Gallium-67-Labeled Lactam Bridge-Cyclized Alpha-MSH Peptides with Enhanced Melanoma Uptake and Reduced Renal Uptake

Abstract

The purpose of this study was to examine the melanoma targeting and pharmacokinetic properties of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex {⁶⁷Ga-1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid-Gly-Gly-Nle-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH₂} and ⁶⁷Ga-NOTA-GGNle-CycMSH_hex {⁶⁷Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid-Gly-Gly-Nle-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH₂} and compare with ⁶⁷Ga-DOTA-GlyGlu-CycMSH {⁶⁷Ga-DOTA-Gly-Glu-c[Lys-Nle-Glu-His-dPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]} we previously reported. DOTA-GGNle-CycMSH_hex and NOTA-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 pharmacokinetic properties of ⁶⁷Ga-NOTA-GGNle-CycMSH_hex and ⁶⁷Ga-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) in B16/F1 melanoma cells. Both ⁶⁷Ga-NOTA-GGNle-CycMSH_hex and ⁶⁷Ga-DOTA-GGNle-CycMSH_hex exhibited dramatically enhanced melanoma uptake and reduced renal uptake than ⁶⁷Ga-DOTA-GlyGlu-CycMSH in B16/F1 melanoma-bearing C57 mice. Furthermore, ⁶⁷Ga-NOTA-GGNle-CycMSH_hexexhibited more favorable radiolabeling conditions (> 85% radiolabeling yields started at 37°C), as well as higher tumor/kidney uptake ratios than ⁶⁷Ga-DOTA-GGNle-CycMSH_hex at 0.5, 2 and 24 h post-injection. High melanoma uptake coupled with low renal uptake highlighted the potential of ⁶⁷Ga-NOTA-GGNle-CycMSH_hexfor melanoma imaging and therapy.

INTRODUCTION

It is highly desirable to develop novel melanoma-specific diagnostic and therapeutic agents^1–18 for melanoma since no curative treatment is available for patients with metastatic melanoma. Over the past several years, we and others have been interested in developing lactam bridge-cyclized alpha-melanocyte stimulating hormone (α-MSH) peptide radiopharmaceuticals targeting melanocortin-1 (MC1) receptors for melanoma imaging.^19–26 Specifically, we have developed two generations of ¹¹¹In-labeled novel lactam bridge-cyclized α-MSH peptides for melanoma targeting.^19–24 The first-generation peptides were designed based on the construct of CycMSH peptide {c[Lys-Nle-Glu-His-dPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp}], which was a 12-amino acid-peptide cyclized by a Lys-Asp lactam bridge. The second-generation peptides built upon the backbone of CycMSH_hex peptide {c[Asp-His-dPhe-Arg-Trp-Lys]-CONH₂}, which was a 6-amino acid-peptide cyclized by an Asp-Lys lactam bridge. The structural modifications from the first-generation peptides to the second-generation peptides enhanced melanoma uptake and reduced renal uptake of ¹¹¹In-labeled CycMSH_hex peptides.^19–24 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) than ¹¹¹In-DOTA-GlyGlu-CycMSH in B16/F1 melanoma-bearing C57 mice.^19,23

In 2009, we reported ⁶⁷Ga-DOTA-GlyGlu-CycMSH²² as a potential melanoma imaging agent taking advantage of the diagnostic properties of ⁶⁷Ga (T_1/2= 78.3 h, 93, 185 and 300 keV γ-emissions). Both flank primary B16/F1 melanoma and B16/F10 pulmonary melanoma metastases were clearly visualized by SPECT/CT using ⁶⁷Ga-DOTA-GlyGlu-CycMSH as an imaging probe 2 h post-injection.²² However, despite the substantial tumor uptake (12.93 ± 1.63 %ID/g at 2 h post-injection), ⁶⁷Ga-DOTA-GlyGlu-CycMSH also exhibited relatively high renal uptake (27.55 ± 7.87 %ID/g at 2 h post-injection) in B16/F1 melanoma-bearing C57 mice.²² It is important to note that ⁶⁷Ga is also a potential therapeutic radionuclide because of its emissions of Auger and conversion electrons.²⁷ Hence, we managed to reduce the renal uptake of ⁶⁷Ga-DOTA-GlyGlu-CycMSH via L-lysine co-injection to facilitate its potential therapeutic application. L-lysine co-injection successfully decreased the renal uptake of ⁶⁷Ga-DOTA-GlyGlu-CycMSH by 69.8% without affecting the melanoma uptake at 2 h post-injection.²²

