Novel [^99mTc]-Tricarbonyl-NOTA-Conjugated Lactam-Cyclized Alpha-MSH Peptide with Enhanced Melanoma Uptake and Reduced Renal Uptake

Abstract

The purpose of this study was to examine the melanoma targeting and imaging properties of 99mTc(CO)₃-NOTA-GGNle-CycMSH_hex {1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-GlyGlyNle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH₂} and ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex {1,4,7-triazacyclononane,1-gluteric acid-4,7-acetic acid-GlyGlyNle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH₂} on B16/F10 melanoma-bearing C57 mice to demonstrate the feasibility of NOTA/NODAGA as metal chelators for ^99mTc(CO)₃⁺ radiolabeling.

Methods:

NOTA/NODAGA-GGNle-CycMSH_hex were synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined on B16/F10 melanoma cells. The biodistribution of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex and ^99mTc(CO)_3-NODAGA-GGNle-CycMSH_hex were determined on B16/F10 melanoma-bearing C57 mice at 2 h post-injection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex and ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex were determined on B16/F10 melanoma-bearing C57 mice.

Results:

The IC₅₀ values of NOTA/NODAGA-GGNle-CycMSH_hex were 0.9 ± 0.1 and 0.8 ± 0.05 nM on B16/F10 cells. ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex and ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex were readily prepared using via [^99mTc(CO)₃(OH₂)₃]⁺ intermediate, and displayed MC1R-specific binding on B16/F10 cells. ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex was further evaluated as a lead peptide because of its higher tumor uptake (19.76 ± 3.62 %ID/g) and lower kidney uptake (1.59 ± 0.52 %ID/g) at 2 h post-injection than ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex. The B16/F10 melanoma uptake of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex was 16.07 ± 4.47, 19.76 ± 3.62, 11.30 ± 2.81 and 3.16 ± 2.28% ID/g at 0.5, 2, 4 and 24 h post-injection, respectively. ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex showed high tumor to normal organ uptake ratios after 2 h post-injection. The B16/F10 melanoma lesions were clearly visualized by SPECT/CT using ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex as an imaging probe at 2 h post-injection.

Conclusions:

High tumor uptake, low kidney uptake and fast urinary clearance of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex highlighted its potential for melanoma imaging and facilitated the evaluation of 188Re(CO)₃-NOTA-GGNle-CycMSH_hex for melanoma therapy.

Graphical Abstract

INTRODUCTION

Malignant melanoma is the most lethal form of skin cancer due to the extreme aggressiveness of melanoma metastasis. It is estimated that approximately 100,350 new cases and 6,850 fatalities will occur in the United States in 2020.¹ Traditional overall survival of patients with metastatic melanoma is only 6–9 months. New molecular treatments including Vemurafenib (BRAF inhibitor), ipilimumab (targeting CTLA-4) and Nivolumab (PD-1 inhibitor) have demonstrated encouraging clinical benefits for metastatic melanoma patients by improving the overall survival by months.^2–6 However, the treatments are still far from satisfactory because the 5-year survival is approximately 35% for patients with metastatic melanoma.⁷ Therefore, it is highly desirable to develop new therapeutic molecular approaches for metastatic melanoma.

We have been targeting melanocortin-1 receptors (MC1Rs) for melanoma imaging and therapy due to the over-expression of MC1Rs on both melanotic and amelanotic human melanoma samples.^{8, 9} Our first-in-human study underscored the clinical relevance of MC1R as an attractive molecular target for developing theranostic peptides for melanoma.¹⁰ Over the past several years, we have developed a novel class of radiolabeled lactam-cyclized α-melanocyte-stimulating hormone (α-MSH) peptides,^11–20 taking advantage of the nanomolar MC1R binding affinity of key construct of GGNle-CycMSH_hex {Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH₂} and various radiometal chelators such as HYNIC, DOTA and NOTA for radiolabeling of theranostic radionuclides. For instance, HYNIC was used for ^99mTc radiolabeling,^{15, 16} whereas DOTA and NOTA were utilized for radiolabeling of diagnostic ¹¹¹In,^{11, 12 203}Pb,^{18 67/68}Ga,^{10, 13 64}Cu¹⁴ and therapeutic ¹⁷⁷Lu¹⁷ and ⁹⁰Y.¹⁹ These radiolabeled α-MSH peptides generally yielded high melanoma uptake and rapid urinary clearance, highlighting their potential for melanoma imaging and therapy. For example, ¹¹¹In-DOTA-GGNle-CycMSH_hex and ¹⁷⁷Lu-DOTA-GGNle-CycMSH_hex displayed high tumor uptake of 19.05 ± 5.04 and 21.63 ± 6.27 %ID/g at 2 h post-injection, respectively.^{12, 17}

