Melanoma-Targeting Property of Y-90-Labeled Lactam-Cyclized α-Melanocyte-Stimulating Hormone Peptide

Abstract

Purpose: The purpose of this study was to evaluate melanoma-targeting property of ⁹⁰Y-DOTA-GGNle-CycMSH_hex to facilitate its potential therapeutic application.

Materials and Methods: DOTA-GGNle-CycMSH_hex was synthesized and readily labeled with ⁹⁰Y in 0.25 M NH₄Ac-buffered solution to generate ⁹⁰Y-DOTA-GGNle-CycMSH_hex. The specific receptor binding, internalization, and efflux of ⁹⁰Y-DOTA-GGNle-CycMSH_hex were determined on B16/F10 murine melanoma cells. The biodistribution property of ⁹⁰Y-DOTA-GGNle-CycMSH_hex was examined on B16/F10 melanoma-bearing C57 mice.

Results: ⁹⁰Y-DOTA-GGNle-CycMSH_hex displayed receptor-specific binding, rapid internalization, and prolonged efflux on B16/F10 melanoma cells. ⁹⁰Y-DOTA-GGNle-CycMSH_hex exhibited high uptake and prolonged retention in melanoma, and fast urinary clearance on B16/F10 melanoma-bearing C57 mice. The B16/F10 tumor uptake was 20.73% ± 7.99%, 19.93% ± 5.73%, 14.8% ± 4.61%, and 6.69% ± 1.85% ID/g at 0.5, 2, 4, and 24 h postinjection, respectively.

Conclusions: ⁹⁰Y-DOTA-GGNle-CycMSH_hex displayed melanocortin-1 receptor (MC1R) targeting and specificity on B16/F10 melanoma cells and tumors. The favorable melanoma-targeting property and fast urinary clearance of ⁹⁰Y-DOTA-GGNle-CycMSH_hex warranted its evaluation for melanoma therapy in future studies.

Introduction

Malignant melanoma is the most lethal skin cancer and fifth most commonly diagnosed cancer in the United States with ∼96,480 new cases in 2019.¹ Extreme aggressiveness of metastatic melanoma leads to high mortality among metastatic melanoma patients. Despite the promising result that the median overall survival of metastatic melanoma patients has been improved by months through new treatments such as Vemurafenib (BRAF inhibitor), ipilimumab (targeting CTLA-4), and Nivolumab (PD-1 inhibitor),^2–6 the treatments are far from satisfactory due to the low long-term survival (<10%) for metastatic melanoma patients. There is an urgent need to develop new therapeutic agents for melanoma.

Melanocortin-1 receptor (MC1R) is an attractive G protein-coupled receptor (GPCR), which overexpresses on both murine and human melanoma cells.^7–12 Importantly, >80% of amelanotic and melanotic human metastatic melanoma samples exhibit MC1Rs.⁷ Recently, we have demonstrated the clinical relevance of MC1R for melanoma imaging through the first-in-human study of ⁶⁸Ga-DOTA-GGNle-CycMSH_hex (1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH₂), which targets MC1Rs.¹³ Remarkably, ⁶⁸Ga-DOTA-GGNle-CycMSH_hex positron emission tomography (PET) could clearly visualize the melanoma metastases in brain, lung, connective tissue, and small intestine of melanoma patients.¹³ These exciting first-in-human images of melanoma metastases highlighted the potential of MC1R as a melanoma target for targeted radionuclide therapy.

We have been interested in utilizing therapeutic radionuclides to target MC1Rs for melanoma therapy. In our previous report, we determined the melanoma-targeting property of ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex on B16/F1 melanoma-bearing C57 mice.¹⁴ Interestingly, ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex displayed high B16/F1 melanoma uptake of 20.25% ± 4.59% and 21.63% ± 6.27% ID/g at 0.5 and 2 h postinjection, respectively. Meanwhile, the melanoma lesions were clearly visualized using ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex as an imaging probe.¹⁴ In this study, we are interested in replacing ¹⁷⁷Lu with ⁹⁰Y to examine whether the change of therapeutic radionuclide could still maintain favorable melanoma-targeting property of ⁹⁰Y-DOTA-GGNle-CycMSH_hex. ⁹⁰Y is a high-energy β-particle emitter with a maximum β energy of 2.3 MeV and a half-life of 2.7 days. Specifically, we prepared ⁹⁰Y-DOTA-GGNle-CycMSH_hex and determined its MC1R-targeting property on B16/F10 melanoma cells and tumor-bearing mice.

