Novel ⁶⁴Cu-Labeled NOTA-Conjugated Lactam-Cyclized Alpha-Melanocyte-Stimulating Hormone Peptides with Enhanced Tumor to Kidney Uptake Ratios

Abstract

The aim of this study was to evaluate the effect of linker on tumor targeting and biodistribution of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex {⁶⁴Cu-1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-polyethylene glycol-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH₂} and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex {⁶⁴Cu-NOTA-8-aminooctanoic acid-Nle-CycMSH_hex} on melanoma-bearing mice. NOTA-PEG₂Nle-CycMSH_hex and NOTA-AocNle-CycMSH_hex were synthesized and purified by HPLC. The melanocortin-1 (MC1) receptor binding affinities of the peptides were examined on B16/F10 melanoma cells. The biodistribution of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex were determined on B16/F10 melanoma-bearing C57 mice. The melanoma imaging property of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex was further examined on B16/F10 melanoma-bearing C57 mice because of its higher melanoma uptake than ⁶⁴Cu-NOTA-AocNle-CycMSH_hex. The IC₅₀ values of NOTA- PEG₂Nle-CycMSH_hex and NOTA-AocNle-CycMSH_hex were 1.24 ± 0.07 and 2.75 ± 0.48 nM on B10/F10 melanoma cells. ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex were readily prepared with more than 90% radiolabeling yields and showed MC1R-specific binding on B16/F10 cells. ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex exhibited higher tumor uptake than ⁶⁴Cu-NOTA-AocNle-CycMSH_hex at 0.5, 2, 4 and 24 h post-injection. The tumor uptake of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex was 16.23 ± 0.42, 19.59 ± 1.48, 12.83 ± 1.69 and 8.78 ± 2.29% ID/g at 0.5, 2, 4 and 24 h post-injection, respectively. Normal organ uptake of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex was lower than 2% ID/g at 2 h post-injection except for kidney uptake. The renal uptake of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex was 3.66 ± 0.52, 3.27 ± 0.52 and 1.47 ± 0.56 ID/g at 2, 4 and 24 h post-injection, respectively. ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex showed high tumor to normal organ uptake ratios after 2 h post-injection. The B16/F10 melanoma lesions could be clearly visualized by positron emission tomography (PET) using ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex as an imaging probe at 2 h post-injection. High tumor uptake and low kidney uptake of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex underscored its potential as an MC1R-targeted theranostic peptide for melanoma imaging and therapy.

Graphical Abstract

INTRODUCTION

As the most lethal form of skin cancer, the financial burden of treating malignant melanoma continues to increase. Approximately 106,110 newly diagnosed cases and 7,180 deaths occurred in the United States in 2021. The 5-year survival of metastatic melanoma patients is only 35% although the new treatments (Vemurafenib, Ipilimumab and Nivolumab) have increased the overall survival of by months (2–7). Melanocortin-1 receptor (MC1R) is a G protein-coupled receptor which over-expresses on both amelanotic and melanotic human melanomas (8–10). Alpha-melanocyte-stimulating hormone (α-MSH) peptides can bind to MC1Rs with nanomolar binding affinities (11–23). Thus, numerous research efforts have been dedicated to the development of theranostic MC1R-targeted α-MSH peptides for melanoma imaging and therapy (11–23).

Building upon the lactam-cyclized key sequence of Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH₂ (GGNle-CycMSH_hex), we have conjugated both diagnostic and therapeutic radionuclides to yield a novel class of MC1R-targeted radiolabeled α-MSH peptides. For instance, our ⁶⁸Ga-DOTA-GGNle-CycMSh_hex displayed a B16/F10 melanoma uptake of 24.27 ± 3.74% ID/g at 1 h post-injection (10). Furthermore, ⁶⁸Ga-DOTA-GGNle-CycMSH_hex clearly detected human metastatic melanoma lesions in brain, lung, connective tissue and intestines (10). The remarkable images of melanoma metastases in patients by ⁶⁸Ga-DOTA-GGNle-CycMSH_hex highlighted the clinical relevance of MC1R for melanoma imaging and therapy.