Technetium-99m is an attractive single photon emission computed tomography (SPECT) radionuclide due to its 6-h half life, 140 keV gamma ray and high availability through a cost-effective ⁹⁹Mo/^99mTc generator. In terms of ^99mTc-labeled lactam-cyclized α-MSH peptides, we have used mercaptoacetyltriglycine (MAG₃)-GGNle-CycMSH_hex, Ac-Cys-Gly-Gly-Gly (AcCG₃)-GGNle-CycMSH_hex to form stable complexes with ^99mTc=O³⁺ core, and hydrazinonicotinamide (HYNIC)-GGNle-CycMSH_hex to yield stable ^99mTc-conjugates via [^99mTc(CO)₃(OH₂)₃]⁺ tricarbonyl intermediate and EDDA/tricine coligands. Interestingly, 99mTc-MAG₃-GGNle-CycMSH_hex and 99mTc-AcCG₃-GGNle-CycMSH_hex displayed 4.64 ± 1.06 and 9.76 ± 4.90 %ID/g on B16/F1 melanoma at 2 h post-injection.¹⁵ Although 99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex exhibited comparable tumor uptake of 5.84 ± 1.26 %ID/g, it also showed high liver and kidney uptake of 38.11 ± 2.31 and 17.69 ± 4.06 %ID/g at 2 h post-injection, respectively.¹⁵ Meanwhile, ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex exhibited higher tumor uptake (14.14 ± 4.90 %ID/g) and lower liver and renal uptake (0.52 ± 0.05 and 7.52 ± 0.96 %ID/g) than ^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex at 2 h post-injection¹⁵. The tremendous differences in biodistribution properties of these above-mentioned ^99mTc-CycMSH_hex peptides suggested that the coordination of ^99mTc core could potentially affect the tumor targeting and clearance properties of ^99mTc-labeled lactam-cyclized α-MSH peptides on melanoma-bearing mice.

We demonstrated that 1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid (NOTA)-GGNle-CycMSH_hex could form stable hydrophilic complexes with ⁶⁷Ga and ⁶⁴Cu in our previous report.^{13, 14} Both ⁶⁷Ga-NOTA-GGNle-CycMSH_hex and ⁶⁴Cu-NOTA-GGNle-CycMSH_hex exhibited high melanoma uptake, highlighting their potential for SPECT and positron emission tomography (PET) imaging of melanoma.^{13, 14} Meanwhile, the tridentate NOTA and NODAGA could coordinate [^99mTc(CO)₃]⁺ tricarbonyl core via three N atoms to yield stable bombesin and somatostatin peptide conjugates for tumor targeting.^20–24 Recently, an interesting report found that ^99mTc(CO)₃-NOTA-sst2-ANT exhibited 7 to 8-fold higher tumor uptake than ^99mTc(CO)₃-NODAGA-sst2-ANT on AR42J tumor-bearing mice.²⁴ Thus we were interested whether and how this new coordination chemistry of [^99mTc(CO)₃]⁺ tricarbonyl core could affect the biodistribution properties of NOTA/NODAGA-conjugated lactam-cyclized α-MSH peptides. In this study, we synthesized NOTA/NODAGA-GGNle-CycMSH_hex using fluorenylmethoxycarbonyl (Fmoc) chemistry, determined their MC1R binding affinities on B16/F10 melanoma cells, prepared their ^99mTc(CO)₃-conjugates using [^99mTc(CO)₃(OH₂)₃]⁺ tricarbonyl intermediate and examined their biodistribution and tumor targeting properties on B16/F10 melanoma-bearing C57 mice.