Structural modification on peptide sequence is another way to reduce the renal uptake of radiolabeled α-MSH peptides.^22–24 Recently, we found that ¹¹¹In-DOTA-GGNle-CycMSH_hex displayed higher B16/F1 melanoma uptake (19.05 ± 5.04 % ID/g at 2 h post-injection) and reduced renal uptake (6.84 ± 0.92 % ID/g at 2 h post-injection) compared to ¹¹¹In-DOTA-GlyGlu-CycMSH.^22–24 Therefore, we hypothesized that ⁶⁷Ga-DOTA-GGNle-CycMSH_hex would exhibit enhanced melanoma uptake and reduced renal uptake than ⁶⁷Ga-DOTA-GlyGlu-CycMSH. To examine our hypothesis, we determined the biodistribution of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex in B16/F1 melanoma-bearing C57 mice in this study. Despite the fact that DOTA can form stable complexes with a variety of radionuclides including ⁶⁷Ga, NOTA can also form stable complex with ⁶⁷Ga at lower reaction temperature.^28–29 Thus, we also determined the melanoma targeting and pharmacokinetic properties of ⁶⁷Ga-NOTA-GGNle-CycMSH_hexin B16/F1 melanoma-bearing C57 mice in this study.

EXPERIMENTAL PROCEDURES

Chemicals and Reagents

Amino acid and resin were purchased from Advanced ChemTech Inc. (Louisville, KY) and Novabiochem (San Diego, CA). NO2AtBu 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. ⁶⁷GaCl₃ was purchased from MDS Nordion, Inc. (Vancouver, Canada) 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

DOTA-GGNle-CycMSH_hex was synthesized, purified by RP-HPLC and characterized by liquid chromatography-mass spectrometry (LC-MS) according to our published procedure.²⁴ New NOTA-GGNle-CycMSH_hex was synthesized using 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 dimethylformamide (DMF) using benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium-hexafluorophosphate (PyBOP) as a coupling agent in the presence of N,N-diisopropylethylamine (DIEA). 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 RP-HPLC on a Grace Vydac C-18 reverse phase analytical column (Vydac 218TP54: 5 µm, 4.6 mm i.d. × 250 mm), and characterized by LC-MS. The MC1 receptor binding affinity (IC₅₀ value) of NOTA-GGNle-CycMSH_hexwas determined in B16/F1 melanoma cells by in vitro competitive receptor binding assay according to our published procedure²⁴ and compared with DOTA-GGNle-CycMSH_hex.

Effects of Reaction Time and Temperature on ⁶⁷Ga Radiolabeling Yield

NOTA can form stable complex with ⁶⁷Ga at lower temperature than DOTA.^28–29 To examine the effects of reaction time and temperature on radiolabeling yield, DOTA-GGNle-CycMSH_hexand NOTA-GGNle-CycMSH_hex were radiolabeled with ⁶⁷Ga in a 0.5 M NH₄OAc-buffered solution (pH 3.5) at different temperatures for different reaction time points, respectively. Briefly, 10 µL of ⁶⁷GaCl₃ (18.5–37.0 MBq in 0.05 M HCl), 10 µL of 1 mg/mL DOTA-GGNle-CycMSH_hexor NOTA-GGNle-CycMSH_hex aqueous solution and 100 µL of 0.5 M NH₄OAc (pH 3.5) were added into a reaction vial and incubated at 25, 37, 45 and 75°C for 5, 15, 30 and 45 min, respectively. After the incubation, 10 µL of 0.5% EDTA aqueous solution was added into each reaction vial to bind unbound ⁶⁷Ga³⁺. The radiolabeling yield of each peptide was determined by Waters RP-HPLC (Milford, MA) on a Grace Vadyc C-18 reverse phase analytical column (Deerfield, IL) using the following gradient at a flow rate of 1 mL/min. 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.

