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[111In-Diethylenetriamine pentaacetic acid-ACMpip5,Tha6,βAla11,Tha13,NIe14]bombesin(5-14)

[111In-DTPA-ACMpip5,Tha6,βAla11,Tha13,NIe14]BN(5-14)
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD, vog.hin.mln.ibcn@dacim

Created: ; Last Update: January 14, 2008.

Chemical name:[111In-Diethylenetriamine pentaacetic acid-ACMpip5,Tha6,βAla11,Tha13,NIe14]bombesin(5-14)
Image ACMpip111In.jpg
Abbreviated name:[111In-DTPA- ACMpip5,Tha6,βAla11,Tha13,NIe14]BN(5-14)
Synonym:111In-BN analog, 111In-BBN analog
Agent Category:Peptide
Target:Gastrin-releasing peptide receptor (GRP-R)
Target Category:Receptor binding
Method of detection:Single-photon emission computed tomography (SPECT), planar gamma imaging
Source of signal:111In
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents

[111In-DTPA- ACMpip5,Tha6,βAla11,Tha13,NIe14]BN(5-14). The 111In coordination configuration has not been established by experiments.
Click on PubChem (SID​:46501389) for additional information.
Click on protein, nucleotide (RefSeq), and gene for more information about BN and GRP.

Background

[PubMed]

[111In-Diethylenetriamine pentaacetic acid-ACMpip5,Tha6,βAla11,Tha13,NIe14]-bombesin(5-14) ([111In-DTPA-ACMpip5,Tha6,βAla11,Tha13,NIe14]-BN(5-14)) is a peptide analog of the human gastrin-releasing peptide (GRP) conjugated with 111In, and it was developed for single-photon emission computed tomography (SPECT) imaging of tumors with overexpressed GRP receptors (GRP-R) (1). 111In is a gamma emitter with a physical half-life (t½) of 2.8 days.

The amphibian bombesin (BN or BBN), a peptide of 14 amino acids, is an analog of human GRP, a peptide of 27 amino acids, which 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 in 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 (8). 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, 6-9).

There have been varying degrees of success in the development of GRP-R–targeted radiopharmaceuticals for diagnostic or therapeutic applications (9). Various BN analogs have been labeled with 99mTc and 111In for SPECT imaging (13, 14). Despite some concern about the possible tumor growth–stimulatory effect of BN, Breeman et al. (10) synthesized the BN-R agonist [111In-DTPA-Pro1,Tyr4]BN. The study reported that this agonist had a high specific localization in GRP-R–positive tissues and tumors. [111In-DTPA-Pro1,Tyr4]BN differs from native BN in that pGlu1 is replaced with DTPA-Pro1 and Leu4 is replaced with Tyr4. Breeman et al. (10) showed that this agonist was internalized by GRP-R–expressing cells, whereas the antagonist ([111In-DTPA-Tyr5,d-Phe6]BN(5-13)NHEt) was not. On the basis of this finding, de Visser et al. (1) developed new DTPA-couple BN analogs with shortened amino acid sequences containing non-natural amino acid derivatives to improve the receptor binding affinity of the compounds. [111In-DTPA-ACMpip5,Tha6,βAla11,Tha13,NIe14]-BN(5-14) is one of the tested radioactive probes that appears to be a promising candidate for use in molecular imaging of tumors expressing GRP-R (1).

Synthesis

[PubMed]

The BN peptide analog 4-aminocarboxymethylpiperidine [ACMpip5,Tha6,βAla11,Tha13,NIe14]-BN(5-14), was synthesized using the solid-phase method according to the Fmoc strategy and Rink amide resin (1). A solid-phase peptide synthesizer was used. The protocol required 25 μmol of starting resin and 75 μmol of subsequent Fmoc-protected amino acids activated by the combination of N-hydroxybenzotriazole and (2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. Coupling of DTPA to [ACMpip5,Tha6,βAla11,Tha13,NIe14]-BN(5-14) was performed by placing tributyl-DTPA at the appropriate location in an amino acid column. Cleavage and deprotection were accomplished using a mixture of 85% trifluoroacetic acid, 5% thioanisole, 5% phenol, and 5% water. The crude peptide was precipitated with t-butyl methyl ether and purified by reverse-phase, high-performance liquid chromatography (HPLC). The molecular weight of the peptide was determined by mass spectrometry to be 1,644.7 (M+H) as compared to the theoretical value of 1,644. Radiolabeling of [ACMpip5,Tha6,βAla11,Tha13,NIe14]-BN(5-14) with 111In used the same procedure as described by Bakker et al. (11). A 40- to 70-molar excess of peptide over 111In was used. The peptide in 0.01–0.1 M acetic acid was mixed with 111In in 0.04–0.05 M hydrochloric acid. The reaction mixture was incubated at room temperature for 10 min. The labeling yield and radiochemical purity were >95% as determined by instant thin-layer chromatography and reverse-phase chromatography on C18 Sep-Pak column. The maximum specific activity was 200 MBq/nmol (5.4 mCi/nmol).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