EXPERIMENTAL SECTION

Chemicals and reagents

Amino acid and resin were purchased from Advanced ChemTec Inc. (Louisville, KY) and Novabiochem (San Diego, CA). NOTA(OtBu)₂ and NODAGA(OtBu)₃ were 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 in vitro binding assay. [^99mTcO₄]⁻ was purchased from Cardinal Health (Denver, CO) and IsoLink kit was obtained from Mallinckrodt (St. Louis, MO) for ^99mTc labeling. B16/F10 murine melanoma cells were received from American Type Culture Collection (Manassas, VA). All other chemicals used in this study were purchased from Thermo Fisher Scientific (Waltham, MA) and used without further purification.

Peptide synthesis

NOTA-GGNle-CycMSH_hex and NODAGA-GGNle-CycMSH_hex were synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry. Briefly, the linear intermediate scaffolds of NOTA(OtBu)₂/NODAGA(OtBu)₃-Nle-Gly-Gly-Asp(O-2-PhiPr)-His(Trt)-DPhe-Arg(Pbf)-Trp(Boc)-Lys(Mtt) were synthesized on Sieber amide resin by an Advanced ChemTech multiple-peptide synthesizer (Louisville, KY). Generally, 70 μmol of resin, 210 μmol of each Fmoc-protected amino acid and NOTA(OtBu)₂/NODAGA(OtBu)₃ 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 at 25 °C in dimethylformamide (DMF) using benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium-hexafluorophosphate (PyBOP) as a coupling agent in the presence of N,N-diisopropylethylamine (DIPEA). 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 and characterized by LC-MS.

In vitro competitive binding assay

The MC1R binding affinities of NOTA/NODAGA-GGNle-CycMSH_hex were determined on B16/F10 melanoma cells by in vitro competitive receptor binding assay. The receptor binding assay was replicated in triplicate. The B16/F10 cells (0.5×10⁵ cells/well, n = 3) were incubated at room temperature (25 °C) for 2 h with approximately 30,000 counts per minute (cpm) of ¹²⁵I-(Tyr²)-NDP-MSH in the presence of 10⁻¹² to 10⁻⁵ M of the peptide 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 twice with 0.5 mL of ice-cold pH 7.4, 0.2% BSA/0.01 M phosphate buffered saline (PBS) and lysed in 0.5 mL of 1 N NaOH for 5 min. The cells were harvested and measured in a Wallac 1480 automated gamma counter (PerkinElmer, NJ). The IC₅₀ value was calculated using the Prism software (GraphPad Software, La Jolla, CA, USA).

Radiolabeling

^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex and 99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex were prepared using IsoLink kit according to our previous publication¹⁵ with modifications. Briefly, 2 mL of freshly eluted ^99mTcO₄⁻ (~383 MBq) solution was added into an IsoLink kit, followed by heating at 100 °C for 20 min to yield [^99mTc(CO)₃(OH₂)₃]⁺ intermediate. The kit vial was removed from the water bath and allowed to cool down to room temperature. For each radiolabeling reaction, 60 μL of 1 M HCl was added into 0.7 mL of [^99mTc(CO)₃(OH₂)₃]⁺ (~126 MBq) solution in a reaction vial to adjust the pH to 7 first, then 50 μL of 1 mg/mL peptide aqueous solution was added into the reaction vial. After adjusting the pH to 5–6 using 0.5 M NH₄OAc-buffered solution (pH 5.3), the reaction mixture was heated at 95 °C for 30 min and cooled down to room temperature.

The radiolabeled complexes were purified to single species by a Waters RP-HPLC (Milford, MA) on a Grace Vydac C-18 reverse phase analytical column (Deerfield, IL) using the following gradient at a 1 mL/min flowrate. The mobile phase consisted of solvent A (20 mM HCl aqueous solution) and solvent B (100% CH₃CN). The gradient was initiated and kept at 78:22 A/B for 3 min followed by a linear gradient of 78:22 A/B to 68:32 A/B over 20 min. Then, the gradient was changed from 68:32 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 78:22 A/B over 3 min. The purified peptide sample was purged with N₂ gas for 15 min to remove the acetonitrile. The pH of the final solution was adjusted to 7.4 with 0.1 N NaOH and sterile saline.

