Melanoma targeting with DOTA-alpha-melanocyte-stimulating hormone analogs: structural parameters affecting tumor uptake and kidney uptake.

UNLABELLED: Radiolabeled analogs of alpha-melanocyte-stimulating hormone (alpha-MSH) are potential candidates for the diagnosis and therapy of melanoma metastases. After our recent observation that a linear octapeptide alpha-MSH analog incorporating the metal chelator 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) at the C-terminal lysine, [Nle(4),Asp(5),d-Phe(7),Lys(11)(DOTA)]-alpha-MSH(4-11) (DOTA-NAPamide), showed high accumulation in melanomas in a mouse model, low uptake in normal tissues, and moderate uptake in the kidneys, we attempted to identify the structural parameters influencing tumor uptake versus kidney uptake. METHODS: We designed a series of novel DOTA-alpha-MSH analogs differing from DOTA-NAPamide by small alterations, such as the position of DOTA in the peptide, hydrophobicity, and charge, by modifying the C-terminal Lys(11) residue. They were evaluated both for their melanocortin type 1 receptor (MC1R)-binding potency and for their biodistribution by use of the B16F1 melanoma mouse model. RESULTS: When DOTA was shifted to the N terminus of the peptide, a 3-fold increase in kidney retention was obtained. However, when the epsilon-amino group of the Lys(11) residue was acetylated in addition to the DOTA relocation, kidney uptake returned to the low values obtained with DOTA-NAPamide; this result indicated that neutralization of the epsilon-amino group positive charge of the Lys(11) residue rather than the position of DOTA accounted for the low kidney retention. Unexpectedly, no further reduction in kidney uptake was obtained by the introduction of 1 or 2 negative charges on Lys(11). Melanoma uptake was in accordance with MC1R affinity; the highest values were obtained for peptides bearing carboxy-terminal amidation and positioning of DOTA. CONCLUSION: The kidney uptake of DOTA-alpha-MSH analogs could be considerably reduced, without affecting MC1R affinity, by altering (neutralizing) the charge of the Lys(11) residue. Accordingly, the resulting peptides exhibited a high ratio of tumor uptake to kidney uptake that is favorable for diagnostic and therapeutic applications. These structure-activity data may help to improve the performance of DOTA-alpha-MSH analogs and other radiopeptides.

A gallium-labeled DOTA-alpha-melanocyte- stimulating hormone analog for PET imaging of melanoma metastases.

UNLABELLED: Although (18)F-FDG PET is widely used for metastatic melanoma diagnosis, it is less accurate than desirable, particularly for small foci. Since both melanotic and amelanotic melanomas overexpress receptors for alpha-melanocyte-stimulating hormone (alpha-MSH; receptor name, melanocortin type 1 receptor [MC1R]), radiolabeled alpha-MSH analogs are potential candidates for melanoma diagnosis. The aim of this study was to develop a positron emitter-labeled alpha-MSH analog suitable for PET imaging of melanoma metastases. METHODS: A short linear alpha-MSH analog, [Nle(4),Asp(5),D-Phe(7)]-alpha-MSH(4-11) (NAPamide), was newly designed and conjugated to the metal chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to enable radiometal incorporation. Compared with our previously reported DOTA-alpha-MSH analog, DOTA-MSH(oct) ([DOTA-betaAla(3),Nle(4),Asp(5),D-Phe(7),Lys(10)]-alpha-MSH(3-10)), the major modification lies in the conjugation of DOTA to the C-terminal end of the peptide via the epsilon-amino group of Lys(11), as opposed to the N-terminal alpha-amino group. After labeling with (111)In, (67)Ga, and the short-lived positron emitter (68)Ga, DOTA-NAPamide was characterized in vitro and in vivo using the mouse melanoma B16F1cell line. RESULTS: DOTA-NAPamide exhibited an almost 7-fold higher MC1R binding potency as compared with DOTA-MSH(oct). In B16F1 melanoma-bearing mice, both (111)In-DOTA-NAPamide and (67)Ga-DOTA-NAPamide behaved more favorably than (111)In-DOTA-MSH(oct). Both radiopeptides exhibited higher tumor and lower kidney uptake leading to tumor-to-kidney ratios of the 4- to 48-h area under the curve that were 4.6 times ((111)In) and 7.5 times ((67)Ga) greater than that obtained with (111)In-DOTA-MSH(oct). In addition, the 4-h kidney uptake of (67)Ga-DOTA-NAPamide could be reduced by 64% by coinjection of 15 mg L-lysine, without affecting tumor uptake. Skin primary melanoma as well as lung and liver melanoma metastases could be easily visualized on tissue section autoradiographs after systemic injection of (67)Ga-DOTA-NAPamide. The melanoma selectivity of DOTA-NAPamide was confirmed by PET imaging studies using (68)Ga-DOTA-NAPamide. Tumor uptake was found to be highest when the smallest amount of peptide was administered. CONCLUSION: DOTA-NAPamide labeled with either (111)In or (67)Ga/(68)Ga is in every way superior to (111)In-DOTA-MSH(oct) in murine models of primary and metastatic melanoma, which makes it a promising agent for melanoma targeting. High-contrast images obtained in PET studies with an experimental tumor model 1 h after injection augurs well for its clinical potential as an imaging tool.

