Evaluation of A Novel Pb-203-Labeled Lactam-Cyclized Alpha-Melanocyte-Stimulating Hormone Peptide for Melanoma Targeting

Abstract

The purpose of this study is to examine the melanocortin-1 receptor (MC1R) targeting and specificity of ²⁰³Pb-DOTA-GGNle-CycMSH_hex in melanoma cells and tumors to facilitate its potential therapeutic application when labeled with ²¹²Pb. The melanocortin-1 receptor (MC1R)-specific targeting and imaging properties of ²⁰³Pb-DOTA-GGNle-CycMSH_hex were determined on B16/F1 and B16/F10 murine melanoma cells, and in B16/F1 flank melanoma-, B16/F10 flank melanoma- and B16/F10 pulmonary metastatic melanoma-bearing C57 mice. ²⁰³Pb-DOTA-GGNle-CycMSH_hex displayed MC1R-specific binding on B16/F1 and B16/F10 melanoma cells and tumors. B16/F1 flank melanoma, B16/F10 flank melanoma and B16/F10 pulmonary metastatic melanoma lesions could be clearly imaged by single photon emission computed tomography (SPECT) using ²⁰³Pb-DOTA-GGNle-CycMSH_hex as an imaging probe. The favorable melanoma targeting and imaging properties highlighted the potential of ²⁰³Pb-DOTA-GGNle-CycMSH_hex as a MC1R-targeting melanoma imaging probe, and warranted the evaluation of ²¹²Pb-DOTA-GGNle-CycMSH_hex for melanoma therapy in future studies.

Graphical Abstract

INTRODUCTION

Malignant melanoma is the most deadly skin cancer with an increasing incidence. Approximately 91,270 new cases and 9,320 deaths will occur in the United States in 2018.¹ Malignant melanoma accounts for 75% of all skin cancer deaths despite that melanoma is only less than 5% of skin cancer cases.¹ High mortality of melanoma is attributed to the extreme aggressiveness associated with metastatic melanoma. Traditional median overall survival is about 6–9 months for metastatic melanoma patients.^{2, 3} Although new treatments including Vemurafenib (BRAF inhibitor), ipilimumab (targeting CTLA-4) and Nivolumab (PD-1 inhibitor) have improved median overall survivals of metastatic melanoma patients by months,^4–8 the long-term survival remains less than 10% for metastatic melanoma patients. Therefore, there is a need to develop new approaches to treatmetastatic melanoma.

Due to the over-expression of melanocortin-1 receptors (MC1Rs) on more than 80% of melanotic and amelanotic human melanoma metastases,^9–14 we have been developing radiolabeled alpha-melanocyte-stimulating hormone (α-MSH) peptides to target MC1Rs for melanoma imaging and therapy. Our radiolabeled lactam-cyclized α-MSH peptides, building upon the structure of 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},^15–23 were readily radiolabeled with both single photon emission tomography (SPECT) radionuclides (i.e. ¹¹¹In and ⁶⁷Ga)^{16, 19} and positron emission tomography (PET) radionuclides (i.e. ⁶⁴Cu and ⁶⁸Ga)^{20, 23} for melanoma detection, and ¹⁷⁷Lu for potential treatment of melanoma using targeted radionuclide.²¹

¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex displayed promising melanoma targeting property in B16/F1 melanoma-bearing mice.²¹ Thus we are interested in replacing ¹⁷⁷Lu with matched-pair theranostic radionuclides ²⁰³Pb/²¹²Pb due to their attractive decay properties. ²⁰³Pb is a cyclotron-produced radionuclide with 51.9 h half-life. It generates a 279 keV gamma ray with 81% abundance which is suitable for SPECT imaging. ²¹²Pb (T_1/2 = 10.6 h) can be easily obtained from a ²²⁴Ra-²¹²Pb generator. ²¹²Pb decays to ²¹²Bi via a beta-decay (0.57 MeV), then ²¹²Bi eventually decays to stable ²⁰⁸Pb through two beta-decays (1.8 and 2.2 MeV) and two alpha-decays (6.1 and 8.8 MeV). Therefore, ²¹²Pb can serve as an in vivo generator that can be delivered by MC1R-binding DOTA-GGNle-CycMSH_hex peptide. Moreover, ²⁰³Pb/²¹²Pb share identical radiolabeling chemistry. Thus, the radiolabeling of ²⁰³Pb/²¹²Pb can be conducted under identical radiolabeling conditions. The combination of ²⁰³Pb/²¹²Pb-DOTA-GGNle-CycMSH_hex could potentially open the avenue for imaging-guided alpha radionuclide therapy by identifying MC1R-positive melanoma patients.