Copper-64 (t_1/2=12.7 h, 17.4% β⁺, 40% β^-) is an attractive theranostic radionuclide because of the emissions of positrons and beta-particles. In our previous work, ⁶⁴Cu-NOTA-GGNle-CycMSH_hex {⁶⁴Cu-1,4,7-triazacyclononane-1,4,7-triacetic acid- GGNle-CycMSH_hex) displayed B16/F1 melanoma uptake of 12.39 ± 1.61% ID/g and 12.71 ± 2.68% ID/g at 2 h and 4 h post-injection (14). The melanoma lesions could be clearly imaged by positron emission tomography (PET) using ⁶⁴Cu-NOTA-GGNle-CycMSH_hex as an imaging probe (14). Furthermore, we reported that the replacement of the -GG- linker with 8-aminooctanoic acid (Aoc) linker increased the uptake of ^99mTc(EDDA)-hydrazinonicotinamide (HYNIC)-AocNle-CycMSH_hex in melanoma by more than 60% at 2 h and 4 h post-injection (15, 16). Therefore, we were interested whether the replacement of the -GG- linker with Aoc or polyethylene glycol (PEG) linker could affect the melanoma uptake of the ⁶⁴Cu-labeled NOTA-conjugated CycMSH_hex peptides.

In this study, we synthesized NOTA-AocNle-CycMSH_hex and NOTA-PEG₂Nle-CycMSH_hex using standard Fluorenylmethyloxycarbonyl (Fmoc) chemistry, radiolabeled both peptides with ⁶⁴Cu, and determined their melanoma targeting and biodistribution properties on B16/F10 melanoma-bearing C57 mice. Because ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex displayed higher tumor uptake than ⁶⁴Cu-NOTA-AocNle-CycMSH_hex at all time points investigated, we further examined the melanoma imaging of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex on B16/F10 melanoma-bearing C57 mice.

Experimental Section

Chemicals and Reagents

Amino acids and resin were purchased from Advanced ChemTech Inc. (Louisville, KY) and Novabiochem (San Diego, CA). NOTA(OtBu)₂ was purchased from CheMatech Inc. (Dijon, France) for peptide synthesis. ¹²⁵I-Tyr²-[Nle⁴, D-Phe⁷]-α-MSH {¹²⁵I-(Tyr²)-NDP-MSH} was obtained from PerkinElmer, Inc. (Waltham, MA) for competitive receptor binding assay. ⁶⁴CuCl₂ was purchased from Washington University School of Medicine (St. Louis, MO) for radiolabeling. B16/F10 murine melanoma cells were received from American Type Culture Collection (Manassas, VA). All other chemicals used in this study were purchased from Thermo Fisher Scientific (Waltham, MA) and used as received without further purification.

Peptide Synthesis and Receptor Binding Assay

NOTA-PEG₂Nle-CycMSH_hex and NOTA-AocNle-CycMSH_hex were synthesized on Sieber amide resin using standard Fmoc chemistry according to the procedure described in our previous publication (14). The peptides were purified by reverse phase-high performance liquid chromatography (RP-HPLC) and characterized by liquid chromatography-mass spectrometry (LC-MS). The MC1 receptor binding affinities of NOTA-PEG₂Nle-CycMSH_hex and NOTA-AocNle-CycMSH_hex were determined on B16/F10 melanoma cells by in vitro competitive receptor binding assay in the presence of 10⁻¹³ to 10⁻⁵ M of each peptide according to our published procedure (14). The IC₅₀ values were calculated using the Prism software (GraphPad Software, La Jolla, CA, USA).

