J. Med. Chem. 2005, 48, 2985-2992
Imaging of Melanoma Using Peptide Analogue of r-MSH
64
Cu- and
86
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Y-DOTA-ReCCMSH(Arg11), a Cyclized
Paul McQuade,† Yubin Miao,‡ Jeongsoo Yoo,† Thomas P. Quinn,‡ Michael J. Welch,†,§ and Jason S. Lewis*,†,§ Division of Radiological Sciences, Mallinckrodt Institute of Radiology and the Alvin J. Siteman Cancer Center, Washington University School of Medicine, St. Louis, Missouri 63110, and Department of Biochemistry, University of MissourisColumbia, Columbia, Missouri 65212 Received November 30, 2004
Early detection of melanoma is essential, since a patient’s prognosis with metastatic melanoma is poor. Previous studies showed that 111In-DOTA-ReCCMSH(Arg11), a cyclic analogue of R-melanocyte stimulating hormone (R-MSH), exhibited high tumor concentration and rapid clearance from nontarget tissue. The goal of this current study was to label DOTA-ReCCMSH(Arg11) with β+-emitting radionuclides, to determine if the high sensitivity of positron emission tomography (PET) imaging would aid in the detection of malignant melanoma. DOTAReCCMSH(Arg11) was labeled with 64Cu and 86Y. Biodistribution and small animal PET imaging were carried out in mice implanted with B16/F1 murine melanoma tumor and compared with data obtained in the same animal model with [18F]FDG. In both cases a subset of animals were co-injected with 20 µg of DOTA-ReCCMSH(Arg11) to determine if tumor concentration was receptor mediated. Tumor concentration for both the 86Y- and 64Cu-complexes reached a maximum at 30 min, while coadministering 20 µg of unlabeled complex reduced tumor uptake significantly. Nontarget organ concentration was considerably lower with 86Y-DOTAReCCMSH(Arg11) than its 64Cu analogue, except in the kidneys, where the 64Cu complex had lower accumulation at all time points. Small animal PET images for both complexes showed the tumor could be visualized after 30 min, with the standardized uptake value (SUV) analysis following a similar trend as the biodistribution data. The data obtained suggests that DOTAReCCMSH(Arg11), when labeled with β+-emitting radionuclides, has the potential for early detection of malignant melanoma by exploiting the sensitivity and high resolution of PET. Introduction Malignant melanoma is the most serious form of skin cancer. In the United States alone there was expected to be more than 55 000 cases of invasive melanoma and 41 000 cases of in situ melanoma diagnosed in 2004, with invasive melanoma accounting for almost 2% of the total number of cancer deaths.1 It is the most common malignancy among young adults with the incidence and mortality rates increasing in western countries;2 along with this, the likelihood of being diagnosed increases with age.3 Early detection is essential as, unless the primary tumor is excised, typically through surgery, patient prognosis is poor.4 At present the compound most commonly used for diagnosing and staging of cancer via positron emission tomography (PET) is 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG).5 [18F]FDG works by being transported into cells that are metabolically active before becoming trapped intercellularly,6 and as cancerous cells typically have a higher metabolic rate than normal cells, they exhibit increased accumulation of [18F]FDG.7 However, for detecting melanoma tumors with small foci [18F]FDG has its limitations.8 Also some melanoma cells use * Corresponding author. Phone: (314) 362-4696. Fax: (314) 3629940. E-mail:
[email protected]. † Mallinckrodt Institute of Radiology, Washington University School of Medicine. ‡ University of MissourisColumbia. § Alvin J. Siteman Cancer Center, Washington University School of Medicine.
substrates other than glucose as an energy source, making them undetectable with [18F]FDG.9 There is therefore a need to develop new PET agents that would be better at both detecting and treating melanoma, as melanoma metastases can also be resistant to conventional therapeutic agents.10 Melanocortin-1 receptors (MC1R) belong to a family of G-protein-coupled receptors of which five (MC1R to MC5R) have been isolated in both mice and humans.11-14 These receptors are normally expressed on the surface of melanocytes but have been found to be overexpressed in melanoma cells and tissue.15-21 R-Melanotropinstimulating hormone (R-MSH), a tridecapeptide produced in the brain and pituitary gland,22 has been found to bind avidly to MC1R, and as a consequence, peptide analogues may be useful in both the detection and treatment of melanoma. Structure-bioactivity studies of R-MSH have shown that the amino acid sequence HisPhe-Arg-Trp (HFRW) is sufficient for receptor recognition,22 with replacement of phenylalanine with its stereoisomer (D-phenylalanine) resulting in up to a 1500-fold increase in receptor binding.23 Peptide analogues containing this core sequence have been prepared, with some analogues possessing subnanomolar affinities for MC1R receptors while being internalized quickly upon binding,24 thus making analogues of this type useful targets in the targeting and treatment of malignant melanoma. One such peptide analogue that was examined and found to have nanomolar affinity for MC1R was the
10.1021/jm0490282 CCC: $30.25 © 2005 American Chemical Society Published on Web 03/08/2005
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Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8
McQuade et al.
