Design and Evaluation of New Tc-99m-Labeled Lactam Bridge-Cyclized Alpha-MSH Peptides for Melanoma Imaging

Abstract

The purpose of this study was to examine the melanoma targeting and imaging properties of new ^99mTc-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone (α-MSH) peptides using bifunctional chelating agents. MAG₃-GGNle-CycMSH_hex, AcCG₃-GGNle-CycMSH_hex and HYNIC-GGNle-CycMSH_hex peptides were synthesized and their melanocortin-1 (MC1) receptor binding affinities were determined in B16/F1 melanoma cells. The biodistribution of ^99mTc-MAG₃-GGNle-CycMSH_hex, ^99mTc-AcCG₃-GGNle-CycMSH_hex, ^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex and ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex were determined in B16/F1 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(EDDA)-HYNIC-GGNle-CycMSH_hex were further examined because of its high melanoma uptake and fast urinary clearance. The IC₅₀ values of MAG₃-GGNle-CycMSH_hex, AcCG₃-GGNle-CycMSH_hex and HYNIC-GGNle-CycMSH_hex were 1.0 ± 0.05, 1.2 ± 0.19 and 0.6 ± 0.04 nM in B16/F1 melanoma cells, respectively. Among these four ^99mTc-peptides, ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex exhibited the highest melanoma uptake (14.14 ± 4.90% ID/g) and fastest urinary clearance (91.26 ± 1.96% ID) at 2 h post-injection. ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex showed high tumor to normal organ uptake ratios except for the kidneys. The tumor/kidney uptake ratios of ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex were 2.50 and 3.55 at 4 and 24 h post-injection. The melanoma lesions were clearly visualized by SPECT/CT using ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex as an imaging probe at 2 h post-injection. Overall, high melanoma uptake coupled with fast urinary clearance of ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex highlighted its potential for metastatic melanoma detection in the future.

INTRODUCTION

Skin cancer is the most commonly diagnosed cancer in the United States. Approximately 3.5 millions skin cancers are diagnosed annually. Among the three major types of skin cancer (basal cell carcinoma, squamous cell carcinoma and melanoma), melanoma is the most deadly skin cancer with an increasing incidence rate.¹ Although melanoma only accounts for less than 5% of skin cancer cases, it results in greater than 75% of deaths from skin cancer. The cancer st11atistics from American Cancer Society predicted that 9,180 deaths of melanoma would occur in the United States in 2012.¹ Unfortunately, no curative treatment is available for metastatic melanoma. Early diagnosis followed by a prompt surgical removal offers patients the best opportunities for cures or prolonged survivals. Thus, it has been of great interest to develop receptor-targeting peptide radiopharmaceuticals for melanoma imaging and therapy.^2–19

We and others have reported radiolabeled lactam bridge-cyclized α-MSH peptides targeting melanocotin-1 (MC1) receptors for melanoma imaging over the past a few years.^20–29 We have developed two generations of α-MSH peptides cyclized either via a Lys-Asp or an Asp-Lys lactam bridge. The first-generation peptides were built on the backbone of CycMSH peptide {c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp}], whereas the second-generation peptides were designed based upon the construct of CycMSH_hex peptide {c[Asp-His-DPhe-Arg-Trp-Lys]-CONH₂}. DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) was attached to the CycMSH and CycMSH_hex peptides for ¹¹¹In labeling. Compared to the first-generation ¹¹¹In-labeled CycMSH peptides, the second-generation ¹¹¹In-labeled CycMSH_hex peptides exhibited enhanced melanoma uptake and decreased renal uptake.^20–25 Among the ¹¹¹In-labeled CycMSH_hex peptides, we identified ¹¹¹In-DOTA-GGNle-CycMSH_hex as a lead peptide because of its high melanoma uptake (19.05 ± 5.04 % ID/g at 2 h post-injection) and relatively low renal uptake (6.84 ± 0.92 % ID/g at 2 h post-injection) in B16/F1 melanoma-bearing C57 mice.²⁵ The melanoma lesions could be clearly visualized by single photon emission computed tomography (SPECT) using ¹¹¹In-DOTA-GGNle-CycMSH_hex as an imaging probe.²⁵