Materials and Methods

Chemicals and reagents

Amino acids, DOTA-tri-tert-butyl ester, and resin were purchased from Advanced ChemTech, Inc. (Louisville, KY), Macrocyclics, Inc. (Richardson, TX) and Novabiochem (San Diego, CA) for peptide synthesis, respectively. ⁹⁰YCl₃ was purchased from PerkinElmer Health Sciences, Inc. (Waltham, MA) for radiolabeling and biodistribution. MC1R antibody (Rabbit/IgG) and FITC-conjugated antirabbit secondary antibody were purchased from Thermo Scientific (Rockford, IL) for MC1R staining on melanoma cells and tumors. All other chemicals used in this study were purchased from Thermo Fisher Scientific (Waltham, MA) and used without further purification. Four percent paraformaldehyde (PFA) in phosphate-buffered saline (PBS) was obtained from Alfa Aesar (Ward Hill, MA), xylene was obtained from Fisher Chemical (Fair Lawn, NJ), 4′,6-diamidino-2-phenylindole (DAPI) Fluoromount-G mounting medium was obtained from SouthernBiotech (Birmingham, AL), and Prolong Diamond antifade mounting reagent with DAPI was obtained from Life Technologies (Eugene, OR). B16/F10 murine melanoma cells were obtained from American Type Culture Collection (Manassas, VA).

Preparation, serum stability, and specific binding of ⁹⁰Y-DOTA-GGNle-CycMSH_hex

DOTA-GGNle-CycMSH_hex was synthesized using standard fluorenylmethyloxycarbonyl (Fmoc) chemistry and characterized by liquid chromatography-mass spectrometry.^{15 90}Y-DOTA-GGNle-CycMSH_hex was prepared in a 0.25 M NH₄OAc-buffered solution (pH 4.5). In brief, 30 μL of ⁹⁰YCl₃ (37–74 MBq in 0.05 M HCl aqueous solution), 10 μL of 1 mg/mL peptide aqueous solution, and 200 μL of 0.25 M NH₄OAc were added to a reaction vial and incubated at 75°C for 30 min. After the incubation, 10 μL of 0.5% EDTA (ethylenediaminetetraacetic acid) aqueous solution was added to the reaction vial to scavenge potential unbound ⁹⁰Y³⁺ ions. The radiolabeled complexes were purified to single species by Waters RP-HPLC (Milford, MA) on a Grace Vydac C-18 reverse-phase analytical column (Deerfield, IL) using the following gradient at a 1 mL/min flowrate. The mobile phase consisted of solvent A (20 mM HCl aqueous solution) and solvent B (100% CH₃CN). The gradient was initiated and kept at 82:18 A/B for 3 min followed by a linear gradient of 82:18 A/B to 72:28 A/B over 20 min. Then, the gradient was changed from 72:28 A/B to 10:90 A/B over 3 min followed by an additional 5 min at 10:90 A/B. Thereafter, the gradient was changed from 10:90 A/B to 82:18 A/B over 3 min. The purified peptide was purged with N₂ gas for 15 min to remove the acetonitrile. The pH of the final solution was adjusted to 7.4 with 0.1 N NaOH and sterile saline for animal studies.

In vitro serum stability of ⁹⁰Y-DOTA-GGNle-CycMSH_hex was determined by incubation in mouse serum at 37°C for 4 h and monitored for degradation by Reversed-phase high-performance liquid chromatography (RP-HPLC). The specific binding of ⁹⁰Y-DOTA-GGNle-CycMSH_hex was determined on B16/F10 melanoma cells. In brief, the B16/F10 cells (1 × 10⁶ cells/tube, n = 3) were incubated at 25°C for 2 h with ∼0.037 MBq of ⁹⁰Y-DOTA-GGNle-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 [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]. After the incubation, the cells were rinsed three times with 0.5 mL of ice-cold pH 7.4, 0.2% BSA/0.01 M PBS and measured in a Wallac 1480 automated γ counter (PerkinElmer, NJ).

