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Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.

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Molecular Imaging and Contrast Agent Database (MICAD) [Internet].

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64Cu-1,4,7-Triazacyclononane-1,4-diacetic acid-para-aminobenzoic acid-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2

64Cu-NO2A-(AMBA)-BBN(7–14)NH2
, PhD
National Center for Biotechnology Information, NLM, NIH

Created: ; Last Update: January 25, 2011.

Chemical name:64Cu-1,4,7-Triazacyclononane-1,4-diacetic acid-para-aminobenzoic acid-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2Image CuNO2AAMBABBN.jpg
Abbreviated name:64Cu-NO2A-(AMBA)-BBN(7–14)NH2
Synonym:64Cu-NO2A-(X)-BBN(7–14)NH2
Agent Category:Peptides
Target:Gastrin-releasing peptide receptors (GRPR)
Target Category:Receptors
Method of detection:Positron emission tomography (PET)
Source of signal / contrast:64Cu
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Structure of Cu-NO2A-(AMBA)-BBN(7-14)NH2 by Lane et al (1).

Background

[PubMed]

64Cu-1,4,7-Triazacyclononane-1,4-diacetic acid (NO2A)-para-aminobenzoic acid (AMBA)-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (BBN(7–14)NH2), abbreviated as 64Cu-NO2A-(AMBA)-BBN(7–14)NH2, is a bombesin (BBN)-based, 64Cu-NO2A-conjugated peptide that was synthesized by Lane et al. for use in positron emission tomography (PET) of tumors expressing gastrin-releasing peptide receptor (GRPR) (1, 2).

GRPR is a glycosylated G-protein–coupled receptor that is normally expressed in non-neuroendocrine tissues of the breast and pancreas and in neuroendocrine cells of the brain, gastrointestinal tract, lung, and prostate (3, 4). GRPR has been found to be overexpressed in various human tumors, and a large number of BBN analogs have been investigated for GRPR-targeted imaging and therapy (5, 6). These analogs have been synthesized on the basis of either truncated BBN ((BBN(6–14) or BBN(7–14)) or full-length BBN(1–14) (7, 8). Chelators and spacers have been used frequently for chelating metals and for improving the kinetics of conjugates (9-11).

64Cu is a radiometal with potential applications in diagnostic and therapeutic nuclear medicine. The half-life for 64Cu (t1/2 = 12.7 h) is long enough for drug preparation, quality control, imaging, and therapy (12, 13). However, use of 64Cu is limited by issues of in vivo transchelation to proteins found in blood and liver (such as superoxide dismutase) (1). A variety of chelators have been investigated for the purpose of stably chelating 64Cu (13). In general, 64Cu-labeled 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (64Cu-DOTA) and 64Cu-labeled 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (64Cu-TETA) exhibit high uptake and retention in nontarget organs, which limits their application. Cross-bridged (CB) analogs, such as CB-DO2A ((1,4,7,10-tetraazabicyclo[5.5.2]tetradecane-4,10-diyl)diacetic acid), CB-TE2A ((1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diyl)diacetic acid), SarAr (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexa-aza-bicyclo-[6.6.6]eichosane-1,8-diamine), and NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), demonstrate improved copper containment by enhancing the ligand's rigidity (2, 14).

Prasanphanich et al. recently reported that the NOTA-based 64Cu-NOTA-8-Aoc-BBN(7–14)NH2 conjugate (where 8-Aoc = 8-aminooctanoic acid) exhibited decreased accumulation in hepatic tissue as compared with other chelator-based (DOTA, TETA and CB-TE2A) conjugates (2, 14). To improve the tumor uptake and maintain the good pharmacokinetic properties of the 64Cu-NOTA-8-Aoc-BBN(7–14)NH2 conjugate, Lane et al. synthesized a new group of conjugates with the NOTA derivative NO2A and replaced the spacer 8-Aoc with an aliphatic or aromatic linking (1). These conjugates were abbreviated as 64Cu-NO2A-(X)-BBN(7–14)NH2, where X denotes the pharmacokinetic modifier, such as AMBA, β-Ala (beta-alanine), 5-Ava (5-aminovaleric acid), 6-Ahx (6-aminohexanoic acid), 8-Aoc, and 9-Anc (9-aminonanoic acid). The β-Ala, 5-Ava, 6-Ahx, and 9-Anc are aliphatic pharmacokinetic modifiers, ranging from three to nine carbons in length, whereas AMBA is an aromatic pharmacokinetic modifier and is more rigid than the aliphatic modifiers. Evidence indicates that a spacing moiety, ranging from three to eight carbons in length, can assist in receptor-mediated uptake (15). Conjugates containing an aromatic linker have significantly higher uptake and retention in PC-3 tumor tissue than those containing hydrocarbon or ether linkers (15, 16). Studies by Lane et al. have shown that the spacer X in the 64Cu-NO2A-(X)-BBN(7–14)NH2 conjugates has a significant role in clearance, accumulation, and retention of the conjugates in tumor tissue (1). 64Cu-NO2A-(AMBA)-BBN(7–14)NH2 exhibited the highest accumulation in tumor tissue and the most efficient clearance from whole-body tissues via the renal–urinary excretion pathway. PET imaging with 64Cu-NO2A-(AMBA)-BBN(7–14)NH2 produced high-contrast images of PC-3 tumor xenografts in severe combined immunodeficient (SCID) mice (1). This chapter describes the data obtained with 64Cu-NO2A-(AMBA)-BBN(7–14)NH2. Detailed information for other 64Cu-NO2A-(X)-BBN(7–14)NH2 conjugates is available in MICAD (http://www.ncbi.nlm.nih.gov/books/NBK5330/).

