Background

[PubMed]

^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 (t_½) 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).

Synthesis

[PubMed]

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 (Na^99mTcO₄) 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

[PubMed]

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

Human Studies

[PubMed]

No publication is currently available.

References

1.

1.Laverman, P., M. Behe, W.J. Oyen, P.H. Willems, F.H. Corstens, T.M. Behr, and O.C. Boerman, Two technetium-99m-labeled cholecystokinin-8 (CCK8) peptides for scintigraphic imaging of CCK receptors. Bioconjug Chem, 2004. 15(3): p. 561-8. [PubMed: 15149184]

2.

Reubi J. C. , Schaer J. C. , Waser B. Cholecystokinin(CCK)-A and CCK-B/gastrin receptors in human tumors. 1997. pp. 1377–86. [PubMed: 9102227]

3.

Aly A. , Shulkes A. , Baldwin G. S. Gastrins, cholecystokinins and gastrointestinal cancer. 2004. pp. 1–10. [PubMed: 15238241]

4.

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. 2007. pp. 615–22. [PMC free article: PMC2246928] [PubMed: 17401100]

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Wang H. , Wong P. T. , Spiess J. , Zhu Y. Z. Cholecystokinin-2 (CCK2) receptor-mediated anxiety-like behaviors in rats. 2005. pp. 1361–73. [PubMed: 16120463]

6.

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. 2002. pp. 97–109. [PubMed: 11965605]

7.

Reubi J. C. , Waser B. Unexpected high incidence of cholecystokinin-B/gastrin receptors in human medullary thyroid carcinomas. 1996. pp. 644–7. [PubMed: 8782652]

8.

Aloj L. , Panico M. R. , Caraco C. , Zannetti A. , Del Vecchio S. , Di Nuzzo C. , Arra C. , Morelli G. , Tesauro D. , De Luca S. , Pedone C. , Salvatore M. Radiolabeling approaches for cholecystokinin B receptor imaging. 2002. pp. 370–80. [PubMed: 12658724]

9.

9.Liu, S. and D.S. Edwards, 99mTc-Labeled Small Peptides as Diagnostic Radiopharmaceuticals. Chem Rev, 1999. 99(9): p. 2235-68. [PubMed: 11749481]

10.

10.Decristoforo, C., L. Melendez-Alafort, J.K. Sosabowski, and S.J. Mather, 99mTc-HYNIC-[Tyr3]-octreotide for imaging somatostatin-receptor-positive tumors: preclinical evaluation and comparison with 111In-octreotide. J Nucl Med, 2000. 41(6): p. 1114-9. [PubMed: 10855644]

11.

11.Barrett, J.A., A.C. Crocker, D.J. Damphousse, S.J. Heminway, S. Liu, D.S. Edwards, J.L. Lazewatsky, M. Kagan, T.J. Mazaika, and T.R. Carroll, Biological evaluation of thrombus imaging agents utilizing water soluble phosphines and tricine as coligands when used to label a hydrazinonicotinamide-modified cyclic glycoprotein IIb/IIIa receptor antagonist with 99mTc. Bioconjug Chem, 1997. 8(2): p. 155-60. [PubMed: 9095355]

12.

12.Harris, T.D., M. Sworin, N. Williams, M. Rajopadhye, P.R. Damphousse, D. Glowacka, M.J. Poirier, and K. Yu, Synthesis of stable hydrazones of a hydrazinonicotinyl-modified peptide for the preparation of 99mTc-labeled radiopharmaceuticals. Bioconjug Chem, 1999. 10(5): p. 808-14. [PubMed: 10502347]

13.

13.Smeets, R.L., M.A. Fouraux, S.E. van Emst-de Vries, J.J. De Pont, and P.H. Willems, Protein kinase C-mediated inhibition of transmembrane signalling through CCK(A) and CCK(B) receptors. Br J Pharmacol, 1998. 123(6): p. 1189-97. [PMC free article: PMC1565266] [PubMed: 9559904]

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.