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99mTc-Nicotinic acid/tricine/hydrazinonicotinamide-sulfated cholecystokinin-8


, PhD and , PhD.

Author Information
, PhD
National Center for Biotechnology Information, NLM, NIH, vog.hin.mln.ibcn@dacim
, PhD
Department of Nuclear Medicine, Radboud University Nijmegen Medical Center, Nijmegen,
The Netherlands, ln.ncmu.demcun@namreval.p

Created: ; Last Update: September 13, 2007.

Chemical name:99mTc-Nicotinic acid/tricine/hydrazinonicotinide-sulfated cholecystokinin-8image 26681507 in the ncbi pubchem database
Abbreviated name:99mTc-NA/tricine/HYNIC-sCCK-8
Agent Category:Peptide
Target:Gastrin/cholecystokinin-2 (CCK-2, CCK-B) receptor and CCK-1 (CCK-A) receptor
Target Category:Receptor binding
Method of detection:Single-photon emission computed tomography (SPECT), planar gamma imaging
Source of signal:99mTc
  • Checkbox In vitro
  • Checkbox Rodents

Click on the above structure for additional information in PubChem.



99mTc-Nicotinic acid/tricine/hydrazinonicotinamide-sulfated cholecystokinin-8 (99mTc-NA/tricine/HYNIC-sCCK-8) is a radiolabeled peptide developed for single-photon emission computed tomography (SPECT) imaging of tumors that express the gastrin/cholecystokinin-2 (CCK-2) receptor (1). 99mTc is a gamma emitter with a physical half-life (t½) of 6.01 h.

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 of 34 amino acids that also exists in several C-terminal truncated forms (4), which include the minigastrin, a 13-residue peptide with the sequence of LEEEEEAYGWMDF-NH2. CCK exists in a variety of biologically active molecular forms that are derived from a precursor molecule of 115 amino acids (5). They range from 4 to 58 amino acids in length and include sulfated (Tyr residue) and unsulfated CCK-8, which has the structure DYMGWMDF-NH2. They bind to and act through transmembrane G-protein–coupled receptors (6). Two different CCK receptor subtypes have been identified in normal tissue. CCK-1 (CCK-A, alimentary) receptors have low affinity for gastrin, and CCK-2 (CCK-B, brain) receptors have high affinity for gastrin (5). They also differ in terms of molecular structure, distribution, and affinity for CCK. These receptors have also been found to be expressed or overexpressed on a multitude of tumor types (6). 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. (7) designed a series of radiolabeled CCK-8 (cholecystokinin fragment 26-33) peptides that showed high specificity for potential in vivo imaging of tumors expressing CCK-2 receptors. Because of its favorable physical properties, 99mTc is still the radionuclide of choice for routine clinical applications (8). Hydrazinonicotinamide (HYNIC) is a bifunctional coupling agent for 99mTc labeling of peptides and proteins that can achieve high specific activities without interfering with the amino acid sequence responsible for receptor binding (9-11). In this approach, it is suggested that 99mTc is bound to the hydrazine group by forming a 99mTc(V)=N bond, and other coordination sites are occupied by one or more coligands (1, 12). The choice of coligand can influence the stability and hydrophilicity of the radiolabeled peptide. Using the HYNIC labeling strategy and nicotinic acid (NA)/tris(hydroxymethyl)-methylglycine (tricine) as the coligands, Laverman et al. (1) successfully labeled sCCK-8 for CCK receptor imaging in mice bearing tumors that express CCK. It has been shown that the sCCK-8 peptide displays high affinity for both the CCK-1 and CCK-2 receptors. Nonsulfated CCK-8 shows a 1,000-fold lower affinity for the CCK-1 receptor than for the CCK-2 receptor (1, 13).