In this study, DOTA-GGNle-CycMSH_hex was prepared using fluorenylmethyloxycarbonyl (Fmoc) chemistry and radiolabeled with ²⁰³Pb. The specific binding of ²⁰³Pb-DOTA-GGNle-CycMSH_hex was determined on B16/F1 and B16/F10 murine melanoma cells. The selection of B16/F1 and B16/F10 cells for this study was based on two reasons. First, B16/F1 murine melanoma cells are commonly used by research groups to generate flank melanoma-bearing miceto evaluate the melanoma targeting and biodistribution properties of radiolabeled α-MSH peptides.^{12, 15, 16} Second, B16/F10 murine melanoma cells are highly metastatic and thus are used to generate pulmonary melanoma metastases by tail vein injection of cells.^{14, 24, 25} Hence, we generated B16/F1 flank melanoma-, B16/F10 flank melanoma- and B16/F10 pulmonary metastatic melanoma-bearing C57 mice to determine the tumor targeting and biodistribution of ²⁰³Pb-DOTA-GGNle-CycMSH_hex in this study. Thereafter, the biodistribution and imaging properties of ²⁰³Pb-DOTA-GGNle-CycMSH_hex were examined using B16/F1 flank melanoma-, B16/F10 flank melanoma- and pulmonary metastatic melanoma-bearing mice.

EXPERIMENTAL SECTION

Chemicals and reagents

Amino acids and resin were purchased from Advanced ChemTech Inc. (Louisville, KY) and Novabiochem (San Diego, CA). DOTA-tri-t-butyl ester was purchased from Macrocyclics Inc. (Richardson, TX) for peptide synthesis. ²⁰³PbCl₂ was purchased from Lantheus Medical Imaging (North Billerica, MA) for radiolabeling. All other chemicals used in this study were purchased from Thermo Fisher Scientific (Waltham, MA) and used without further purification. B16/F1 and B16/F10 murine melanoma cells were obtained from American Type Culture Collection (Manassas, VA).

Preparation of ²⁰³Pb-DOTA-GGNle-CycMSH_hex and its serum stability and urine metabolites

DOTA-GGNle-CycMSH_hex was synthesized using standard fluorenylmethyloxycarbonyl (Fmoc) chemistry according to our published procedure.¹⁶ The peptide was purified by reverse phase-high performance liquid chromatography (RP-HPLC) and characterized by liquid chromatography-mass spectrometry (LC-MS). ²⁰³Pb-DOTA-GGNle-CycMSH_hex was prepared in a 0.25 M NH₄OAc-buffered solution (pH 5.3). Briefly, 50 µL of ²⁰³PbCl₂ (37–74 MBq in 0.5 M HCl aqueous solution), 10 μL of peptide aqueous solution (1 mg/mL) and 300 μL of 0.25 M NH₄OAc were added into a reaction vial and incubated at 75 °C for 30 min. After the reaction, 10 μL of 0.5% EDTA (ethylenediaminetetraacetic acid) aqueous solution was added into the reaction vial to scavenge potential unbound ²⁰³Pb²⁺ 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 included 20 mM HCl aqueous solution as solvent A () and 100% CH₃CN as solvent B. 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. Thereafter, 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. Then the gradient was changed from 10:90 A/B to 82:18 A/B over 3 min. The purified peptide sample was purged with N₂ gas to remove the acetonitrile for 15 min. The pH of the final solution was adjusted to 7.4 using 0.1 N NaOH and sterile saline for animal studies. ²⁰³Pb-DOTA-GGNle-CycMSH_hex was incubated in mouse serum at 37 °C for 4 h and monitored for degradation by RP-HPLC to determine its in vitro serum stability. Furthermore, one hundred microliter of HPLC-purified ²⁰³Pb-DOTA-GGNle-CycMSH_hex (0.74 MBq) was injected into a normal C57 mouse through the tail vein to determine urine metabolites.. The mouse was sacrificed at 2 h post-injection and the urine was collected. The radioactive urine metabolites were analyzed by injecting aliquots of urine into HPLC. A 20-minute gradient of 18–28% acetonitrile / 20 mM HCl was used to analyze the urine metabolites.

Specific cellular binding, internalization and efflux of ²⁰³Pb-DOTA-GGNle-CycMSH_hex

The specific binding of ²⁰³Pb-DOTA-GGNle-CycMSH_hex was determined on B16/F1 and B16/F10 melanoma cells. The B16/F1 and B16/F10 cells (1×10⁶ cells pertube, n = 3) were incubated at 25 °C for 2 h with approximately 0.037 MBq of ²⁰³Pb-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}. The binding medium was aspirated 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 phosphate buffered saline (PBS) and measured in a Wallac 1480 automated gamma counter (PerkinElmer, NJ).

The internalization and efflux properties of ²⁰³Pb-DOTA-GGNle-CycMSH_hex were examined on B16/F1 and B16/F10 melanoma cells. B16/F1 or B16/F10 cells (3×10⁵/well) were seeded into a 24-well cell culture plate and incubated at 37°C overnight. After being washed once with binding media (MEM with 25 mM HEPES, pH 7.4, 0.2% BSA, 0.3 mM 1,10-phenathroline), the cells were incubated at 25°C for 20, 40, 60, 90 and 120 min (n = 3) with approximately 100,000 counts per minute (cpm) of HPLC-purified ²⁰³Pb-DOTA-GGNle-CycMSH_hex. After incubation, the reaction medium was aspirated and cells were rinsed with 2 × 0.5 mL of ice-cold pH 7.4, 0.2% BSA / 0.01 M PBS. Cellular internalization of ²⁰³Pb-DOTA-GGNle-CycMSH_hex was evaluated 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 lysing the cells with 0.5 mL of 1N NaOH for 5 min. Membrane-bound and internalized ²⁰³Pb activity was counted in a gamma counter. Cellular efflux of ²⁰³Pb-DOTA-GGNle-CycMSH_hex was determined by incubating cells with ²⁰³Pb-DOTA-GGNle-CycMSH_hex at 25 °C for 2 h, removing non-specific 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 media.The radioactivity in media, on cell surfaces and in cells were separately collected and counted in a gamma counter 20, 40, 60, 90 and 120 min post incubation.