Biodistribution Studies

All animal studies were conducted in compliance with Institutional Animal Care and Use Committee approval. The melanoma targeting and pharmacokinetic properties of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex and ⁶⁷Ga-NOTA-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 ⁶⁷Ga-DOTA-GGNle-CycMSH_hex or ⁶⁷Ga-NOTA-GGNle-CycMSH_hex via the tail vein. Groups of 5 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 specificity of tumor uptake of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex or ⁶⁷Ga-NOTA-GGNle-CycMSH_hex was determined by co-injecting 10 µg (6.07 nmol) of unlabeled NDP-MSH peptide at 2 h post-injection.

Melanoma Imaging with ⁶⁷Ga-NOTA-GGNle-CycMSH_hex

⁶⁷Ga-NOTA-GGNle-CycMSH_hexexhibited more favorable radiolabeling conditions (> 85% radiolabeling yields at 37°C for 30 min), as well as higher tumor/kidney uptake ratio than ⁶⁷Ga-DOTA-GGNle-CycMSH_hex at 2 h post-injection. Thus, we further evaluated the melanoma imaging property of ⁶⁷Ga-NOTA-GGNle-CycMSH_hex. Approximately 11.1 MBq of ⁶⁷Ga-NOTA-GGNle-CycMSH_hex was injected into a B16/F1 melanoma-bearing mouse via the tail vein. The mouse was euthanized for small animal SPECT/CT (Nano-SPECT/CT^®, Bioscan) imaging at 2 h post-injection. The CT imaging was immediately followed by single photon emission computed tomography (SPECT) imaging of whole-body. The SPECT scans of 24 projections were acquired. Reconstructed SPECT and CT data were visualized and co-registered 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 and renal uptake between ⁶⁷Ga-NOTA-GGNle-CycMSH_hex and ⁶⁷Ga-DOTA-GGNle-CycMSH_hex, as well as the significance of the difference in tumor uptake between ⁶⁷Ga-NOTA-GGNle-CycMSH_hex with/without NDP-MSH co-injection, and between ⁶⁷Ga-DOTA-GGNle-CycMSH_hex with/without NDP-MSH co-injection. The differences at the 95% confidence level (p<0.05) were considered significant.

RESULTS

New NOTA-GGNle-CycMSH_hex was synthesized and purified by RP-HPLC. NOTA-GGNle-CycMSH_hex displayed greater than 90% chemical purity after HPLC purification with 30% synthetic yield. 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 procedure²⁴ for comparison. The calculated and found molecular weights of DOTA-GGNle-CycMSH_hex were 1368 and 1368, respectively. The schematic structures of NOTA-GGNle-CycMSH_hex and DOTA-GGNle-CycMSH_hex are shown in Figure 1. The IC₅₀ value of NOTA-GGNle-CycMSH_hex was 1.6 nM in B16/F1 melanoma cells, which was comparable to that of DOTA-GGNle-CycMSH_hex (2.1 nM).²⁴

Figure 1.

Schematic structures of DOTA-GGNle-CycMSH_hex (A) and NOTA-GGNle-CycMSH_hex(B).