Specific binding of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex and ^99mTc(CO)_3-NODAGA-GGNle-CycMSH_hex

The specific binding of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex and ^99mTc(CO)_3-NODAGA-GGNle-CycMSH_hex was determined on B16/F10 melanoma cells, respectively. Briefly, the B16/F10 cells (1×10⁶ cells/tube, n = 3) were incubated at 25 °C for 2 h with approximately 0.22 MBq of either ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex or ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex with or without 10 μg (6.07 nmol) of unlabeled [Nle⁴, D-Phe⁷]-α-MSH (NDP-MSH) in 0.3 mL of binding medium {Modified Eagle’s medium with 25 mM N-(2-hydroxyethyl)-piperazine-N’-(2-ethanesulfonic acid), pH 7.4, 0.2% bovine serum albumin (BSA), 0.3 mM 1,10-phenathroline}. After the incubation, the cells were rinsed twice with 0.5 mL of ice-cold pH 7.4, 0.2% BSA/0.01 M phosphate buffered saline (PBS), lysed with 0.5 mL of 1 M NaOH for 5 min, collected and measured in a Wallac 1480 automated gamma counter (PerkinElmer, NJ).

Biodistribution and melanoma imaging studies

All animal studies were conducted in compliance with Institutional Animal Care and Use Committee approval. Each C57 mouse was subcutaneously inoculated with 1×10⁶ B16/F10 cells on the right flank to generate melanoma tumors. Ten days post inoculation, the tumor weights reached approximately 0.2 g and the melanoma-bearing mice were used for biodistribution and imaging studies. The biodistribution properties of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex and ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex were determined on B16/F10 melanoma-bearing C57 mice at 2 h post-injection first to select one peptide for further evaluation. Because ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex displayed more favorable tumor targeting property than 99mTc(CO)_3-NODAGA-GGNle-CycMSH_hex, the full biodistribution of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex was further examined on B16/F10 melanoma-bearing C57 mice.

For full biodistribution of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex, each melanoma-bearing mouse was injected with 0.037 MBq of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex via the tail vein. The specificity of the tumor uptake of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex was determined by co-injecting 10 μg (6.07 nmol) of unlabeled NDP-MSH which is a linear α-MSH peptide analogue with sub-nanomolar MC1R binding affinity. 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 melanoma imaging property of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex was examined on B16/F10 melanoma-bearing C57 mice and compared with that of ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex. Approximately 11.1 MBq of ^99mTc(CO)_3-NOTA-GGNle-CycMSH_hex or ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex was injected in B16/F10 melanoma-bearing C57 mice via the tail veins for melanoma imaging. Each mouse was sacrificed at 2 h post-injection for small animal SPECT/CT (nanoScan SPECT/CT, Mediso) imaging. The CT scan was immediately followed by the whole-body SPECT scan. Reconstructed SPECT and CT data were visualized and co-registered using VivoQuant (inviCRO, Boston, MA).

Statistical analysis

Statistical analysis was performed using the Student’s t-test for unpaired data. A 95% confidence level was chosen to determine the significance of difference in cellular binding, in tumor, blood, liver and kidney uptake between ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex and ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex, in tumor and renal uptake of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex with or without NDP-MSH co-injection. The differences at the 95% confidence level (p<0.05) were considered significant.

RESULTS

NOTA/NODAGA-GGNle-CycMSH_hex (Figure 1) were synthesized and purified by reverse phase high pressure liquid chromatography (RP-HPLC). After the HPLC purification, NOTA/NODAGA-GGNle-CycMSH_hex displayed greater than 90% purity. The identities of NOTA/NODAGA-GGNle-CycMSH_hex was confirmed by electrospray ionization mass spectrometry. The calculated and found molecular weights of NOTA/NODAGA-GGNle-CycMSH_hex were 1381, 1381, 1453 and 1453, respectively. The IC₅₀ values of NOTA/NODAGA-GGNle-CycMSH_hex were 0.9 ± 0.1 and 0.8 ± 0.05 nM on B16/F10 melanoma cells (Figure 2).

Figure 1.

The proposed schematic structures of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex and ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex.

Figure 2.