B16/F1 and B16/F10 melanoma-bearing mice for biodistribution and imaging studies

All animal studies were performed in compliance with Institutional Animal Care and Use Committee approval. B16/F1 flank melanoma-, B16/F10 flank melanoma- and pulmonary metastatic melanoma-bearing mice were generated for biodistribution and imaging studies. bearing mice Each C57 mouse was subcutaneously inoculated with 1×10⁶ B16/F1 or B16/F10 cells on the right flank to generate flank tumors. The flank tumor weights reached approximately 0.2 g after 10 days and the tumor-bearing mice were used for biodistribution and imaging studies. To generate B16/F10 pulmonary melanoma metastases, each C57 mouse was intravenously injected with 2 × 10⁵ B16/F10 cells into the tail vein. The mice were used for biodistribution and imaging studies 16 days post-injection.

Biodistribution and imaging studies of ²⁰³Pb-DOTA-GGNle-CycMSH_hex

The biodistribution property of ²⁰³Pb-DOTA-GGNle-CycMSH_hex were determined on B16/F1 flank melanoma-, B16/F10 flank melanoma- and pulmonary metastatic melanoma-bearing C57 mice (Charles River, Wilmington, MA). Each tumor-bearing mouse was injected with 0.056 MBq of ²⁰³Pb-DOTA-GGNle-CycMSH_hex through the tail vein. Tumor-bearing mice were sacrificed at 0.5, 2, 4 and 24 h post-injection. Tumors and organs of interest were collected, weighed and counted. Blood values were calculated as 6.5% of the whole-body weight. The specificity of the tumor uptake of ²⁰³Pb-DOTA-GGNle-CycMSH_hex was examined by co-injecting 10 µg (6.07 nmol) of unlabeled NDP-MSH peptide.

Flank melanoma- and pulmonary metastatic melanoma-bearing mice were used to determine the melanoma imaging property of ²⁰³Pb-DOTA-GGNle-CycMSH_hex. Each tumor-bearing mouse was injected with 7.4 MBq of ²⁰³Pb-DOTA-GGNle-CycMSH_hex through the tail vein. SPECT imaging studies were performed at 2 h post-injection. CT data was collected followed by SPECT data acquisition. Reconstructed SPECT/CT data were visualized using Vivoquant (Invicro, Boston, MA).

Statistical Analysis

Student’s t-test for unpaired data was performed for statistical analysis. A 95% confidence level was chosen to determine the difference between groups in cellular binding of ²⁰³Pb-DOTA-GGNle-CycMSH_hex, difference in tumor and kidney uptake between ²⁰³Pb-DOTA-GGNle-CycMSH_hex with/without NDP-MSH blockade, and the difference in lung uptake in normal lung and metastatic melanoma-bearing lung. The differences at the 95% confidence level (p<0.05) were considered significant.

RESULTS

DOTA-GGNle-CycMSH_hex (Fig. 1) was synthesized and purified by reverse phase high pressure liquid chromatography (RP-HPLC) and displayed greater than 90% purity after HPLC purification. The identity of DOTA-GGNle-CycMSH_hex was confirmed by electrospray ionization mass spectrometry. ²⁰³Pb-DOTA-GGNle-CycMSH_hex was readily prepared with greater than 95% radiolabeling yield, and was completely separated from its excess non-labeled peptide by RP-HPLC. The retention time of ²⁰³Pb-DOTA-GGNle-CycMSH_hex was 18.3 min. ²⁰³Pb-DOTA-GGNle-CycMSH_hex was stable in mouse serum at 37 °C for 4 h. The urine analysis revealed that approximately 65% of ²⁰³Pb-DOTA-GGNle-CycMSH_hex remained intact in urine at 2 h post-injection (Fig. 2). ²⁰³Pb-DOTA-GGNle-CycMSH_hex displayed MC1R-specific binding on B16/F1 and B16/F10 cells. Approximately 79% and 84% of ²⁰³Pb-DOTA-GGNle-CycMSH_hex uptake were blocked by peptide blockade on B16/F1 and B16/F10 cells (p<0.05).

Figure 1.

Schematic structure of ²⁰³Pb-DOTA-GGNle-CycMSH_hex.

Figure 2.