The effects of reaction time and temperature on radiolabeling yields are presented in Figure 2. The radiolabeling yield of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex or ⁶⁷Ga-NOTA-GGNle-CycMSH_hex was reaction time-dependent at 25°C, 37°C, 45°C and 75°C, respectively. The radiolabeling yield improved when the reaction time prolonged from 5 min to 45 min. For instance, as showed in Figure 2 at 25°C, the radiolabeling yield of ⁶⁷Ga-NOTA-GGNle-CycMSH_hexwas 27% after 5 min incubation, and was 70% after 45 min incubation. Meanwhile, the time-dependent fashion was observed for ⁶⁷Ga-DOTA-GGNle-CycMSH_hex as well despite the fact that the radiolabeling yield of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex was lower than 5% at 25°C even after 45 min incubation. The radiolabeling yield of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex or ⁶⁷Ga-NOTA-GGNle-CycMSH_hex was reaction temperature-dependent after 5, 15, 30 and 45 min incubation, respectively. The radiolabeling yield increased when the reaction temperature elevated from 25°C to 75°C. For example, as showed in Figure 2 after 30 min incubation, the radiolabeling yield of ⁶⁷Ga-NOTA-GGNle-CycMSH_hexwas 67% at 25°C and was 91% at 75°C. Similarly, the radiolabeling yield of ⁶⁷Ga-DOTA-GGNle-CycMSH_hexwas less than 5% at 25°C and was 97% at 75°C. Overall, the radiolabeling yield of ⁶⁷Ga-NOTA-GGNle-CycMSH_hex could reach greater than 85% started from 37°C, whereas the radiolabeling yield of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex could achieve greater than 84% only at 75°C. After we examined the effects of reaction time and temperature on radiolabeling yield, we performed peptide radiolabeling at 75°C for 30 min for biodistribution and imaging studies. DOTA-GGNle-CycMSH_hex and NOTA-GGNle-CycMSH_hex were readily labeled with ⁶⁷Ga in a 0.5 M NH₄OAc-buffered solution (pH 3.5) with greater than 90% radiolabeling yields. ⁶⁷Ga-DOTA-GGNle-CycMSH_hex and ⁶⁷Ga-NOTA-GGNle-CycMSH_hex were completely separated from their excess non-labeled peptides by RP-HPLC. The retention times of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex and ⁶⁷Ga-NOTA-GGNle-CycMSH_hex were 16.3 and 16.4 min, respectively.

Figure 2.

Effects of reaction time and temperature on radiolabeling yields of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex and ⁶⁷Ga-NOTA-GGNle-CycMSH_hex.

The melanoma targeting and pharmacokinetic properties of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex and ⁶⁷Ga-NOTA-GGNle-CycMSH_hex were determined in B16/F1 melanoma-bearing C57 mice. The biodistribution results of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex and ⁶⁷Ga-NOTA-GGNle-CycMSH_hexb are presented in Table 1. ⁶⁷Ga-DOTA-GGNle-CycMSH_hex exhibited rapid high melanoma uptake and prolonged tumor retention. The tumor uptake of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex was 26.96 ± 3.82 % ID/g at 0.5 h post-injection. ⁶⁷Ga-DOTA-GGNle-CycMSH_hex displayed similar high tumor uptake (25.53 ± 2.22 and 25.13 ± 4.13 % ID/g) at 2 and 4 h post-injection. Even at 24 h post-injection, there was 7.54 ± 1.19 % ID/g of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex activity remained in the tumor. Approximately 96.3% of the tumor uptake of ⁶⁷Ga-DOTA-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 ⁶⁷Ga-DOTA-GGNle-CycMSH_hex was rapid, with 84.9% of the injected radioactivity cleared through the urinary system by 2 h post-injection. Normal organ uptake of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex was lower than 1.68 % ID/g except for the kidneys at 2, 4 and 24 h post-injection. The kidney uptake was 8.90 ± 1.81 % ID/g at 2 h post-injection and decreased to 5.60 ± 1.24 % ID/g at 24 h post-injection. 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/kidney uptake ratio of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex was 1.64, 2.87, 2.98 and 1.35 at 0.5, 2, 4 and 24 h post-injection, respectively.

Table 1.

