
Table
In vitro Rodents
Background
[PubMed]
64Cu-DOTA-[Pro1,Tyr4]-Bombesin[1-14] (64Cu-MP2346) is a peptide analog of human gastrin-releasing peptide (GRP) conjugated with 64Cu, and it was developed for positron emission tomography (PET) imaging of tumors with overexpressed GRP receptors (GRP-R) (1). 64Cu is a positron emitter with a 19.3% abundance and a physical half-life (t½) of 12.7 h.
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-R with high affinity and specificity (2, 3). Both GRP and BN share an amidated C-terminus sequence homology of seven 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 in CNS and GI tissues and a number of tumor cell lines. The BN-R superfamily includes at least four different subtypes, namely the GRP-R subtype (BB2), the neuromedin B receptor subtype (BB1), the BB3 subtype, and the BB4 subtype (6). Overexpression of GRP-R in various human tumors (e.g., breast, prostate, lung, colon, ovarian, and pancreatic cancers) provides opportunities to image tumors with the use of specific molecular imaging agents designed to target the GRP-R (1, 3, 7-9).
There have been varying degrees of success in the current development of GRP-R–targeted radiopharmaceuticals for diagnostic or therapeutic applications (9). Various BN analogs have been labeled with 99mTc and 111In for single-photon emission computed tomography (SPECT) imaging (10, 11). BN Analogs labeled with 68Ga, 18F, 86Y, or 64Cu have been studied for PET imaging (1, 12, 13). Breeman et al. (14, 15) prepared two GRP-R agonists, diethylenetriamine pentaacetic acid (DTPA)-[Pro1,Tyr4]BN[1-14] and 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-[Pro1,Tyr4]BN[1-14] (MP2346), for radiometal labeling by replacing pGlu1 and Leu4 in the native BN with DTPA-Pro or DOTA-Pro and with Tyr, respectively. Both MP2346 and DTPA-[Pro1,Tyr4]BN[1-14] were readily labeled with 111In and appeared to be promising radioligands for SPECT imaging. For PET imaging, Biddlecombe et al. (1) prepared and evaluated 64Cu-MP2346 and 86Y-MP2346. The investigators reported distinct differences in the in vivo pharmacokinetics and tumor uptake of these two radioligands; the authors suggested that this may be the result of their differences in peptide-to-receptor affinity, overall chemical charge, and radiometal-chelate stability.
Synthesis
[PubMed]
Biddlecombe et al. (1) reported the synthesis of 64Cu-MP2346. The [Pro1,Tyr4]BN[1-14] peptide was synthesized by the standard 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase chemistry and Rink amide resin. The DOTA conjugation procedure with [Pro1,Tyr4]BN[1-14] was not discussed. The standard method that involved the sulfosuccinimidyl ester of DOTA was used (16). The resulting MP2346 was purified with high-performance liquid chromatography (HPLC), and the mass was confirmed by mass spectrometry. The radiolabeling involved mixing MP2346 with 64Cu chloride (64CuCl2) in ammonium acetate (pH 5.5). The reaction mixture was incubated at 80ºC for 30 min. Reaction progress and purity were analyzed by analytical reverse-phase HPLC. On the basis of the HPLC analysis, the radiochemical yield and radiochemical purity were >99%. The specific activity was 36.6 MBq/μg (0.99 mCi/μg) or 72.5 GBq/μmol (1.958 Ci/μmol).
In Vitro Studies: Testing in Cells and Tissues
[PubMed]
Biddlecombe et al. (1) performed in vitro internalization studies (n = 3) of 64Cu-MP2346 in human prostate adenocarcinoma PC-3 cells. The amount of specifically internalized 64Cu-MP2346 was 121.83 ± 33.74 fmol/mg at 15 min after incubation and increased steadily to 512.04 ± 83.41 fmol/mg at 20 h. The amount of surface-bound radioactivity was <100 fmol/mg at all times, and the maximum internalized radioactivity was 535 fmol/mg. In comparison, the 86Y-MP2346 had a maximum internalization of 1,945 fmol/mg.
Animal Studies
Rodents
[PubMed]
Biodistribution studies of 64Cu-MP2346 were performed in nude mice (n = 5) bearing human prostate adenocarcinoma PC-3 tumors in their flanks (1). Each mouse received 0.22–0.27 kBq (8–10 μCi) radioactivity in 8–10 ng of 64Cu-MP2346 by i.v. administration. 64Cu-MP2346 showed rapid clearance of radioactivity from the blood over 24 h. The tumor radioactivity levels in percentage injected dose per gram (% ID/g) were 0.793 ± 0.181 (1 h), 0.421 ± 0.214 (4 h), and 0.309 ± 0.199 (24 h). The tumor/blood ratios were 6.192 (1 h), 5.511 (4 h), and 4.487 (24 h), and the tumor/muscle ratios were 2.718 (1 h), 4.996 (4 h), and 9.056 (24 h). In comparison, the tumor radioactivity levels and tumor/organ ratios of 86Y-MP2346 were significantly higher (P < 0.0005). At 1 h, the radioactivity levels (% ID/g) for major organs were 0.128 ± 0.0015 (blood), 0.426 ± 0.117 (lung), 1.329 ± 0.304 (liver), 6.148 ± 1.067 (kidney), 0.316 ± 0.255 (muscle), 0.160 ± 0.048 (heart), 0.885 ± 0.928 (bone), and 4.387 ± 1.090 (pancreas). At 24 h, these levels changed to 0.069 ± 0.007 (blood), 0.218 ± 0.043 (lung), 1.089 ± 0.429 (liver), 1.222 ± 0.296 (kidney), 0.034 ± 0.013 (muscle), 0.1865 ± 0.024 (heart), 0.038 ± 0.026 (bone), and 1.155 ± 0.327 (pancreas). Blocking studies with preinjection of 100 μg of [Tyr4]-BN immediately before radioligand injection were performed, and the mice were euthanized 1 h after injection. Radioactivity levels of both the tumor and GRP-R–rich pancreas decreased significantly (P < 0.0005) to 0.099 ± 0.134% ID/g and 0.464 ± 0.259% ID/g, respectively. Similar trends were observed in other GRP-R–rich organs.
PET imaging with computed tomography coregistration of 64Cu-MP2346 was conducted in mice bearing PC-3 tumors (n = 3) with a dose of 4.625 MBq (0.125 mCi) in 125 ng peptide by i.v. administration (1). The images showed that the radioactivity distribution pattern was similar to that of the biodistribution studies. The tumor was clearly visualized for 1–24 h. There was significant activity in the liver as shown by the biodistribution studies. The blocking study of mice coinjected with 100 μg [Tyr4]-BN and imaged side by side with mice that received only 64Cu-MP2346 showed a significant reduction of tumor radioactivity. The measured standard uptake value of 64Cu-MP2346 for the tumor at 1 h after injection decreased from ~1 (range, 0.8-1) in the study without blocking to ~0.4 (0.2–0.5) with blocking (extrapolated from Figure 4).
NIH Support
NCI R24 CA86307, NIH/NCI SAIRP grant R24 CA83060, NCI Cancer Center Support Grant 1 P30 CA91842.
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.
Publication Details
Author Information and Affiliations
Publication History
Created: August 16, 2007; Last Update: January 8, 2008.
Copyright
Publisher
National Center for Biotechnology Information (US), Bethesda (MD)
NLM Citation
Cheng KT, Lewis JS, Biddlecombe GB. 64Cu-DOTA-[Pro1,Tyr4]-Bombesin[1-14] 2007 Aug 16 [Updated 2008 Jan 8]. In: Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.