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Article

Introduction of a Polyethylene Glycol Linker Improves Uptake of 67Cu-NOTA-Conjugated Lactam-Cyclized Alpha-Melanocyte-Stimulating Hormone Peptide in Melanoma

by
Zheng Qiao
1,
Jingli Xu
1,
Darrell R. Fisher
2,
Rene Gonzalez
3 and
Yubin Miao
1,*
1
Department of Radiology, University of Colorado Denver, Aurora, CO 80045, USA
2
Versant Medical Physics and Radiation Safety, Richland, WA 99354, USA
3
Department of Medical Oncology, University of Colorado Denver, Aurora, CO 80045, USA
*
Author to whom correspondence should be addressed.
Cancers 2023, 15(10), 2755; https://doi.org/10.3390/cancers15102755
Submission received: 23 April 2023 / Revised: 10 May 2023 / Accepted: 12 May 2023 / Published: 14 May 2023
(This article belongs to the Special Issue Diagnosis of Melanoma and Non-melanoma Skin Cancer)
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Abstract

:

Simple Summary

There is a need to develop new theranostic approaches for malignant melanoma. Only 35% of patients with metastatic melanoma reach the milestone of 5-year survival, despite the success of new immunotherapy. We have developed a new class of peptides to target melanocortin-1 receptors (MC1Rs) that display elevated levels in human melanoma. In this study, we examined the melanoma targeting and biodistribution properties of two 67Cu-tagged peptides in tumor-bearing mice. We found that one of the peptides, namely 67Cu-NOTA-PEG2Nle-CycMSHhex, exhibited favorable melanoma targeting and biodistribution properties that underscored its potential as an MC1R-targeted therapeutic peptide for melanoma treatment in the future.

Abstract

The aim of this study was to evaluate the effect of linker on tumor targeting and biodistribution of 67Cu-NOTA-PEG2Nle-CycMSHhex {67Cu-1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid-polyethylene glycol-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and 67Cu-NOTA-GGNle-CycMSHhex {67Cu-NOTA-GlyGlyNle-CycMSHhex} on melanoma-bearing mice. NOTA-PEG2Nle-CycMSHhex and NOTA-GGNle-CycMSHhex were synthesized and purified by HPLC. The biodistribution of 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex was determined in B16/F10 melanoma-bearing C57 mice. The melanoma imaging property of 67Cu-NOTA-PEG2Nle-CycMSHhex was further examined in B16/F10 melanoma-bearing C57 mice. 67Cu-NOTA-PEG2Nle-CycMSHhex exhibited higher tumor uptake than 67Cu-NOTA-GGNle-CycMSHhex at 2, 4, and 24 h post-injection. The tumor uptake of 67Cu-NOTA-PEG2Nle-CycMSHhex was 27.97 ± 1.98, 24.10 ± 1.83, and 9.13 ± 1.66% ID/g at 2, 4, and 24 h post-injection, respectively. Normal organ uptake of 67Cu-NOTA-PEG2Nle-CycMSHhex was lower than 2.6% ID/g at 4 h post-injection, except for kidney uptake. The renal uptake of 67Cu-NOTA-PEG2Nle-CycMSHhex was 6.43 ± 1.31, 2.60 ± 0.79, and 0.90 ± 0.18% ID/g at 2, 4, and 24 h post-injection, respectively. 67Cu-NOTA-PEG2Nle-CycMSHhex showed high tumor to normal organ uptake ratios after 2 h post-injection. The B16/F10 melanoma lesions could be clearly visualized by single photon emission computed tomography (SPECT) using 67Cu-NOTA-PEG2Nle-CycMSHhex as an imaging probe at 4 h post-injection. The favorable tumor targeting and biodistribution properties of 67Cu-NOTA-PEG2Nle-CycMSHhex underscored its potential as an MC1R-targeted therapeutic peptide for melanoma treatment.