Biodistribution of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex and ⁶⁷Ga-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)

	67Ga-DOTA-GGNle-CycMSHhex				67Ga-NOTA-GGNle-CycMSHhex
Tissues	0.5 h	2 h	4 h	24 h	0.5 h	2 h	4 h	24 h
	Percent injected dose/gram (%ID/g)
Tumor	26.96±3.82	25.53±2.22	25.13±4.13	7.54±1.19	20.59±1.97*	25.12±1.03	18.17±4.89	7.95±2.58
Brain	0.25±0.07	0.15±0.12	0.08±0.03	0.06±0.03	0.21±0.01	0.11±0.07	0.08±0.03	0.08±0.02
Blood	3.33±1.08	0.97±0.91	0.53±0.25	0.29±0.05	2.25±0.09	0.40±0.19	1.02±0.06	0.55±0.12
Heart	1.64±0.18	0.23±0.09	0.24±0.04	0.37±0.08	1.52±0.28	0.45±0.09	0.39±0.17	0.96±0.39
Lung	3.23±0.07	0.40±0.15	0.36±0.08	0.32±0.19	3.71±0.25	0.54±0.04	0.30±0.10	0.35±0.08
Liver	1.82±0.13	0.86±0.19	0.85±0.02	0.54±0.14	1.28±0.14	0.59±0.09	0.54±0.05	0.50±0.12
Spleen	1.39±0.13	0.44±0.09	0.64±0.06	0.52±0.16	1.25±0.11	0.67±0.19	0.57±0.14	0.85±0.23
Stomach	2.51±0.50	1.68±0.50	1.26±0.11	0.62±0.23	1.79±0.22	1.62±0.63	1.45±0.23	1.21±0.40
Kidneys	16.42±2.99	8.90±1.81	8.44±0.11	5.60±1.24	11.67±0.06*	8.34±3.25	7.58±2.70	2.74±0.64*
Muscle	0.75±0.20	0.34±0.10	0.32±0.07	0.30±0.18	0.73±0.26	0.34±0.12	0.33±0.11	0.99±0.56
Pancreas	0.83±0.26	0.34±0.07	0.23±0.06	0.37±0.06	1.03±0.16	0.54±0.13	0.36±0.04	0.88±0.31
Bone	1.44±0.63	1.04±0.24	0.87±0.09	0.72±0.70	1.95±0.08	0.91±0.56	0.96±0.87	0.71±0.24
Skin	5.31±2.35	0.84±0.25	0.81±0.22	0.76±0.21	4.00±0.32	0.54±0.35	0.65±0.24	0.96±0.24
	Percent injected dose (%ID)
Intestines	1.60±0.30	1.52±1.14	0.98±0.18	0.46±0.06	1.86±0.24	1.22±0.97	0.91±0.05	1.23±0.35
Urine	59.37±5.31	84.91±2.81	87.75±1.44	92.72±1.09	54.16±13.08	84.02±5.71	86.39±3.94	94.57±0.25
	Uptake ratio of tumor/normal tissue
Tumor/liver	14.84	29.73	29.66	13.86	16.09	42.39	33.40	15.75
Tumor/kidney	1.64	2.87	2.98	1.35	1.76	3.01	2.40	2.90
Tumor/lung	8.35	63.55	69.97	23.35	5.54	46.37	60.24	22.52
Tumor/muscle	35.77	76.04	78.34	25.47	28.19	74.55	54.71	8.05
Tumor/blood	8.10	26.21	47.74	25.56	9.14	62.93	17.75	14.46
Tumor/skin	5.08	30.44	31.10	9.95	5.15	46.35	28.10	8.27

^*P<0.05 for determining significance of differences in tumor and kidney uptake between ⁶⁷Ga-DOTA-GGNle-CycMSH_hex and ⁶⁷Ga-NOTA-GGNle-CycMSH_hex at the same time point.

Figure 3.

The tumor uptake of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex (DOTA) and ⁶⁷Ga-NOTA-GGNle-CycMSH_hex (NOTA) with () or without () 10 µg of NDP-MSH blockade at 2 h post-injection. *p<0.05.