Radiolabeling

⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex were prepared as described in our previous publication (14). The proposed schematic structures of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex are shown in Figure 1. Briefly, 10 μL of ⁶⁴CuCl₂ (37–74 MBq in 0.05 M HCl aqueous solution), 10 μL of 1 mg/mL peptide aqueous solution, and 200 μL of 0.5 M NH₄OAc (pH 5.4) were added into a reaction vial and incubated at 75 °C for 1 h. After incubation, 10 μL of 0.5% EDTA aqueous solution was added to scavenge potentially unbound ⁶⁴Cu²⁺. ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex complexes were purified by Waters RP-HPLC (Milford, MA) on a Grace Vydac C-18 reverse phase analytical column (Deerfield, IL) with a flow rate of 1 mL/min. A 20 min gradient of 20–30% acetonitrile in 20 mM HCl aqueous solution was used for ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex, whereas a 20 min gradient of 24–34% acetonitrile in 20 mM HCl aqueous solution was utilized for ⁶⁴Cu-NOTA-AocNle-CycMSH_hex. Each purified peptide solution was purged with N₂ gas for 15 min to remove the acetonitrile, then adjusted to pH 7.4 with 0.1 M NaOH and sterile saline for animal studies.

Figure 1.

Proposed schematic structures of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex.

Specific Binding

Specific binding of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex was determined on B16/F10 cells seeded on 24-well plates. The B16/F10 melanoma cells (1 × 10⁶ cells per well, n = 3) were incubated at 25 °C for 1 h with approximately 22.2 KBq of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex or ⁶⁴Cu-NOTA-AocNle-CycMSH_hex with or without 10 μg (6.07 nmol) of unlabeled [Nle⁴, D-Phe⁷]-α-MSH (NDP-MSH) in 0.3 mL of binding medium {Dulbecco’s modified Eagle’s medium with 25 mM N-(2-hydroxyethyl)-piperazine-N’-(2-ethanesulfonic acid), pH 7.4, 0.2% bovine serum albumin (BSA), 0.3 mM 1,10-phenathroline}. The binding medium was aspirated after incubation. The cells were washed twice with 0.5 mL of ice-cold 0.01 M phosphate buffered saline (PBS) buffer containing 0.2% BSA (pH = 7.4), and lysed with 0.5 mL of 1 M NaOH for 5 min, collected and measured in a Wallac 1480 automated gamma counter (PerkinElmer, NJ).

Biodistribution and Imaging Studies

All animal studies were conducted in compliance with Institutional Animal Care and Use Committee approval. C57 mice were purchased from Charles River Laboratory (Wilmington, MA). Each C57 mouse was subcutaneously inoculated with 1 × 10⁶ B16/F10 cells on the right flank to generate melanoma tumors. Ten days post inoculation, the tumor weights reached about 0.2 g and the melanoma-bearing C57 mice were used for biodistribution and PET imaging studies. Each melanoma-bearing mouse was injected with 0.37 MBq of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex or ⁶⁴Cu-NOTA-AocNle-CycMSH_hex via the tail vein. The specificity of the tumor uptake of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex was determined by co-injecting 10 μg (6.07 nmol) of unlabeled NDP-MSH. Mice were sacrificed at 0.5, 2, 4 and 24 h post-injection, and tumors and organs of interest were harvested, weighted and counted. Blood value was taken as 6.5% of the whole-body weight.

Since ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex displayed higher tumor uptake than ⁶⁴Cu-NOTA-AocNle-CycMSH_hex, the melanoma imaging property of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex was examined on B16/F10 melanoma-bearing C57 mice. Each mouse was injected with 7.4 MBq of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex via the tail vein. PET imaging studies of melanoma-bearing mice were performed at 2 h post-injection. Reconstructed PET data was visualized using VivoQuant (Invicro, Boston, MA).

Statistical Analysis

Statistical analysis was performed using the Student’s t test for unpaired data. A 95% confidence level was chosen to determine the significance of difference in tumor and kidney uptake between ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex with/without NDP-MSH blockade, tumor and kidney uptake between ⁶⁴Cu-NOTA-AocNle-CycMSH_hex with/without NDP-MSH blockade. The differences at the 95% confidence level (p < 0.05) were considered significant.

Results

The schematic structures of NOTA-PEG₂Nle-CycMSH_hex and NOTA-AocNle-CycMSH_hex are presented in Figure 1. Both peptides were synthesized, purified by HPLC and displayed greater than 90% purities after HPLC purification. The identities of the peptides were confirmed by mass spectrometry. As shown in Table 1, the measured molecular weights of NOTA-PEG₂Nle-CycMSH_hex and NOTA-AocNle-CycMSH_hex matched their calculated molecular weights. The molecular weights of NOTA-PEG₂Nle-CycMSH_hex and NOTA-AocNle-CycMSH_hex were 1412 and 1408. The IC₅₀ values of NOTA-PEG₂Nle-CycMSH_hex and NOTA-AocNle-CycMSH_hex were 1.24 ± 0.07 and 2.75 ± 0.48 nM on B10/F10 cells.