1. Introduction

Malignant melanoma continues to be the most deadly form of skin cancer, with approximately 97,610 new cases and 7990 deaths in the United States in 2023 [1]. Molecularly-targeted melanoma therapies, including BRAF inhibitors (Vemurafenib), cytotoxic T-lymphocyte antigen 4 (CTLA-4) inhibitors (Ipilimumab), and programmed death-1 receptor (PD-1) inhibitors (Nivolumab), have significantly extended the overall survival of metastatic melanoma patients by months over the past decade [2,3,4,5,6,7]. However, the 5-year survival of metastatic melanoma patients remains at only 35% despite the successes of the aforementioned new treatments [7]. There is a need to develop new theranostic approaches for malignant melanoma.
Melanocortin-1 receptor (MC1R) is a G protein-coupled receptor that expresses at elevated levels in human melanotic and amelanotic melanoma [8,9,10]. Meanwhile, α-melanocyte-stimulating hormone (α-MSH) peptides can bind to MC1Rs with nanomolar binding affinities [11,12,13,14,15,16,17,18,19,20]. Therefore, MC1R is an attractive molecular target for developing MC1R-targeted theranostic α-MSH peptides. Over the past years, we have developed a new class of MC1R-targeted peptide radiopharmaceuticals that include the core structure of Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2 (GGNle-CycMSHhex) for melanoma imaging and therapy [11,12,13,14,15,16,17,18,19,20]. Our first-in-human melanoma imaging work using 68Ga-DOTA-GGNle-CycMSHhex clearly detected human metastatic melanoma lesions in the brain, lung, connective tissue, and intestines [10], demonstrating the clinical relevance of MC1R for melanoma imaging, as well as for potential MC1R-targeted radionuclide therapy.
Due to the emissions of positrons and beta-particles, 64Cu (T1/2 = 12.7 h, 17.4% β+, 40% β−) is an attractive theranostic radionuclide. NOTA is a better chelator for 64Cu than DOTA because of the superior in vivo stability of 64Cu-NOTA compared to 64Cu-DOTA. In our previous work, 64Cu-NOTA-GGNle-CycMSHhex {64Cu-1,4,7-triazacyclononane-1,4,7-triacetic acid-GGNle-CycMSHhex) showed higher B16/F1 melanoma uptake by 2.4 times and less liver uptake by 93% than 64Cu-DOTA-GGNle-CycMSHhex at 2 h post-injection [14]. Meanwhile, we reported that the substitution of the GG linker with an Aoc or polyethylene glycol (PEG) linker could dramatically affect the melanoma uptake of 64Cu-NOTA-AocNle-CycMSHhex and 64Cu-NOTA-PEG2Nle-CycMSHhex peptides [21]. Interestingly, the B16/F10 melanoma uptake of 64Cu-NOTA-PEG2Nle-CycMSHhex was 2.5 times the tumor uptake of 64Cu-NOTA-AocNle-CycMSHhex at 2 h post-injection [21].
Recently, 67Cu (T1/2 = 61.8 h, 100% β−, Emax = 562 keV, Emean = 141 keV) has obtained renewed interest as an attractive therapeutic radionuclide [22,23,24,25,26,27,28]. 67Cu and 64Cu are matched-pair theranostic radionuclides that share identical coordination chemistry. Moreover, 67Cu emits γ-photons (93 keV, 16% and 185 keV, 49%) that are suitable for SPECT imaging. Therefore, the follow-up 67Cu SPECT imaging provides the opportunity for dosimetry calculation after treatment. In this study, we prepared 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex peptides and determined their melanoma targeting and biodistribution properties in B16/F10 melanoma-bearing C57 mice. Since 67Cu-NOTA-PEG2Nle-CycMSHhex displayed higher tumor uptake than 67Cu-NOTA-GGNle-CycMSHhex after 2 h post-injection, we further examined the melanoma imaging of 67Cu-NOTA-PEG2Nle-CycMSHhex in B16/F10 melanoma-bearing C57 mice.