Synthesis

[PubMed]

Lane et al. described the synthesis of 64Cu-NO2A-(X)-BBN(7–14)NH2 conjugates in detail (1). For 64Cu-NO2A-(AMBA)-BBN(7–14)NH2, the peptide AMBA-BBN(7–14)NH2 was synthesized with traditional F-moc chemistry. Crude peptides were obtained in ~60% yield. NOTA was conjugated to the peptide via an active ester to produce the NOTA derivative conjugate, NO2A-(AMBA)-BBN(7–14)NH2, with ~80% yield after purification. The calculated and observed molecular weights were both 1,344.7 for the NO2A-(AMBA)-BBN peptide conjugate64Cu-NO2A-(AMBA)-BBN(7–14)NH2 was synthesized with the reaction of 64CuCl2 and peptide conjugate in the presence of ammonium acetate. The radiochemical yield was >90%. All of the unconjugated peptide precursors, peptide conjugates, and metallated peptide conjugates were purified with reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized with mass spectrometry. The radiochemical purity and specific activity were not described.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The stability of 64Cu-NO2A-(AMBA)-BBN(7–14)NH2 in human serum albumin was determined with quantification of the 64Cu-NO2A-(AMBA)-BBN(7–14)NH2 peak area in the radiometric RP-HPLC chromatogram. The percent of intact conjugate was >80% after 24 h incubation (37°C, 5% CO2-enriched atmosphere) with human serum albumin (1).

A competitive displacement binding assay was performed in PC-3 cells with 125I-(Tyr4)-BBN as the radioligand (1). High binding affinities were observed for both unlabeled NO2A-(AMBA)-BBN(7–14)NH2 (inhibition constant (Ki) = 4.61 ± 1.54 nM) and natCu-NO2A-(AMBA)-BBN(7–14)NH2 (natCu = natural copper) (Ki = 15.45 ± 2.88 nM).

Internalization was determined after incubation of the PC-3 cells with 64Cu-NO2A-(AMBA)-BBN(7–14)NH2 for different times. Externalization was determined after an initial 40-min internalization period in PC-3 cells. The results revealed GRPR-mediated trapping of radioactivity inside the cells with very little externalization of 64Cu with cells washed after the internalization period (1).

Animal Studies

Rodents

[PubMed]

Biodistribution studies of 64Cu-NO2A-(AMBA)-BBN(7–14)NH2 were performed in both normal CF-1 mice (n = 4 or 5/group) and PC-3 xenograft-bearing SCID mice (n = 5 mice/group per time point). The biodistribution data were compared among the 64Cu-NO2A-(X)-BBN(7–14)NH2 conjugates (where X = β-Ala, 5-Ava, 6-Ahx, 8-Aoc, 9-Anc, or AMBA) (1, 2).

In normal mice, rapid clearance from blood was observed for all conjugates, with ≤0.5% injected dose per gram tissue (% ID/g) remaining in circulation at 1 h after injection (for 64Cu-NO2A-(AMBA)-BBN(7–14)NH2, 0.28 ± 0.05% ID/g). Excretion properties of the conjugates were directly related to the length of X. Increasing the hydrophobicity of the conjugate (increasing the aliphatic length of X) led to higher excretion rates via the hepatobiliary system. The more hydrophilic conjugate, 64Cu-NO2A-(AMBA)-BBN(7–14)NH2, had a lesser degree of accumulation in hepatic tissue (0.70 ± 0.08% ID/g at 1 h after injection). Uptake of the conjugates in GRPR-expressing pancreas ranged from 15.10 to 30.63% ID/g at 1 h after injection with 30.63 ± 3.26% ID/g for 64Cu-NO2A-(AMBA)-BBN(7–14)NH2, indicating effective GRPR targeting for the radiolabeled conjugates. The four conjugates showing the most favorable pharmacokinetic properties and the highest degree of pancreas accumulation were those in which X = 6-Ahx, 8-Aoc, 9-Anc, or AMBA, which were further studied in PC-3 tumor-bearing SCID mice (1).