Table 1.

Molecular weights (MW) and IC₅₀ values of NOTA-PEG₂Nle-CycMSH_hex and NOTA-AocNle-CycMSH_hex peptides.

Peptide	Calculated MW	Measured MW	IC50 (nM)
NOTA-PEG2Nle-CycMSHhex	1412.6	1412.1	1.24 ± 0.07
NOTA-AocNle-CycMSHhex	1408.8	1408.1	2.75 ± 0.48

⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex were prepared in 0.5 M NH₄OAc-buffered solution with the greater than 90% radiochemical yields. Radioactive HPLC profiles of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex are shown in Figure 2. The retention time of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex was 12.3 and 12.7 min, respectively. The specific activity was 2.36 × 10⁴ mCi/μmol for ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex. As shown in Figure 2B, both ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex exhibited MC1R-specific binding. The peptide blockade reduced 92% of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and 70% of ⁶⁴Cu-NOTA-AocNle-CycMSH_hex cellular uptake.

Figure 2.

A. Radioactive HPLC profiles of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex. B. Specific binding of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex (Cu-64-PEG₂) and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex (Cu-64-Aoc) on B16/F10 melanoma cells with (black) and without (white) peptide blockade, respectively.

Table 2 and Table 3 showed the biodistribution results of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex . ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex displayed rapid melanoma uptake and prolonged tumor retention. The tumor uptake of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex was 16.23 ± 0.42, 19.59 ± 1.48, 12.83 ± 1.69 and 8.78 ± 2.29% ID/g at 0.5, 2, 4 and 24 h post-injection, respectively. Approximately 88% of tumor uptake of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex was blocked by 10 μg (6.07 nmol) of NDP-MSH (P<0.05), suggesting that the tumor uptake was MC1R-mediated. Normal organ uptake of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex was lower than 2% ID/g at 2 h post-injection except for kidney uptake (3.66 ± 0.52% ID/g).

Table 2.

Biodistribution of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex on B16/F10 melanoma-bearing C57 mice. The data are presented as percent injected dose per gram (%ID/g) or as percent injected dose (%ID) (means ± SD, n = 4)

Tissues	0.5 h	2 h	4 h	24 h	2 h NDP blockade
Percent injected dose/gram (%ID/g)
Tumor	16.23 ± 0.42	19.59 ± 1.48	12.83 ± 1.69	8.78 ± 2.29	2.37 ± 0.32*
Brain	0.24 ± 0.06	0.03 ± 0.01	0.02 ±.0.01	0.04 ± 0.03	0.02 ± 0.02
Blood	3.48 ± 0.95	0.18 ± 0.06	0.20 ± 0.03	0.27 ± 0.10	0.29 ± 0.14
Heart	2.29 ± 0.54	0.19 ± 0.01	0.18 ± 0.06	0.17 ± 0.05	0.21 ± 0.06
Lung	4.04 ± 0.12	0.70 ± 0.04	0.62 ± 0.18	0.69 ± 0.17	0.52 ± 0.14
Liver	2.61 ± 0.55	1.94 ± 0.26	1.95 ± 0.43	1.21 ± 0.23	1.27 ± 0.18
Spleen	1.88 ± 0.67	0.26 ± 0.12	0.41 ± 0.25	0.36 ± 0.17	0.33 ± 0.08
Stomach	2.21 ± 0.56	0.99 ± 0.17	0.92 ± 0.27	0.32 ± 0.15	0.67 ± 0.22
Kidneys	12.14 ± 1.51	3.66 ± 0.52	3.27 ± 0.52	1.47 ± 0.56	4.74 ± 0.48*
Muscle	1.28 ± 0.39	0.08 ± 0.06	0.03 ± 0.05	0.01 ± 0.01	0.12 ± 0.19
Pancreas	1.10 ± 0.20	0.21 ± 0.13	0.17 ± 0.11	0.01 ± 0.01	0.06 ± 0.05
Bone	2.00 ± 0.42	0.22 ± 0.07	0.19 ± 0.13	0.01 ± 0.01	0.19 ± 0.09
Skin	4.37 ± 1.12	0.40 ± 0.07	0.46 ± 0.22	0.18 ± 0.16	0.43 ± 0.13
Percent injected dose (%ID)
Intestines	3.26 ± 1.86	1.51 ± 0.23	1.63 ± 0.26	1.24 ± 0.38	1.38 ± 0.44
Urine	44.12 ± 1.85	85.24 ± 3.75	84.17 ± 2.73	88.69 ± 3.18	87.11 ± 2.62
Uptake ratio of tumor to normal tissue
Tumor/blood	4.66	108.83	64.15	32.52	8.17
Tumor/kidney	1.34	5.35	3.92	5.97	0.50
Tumor/lung	4.02	27.99	20.69	12.72	4.56
Tumor/liver	6.22	10.10	6.58	7.26	1.87
Tumor/muscle	12.68	244.88	427.67	878	19.75
Tumor/skin	3.71	48.98	27.89	48.78	5.51