Internalization and efflux of ⁹⁰Y-DOTA-GGNle-CycMSH_hex

Cellular internalization and efflux of ⁹⁰Y-DOTA-GGNle-CycMSH_hex were evaluated on B16/F10 melanoma cells. The B16/F10 cells (3 × 10⁵/well) were seeded onto a 24-well cell culture plate and incubated at 37°C overnight. After being washed once with binding medium, the cells were incubated at 25°C for 20, 40, 60, 90, and 120 min (n = 3) in the presence of ∼130,000 counts per minute of HPLC purified ⁹⁰Y-DOTA-GGNle-CycMSH_hex. After incubation, the cells were rinsed with 2 × 0.5 mL of ice-cold pH 7.4, 0.2% BSA/0.01 M PBS. Cellular internalization of ⁹⁰Y-DOTA-GGNle-CycMSH_hex was assessed by washing the cells with acidic buffer (40 mM sodium acetate [pH 4.5] containing 0.9% NaCl and 0.2% BSA) to remove the membrane-bound radioactivity. The remaining internalized radioactivity was obtained by lyzing the cells with 0.5 mL of 1 N NaOH for 5 min. Membrane-bound and internalized ⁹⁰Y activity was counted in a γ counter. Cellular efflux of ⁹⁰Y-DOTA-GGNle-CycMSH_hex was determined by incubating cells with ⁹⁰Y-DOTA-GGNle-CycMSH_hex at 25°C for 2 h, removing nonspecific bound activity with 2 × 0.5 mL of ice-cold pH 7.4, 0.2% BSA/0.01 M PBS rinse, and monitoring radioactivity released into cell culture medium. At time points of 20, 40, 60, 90, and 120 min, the radioactivity on cell surface and in cells was separately collected and counted in a γ counter.

MC1R staining on B16/F10 melanoma cells and lesions

The B16/F10 cells (1 × 10⁵ cells/well) were seeded onto a 4-well Lab-Tek Chamber Glass Slide System (Thermo Scientific, MA) and incubated at 37° overnight. After 24 h, the cells were fixed with 4% PFA in PBS and incubated at room temperature for 15 min, washed with PBS three times, treated with 0.5% Triton X-100 at room temperature for 15 min, and washed with PBS three times. The cells were incubated with MC1R antibody (1:300 dilution) at room temperature for 1 h followed by PBS wash three times, then incubated with FITC-conjugated antirabbit secondary antibody (1:100 dilution) at room temperature for 30 min followed by PBS wash three times. The cells were stained for nuclei and mounted with DAPI Fluoromount-G mounting medium (SouthernBiotech) and stayed in the dark at room temperature for 24 h. The fluorescent signal was observed and recorded at 100 × magnification under an Olympus FV1000 confocal microscope.

All animal studies were conducted in compliance with Institutional Animal Care and Use Committee approval. B16/F10 flank melanoma-bearing mice were generated for MC1R staining and biodistribution studies. In brief, each C57 mouse was subcutaneously inoculated with 1 × 10⁶ B16/F10 cells on the right flank. Ten days postinoculation, the tumor weights reached ∼0.2 g. The B16/F10 tumor was used to generate paraffin-embedded tumor sections (5 μm thickness) for MC1R staining. The paraffin-embedded tumor sections were deparaffinized with xylene first, and incubated with MC1R antibody (1:300 dilution) at room temperature for 1 h followed by PBS wash three times, then incubated with FITC-conjugated antirabbit secondary antibody (1:100 dilution) at room temperature for 30 min followed by PBS wash three times. Tissue samples were stained and mounted with Prolong Diamond antifade mounting reagent with DAPI (Life Technologies). The fluorescent signal was observed and recorded at 100 × magnification under an Olympus FV1000 confocal microscope.

Biodistribution and bremsstrahlung imaging of ⁹⁰Y-DOTA-GGNle-CycMSH_hex

The biodistribution property of ⁹⁰Y-DOTA-GGNle-CycMSH_hex was determined on B16/F10 flank melanoma-bearing C57 mice (Charles River, Wilmington, MA). Each melanoma-bearing mouse was injected with 0.037 MBq of ⁹⁰Y-DOTA-GGNle-CycMSH_hex through the tail vein. Mice were sacrificed at 0.5, 2, 4, and 24 h postinjection, and tumors and organs of interest were harvested, weighed, and counted. Blood values were taken as 6.5% of the whole-body weight. The specificity of the tumor uptake of ⁹⁰Y-DOTA-GGNle-CycMSH_hex was determined by coinjecting 10 μg (6.07 nmol) of unlabeled NDP-MSH, which is a linear α-MSH peptide analog with subnanomolar MC1R binding affinity.