Radioactive HPLC profile of ²⁰³Pb-DOTA-GGNle-CycMSH_hex (A, T_R = 18.3 min) and its mouse serum stability (B) after 4 h incubation at 37 °C. Radioactive HPLC profile of the urine sample of a normal C57 mouse at 2 h post-injection of ²⁰³Pb-DOTA-GGNle-CycMSH_hex (C). Arrows indicate the original compound of ²⁰³Pb-DOTA-GGNle-CycMSH_hex. Specific binding of ²⁰³Pb-DOTA-GGNle-CycMSH_hex on B16/F1 (D) and B16/F10 (E) melanoma cells with or without peptide blockade.

Figure 3 illustrates the internalization and efflux of ²⁰³Pb-DOTA-GGNle-CycMSH_hex on B16/F1 and B16/F10 cells. ²⁰³Pb-DOTA-GGNle-CycMSH_hex exhibited rapid cellular internalization property on B16/F1 cells. Approximately 52% and 62% of ²⁰³Pb-DOTA-GGNle-CycMSH_hex activity were internalized in the B16/F1 cells after 40 min and 2 h incubation, respectively. Cellular efflux of ²⁰³Pb-DOTA-GGNle-CycMSH_hex demonstrated that 90% and 80% of the ²⁰³Pb activity remained inside the B16/F1 cells 40 min and 2 h after incubating cells in culture medium at 25 °C, respectively. ²⁰³Pb-DOTA-GGNle-CycMSH_hex exhibited similar internalization and efflux patterns on B16/F10 cells. Approximately 48% and 67% of ²⁰³Pb-DOTA-GGNle-CycMSH_hex activity were internalized in the B16/F10 cells after 40 min and 2 h incubation, respectively. Cellular efflux of ²⁰³Pb-DOTA-GGNle-CycMSH_hex indicated that 96% and 91% of the ²⁰³Pb activity remained inside the B16/F10 cells 40 min and 2 h after cell incubation in culture medium at 25 °C, respectively.

Figure 3.

Cellular internalization (A, C) and efflux (B, D) of ²⁰³Pb-DOTA-GGNle-CycMSH_hex on B16/F1 (A, B) and B16/F10 (C, D) melanoma cells. Total bound radioactivity (♦), internalized radioactivity (■) and cell membrane radioactivity (▲) are presented as counts per minute (cpm).

The tumor targeting and biodistribution properties of ²⁰³Pb-DOTA-GGNle-CycMSH_hex were determined on B16/F1 flank melanoma-, B16/F10 flank melanoma- and pulmonary metastatic melanoma-bearing C57 mice. The biodistribution results of ²⁰³Pb-DOTA-GGNle-CycMSH_hex are presented in Tables 1–3. The B16/F1 tumor uptake was 14.37 ± 3.43 and 12.61 ± 2.28% ID/g at 0.5 and 2 h post-injection, respectively. ²⁰³Pb-DOTA-GGNle-CycMSH_hex exhibited prolonged B16/F1 tumor retention, with 9.37 ± 2.23 and 6.4 ± 0.37% ID/g at 4 and 24 h post-injection, respectively. The co-injection of non-radioactive NDP-MSH blocked 94% of the B16/F1 tumor uptake at 2 h post-injection, demonstrating that the B16/F1 tumor uptake was MC1R-mediated. Whole-body clearance of ²⁰³Pb-DOTA-GGNle-CycMSH_hex was rapid, with approximately 92% of the injected dose being washed out of the body via urinary system by 2 h post-injection. Kidneys are the normal organs with the highest uptake of ²⁰³Pb-DOTA-GGNle-CycMSH_hex. The renal uptake was 9.3 ± 1.75, 4.99 ± 1.48 and 4.82 ± 0.59% ID/g at 0.5, 2 and 4 h post-injection, respectively, and decreased to 2.77 ± 0.81% ID/g at 24 h post-injection. The co-injection of NDP-MSH didn’t significantly decrease the renal uptake (p>0.05), suggesting that the renal uptake of ²⁰³Pb-DOTA-GGNle-CycMSH_hex was not receptor-specific. The accumulation of ²⁰³Pb-DOTA-GGNle-CycMSH_hex in other normal organs was much lower than kidneys. ²⁰³Pb-DOTA-GGNle-CycMSH_hex displayed high tumor/blood and tumor/normal organ uptake ratios as early as 0.5 h post-injection.

Table 1.

Biodistribution of ²⁰³Pb-DOTA-GGNle-CyCMSH_hex in B16/F1 murine melanoma-bearing C57 mice. The data were presented as percent injected dose/gram or as percent injected dose (Mean ± SD, n = 5)

