MSH radiopeptides for targeting melanoma metastases.

Radiolabeled peptides have become important tools for preclinical cancer research and in nuclear oncology they serve as diagnostic and more recently also as therapeutic agents. Whereas the development of receptor-mediated targeting for therapy has been confined to some radiolabeled antibodies and somatostatin/SRIF analogs, recent research into radiolabeled α-Melanocyte-stimulating hormone (α-MSH) and its receptor MC1R (over-)expressed by melanoma tumor cells has demonstrated that small metastatic melanoma lesions in experimental animals are specifically targeted by MSH radiopeptides. Thus MSH radiopharmaceuticals will eventually open a new avenue for the treatment of melanoma metastases in man, provided that the targeting efficiency can be further enhanced and nonspecific incorporation into nontarget organs, e.g., the kidneys, minimized. Some novel MSH lead compounds containing a glyco moiety, added negatively charged groups or a cyclic structure show very promising in vivo targeting characteristics.

Glycosylated DOTA-alpha-melanocyte-stimulating hormone analogues for melanoma targeting: influence of the site of glycosylation on in vivo biodistribution.

alpha-Melanocyte-stimulating hormone (alpha-MSH) is known to bind to the melanocortin receptor 1 (MC1R) which is overexpressed on melanotic and amelanotic melanoma cells. alpha-MSH analogues are potential candidates for specific targeting of melanoma metastases. Several linear and cyclic radiolabeled MSH peptides have been designed and tested in the past, showing both high affinity for the MC1R in vitro and good incorporation in tumor xenografts in vivo. However, considerable kidney reabsorption of the radiopeptides could not be avoided. With the aim to increase the tumor-to-kidney ratio, we synthesized six glycosylated derivatives of NAPamide, an alpha-MSH octapeptide analogue with high tumor selectivity and coupled them to the chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). The peptides were evaluated in vitro for MC1R binding and bioactivity and, after labeling with (111)In, for in vitro cellular uptake and in vivo tissue distribution in mice carrying B16F1 melanoma tumors. The glycopeptides showed excellent binding affinities in the low nanomolar to subnanomolar range using both murine and human melanoma cell lines. However, five glycopeptides displayed lower selectivity in vivo than the parent DOTA-NAPamide, because of either a lower tumor uptake or a higher kidney uptake. In particular C-terminal extension of the amide group by a galactosyl moiety increased the kidney retention dramatically. By contrast, an N-terminally positioned galactose residue in DOTA-Gal-NAPamide improved the tumor-to-kidney ratio (4-48 h AUC of 1.34) by a factor of about 1.2 as compared to the parent DOTA-NAPamide (4-48 h AUC of 1.11), thus serving as new lead compound for MC1R-targeting molecules.

Dimeric DOTA-alpha-melanocyte-stimulating hormone analogs: synthesis and in vivo characteristics of radiopeptides with high in vitro activity.

Dimeric analogs of alpha-melanocyte-stimulating hormone (alpha-MSH) labeled with radiometals are potential candidates for diagnosis and therapy of melanoma by receptor-mediated tumor targeting. Both melanotic and amelanotic melanomas (over-)express the melanocortin-1 receptor (MC1-R), the target for alpha-MSH. In the past, dimerized MSH analogs have been shown to display increased receptor affinity compared to monomeric MSH, offering the possibility of improving the ratio between specific uptake of radiolabeled alpha-MSH by melanoma and nonspecific uptake by the kidneys. We have designed three linear dimeric analogs containing a slightly modified MSH hexapeptide core sequence (Nle-Asp-His-d-Phe-Arg-Trp) in parallel or antiparallel orientation, a short spacer, and the DOTA chelator for incorporation of the radiometal. In vitro, all three peptides were more potent ligands of the mouse B16-F1 melanoma cell melanocortin-1 receptor (MC1-R) than DOTA-NAPamide, which served as standard. The binding activity of DOTA-diHexa(NC-NC)-amide was 1.75-fold higher, that of diHexa(NC-NC)-Gly-Lys(DOTA)-amide was 3.37-fold higher, and that of DOTA-diHexa(CN-NC)-amide was 2.34-fold higher. Using human HBL melanoma cells, the binding activity of diHexa(NC-NC)-Gly-Lys(DOTA)-amide was sixfold higher than that of DOTA-NAPamide. Uptake by cultured B16-F1 cells was rapid and almost quantitative. In vivo, however, the data were less promising: tumor-to-kidney ratios 4 hr postinjection were 0.11 for [(111)In]DOTA-diHexa(NC-NC)-amide, 0.26 for diHexa(NC-NC)-Gly-Lys([(111)In]DOTA)-amide, and 0.36 for [(111)In]DOTA-diHexa(CN-NC)-amide, compared to 1.67 for [(111)In]DOTA-NAPamide. It appears that despite the higher affinity to the MC1-R of the peptide dimers and their excellent internalization in vitro, the uptake by melanoma tumors in vivo was lower, possibly because of reduced tissue penetration. More striking, however, was the marked increase of kidney uptake of the dimers, explaining the unfavorable ratios. In conclusion, although radiolabeled alpha-MSH dimer peptides display excellent receptor affinity and internalization, they are no alternative to the monomeric DOTA-NAPamide for in vivo application.

