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. Author manuscript; available in PMC: 2021 Jun 15.
Published in final edited form as: Bioorg Med Chem Lett. 2020 Oct 24;30(24):127627. doi: 10.1016/j.bmcl.2020.127627

Facile preparation of a novel Ga-67-labeled NODAGA-conjugated lactam-cyclized alpha-MSH peptide at room temperature for melanoma targeting

Jingli Xu a, Zheng Qiao a, Rene Gonzalez b, Yubin Miao a,*
PMCID: PMC7704577  NIHMSID: NIHMS1639515  PMID: 33141072

Abstract

In this study, the melanoma targeting property of 67Ga-NODAGA-GGNle-CycMSHhex {1,4,7-triazacyclononane,1-gluteric acid-4,7-acetic acid-GlyGlyNle-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2} was determined on B16/F10 melanoma-bearing C57 mice to demonstrate the feasibility of NODAGA as a radiometal chelator for facile room temperature radiolabeling of NODAGA-GGNle-CycMSHhex. The IC50 value of NODAGA-GGNle-CycMSHhex was 0.87 ± 0.12 nM on B16/F10 melanoma cells. 67Ga-NODAGA-GGNle-CycMSHhex was readily prepared at room temperature with greater than 98% radiolabeling yield and displayed MC1R-specific binding on B16/F10 melanoma cells. The B16/F10 melanoma uptake of 67Ga-NODAGA-GGNle-CycMSHhex was 10.31 ± 0.78, 14.96 ± 1.34, 13.7 ± 3.33 and 10.4 ± 2.2% ID/g at 0.5, 2, 4 and 24 h post-injection, respectively. Approximately 85% of injected dose cleared out the body via urinary system at 2 h post-injection. 67Ga-NODAGA-GGNle-CycMSHhex showed high tumor/blood, tumor/muscle and tumor/skin uptake ratios after 2 h post-injection. Overall, 67Ga-NODAGA-GGNle-CycMSHhex could be easily prepared at room temperature and exhibited favorable melanoma targeting property, suggesting the potential use of NODAGA as a radiometal chelator for facile room temperature radiolabeling of α-MSH peptides.

Keywords: Room temperature radiolabeling, 67Ga-labeled lactam bridge-cyclized peptide, alpha-melanocyte stimulating hormone, melanoma targeting

Malignant melanoma is the most lethal form of skin cancer due to the extreme aggressiveness associated with melanoma metastasis. It was estimated that approximately 96,480 new cases and 7,230 fatalities occurred in the United States in 2019.1 We have been developing peptide radiopharmaceuticals2–11 to target melanocortin-1 receptors (MC1Rs) for melanoma imaging and therapy due to the over-expression of MC1Rs on both melanotic and amelanotic human melanoma samples.12 Specifically, we have developed a novel class of radiolabeled lactam bridge-cyclized α-melanocyte-stimulating hormone (α-MSH) peptides building upon the construct of GGNle-CycMSHhex {Gly-Gly-Nle-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2}. Different radiometal chelators, such as hydrazinonicotinamide (HYNIC), 1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), were conjugated to GGNle-CycMSHhex for radiolabeling of diagnostic 111In,2,3 99mTc,6,7 67/68Ga,4,9 203Pb,10 64Cu5 and therapeutic 177Lu8 and 90Y.11 Our first-in-human study further highlighted the clinical relevance of MC1R and underscored it as an attractive molecular target for developing theranostic peptides for melanoma.9