In vitro competitive binding curves of NOTA-GGNle-CycMSH_hex (●) and NODAGA-GGNle-CycMSH_hex (▲). The IC₅₀ value of NOTA-GGNle-CycMSH_hex and NODAGA-GGNle-CycMSH_hex was 0.9 ± 0.1 and 0.8 ± 0.05 nM on B10/F10 melanoma cells, respectively.

^99mTc(CO)₃-NOTA-GGNle-CycMSH and ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex were readily prepared using IsoLink kit and collected by HPLC. The radiochemical purity of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex and ^99mTc(CO)_3-NODAGA-GGNle-CycMSH_hex was greater than 95%, respectively (Figure 3). Both ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex and ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex displayed receptor-mediated binding on B16/F10 melanoma cells. Approximately 75% of cellular uptake of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex was blocked by peptide blockade, whereas 76% of cellular uptake of ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex was blocked by peptide blockade (Figure 3). Interestingly, ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex exhibited higher receptor binding than ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex on B16/F10 cells.

Figure 3.

Radioactive HPLC profiles (A) of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex (NOTA) and ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex (NODAGA). The retention time of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex and ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex was 15.6 and 12.8 min, respectively. Specific binding (B) of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex and ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex on B16/F10 melanoma cells with (white) and without (black) peptide blockade.

The biodistribution comparison between ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex and ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex at 2 h post-injection is presented in Table 1. The tumor uptake of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex was higher than that of ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex. The tumor uptake of ^99mTc(CO)₃-NOTAGGNle-CycMSH_hex and ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex was 19.76 ± 3.62 and 7.19 ± 1.80 %ID/g at 2 h post-injection, respectively. Although the uptake of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex in blood and liver was higher than that of ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex, the uptake of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex in other normal organs was similar to that of ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex except for kidneys. Interestingly, the renal uptake of ^99mTc(CO)₃-NOTAGGNle-CycMSH_hex was much lower than that of 99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex by 5-fold. The renal uptake of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex was as low as 1.59 ± 0.52 %ID/g at 2 h post-injection. Therefore, the full biodistribution of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex at 0.5, 4 and 24 h post-injection was further evaluated in this study.

Table 1.

Biodistribution comparison between ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex (NOTA) and ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex (NODAGA) on B16/F10 melanoma-bearing C57 mice at 2 h post-injection. The data were presented as percent injected dose/gram or as percent injected dose (Mean ± SD, n = 4)

Tissues	NOTA	NODAGA
	Percent injected dose/gram (%ID/g)
Tumor	19.76 ± 3.62	7.19 ± 1.80*
Brain	0.03 ± 0.01	0.02 ± 0.01
Blood	2.19 ± 0.73	0.55 ± 0.28*
Heart	0.11 ± 0.07	0.15 ± 0.02
Lung	0.28 ± 0.17	0.30 ± 0.13
Liver	1.57 ± 0.32	0.34 ± 0.03*
Spleen	0.38 ± 0.20	0.13 ± 0.03
Stomach	0.50 ± 0.37	0.31 ± 0.09
Kidneys	1.59 ± 0.52	8.66 ± 2.59*
Muscle	0.09 ± 0.06	0.09 ± 0.03
Pancreas	0.11 ± 0.06	0.10 ± 0.07
Bone	0.29 ± 0.09	0.20 ± 0.06
Skin	0.35 ± 0.14	0.29 ± 0.03
	Percent injected dose (%ID)
Intestines	2.95 ± 0.25	0.56 ± 0.15
Urine	89.50 ± 2.10	92.02 ± 1.36
	Uptake ratio of tumor/normal tissue
Tumor/blood	9.02	13.07
Tumor/kidney	12.43	0.83
Tumor/lung	70.57	23.97
Tumor/liver	12.59	21.15
Tumor/muscle	219.56	79.89
Tumor/skin	56.46	24.79

^*p<0.05 for determining significance of differences in tumor, blood, liver and kidney uptake between ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex and ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex.