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.

Different structural requirements for melanin-concentrating hormone (MCH) interacting with rat MCH-R1 (SLC-1) and mouse B16 cell MCH-R.

Melanin-concentrating hormone (MCH) is a neuropeptide occurring in all vertebrates and some invertebrates and is now known to stimulate pigment aggregation in teleost melanophores and food-intake in mammals. Whereas the two MCH receptor subtypes hitherto cloned, MCH-R1 and MCH-R2, are thought to mediate mainly the central effects of MCH, the MCH-R on pigment cells has not yet been identified, although in some studies MCH-R1 was reported to be expressed by human melanocytes and melanoma cells. Here we present data of a structure-activity study in which 12 MCH peptides were tested on rat MCH-R1 and mouse B16 melanoma cell MCH-R, by comparing receptor binding affinities and biological activities. For receptor binding analysis with HEK-293 cells expressing rat MCH-R1 (SLC-1), the radioligand was [125I]-[Tyr13]-MCH with the natural sequence. For B16 cells (F1 and G4F sublines) expressing B16 MCH-R, the analog [125I]-[D-Phe13, Tyr19]-MCH served as radioligand. The bioassay used for MCH-R1 was intracellular Ca2+ mobilization quantified with the FLIPR instrument, whereas for B16 MCH-R the signal determined was MAP kinase activation. Our data show that some of the peptides displayed a similar relative increase or decrease of potency in both cell types tested. For example, linear MCH with Ser residues at positions 7 and 16 was almost inactive whereas a slight increase in side-chain hydrophilicity at residues 4 and 8, or truncation of MCH at the N-terminus by two residues hardly changed binding affinity or bioactivity. On the other hand, salmonic MCH which also lacks the first two residues of the mammalian sequence but in addition has different residues at positions 4, 5, 9, and 18 exhibited a 5- to 10-fold lower binding activity than MCH in both cell systems. A striking difference in ligand recognition between MCH-R1 and B16 MCH-R was however observed with modifications at position 13 of MCH: whereas L-Phe13 in [Phe13, Tyr19]-MCH was well tolerated by both MCH-R1 and B16 MCH-R, change of configuration to D-Phe13 in [D-Phe13, Tyr19]-MCH or [D-Phe13]-MCH led to a complete loss of biological activity and to a 5- to 10-fold lower binding activity with MCH-R1. By contrast, the D-Phe13 residue increased the affinity of [D-Phe13, Tyr19]-MCH to B16 MCH-R about 10-fold and elicited MAP kinase activation as observed with [Phe13, Tyr19]-MCH or MCH. These data demonstrate that ligand recognition by B16 MCH-R differs from that of MCH-R1 in several respects, indicating that the B16 MCH-R represents an MCH-R subtype different from MCH-R1.

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.

Endogenous receptor for melanin-concentrating hormone in human neuroblastoma Kelly cells.

Melanin-concentrating hormone (MCH), a cyclic nonadecapeptide, is predominantly expressed in mammalian neurons located in the zona incerta and lateral hypothalamus. Current interest in MCH relates to its role in the control of feeding behaviour. Two receptors for MCH were recently found: MCH-R(1) and MCH-R(2). We show here by RT-PCR analysis and immunofluorescence studies that the human neuroblastoma cell line Kelly expresses MCH and MCH-R(1) but not MCH-R(2). In competition assays using 125I-labelled MCH an inhibitory concentration 50% (IC(50)) of 76nM was determined for MCH, indicating a high affinity of Kelly cells for MCH. MCH induces mitogen-activated protein kinase (MAPK) phosphorylation in Kelly cells but no increase in the intracellular free Ca(2+) concentration. This suggests that MCH signals via Galpha(i)/Galpha(0) in these cells. The presence and functionality of MCH-R(1) renders this neuronal cell a very useful model for future structure-activity studies in a physiological environment mimicking the human brain for the evaluation of potential appetite-regulating drugs.