2. Materials and Methods

2.1. Chemicals and Reagents

Amino acids and resin were purchased from Advanced ChemTec Inc. (Louisville, KY, USA) and Novabiochem (San Diego, CA, USA). NOTA(OtBu)2 was purchased from the CheMatech Inc. (Dijon, France) for peptide synthesis. 67CuCl2 was purchased from Idaho Accelerator Center at Idaho State University (Pocatello, ID, USA) for radiolabeling. B16/F10 murine melanoma cells were received from the American Type Culture Collection (Manassas, VA, USA). All other chemicals used in this study were purchased from Thermo Fisher Scientific (Waltham, MA, USA) and used without further purification.

2.2. Peptide Synthesis and Radiolabeling

NOTA-PEG2Nle-CycMSHhex and NOTA-GGNle-CycMSHhex were synthesized and characterized by liquid chromatography-mass spectrometry (LC-MS) according to our previous publication [21]. Generally, 210 µmol of each fluorenylmethoxycarbonyl (Fmoc)-protected amino acid and NOTA(OtBu)2 and 70 µmol of resin were used for the synthesis. 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex were prepared using 0.5 M NH4OAc (pH 5.4). Briefly, 10 μL of 67CuCl2 (37–74 MBq in 0.01 M HCl aqueous solution), 10 μL of 1 mg/mL peptide 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. Thereafter, 10 μL of 0.5% EDTA aqueous solution was added to scavenge potentially unbound 67Cu2+. 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex complexes were purified by Waters RP-HPLC (Milford, MA, USA) on a Grace Vydac C-18 reverse-phase analytical column (Deerfield, IL, USA) with a flow rate of 1 mL/min. A 20 min gradient of 20–30% acetonitrile in a 20 mM HCl aqueous solution was used for peptide purification. Each collected peptide solution was purged with N2 gas for 15 min to remove the acetonitrile, then adjusted to pH 7.4 with 0.1 M NaOH and sterile saline for cellular binding and animal studies.

2.3. Specific Binding

Specific binding of 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex was determined on B16/F10 melanoma cells according to our previous publication [21]. Briefly, the cells were incubated at 25 °C for 1 h with approximately 0.01 MBq of 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex with or without 10 μg (6.07 nmol) of unlabeled [Nle4, D-Phe7]-α-MSH (NDP-MSH). The binding medium was aspirated after incubation. The cells were washed twice with 0.5 mL of ice-cold 0.01 M phosphate-buffered saline (PBS) buffer containing 0.2% BSA (pH = 7.4) and collected and measured in a Wallac 1480 automated gamma counter (PerkinElmer, NJ, USA).

2.4. Biodistribution and Imaging Studies

All animal studies were conducted in compliance with Institutional Animal Care and Use Committee approval. C57 mice were purchased from Charles River Laboratory (Wilmington, NC). B16/F10 melanoma-bearing C57 mice were generated according to our previous publication [21]. Each melanoma-bearing mouse was injected with 0.037 MBq of 67Cu-NOTA-PEG2Nle-CycMSHhex or 67Cu-NOTA-GGNle-CycMSHhex via the tail vein. The tumor uptake specificity of 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex was determined by co-injecting 10 μg (6.07 nmol) of unlabeled NDP-MSH. Mice were sacrificed at 0.5, 2, 4, and 24 h post-injection, and tumors and organs of interest were harvested, weighed, and counted. The blood value was taken as 6.5% of the whole-body weight.
The melanoma imaging property of 67Cu-NOTA-PEG2Nle-CycMSHhex was further examined in B16/F10 melanoma-bearing C57 mice. Each mouse was injected with 7.4 MBq of 67Cu-NOTA-PEG2Nle-CycMSHhex via the tail vein. Imaging studies were performed 4 h post-injection. Reconstructed SPECT data was visualized using VivoQuant (Invicro, Boston, MA, USA).

2.5. Statistical Analysis

Statistical analysis was performed using the Microsoft Office Excel 2007 Student’s t test for unpaired data. A 95% confidence level was chosen to determine the significance of differences in tumor and kidney uptake between 67Cu-NOTA-PEG2Nle-CycMSHhex with/without NDP-MSH blockade and tumor and kidney uptake between 67Cu-NOTA-GGNle-CycMSHhex with/without NDP-MSH blockade. The differences at the 95% confidence level (p < 0.05) were considered significant.