⁶⁷Ga-NOTA-GGNle-CycMSH_hex showed comparable high melanoma uptake with ⁶⁷Ga-DOTA-GGNle-CycMSH_hex at 2 and 24 h post-injection. The tumor uptake of ⁶⁷Ga-NOTA-GGNle-CycMSH_hex was 20.59 ± 1.57 % ID/g at 0.5 h post-injection. ⁶⁷Ga-NOTA-GGNle-CycMSH_hex reached its peak tumor uptake of 25.12 ± 1.03 % ID/g at 2 h post-injection. The tumor uptake of ⁶⁷Ga-NOTA-GGNle-CycMSH_hex was 18.17 ± 4.89 and 7.95 ± 2.58 % ID/g at 4 and 24 h post-injection. Approximately 90.8% of the tumor uptake of ⁶⁷Ga-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 ⁶⁷Ga-NOTA-GGNle-CycMSH_hex was rapid, with 84.1% of the injected radioactivity cleared through the urinary system by 2 h post-injection. Normal organ uptakes of ⁶⁷Ga-NOTA-GGNle-CycMSH_hex were lower than 1.62 % ID/g except for the kidneys at 2, 4 and 24 h post-injection. The kidney uptake was 8.34 ± 3.25 % ID/g at 2 h post-injection and decreased to 2.74 ± 0.64 % ID/g at 24 h post-injection. 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/kidney uptake ratio of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex was 1.76, 3.01, 2.40 and 2.90 at 0.5, 2, 4 and 24 h post-injection, respectively.

⁶⁷Ga-NOTA-GGNle-CycMSH_hexexhibited more favorable radiolabeling conditions (> 85% radiolabeling yields started at 37°C), as well as higher tumor/kidney uptake ratio than ⁶⁷Ga-DOTA-GGNle-CycMSH_hex at 2 h post-injection. Therefore, we further evaluated the melanoma imaging properties of ⁶⁷Ga-NOTA-GGNle-CycMSH_hex at 2 h post-injection. The whole-body, coronal and transversal SPECT/CT images are presented in Figure 5. The melanoma tumors were clearly visualized by SPECT/CT using ⁶⁷Ga-NOTA-GGNle-CycMSH_hex as an imaging probe. ⁶⁷Ga-NOTA-GGNle-CycMSH_hex exhibited high tumor to normal organ uptake ratios except for the kidneys, which was consistent with the biodistribution results.

Figure 5.

Representative whole-body (A), coronal (B) and transversal (C) SPECT/CT images of a B16/F1 melanoma-bearing C57 mouse at 2 h post-injection of ⁶⁷Ga-NOTA-GGNle-CycMSH_hex. The tumor lesions (T) were highlighted with arrows on the images.

DISCUSSION

Gallium-67 is an attractive SPECT radionuclide with a half-life of 78.3 h and three gamma-emissions (38% 93 keV, 24% 185 keV and 16% 300 keV) (30–33). Meanwhile, ⁶⁷Ga is also a potential therapeutic radionuclide due to its emissions of Auger and conversion electrons.²⁷ Both linear and cyclic ⁶⁷Ga-labeled α-MSH peptides^8,22 have been reported for melanoma imaging over the past several years. Initially, Froidevaux et al. reported the linear ⁶⁷Ga-DOTA-NAPamide, which exhibited 9.43 ± 1.06 and 3.10 ± 0.36 %ID/g of B16/F1 melanoma uptake at 4 and 24 post-injection, respectively.⁸ Thereafter, we reported the lactam bridge-cyclized ⁶⁷Ga-DOTA-GlyGlu-CycMSH.²² The B16/F1 melanoma uptake of ⁶⁷Ga-DOTA-GlyGlu-CycMSH was 14% less than that of ⁶⁷Ga-DOTA-NAPamide at 4 h post-injection, whereas the B16/F1 melanoma uptake of ⁶⁷Ga-DOTA-GlyGlu-CycMSH was 1.6 times the melanoma uptake of ⁶⁷Ga-DOTA-NAPamide at 24 h post-injection. Surprisingly, the renal uptake of ⁶⁷Ga-DOTA-GlyGlu-CycMSH was 5.7 and 10.3 times the renal uptake of ⁶⁷Ga-DOTA-NAPamide at 4 and 24 h post-injection, respectively. Dramatic difference in renal uptake between ⁶⁷Ga-DOTA-GlyGlu-CycMSH and ⁶⁷Ga-DOTA-NAPamide was likely due to their structural differences.