^*p<0.05 for determining significance of differences in tumor and kidney uptake between ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex with or without peptide blockade at 2 h post-injection.

Table 3.

Biodistribution of ⁶⁴Cu-NOTA-AocNle-CycMSH_hex on B16/F10 melanoma-bearing C57 mice. The data are presented as percent injected dose per gram (%ID/g) or as percent injected dose (%ID) (means ± SD, n = 4)

Tissues	0.5 h	2 h	4 h	24 h	2 h NDP blockade
Percent injected dose/gram (%ID/g)
Tumor	5.69 ± 0.23	7.71 ± 0.67	5.47 ± 0.52	1.54 ± 0.16	2.03 ± 0.59*
Brain	0.20 ± 0.08	0.04 ± 0.01	0.02 ±.0.02	0.01 ± 0.01	0.02 ± 0.03
Blood	2.01 ± 0.60	0.27 ± 0.05	0.18 ± 0.07	0.02 ± 0.03	0.38 ± 0.09
Heart	1.49 ± 0.33	0.18 ± 0.05	0.12 ± 0.06	0.22 ± 0.04	0.28 ± 0.11
Lung	3.88 ± 0.71	0.72 ± 0.17	0.36 ± 0.08	0.38 ± 0.06	0.82 ± 0.24
Liver	3.42 ± 0.58	2.19 ± 0.14	0.95 ± 0.21	0.73 ± 0.01	2.20 ± 0.33
Spleen	0.99 ± 0.25	0.17 ± 0.12	0.18 ± 0.09	0.10 ± 0.14	0.34 ± 0.27
Stomach	1.67 ± 0.68	1.69 ± 0.57	0.43 ± 0.09	0.10 ± 0.05	0.74 ± 0.53
Kidneys	30.67 ± 0.94	3.29 ± 0.61	1.08 ± 0.22	0.83 ± 0.11	3.66 ± 0.50
Muscle	0.70 ± 0.18	0.11 ± 0.06	0.03 ± 0.01	0.08 ± 0.02	0.06 ± 0.05
Pancreas	0.75 ± 0.19	0.20 ± 0.18	0.07 ± 0.05	0.02 ± 0.04	0.14 ± 0.13
Bone	1.23 ± 0.34	0.13 ± 0.13	0.08 ± 0.10	0.08 ± 0.14	0.02 ± 0.01
Skin	2.93 ± 0.22	0.39 ± 0.05	0.11 ± 0.06	0.02 ± 0.02	0.21 ± 0.05
Percent injected dose (%ID)
Intestines	2.12 ± 0.45	3.78 ± 0.51	2.04 ± 0.25	0.74 ± 0.23	2.72 ± 0.45
Urine	56.53 ± 1.07	85.54 ± 3.69	89.41 ± 0.73	96.08 ± 0.39	87.14 ± 1.78
Uptake ratio of tumor to normal tissue
Tumor/blood	2.82	28.16	30.39	77.0	5.34
Tumor/kidney	0.19	2.34	5.06	1.86	0.55
Tumor/lung	1.47	10.71	15.19	4.05	2.48
Tumor/liver	1.66	3.52	5.76	2.11	0.92
Tumor/muscle	8.13	70.09	182.33	19.25	33.83
Tumor/skin	1.94	19.77	49.73	77.0	9.67

