Cu-64-Labeled Lactam Bridge-Cyclized α-MSH Peptides for PET Imaging of Melanoma

Abstract

The purpose of this study was to examine and compare the melanoma targeting and imaging properties of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex {⁶⁴Cu-1,4,7-triazacyclononane-1,4,7-triacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH₂} and ⁶⁴Cu-DOTA-GGNle-CycMSH_hex {⁶⁴Cu-1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid-GGNle-CycMSH_hex}. Two lactam bridge-cyclized peptides, NOTA-GGNle-CycMSH_hex and DOTA-GGNle-CycMSH_hex, were synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinity of NOTA-GGNle-CycMSH_hex was determined in B16/F1 melanoma cells and compared with DOTA-GGNle-CycMSH_hex. The melanoma targeting and imaging properties of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex and ⁶⁴Cu-DOTA-GGNle-CycMSH_hex were determined in B16/F1 melanoma-bearing C57 mice. NOTA-GGNle-CycMSH_hex and DOTA-GGNle-CycMSH_hex displayed comparable MC1 receptor binding affinities (1.6 vs. 2.1 nM). The substitution of DOTA with NOTA dramatically increased the melanoma uptake and decreased the renal and liver uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex. The tumor uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex was between 12.39 ± 1.61 and 12.71 ± 2.68 % ID/g at 0.5, 2 and 4 h post-injection. The accumulation of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex activity in normal organs was lower than 1.02 % ID/g except for the kidneys 2, 4 and 24 h post-injection. The tumor/liver uptake ratios of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex were 17.96, 16.95 and 8.02, whereas the tumor/kidney uptake ratios of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex were 2.52, 3.60 and 5.74 at 2, 4 and 24 h post-injection, respectively. Greater than 91% of the injected radioactivity cleared through the urinary system by 2 h post-injection. The substitution of DOTA with NOTA resulted in a dramatic increase in melanoma uptake and decrease in renal and liver uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex compared to ⁶⁴Cu-DOTA-GGNle-CycMSH_hex. High melanoma uptake coupled with low accumulation in non-target organs suggested ⁶⁴Cu-NOTA-GGNle-CycMSH_hex as a lead radiolabeled peptide for melanoma imaging and therapy.

INTRODUCTION

Melanoma is the most deadly skin cancer with an increased incidence worldwide over the past decade. The cancer statistics from the American Cancer Society predicted that 68,130 new melanoma cases would be diagnosed and 8,700 deaths of melanoma would occur in 2010 in the United States.¹ There is a great need for better diagnostic strategies and more effective treatments for melanoma since no curative treatment exists for patients with metastatic melanoma. At the present time, the utilization of melanocortin-1 (MC1) receptor-targeting radiolabeled alpha-melanocyte stimulating hormone (α-MSH) peptides represents a very promising strategy for melanoma detection and treatment. This strategy takes advantage of fast distribution via blood circulation, receptor-targeting cancer tissue localization and rapid whole-body clearance of the radiolabeled α-MSH peptides. Both linear and cyclized α-MSH peptide radiopharmaceuticals have been reported to target the MC1 receptors^2–6 for melanoma imaging and therapy.^7–23

Over the past several years, we have developed two generations of novel lactam bridge-cyclized ¹¹¹In-labeled α-MSH peptides for melanoma imaging using single photon emission computed tomography (SPECT).^18–23 The first-generation peptides built upon the backbone of CycMSH {c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp], 12 amino acids} peptide,^18–21 whereas the second-generation peptides were based on the construct of CycMSH_hex {c[Asp-His-DPhe-Arg-Trp-Lys]-CONH₂, 6 amino acids} peptide. ^22–23 The MC1 receptor binding motif (His-DPhe-Arg-Trp) was retained in the 12-amino acid CycMSH moiety cyclized by a Lys-Asp lactam bridge, or in the 6-amino acid CycMSH_hex moiety cyclized via an Asp-Lys lactam bridge. The radiometal chelator DOTA (1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid) was conjugated to the N-terminus of the CycMSH or CycMSH_hex moiety with or without an amino acid linker for radiolabeling. The second-generation ¹¹¹In-labeled CycMSH_hex peptides exhibited enhanced melanoma uptake and reduced renal uptake than the first-generation ¹¹¹In-labeled CycMSH peptides.^18–23 For instance, ¹¹¹In-DOTA-Nle-CycMSH_hex displayed higher melanoma uptake (19.39 ± 1.65 % ID/g at 2 h post-injection) and lower renal uptake (9.52 ± 0.44 % ID/g at 2 h post-injection) compared to ¹¹¹In-DOTA-GlyGlu-CycMSH in B16/F1 melanoma-bearing C57 mice.^18–22 Recently, we have found that the introduction of the -GlyGly- linker between the DOTA and Nle-CycMSH_hex moiety maintained high melanoma uptake (19.05 ± 5.04 % ID/g at 2 h post-injection) while reducing the renal and liver uptake of ¹¹¹In-DOTA-GGNle-CycMSH_hex by 42 and 68% compared to ¹¹¹In-DOTA-Nle-CycMSH_hex.²³ The melanoma lesions could be clearly visualized by SPECT/CT using ¹¹¹In-DOTA-GGNle-CycMSH_hex as an imaging probe.²³

