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99mTc-Ethylenenediamine-N,N'-diacetic acid/hydrazinonicotinamide[Lys3]-bombesin

99mTc-EDDA/HYNIC-[Lys3]-BN

, PhD, , PhD, and , PhD.

Author Information

Created: ; Last Update: September 19, 2007.

Chemical name:99mTc-Ethylenediamine-N,N'-diacetic acid/hydrazinonicotinamide-[Lys3]-bombesinimage 26683897 in the ncbi pubchem database
Abbreviated name:99mTc-EDDA/HYNIC-[Lys3]-BN
Synonym:99mTc-EDDA/HYNIC-[Lys3]-BBN, 99mTc-BN, 99mTc-BNN
Agent Category:Peptide
Target:Gastrin-releasing peptide receptor (GRP-R)
Target Category:Receptor binding
Method of detection:Single-photon emission computed tomography (SPECT) imaging, planar gamma imaging
Source of signal:99mTc
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents

Click on the above structure for additional information in PubChem.
Click on protein, nucleotide (RefSeq), and gene for more information about BN and GRP.

Background

[PubMed]

99mTc-Ethylenediamine-N,N’-diacetic acid/hydrazinonicotinamide-[Lys3]-bombesin (99mTc-EDDA/HYNIC-[Lys3]-BN) is a peptide analog of human gastrin-releasing peptide (GRP) conjugated with 99mTc, and it was developed for planar gamma and single-photon emission computed tomography (SPECT) imaging of tumors with overexpressed GRP receptors (GRP-R) (1). 99mTc is a gamma emitter with a physical half-life (t½) of 6.02 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 (BB2) 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, 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 (3, 7-10).

There have been varying degrees of success in the current development of GRP-R–targeted radiopharmaceuticals for diagnostic or therapeutic applications (10). Various BN analogs have been labeled with 99mTc and 111In for SPECT imaging (1, 11-13). Baidoo et al. (12) synthesized and radiolabeled [Lys3]-BN (Pyr-Gln-Lys-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) with 99mTc through the diaminedithiol (DADT) bifunctional chelating agent. The 99mTc-DADT-[Lys3]-BN showed high in vitro affinity to the BN-R in rat brain membrane preparations. Because of its favorable physical properties, 99mTc is the radionuclide of choice for routine clinical applications of SPECT imaging (14). HYNIC is a bifunctional coupling agent for 99mTc-labeling of peptides that can achieve high specific activities without interfering with the amino acid sequence responsible for receptor binding (15-17). In this approach, 99mTc is bound to the hydrazine group, and other coordination sites are occupied by one or more coligands . The choice of coligand can influence the stability and hydrophilicity of the radiolabeled peptide (15, 18). Convenient freeze-dried kit formulations with HYNIC for labeling peptides with 99mTc have been achieved (19). Using the HYNIC labeling strategy and EDDA/N-tris(hydroxymethyl)-methylglycine (tricine) as the coligands, Ferro-Flores et al. (1) successfully prepared 99mTc-EDDA/HYNIC-[Lys3]-BN as a potential molecular imaging probe for GRP-R.

Synthesis

[PubMed]

Ferro-Flores et al. (1) reported the synthesis of 99mTc-EDDA/HYNIC-[Lys3]-BN. The [Lys3]-BN peptide was obtained commercially and could be synthesized by the standard 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase chemistry (2). Conjugation of HYNIC to the Є-amino group of the Lys3 residue at the N-terminal region of the BN peptide was achieved by the use of succinimidyl-N-Boc-HYNIC. Briefly, [Lys3]-BN in N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) buffer (pH 9.0) was mixed with succinimidyl-N-Boc-HYNIC in dimethylformamide in the HYNIC:peptide molar ratio of 5:1. The reaction mixture was incubated at room temperature for 60 min. The resulting Boc-HYNIC-[Lys3]-BN was purified by solid-phase extraction and then deprotected by the addition of trifluoroacetic acid. The final product of HYNIC-[Lys3]-BN was purified by high-performance liquid chromatography (HPLC). HYNIC-[Lys3]-BN was then lyophilized for 24 h in a mixture of EDDA-tricine-mannitol in the weight ratio (g) 0.4:0.8:2 and stannous chloride under a nitrogen atmosphere. Radiolabeling was performed by adding phosphate buffer (pH 7.0) and 99mTc sodium pertechnetate immediately after the buffer addition to the lyophilized peptide conjugate. The mixture was incubated at 92ºC for 15 min. On the basis of the analyses of instant thin-layer chromatography, SepPak C-18 solid phase extraction, and HPLC, the radiochemical yield and purity was 95 ± 2% (n > 30). The specific activity was ~0.1 GBq/nmol (2.7 mCi/nmol).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

In vitro stability in human serum at 37ºC showed that the radiochemical purity of 99mTc-EDDA/HYNIC-[Lys3]-BN remained >90% after 24 h incubation (1). Serum protein binding was found to be 29 ± 1.4% and 34 ± 2.1% at 1 and 24 h, respectively. When 99mTc-EDDA/HYNIC-[Lys3]-BN was challenged with cysteine in the cysteine:peptide molar ratios of 500:1, <9% of radioactivity was dissociated. Cell binding and internalization studies were performed in human prostate cancer PC-3 cells. The fractions of radioactivity internalized were 8% at 2 h and 11.5% at 4 h. When the cells were blocked with [Tyr4]-BN, the internalized fractions decreased to <2–3% (extrapolated from Figure 4).