Recently, we have identified a novel class of lactam bridge-cyclized α-MSH peptides with enhanced melanoma uptake and reduced renal uptake.^23,24 Specifically, ¹¹¹In-DOTA-GGNle-CycMSH_hex exhibited the highest tumor/kidney uptake ratios (2.8 and 2.7 at 2 and 4 h post-injection) among all reported ¹¹¹In-labeled cyclic peptides.^23,24 Thus, we were interested in determining whether or not ⁶⁷Ga-DOTA-GGNle-CycMSH_hex could display higher melanoma uptake and lower renal uptake compared to ⁶⁷Ga-DOTA-GlyGlu-CycMSH in this study. Meanwhile, we also evaluated ⁶⁷Ga-NOTA-GGNle-CycMSH_hex because NOTA can also form a stable complex with ⁶⁷Ga even at lower reaction temperature than DOTA.^28,29 As we anticipated, NOTA-GGNle-CycMSH_hex showed comparable MC1 receptor binding affinity with DOTA-GGNle-CycMSH_hex (1.6 vs. 2.1 nM). In terms of radiolabeling yield, ⁶⁷Ga-NOTA-GGNle-CycMSH_hex could reach 70% and 85% radiolabeling yields even at 25°C and 37°C, respectively. Meanwhile, the radiolabeling yield of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex could be greater than 85% only at 75°C. At 75°C, both ⁶⁷Ga-NOTA-GGNle-CycMSH_hex and ⁶⁷Ga-DOTA-GGNle-CycMSH_hex could achieve greater than 90% radiolabeling yield after 30 min incubation. It was important to note that high radiolabeling yield of ⁶⁷Ga-NOTA-GGNle-CycMSH_hexat 37°C represented a distinct practical advantage compared to ⁶⁷Ga-DOTA-GGNle-CycMSH_hex.

The biodistribution results supported our hypothesis. ⁶⁷Ga-DOTA-GGNle-CycMSH_hex exhibited higher melanoma uptake and lower renal uptake compared to ⁶⁷Ga-DOTA-GlyGlu-CycMSH. The tumor uptake of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex was 3.1, 2.0 and 3.1 times the tumor uptake of ⁶⁷Ga-DOTA-GlyGlu-CycMSH at 0.5, 2 and 4 h post-injection, respectively. Meanwhile, the renal uptake of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex was only 69%, 32%, 37% and 27% of the renal uptake of ⁶⁷Ga-DOTA-GlyGlu-CycMSH 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 ⁶⁷Ga-DOTA-GGNle-CycMSH_hex at all time points investigated in this study (Table 1). The tumor to kidney uptake ratio of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex was 4.6, 6.2, 8.3 and 5.6 times those of ⁶⁷Ga-DOTA-GlyGlu-CycMSH at 0.5, 2, 4 and 24 h post-injection, respectively.