3. Results

The schematic structures of NOTA-PEG2Nle-CycMSHhex and NOTA-GGNle-CycMSHhex are presented in Figure 1. Both peptides were synthesized, purified by HPLC, and displayed greater than 90% purities after HPLC purification. 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex were prepared in a 0.5 M NH4OAc-buffered solution with greater than 90% radiochemical yields. The radioactive HPLC profiles of 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex are shown in Figure 2. The retention times of 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex were 18.6 and 11.6 min, respectively. The specific activity was 560 mCi/µg for 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex. As shown in Figure 2B, both 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex exhibited MC1R-specific binding. The peptide blockade reduced 92% of 67Cu-NOTA-PEG2Nle-CycMSHhex and 72% of 67Cu-NOTA-GGNle-CycMSHhex cellular uptake.
Table 1 and Table 2 show the biodistribution results of 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex. 67Cu-NOTA-PEG2Nle-CycMSHhex displayed rapid melanoma uptake and prolonged tumor retention. The tumor uptake of 67Cu-NOTA-PEG2Nle-CycMSHhex was 8.83 ± 2.19, 27.97 ± 1.98, 24.10 ± 1.83, and 9.13 ± 1.66% ID/g at 0.5, 2, 4, and 24 h post-injection, respectively. Approximately 94% of tumor uptake of 67Cu-NOTA-PEG2Nle-CycMSHhex was blocked by 10 μg (6.07 nmol) of NDP-MSH (p < 0.05), suggesting that the tumor uptake was MC1R-mediated. Normal organ uptake of 67Cu-NOTA-PEG2Nle-CycMSHhex was lower than 2% ID/g at 2 h post-injection, except for kidney uptake (6.34 ± 1.63% ID/g). The renal uptake of 67Cu-NOTA-PEG2Nle-CycMSHhex decreased to 2.60 ± 0.79 and 0.90 ± 0.18% ID/g at 4 and 24 h post-injection.
67Cu-NOTA-GGNle-CycMSHhex exhibited lower tumor uptake than 67Cu-NOTA-PEG2Nle-CycMSHhex at 2, 4, and 24 h post-injection. The tumor uptake of 67Cu-NOTA-GGNle-CycMSHhex was 16.58 ± 1.40, 11.66 ± 1.94, 8.79 ± 1.31, and 4.92 ± 1.58% ID/g at 0.5, 2, 4, and 24 h post-injection, respectively. Approximately 95% of tumor uptake of 67Cu-NOTA-GGNle-CycMSHhex was decreased by 10 μg (6.07 nmol) of NDP-MSH blockade (p < 0.05), indicating that the tumor uptake was MC1R-specific. Normal organ uptake of 67Cu-NOTA-GGNle-CycMSHhex was lower than 2% ID/g at 2 h post-injection, except for kidney uptake (5.08 ± 1.52% ID/g). The renal uptake of 67Cu-NOTA-GGNle-CycMSHhex was 4.21 ± 0.92 and 1.19 ± 0.28% ID/g at 4 and 24 h post-injection.
67Cu-NOTA-PEG2Nle-CycMSHhex showed higher tumor/kidney and tumor/liver ratios than 67Cu-NOTA-GGNle-CycMSHhex. Thus, we further performed SPECT imaging of 67Cu-NOTA-PEG2Nle-CycMSHhex in B16/F10 melanoma-bearing mice. The representative maximum intensity projection SPECT/CT image of 67Cu-NOTA-PEG2Nle-CycMSHhex in a B16/F10 melanoma-bearing C57 mouse is presented in Figure 3. In agreement with the biodistribution result, the B16/F10 tumor lesions were clearly imaged at 4 h post-injection using 67Cu-NOTA-PEG2Nle-CycMSHhex as an imaging probe.