^*p<0.05 for determining significance of differences in tumor and kidney uptake between ⁶⁴Cu-NOTA-AocNle-CycMSH_hex with or without peptide blockade at 2 h post-injection.

⁶⁴Cu-NOTA-AocNle-CycMSH_hex exhibited lower tumor uptake than ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex at all time points investigated. The tumor uptake of ⁶⁴Cu-NOTA-AocNle-CycMSH_hex was 5.69 ± 0.23, 7.71 ± 0.67, 5.47 ± 0.52 and 1.54 ± 0.16% ID/g at 0.5, 2, 4 and 24 h post-injection, respectively. Approximately 74% of tumor uptake of ⁶⁴Cu-NOTA-AocNle-CycMSH_hex was decreased by 10 μg (6.07 nmol) of NDP-MSH blockade (P<0.05), indicating that the tumor uptake was MC1R-specific. Normal organ uptake of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex was lower than 2.2% ID/g at 2 h post-injection except for kidney uptake (3.29 ± 0.61% ID/g).

⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex showed higher tumor/kidney and tumor/liver ratios than ⁶⁴Cu-NOTA-AocNle-CycMSH_hex. Thus, we further performed PET imaging of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex on B16/F10 melanoma-bearing mice. The representative maximum intensity projection (MIP) and coronal PET images of Al¹⁸F-NOTA-PEG₂Nle-CycMSH_hex on B16/F10 melanoma-bearing C57 mice are presented in Figure 3. In agreement with biodistribution result, the B16/F10 tumor lesions were clearly imaged at 2 h post-injection using ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex as imaging probe.

Figure 3.

Representative maximum intensity projection (MIP) and coronal PET images of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex on a B16/F10 melanoma-bearing C57 mouse at 2 h post-injection. The melanoma lesions (T) are highlighted with arrows on the images.

Discussion

We demonstrated that the substitution of DOTA with NOTA dramatically improved the uptake in melanoma and decreased the kidney uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex as compared to ⁶⁴Cu-DOTA-GGNle-CycMSH_hex (14). The higher melanoma uptake and lower renal uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex improved the tumor to kidney uptake ratios of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex (14). Thus, NOTA is a better suited for ⁶⁴Cu chelation. Interestingly, we also reported that the switch from the -GG- linker to Aoc linker improved the melanoma uptake of ^99mTc(EDDA)-HYNIC-AocNle-CycMSH_hex by greater than 60% at 2 h and 4 h post-injection as compared to ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex (15, 16). Meanwhile, ^99mTc(EDDA)-HYNIC-PEG₂Nle-CycMSH_hex exhibited similar melanoma and renal uptake as ^99mTc(EDDA)-HYNIC-AocNle-CycMSH_hex (15, 16). Therefore, we were interested in the effect of PEG₂ and Aoc linkers on tumor targeting and biodistribution of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex on melanoma-bearing mice in this study.

NOTA-PEG₂Nle-CycMSH_hex exhibited slightly stronger MC1R binding affinity than NOTA-AocNle-CycMSH_hex on B16/F10 cells (1.24 vs. 2.75 nM). Similarly, ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex displayed greater MC1R-speific cellular uptake than ⁶⁴Cu-NOTA-AocNle-CycMSH_hex on B16/F10 cells. The cellular uptake of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex was 2.8 times the uptake of ⁶⁴Cu-NOTA-AocNle-CycMSH_hex (Fig. 2). Furthermore, ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex exhibited more tumor uptake than ⁶⁴Cu-NOTA-AocNle-CycMSH_hex on B16/F10 melanoma-bearing mice. The tumor uptake of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex was 2.9, 2.5, 2.3 and 5.7 times the uptake of ⁶⁴Cu-NOTA-AocNle-CycMSH_hex at 0.5, 2, 4 and 24 h post-injection (Tables 2 and 3). The uptake in kidneys and liver was comparably low for both ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex after 2 h post-injection. Interestingly, ^99mTc(EDDA)-HYNIC-AocNle-CycMSH_hex showed 60% higher melanoma uptake than ^99mTc(EDDA)-HYNIC-PEG₂Nle-CycMSH_hex at 2 h post-injection in our previous publications (15, 16), suggesting that the moiety of radiometal-chelator and the linker attached to the receptor-targeted peptide could affect the tumor uptake of radiolabeled peptides.