The full biodistribution results of 99mTc(CO)₃-NOTA-GGNle-CycMSH_hex are presented in Table 2. The accumulation of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex in the tumor was rapid and high, with 16.07 ± 4.47 %ID/g at 0.5 h post-injection. The tumor uptake of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex was 19.76 ± 3.62 and 11.30 ± 2.81 %ID/g at 2 and 4 h post-injection, and gradually decreased to 3.16 ± 2.28 %ID/g at 24 h post-injection. Approximately 96% of the tumor uptake was blocked at 2 h post-injection, demonstrating that the tumor uptake was MC1R-medicated. The blood uptake was 2.19 ± 0.73 %ID/g at 2 h post-injection and quickly reduced to 0.11 ± 0.06 %ID/g at 4 h post-injection. Interestingly, the renal uptake dramatically decreased from 7.16 ± 0.79 %ID/g at 0.5 h post-injection to 1.59 ± 0.52 %ID/g at 2 h post-injection, and eventually reached 0.81 ± 0.21 and 0.23 ± 0.19 %ID/g at 4 and 24 h post-injection. The peptide blockade didn’t decrease the renal uptake, indicating that the renal uptake wasn’t MC1R-specific. ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex displayed a rapid urinary excretion, approximately 90% of the injected activity being cleared at 2 h post-injection. Fast clearance from normal organs resulted in high tumor to normal organ uptake rations as early as 0.5 h post-injection.

Table 2.

Biodistribution of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex on B16/F10 melanoma-bearing C57 mice. The data were presented as percent injected dose/gram or as percent injected dose (Mean ± SD, n = 4)

Tissues	0.5 h	2 ha	4 h	24 h	2 h NDP blockade
	Percent injected dose/gram (%ID/g)
Tumor	16.07 ± 4.47	19.76 ± 3.62	11.30 ± 2.81	3.16 ± 2.28	0.80 ± 0.11*
Brain	0.13 ± 0.02	0.03 ± 0.01	0.01 ± 0.01	0.01 ± 0.01	0.04 ±.0.03
Blood	1.44 ± 0.33	2.19 ± 0.73	0.11 ± 0.06	0.03 ± 0.05	0.22 ± 0.18
Heart	0.74 ± 0.20	0.11 ± 0.07	0.07 ± 0.03	0.08 ± 0.07	0.17 ± 0.05
Lung	2.11 ± 0.65	0.28 ± 0.17	0.18 ± 0.05	0.03 ± 0.03	0.44 ± 0.26
Liver	2.32 ± 0.20	1.57 ± 0.32	1.43 ± 0.31	0.53 ± 0.17	4.32 ± 1.34
Spleen	0.76 ± 0.48	0.38 ± 0.20	0.11 ± 0.05	0.19 ± 0.25	0.78 ± 0.44
Stomach	1.11 ± 0.42	0.50 ± 0.37	0.25 ± 0.06	0.06 ± 0.07	1.02 ± 0.36
Kidneys	7.16 ± 0.79	1.59 ± 0.52	0.81 ± 0.21	0.23 ± 0.19	3.07 ± 0.61*
Muscle	0.19 ± 0.06	0.09 ± 0.06	0.01 ± 0.01	0.03 ± 0.04	0.08 ± 0.08
Pancreas	0.29 ± 0.10	0.11 ± 0.06	0.01 ± 0.01	0.03 ± 0.05	0.14 ± 0.17
Bone	0.82 ± 0.41	0.29 ± 0.09	0.07 ± 0.06	0.01 ± 0.00	0.16 ± 0.05
Skin	1.89 ± 0.44	0.35 ± 0.14	0.07 ± 0.07	0.12 ± 0.12	0.12 ± 0.18
	Percent injected dose (%ID)
Intestines	2.41 ± 0.31	2.95 ± 0.25	2.60 ± 0.28	0.40 ± 0.40	4.18 ± 1.90
Urine	80.18 ± 2.97	89.50 ± 2.10	92.88 ± 0.42	97.76 ± 0.56	89.26 ± 2.78
	Uptake ratio of tumor/normal tissue
Tumor/blood	11.16	9.02	102.64	105.33	3.64
Tumor/kidney	2.24	12.43	13.94	13.74	0.26
Tumor/lung	7.62	70.57	62.72	105.33	1.82
Tumor/liver	6.93	12.59	7.9	5.96	0.19
Tumor/muscle	84.58	219.56	1129.0	105.33	10.0
Tumor/skin	8.50	56.46	161.29	26.33	6.67

^a2 h Data was cited from Table 1 for comparison.