4. Discussion

The attractive decay properties and availability of high-quality 177Lu are key factors that contribute to the success of 177Lu radionuclide therapy [29]. 67Cu shares some similar decay properties with 177Lu. 177Lu is a medium-energy (0.497 MeV) β-emitter with a half-life of 6.7 days, whereas 67Cu emits 0.562 MeV β-particles with a half-life of 2.6 days. Meanwhile, the specific activity of 67Cu can reach 5.55 GBq/μg (150 mCi/μg) by irradiating an enriched 68Zn target via a 68Zn(γ,p)67Cu reaction [30,31]. From a matched-pair perspective, the identical coordination chemistry and theranostic features of 67Cu/64Cu make 67Cu attractive for potential therapeutic application. Furthermore, both 177Lu and 67Cu emit imageable γ-photons for SPECT that can be utilized to calculate dosimetry and monitor therapeutic response.
We reported the effects of GG, PEG2, and Aoc linkers on melanoma uptake of 64Cu-NOTA-GGNle-CycMSHhex, 64Cu-NOTA-PEG2Nle-CycMSHhex, and 64Cu-NOTA-AocNle-CycMSHhex [14,21]. Interestingly, both 64Cu-NOTA-GGNle-CycMSHhex and 64Cu-NOTA-PEG2Nle-CycMSHhex exhibited higher melanoma uptake than 64Cu-NOTA-AocNle-CycMSHhex [14,21]. Thus, we further evaluated the tumor targeting and clearance properties of 67Cu-NOTA-GGNle-CycMSHhex and 67Cu-NOTA-PEG2Nle-CycMSHhex in B16/F10 melanoma-bearing C57 mice in this study. First of all, 67Cu-NOTA-PEG2Nle-CycMSHhex displayed higher MC1R-specific cellular uptake than 67Cu-NOTA-GGNle-CycMSHhex on B16/F10 melanoma cells. The cellular uptake of 64Cu-NOTA-PEG2Nle-CycMSHhex was 2.5 times that of 64Cu-NOTA-GGNle-CycMSHhex (Figure 2). Furthermore, 67Cu-NOTA-PEG2Nle-CycMSHhex exhibited higher MC1R-specific uptake than 67Cu-NOTA-GGNle-CycMSHhex on B16/F10 melanoma. The tumor uptake of 67Cu-NOTA-PEG2Nle-CycMSHhex was 2.4, 2.7, and 1.9 times the tumor uptake of 67Cu-NOTA-GGNle-CycMSHhex at 2, 4, and 24 h post-injection, respectively (Figure 4). The B16/F10 melanoma lesions could be clearly visualized using 67Cu-NOTA-PEG2Nle-CycMSHhex as an imaging probe (Figure 3). It is worthwhile to note that NOTA-PEG2Nle-CycMSHhex and NOTA-GGNle-CycMSHhex showed similar nanomolar MC1R binding affinities (1.2 vs. 1.6 nM) [14,21]. Therefore, it is necessary to perform biodistribution studies to fully appreciate the potential difference in tumor uptake of the peptides with similar in vitro binding affinities.
The renal and liver uptake of 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex were in a similar range (Figure 4). As compared to 67Cu-NOTA-GGNle-CycMSHhex, higher tumor uptake of 67Cu-NOTA-PEG2Nle-CycMSHhex yielded higher tumor/kidney and tumor/liver uptake ratios. As shown in Table 1 and Table 2, the tumor/kidney ratio of 67Cu-NOTA-PEG2Nle-CycMSHhex was 2.4, 2.7, and 1.9 times the tumor/kidney ratio of 67Cu-NOTA-GGNle-CycMSHhex at 2, 4, and 24 h post-injection, respectively. The tumor/liver ratio of 67Cu-NOTA-PEG2Nle-CycMSHhex was 3.3, 2.0, and 1.8 times the tumor/liver ratio of 67Cu-NOTA-GGNle-CycMSHhex at 2, 4, and 24 h post-injection, respectively. The enhanced tumor/kidney and tumor/liver uptake ratios of 67Cu-NOTA-PEG2Nle-CycMSHhex would facilitate its application in melanoma treatment.
A recent study reported the effectiveness of gastrin-releasing peptide receptor (GRPR)-targeted 67Cu-SAR-BBN in PC-3 prostate tumor-bearing mice [26]. Huynh et al. examined the efficacy of the treatment of 6 × 24 MBq of 67Cu-SAR-BBN and concluded that the treatment inhibited tumor growth and extended median survival from 34.5 days for the control group to greater than 54 days for the treatment group [26]. Another report directly compared the therapeutic efficacy between somatostatin receptor 2 (SST-2)-targeted 67Cu-SarTATE and 177Lu-DOTA-TATE in AR42J pancreatic tumor-bearing mice [22]. Specifically, Cullinane et al. examined the effectiveness of a single injection of 5 MBq of 67Cu-SarTATE or 177Lu-DOTA-TATE and found that the treatment of 67Cu-SarTATE and 177Lu-DOTA-TATE inhibited tumor growth and extended survival from 12 days in the control group to 21 days in the treatment group [22]. Furthermore, they compared the efficacy of 30 MBq of 67Cu-SarTATE or 177Lu-DOTA-TATE either as a single administration or as two fractions of even activity 2 weeks apart. The results revealed that the treatment of 2 × 15 MBq of 67Cu-SarTATE or 177Lu-DOTA-TATE significantly improved the survival over the single dose of 30 MBq by 11 days and 17 days, respectively [22]. These promising preclinical results of 67Cu-SarTATE and 67Cu-SAR-BBN clearly underscore the suitability and feasibility of receptor-targeted 67Cu-peptides for targeted radionuclide therapy.