We have demonstrated the unique radiolabeling feature of NOTA for 67Ga as compared to DOTA in our previous report.4 67Ga-NOTA-GGNle-CycMSHhex exhibited more favorable radiolabeling conditions than 67Ga-DOTA-GGNle-CycMSHhex. For instance, the labeling yield of 67Ga-NOTA-GGNle-CycMSHhex easily reached greater than 85% at 37 °C, while the labeling yield of 67Ga-DOTA-GGNle-CycMSHhex only achieved that at 75 °C. Meanwhile, 67Ga-NOTA-GGNle-CycMSHhex displayed similar melanoma targeting property as 67Ga-DOTA-GGNle-CycMSHhex.4 NODAGA is a similar metal chelator as NOTA which could form stable hydrophilic complexes with 64Cu and 68Ga.13 NOTA and NODAGA were conjugated to a gastrin-releasing peptide receptor (GRPr) antagonist MJ9. Interestingly, NODAGA-MJ9 easily reached the radiolabeling yield of 98% at room temperature in 10 min whereas heat and more peptide were necessary for NOTA-MJ9 to achieve such high radiolabeling yield.13 Thus, we were interested in whether the replacement of NOTA with NODAGA could facilitate high radiolabeling yield of NODAGA-GGNle-CycMSHhex at room temperature, and how such replacement would affect the melanoma targeting property of 67Ga-NODAGA-GGNle-CycMSHhex. In this study, we synthesized NODAGA-GGNle-CycMSHhex and determined its MC1R binding affinity, prepared 67Ga-NODAGA-GGNle-CycMSHhex at room temperature and examined its cellular binding specificity on B16/F10 melanoma cells and biodistribution property on B16/F10 melanoma-bearing C57 mice.

Firstly, NODAGA-GGNle-CycMSHhex (Figure 1) was synthesized and purified by reverse phase high pressure liquid chromatography (RP-HPLC) according to our published procedure with modifications.4 After the HPLC purification, NODAGA-GGNle-CycMSHhex displayed greater than 90% purity. The identity of NODAGA-GGNle-CycMSHhex was confirmed by electrospray ionization mass spectrometry. The calculated and found molecular weights of NODAGA-GGNle-CycMSHhex were 1453 and 1453, respectively. The IC50 value of NODAGA-GGNle-CycMSHhex was 0.87 ± 0.12 nM on B16/F10 melanoma cells (Figure 2). 67Ga-NODAGA-GGNle-CycMSHhex was readily prepared with greater than 98% radiolabeling yield at room temperature or at 95 °C for 20 min (Figure 3A). Then we examined the effects of reaction time and peptide amount on the radiolabeling yield at room temperature (Figure 3B and 3C). In fact, the radiolabeling yield of 67Ga-NODAGA-GGNle-CycMSHhex was more than 98% after 5 min incubation at room temperature using 10 μg of NODAGA-GGNle-CycMSHhex (Figure 3B). We previously reported that the radiolabeling yield of 67Ga-NOTA-GGNle-CycMSHhex was approximately 30% after 5 min incubation at room temperature using 10 μg of NOTA-GGNle-CycMSHhex, whereas DOTA-GGNle-CycMSHhex couldn’t be labeled with 67Ga under the same radiolabeling condition.4 Furthermore, the low amount of 1 μg of NODAGA-GGNle-CycMSHhex also resulted in greater than 90% radiolabeling yield at room temperature for 5 min (Figure 3C).

Figure 1.

Figure 1.

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Schematic structure of NODAGA-GGNle-CycMSHhex.

Figure 2.

Figure 2.

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In vitro competitive binding curve of NODAGA-GGNle-CycMSHhex. The IC50 value of NODAGA-GGNle-CycMSHhex was 0.87 ± 0.12 nM on B10/F10 melanoma cells.

Figure 3.

Figure 3.

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Effects of reaction temperature (A), reaction time (B) and peptide amount (C) on radiolabeling yield of 67Ga-NODAGA-GGNle-CycMSHhex.

Secondly, the cellular binding specificity of 67Ga-NODAGA-GGNle-CycMSHhex was determined on B16/F10 melanoma cells. As shown in Figure 4, there were two radiolabeled shoulder peaks in similar ratio in the reaction mixture. Two shoulder peaks were likely isomers, thus we separately collected them and determined their specific MC1R binding on B16/F10 melanoma cells using [Nle4, d-Phe7]-α-MSH (NDP-MSH) as a block peptide due to its sub-nanomolar MC1R binding affinity. The retention time of peak 1 and peak 2 was 13.4 and 14.5 min, respectively. Interestingly, both peaks exhibited MC1R-specific binding on melanoma cells. However, Peak 2 displayed higher MC1R binding than peak 1. Approximately 90% of peak 2 binding was blocked by NDP-MSH, whereas about 70% of peak 1 binding was reduced by NDP-MSH. Therefore, we further evaluated the biodistribution property of peak 2 of 67Ga-NODAGA-GGNle-CycMSHhex on B16/F10 melanoma-bearing C57 mice.