We were interested whether B16/F10 melanoma lesions could be imaged by single photon emission computed tomography (SPECT) by collecting ⁹⁰Y bremsstrahlung photons. As an exploratory effort, we examined the bremsstrahlung imaging property of ⁹⁰Y-DOTA-GGNle-CycMSH_hex on B16/F10 flank melanoma-bearing C57 mice using a small energy window of 126.5–155.7 keV. Each melanoma-bearing mouse was injected with 7.4 MBq of ⁹⁰Y-DOTA-GGNle-CycMSH_hex through the tail vein. SPECT imaging study was performed at 2 h postinjection. CT data were collected followed by SPECT data acquisition. Reconstructed SPECT/CT data were visualized using Vivoquant (Invicro, Boston, MA).

Results

⁹⁰Y-DOTA-GGNle-CycMSH_hex (Fig. 1) was readily prepared with >95% radiolabeling yield, and was completely separated from its excess nonlabeled peptide by RP-HPLC. The retention time of ⁹⁰Y-DOTA-GGNle-CycMSH_hex and DOTA-GGNle-CycMSH_hex was 19.3 and 17.1 min, respectively. The specific activity of ⁹⁰Y-DOTA-GGNle-CycMSH_hex was ∼4.8962 × 10⁴ mCi/μmol. ⁹⁰Y-DOTA-GGNle-CycMSH_hex was stable in mouse serum at 37°C for 4 h (Fig. 2). ⁹⁰Y-DOTA-GGNle-CycMSH_hex displayed receptor-mediated binding on B16/F10 cells. Approximately 94% of ⁹⁰Y-DOTA-GGNle-CycMSH_hex uptake was blocked on B16/F10 cells (p < 0.05) (Fig. 2). The MC1R expression on B16/F10 cells was examined through fluorescence staining using MC1R antibody. As shown in Figure 2, MC1R-binding antibody showed substantial binding on B16/F10 cells.

FIG. 1.

Schematic structure of ⁹⁰Y-DOTA-GGNle-CycMSH_hex.

FIG. 2.

Fluorescence staining of MC1Rs on B16/F10 melanoma cells (A) using MC1R antibody (white). The nuclei were stained with DAPI (grey). The microscopic images were acquired by confocal laser microscopy at 100 × magnification. Scale bar, 20 μm. Radioactive HPLC profile of ⁹⁰Y-DOTA-GGNle-CycMSH_hex (B, T_R = 19.3 min) and its mouse serum stability (C) after 4 h incubation at 37°C. Arrows indicate the original compound of ⁹⁰Y-DOTA-GGNle-CycMSH_hex. Specific binding of ⁹⁰Y-DOTA-GGNle-CycMSH_hex on B16/F10 (D) cells with or without peptide blockade. DAPI, 4′,6-diamidino-2-phenylindole; HPLC, high-performance liquid chromatography; MC1R, melanocortin-1 receptor.

The cellular internalization and efflux of ⁹⁰Y-DOTA-GGNle-CycMSH_hex on 16/F10 cells are presented in Figure 3. ⁹⁰Y-DOTA-GGNle-CycMSH_hex exhibited rapid cellular internalization on B16/F1 cells. Approximately 49% and 68% of ⁹⁰Y-DOTA-GGNle-CycMSH_hex activity were internalized in the B16/F10 cells after 40 min and 2 h incubation, respectively. Cellular efflux of ⁹⁰Y-DOTA-GGNle-CycMSH_hex demonstrated that 92% of the ⁹⁰Y activity remained inside the B16/F10 cells 2 h after incubating cells in culture medium at 25°C.

FIG. 3.

Cellular internalization (A) and efflux (B) of ⁹⁰Y-DOTA-GGNle-CycMSH_hex on B16/F10 melanoma cells. Total bound radioactivity (♦), internalized radioactivity (▪), and cell membrane radioactivity (▴) are presented as counts per minute.