5. Conclusions

The melanoma targeting and biodistribution properties of 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-AocNle-CycMSHhex were determined in B16/F10 melanoma-bearing C57 mice. 67Cu-NOTA-PEG2Nle-CycMSHhex showed higher tumor uptake than 64Cu-NOTA-AocNle-CycMSHhex after 2 h post-injection. The favorable tumor targeting and biodistribution properties of 67Cu-NOTA-PEG2Nle-CycMSHhex underscored its potential as an MC1R-targeted therapeutic peptide for melanoma treatment.

Author Contributions

Conceptualization, Y.M.; methodology, Y.M., R.G. and D.R.F.; investigation, Z.Q. and J.X.; writing—original draft preparation, Z.Q., J.X. and Y.M.; writing—review and editing, all authors; project administration, Y.M.; funding acquisition, Y.M. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by NIH Grants R01CA225837 and R01CA269221.

Institutional Review Board Statement

All animal studies were conducted in compliance with Institutional Animal Care and Use Committee approval of the University of Colorado Anschutz Medical Campus, Animal Protocol # 00351.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data are contained in the manuscript.

Acknowledgments

The authors thank Fabio Gallazzi for his technical assistance and Jon Stoner (ISU Idaho Accelerator Center) for 67CuCl2 production.

Conflicts of Interest

The authors declare no conflict of interest.