⁶⁷Ga-NOTA-GGNle-CycMSH_hex displayed comparable high melanoma uptake with ⁶⁷Ga-DOTA-GGNle-CycMSH_hex at 2 and 24 h post-injection. Meanwhile, ⁶⁷Ga-NOTA-GGNle-CycMSH_hex exhibited comparable renal uptake with ⁶⁷Ga-DOTA-GGNle-CycMSH_hex at 2 and 4 h post-injection. Interestingly, the renal uptake of with ⁶⁷Ga-NOTA-GGNle-CycMSH_hex was only 71% and 49% of that of ⁶⁷Ga-DOTA-GGNle-CycMSH_hex at 0.5 and 24 h post-injection. The further reduced renal uptake resulted in higher tumor/kidney uptake ratios of ⁶⁷Ga-NOTA-GGNle-CycMSH_hex than ⁶⁷Ga-DOTA-GGNle-CycMSH_hex at 0.5 and 24 h post-injection. Moreover, ⁶⁷Ga-NOTA-GGNle-CycMSH_hex showed slightly higher tumor/kidney uptake ratio than ⁶⁷Ga-DOTA-GGNle-CycMSH_hex at 2 h post-injection (Figure 4). We have demonstrated that L-lysine co-injection decreased the renal uptake of ⁶⁷Ga-DOTA-GlyGlu-CycMSH by 69.8% at 2 h post-injection in our previous report.²² In this study, the renal uptake of ⁶⁷Ga-NOTA-GGNle-CycMSH_hex and ⁶⁷Ga-DOTA-GGNle-CycMSH_hex was only 30% and 32% of the renal uptake of ⁶⁷Ga-DOTA-GlyGlu-CycMSH at 2 h post-injection (Figure 4). Dramatic difference in renal uptake among ⁶⁷Ga-DOTA-GlyGlu-CycMSH, ⁶⁷Ga-NOTA-GGNle-CycMSH_hex and ⁶⁷Ga-DOTA-GGNle-CycMSH_hex was likely due to their structural differences.

Figure 4.

The comparisons of tumor (A) and kidney (B) uptake, and tumor/kidney uptake ratios (C) at 2 h post-injection among ⁶⁷Ga-DOTA-GlyGlu-CycMSH (), ⁶⁷Ga-DOTA-GGNle-CycMSH_hex () and ⁶⁷Ga-NOTA-GGNle-CycMSH_hex ().

Despite the fact that the melanoma lesions could be visualized by SPECT/CT using ⁶⁷Ga-DOTA-GlyGlu-CycMSH as an imaging probe,^{22 67}Ga-NOTA-GGNle-CycMSH_hex exhibited higher tumor imaging contrast (Figure 5) than ⁶⁷Ga-DOTA-GlyGlu-CycMSH. Higher melanoma uptake coupled with lower renal uptake underscored the potential therapeutic application of ⁶⁷Ga-NOTA-GGNle-CycMSH_hex. It will be interesting to examine the therapeutic efficacy of ⁶⁷Ga-NOTA-GGNle-CycMSH_hex in melanoma-bearing mouse model taking advantage of the therapeutic properties of ⁶⁷Ga in the future. Another potential application of NOTA-GGNle-CycMSH_hex peptide is to develop ⁶⁸Ga-NOTA-GGNle-CycMSH_hex for positron emission tomography (PET) imaging of melanoma. Gallium-68 is an attractive PET radionuclide with a half-life of 68 min and can be easily obtained via an in-house commercial ⁶⁸Ge-⁶⁸Ga generator. The combination of ⁶⁸Ga-NOTA-GGNle-CycMSH_hex with PET, taking advantage of the outstanding imaging properties of PET and receptor-targeting properties of ⁶⁸Ga-NOTA-GGNle-CycMSH_hex, will offer an exciting opportunity for sensitive tumor-specific imaging of melanoma metastases.

In conclusion, both ⁶⁷Ga-NOTA-GGNle-CycMSH_hex and ⁶⁷Ga-DOTA-GGNle-CycMSH_hex exhibited dramatically enhanced melanoma uptake and reduced renal uptake than ⁶⁷Ga-DOTA-GlyGlu-CycMSH in B16/F1 melanoma bearing C57 mice. ⁶⁷Ga-NOTA-GGNle-CycMSH_hexexhibited more favorable radiolabeling conditions (> 85% radiolabeling yields started at 37°C), as well as higher tumor/kidney uptake ratio than ⁶⁷Ga-DOTA-GGNle-CycMSH_hex at 0.5, 2 and 24 h post-injection. High melanoma uptake coupled with low renal uptake highlighted the potential of ⁶⁷Ga-NOTA-GGNle-CycMSH_hexfor melanoma imaging and therapy.