The biodistribution result of ⁹⁰Y-DOTA-GGNle-CycMSH_hex on B16/F10 melanoma-bearing mice is presented in Table 1. The B16/F10 tumor uptake was 20.73% ± 7.99% and 19.93% ± 5.73% ID/g at 0.5 and 2 h postinjection, respectively. ⁹⁰Y-DOTA-GGNle-CycMSH_hex exhibited prolonged retention in B16/F10 tumor, with 14.8% ± 4.61% and 6.69% ± 1.85% ID/g at 4 and 24 h postinjection, respectively. The coinjection of nonradioactive NDP-MSH blocked 94% of the tumor uptake at 2 h postinjection, demonstrating that the tumor uptake was MC1R mediated. As shown in Figure 4, MC1R-binding antibody displayed substantial binding on B16/F10 tumor sections. ⁹⁰Y-DOTA-GGNle-CycMSH_hex displayed a rapid urinary clearance, with ∼91% of the injected activity being washed out of the body by 2 h postinjection. The accumulation of ⁹⁰Y-DOTA-GGNle-CycMSH_hex in normal organs was <1% ID/g except in kidneys. The renal uptake was 12.68% ± 5.2%, 7.44% ± 1.85%, and 7.75% ± 1.59% ID/g at 0.5, 2, and 4 h postinjection, respectively, and decreased to 5.23% ± 1.76% ID/g at 24 h postinjection. The coinjection of NDP-MSH did not significantly reduce the renal uptake (p > 0.05), indicating that the renal uptake of ⁹⁰Y-DOTA-GGNle-CycMSH_hex was not receptor mediated. ⁹⁰Y-DOTA-GGNle-CycMSH_hex exhibited high tumor/blood and tumor/normal organ uptake ratios were demonstrated as early as 0.5 h postinjection.

Table 1.

Biodistribution of ⁹⁰Y-DOTA-GGNle-CyCMSH_hex on B16/F10 Murine Melanoma-Bearing C57 Mice

Tissues	0.5 h	2 h	2 h blockade	4 h	24 h
Percent injected dose/gram (% ID/g)
Tumor	20.73 ± 7.99	19.93 ± 5.73	1.20 ± 1.04a	14.8 ± 4.61	6.69 ± 1.85
Brain	0.10 ± 0.02	0.05 ± 0.03	0.03 ± 0.03	0.03 ± 0.02	0.03 ± 0.02
Blood	2.76 ± 0.69	0.36 ± 0.15	0.25 ± 0.12	0.22 ± 0.12	0.06 ± 0.07
Heart	1.28 ± 0.51	0.14 ± 0.10	0.13 ± 0.05	0.16 ± 0.09	0.11 ± 0.07
Lung	3.06 ± 1.05	0.42 ± 0.08	0.19 ± 0.12	0.27 ± 0.05	0.13 ± 0.15
Liver	0.99 ± 0.19	0.63 ± 0.14	0.39 ± 0.12	1.20 ± 1.34	0.60 ± 0.20
Skin	2.48 ± 1.05	0.78 ± 0.29	0.10 ± 0.12	0.53 ± 0.29	0.37 ± 0.18
Spleen	1.4 ± 1.36	0.24 ± 0.17	0.15 ± 0.12	0.31 ± 0.17	0.53 ± 0.42
Stomach	1.36 ± 0.4	0.87 ± 0.47	0.15 ± 0.06	0.61 ± 0.27	0.76 ± 0.52
Kidneys	12.68 ± 5.2	7.44 ± 1.85	4.99 ± 0.69	7.75 ± 1.59	5.23 ± 1.76
Muscle	0.73 ± 0.51	0.19 ± 0.13	0.10 ± 0.10	0.22 ± 0.31	0.15 ± 0.14
Pancreas	0.61 ± 0.43	0.18 ± 0.05	0.06 ± 0.07	0.14 ± 0.09	0.16 ± 0.14
Bone	1.37 ± 0.44	0.41 ± 0.18	0.15 ± 0.14	0.44 ± 0.34	0.2 ± 0.04
Percent injected dose (% ID)
Intestines	1.42 ± 0.38	0.57 ± 0.22	0.33 ± 0.15	1.36 ± 1.16	1.92 ± 3.51
Urine	75.42 ± 6.72	90.62 ± 1.79	96.77 ± 0.37	89.45 ± 4.25	93.01 ± 4.43
Uptake ratio of tumor/normal tissue
Tumor/blood	7.51	55.36	4.8	67.27	111.5
Tumor/kidney	1.63	2.68	0.24	1.91	1.28
Tumor/lung	6.77	47.45	6.32	54.81	51.46
Tumor/liver	20.94	31.63	3.08	12.33	11.15
Tumor/muscle	28.4	104.89	12.0	67.27	44.6

The data were presented as percentage injected dose/gram or as percentage injected dose (mean ± SD, n = 5).