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Cancers 15 02755 g001 550
Figure 1. Schematic structures of 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex.
Figure 1. Schematic structures of 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex.
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Figure 2. Radioactive HPLC profiles of 67Cu-NOTA-PEG2Nle-CycMSHhex (A) and 67Cu-NOTA-GGNle-CycMSHhex (B). The retention times of 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex were 18.6 and 11.6 min, respectively. Specific binding (C) of 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex on B16/F10 melanoma cells with (black) and without (white) peptide blockade, respectively.
Figure 2. Radioactive HPLC profiles of 67Cu-NOTA-PEG2Nle-CycMSHhex (A) and 67Cu-NOTA-GGNle-CycMSHhex (B). The retention times of 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex were 18.6 and 11.6 min, respectively. Specific binding (C) of 67Cu-NOTA-PEG2Nle-CycMSHhex and 67Cu-NOTA-GGNle-CycMSHhex on B16/F10 melanoma cells with (black) and without (white) peptide blockade, respectively.
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Figure 3. Representative maximum intensity projection SPECT/CT image of a B16/F10 melanoma-bearing C57 mouse using 67Cu-NOTA-PEG2Nle-CycMSHhex as an imaging probe at 4 h post-injection. The melanoma lesions (T) are highlighted with an arrow on the image.
Figure 3. Representative maximum intensity projection SPECT/CT image of a B16/F10 melanoma-bearing C57 mouse using 67Cu-NOTA-PEG2Nle-CycMSHhex as an imaging probe at 4 h post-injection. The melanoma lesions (T) are highlighted with an arrow on the image.
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Figure 4. Comparison of uptake in tumor, kidneys, and liver between 67Cu-NOTA-PEG2Nle-CycMSHhex (PEG2Nle) and 67Cu-NOTA-GGNle-CycMSHhex (GGNle) at 2, 4, and 24 h post-injection.
Figure 4. Comparison of uptake in tumor, kidneys, and liver between 67Cu-NOTA-PEG2Nle-CycMSHhex (PEG2Nle) and 67Cu-NOTA-GGNle-CycMSHhex (GGNle) at 2, 4, and 24 h post-injection.
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Table
Table 1. Biodistribution of 67Cu-NOTA-PEG2Nle-CycMSHhex in B16/F10 melanoma-bearing C57 mice. The data were presented as percent injected dose/gram or as percent injected dose (Mean ± SD, n = 4).
Table 1. Biodistribution of 67Cu-NOTA-PEG2Nle-CycMSHhex in B16/F10 melanoma-bearing C57 mice. The data were presented as percent injected dose/gram or as percent injected dose (Mean ± SD, n = 4).
Tissues0.5 h2 h4 h24 h2 h NDP Blockade
Percent injected dose/gram (%ID/g)
Tumor8.83 ± 2.1927.97 ± 1.9824.10 ± 1.839.13 ± 1.661.66 ± 0.28 *
Brain0.04 ± 0.020.01 ± 0.010.01 ± 0.010.04 ± 0.030.02 ± 0.01
Blood0.63 ± 0.260.20 ± 0.110.05 ± 0.020.02 ± 0.010.01 ± 0.01
Heart0.58 ± 0.180.79 ± 0.060.34 ± 0.080.05 ± 0.050.11 ± 0.09
Lung0.76 ± 0.160.53 ± 0.190.62 ± 0.180.35 ± 0.130.68 ± 0.29
Liver0.95 ± 0.191.09 ± 0.141.41 ± 0.260.84 ± 0.151.70 ± 0.39
Spleen0.37 ± 0.100.02 ± 0.010.01 ± 0.010.02 ± 0.010.03 ± 0.02
Stomach1.13 ± 0.361.40 ± 0.510.77 ± 0.090.21 ± 0.090.65 ± 0.07
Kidneys6.43 ± 1.316.34 ± 1.632.60 ± 0.790.90 ± 0.184.83 ± 0.72
Muscle0.48 ± 0.120.81 ± 0.120.03 ± 0.030.01 ± 0.010.01 ± 0.01
Pancreas0.10 ± 0.050.47 ± 0.060.01 ± 0.010.01 ± 0.010.08 ± 0.06
Bone0.60 ± 0.350.07 ± 0.040.03 ± 0.010.03 ± 0.010.03 ± 0.01
Skin1.77 ± 0.280.59 ± 0.110.18 ± 0.070.03 ± 0.020.16 ± 0.04