DOTA alpha-melanocyte-stimulating hormone analogues for imaging metastatic melanoma lesions.

Scintigraphic imaging of metastatic melanoma lesions requires highly tumor-specific radiopharmaceuticals. Because both melanotic and amelanotic melanomas overexpress melanocortin-1 receptors (MC1R), radiolabeled analogues of alpha-melanocyte-stimulating hormone (alpha-MSH) are potential candidates for melanoma diagnosis. Here, we report the in vivo performance of a newly designed octapeptide analogue, [betaAla(3), Nle(4), Asp(5), D-Phe(7), Lys(10)]-alpha-MSH(3-10) (MSH(OCT)), which was conjugated through its N-terminal amino group to the metal chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to enable incorporation of radiometals (e.g., indium-111) into the peptide. DOTA-MSH(OCT) displayed high in vitro MC1R affinity (IC(50) 9.21 nM). In vivo [(111)In]DOTA-MSH(OCT) exhibited a favorable biodistribution profile after injection in B16-F1 tumorbearing mice. The radiopeptide was rapidly cleared from blood through the kidneys and, most importantly, accumulated preferentially in the melanoma lesions. Lung and liver melanoma metastases could be clearly imaged on tissue section autoradiographs 4 h after injection of [(111)In]DOTA-MSH(OCT). A comparative study of [(111)In]DOTA-MSH(OCT) with [(111)In]DOTA-[Nle(4), D-Phe(7)]-alpha-MSH ([(111)In]-DOTA-NDP-MSH) demonstrated the superiority of the DOTA-MSH(OCT) peptide, particularly for the amount of radioactivity taken up by nonmalignant organs, including bone, the most radiosensitive tissue. These results demonstrate that [(111)In]DOTA-MSH(OCT) is a promising melanoma imaging agent.

A novel DOTA-alpha-melanocyte-stimulating hormone analog for metastatic melanoma diagnosis.

UNLABELLED: Scintigraphic imaging of metastatic melanoma lesions requires highly tumor-specific radiolabeled compounds. Because both melanotic and amelanotic melanomas overexpress receptors for alpha-melanocyte-stimulating hormone (alpha-MSH; receptor name: melanocortin type 1 receptor, or MC1R), radiolabeled alpha-MSH analogs are potential candidates for melanoma diagnosis. The aim of this study was to develop a melanoma-selective radiolabeled alpha-MSH analog suitable for melanoma diagnosis. METHODS: The very potent alpha-MSH analog [Nle(4), D-Phe(7)]-alpha-MSH (NDP-MSH) and a newly designed alpha-MSH octapeptide analog, [betaAla(3), Nle(4), Asp(5), D-Phe(7), Lys(10)]-alpha-MSH(3-10) (MSH(oct)), were conjugated to the metal chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to enable radiometal incorporation. The resulting DOTA conjugates were evaluated in vitro for their MC1R-binding affinity and melanogenic activity in isolated mouse B16F1 cells and in vivo for their biodistribution in mouse models of primary and metastatic melanoma after labeling with (111)In. RESULTS: DOTA-MSH(oct) was shown to bind with high affinity (inhibitory concentration of 50% [IC(50)] = 9.21 nmol/L) to the MC1R, although with lower potency than does DOTA-NDP-MSH (IC(50) = 0.25 nmol/L). In B16F1 melanoma-bearing mice, both (111)In-DOTA-NDP-MSH and (111)In-DOTA-MSH(oct) exhibited high MC1R-mediated uptake by melanoma, which differed by a factor of only 1.5 at 4 h after injection. The main route of excretion for both radioconjugates was the kidneys, whereby (111)In-DOTA-MSH(oct) led to somewhat higher kidney values than did (111)In-DOTA-NDP-MSH. In contrast, the latter was much more poorly cleared from other nonmalignant tissues, including bone, the most radiosensitive organ. Therefore, (111)In-DOTA-MSH(oct) displayed higher uptake ratios of tumor to nontarget tissue (e.g., tumor-to-bone ratio 4 h after injection was 4.9 for (111)In-DOTA-NDP-MSH and 53.9 for (111)In-DOTA-MSH(oct)). Lung and liver melanoma metastases could easily be visualized on tissue section autoradiographs after injection of (111)In-DOTA-MSH(oct). Radio-reversed-phase high-performance liquid chromatography analysis of urine samples revealed that most (111)In-DOTA-MSH(oct) is excreted intact 4 h after injection, indicating good in vivo stability. CONCLUSION: (111)In-DOTA-MSH(oct) exhibits more favorable overall performance than does (111)In-DOTA-NDP-MSH in murine models of primary and metastatic melanoma, making it a promising melanoma imaging agent.