We have been interested in expanding our melanoma imaging modality from SPECT to positron emission tomography (PET) because PET has some distinct advantages over other functional imaging modalities in terms of sensitivity, resolution and quantification. The combination of the outstanding imaging properties of PET with specific receptor-targeting properties of peptide radiopharmaceuticals offers an exciting opportunity for sensitive tumor-specific imaging. Copper-64 (t_1/2=12.7 h, 17.4% β⁺, 40% β⁻) is an attractive PET radionuclide coupled with therapeutic properties due to its positron- and electron-emissions.^24–26 Despite the fact that DOTA can form stable complexes with a variety of diagnostic and therapeutic radionuclides, DOTA can only form a moderately stable complex with ⁶⁴Cu due to the demetallation of ⁶⁴Cu-DOTA moiety in vivo. The dissociation of ⁶⁴Cu from DOTA chelator generally resulted in high accumulation in non-target tissues such as liver.^{13,15,27–30} Alternatively, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) could form a more stable complex with ⁶⁴Cu than DOTA in vivo. It was reported that the ⁶⁴Cu-NOTA-8-Aoc-BBN(7-14)NH₂ displayed higher resistance to transmetallation reactions in vivo than ⁶⁴Cu-DOTA-8-Aoc-BBN(7-14)NH₂.^{27 64}Cu-NOTA-8-Aoc-BBN(7-14)NH₂ displayed considerably lower liver uptake than ⁶⁴Cu-DOTA-8-Aoc-BBN(7-14)NH₂^27,28 in PC-3 tumor-bearing mice, underscoring the advantage of NOTA for ⁶⁴Cu conjugation compared to DOTA.

¹¹¹In-DOTA-GGNle-CycMSH_hex exhibited more favorable pharmacokinetic properties (comparable high tumor uptake, less renal and liver uptake) than ¹¹¹In-DOTA-Nle-CycMSH_hex in our previous reports.^22,23 Hence, we managed to develop PET imaging probes building upon the GGNle-CycMSH_hex peptide construct. In this study, we hypothesized that ⁶⁴Cu-NOTA-GGNle-CycMSH_hex would display more favorable melanoma targeting and pharmacokinetic properties than ⁶⁴Cu-DOTA-GGNle-CycMSH_hex. To examine the hypothesis, we synthesized NOTA-GGNle-CycMSH_hex and DOTA-GGNle-CycMSH_hex peptides. The MC1 receptor binding affinity of NOTA-GGNle-CycMSH_hex was determined in B16/F1 melanoma cells and compared with DOTA-GGNle-CycMSH_hex. Thereafter, we examined the biodistribution properties of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex and ⁶⁴Cu-DOTA-GGNle-CycMSH_hex in B16/F1 melanoma-bearing C57 mice. Furthermore, we determined the melanoma imaging properties of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex and ⁶⁴Cu-DOTA-GGNle-CycMSH_hex in B16/F1 melanoma-bearing C57 mice using small animal PET/CT.

EXPERIMENTAL SECTION

Chemicals and Reagents

Amino acid and resin were purchased from Advanced ChemTech Inc. (Louisville, KY) and Novabiochem (San Diego, CA). NO2AtBu ester 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 receptor binding assay. ⁶⁴CuCl₂ was purchased from Trace Life Sciences, Inc. (Dallas, TX) for radiolabeling. All other chemicals used in this study were purchased from Thermo Fischer Scientific (Waltham, MA) and used without further purification. B16/F1 murine melanoma cells were obtained from American Type Culture Collection (Manassas, VA).

Peptide Synthesis and Receptor Binding Assay

New NOTA-GGNle-CycMSH_hex was synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. Briefly, the intermediate scaffold of Fmoc-Asp(O-2-PhiPr)-His(Trt)-DPhe-Arg(Pbf)-Trp(Boc)-Lys(Mtt) was synthesized on H₂N-Novagel resin by an Advanced ChemTech multiple-peptide synthesizer (Louisville, KY). Generally, 70 μmol of resin, 210 μmol of each Fmoc-protected amino acid and NO2AtBu were used for the synthesis. The protecting groups of Mtt and 2-phenylisopropyl were removed by 2.5% of trifluoroacetic acid (TFA) for peptide cyclization. The cyclization reaction was achieved on the resin by an overnight reaction in N,N-dimethylformamide (DMF) using benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium-hexafluorophosphate (PyBOP) as a coupling agent in the presence of N,N-diisopropylethylamine (DIPEA). After the cyclization, the moiety of NO2AtBu-CH₂-Gly-Gly-Nle was coupled to the cyclic intermediate scaffold to yield NO2AtBu-CH₂-Gly-Gly-Nle-Cyclic[Asp-His(Trt)-DPhe-Arg(Pbf)-Trp(Boc)-Lys] on the resin. All protecting groups were totally removed and the peptide was cleaved from the resin by treating with a mixture of trifluoroacetic acid (TFA), thioanisole, phenol, water, ethanedithiol and triisopropylsilane (87.5:2.5:2.5:2.5:2.5:2.5) for 2 h at 25 °C. The peptide was precipitated and washed with ice-cold ether four times, purified by reverse phase-high performance liquid chromatography (RP-HPLC) and characterized by liquid chromatography-mass spectrometry (LC-MS). DOTA-GGNle-CycMSH_hex was synthesized, purified by RP-HPLC and characterized by LC-MS according to our published procedure.²³ The MC1 receptor binding affinity (IC₅₀ value) of NOTA-GGNle-CycMSH_hex was determined in B16/F1 melanoma cells by in vitro competitive receptor binding assay according to our published procedure.²³ The IC₅₀ value of DOTA-GGNle-CycMSH_hex was cited from our previous report²³ for direct comparison.

