Background

[PubMed]

^99mTc-[Cys^3,4,10,d-Phe⁷,Arg¹¹]αMSH_3-13 (^99mTc-(Arg¹¹)CCMSH) is a radioligand developed as a single-photon emission computed tomography (SPECT) imaging probe for primary and metastatic melanoma (1). ^99mTc-(Arg¹¹)CCMSH is an α-melanocyte−stimulating hormone (MSH) peptide labeled with ^99mTc, a gamma emitter with a physical t_½ of 6 h.

Malignant melanoma is the sixth most common cancer in the United States (1). Early diagnosis and prompt surgical removal comprise the best approach for a possible cure (2). The melanocortin (MC) system is the best characterized neuropeptide network of the skin, and it is involved in pigmentation regulation, cortisol production and many other physiological processes (3). Most cutaneous cell types express MC receptors, proopiomelanocortin (POMC), prohormone convertases, and also release MCs. Five MC receptors (MC-1 to MC-5) have been cloned and characterized as receptors that belong to the G-protein−coupled receptors superfamily. MSHs (α-, β-, and γ-MSH) are derived from POMC by the proteolytic action of prohormone convertases. α-MSH (Ac-Ser¹-Tyr²-Ser³-Met⁴-Glu⁵-His⁶-Phe⁷-Arg⁸-Trp⁹-Gly¹⁰-Lys¹¹-Pro¹²-Val¹³-NH₂) is a 13−amino acid peptide and is the most potent naturally occurring melanotropic peptide (4). The biological effects of α-MSH are mediated via MC receptors.

Although positron emission tomography (PET) imaging with [¹⁸F]fluoro-2-deoxy-2-d-glucose ([¹⁸F]FDG) is effective in the detection of melanoma, it is not melanoma-specific and some melanoma cells do not take up [¹⁸F]FDG (5, 6). Radiolabeled α-MSH peptide analogs have been shown to specifically bind to MC-1 receptors which are overexpressed on human and mouse melanoma cells (5, 7-10). Some studies have successfully used 1,4,7,10-tetraazacyclodecane-N,N',N'',N'''-tetraacetic acid (DOTA) coupled to the α-MSH peptide analogs for radiolabeling. These α-MSH derivatives (DOTA-α-MSH) can be labeled with a variety of radionuclides (5). To improve the in vivo pharmacokinetics of these radiolabeled peptides, a number of different α-MSH analogs have been designed (11, 12). Froidevaux et al. (11) showed that the kidney uptake of a short linear DOTA−α-MSH analog (DOTA-NAPamide) could be considerably reduced by neutralizing the charge of the Lys¹¹ residue. Giblin et al. (13) used metal cyclization to design a new class of α-MSH peptide analogs that are resistant to chemical and proteolytic degradation in vivo (10). ^99mTc-CCMSH analogs, cyclized through site-specific coordination of ^99mTcO, showed excellent tumor uptake and retention properties in B16/F1 murine melanoma-bearing C57 mice (13, 14). Miao et al. (12) replaced the Lys¹¹ with Arg¹¹ in the CCMSH analog to form (Arg¹¹)CCMSH in an effort to reduce nonspecific kidney uptake and minimize the kidney radiation dose.

Synthesis

[PubMed]

Miao et al. (12) reported the synthesis of the (Arg¹¹)CCMSH by using the standard fluorenylmethoxycarbonyl (Fmoc)/HBTU chemistry on amide resin. A solid-phase peptide synthesizer was used. The peptide was acetylated with glacial acetic acid at the N-terminus. The peptide was deprotected and cleaved from the resin by a mixture of trifluoroacetic acid, thioanisol, ethanedithiol, and water at room temperature for 3 h. The peptide was then purified by high-performance liquid chromatography (HPLC), and the identity was confirmed by electrospray ionization mass spectrometry. The ^99mTc radiolabeling was prepared via a glucoheptonate transchelation reaction (14).In this procedure, stannous chloride (SnCl₂) was used as a reducing agent and glucoheptonate was used as a transfer ligand. Briefly, 10 μg of the peptide was added to the mixture of SnCl₂, ^99mTc-pertechnetate, and glucoheptonate. The mixture was incubated at 75ºC for 30 min. The final ^99mTc-(Arg¹¹)CCMSH product was purified by HPLC. The specific activity was 1.2135 x 10¹⁰ MBq/g (3.280 x 10⁸ mCi/g). Based on a molecular weight of 1,467, the specific activity can be estimated to be 1.79 x 10⁷ MBq/μmol (48.4 x 10⁴ mCi/μmol).The radiochemical stability was evaluated in phosphate- buffered saline (pH 7.4). Over a 24-h period of incubation at 25ºC in phosphate-buffered saline, only radiolabeled peptide was detected by HPLC analysis with no detectable free radioactivity (14).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Giblin et al. (13) performed in vitro quantitative receptor-binding assays of unlabeled CCMSH on B16/F1 murine melanoma cells. The inhibition constant (IC₅₀) was determined to be 7.6 x 10−⁹ M. Miao et al. (12) determined the IC₅₀ of unlabeled (Arg¹¹)CCMSH to be 1.7 X 10−⁹ M, and the molecular weight was measured to be 1,476.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

Supplemental Information

[Disclaimers]

NIH Support

NCI P50-CA-103130

References

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2.

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Tatro J.B., Reichlin S. Specific receptors for alpha-melanocyte-stimulating hormone are widely distributed in tissues of rodents. Endocrinology. 1987;121(5):1900–7. [PubMed: 2822378]

8.

Siegrist W., Solca F., Stutz S., Giuffre L., Carrel S., Girard J., Eberle A.N. Characterization of receptors for alpha-melanocyte-stimulating hormone on human melanoma cells. Cancer Res. 1989;49(22):6352–8. [PubMed: 2804981]

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Chen J., Cheng Z., Owen N.K., Hoffman T.J., Miao Y., Jurisson S.S., Quinn T.P. Evaluation of an (111)In-DOTA-rhenium cyclized alpha-MSH analog: a novel cyclic-peptide analog with improved tumor-targeting properties. J Nucl Med. 2001;42(12):1847–55. [PubMed: 11752084]

11.

Froidevaux S., Calame-Christe M., Tanner H., Eberle A.N. Melanoma targeting with DOTA-alpha-melanocyte-stimulating hormone analogs: structural parameters affecting tumor uptake and kidney uptake. J Nucl Med. 2005;46(5):887–95. [PubMed: 15872364]

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Miao Y., Owen N.K., Whitener D., Gallazzi F., Hoffman T.J., Quinn T.P. In vivo evaluation of 188Re-labeled alpha-melanocyte stimulating hormone peptide analogs for melanoma therapy. Int J Cancer. 2002;101(5):480–7. [PubMed: 12216078]

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Giblin M.F., Wang N., Hoffman T.J., Jurisson S.S., Quinn T.P. Design and characterization of alpha-melanotropin peptide analogs cyclized through rhenium and technetium metal coordination. Proc Natl Acad Sci U S A. 1998;95(22):12814–8. [PMC free article: PMC23606] [PubMed: 9788997]

14.

Chen J., Cheng Z., Hoffman T.J., Jurisson S.S., Quinn T.P. Melanoma-targeting properties of (99m)technetium-labeled cyclic alpha-melanocyte-stimulating hormone peptide analogues. Cancer Res. 2000;60(20):5649–58. [PubMed: 11059756]