Animal Studies

Rodents

[PubMed]

Biodistribution studies of 99mTc-EDDA/HYNIC-[Lys3]-BN were performed in nude mice (n = 4) bearing PC-3 tumors (0.2–2.3 g) in their flanks (1). Each mouse received 1.11 MBq (30 μCi) radioactivity by i.v. administration and were then euthanized at 2 h after injection. 99mTc-EDDA/HYNIC-[Lys3]-BN appeared to have a rapid clearance of radioactivity from the blood and most tissues in 2 h. Liver and intestine radioactivity levels were relatively low. Renal excretion was the predominant route of radioactivity elimination. The radioactivity levels (n = 4) of major organs in percentage injected dose per gram (% ID/g) were 0.30 ± 0.11 (tumor), 0.08 ± 0.03 (blood), 0.05 ± 0.03 (heart), 0.17 ± 0.04 (liver), 0.10 ± 0.04 (lung), 0.08 ± 0.04 (spleen), 4.70 ± 1.20 (kidney), 1.29 ± 0.31 (pancreas), 0.17 ± 0.10 (intestine), 0.05 ± 0.03 (muscle), and 0.05 ± 0.02 (heart). The tumor/blood, tumor/muscle, and pancreas/blood ratios were 3.75, 7.5, and 16, respectively. Blocking studies with a dose of 50 μg [Tyr4]-BN 30 min before radioligand injection reduced the radioactivity levels in the tumor (0.09 ± 0.03% ID/g) by 70% and in the pancreas (0.34 ± 0.16% ID/g) by 73%. The radioactivity levels in other non-targeted tissues were not significantly affected by the blocking dose. Gamma imaging of 99mTc-EDDA/HYNIC-[Lys3]-BN in mice bearing the PC-3 tumors clearly visualized the tumors (1).

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

References

1.
Ferro-Flores G. , Arteaga de Murphy C. , Rodriguez-Cortes J. , Pedraza-Lopez M. , Ramirez-Iglesias M.T. Preparation and evaluation of 99mTc-EDDA/HYNIC-[Lys 3]-bombesin for imaging gastrin-releasing peptide receptor-positive tumours. Nucl Med Commun. 2006;27(4):371–6. [PubMed: 16531924]
2.
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]
3.
Ma L. , Yu P. , Veerendra B. , Rold T.L. , Retzloff L. , Prasanphanich A. , Sieckman G. , Hoffman T.J. , Volkert W.A. , Smith C.J. In Vitro and In Vivo Evaluation of Alexa Fluor 680-Bombesin[7-14]NH(2) Peptide Conjugate, a High-Affinity Fluorescent Probe with High Selectivity for the Gastrin-Releasing Peptide Receptor. Mol Imaging. 2007;6(3):171–80. [PubMed: 17532883]
4.
Mantey S. , Frucht H. , Coy D.H. , Jensen R.T. Characterization of bombesin receptors using a novel, potent, radiolabeled antagonist that distinguishes bombesin receptor subtypes. Mol Pharmacol. 1993;43(5):762–74. [PubMed: 7684815]
5.
Benya R.V. , Kusui T. , Pradhan T.K. , Battey J.F. , Jensen R.T. Expression and characterization of cloned human bombesin receptors. Mol Pharmacol. 1995;47(1):10–20. [PubMed: 7838118]
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Reubi J.C. , Wenger S. , Schmuckli-Maurer J. , Schaer J.C. , Gugger M. Bombesin receptor subtypes in human cancers: detection with the universal radioligand (125)I-[D-TYR(6), beta-ALA(11), PHE(13), NLE(14)] bombesin(6-14) Clin Cancer Res. 2002;8(4):1139–46. [PubMed: 11948125]
7.
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]
8.
Biddlecombe G.B. , Rogers B.E. , de Visser M. , Parry J.J. , de Jong M. , Erion J.L. , Lewis J.S. Molecular imaging of gastrin-releasing peptide receptor-positive tumors in mice using 64Cu- and 86Y-DOTA-(Pro1,Tyr4)-bombesin(1-14) Bioconjug Chem. 2007;18(3):724–30. [PubMed: 17378600]
9.
Nock B. , Nikolopoulou A. , Chiotellis E. , Loudos G. , Maintas D. , Reubi J.C. , Maina T. [99mTc]Demobesin 1, a novel potent bombesin analogue for GRP receptor-targeted tumour imaging. Eur J Nucl Med Mol Imaging. 2003;30(2):247–58. [PubMed: 12552343]
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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]
  • 11. de Visser, M., H.F. Bernard, J.L. Erion, M.A. Schmidt, A. Srinivasan, B. Waser, J.C. Reubi, E.P. Krenning, and M. de Jong, Novel (111)In-labelled bombesin analogues for molecular imaging of prostate tumours. Eur J Nucl Med Mol Imaging, 2007. [PubMed: 17287960]
  • 12.
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    13.
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    14.
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    15.
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    16.
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    17.
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    18.
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    19.
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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.

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