Peptide Radiolabeling with ⁶⁴Cu

Both ⁶⁴Cu-NOTA-GGNle-CycMSH_hex and ⁶⁴Cu-DOTA-GGNle-CycMSH_hex were prepared in a 0.5 M NH₄OAc-buffered solution at pH 5.4. Briefly, 10 μL of ⁶⁴CuCl₂ (37–74 MBq in 0.05 M HCl aqueous solution), 10 μL of 1 mg/mL NOTA-GGNle-CycMSH_hex or DOTA-GGNle-CycMSH_hex 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 the incubation, 10 μL of 0.5% EDTA aqueous solution was added into the reaction vial to scavenge potential unbound ⁶⁴Cu²⁺ 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 a 20-minute gradient of 18–28% acetonitrile in 20 mM HCl aqueous solution with a flow rate of 1.0 mL/min. Each purified peptide sample was purged with N₂ gas at 25 °C for 20 min to remove the acetonitrile. The pH of final solution was adjusted to 7.4 with 0.1 N NaOH and sterile normal saline for animal studies. In vitro serum stabilities of both ⁶⁴Cu-NOTA-GGNle-CycMSH_hex and ⁶⁴Cu-DOTA-GGNle-CycMSH_hex were determined by incubation in mouse serum at 37 °C for 2 h and monitored for degradation by RP-HPLC.

Biodistribution Studies

All the animal studies were conducted in compliance with Institutional Animal Care and Use Committee approval. The melanoma targeting and pharmacokinetic properties of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex and ⁶⁴Cu-DOTA-GGNle-CycMSH_hex were determined in B16/F1 melanoma-bearing C57 female mice (Harlan, Indianapolis, IN). The C57 mice were subcutaneously inoculated with 1×10⁶ B16/F1 cells on the right flank for each mouse to generate B16/F1 tumors. The weights of tumors reached approximately 0.2 g 10 days post cell inoculation. Each melanoma-bearing mouse was injected with 0.037 MBq of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex or ⁶⁴Cu-DOTA-GGNle-CycMSH_hex via the tail vein. Groups of five mice were sacrificed at 0.5, 2, 4 and 24 h post-injection, and tumors and organs of interest were harvested, weighed and counted. Blood values were taken as 6.5% of the whole-body weight. The specificities of the tumor uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex and ⁶⁴Cu-DOTA-GGNle-CycMSH_hex were determined by co-injecting 10 μg (6.07 nmol) of unlabeled NDP-MSH peptide at 2 h post-injection.

Melanoma Imaging with ⁶⁴Cu-NOTA-GGNle-CycMSH_hex and ⁶⁴Cu-DOTA-GGNle-CycMSH_hex

The melanoma imaging properties of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex or ⁶⁴Cu-DOTA-GGNle-CycMSH_hex were determined in B16/F1 melanoma-bearing C57 mice using small animal PET/CT. Approximately 37.0 MBq of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex or ⁶⁴Cu-DOTA-GGNle-CycMSH_hex was injected into each mouse via the tail vein. The mice were sacrificed for small animal PET and CT imaging 2 and 4 h post-injection. The 9-min CT imaging performed by Nano-SPECT/CT (Bioscan, Washington DC) was immediately followed by the PET imaging conducted by LabPET (Gamma Media-Ideas, Quebec, Canada) using the same animal bed. The PET data were reconstructed by maximum-likelihood expectation maximization (MLEM) reconstruction algorithms. The CT data were reconstructed, visualized and fused with the PET data using InVivoScope (Bioscan, Washington DC).

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 the difference in tumor, kidney and liver uptake between ⁶⁴Cu-NOTA-GGNle-CycMSH_hex with/without NDP-MSH co-injection, and between ⁶⁴Cu-DOTA-GGNle-CycMSH_hex with/without NDP-MSH co-injection. The differences at the 95% confidence level (p<0.05) were considered significant.

RESULTS

Novel NOTA-GGNle-CycMSH_hex was synthesized and purified by RP-HPLC. NOTA-GGNle-CycMSH_hex displayed greater than 90% chemical purity after HPLC purification. The identity of NOTA-GGNle-CycMSH_hex was confirmed by electrospray ionization mass spectrometry. The calculated and found molecular weights of NOTA-GGNle-CycMSH_hex were 1381 and 1381, respectively. Meanwhile, DOTA-GGNle-CycMSH_hex was synthesized and characterized according to our published procedure²³ for direct comparison. The schematic structures of NOTA-GGNle-CycMSH_hex and DOTA-GGNle-CycMSH_hex are shown in Figure 1. The competitive binding curve of NOTA-GGNle-CycMSH_hex in B16/F1 cells is presented in Figure 2 and compared with DOTA-GGNle-CycMSH_hex. The IC₅₀ of NOTA-GGNle-CycMSH_hex was 1.6 nM, which was comparable to that of DOTA-GGNle-CycMSH_hex (2.1 nM).²³

Figure 1.