At the present time, ⁶⁴Cu-NOTA-GGNle-CycMSH_hex displayed the highest tumor/kidney ratios among all reported MC1R-targeted ⁶⁴Cu-labeled α-MSH peptides (14, 21–23). As shown in Fig. 4, ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex displayed higher tumor/kidney ratios than ⁶⁴Cu-NOTA-GGNle-CycMSH_hex at 2, 4 and 24 h post-injection. The tumor uptake of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex was 1.6 and 2.1 times the tumor uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex at 2 h and 4 h post-injection. The B16/F10 melanoma lesions could be clearly visualized using ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex as an imaging probe (Fig. 3). It is worthwhile to note that ⁶⁴Cu is also a therapeutic radionuclide with beta-emissions. The improved tumor/kidney uptake ratios of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex would potentially facilitate its application in melanoma therapy.

Figure 4.

Comparison of uptake in tumor and kidneys between ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex (PEG₂Nle) and ⁶⁴Cu-NOTA-GGNle-CycMSH_hex (GGNle) at 2, 4 and 24 h post-injection. The data of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex was cited from our previous publication (ref. 14) for comparison. *p<0.05

Over the past several years, various VLA-4-targeted (integrin α₄β₁-targeted) ⁶⁴Cu-labeled LLP2A peptides were developed and evaluated for melanoma targeting (24, 25). For instance, CB-TE1A1P (1,4,8,11-tetraazacyclotetradecane-1-(methane phosphonic acid)-8-(methane carboxylic acid), 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentanedioic acid (NODAGA) and 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A) were conjugated to the LLP2A peptide for ⁶⁴Cu chelation (24, 25). Among these reported ⁶⁴Cu-labeled LLP2A peptides, ⁶⁴Cu-CB-TE1A1P-PEG₄-LLP2A showed the highest B16/F10 melanoma uptake, with 15.1 ± 1.0% ID/g and 16.9 ± 2.2% ID/g at 2 h and 4 h post-injection (25). As demonstrated in this study, ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex exhibited similar uptake in tumor, liver and kidneys as ⁶⁴Cu-CB-TE1A1P-PEG₄-LLP2A. Meanwhile, ⁶⁴Cu-CB-TE1A1P-PEG₄-LLP2A also displayed high VLA-4-specific uptake in normal lung, bone and spleen (4.6–18.1% ID/g at 2 h post-injection) (25), whereas the uptake of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex was extremely low in these normal organs (<0.7% ID/g at 2 h post-injection). Low accumulation of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex in normal lung and bone would potentially facilitate the imaging of melanoma metastases in these organs. From the therapeutic point of view, the enhanced tumor/kidney and tumor/liver uptake ratios of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex would potentially increase the absorbed dose to tumor while minimizing the absorbed dose to liver and kidneys when treating the melanoma with ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex in future studies.

Conclusions

The melanoma targeting and biodistribution properties of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex and ⁶⁴Cu-NOTA-AocNle-CycMSH_hex were determined on B16/F10 melanoma-bearing C57 mice. ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex showed higher tumor uptake than ⁶⁴Cu-NOTA-AocNle-CycMSH_hex at all time points investigated. The favorable biodistribution and imaging properties of ⁶⁴Cu-NOTA-PEG₂Nle-CycMSH_hex underscored its potential as an MC1R-targeted theranostic peptide for melanoma imaging and therapy.