Laverman et al. (1) reported the radiosynthesis of 99mTc-HYNIC-sCCK-8 by using ethylenediaminediacetic acid (EDDA), tricine, or a combination of NA/tricine as the coligand. Tricine, NA, EDDA, and sCCK-8 (Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2) were obtained commercially. The sCCK-8 peptide was conjugated to the bifunctional chelator N-hydroxysuccinimidyl hydrazino nicotinate (s-HYNIC) by adding s-HYNIC (in dimethyl sulfoxide) to the peptide in sodium bicarbonate (pH 8.2). The mixture was incubated for 30 min at room temperature, and purified HYNIC-sCCK-8 was obtained by preparative high-performance liquid chromatography (HPLC). The conjugation efficiency was 28 ± 3% (n = 3). In the NA/tricine procedure, tricine in phosphate-buffered saline (PBS) and NA in benzoate buffer (pH 5.0) were first mixed with HYNIC-sCCK-8. Freshly prepared stannous sulfate and 100–370 MBq (2.7–10 mCi) of sodium 99mTc-pertechnetate (Na99mTcO4) were then added. The mixture was incubated at 75ºC for 30 min. Solid-phase extraction (SPE) was used for purification of the radiolabeled peptide for the stability studies. The labeling efficiency with EDDA as the coligand was only 19 ± 1%. Although using tricine as the coligand alone gave a high labeling efficiency of 98 ± 1%, the obtained 99mTc-tricine/HYNIC-sCCK-8 was highly unstable. The combination of NA/tricine gave a high labeling efficiency of 96 ± 3% with a specific activity of 28.3 GBq/μmol (0.76 Ci/μmol). The final radioligand 99mTc-NA/tricine/HYNIC-sCCK-8 was much more stable. The chemical structure of this HYNIC compound was not determined.

In Vitro Studies: Testing in Cells and Tissues


The in vitro stability of 99mTc-NA/tricine/HYNIC-sCCK-8 was tested by incubating the radiolabeled peptide in PBS at 37ºC for 16 h and the radiochemical purity (RCP) was assessed by HPLC (1). The RCP values (n = 3) of 99mTc-NA/tricine/HYNIC-sCCK-8 were 83 ± 3% and 77 ± 2% at 5 min and 16 h, respectively. In comparison, the RPC of 99mTc-tricine/HYNIC-sCCK-8 prepared by tricine alone gave RCP values of 88 ± 2% and 42 ± 3% at 5 min and 16 h, respectively.

The in vitro binding affinity of 99mTc-NA/tricine/HYNIC-sCCK-8 was determined on Chinese hamster ovary (CHO) cells expressing a CCK receptor (CCK-1R or CCK-2R) (1). The 50% inhibitory concentration (IC50) values of 99mTc-NA/tricine/HYNIC-sCCK-8 were 8 nM for the CCK-1 receptor and 3 nM for the CCK-2 receptor. The radioligand showed rapid binding and time-dependent internalization. After incubation at 37ºC for 2 h, CCK-1R and CCK-2R CHO cells internalized 2.7% and 27% of 99mTc-NA/tricine/HYNIC-sCCK-8, respectively. Addition of excess unlabeled peptide blocked both the binding and internalization of the radioligand. The internalized radioactivity was also rapidly excreted by the CHO-CCK-2R cells, with 36% of the internalized activity excreted after 2 h of incubation. The SPE analysis showed that about 14 ± 4% of the excreted activity was 99mTcO4.

Animal Studies



Laverman et al. (1) conducted biodistribution studies of 99mTc-NA/tricine/HYNIC-sCCK-8 in nude mice bearing s.c. CCK receptor–transfected CHO tumors (~0.2 g). Each mouse received 3.7 MBq (100 μCi) 99mTc-NA/tricine/HYNIC-sCCK-8 (17 ng) radioactivity. The radiolabeled peptide was rapidly cleared from the blood with high radioactivity levels in the tumor and CCK-1R positive pancreas. At 1 h, the radioactivity levels (n = 5) of 99mTc-NA/tricine/HYNIC-sCCK-8 in percentage injected dose per g (% ID/g) for the major organs were 4.15 ± 0.28 (CCK-2R tumor), 2.04 ± 0.65 (CCK-1R tumor), 11.7 ± 0.93 (pancreas), 2.14 ± 0.23 (kidney), 2.01 ± 0.46 (stomach), 0.29 ± 0.06 (liver), 0.38 ± 0.08 (intestine), 0.31 ± 0.13 (blood), and 0.08 ± 0.04 (muscle). The tumor/blood ratios were 14.7 ± 4.3 and 7.1 ± 2.6 for the CCK-2R tumor and CCK-1R tumor, respectively. In comparison, the radioactivity levels of 99mTc-NA/tricine/HYNIC-nonsulfated CCK-8 peptide were only 1.02 ± 0.12% ID/g and 0.57 ± 0.13% ID/g for the CCK-2R tumor and CCK-1R tumor, respectively. With coinjection of a 1,000 molar excess of unlabeled sCCK-8, the tumor radioactivity levels were decreased to 1.02 ± 0.12 and 0.57 ± 0.13 for the CCK-2R tumor and CCK-1R tumor, respectively. The pancreas radioactivity level was also reduced to 0.56 ± 0.09, but the blood level was not affected by the blocking dose.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.


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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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