Schematic structures of NOTA-GGNle-CycMSH_hex and DOTA-GGNle-CycMSH_hex.

Figure 2.

The in vitro competitive binding curves of NOTA-GGNle-CycMSH_hex (■) and DOTA- GGNle-CycMSH_hex (▲) in B16/F1 melanoma cells. The IC₅₀ values of NOTA-GGNle- CycMSH_hex and DOTA-GGNle-CycMSH_hex were 1.6 and 2.1 nM, respectively. The data of DOTA-GGNle-CycMSH_hex was cited from our previous report23 for comparison.

Both NOTA-GGNle-CycMSH_hex and DOTA-GGNle-CycMSH_hex were readily labeled with ⁶⁴Cu in 0.5 M ammonium acetate at pH 5.4 with greater than 95% radiolabeling yields. ⁶⁴Cu-NOTA-GGNle-CycMSH_hex and ⁶⁴Cu-DOTA-GGNle-CycMSH_hex were completely separated from their excess non-labeled peptides by RP-HPLC. The specific activities of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex and ⁶⁴Cu-DOTA-GGNle-CycMSH_hex were approximately 20,000 Ci/g and 20,000 Ci/g, respectively. The retention times of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex and ⁶⁴Cu-DOTA-GGNle-CycMSH_hex were 18.5 and 18.8 min, respectively. Both ⁶⁴Cu-NOTA-GGNle-CycMSH_hex and ⁶⁴Cu-DOTA-GGNle-CycMSH_hex showed greater than 98% radiochemical purities after HPLC purification, and were stable in mouse serum at 37°C for 2 h. Only intact ⁶⁴Cu-NOTA-GGNle-CycMSH_hex and ⁶⁴Cu-DOTA-GGNle-CycMSH_hex were detected by RP-HPLC after 2 h of incubation in mouse serum.

The melanoma targeting and pharmacokinetic properties of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex and ⁶⁴Cu-DOTA-GGNle-CycMSH_hex were determined in B16/F1 melanoma-bearing C57 mice. The biodistribution results of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex and ⁶⁴Cu-DOTA-GGNle-CycMSH_hex are presented in Tables 1 and 2. ⁶⁴Cu-NOTA-GGNle-CycMSH_hex exhibited rapid high melanoma uptake and prolonged tumor retention. The tumor uptake value of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex was 12.51 ± 0.44 % ID/g at 0.5 h post-injection. ⁶⁴Cu-NOTA-GGNle-CycMSH_hex displayed similar high tumor uptake (12.39 ± 1.61 and 12.71 ± 2.68 % ID/g) at 2 and 4 h post-injection. Even at 24 h post-injection, there was 4.25 ± 0.32 % ID/g of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex activity remained in the tumor. Approximately 94.2% of the tumor uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex was blocked by 10 μg (6.07 nmol) of non-radiolabeled NDP-MSH (p<0.05) (Figure 3), demonstrating that the tumor uptake was specific and MC1 receptor-mediated. Whole-body clearance of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex was rapid, with 91.7% of the injected radioactivity cleared through the urinary system by 2 h post-injection. Normal organ uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex was lower than 1.02 % ID/g except for the kidneys at 2, 4 and 24 h post-injection. The liver uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex was only 0.69 ± 0.06 and 0.75 ± 0.17 % ID/g at 2 and 4 h post-injection. The kidney uptake was 9.28 ± 0.77 % ID/g at 0.5 h post-injection and decreased to 4.92 ± 1.43 % ID/g at 2 h post-injection. The renal uptake was only 0.74 ± 0.18 % ID/g at 24 h post-injection. Co-injection of NDP-MSH didn’t significantly reduce the renal uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex activity at 2 h post-injection, indicating that the renal uptake was not MC1 receptor-mediated. High tumor uptake and prolonged retention coupled with rapid whole-body clearance resulted in high tumor/blood and high tumor/normal organ uptake ratios achieved as early as 0.5 h post-injection. The tumor/liver uptake ratios of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex were 11.37, 17.96, 16.95 and 8.02 at 0.5, 2, 4 and 24 h post-injection, whereas the tumor/kidney uptake ratios of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex were 1.35, 2.52, 3.60 and 5.74 at 0.5, 2, 4 and 24 h post-injection.

Table 1.