Table 1. Biodistribution (% ID/g ( SD, n ) 4) in C57 Mice Bearing Day-10 B16/F1 Melanoma Tumors at 30 min and 2, 4, and 24 h after 86Y-DOTA-ReCCMSH(Arg11) and 64Cu-DOTA-ReCCMSH(Arg11) Administrationa
tissue
30 min
blood lung liver spleen kidney muscle heart skin B16/F1 tumor
2.18 ( 0.83 2.86 ( 0.85 7.35 ( 1.49 1.08 ( 0.29 12.43 ( 3.62 1.13 ( 0.44 1.64 ( 0.67 2.07 ( 0.90 9.68 ( 1.51
blood lung liver spleen kidney muscle heart skin B16/F1 tumor
1.69 ( 0.28 1.81 ( 0.44 0.66 ( 0.09 0.57 ( 0.08 21.21 ( 4.18 0.82 ( 0.13 0.83 ( 0.14 2.10 ( 0.39 11.87 ( 3.31
2h
2h with blockade
64Cu-DOTA-ReCCMSH(Arg11)
0.71 ( 0.10 2.67 ( 0.53 7.25 ( 1.02 0.73 ( 0.13 9.88 ( 1.26 0.49 ( 0.17 1.11 ( 0.21 0.68 ( 0.25 8.80 ( 1.70
0.80 ( 0.18 2.84 ( 0.69 8.34 ( 1.39 1.01 ( 0.49 9.33 ( 1.36 0.79 ( 0.45 1.21 ( 0.30 0.88 ( 0.27 2.22 ( 0.65
86Y-DOTA-ReCCMSH(Arg11) 0.06 ( 0.01 0.07 ( 0.02 0.20 ( 0.04 0.15 ( 0.01 0.28 ( 0.04 0.23 ( 0.01 0.13 ( 0.05 0.12 ( 0.03 18.63 ( 3.62 13.86 ( 1.85 0.06 ( 0.02 0.05 ( 0.01 0.07 ( 0.02 0.06 ( 0.02 0.21 ( 0.11 0.14 ( 0.03 9.83 ( 2.27 0.53 ( 0.31
4h
24 h
0.65 ( 0.16 3.17 ( 0.57 7.34 ( 1.79 0.81 ( 0.16 9.75 ( 2.88 0.32 ( 0.04 1.15 ( 0.29 0.48 ( 0.12 7.35 ( 1.47
0.45 ( 0.19 2.16 ( 0.73 4.85 ( 1.52 0.82 ( 0.35 5.05 ( 1.37 0.30 ( 0.15 1.15 ( 0.54 0.80 ( 1.04 3.11 ( 0.68
0.04 ( 0.00 0.16 ( 0.12 0.25 ( 0.03 0.14 ( 0.04 16.92 ( 3.18 0.04 ( 0.01 0.04 ( 0.02 0.15 ( 0.03 9.98 ( 2.05
0.06 ( 0.02 0.08 ( 0.02 0.48 ( 0.17 0.92 ( 0.11 11.67 ( 2.78 0.03 ( 0.00 0.04 ( 0.01 0.09 ( 0.01 0.78 ( 0.27
a Two additional groups were coadministered blockade (20 µg DOTA-ReCCMSH(Arg11)), and one additional group of tumor-bearing animals was administered 10 µCi [18F]FDG. [18F]FDG (% ID/g ( SD, n ) 4) at 2 h: blood, 0.24 ( 0.06; lung, 2.19 ( 0.06; liver, 0.56 ( 0.05; spleen, 1.65 ( 0.19; kidney, 0.65 ( 0.17; muscle, 4.83 ( 0.75; heart, 16.68 ( 6.38; tumor, 5.08 ( 1.09; skin, 1.29 ( 0.67.