^*p<0.05 for determining significance of differences in tumor and kidney uptake between ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex with or without peptide blockade at 2 h post-injection.

Figure 4 illustrates the whole-body SPECT/CT images of B16/F10 melanoma-bearing mice at 2 h post injection of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex and ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex, respectively. In agreement with the biodistribution results, the tumor lesions were clearly visualized by both peptides. However, as compared to ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex, ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex exhibited higher melanoma uptake and lower renal uptake that were demonstrated in the biodistribution results in Table 1.

Figure 4.

Representative maximum intensity projection SPECT/CT images of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex (left) and ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex (right) on B16/F10 melanoma-bearing C57 mice at 2 h post-injection. The melanoma lesions (T) are highlighted with arrows.

DISCUSSION

^99mTc is widely used in nuclear medicine due to its ideal decay properties, low-cost and high availability from commercial ⁹⁹Mo/^99mTc generator. Most ^99mTcradiopharmaceuticals are readily prepared through the coordination of ^99mTc=O³⁺ core (reduced from ^99mTcO₄⁻) by N and S atoms in bifunctional chelators attached to biomolecules. Subsequently, the development of [^99mTc(CO)₃(OH₂)₃]⁺ tricarbonyl kit offers another attractive and robust method for designing new ^99mTcradiopharmaceuticals via replacing the labile water molecules with bifunctional chelators. Our previous study on ^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex demonstrated the feasibility of HYNIC to coordinate ^99mTc(CO)₃ core. However, high liver and kidney uptake and moderate tumor uptake limited further evaluation of ^99mTc(CO)₃-HYNICGGNle-CycMSH_hex. We previously reported that both ⁶⁷Ga-NOTA-GGNle-CycMSH_hex and ⁶⁴Cu-NOTA-GGNle-CycMSH_hex exhibited high melanoma uptake and contrast to normal organs, highlighting their potential for SPECT and PET imaging of melanoma.^{13, 14} Recent publication regarding high tumor uptake of ^99mTc(CO)₃-NOTA-sst2-ANT on AR42J tumor-bearing mice²⁴ provided further impetus for us to explore the preparation of ^99mTc(CO)₃-NOTA/NODAGA-GGNle-CycMSH_hex using [^99mTc(CO)₃(OH₂)₃]⁺ intermediate, and examine the tumor targeting and biodistribtution properties of ^99mTc(CO)₃-NOTA/NODAGA-GGNle-CycMSH_hex in this study.

^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex displayed slightly higher MC1R-specific cellular uptake than ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex on B16/F10 cells. However, the tumor targeting and biodistribution properties were dramatically different between ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex and ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex. The B16/F10 melanoma uptake of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex was 19.76 ± 3.62% ID/g at 2 h post-injection which was 2.7 times the tumor uptake of ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex. Meanwhile, the renal uptake of ^99mTc(CO)_3-NOTA-GGNle-CycMSH_hex was as low as 1.59 ± 0.52% ID/g at 2 h post-injection which was only 18% of that of ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex. High tumor uptake and low renal uptake clearly suggested ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex as a lead peptide for further evaluation. Interestingly, as compared to our reported ^99mTc(CO)_3-HYNIC-GGNle-CycMSH_hex,^{15 99m}Tc(CO)₃-NODAGA-GGNle-CycMSH_hex exhibited tremendous improvement in tumor, kidney and liver uptake. The tumor uptake of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex was 3.4 times the tumor uptake of ^99mTc(CO)_3-HYNIC-GGNle-CycMSHhex, whereas the renal and liver uptake of ^99mTc(CO)₃-NOTAGGNle-CycMSH_hex was only 9% and 4% of that of ^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex at 2 h post-injection,¹⁵ respectively. The tremendous differences in tumor targeting and biodistribution properties between ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex and 99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex were attributed to the difference in coordination chemistry of ^99mTc(CO)₃ between NOTA and HYNIC. The switch from HYNIC to NOTA dramatically increased the tumor uptake and decreased the renal and liver uptake of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex as compared to ^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex.