^ap < 0.05 for determining significance of differences in tumor and kidney uptake between ⁹⁰Y-DOTA-GGNle-CyCMSH_hex with or without peptide blockade at 2 h postinjection.

FIG. 4.

Fluorescence staining of MC1Rs on B16/F10 tumor section using MC1R antibody (white). The nuclei were stained with DAPI (grey). The microscopic images were acquired by confocal laser microscopy at 100 × magnification. Scale bar, 20 μm. DAPI, 4′,6-diamidino-2-phenylindole; MC1R, melanocortin-1 receptor.

The representative maximum intensity projection bremsstrahlung SPECT image of B16/F10 melanoma-bearing mouse is presented in Figure 5. The B16/F10 flank melanoma lesions could be visualized by collecting bremsstrahlung photons from ⁹⁰Y-DOTA-GGNle-CycMSH_hex at 2 h postinjection. However, as shown in Figure 5, the scattered photons over the body were substantially collected by SPECT, thus decreasing the contrast of tumor to normal organ.

FIG. 5.

Representative maximum intensity projection Bremsstralung SPECT/CT image of a B16/F10 flank melanoma-bearing C57 mouse using ⁹⁰Y-DOTA-GGNle-CycMSH_hex as an imaging probe at 2 h postinjection. Melanoma lesions are highlighted with an arrow on the image. white and black indicate high and low activity on the scale bar, respectively. SPECT, single photon emission computed tomography.

Discussion

The remarkable first-in-human images of patients with metastatic melanomas highlighted the clinical relevance of MC1R as a molecular target for melanoma imaging and therapy.¹³ We have been interested in developing MC1R-targeted therapeutic peptides for melanoma therapy.^14,16 Both ¹⁷⁷Lu and ⁹⁰Y are attractive β-emitters with different half-lives and β-energy levels. ¹⁷⁷Lu has a half-life of 6.7 days with low-energy β-particles (0.479 MeV), whereas ⁹⁰Y has a half-life of 2.7 days with high-energy β-particles (2.3 MeV). Meanwhile, ¹⁷⁷Lu also emits γ-rays (113 and 208 keV) that can be used for imaging, while ⁹⁰Y is a pure β-emitter. We previously reported promising melanoma-targeting and imaging properties of ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex.¹⁴ In this study, we examined the biodistribution of ⁹⁰Y-DOTA-GGNle-CycMSH_hex on B16/F10 melanoma-bearing C57 mice. We selected B16/F10 melanoma cells for this study because they are highly metastatic and can readily form pulmonary melanoma metastases when injected into the tail veins of C57 mice.¹⁷ Favorable melanoma-targeting property of ⁹⁰Y-DOTA-GGNle-CycMSH_hex will underscore the potential of utilizing DOTA-GGNle-CycMSH_hex to deliver both ¹⁷⁷Lu and ⁹⁰Y to address the tumor size and burden when needed.

⁹⁰Y-DOTA-GGNle-CycMSH_hex displayed MC1R-specific binding, and exhibited rapid cellular internalization and prolonged efflux on B16/F10 melanoma cells. The change of radionuclide from ¹⁷⁷Lu to ⁹⁰Y maintained similar melanoma-targeting property. For instance, the tumor uptake of ⁹⁰Y-DOTA-GGNle-CycMSH_hex was 20.73% ± 7.99% and 19.93% ± 5.73% ID/g at 0.5 and 2 h postinjection, respectively, whereas the tumor uptake of ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex was 20.25% ± 4.59% and 20.63% ± 6.27% ID/g at 0.5 and 2 h postinjection, respectively. Meanwhile, ⁹⁰Y-DOTA-GGNle-CycMSH_hex and ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex displayed similar fast urinary clearance. The accumulation of both peptides was low in normal organs except in kidneys that could be potential dose-limiting organs in melanoma therapy studies. Nonetheless, the rapid accumulation and prolonged retention of ⁹⁰Y-DOTA-GGNle-CycMSH_hex highlighted its potential for melanoma therapy in future studies.