Tissues	0.5 h	2 h	2 h blockade	4 h	24 h
	Percent injected dose/gram (% ID/g)
Tumor	14.37 ± 3.43	12.61 ± 2.28	0.68 ± 0.21*	9.37 ± 2.23	6.40 ± 0.37
Brain	0.11 ± 0.04	0.01 ± 0.01	0.01 ± 0.01	0.03 ± 0.02	0.01 ± 0.02
Blood	2.40 ± 0.57	0.25 ± 0.11	0.27 ± 0.12	0.14 ± 0.07	0.09 ± 0.04
Heart	1.0 ± 0.19	0.08 ± 0.09	0.11 ± 0.04	0.07 ± 0.07	0.02 ± 0.03
Lung	2.16 ± 0.29	0.27 ± 0.07	0.24 ± 0.15	0.18 ± 0.04	0.09 ± 0.02
Liver	1.08 ± 0.10	0.64 ± 0.11	0.61 ± 0.11	0.54 ± 0.08	0.52 ± 0.02
Skin	2.16 ± 0.79	0.12 ± 0.08	0.12 ± 0.17	0.11 ± 0.08	0.18 ± 0.13
Spleen	0.61 ± 0.22	0.15 ± 0.15	0.07 ± 0.12	0.13 ± 0.06	0.17 ± 0.10
Stomach	1.10 ± 0.37	0.97 ± 1.09	0.70 ± 0.53	0.49 ± 0.10	1.32 ± 0.49
Kidneys	9.3 ± 1.75	4.99 ± 1.48	4.61 ± 0.75	4.82 ± 0.59	2.77 ± 0.81
Muscle	0.38 ± 0.19	0.07 ± 0.09	0.01 ± 0.01	0.01 ± 0.01	0.01 ± 0.01
Pancreas	0.31 ± 0.18	0.05 ± 0.06	0.04 ± 0.04	0.01 ± 0.01	0.01 ± 0.01
Bone	1.17 ± 0.46	0.35 ± 0.34	0.20 ± 0.26	0.31 ± 0.27	0.46 ± 0.14
	Percent injected dose (% ID)
Intestines	1.04 ± 0.13	1.25 ± 0.65	1.21 ± 0.52	2.13 ± 1.13	2.09 ± 1.56
Urine	78.62 ± 0.59	92.11 ± 3.55	94.25 ± 1.99	93.45 ± 1.0	94.45 ± 1.58
	Uptake ratio of tumor/normal tissue
Tumor/blood	5.99	50.44	2.52	66.93	71.11
Tumor/kidney	1.55	2.53	0.15	1.94	2.31
Tumor/liver	6.65	46.70	2.83	52.06	71.11
Tumor/lung	13.31	19.70	1.11	17.35	12.31
Tumor/muscle	37.82	180.14	68.0	937.0	640.0

^*p<0.05 for determining significance of differences in tumor and kidney uptake between ²⁰³Pb-DOTA-GGNle-CyCMSH_hex with or without peptide blockade at 2 h post-injection.

Table 3.

Biodistribution of ²⁰³Pb-DOTA-GGNle-CyCMSH_hex in B16/F10 pulmonary metastatic melanoma-bearing and normal C57 mice. The data was presented as percent injected dose/gram or as percent injected dose (Mean ± SD, n = 3).

	2 h		4 h		24 h
Tissues	Lung Met.	Normal	Lung Met.	Normal	Lung Met.	Normal
	Percent injected dose/gram (% ID/g)
Lung	7.32 ± 2.13*	0.42 ± 0.16	9.27 ± 1.13*	0.28 ± 0.15	3.90 ± 0.18*	0.29 ± 0.12
Brain	0.04 ± 0.02	0.05 ± 0.01	0.02 ± 0.01	0.04 ± 0.01	0.01 ± 0.01	0.01 ± 0.02
Blood	0.15 ± 0.04	0.72 ± 0.12	0.09 ± 0.04	0.59 ± 0.16	0.15 ± 0.10	0.30 ± 0.05
Heart	0.47 ± 0.46	0.27 ± 0.18	0.46 ± 0.28	0.28 ± 0.08	0.15 ± 0.07	0.16 ± 0.07
Liver	0.60 ± 0.05	1.03 ± 0.13	0.65 ± 0.06	1.08 ± 0.34	0.45 ± 0.09	1.04 ± 0.21
Skin	0.39 ± 0.05	0.27 ± 0.10	0.32 ± 0.15	0.22 ± 0.17	0.35 ± 0.05	0.17 ± 0.11
Spleen	0.25 ± 0.08	0.08 ± 0.09	0.24 ± 0.09	0.26 ± 0.20	0.24 ± 0.03	0.28 ± 0.11
Stomach	0.90 ± 0.49	0.55 ± 0.16	0.49 ± 0.13	0.53 ± 0.20	0.32 ± 0.16	1.67 ± 0.93
Kidneys	8.13 ± 3.29	5.84 ± 2.86	6.16 ± 0.31	6.66 ± 1.26	3.55 ± 0.36	4.48 ± 1.14
Muscle	0.04 ± 0.05	0.08 ± 0.08	0.08 ± 0.06	0.01 ± 0.01	0.09 ± 0.10	0.06 ± 0.05
Pancreas	0.01 ± 0.01	0.16 ± 0.12	0.05 ± 0.01	0.07 ± 0.11	0.09 ± 0.01	0.09 ± 0.03
Bone	0.13 ± 0.08	1.15 ± 0.36	0.15 ± 0.08	0.77 ± 0.35	0.31 ± 0.24	1.06 ± 0.54
	Percent injected dose (% ID)
Intestine	0.61 ± 0.52	1.43 ± 0.58	1.66 ± 1.49	1.68 ± 0.96	0.76 ± 0.57	1.86 ± 1.02
Urine	90.66 ± 1.33	83.92 ± 11.77	88.45 ± 1.9	92.02 ± 2.16	93.18 ± 1.47	92.07 ± 2.14

^*p<0.05, significance comparison between ²⁰³Pb-DOTA-GGNle-CycMSH_hex in pulmonary metastatic melanoma-bearing and normal C57 mice.