De Visser et al. (1) determined the receptor affinity of [111In-DTPA-ACMpip5,Tha6,βAla11,Tha13,NIe14]-BN(5-14) using frozen sections of the GRP-R–expressing human prostate tumor xenograft PC-295 and rat colon. In vitro autoradiography was conducted by incubating 10-μm sections for 1 h at room temperature with 0.1 nM of the radiolabeled BN analog. The sections were incubated with increasing amounts of the nonradioactive BN analog to generate the competitive inhibition curve. The 50% inhibitory concentration (IC50) was determined to be 0.08 ± 0.01 nM on the rat colon tissue and 0.36 ± 0.01 nM on the human PC-295 tissue. De Visser et al. (1) also reported an IC50 value of 1.3 nM for a primary human prostate cancer section. In comparison, the IC50 values for [111In-DTPA-Pro1,Tyr4]BN were 2.28 ± 0.57 nM and 1.40 ± 0.07 nM for the rat colon and human PC-295, respectively.

De Visser et al. (1) studied the cell internalization of [111In-DTPA-ACMpip5,Tha6,βAla11,Tha13,NIe14]-BN(5-14) in the rat pancreatic tumor cell line CA20948 and human prostate tumor cell line PC3. The radioligand had the highest rate of internalization in the first hour of incubation. Extrapolated from Figure 2a of de Visser et al. (1), the internalization, expressed as a percentage of the injected dose per mg cellular protein (% ID/mg) of the radioligand, in the CA20948 cells was ~26% ID/mg at 60 min and ~32% ID/mg at 180 min. In comparison, the internalization of [111In-DTPA-Pro1,Tyr4]BN was ~11% ID/mg and ~23% ID/mg at 60 min and 180 min, respectively. The cell internalization was different in the PC3 cells. Extrapolated from Figure 2b of de Visser et al. (1), [111In-DTPA-ACMpip5,Tha6,βAla11,Tha13,NIe14]-BN(5-14) was internalized ~6% ID/mg at 60 min and ~7.75% ID/mg at 180 min. In comparison, the internalization of [111In-DTPA-Pro1,Tyr4]BN was ~5% ID/mg and ~7% ID/mg at 60 min and 180 min, respectively. When a blocking study was performed by adding increased amounts of unlabeled [Tyr4]BN to the incubating medium that contained 10–10 M of the radioactive probe, the results showed that the addition of 1–10 nM of unlabeled [Tyr4]BN effectively blocked the internalization of [111In-DTPA-ACMpip5,Tha6,βAla11,Tha13,NIe14]-BN(5-14).

An in vitro serum stability study of [111In-DTPA-ACMpip5,Tha6,βAla11,Tha13,NIe14]-BN(5-14) using HPLC analysis showed that the percentage of intact radiolabeled peptide was 74% after incubation in human serum for 4 h at 37ºC (1).

Animal Studies

Rodents

[PubMed]