In tumor-bearing SCID mice, all four conjugates were cleared effectively from the bloodstream, with 0.93–1.35% ID/g remaining at 1 h after injection (for 64Cu-NO2A-(AMBA)-BBN(7–14)NH2, 1.35 ± 0.51% ID/g). Excretion routes were similar to those observed in normal CF-1 mice, with more hydrophobic conjugates excreted through the hepatobiliary system. Minimal accumulation of radioactivity was observed in the liver at 24 h, ranging from 0.68 to 1.37% ID/g (for 64Cu-NO2A-(AMBA)-BBN(7–14)NH2, 1.23 ± 0.25% ID/g), suggesting that the metal complex was effectively stable under in vivo conditions. High receptor-mediated accumulation was observed in both the pancreas and tumor for all conjugates. Tumor uptake of the 64Cu-NO2A-(AMBA)-BBN(7–14)NH2 was 6.05 ± 1.15, 3.66 ± 0.54, and 2.54 ± 0.48% ID/g at 1 h, 4 h, and 24 h after injection, respectively. Pancreas uptake of the 64Cu-NO2A-(AMBA)-BBN(7–14)NH2 was 37.34 ± 4.83, 22.50 ± 3.13, and 8.93 ± 1.81% ID/g at 1 h, 4 h, and 24 h after injection, respectively. When comparing the tumor/tissue ratios at 24 h after injection, 64Cu-NO2A-(AMBA)-BBN(7–14)NH2 showed optimal tumor/blood, tumor/kidney, and tumor/liver ratios, with values of 2.6, 2.8, and 2.1, respectively (1).

PET imaging was performed at 18 h after tail-vein injection of 64Cu-NO2A-(X)-BBN(7–14)NH2 (where X = 6-Ahx, 8-Aoc, 9-Anc, or AMBA) (1). The PC-3 tumors were clearly identifiable in PET images for all conjugates. Tumor/background ratios decreased with increased length of X, which appears to be a function of increasing the hydrophobicity of the conjugate. 64Cu-NO2A-(AMBA)-BBN(7–14)NH2 demonstrated superior tumor targeting and retention, with clear tumor contrast from surrounding tissue and minimal abdominal accumulation. In comparison, for 64Cu-NO2A-(X)-BBN(7–14)NH2 where X = 6-Ahx, high tumor uptake with relatively high kidney accumulation was observed. For 64Cu-NO2A-(X)-BBN(7–14)NH2 where X = 8-Aoc, high tumor and liver uptake values were observed. For 64Cu-NO2A-(X)-BBN(7–14)NH2 where X = 9-Anc, moderate tumor uptake with significant abdominal accumulation (liver and gastrointestinal tract) was observed.

Other Non-Primate Mammals

[PubMed]

No references are currently available.

Non-Human Primates

[PubMed]

No references are currently available.

Human Studies

[PubMed]

No references are currently available.