²⁰³Pb-DOTA-GGNle-CycMSH_hex displayed similar uptake in B16/F10 flank melanoma as the uptake in B16/F1 flank melanoma. The B16/F10 tumor uptake was 11.29 ± 4.42, 16.81 ± 5.48, 12.67 ± 1.48, 4.57 ± 2.17% ID/g at 0.5, 2, 4 and 24 h post-injection, respectively. The co-injection of non-radioactive NDP-MSH blocked 95% of the B16/F10 tumor uptake at 2 h post-injection, demonstrating that the tumor uptake was MC1R-specific. ²⁰³Pb-DOTA-GGNle-CycMSH_hex exhibited similar urinary clearance pattern in B16/F10 and B16/F1 flank melanoma-bearing mice. The kidney uptake was 12.9 ± 4.98, 5.43 ± 2.0 and 5.31 ± 1.1% ID/g in B16/F10 flank melanoma-bearing mice at 0.5, 2 and 4 h post-injection, respectively. The renal uptake gradually decreased to 3.87 ± 2.12% ID/g at 24 h post-injection. Similarly, the co-injection of NDP-MSH didn’t significantly reduce the renal uptake (p>0.05), indicating that the renal uptake of ²⁰³Pb-DOTA-GGNle-CycMSH_hex was not receptor-specific. Because of low accumulation in other normal organs, ²⁰³Pb-DOTA-GGNle-CycMSH_hex exhibited high tumor/blood and tumor/normal organ uptake ratios as early as 0.5 h post-injection.

As shown in Table 3, ²⁰³Pb-DOTA-GGNle-CycMSH_hex exhibited rapid uptake in pulmonary metastatic melanoma-bearing lung. The uptake in metastatic melanoma-bearing lung was 7.32 ± 2.13, 9.27 ± 1.13 and 3.9 ± 0.18% ID/g at 2, 4 and 24 h post-injection, respectively. The uptake in metastatic melanoma-bearing lung was 13–33 times the uptake of normal lung. ²⁰³Pb-DOTA-GGNle-CycMSH_hex displayed similar accumulation pattern in normal organs of pulmonary metastatic melanoma-bearing mice as compared to the accumulation in normal organs of flank melanoma-bearing mice. The representative maximum intensity projection SPECT images in flank melanoma- and pulmonary metastatic melanoma-bearing mice are presented in Figure 4. Both B16/F1 and B16/F10 flank melanoma lesions could be visualized by SPECT using ²⁰³Pb-DOTA-GGNle-CycMSH_hex as an imaging probe at 2 h post-injection. Moreover, the pulmonary melanoma metastases could be clearly imaged by SPECT using ²⁰³Pb-DOTA-GGNle-CycMSH_hex as an imaging probe at 2 h post-injection.

Figure 4.

Representative maximum intensity projection SPECT/CT images of B16/F1 (A) and B16/F10 (B) flank melanoma-bearing C57 mice, maximum intensity projection SPECT/CT and coronal images of B16/F10 pulmonary metastatic melanoma-bearing (C, D) C57 mice using ²⁰³Pb-DOTA-GGNle-CycMSH_hex as an imaging probe at 2 h post-injection.

DISCUSSION

We have been developing MC1R-targeting α-MSH peptide radiopharmaceuticals for melanoma imaging and therapy because of the expression of MC1Rs on both melanotic and amelanotic human melanoma metastases.⁹ Our recent first-in-human images of melanoma metastases in clearly demonstrated the clinical relevance of MC1R as a molecular target for human melanoma imaging.²³ The melanoma metastases in brain, lung, connective tissue and small intestine of patients could be clearly visualized by positron emission tomography (PET) using ⁶⁸Ga-DOTA-GGNle-CycMSH_hex as an imaging probe.²³ The remarkable PET images of melanoma metastases in patients highlighted the need to develop MC1R-targeting therapeutic peptide for treating patients with melanoma metasases. Therefore, we have committed efforts to develop ²⁰³Pb/²¹²Pb-DOTA-GGNle-CycMSH_hex peptides for potential imaging-guided melanoma therapy because of the attractive theranostic properties of matched-pair ²⁰³Pb/²¹²Pb and nanomolar MC1R binding affinity of DOTA-GGNle-CycMSH_hex with B16/F1 and B16/F10 melanoma cells.^{16, 23} In this study, we prepared ²⁰³Pb-DOTA-GGNle-CycMSH_hex and evaluated its MC1R binding specificity with melanoma cells, and its melanoma targeting and imaging properties in flank melanoma-bearing and pulmonary metastatic melanoma-bearing mice.