Biodistribution studies of [111In-DTPA-ACMpip5,Tha6,βAla11,Tha13,NIe14]-BN(5-14) were performed in Lewis rats bearing a rat pancreatic tumor CA20948 (area = 2–4 cm2) and nude mice bearing a human prostate tumor PC3 (area = 1 cm2) (1). The dose of [111In-DTPA-ACMpip5,Tha6,βAla11,Tha13,NIe14]-BN(5-14) for each rat or mouse was 2–4 MBq/0.1 μg (54–108 μCi/0.1 μg) of radiolabeled peptide or 2–4 MBq/0.06 pmol (54–108 μCi/0.06 pmol) of radiolabeled peptide on the basis of the peptide’s molecular weight of 1,644. In the rats with a CA20948 tumor, the tumor radioactivity levels (n = 3–27) in percentage injected dose per gram (% ID/g) were 0.583 ± 0.244 (4 h), 0.521 ± 0.115 (24 h), 0.277 ± 0.167 (48 h), and 0.266 ± 0.082 (72 h). The pancreas and bowel (stomach, small intestine, caecum, and colon) also had relatively high radioactivity levels. The tumor/blood ratios were 49, 74, 92, and 133 for 4 h, 24 h, 48 h, and 72 h, respectively. The tumor/muscle ratios were 146, 130, 69, and 89 for 4 h, 24 h, 48 h, and 72 h, respectively. The tumor/kidney ratios were 0.5, 0.4, 0.3, and 0.4 for 4 h, 24 h, 48 h, and 72 h, respectively. In comparison, the tumor/normal tissue radioactivity ratios (n = 6) at 4 h for 111In-DTPA-Pro1,Tyr4]BN were 50 (tumor/blood), 126 (tumor/muscle), and 0.2 (tumor/kidney). When a blocking dose of 100 μg [Tyr4]BN (n = 20) was co-injected, the tumor radioactivity level decreased from 0.583 ± 0.244% ID/g to 0.067 ± 0.088% ID/g at 4 h, and the pancreas radioactivity level was reduced from 2.883 ± 1.135% ID/g to 0.185 ± 0.520% ID/g. The bowel radioactivity levels were all decreased. For the mice with a PC3 tumor (n = 11), the tumor radioactivity level was 0.839 ± 0.577% ID/g at 4 h and was reduced to 0.120 ± 0.122% ID/g (n = 7) with a co-injection dose of 50 μg [Tyr4]BN. The tumor/normal tissue radioactivity ratios at 4 h were 18 (tumor/blood), 49 (tumor/muscle), and 0.8 (tumor/kidney). The pancreas radioactivity level was 15.010 ± 3.930% ID/g at 4 h and was reduced to 0.553 ± 0.312% ID/g by [Tyr4]BN. The bowel radioactivity levels were also reduced by the blocking dose. In comparison, the [111In-DTPA-Pro1,Tyr4]BN had 4-h tumor/normal tissue ratios of 28 (tumor/blood), 36 (tumor/muscle), and 0.1 (tumor/kidney).

A tumor-imaging study was conducted in one rat bearing a CA20948 tumor (right flank) and a GRP-R–positive rat pancreatic tumor AR42J (left flank) (1). A rat received a [111In-DTPA-ACMpip5,Tha6,βAla11,Tha13,NIe14]-BN(5-14) dose of 4 MBq/0.1 μg (108 μCi/0.1 μg) radiolabeled peptide or 4 MBq/0.06 pmol radiolabeled peptide on the basis of the peptide’s molecular weight of 1,644. In this study, the radioactivity level in the AR42J tumor was readily detectable at 1 min, and the background radioactivity rapidly decreased after 1 h. At 4 h, the tumor/normal tissue radioactivity ratios were 85 (tumor/blood), 145 (tumor/muscle), and 0.58 (tumor/kidney). However, the radioactivity in the CA20948 tumor was relatively lower with tumor/normal tissue ratios of 34 (tumor/blood), 58 (tumor/muscle), and 0.23 (tumor/kidney) at 4 h. De Visser et al. (1) suggested that the lower radioactivity tumor level was a result of necrosis in the large CA20948 tumor and a higher GRP-R density for the AR42J tumor. When a microSPECT/CT study was performed in one nude mouse bearing a CA20948 tumor on the shoulder with a [111In-DTPA-ACMpip5,Tha6,βAla11,Tha13,NIe14]-BN(5-14) dose of 10 MBq/0.1 μg (0.27 mCi/μg) or 10 MBq/0.06 pmol (0.27 mCi/0.06 pmol), the tumor radioactivity level was much higher than that in the rat bearing the tumor. At 4 h, the tumor/blood ratio was 99, the tumor/muscle ratio was 234, and the tumor/kidney ratio was 3.5. The sagittal, coronal, and transaxial slices showed a clear localization of the tumor with no interfering background.

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. 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]
  • 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]
    6.
    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]
    7.
    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]
    8.
    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]
    9.
    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]
    10.
    Breeman W.A., Hofland L.J., de Jong M., Bernard B.F., Srinivasan A., Kwekkeboom D.J., Visser T.J., Krenning E.P. Evaluation of radiolabelled bombesin analogues for receptor-targeted scintigraphy and radiotherapy. Int J Cancer. 1999;81(4):658–65. [PubMed: 10225459]
    11.
    Bakker W.H., Albert R., Bruns C., Breeman W.A., Hofland L.J., Marbach P., Pless J., Pralet D., Stolz B., Koper J.W. et al. [111In-DTPA-D-Phe1]-octreotide, a potential radiopharmaceutical for imaging of somatostatin receptor-positive tumors: synthesis, radiolabeling and in vitro validation. Life Sci. 1991;49(22):1583–91. [PubMed: 1658515]

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