Both ⁹⁰Y-DOTA-Re(Arg¹¹)CCMSH and ⁹⁰Y-DOTA-Re(Glu², Arg¹¹)CCMSH were reported to target MC1Rs for potential melanoma therapy.^18,19 Their B16/F1 melanoma uptake was 25.7% ± 4.64% and 11.71% ± 1.32% ID/g at 2 h postinjection, respectively. Although the tumor uptake of ⁹⁰Y-DOTA-Re(Glu², Arg¹¹)CCMSH was lower than that of ⁹⁰Y-DOTA-Re(Arg¹¹)CCMSH, ⁹⁰Y-DOTA-Re(Glu², Arg¹¹)CCMSH displayed higher tumor to kidney uptake ratio than ⁹⁰Y-DOTA-Re(Arg¹¹)CCMSH. Interestingly, ⁹⁰Y-DOTA-GGNle-CycMSH_hex exhibited the highest tumor to kidney uptake ratio (2.68 at 2 h postinjection) among these peptides because of its high tumor uptake and low renal uptake. The improved tumor to kidney uptake ratio of ⁹⁰Y-DOTA-GGNle-CycMSH_hex would potentially deliver more radiation to tumor without increasing the radiation to kidneys.

The melanoma lesions could be clearly visualized by SPECT using ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex as an imaging agent due to the γ-rays from ¹⁷⁷Lu.¹⁴ Although ⁹⁰Y is a pure β-emitter, Bremsstrahlung photons could be produced by interaction of the β-particles of ⁹⁰Y with tissue. Thus, both Bremsstrahlung SPECT and PET of ⁹⁰Y have been investigated and reported in the literature.^20–23 In this study, we examined the Bremsstrahlung SPECT of ⁹⁰Y-DOTA-GGNle-CycMSH_hex on melanoma-bearing mice using a small energy window of 126.5–155.7 keV. Although the flank melanoma lesions could be visualized by collecting bremsstrahlung photons from ⁹⁰Y-DOTA-GGNle-CycMSH_hex, the presence of scattered photons in the image resulted in degradation in contrast to tumor to normal organ. The challenge in imaging ⁹⁰Y using SPECT is due to its continuous Bremsstrahlung spectrum with energies up to 2.3 MeV and the absence of a photopeak, making the traditional small energy window-based scatter rejection ineffective.

Yttrium-86 is an attractive PET radionuclide that can be produced by a cyclotron through the ⁸⁶Sr (p,n) ⁸⁶Y reaction. It has a half-life of 14.7 h and can serve as an imaging surrogate for ⁹⁰Y to form a true matched-pair theranostic radionuclide. ⁸⁶Y-DOTA-ReCCMSH(Arg¹¹) has been developed to target MC1Rs for melanoma imaging. It exhibited rapid high B16/F1 melanoma uptake (11.87% ± 3.31% ID/g at 0.5 h postinjection) and low accumulation in normal organs expect in kidneys.²⁴ The PET images of melanoma-bearing mice using ⁸⁶Y-DOTA-ReCCMSH(Arg¹¹) clearly demonstrated its melanoma imaging potential due to its high tumor concentration and low nontarget tissue accumulation. However, the availability of ⁸⁶Y can potentially be a limiting factor since it is not a commercial radionuclide. Alternatively, ⁶⁸Ga-DOTA-GGNle-CycMSH_hex can be used to monitor the response when examining the therapeutic efficacy of ⁹⁰Y-DOTA-GGNle-CycMSH_hex in the future.

Conclusions

⁹⁰Y-DOTA-GGNle-CycMSH_hex displayed MC1R targeting and specificity on B16/F10 melanoma cells and tumors. The favorable melanoma-targeting property and fast urinary clearance of ⁹⁰Y-DOTA-GGNle-CycMSH_hex warranted its evaluation for melanoma therapy in future studies.

Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported in part by the NIH grant R01CA225837 and University of Colorado Denver start-up fund. Microscopy imaging experiments were performed in the University of Colorado Anschutz Medical Campus Advance Light Microscopy Core supported in part by NIH/NCATS Colorado CTSI Grant Number UL1 TR001082.