Figure 4.

Figure 4.

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Radioactive HPLC profiles (A-C) of two peaks of 67Ga-NODAGA-GGNle-CycMSHhex. The retention time of peak 1 and peak 2 was 13.4 and 14.5 min, respectively. Specific binding (D) of two peaks of 67Ga-NODAGA-GGNle-CycMSHhex on B16/F10 melanoma cells with (black) and without (white) peptide blockade.

Thirdly, the melanoma targeting and pharmacokinetic properties of 67Ga-NODAGA-GGNle-CycMSHhex were determined on B16/F10 melanoma-bearing C57 mice. The biodistribution results of 67Ga-NODAGA-GGNle-CycMSHhex are presented in Table 1. 67Ga-NODAGA-GGNle-CycMSHhex displayed rapid melanoma uptake. The tumor uptake was 10.31 ± 0.78, 14.96 ± 1.34, 13.7 ± 3.33% ID/g at 0.5, 2 and 4 h post-injection, respectively. 67Ga-NODAGA-GGNle-CycMSHhex exhibited prolonged tumor retention, with 10.4 ± 2.2% ID/g of tumor uptake at 24 h post-injection. The co-injection of non-radioactive NDP-MSH blocked 91% of the tumor uptake at 2 h post-injection, demonstrating that the tumor uptake was MC1 receptor-mediated. Whole-body clearance of 67Ga-NODAGA-GGNle-CycMSHhex was rapid, with approximately 85% of the injected dose being washed out of the body via urinary system by 2 h post-injection.

Table 1.

Biodistribution of 67Ga-NODAGA-GGNle-CycMSHhex 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 h 4 h 24 h 2 h NDP blockade
Percent injected dose/gram (%ID/g)
Tumor 10.31 ± 0.78 14.96 ± 1.34 13.7 ± 3.33 10.4 ± 2.2 1.34 ± 0.58*
Brain 0.18 ± 0.04 0.17 ± 0.03 0.03 ± 0.02 0.01 ± 0.02 0.02 ± 0.02
Blood 2.65 ± 1.15 1.18 ± 0.68 0.03 ± 0.04 0.05 ± 0.04 0.34 ± 0.22
Heart 1.21 ± 0.4 0.73 ± 0.22 0.11 ± 0.05 0.03 ± 0.04 0.07 ± 0.05
Lung 1.96 ± 0.52 0.67 ± 0.35 0.27 ± 0.12 0.08 ± 0.05 0.27 ± 0.25
Liver 1.42 ± 0.31 0.67 ± 0.22 0.63 ± 0.19 0.54 ± 0.19 0.61 ± 0.13
Spleen 0.79 ± 0.49 0.96 ± 0.19 0.13 ± 0.08 0.18 ± 0.16 0.17 ± 0.14
Stomach 1.25 ± 0.43 1.1 ± 0.06 0.98 ± 0.34 0.58 ± 0.3 0.24 ± 0.13
Kidneys 14.03 ± 5.77 7.06 ± 2.72 8.26 ± 3.84 3.44 ± 2.86 7.16 ± 2.19a
Muscle 0.66 ± 0.59 0.59 ± 0.09 0.06 ± 0.07 0.15 ± 0.11 0.03 ± 0.02
Pancreas 0.3 ± 0.15 0.82 ± 0.17 0.01 ± 0.01 0.09 ± 0.11 0.08 ± 0.09
Bone 1.38 ± 0.7 1.62 ± 0.81 0.18 ± 0.19 0.09 ± 0.12 0.14 ± 0.12
Skin 4.0±0.90 1.41 ± 0.46 0.42 ± 0.33 0.24 ± 0.18 0.28 ± 0.26
Percent injected dose (%ID)
Intestines 1.68 ± 0.62 0.93 ± 0.2 0.8 ± 0.24 0.77 ± 0.35 0.69 ± 0.23
Urine 63.03 ± 11.41 84.56 ± 4.1 83.22 ± 1.65 90.67 ± 2.9 92.4 ± 4.58
Uptake ratio of tumor/normal tissue
Tumor/blood 3.89 12.68 456.67 208.0 3.94
Tumor/kidney 0.73 2.12 1.66 3.02 0.19
Tumor/liver 7.26 22.33 21.75 19.26 2.20
Tumor/lung 5.26 22.33 50.74 130.0 4.96
Tumor/muscle 15.62 25.36 44.67 228.33 69.33
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*