cyclic peptide Re-[Cys3,4,10,D-Phe7]R-MSH3-13 (ReCCMSH), in which the 99mTc analogue was able to target melanoma in a murine-tumor model system.25 Further studies with this peptide have conjugated 1,4,7,10tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA) to the terminal amine, allowing attachment of radiometals such as 111In.26 This strategy, however, increased nonspecific concentration in the kidneys, which was compensated for by replacing the lysine present with arginine [DOTA-ReCCMSH(Arg11)]. 111In studies with this DOTA-conjugated peptide carried out on the same melanoma murine-tumor model that was used for 99mTcCCMSH show the 111In-labeled peptide exhibiting high tumor concentration with low uptake and rapid clearance from nontarget tissue.26 This report describes our studies with DOTAReCCMSH(Arg11) labeled with β+-emitting radiometals to determine if it is a viable agent for the detection of malignant melanoma via PET imaging. The divalent metal ion 64Cu (t1/2 ) 12.7 h, 17.4% β+, 41% EC, 40% β-, Eavg ) 278 keV) and trivalent metal ion 86Y (t1/2 ) 14.7 h, β+ ) 33%, Eavg ) 664 keV), both of which can be prepared on a biomedical cyclotron utilizing the 64Ni(p,n)64Cu and 86Sr(p,n)86Y reactions, respectively,27,28 were chosen for evaluation. This was due to the fact that the half-life and unique decay scheme of 64Cu make it the most versatile copper radionuclide with both diagnostic and therapeutic applications.29 86Y finds use not only as a PET emitting isotope but also as an imaging surrogate for 90Y, which has been used extensively in nuclear medicine as a therapeutic isotope. Results Radiolabeling of DOTA-ReCCMSH(Arg11). DOTA-ReCCMSH(Arg11) was successfully labeled with 64Cu and 86Y at 65 and 85 °C, respectively. Upon isolation by RP-HPLC, 64Cu-DOTA-ReCCMSH(Arg11) (tR ) 27 min) was obtained with a radiochemical purity of >95% and a specific activity of 300 µCi/µg, while 86Y-
DOTA-ReCCMSH(Arg11) (tR ) 34 min) was recovered with a radiochemical purity of >98% and a specific activity approaching 3000 µCi/µg. As a comparison, the unlabeled DOTA-ReCCMSH was eluted under the same HPLC conditions with a tR of 27 min. Receptor Quantitation Assay. The MC1 receptor density on B16/F1 cells was determined in order for a complete comparison to be made with previous studies.30 A greater than 2-fold increase in the number of MC1 receptors per B16/F1 cell was observed when the cell line was incubated in DMEM-HG media used for the 111In studies31 (8009 ( 1606 receptors/cell) compared with the cells cultured in RPMI 1640 media that was used with the 86Y and 64Cu described in this report (3191 ( 139 receptors/cell). Biodistribution Studies. In the acute biodistribution experiments female C57 mice bearing 10-day B16/ F1 murine melanoma tumors were used. Data at 30 min, 2, 4, and 24 h was obtained for 64Cu-DOTAReCCMSH(Arg11) (5 µCi, 16.6 ng, 8.3 pmol) and 86YDOTA-ReCCMSH(Arg11) (5 µCi, 1.7 ng, 0.8 pmol). The results represent biodistribution performed with a similar mass of each radiolabeled peptide (1.7-16.6 ng). Two additional groups were coadministered with unlabeled peptide to act as a blockade (20 µg of DOTAReCCMSH(Arg11), 9.9 nmol) and were sacrificed at 2-h postinjection. For comparison, an additional group of animals received [18F]FDG and were sacrificed at 2-h postinjection. The 64Cu- and 86Y-DOTA-ReCCMSH(Arg11) data along with the [18F]FDG data obtained at 2 h are summarized in Table 1. At 2 h postinjection, the tumor concentration of 86YDOTA-ReCCMSH(Arg11) (9.83 ( 2.27% ID/g) and 64Cu-DOTA-ReCCMSH(Arg11) (8.80 ( 1.70% ID/g) compared favorably with that of [18F]FDG (5.08 ( 1.09% ID/g). This represents an approximate 2-fold increase in tumor concentration for the MC1-targeting agents compared to that of the metabolic agent [18F]FDG. For both 86Y-DOTA-ReCCMSH(Arg11) and 64Cu-DOTA-
Imaging of Melanoma with an Analogue of R-MSH
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 2987
Figure 1. Tumor concentration (table insert) and selected tumor/tissue ratios for 86Y-DOTA-ReCCMSH(Arg11), 64Cu-DOTAReCCMSH(Arg11), 111In-DOTA-ReCCMSH(Arg11),26 and [18F]FDG in C57 mice implanted with B16F1 tumors. Tumor concentration of 67/68Ga-DOTA-NAPamide in B6D2F1 mice implanted with B16F1 tumors.32 Data presented as % ID/g ( SD (n ) 4). *Ratios calculated from mean values reported; #Data not available.
ReCCMSH(Arg11), tumor concentration reached a maximum after only 30 min (9.68 ( 1.51% ID/g and 11.87 ( 3.31% ID/g, respectively, p ) not significant (NS)) that gradually decreased over 24 h (64Cu, 3.11 ( 0.68% ID/g vs 86Y, 0.78 ( 0.27% ID/g, p < 0.001). However, the values obtained were lower than those reported for 111In-DOTA-ReCCMSH at 4 h (17.4 ( 5.6% ID/g) in the same tumor model.30 To fully place the tumor data in context, a comparison of the data obtained from 64Cu-DOTA-ReCCMSH(Arg11) and 86Y-DOTA-ReCCMSH(Arg11) with tumor concentration and selected tumor-tissue ratios obtained for [18F]FDG, 111In-DOTAReCCMSH(Arg11),26 and 67/68Ga-DOTA-NAPamide32 is shown in Figure 1. It is apparent that there is a significant increase in the tumor/tissue ratios for 86YDOTA-ReCCMSH(Arg11) when compared with 64CuDOTA-ReCCMSH(Arg11) (tumor/blood at 4 h, 64Cu, 11.4 ( 1.0 vs 86Y, 259.4 ( 37.6, p < 0.005; tumor/muscle at 4 h, 64Cu, 22.8 ( 1.7 vs 86Y, 228.1 ( 41.3, p