Biodistribution of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex in B16/F1 melanoma-bearing C57 mice. The data were presented as percent injected dose/gram or as percent injected dose (mean ± SD, n=5)

Tissue	0.5 h	2 h	4 h	24 h
	Percent injected dose/gram (%ID/g)
Tumor	12.51±0.44	12.39±1.61	12.71±2.68	4.25±0.32
Brain	0.10±0.03	0.04±0.02	0.07±0.04	0.04±0.03
Blood	1.59±0.19	0.33±0.10	0.30±0.11	0.10±0.04
Heart	0.89±0.29	0.24±0.13	0.41±0.24	0.13±0.09
Lung	2.05±0.26	0.56±0.09	0.71±0.17	0.37±0.11
Liver	1.10±0.18	0.69±0.06	0.75±0.17	0.53±0.07
Spleen	0.51±0.07	0.25±0.09	0.17±0.08	0.15±0.06
Stomach	0.65±0.15	0.76±0.27	1.02±0.17	0.22±0.04
Kidneys	9.28±0.77	4.92±1.43	3.53±0.57	0.74±0.18
Muscle	0.31±0.08	0.29±0.16	0.36±0.09	0.04±0.03
Pancreas	0.71±0.04	0.29±0.13	0.34±0.04	0.30±0.14
Bone	0.41±0.19	0.27±0.05	0.19±0.06	0.09±0.07
Skin	2.23±0.21	0.35±0.11	0.71±0.21	0.35±0.27
	Percent injected dose (%ID)
Intestines	1.27±0.11	1.41±0.70	1.19±0.23	0.50±0.20
Urine	76.82±1.19	91.67±1.57	91.47±1.66	97.05±0.55
	Tumor-to-normal-tissue uptake ratio
Tumor/Blood	7.87	37.55	42.37	42.50
Tumor/Heart	14.06	51.63	31.00	32.69
Tumor/Kidneys	1.35	2.52	3.60	5.74
Tumor/Lung	6.10	22.13	17.90	11.49
Tumor/Liver	11.37	17.96	16.95	8.02
Tumor/Stomach	19.25	16.30	12.46	19.32
Tumor/Muscle	40.35	42.72	35.31	106.25

Table 2.

Biodistribution of ⁶⁴Cu-DOTA-GGNle-CycMSH_hex in B16/F1 melanoma-bearing C57 mice. The data were presented as percent injected dose/gram or as percent injected dose (mean ± SD, n=5)

Tissue	0.5 h	2 h	4 h	24 h
	Percent injected dose/gram (%ID/g)
Tumor	5.61±0.91	5.20±1.28	5.25±1.22	5.96±0.36
Brain	0.27±0.03	0.39±0.22	0.17±0.06	0.29±0.06
Blood	2.33±0.54	1.03±0.19	0.81±0.33	1.07±0.17
Heart	2.22±0.25	1.75±0.13	1.45±0.13	2.23±0.10
Lung	3.76±0.54	3.93±0.64	4.39±0.36	4.45±0.51
Liver	11.59±1.77	10.33±1.18	9.61±1.34	7.23±0.58
Spleen	1.43±0.58	0.82±0.26	1.12±0.26	2.87±1.00
Stomach	2.83±0.84	4.78±0.96	5.09±0.69	1.21±0.14
Kidneys	14.44±1.85	9.30±2.81	7.45±0.96	5.39±0.42
Muscle	0.80±0.12	0.42±0.24	0.02±0.02	0.34±0.16
Pancreas	1.34±0.28	1.30±0.23	1.26±0.16	1.40±0.28
Bone	1.07±0.24	0.82±0.28	1.95±0.53	0.42±0.11
Skin	2.46±0.40	1.60±0.35	1.27±0.47	0.46±0.40
	Percent injected dose (%ID)
Intestines	7.19±0.35	10.71±1.68	12.52±2.98	6.63±0.51
Urine	46.00±4.07	62.68±3.56	62.22±4.43	67.36±1.55
	Tumor-to-normal-tissue uptake ratio
Tumor/Blood	2.41	5.05	6.48	5.57
Tumor/Heart	2.53	2.97	3.62	2.67
Tumor/Kidneys	0.39	0.56	0.70	1.11
Tumor/Lung	1.49	1.32	1.20	1.34
Tumor/Liver	0.48	0.50	0.55	0.82
Tumor/Stomach	1.98	1.09	1.03	4.93
Tumor/Muscle	7.01	12.38	262.50	17.53

Figure 3.

The tumor, kidney and liver uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex (A) and 64Cu-DOTA-GGNle-CycMSH_hex (B) with ( ) or without ( ) 10 μg of NDP-MSH blockade at 2 h post-injection. *p<0.05.