Despite the success of ¹¹¹In-DOTA-GGNle-CycMSH_hex for melanoma imaging, we managed to develop new ^99mTc-labeled CycMSH_hex peptides for melanoma imaging due to the wide utilization of ^99mTc as a diagnostic radionuclide in nuclear medicine. Technetium-99m is an ideal SPECT radionuclide because of its 140-keV γ-photon emission and a manageable 6 h half-life. Meanwhile, ^99mTc is very cost-effective and can be easily obtained from a commercial ⁹⁹Mo-^99mTc generator. High melanoma uptake of ¹¹¹In-DOTA-GGNle-CycMSH_hex underscored it as a lead peptide construct. Thus, we managed to develop new ^99mTc-labeled GGNle-CycMSH_hex peptides using different ^99mTc chelators for melanoma imaging in this study. Mercaptoacetyltriglycine (MAG₃) and Ac-Cys-Gly-Gly-Gly (AcCG₃) are N₃S chelators for ^99mTc, whereas hydrazinonicotinamide (HYNIC) can form ^99mTc complexes via either an IsoLink^™ carbonyl labeling agent or Ethylenediaminediacetic acid (EDDA)/Tricine solution. Therefore, we replaced the DOTA chelator with three different bifunctional chelating agents, namely MAG₃, AcCG₃ and HYNIC, to generate new MAG₃-GGNle-CycMSH_hex, AcCG₃-GGNle-CycMSH_hex and HYNIC-GGNle-CycMSH_hex peptides to examine which chelating agent was the best in retaining favorable melanoma targeting and pharmacokinetic properties. We determined their MC1 receptor binding affinities in B16/F1 melanoma cells first. Then, we examined the biodistribution of ^99mTc-MAG₃-GGNle-CycMSH_hex, ^99mTc-AcCG₃-GGNle-CycMSH_hex, ^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex and ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex at 2 h post-injection to select a lead ^99mTc-peptide for further evaluation. ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex displayed the highest melanoma uptake and fastest urinary clearance. Therefore, we further determined the biodistribution of ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex and its property for molecular imaging in B16/F1 melanoma-bearing C57 mice in this study.

EXPERIMENTAL SECTION

Chemicals and Reagents

Amino acids and resin were purchased from Advanced ChemTech Inc. (Louisville, KY) and Novabiochem (San Diego, CA). Boc-HYNIC was purchased from VWR International, Inc. (Albuquerque, NM). ¹²⁵I-Tyr²-[Nle⁴, D-Phe⁷]-α-MSH {¹²⁵I-(Tyr²)-NDP-MSH} was obtained from PerkinElmer, Inc. (Waltham, MA) for receptor binding assays. ^99mTcO₄⁻ was purchased from Cardinal Health (Albuquerque, NM) for peptide 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

MAG₃-GGNle-CycMSH_hex, AcCG₃-GGNle-CycMSH_hex and HYNIC-GGNle-CycMSH_hex were synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry, purified by reverse phase-high performance liquid chromatography (RP-HPLC) and characterized by liquid chromatography-mass spectrometry (LC-MS). Generally, 70 μmol of resin, 210 μmol of each Fmoc-protected amino acid and 210 μmol of Boc-HYNIC were used for the synthesis. Briefly, the intermediate scaffolds of Mercaptoacetyl(Trt)-Gly-Gly-Gly-Gly-Gly-Nle-Asp(O-2-PhiPr)-His(Trt)-DPhe-Arg(Pbf)-Trp(Boc)-Lys(Dde), A c-Cys-Gly-Gly-Gly-Gly-Gly-Nle-Asp(O-2-PhiPr)-His(Trt)-DPhe-Arg(Pbf)-Trp(Boc)-Lys(Dde) and HYNIC(Boc)-Gly-Gly-Nle-Asp(O-2-PhiPr)-His(Trt)-DPhe-Arg(Pbf)-Trp(Boc)-Lys(Dde) were synthesized on H₂N-Sieber amide resin using an Advanced ChemTech multiple-peptide synthesizer (Louisville, KY). The protecting group of Dde was removed by 2% hydrazine for peptide cyclization. The protecting group of 2-phenylisopropyl was removed and the protected peptide was cleaved from the resin by treating with a mixture of 2.5% of trifluoroacetic acid (TFA) and 5% of triisopropylsilane. Each protected peptide was cyclized by coupling the carboxylic group from the Asp with the epsilon amino group from the Lys. The cyclization reaction was achieved by an overnight reaction in N,N-dimethylformamide (DMF) using benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium-hexafluorophosphate (PyBOP) as a coupling agent in the presence of N,N-diisopropylethylamine (DIPEA). Each protected cyclic peptide was dissolved in H₂O/CH₃CN (50:50) and lyophilized to remove the reagents. The protecting groups were totally removed by treating with a mixture of TFA, thioanisole, phenol, water, ethanedithiol and triisopropylsilane (87.5:2.5:2.5:2.5:2.5:2.5) for 2 h at room temperature (25 °C). Each peptide was precipitated and washed with ice-cold ether four times, purified by RP-HPLC and characterized by LC-MS. The MC1 receptor binding affinities (IC₅₀ values) of MAG₃-GGNle-CycMSH_hex, AcCG₃-GGNle-CycMSH_hex and HYNIC-GGNle-CycMSH_hex were determined in B16/F1 melanoma cells by in vitro competitive receptor binding assays according to our published procedure.²⁵ The study was carried out in triplicate, the final IC₅₀ value was calculated by averaging two experiments.

