9 ± 1.8% was effluxed in 2 h; most (78%) remained in the form of parent ¹⁷⁷Lu-AMBA as found with HPLC. ¹⁷⁷Lu-AMBA appeared to be very stable; its half-life time was 38.8 h in human plasma and 3.1 h in mouse plasma. The specificity of ¹⁷⁷Lu-AMBA binding to BB-R subtypes was examined with receptor autoradiography in vitro. Tissue sections of human ileal carcinoid (NMB-R), human prostate carcinoma (GRP-R), and human bronchial carcinoid (BB₃-R) were used for studies. ¹⁷⁷Lu-AMBA bound specifically to GRP-R (IC₅₀, 0.8 nmol) and NMB-R (IC₅₀, 0.9 nmol/L) at high affinities, but less exhibited less preference for BB₃-R (IC₅₀ >1,000 nmol).

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

References

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2.

Ohki-Hamazaki H. , Iwabuchi M. , Maekawa F. Development and function of bombesin-like peptides and their receptors. Int J Dev Biol. 2005; 49 (2-3):293–300. [PubMed: 15906244]

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Dijkgraaf I. , Boerman O.C. , Oyen W.J. , Corstens F.H. , Gotthardt M. Development and application of peptide-based radiopharmaceuticals. Anticancer Agents Med Chem. 2007; 7 (5):543–51. [PubMed: 17896914]

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Smith C.J. , Gali H. , Sieckman G.L. , Hayes D.L. , Owen N.K. , Mazuru D.G. , Volkert W.A. , Hoffman T.J. Radiochemical investigations of 177Lu-DOTA-8-Aoc-BBN[7-14]NH2: an in vitro/in vivo assessment of the targeting ability of this new radiopharmaceutical for PC-3 human prostate cancer cells. Nucl Med Biol. 2003; 30 (2):101–9. [PubMed: 12623108]

5.

Lantry L.E. , Cappelletti E. , Maddalena M.E. , Fox J.S. , Feng W. , Chen J. , Thomas R. , Eaton S.M. , Bogdan N.J. , Arunachalam T. , Reubi J.C. , Raju N. , Metcalfe E.C. , Lattuada L. , Linder K.E. , Swenson R.E. , Tweedle M.F. , Nunn A.D. 177Lu-AMBA: Synthesis and characterization of a selective 177Lu-labeled GRP-R agonist for systemic radiotherapy of prostate cancer. J Nucl Med. 2006; 47 (7):1144–52. [PubMed: 16818949]

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Schotzig U. , Schrader H. , Schonfeld E. , Gunther E. , Klein R. Standardisation and decay data of 177Lu and 188Re. Appl Radiat Isot. 2001; 55 (1):89–96. [PubMed: 11339536]

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Dvorakova Z. , Henkelmann R. , Lin X. , Turler A. , Gerstenberg H. Production of 177Lu at the new research reactor FRM-II: Irradiation yield of 176Lu(n,gamma)177Lu. Appl Radiat Isot. 2008; 66 (2):147–51. [PubMed: 17900914]

8.

Waser B. , Eltschinger V. , Linder K. , Nunn A. , Reubi J.C. Selective in vitro targeting of GRP and NMB receptors in human tumours with the new bombesin tracer 177Lu-AMBA. Eur J Nucl Med Mol Imaging. 2007; 34 (1):95–100. [PubMed: 16909223]