As we anticipated, ⁶⁴Cu-DOTA-GGNle-CycMSH_hex showed lower tumor uptake than ⁶⁴Cu-NOTA-GGNle-CycMSH_hex at 0.5, 2 and 4 h post-injection (Figure 4). The tumor uptake of ⁶⁴Cu-DOTA-GGNle-CycMSH_hex was 5.61 ± 0.91, 5.20 ± 1.28 and 5.25 ± 1.22 % ID/g at 0.5, 2 and 4 h post-injection, respectively. Co-injection of non-radioactive NDP-MSH blocked 47.9% of the tumor uptake at 2 h post-injection (p<0.05) (Figure 3), indicating that the partial tumor uptake of ⁶⁴Cu-DOTA-GGNle-CycMSH_hex was MC1 receptor-specific. Besides the lower tumor uptake, ⁶⁴Cu-DOTA-GGNle-CycMSH_hex displayed higher renal uptake than ⁶⁴Cu-NOTA-GGNle-CycMSH_hex at all time points investigated. The renal uptake of ⁶⁴Cu-DOTA-GGNle-CycMSH_hex was 14.44 ± 1.85, 9.30 ± 2.81, 7.45 ± 0.96 and 5.39 ± 0.42 % ID/g at 0.5, 2, 4 and 24 h post-injection, respectively. Co-injection of NDP-MSH didn’t significantly reduce the renal uptake of ⁶⁴Cu-DOTA-GGNle-CycMSH_hex activity at 2 h post-injection, indicating that the renal uptake was non-specific. Not surprisingly, ⁶⁴Cu-DOTA-GGNle-CycMSH_hex also displayed higher accumulation than ⁶⁴Cu-NOTA-GGNle-CycMSH_hex in normal organs such as liver, lung, heart and stomach at all the time points investigated. The liver uptake of ⁶⁴Cu-DOTA-GGNle-CycMSH_hex was 11.59 ± 1.77, 10.30 ± 1.18, 9.61 ± 1.34 and 7.23 ± 0.58 % ID/g at 0.5, 2, 4 and 24 h post-injection, respectively. High liver uptake associated with ⁶⁴Cu-DOTA-GGNle-CycMSH_hex activity was not receptor-mediated because co-injection of NDP-MSH didn’t significantly reduce the liver uptake of ⁶⁴Cu-DOTA-GGNle-CycMSH_hex at 2 h post-injection (Figure 3). In comparison with ⁶⁴Cu-NOTA-GGNle-CycMSH_hex, ⁶⁴Cu-DOTA-GGNle-CycMSH_hex exhibited dramatically lower tumor/kidney and tumor/liver uptake ratios (Figure 5) due to the decreased tumor uptake and increased renal and liver uptake. The tumor/kidney uptake ratios of ⁶⁴Cu-DOTA-GGNle-CycMSH_hex were 0.39, 0.56, 0.70 and 1.11 at 0.5, 2, 4 and 24 h post-injection, whereas the tumor/liver uptake ratios of ⁶⁴Cu-DOTA-GGNle-CycMSH_hex were 0.48, 0.50, 0.55 and 0.82 at 0.5, 2, 4 and 24 h post-injection.

Figure 4.

The comparison of tumor (A), kidney (B) and liver (C) uptake between ⁶⁴Cu-NOTA-GGNle-CycMSH_hex ( ) and ⁶⁴Cu-DOTA-GGNle-CycMSH_hex ( ) at 0.5, 2 and 4 h post-injection. *p<0.05.

Figure 5.

The comparison of tumor/kidney (A) and tumor/liver (B) uptake ratios among ⁶⁴Cu-DOTA- GGNle-CycMSH_hex ( ), ⁶⁴Cu-NOTA-GGNle-CycMSH_hex ( ) and ⁶⁴Cu-CBTE2A- ReCCMSH(Arg¹¹) ( ) at 0.5, 2, 4 and 24 h post-injection. *p<0.05, significance comparison between ⁶⁴Cu-NOTA-GGNle-CycMSH_hex and 64Cu-DOTA-GGNle-CycMSH_hex, and between ⁶⁴Cu-NOTA-GGNle-CycMSH_hex and 64Cu-CBTE2A-ReCCMSH(Arg11). The tumor/kidney and tumor/liver uptake ratios of ⁶⁴Cu-CBTE2A-ReCCMSH(Arg¹¹) ( ) were calculated based on the results published by Wei et al.¹⁴

We further evaluated the melanoma imaging properties (melanoma visualization and imaging contrast) of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex and ⁶⁴Cu-DOTA-GGNle-CycMSH_hex. The coronal and transversal PET/CT images are presented in Figure 6. The melanoma tumors were clearly visualized by PET/CT using either ⁶⁴Cu-NOTA-GGNle-CycMSH_hex or ⁶⁴Cu-DOTA-GGNle-CycMSH_hex as an imaging probe. However, ⁶⁴Cu-NOTA-GGNle-CycMSH_hex displayed higher tumor uptake and lower non-target organ accumulation compared to ⁶⁴Cu-DOTA-GGNle-CycMSH_hex. Both coronal and transversal images of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex exhibited high tumor to normal organ uptake ratios except for the kidneys, which was consistent with the biodistribution results.

Figure 6.

Representative coronal (A, B, C, D) and transversal (E, F, G, H) PET/CT images of B16/F1 melanoma-bearing mice (12 days post cell inoculation) at 2 h (A, E, C, G) and 4 h (B, F, D, H) post-injection of 37.0 MBq of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex (A, B, E, F) or 64Cu-DOTA- GGNle-CycMSH_hex (C, D, G, H). The tumors (T) and kidneys (K) were highlighted with arrows on the images.