References

1.
Lane S.R., Nanda P., Rold T.L., Sieckman G.L., Figueroa S.D., Hoffman T.J., Jurisson S.S., Smith C.J. Optimization, biological evaluation and microPET imaging of copper-64-labeled bombesin agonists, [64Cu-NO2A-(X)-BBN(7-14)NH2], in a prostate tumor xenografted mouse model. Nucl Med Biol. 2010;37(7):751–61. [PubMed: 20870150]
2.
Prasanphanich A.F., Retzloff L., Lane S.R., Nanda P.K., Sieckman G.L., Rold T.L., Ma L., Figueroa S.D., Sublett S.V., Hoffman T.J., Smith C.J. In vitro and in vivo analysis of [(64)Cu-NO2A-8-Aoc-BBN(7-14)NH(2)]: a site-directed radiopharmaceutical for positron-emission tomography imaging of T-47D human breast cancer tumors. Nucl Med Biol. 2009;36(2):171–81. [PMC free article: PMC2756974] [PubMed: 19217529]
3.
Smith C.J., Volkert W.A., Hoffman T.J. Gastrin releasing peptide (GRP) receptor targeted radiopharmaceuticals: a concise update. Nucl Med Biol. 2003;30(8):861–8. [PubMed: 14698790]
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Erspamer V., Erpamer G.F., Inselvini M. Some pharmacological actions of alytesin and bombesin. J Pharm Pharmacol. 1970;22(11):875–6. [PubMed: 4395815]
5.
Ananias H.J., de Jong I.J., Dierckx R.A., van de Wiele C., Helfrich W., Elsinga P.H. Nuclear imaging of prostate cancer with gastrin-releasing-peptide-receptor targeted radiopharmaceuticals. Curr Pharm Des. 2008;14(28):3033–47. [PubMed: 18991717]
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Schroeder R.P., van Weerden W.M., Bangma C., Krenning E.P., de Jong M. Peptide receptor imaging of prostate cancer with radiolabelled bombesin analogues. Methods. 2009;48(2):200–4. [PubMed: 19398012]
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Smith C.J., Volkert W.A., Hoffman T.J. Radiolabeled peptide conjugates for targeting of the bombesin receptor superfamily subtypes. Nucl Med Biol. 2005;32(7):733–40. [PubMed: 16243649]
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Zhang X., Cai W., Cao F., Schreibmann E., Wu Y., Wu J.C., Xing L., Chen X. 18F-labeled bombesin analogs for targeting GRP receptor-expressing prostate cancer. J Nucl Med. 2006;47(3):492–501. [PubMed: 16513619]
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Hohne A., Mu L., Honer M., Schubiger P.A., Ametamey S.M., Graham K., Stellfeld T., Borkowski S., Berndorff D., Klar U., Voigtmann U., Cyr J.E., Friebe M., Dinkelborg L., Srinivasan A. Synthesis, 18F-labeling, and in vitro and in vivo studies of bombesin peptides modified with silicon-based building blocks. Bioconjug Chem. 2008;19(9):1871–9. [PubMed: 18754574]
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Mu L., Honer M., Becaud J., Martic M., Schubiger P.A., Ametamey S.M., Stellfeld T., Graham K., Borkowski S., Lehmann L., Dinkelborg L., Srinivasan A. In vitro and in vivo characterization of novel 18F-labeled bombesin analogues for targeting GRPR-positive tumors. Bioconjug Chem. 2010;21(10):1864–71. [PubMed: 20857927]
11.
Becaud J., Mu L., Karramkam M., Schubiger P.A., Ametamey S.M., Graham K., Stellfeld T., Lehmann L., Borkowski S., Berndorff D., Dinkelborg L., Srinivasan A., Smits R., Koksch B. Direct one-step 18F-labeling of peptides via nucleophilic aromatic substitution. Bioconjug Chem. 2009;20(12):2254–61. [PubMed: 19921791]
12.
Wadas T.J., Wong E.H., Weisman G.R., Anderson C.J. Copper chelation chemistry and its role in copper radiopharmaceuticals. Curr Pharm Des. 2007;13(1):3–16. [PubMed: 17266585]
13.
Hoffman T.J., Smith C.J. True radiotracers: Cu-64 targeting vectors based upon bombesin peptide. Nucl Med Biol. 2009;36(6):579–85. [PubMed: 19647163]
14.
Prasanphanich A.F., Nanda P.K., Rold T.L., Ma L., Lewis M.R., Garrison J.C., Hoffman T.J., Sieckman G.L., Figueroa S.D., Smith C.J. [64Cu-NOTA-8-Aoc-BBN(7-14)NH2] targeting vector for positron-emission tomography imaging of gastrin-releasing peptide receptor-expressing tissues. Proc Natl Acad Sci U S A. 2007;104(30):12462–7. [PMC free article: PMC1914305] [PubMed: 17626788]
15.
Smith C.J., Gali H., Sieckman G.L., Higginbotham C., Volkert W.A., Hoffman T.J. Radiochemical investigations of (99m)Tc-N(3)S-X-BBN[7-14]NH(2): an in vitro/in vivo structure-activity relationship study where X = 0-, 3-, 5-, 8-, and 11-carbon tethering moieties. Bioconjug Chem. 2003;14(1):93–102. [PubMed: 12526698]
16.
Garrison J.C., Rold T.L., Sieckman G.L., Naz F., Sublett S.V., Figueroa S.D., Volkert W.A., Hoffman T.J. Evaluation of the pharmacokinetic effects of various linking group using the 111In-DOTA-X-BBN(7-14)NH2 structural paradigm in a prostate cancer model. Bioconjug Chem. 2008;19(9):1803–12. [PMC free article: PMC2647743] [PubMed: 18712899]
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