²⁰³Pb-DOTA-GGNle-CycMSH_hex displayed MC1R-specific binding on B16/F1 and B16/F10 melanoma cells. The biodistribution and imaging properties of ²⁰³Pb-DOTA-GGNle-CycMSH_hex were examined in B16/F1 flank melanoma-, B16/F10 flank melanoma- and B16/F10 pulmonary metastatic melanoma-bearing micebecause these melanoma models have been used among research groups to evaluate the tumor targeting properties of radiolabeled α-MSH peptides.^{14, 18, 24–30 203}Pb-DOTA-GGNle-CycMSH_hex exhibited similar MC1R-specific uptake in B16/F1 and B16/F10 melanoma lesions. The phantom imaging of ^99mTcO₄⁻ and ²⁰³PbCl₂ exhibited comparable tomographic spatial resolution of 1.6 mm³¹ and highlighted the SPECT imaging potential of ²⁰³Pb. In this study, B16/F1 flank melanoma, B16/F10 flank melanoma and B16/F10 pulmonary metastatic melanoma lesions could be clearly imaged by SPECT using ²⁰³Pb-DOTA-GGNle-CycMSH_hex as an imaging probe.

²⁰³Pb can be produced by irradiating natural or enriched ²⁰³Tl and ²⁰⁵Tl targets with 13.7-MeV deuterons through a ²⁰³Tl (d, 2n) ²⁰³Pb reaction,³² 14.5-MeV protons through a ²⁰³Tl (p, n) ²⁰³Pb reaction,³³ and 26.5-MeV protons through a ²⁰⁵Tl (p, 3n) ²⁰³Pb reaction.³⁴ Non-radioactive Pb and Fe are the major metallic contaminants in the ²⁰³PbCl₂ solution produced by the ²⁰⁵Tl (p, 3n) ²⁰³Pb reaction, thus serve as competitors for ²⁰³Pb in the radiolabeling process. The amount of non-radioactive Pb varies from 0.15 to 0.65 µg/mCi, whereas the amount of non-radioactive Fe ranges from 0.1 to 0.19 µg/mCi. Therefore, excess amount of DOTA-GGNle-CycMSH_hex was used for radiolabeling. Despite that HPLC purification completely separated DOTA-GGNle-CycMSH_hex from ²⁰³Pb-DOTA-GGNle-CycMSH_hex, HPLC purification couldn’t get rid of non-radioactive Pb- and Fe-labeled peptides that could compete with ²⁰³Pb-DOTA-GGNle-CycMSH_hex for MC1R receptors in melanoma lesions. Therefore, in order to minimize the competition of MC1Rs from non-radioactive Pb- and Fe-labeled peptides, it is desirable to improve the specific activity of ²⁰³Pb during the production and processing.

Non-radioactive Re-cyclized ²⁰³Pb-DOTA-Re(Arg¹¹)CCMSH displayed high MC1R-specific uptake (12 ± 3.2% ID/g at 1 h post-injection) in B16/F10 melanoma.³¹ B16/F1 flank melanoma was clearly visualized by SPECT using ²⁰³Pb-DOTA-Re(Arg¹¹)CCMSH as an imaging probe.³¹ In this study, ²⁰³Pb-DOTA-GGNle-CycMSH_hex exhibited similar B16/F1 tumor uptake (14.37 ± 3.43% ID/g at 0.5 h post-injection) and renal uptake (4.99 ± 1.48% ID/g at 2 h post-injection) as compared to ²⁰³Pb-DOTA-Re(Arg¹¹)CCMSH. Importantly, the pulmonary metastatic melanoma lesions could be clearly identified by SPECT using ²⁰³Pb-DOTA-GGNle-CycMSH_hex as an imaging probe in this study, suggesting the potential utilization of ²⁰³Pb-DOTA-GGNle-CycMSH_hex to identify melanoma patients with MC1R-positive tumors for MC1R-targeted radionuclide therapy.

The statistical analysis of tumor and renal uptake of ²⁰³Pb/²¹²Pb-DOTA-Re(Arg¹¹)CCMSH demonstrated the matched-pair properties of ²⁰³Pb/²¹²Pb.^{31, 35} Moreover, ²¹²Pb-DOTA-Re(Arg¹¹)CCMSH exhibited remarkable dose-dependent therapeutic efficacy in extremely aggressive B16/F1 melanoma model. For instance, 20% and 45% of the mice receiving 100 or 200 µCi of ²¹²Pb-DOTA-Re(Arg¹¹)CCMSH survived the 120-d study disease free.³⁵ Interestingly, ²⁰³Pb-DOTA-GGNle-CycMSH_hex displayed comparable B16/F1 tumor uptake and renal uptake in this study. According to the matched-pair melanoma targeting properties of ²⁰³Pb/²¹²Pb-DOTA-Re(Arg11)CCMSH,^{31, 35} we anticipate that ²¹²Pb-DOTA-GGNle-CycMSH_hex would exhibit similar melanoma uptake and biodistribution profile as ²⁰³Pb-DOTA-Re(Arg¹¹)CCMSH in same melanoma model. Favorable tumor targeting and pharmacokinetic properties of ²⁰³Pb-DOTA-GGNle-CycMSH_hex warranted the further evaluation of ²¹²Pb-DOTA-GGNle-CycMSH_hex. It would be interesting to determine the biodistribution property and therapeutic efficacy of ²¹²Pb-DOTA-GGNle-CycMSH_hex in the future.