DISCUSSION

Both ⁶⁴Cu-labeled DOTA-conjugated linear or rhenium-cyclized a-MSH peptides have been reported to target the MC1 receptors for successful melanoma imaging.^13,15 However, the rhenium-cyclized ⁶⁴Cu-DOTA-ReCCMSH(Arg¹¹) {⁶⁴Cu-DOTA-Re-[Cys^3,4,10, D-Phe⁷, Arg¹¹]a-MSH_3–13}¹³ showed higher melanoma uptake than the linear ⁶⁴Cu-DOTA-NAPamide {Ac-Nle-Asp-His-D-Phe-Arg-Trp-Gly-Lys(⁶⁴Cu-DOTA)-NH₂}¹⁵ at 2, 4 and 24 h post-injection. The melanoma uptake (8.80 ± 1.70 % ID/g) of ⁶⁴Cu-DOTA-ReCCMSH(Arg¹¹) was 1.9 times the melanoma uptake (4.63 ± 0.45 % ID/g) of ⁶⁴Cu-DOTA-NAPamide at 2 h post-injection. Not surprisingly, both ⁶⁴Cu-DOTA-ReCCMSH(Arg¹¹) and ⁶⁴Cu-DOTA-NAPamide displayed high liver uptake (7.3 vs. 11.0 % ID/g at 2 h post-injection) that could be attributed to the demetallation of ⁶⁴Cu from the DOTA chelator in vivo. The in vivo stability of ⁶⁴Cu-DOTA moiety was dramatically improved by using the CBTE2A {4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane} chelator. Compared to ⁶⁴Cu-DOTA-ReCCMSH(Arg¹¹), ⁶⁴Cu-CBTE2A-ReCCMSH(Arg¹¹) showed dramatically reduced liver uptake (1.57 ± 0.42 % ID/g) while maintained similar melanoma uptake (7.09 ± 3.20 % ID/g) at 2 h post-injection.¹⁴ Interestingly, the substitution of DOTA with NOTA considerably reduced the liver uptake of ⁶⁴Cu-NOTA-8-Aoc-BBN(7-14)NH₂ compared to ⁶⁴Cu-DOTA-8-Aoc-BBN(7-14)NH₂ in PC-3 tumor-bearing mice,^27,28 suggesting that the NOTA was more suitable than DOTA for ⁶⁴Cu conjugation. Therefore, based on the novel GGNle-CycMSH_hex peptide construct we identified,²³ we were interested in examining whether the replacement of DOTA with NOTA could enhance the melanoma uptake and reduce the liver uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex compared to ⁶⁴Cu-DOTA-GGNle-CycMSH_hex in this study.

In this study, we hypothesized that ⁶⁴Cu-NOTA-GGNle-CycMSH_hex would display more favorable melanoma targeting and pharmacokinetic properties than ⁶⁴Cu-DOTA-GGNle-CycMSH_hex. To examine the hypothesis, we evaluated NOTA-GGNle-CycMSH_hex and DOTA-GGNle-CycMSH_hex both in vitro and in vivo. The substitution of DOTA with NOTA slightly improved the MC1 receptor binding affinity of the peptide in B16/F1 melanoma cells (Figure 2). NOTA-GGNle-CycMSH_hex exhibited 1.6 nM MC1 receptor binding affinity, whereas DOTA-GGNle-CycMSH_hex showed 2.1 nM MC1 receptor binding affinity. Despite the slightly difference in receptor binding affinity between NOTA-GGNle-CycMSH_hex and DOTA-GGNle-CycMSH_hex, we observed dramatic differences in melanoma targeting and pharmacokinetic properties between their ⁶⁴Cu-conjugates. ⁶⁴Cu-NOTA-GGNle-CycMSH_hex exhibited higher melanoma uptake than ⁶⁴Cu-DOTA-GGNle-CycMSH_hex in B16/F1 melanoma-bearing mice. The tumor uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex was 2.2, 2.4 and 2.4 times the tumor uptake of ⁶⁴Cu-DOTA-GGNle-CycMSH_hex at 0.5, 2 and 4 h post-injection, respectively (Tables 1 and 2, Figure 4). The lower melanoma uptake of ⁶⁴Cu-DOTA-GGNle-CycMSH_hex was likely due to the dissociation of ⁶⁴Cu from the DOTA chelator in vivo. Interestingly, the co-injection of 10 μg of NDP-MSH blocked 94.2% of the tumor uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex, whereas the co-injection of 10 μg of NDP-MSH only blocked 47.9% of the tumor uptake of ⁶⁴Cu-DOTA-GGNle-CycMSH_hex (Figure 3). ⁶⁴Cu-DOTA-GGNle-CycMSH_hex showed 2.71 ± 0.42 % ID/g of non-specific melanoma uptake, which was similar to those of ⁶⁴Cu-DOTA-ReCCMSH(Arg¹¹) and ⁶⁴Cu-DOTA-NAPamide.^13,15