p<0.05 and

a

p>0.05 for determining significance of differences in tumor and kidney uptake between 67Ga-NODAGA-GGNle-CycMSHhex with or without peptide blockade at 2 h post-injection.

Kidneys are the normal organs with the highest uptake of 67Ga-NODAGA-GGNle-CycMSHhex. The renal uptake was 14.03 ± 5.77, 7.06 ± 2.72 and 8.26 ± 3.84% ID/g at 0.5, 2 and 4 h post-injection, respectively. At 24 h post-injection, the kidney uptake was 3.44 ± 2.86% ID/g. The co-injection of NDP-MSH didn’t reduce the renal uptake, indicating that the renal uptake of 67Ga-NODAGA-GGNle-CycMSHhex was not receptor-mediated. The accumulation of 67Ga-NODAGA-GGNle-CycMSHhex in other normal organs was much lower than kidneys. High tumor/blood and tumor/normal organ uptake ratios were demonstrated as early as 2 h post-injection.

We reported the melanoma targeting property of 67Ga-NOTA-GGNle-CycMSHhex on B16/F1 melanoma-bearing C57 mice.4 The tumor uptake of 67Ga-NOTA-GGNle-CycMSHhex was 20.59 ± 1.97, 25.12 ± 1.03, 18.17 ± 4.89 and 7.95 ± 2.58% ID/g at 0.5, 2, 4 and 24 h post-injection, respectively.4 The tumor uptake of 67Ga-NODAGA-GGNle-CycMSHhex was less than that of 67Ga-NOTA-GGNle-CycMSHhex by 25-50% from 0.5 to 4 h post-injection. However, the tumor uptake of 67Ga-NODAGA-GGNle-CycMSHhex was slightly higher than that of 67Ga-NOTA-GGNle-CycMSHhex at 24 h post-injection. Meanwhile, both peptides exhibited similar renal uptake at 0.5, 2, 4 and 24 h post-injection, suggesting that the extra carboxymethyl group in NODAGA didn’t result in lower renal uptake. However, the third carboxymethyl group in NODAGA facilitated high 67Ga labeling yield at room temperature as compared to NOTA. One carboxymethyl group in NOTA was conjugated to the N-terminus of GGNle-CycMSHhex, making high 67Ga labeling yield more difficult to achieve if not at high temperature.4 Furthermore, despite the lower tumor uptake, 67Ga-NODAGA-GGNle-CycMSHhex exhibited an attractive feature as compared to 67Ga-NOTA-GGNle-CycMSHhex because of rapid high radiolabeling yield (> 90%) at room temperature in 5 min using low amount of peptide (1 μg). Moreover, both 67Ga and 68Ga share identical radiolabeling chemistry. From practical perspective, the impact of rapid high radiolabeling yield at room temperature would be even greater when switching 67Ga to 68Ga due to the shorter half-life of 68 min for 68Ga.

The experimental details are presented in References and notes.14–19

In conclusion, 67Ga-NODAGA-GGNle-CycMSHhex was easily prepared at room temperature and exhibited favorable melanoma targeting property, suggesting the potential use of NODAGA as a radiometal chelator for facile room temperature radiolabeling of α-MSH peptides in the future.

ACKNOWLEDGMENTS

We thank Dr. Fabio Gallazzi for his technical assistance. This work was supported by NIH grant R01CA225837.