In summary, ²⁰³Pb-DOTA-GGNle-CycMSH_hex exhibited MC1R-targeting and specificity on B16/F1 and B16/F10 melanoma cells and tumors. B16/F1 flank melanoma, B16/F10 flank melanoma and B16/F10 pulmonary metastatic melanoma lesions could be clearly imaged by SPECT using ²⁰³Pb-DOTA-GGNle-CycMSH_hex as an imaging probe. The favorable melanoma targeting and imaging properties highlighted the potential of ²⁰³Pb-DOTA-GGNle-CycMSH_hex as a MC1R-targeting melanoma imaging probe, and warranted the evaluation of ²¹²Pb-DOTA-GGNle-CycMSH_hex for melanoma therapy in future studies.

Table 2.

Biodistribution of ²⁰³Pb-DOTA-GGNle-CyCMSH_hex in B16/F10 murine melanoma-bearing C57 mice. The data were presented as percent injected dose/gram or as percent injected dose (Mean ± SD, n = 5)

Tissues	0.5 h	2 h	2 h blockade	4 h	24 h
	Percent injected dose/gram (% ID/g)
Tumor	11.29 ± 4.42	16.81 ± 5.48	0.79 ± 0.50*	12.67 ± 1.48	4.57 ± 2.17
Brain	0.20 ± 0.06	0.03 ± 0.01	0.01 ± 0.02	0.04 ± 0.03	0.05 ± 0.05
Blood	3.31 ± 1.84	0.23 ± 0.06	0.15 ± 0.03	0.18 ± 0.07	0.38 ± 0.11
Heart	1.88 ± 0.89	0.13 ± 0.05	0.1 ± 0.05	0.09 ± 0.02	0.12 ± 0.05
Lung	3.61 ± 2.20	0.64 ± 0.16	0.62 ± 0.07	0.83 ± 0.13	0.26 ± 0.14
Liver	1.64 ± 0.39	0.77 ± 0.16	0.63 ± 0.02	0.71 ± 0.09	0.50 ± 0.16
Skin	3.16 ± 1.21	0.35 ± 0.16	0.40 ± 0.44	0.35 ± 0.24	0.38 ± 0.21
Spleen	0.87 ± 0.24	0.37 ± 0.09	0.14 ± 0.10	0.39 ± 0.11	0.37 ± 0.17
Stomach	1.58 ± 0.61	0.55 ± 0.12	0.18 ± 0.01	0.52 ± 0.13	0.16 ± 0.06
Kidneys	12.9 ± 4.98	5.43 ± 2.0	4.75 ± 0.48	5.31 ± 1.10	3.87 ± 2.12
Muscle	0.57 ± 0.12	0.21 ± 0.34	0.08 ± 0.09	0.12 ± 0.02	0.13 ± 0.10
Pancreas	0.56 ± 0.25	0.04 ± 0.07	0.04 ± 0.05	0.09 ± 0.07	0.17 ± 0.10
Bone	1.26 ± 0.48	0.38 ± 0.15	0.15 ± 0.13	0.37 ± 0.18	0.54 ± 0.26
	Percent injected dose (% ID)
Intestines	1.25 ± 0.41	0.49 ± 0.08	0.39 ± 0.15	0.53 ± 0.15	0.30 ± 0.09
Urine	59.99 ± 8.23	80.5 ± 10.68	95.39 ± 1.15	89.71 ± 1.06	95.51 ± 1.85
	Uptake ratio of tumor/normal tissue
Tumor/blood	3.41	73.09	5.27	70.39	12.03
Tumor/kidney	0.88	3.10	0.17	2.39	1.18
Tumor/liver	3.13	26.27	1.27	15.27	17.58
Tumor/lung	6.88	21.83	1.25	17.85	9.14
Tumor/muscle	19.81	80.05	9.88	105.58	35.15

^*p<0.05 for determining significance of differences in tumor and kidney uptake between ²⁰³Pb-DOTA-GGNle-CyCMSH_hex with or without peptide blockade at 2 h post-injection.

ABBREVIATIONS USED

α-MSH

α-melanocyte stimulating hormone

MC1R

melanocortin-1 receptor

SPECT

single photon emission computed tomography

PET

positron emission tomography

Fmoc

fluorenylmethyloxycarbonyl

RP-HPLC

reverse phase-high performance liquid chromatography

LC-MS

liquid chromatography-mass spectrometry

DAPI 4’

6-diamidino-2-phenylindole

EDTA

ethylenediaminetetraacetic acid

NDP-MSH

[Nle⁴, D-Phe⁷]-α-MSH

BSA

bovine serum albumin

PBS

phosphate buffered saline

MEM

Modified Eagle’s medium