The substitution of DOTA with NOTA dramatically reduced the renal uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex compared to ⁶⁴Cu-DOTA-GGNle-CycMSH_hex (Figure 4). The renal uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex was only 64.3%, 52.9%, 47.3% and 13.7% of the renal uptake of ⁶⁴Cu-DOTA-GGNle-CycMSH_hex at 0.5, 2, 4 and 24 h post-injection, respectively. The increased tumor uptake and decreased renal uptake dramatically improved the tumor to kidney uptake ratios of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex at all time points investigated in this study (Figure 5). The tumor to kidney uptake ratios of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex were 3.5, 4.5, 5.1 and 5.2 times those of ⁶⁴Cu-DOTA-GGNle-CycMSH_hex at 0.5, 2, 4 and 24 h post-injection, respectively. Meanwhile, the substitution of DOTA with NOTA dramatically reduced the liver uptake by 14-fold to 0.69 ± 0.06 % ID/g at 2 h post-injection. The liver uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex was lower than 0.8 % ID/g after 2 h post-injection. The liver uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex was only 9.5%, 6.7%, 7.8% and 7.3% of the liver uptake of ⁶⁴Cu-DOTA-GGNle-CycMSH_hex at 0.5, 2, 4 and 24 h post-injection, respectively (Figure 4). Higher tumor uptake and lower liver uptake dramatically enhanced the tumor to liver uptake ratios of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex at all time points investigated in this study (Figure 5). The tumor to liver uptake ratios of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex were 23.7, 35.9, 30.8 and 9.8 times those of ⁶⁴Cu-DOTA-GGNle-CycMSH_hex at 0.5, 2, 4 and 24 h post-injection, respectively. Extremely low liver uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex indicated the in vivo stability of ⁶⁴Cu-NOTA moiety in ⁶⁴Cu-NOTA-GGNle-CycMSH_hex peptide, which was consistent with the findings reported for ⁶⁴Cu-NOTA-8-Aoc-BBN(7-14)NH₂^27,28 and ⁶⁴Cu-NOTA-STh peptides.²⁹ As we anticipated, the improved in vivo stability of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex also resulted in its low accumulation (<1.02 % ID/g) in non-target organs such as lung, stomach and heart after 2 h post-injection. Rapid whole-body clearance of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex further confirmed its enhanced in vivo stability compared to ⁶⁴Cu-DOTA-GGNle-CycMSH_hex. Greater than 91% of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex activity cleared out the body through urinary system, whereas only 63% of ⁶⁴Cu-DOTA-GGNle-CycMSH_hex activity excreted out the body via urinary system at 2 h post-injection.

At the present time, ⁶⁴Cu-CBTE2A-ReCCMSH(Arg¹¹) displayed the highest tumor/kidney ratio (1.11 at 24 h post-injection) and tumor/liver uptake ratio (4.52 at 2 h post-injection) among all reported ⁶⁴Cu-labeled linear and cyclic a-MSH peptides.^13–15 Remarkably, ⁶⁴Cu-NOTA-GGNle-CycMSH_hex displayed higher tumor uptake and lower renal and liver uptake than ⁶⁴Cu-CBTE2A-ReCCMSH(Arg¹¹) at all time points investigated in this study. The tumor uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex were 1.5, 1.7 and 1.7 times the tumor uptake of ⁶⁴Cu-CBTE2A-ReCCMSH(Arg¹¹) at 0.5, 2 and 4 h post-injection. The tumor/kidney uptake ratios of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex were 2.0, 4.5, 3.4 and 5.2 times the tumor/kidney uptake ratios of ⁶⁴Cu-CBTE2A-ReCCMSH(Arg¹¹) at 0.5, 2, 4 and 24 h post-injection, whereas The tumor/liver uptake ratios of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex were 3.1, 4.0, 4.1 and 2.0 times the tumor/liver uptake ratios of ⁶⁴Cu-CBTE2A-ReCCMSH(Arg¹¹) at 0.5, 2, 4 and 24 h post-injection (Figure 5). The increase in tumor uptake and decrease in renal and liver uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex were likely due to the structural difference between ⁶⁴Cu-NOTA-GGNle-CycMSH_hex and ⁶⁴Cu-CBTE2A-ReCCMSH(Arg¹¹). Despite the fact that the melanoma lesions could be visualized by PET/CT using either ⁶⁴Cu-NOTA-GGNle-CycMSH_hex or ⁶⁴Cu-DOTA-GGNle-CycMSH_hex as an imaging probe, ⁶⁴Cu-NOTA-GGNle-CycMSH_hex exhibited higher tumor imaging contrast than ⁶⁴Cu-DOTA-GGNle-CycMSH_hex. Higher melanoma uptake coupled with lower accumulation in normal organs suggested ⁶⁴Cu-NOTA-GGNle-CycMSH_hex as a lead radiolabeled peptide for future melanoma imaging and therapy. From the therapeutic perspective, the enhanced tumor/liver and tumor/kidney uptake ratios of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex would potentially increase the absorbed dose to the tumor while keeping the liver and kidneys safe when treating the melanoma with ⁶⁴Cu-NOTA-GGNle-CycMSH_hex in future studies.

In conclusion, the melanoma targeting and imaging properties of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex and ⁶⁴Cu-DOTA-GGNle-CycMSH_hex were determined in B16/F1 melanoma-bearing C57 mice in this study. The substitution of DOTA with NOTA dramatically increased the melanoma uptake and decreased the renal and liver uptake of ⁶⁴Cu-NOTA-GGNle-CycMSH_hex compared to ⁶⁴Cu-DOTA-GGNle-CycMSH_hex. High melanoma uptake coupled with low accumulation in non-target organs suggested ⁶⁴Cu-NOTA-GGNle-CycMSH_hex as a lead radiolabeled peptide for future melanoma imaging and therapy.