Peptide Radiolabeling with ^99mTc

^99mTc-MAG₃-GGNle-CycMSH_hex and ^99mTc-AcCG₃-GGNle-CycMSH_hex were prepared according to the published procedure³⁰ with modifications. Briefly, 10 μL of 1 mg/mL MAG₃-GGNle-CycMSH_hex or AcCG₃-GGNle-CycMSH_hex aqueous solution, 10 μL of pH 9.2 tartrate buffer (50 μg/μL Na₂tartrate·2H₂O, 0.5 M Na₂HCO₃, 0.25 M NH₄OAc, 0.175 M NH₃), 3 μL of freshly prepared tin chloride solution (10 μg/μL SnCl₂·2H₂O, 1 μg/μL sodium ascorbate, 10 mM HCl), and 50 μL of ^99mTcO₄⁻ solution (37–74 MBq) were added into a reaction vial and incubated at 100 °C for 20 min. Each radiolabeled peptide was purified to a single species by Waters RP-HPLC (Milford, MA) on a Grace Vydac C-18 reverse phase analytical column (Deerfield, IL) using a 20-min gradient of 22–32% acetonitrile in 20 mM HCl aqueous solution 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 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. Each purified peptide was purged with N₂ gas for 20 min to remove the acetonitrile. The pH of the final solution was adjusted to 5 with 0.1 N NaOH and normal saline for animal studies.

^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex was prepared using an IsoLink^™ carbonyl labeling agent (DRN4335; Mallinckrodt). First, 1 mL of ^99mTcO₄⁻ (185 MBq) solution was added into an IsoLink^™ kit and incubated at 100°C for 20 min to yield [^99mTc(CO)₃(OH₂)₃]⁺. Second, 200 μL of the [^99mTc(CO)₃(OH₂)₃]⁺ preparation and 10 μL of 1 mg/mL HYNIC-GGNle-CycMSH_hex aqueous solution were added into 200 μL of 0.5 M NH₄OAc buffer (pH 5.44) in a reaction vial and incubated at 75°C for 30 min. The radiolabeled peptide was purified to a single species by Waters RP-HPLC on a Grace Vydac C-18 reverse phase analytical column using a 20-min gradient of 24–34% acetonitrile in 20 mM HCl aqueous solution at a flow rate of 1 mL/min. The purified peptide was purged with N₂ gas for 20 min to remove the acetonitrile. The pH of the final solution was adjusted to 5 with 0.1 N NaOH and normal saline for animal studies.

