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111In-Tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid-dihistidine-norleucine peptide analog

, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD, vog.hin.mln.ibcn@dacim

Created: ; Last Update: December 11, 2007.

Chemical name:111In-Tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid-dihistidine-norleucine peptide analog
Abbreviated name:111In-DOTA-H2-Nle
Synonym:Radiolabeled minigastrin
Agent Category:Peptide
Target:Gastrin/cholecystokinin-2 (CCK-2, CCK-B) receptor
Target Category:Receptor binding
Method of detection:Single-photon emission computed tomography (SPECT), planar gamma imaging
Source of signal:111In
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Click on protein, nucleotide (RefSeq), and gene for more information about.the CCK-2 receptor.



111In-Tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid-dihistidine-norleucine peptide (111In-DOTA-H2-Nle) is a radiolabeled gastrin analog that can be used for single-photon emission computed tomography (SPECT) imaging of tumors that express the gastrin/cholecystokinin-2 (CCK-2) receptor (1). 111In is a gamma emitter with a physical half-life (t½) of 2.8 days.

The gastrointestinal peptides gastrin and CCK have various regulatory functions in the brain and gastrointestinal tract (2). Gastrin and CCK have the same COOH-terminal pentapeptide amide sequence, which is the biologically active site (3). Human gastrin is a peptide composed of 34 amino acids and also exists in several C-terminal–truncated forms (1). These C-terminal–truncated forms include minigastrin, which is a 13-residue peptide with the sequence of LEEEEEAYGWMDF-NH2. CCKs exist in a variety of biologically active molecular forms that are derived from a precursor molecule comprising 115 amino acids (4). These forms range from 4 to 58 amino acids in length and include sulphated and unsulphated CCK-8, which has the structure DYMGWMDF-NH2. They bind to and act through transmembrane G-protein–coupled receptors (5). Two different CCK receptor subtypes have been identified in normal tissues. CCK-1 (CCK-A, alimentary) receptors have low affinity for gastrin, and CCK-2 (CCK-B, brain) receptors have high affinity for gastrin (4). They also differ in terms of molecular structure, distribution, and affinity for CCK. These receptors have also been found to be expressed or overexpressed in a multitude of tumor types (5). CCK-2 receptors have been found most frequently in medullary thyroid carcinomas, small-cell lung cancers, astrocytomas, and stromal ovarian cancers (2). CCK-1 receptors have been identified in gastroenteropancreatic tumors, meningiomas, and neuroblastomas.

Reubi et al. (6) designed a series of radiolabeled CCK-8 peptides that showed high specificity for potential in vivo imaging of tumors expressing CCK-2 receptors. de Jong et al. (7) developed an 111In-labeled unsulphated CCK8 analog that used 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) as a bifunctional chelating agent. The radioligand showed high specific internalization rates in receptor-positive rat pancreatic AR42J tumor cells. von Guggenberg et al. (8) reported the synthesis of 99mTc-hydrazinonicotinic acid (HYNIC)-minigastrin complexes and high tumor uptake in nude mice bearing AR42J tumors. Nock et al. (9) prepared 99mTc-labeled minigastrin analogs and found that they displayed high specific localization in nude mice bearing AR42J tumors. Mather et al. (1) synthesized a library of different peptide sequences based on the C-terminal sequences of CCK-8 or minigastrin. These peptides were labeled with 111In by DOTA or diethylene triamine pentaacetic acid (DTPA) conjugation. The dihistidine analog 111In-DOTA-H2-Nle was evaluated to study the effect of substituting a norleucine (Nle) residue for the methionine (Met) residue near the C-terminus. The study appeared to show that this substitution could result in a reduction in tumor uptake of the radiopeptide analog.



The peptide sequence HHEAYGWMDF-amide was obtained by solid-phase peptide synthesis under standard conditions from commercial sources (1). The N-terminus was capped with a DOTA chelating group to produce DOTA-H2-Nle. The identity and purity were confirmed by matrix-assisted laser desorption/ionization mass spectroscopy and reverse-phase high-performance liquid chromatography. Radiolabeling was performed by mixing 111In-chloride in ammonium acetate and 0.04 M monothioglycerol (MTG), an antioxidant, with DOTA-H2-Nle in 0.01 M phosphate-buffered saline (pH 7.2). The mixture was heated at 98ºC for 15 min, and 0.1 M ethylenediamine tetraacetic acid (EDTA) was then added to quench the reaction. The labeling yield was >90%. The radiochemical purity and specific activity were not reported.