Percent injected dose (%ID)
Intestines0.83 ± 0.090.95 ± 0.161.40 ± 0.340.77 ± 0.122.09 ± 0.96
Urine78.31 ± 3.7989.08 ± 4.9691.05 ± 1.2095.61 ± 0.5989.34 ± 2.19
Uptake ratio of tumor/normal tissue
Tumor/blood14.02139.85482.0456.50166.0
Tumor/kidney1.374.419.2710.140.34
Tumor/lung11.6252.7738.8726.092.44
Tumor/liver9.2925.6617.0910.870.98
Tumor/muscle18.3934.53803.33913.0166.0
* p < 0.05 for determining the significance of differences in tumor and kidney uptake between 67Cu-NOTA-PEG2Nle-CycMSHhex with or without peptide blockade at 2 h post-injection.
Table
Table 2. Biodistribution of 67Cu-NOTA-GGNle-CycMSHhex in B16/F10 melanoma-bearing C57 mice. The data were presented as percent injected dose/gram or as percent injected dose (Mean ± SD, n = 4).
Table 2. Biodistribution of 67Cu-NOTA-GGNle-CycMSHhex in B16/F10 melanoma-bearing C57 mice. The data were presented as percent injected dose/gram or as percent injected dose (Mean ± SD, n = 4).
Tissues0.5 h2 h4 h24 h2 h NDP Blockade
Percent injected dose/gram (%ID/g)
Tumor16.58 ± 1.4011.66 ± 1.948.79 ± 1.314.92 ± 1.580.61 ± 0.15 *
Brain0.07 ± 0.040.03 ± 0.020.02 ± 0.030.02 ± 0.020.01 ± 0.01
Blood1.22 ± 0.080.03 ± 0.050.06 ± 0.070.01 ± 0.010.01 ± 0.01
Heart0.71 ± 0.310.04 ± 0.040.07 ± 0.060.03 ± 0.030.04 ± 0.04
Lung0.54 ± 0.690.41 ± 0.200.37 ± 0.090.27 ± 0.120.25 ± 0.16
Liver1.15 ± 0.071.49 ± 0.561.02 ± 0.070.82 ± 0.120.66 ± 0.23
Spleen0.52 ± 0.320.05 ± 0.030.12 ± 0.080.03 ± 0.010.09 ± 0.08
Stomach1.53 ± 0.530.81 ± 0.140.71 ± 0.220.30 ± 0.170.65 ± 0.22
Kidneys7.18 ± 2.635.08 ± 1.524.21 ± 0.921.19 ± 0.283.19 ± 0.89
Muscle0.51 ± 0.180.04 ± 0.040.01 ± 0.010.01 ± 0.010.01 ± 0.01
Pancreas0.10 ± 0.080.12 ± 0.080.04 ± 0.040.02 ± 0.010.02 ± 0.02
Bone0.68 ± 0.100.21 ± 0.240.06 ± 0.060.13 ± 0.160.01 ± 0.01
Skin2.05 ± 0.460.50 ± 0.240.27 ± 0.190.02 ± 0.010.09 ± 0.06
Percent injected dose (%ID)
Intestines1.46 ± 0.491.30 ± 0.272.70 ± 0.260.94 ± 0.321.48 ± 0.17
Urine79.77 ± 2.2588.97 ± 2.4886.99 ± 1.2296.23 ± 1.0395.14 ± 1.28
Uptake ratio of tumor/normal tissue
Tumor/blood13.59388.67146.50492.061.0
Tumor/kidney2.312.292.094.130.19
Tumor/lung30.7028.4423.7618.222.44
Tumor/liver14.427.838.626.00.92
Tumor/muscle32.51291.50879.0492.061.0
* p < 0.05 for determining the significance of differences in tumor and kidney uptake between 67Cu-NOTA-GGNle-CycMSHhex with or without peptide blockade at 2 h post-injection.
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Qiao, Z.; Xu, J.; Fisher, D.R.; Gonzalez, R.; Miao, Y. Introduction of a Polyethylene Glycol Linker Improves Uptake of 67Cu-NOTA-Conjugated Lactam-Cyclized Alpha-Melanocyte-Stimulating Hormone Peptide in Melanoma. Cancers 2023, 15, 2755. https://doi.org/10.3390/cancers15102755

AMA Style

Qiao Z, Xu J, Fisher DR, Gonzalez R, Miao Y. Introduction of a Polyethylene Glycol Linker Improves Uptake of 67Cu-NOTA-Conjugated Lactam-Cyclized Alpha-Melanocyte-Stimulating Hormone Peptide in Melanoma. Cancers. 2023; 15(10):2755. https://doi.org/10.3390/cancers15102755

Chicago/Turabian Style

Qiao, Zheng, Jingli Xu, Darrell R. Fisher, Rene Gonzalez, and Yubin Miao. 2023. "Introduction of a Polyethylene Glycol Linker Improves Uptake of 67Cu-NOTA-Conjugated Lactam-Cyclized Alpha-Melanocyte-Stimulating Hormone Peptide in Melanoma" Cancers 15, no. 10: 2755. https://doi.org/10.3390/cancers15102755

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