^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex showed the highest tumor uptake at 2 h post-injection among different time points investigated, and high tumor to normal organ ratios after 2 h post-injection. The tumor uptake of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex was MC1R-specific, with 96% of the tumor uptake was blocked at 2 h post-injection. Meanwhile, the B16/F10 melanoma lesions could be clearly visualized by SPECT using ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex as an imaging agent, demonstrating its potential use as an MC1R-targeting agent for melanoma detection. Moreover, ⁶⁷Ga/⁶⁴Cu-NOTAGGNle-CycMSH_hex exhibited high melanoma uptake and potential for imaging of melanoma in our previous work.^{13, 14} Essentially, NOTA-GGNle-CycMSH_hex could serve as a versatile peptide platform for radiolabeling of ^99mTc, ^67/68Ga³⁺ and ⁶⁴Cu²⁺ radiometals for melanoma imaging. Depending upon the availability of radionuclides, generators, SPECT and PET machines, NOTA-GGNle-CycMSH_hex could offer the flexibility to readily prepare the needed peptide radiopharmaceuticals for SPECT and PET imaging of melanoma. Meanwhile, the wide range of half-lives of ⁶⁸Ga (68 min), ^99mTc (6 h), ⁶⁴Cu (12.7 h) and ⁶⁷Ga (78.3 h) would also provide options for selection.

^99mTc-(Arg¹¹)CCMSH, 99mTc(CO)₃-pz-βAla-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH₂ and ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex were promising ^99mTc-α-MSH peptides with high melanoma uptake reported in the literature.^{15, 25, 26} Among these peptides, ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex showed the lowest renal uptake of 7.52 ± 0.96% ID/g at 2 h post-injection.¹⁵ Remarkably, the renal uptake of ^99mTc(CO)_3-NOTA-GGNle-CycMSH_hex was only 21% of the renal uptake of ^99mTc(EDDA)-HYNICGGNle-CycMSH_hex, while the tumor uptake of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex was 40% more than that of ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex at 2 h post-injection. Clearly, high melanoma uptake and low renal uptake of ^99mTc(CO)₃-NOTAGGNle-CycMSH_hex warranted further investigation of ¹⁸⁸Re(CO)₃-NOTA-GGNle-CycMSH_hex for melanoma therapy, taking advantage of similar coordination chemistry between matched-pair ^99mTc/¹⁸⁸Re theranostic radionuclides. The metal-cyclized ^99mTc/¹⁸⁸Re-(Arg¹¹)CCMSH clearly demonstrated the matched-pair properties and promising theranostic results on B16/F1 murine and TXM13 human melanoma models.^26–28 Interestingly, ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex exhibited similar high tumor uptake as ^99mTc-(Arg¹¹)CCMSH but much lower renal uptake than ^99mTc-(Arg¹¹)CCMSH. According to the similar biodistribution results of ^99mTc/¹⁸⁸Re-(Arg¹¹)CCMSH,^26–28 it is reasonable to anticipate similar tumor targeting and biodistribution pattern for ¹⁸⁸Re(CO)₃-NOTA-GGNle-CycMSH_hex. It is worthwhile to note that ¹⁸⁸Re can be easily produced through a ¹⁸⁸W/¹⁸⁸Re generator which can be shipped to a pharmacy/research laboratory for on-site preparation. Therefore, it would be interesting to prepare and evaluate ¹⁸⁸Re(CO)₃-NOTA-GGNle-CycMSH_hex for melanoma therapy in future studies.

CONCLUSIONS

The melanoma targeting and imaging properties of ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex and ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex were determined on B16/F10 melanoma bearing C57 mice. ^99mTc(CO)₃-NOTA-GGNle-CycMSH_hex exhibited higher tumor uptake and lower renal uptake than ^99mTc(CO)₃-NODAGA-GGNle-CycMSH_hex at 2 h post-injection. High tumor uptake, low kidney uptake and fast urinary clearance of 99mTc(CO)₃-NOTA-GGNle-CycMSH_hex highlighted its potential for melanoma imaging and warranted the evaluation of ¹⁸⁸Re(CO)₃-NOTA-GGNle-CycMSH_hex for melanoma therapy.