In Vitro Studies: Testing in Cells and Tissues


An in vitro receptor affinity assay was performed with AGS human gastric tumor cells transfected with the CCK-2 receptor (AGS-CCK2R) and 125I-G17 as the radioligand. The inhibition constant (Ki) and half of the maximum binding fraction (EC50) of unlabeled DOTA-H2-Nle were 16.7 nM and 11.3 nM, respectively. In comparison, the Ki and EC50 of DOTA-H2-Met were 5.7 nM and 3.9 nM, respectively.

Animal Studies



Biodistribution studies of 111In-DOTA-H2-Nle were performed in nude mice bearing rat pancreatic AR42J tumors or rat pancreatic CA20948 tumors (1). Both AR42J and CA20948 tumors expressed gastrin receptors. Each mouse received 0.2 μg of 111In-DOTA-H2-Nle by i.v. injection. The tumor radioactivity levels at 4 h (n = 3–4), represented as the percentage of injected dose per gram (% ID/g), were 0.73 ± 0.14 and 0.53 ± 0.08 for AR42J and CA20948, respectively. The blood radioactivity levels (% ID/g) at 4 h were 0.03 ± 0.00 and 0.03 ± 0.00 for AR42J and CA20948 tumors, respectively. The kidney radioactivity levels (% ID/g) at 4 h were 2.40 ± 2.13 and 1.73 ± 0.31 for AR42J and CA20948 tumors, respectively. No blocking study was performed. In comparison, the tumor radioactivity levels (% ID/g) of 111In-DOTA-H2-Met at 4 h were 0.87 ± 0.21 and 0.59 ± 0.10 for AR42J and CA20948 tumors, respectively.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.


Mather S.J. , McKenzie A.J. , Sosabowski J.K. , Morris T.M. , Ellison D. , Watson S.A. Selection of radiolabeled gastrin analogs for Peptide receptor-targeted radionuclide therapy. J Nucl Med. 2007;48(4):615–22. [PMC free article: PMC2246928] [PubMed: 17401100]
Reubi J.C. , Schaer J.C. , Waser B. Cholecystokinin(CCK)-A and CCK-B/gastrin receptors in human tumors. Cancer Res. 1997;57(7):1377–86. [PubMed: 9102227]
Aly A. , Shulkes A. , Baldwin G.S. Gastrins, cholecystokinins and gastrointestinal cancer. Biochim Biophys Acta. 2004;1704(1):1–10. [PubMed: 15238241]
Wang H. , Wong P.T. , Spiess J. , Zhu Y.Z. Cholecystokinin-2 (CCK2) receptor-mediated anxiety-like behaviors in rats. Neurosci Biobehav Rev. 2005;29(8):1361–73. [PubMed: 16120463]
Behr T.M. , Behe M.P. Cholecystokinin-B/Gastrin receptor-targeting peptides for staging and therapy of medullary thyroid cancer and other cholecystokinin-B receptor-expressing malignancies. Semin Nucl Med. 2002;32(2):97–109. [PubMed: 11965605]
Reubi J.C. , Waser B. Unexpected high incidence of cholecystokinin-B/gastrin receptors in human medullary thyroid carcinomas. Int J Cancer. 1996;67(5):644–7. [PubMed: 8782652]
de Jong M. , Bakker W.H. , Bernard B.F. , Valkema R. , Kwekkeboom D.J. , Reubi J.C. , Srinivasan A. , Schmidt M. , Krenning E.P. Preclinical and initial clinical evaluation of 111In-labeled nonsulfated CCK8 analog: a peptide for CCK-B receptor-targeted scintigraphy and radionuclide therapy. J Nucl Med. 1999;40(12):2081–7. [PubMed: 10616889]
von Guggenberg E. , Behe M. , Behr T.M. , Saurer M. , Seppi T. , Decristoforo C. 99mTc-labeling and in vitro and in vivo evaluation of HYNIC- and (Nalpha-His)acetic acid-modified [D-Glu1]-minigastrin. Bioconjug Chem. 2004;15(4):864–71. [PubMed: 15264875]
Nock B.A. , Maina T. , Behe M. , Nikolopoulou A. , Gotthardt M. , Schmitt J.S. , Behr T.M. , Macke H.R. CCK-2/gastrin receptor-targeted tumor imaging with (99m)Tc-labeled minigastrin analogs. J Nucl Med. 2005;46(10):1727–36. [PubMed: 16204724]


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