REFERENCES AND NOTES

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  • 14.Peptide synthesis: Amino acid and resin were purchased from Advanced ChemTech Inc. (Louisville, KY) and Novabiochem (San Diego, CA) for peptide synthesis. All other chemicals used in this study were purchased from Thermo Fischer Scientific (Waltham, MA) and used without further purification. NODAGA-GGNle-CycMSHhex was synthesized using Fmoc chemistry. Briefly, the linear intermediate scaffold of NODAGA(OtBu)3-Nle-Gly-Gly-Asp(O-2-PhiPr)-His(Trt)-DPhe-Arg(Pbf)-Trp(Boc)-Lys(Mtt) was 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 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.
  • 15.In vitro competitive binding assay: 125I-Tyr2-[Nle4, d-Phe7]-α-MSH {125I-(Tyr2)-NDP-MSH} was obtained from PerkinElmer, Inc. (Waltham, MA) for receptor binding assay. The MC1 receptor binding affinity of NODAGA-GGNle-CycMSHhex was 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).
  • 16.Radiolabeling: 67GaCl3 was purchased from Lantheus (N. Billerica, MA) for peptide radiolabeling. 67Ga-NODAGA-GGNle-CycMSHhex was prepared in a 0.25 M NH4OAc-buffered solution (pH 4.5). Briefly, 20 μL of 67GaCl3 (37-74 MBq in 0.1 M HCl aqueous solution), 10 μL of 1 mg/mL peptide aqueous solution and 200 μL of 0.25 M NH4OAc (pH 4.5) were added into a reaction vial and incubated at room temperature (25 °C) or 95 °C for 20 mins. The pH of the reaction mixture was 4. After the incubation, 10 μL of 0.5% EDTA (ethylenediaminetetraacetic acid) aqueous solution was added into the reaction vial to scavenge potentially unbound 67Ga3+ ions. 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 80:20 A/B for 3 min followed by a linear gradient of 80:20 A/B to 70:30 A/B over 20 min. Then, the gradient was changed from 70:30 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 80:20 A/B over 3 mins. 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 for animal studies.
  • 17.Effects of reaction time and peptide amount on 67Ga radiolabeling yield: NODAGA-GGNle-CycMSHhex was radiolabeled with 67Ga as described above at room temperature for 5 and 20 min and the reactions were quenched with 10 μL of 0.5% EDTA aqueous solution. Then the radiolabeling yields were determined by HPLC using the gradient described above. For effect of peptide amount, NODAGA-GGNle-CycMSHhex was radiolabeled with 67Ga as described above at room temperature for 5 min using 1, 2, 5 and 10 μg of peptide, respectively. The reactions were terminated with 10 μL of 0.5% EDTA aqueous solution and the radiolabeling yields were determined by HPLC using the gradient described above.
  • 18.Specific binding of 67Ga-NODAGA-GGNle-CycMSHhex: The specific binding of two peaks of 67Ga-NODAGA-GGNle-CycMSHhex 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 11.1 KBq of 67Ga-NODAGA-GGNle-CycMSHhex with or without 10 μg (6.07 nmol) of unlabeled NDP-MSH in 0.3 mL of binding medium {Modified Eagle’s medium with 25 mM N-(2-hydroxyethyl)-piperazine-N’-(2-ethanesulfonic acid), pH 7.4, 0.2% bovine serum albumin (BSA), 0.3 mM 1,10-phenathroline}. After the incubation, the cells were rinsed three times with 0.5 ml of ice-cold pH 7.4, 0.2% BSA/0.01 M phosphate buffered saline (PBS) and measured in a Wallac 1480 automated gamma counter (PerkinElmer, NJ).
  • 19.Biodistribution studies: All animal studies were conducted in compliance with Institutional Animal Care and Use Committee approval. The biodistribution property of 67Ga-NODAGA-GGNle-CycMSHhex was determined on B16/F10 flank melanoma-bearing C57 mice (Charles River, Wilmington, MA). Briefly, each C57 mouse was subcutaneously inoculated with 1×106 B16/F10 cells on the right flank. Ten days post inoculation, the tumor weights reached approximately 0.2 g. Each melanoma-bearing mouse was injected with 0.037 MBq of 67Ga-NODAGA-GGNle-CycMSHhex via the tail vein. 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 the tumor uptake of 67Ga-NODAGA-GGNle-CycMSHhex 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. 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 tumor and renal uptake of 67Ga-NODAGA-GGNle-CycMSHhex with/without NDP-MSH co-injection. The differences at the 95% confidence level (p<0.05) were considered significant.

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