^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex was prepared according to the published procedure³¹ with modifications. Briefly, 50 μL of ^99mTcO₄⁻ (37–74 MBq), 10 μL of 1 mg/mL SnCl₂ in 0.1 N HCl solution, 200 μL of a mixture of 5 mg/mL of EDDA and 25 mg/mL of Tricine aqueous solution and 10 μL of 1 mg/mL HYNIC-GGNle-CycMSH_hex aqueous solution were added into 400 μL of 0.5 M NH₄OAc (pH 5.44) in a reaction vial and incubated at 95°C for 30 min. The radiolabeled peptide was purified to a single species using a Waters RP-HPLC on a Grace Vydac C-18 reverse phase analytical column using a 20-min gradient of 18–28% acetonitrile in 20 mM HCl aqueous solution at a flow rate of 1 mL/min. The purified peptide was purged with N₂ gas for 20 min to remove the acetonitrile. The pH of the final solution was adjusted to 5 with 0.1 N NaOH and normal saline for animal studies.

Biodistribution Studies

All animal studies were conducted in compliance with Institutional Animal Care and Use Committee approval. In an attempt to select a lead ^99mTc-peptide for further evaluation, the biodistribution of ^99mTc-MAG₃-GGNle-CycMSH_hex, ^99mTc-AcCG₃-GGNle-CycMSH_hex, ^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex and ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex were examined in B16/F1 melanoma-bearing C57 female mice (Harlan, Indianapolis, IN) at 2 h post-injection, respectively. 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 ^99mTc-MAG₃-GGNle-CycMSH_hex, ^99mTc-AcCG₃-GGNle-CycMSH_hex, ^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex or ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex via the tail vein. Groups of 5 mice were sacrificed at 2 h post-injection, tumor and organs of interest were harvested, weighed and counted in a Wallace 1480 automated gamma counter (PerkinElmer). Meanwhile, intestines and urine were collected and counted to evaluate the clearance pathway of each ^99mTc-labeled peptide. Blood was taken as 6.5% of the body weight.

^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex displayed higher melanoma uptake and faster urinary clearance than the other three ^99mTc-peptides. Therefore, its biodistribution properties were further determined in B16/F1 melanoma-bearing C57 female mice at 0.5, 4 and 24 h post-injection. The B16/F1 melanoma-bearing mice were generated as described above. Each melanoma-bearing mouse was injected with 0.037 MBq of ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex via the tail vein. Groups of 5 mice were sacrificed at 0.5, 4 and 24 h post-injection, and tumors and organs of interest were harvested, weighed and counted. Blood was taken as 6.5% of the body weight. The tumor uptake specificity of ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex was determined by co-injecting 10 μg (6.07 nmol) of unlabeled NDP-MSH peptide at 2 h post-injection. To examine whether L-lysine co-injection could decrease the renal uptake, a group of 5 mice was injected with a mixture of 12 mg of L-lysine and 0.037 MBq of ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex. The mice were sacrificed at 2 h post-injection and the tumors and organs of interest were harvested, weighed and counted.

Melanoma Imaging and Urinary Metabolites of ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex

Approximately 7.4 MBq of ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex was injected into a B16/F1 melanoma-bearing C57 mouse for melanoma imaging and urine metabolites analysis. The mouse was euthanized at 2 h post-injection for small animal SPECT/CT (Nano-SPECT/CT^®, Bioscan) imaging, as well as to collect urine for analyzing the metabolites. The 9-min CT imaging was immediately followed by the whole-body SPECT scan. The SPECT scans of 24 projections were acquired. Reconstructed SPECT and CT data were visualized and co-registered using InVivoScope (Bioscan, Washington DC). The collected urine sample was centrifuged at 16,000 g for 5 min before the HPLC analysis. Thereafter, aliquots of the urine were injected into the HPLC. A 20-minute gradient of 18–28% acetonitrile/20 mM HCl with a flow rate of 1 mL/min was used for urine analysis.

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 tumor and renal uptake of ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex with/without NDP-MSH co-injection, as well as the significance of difference in tumor and renal uptake of ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex with/without L-lysine co-injection in the biodistribution studies described above. The differences at the 95% confidence level (p<0.05) were considered significant.

RESULTS

New MAG₃-GGNle-CycMSH_hex, AcCG₃-GGNle-CycMSH_hex and HYNIC-GGNle-CycMSH_hex were synthesized and purified by RP-HPLC. The overall synthetic yields were 25–30% for MAG₃-GGNle-CycMSH_hex, AcCG₃-GGNle-CycMSH_hex and HYNIC-GGNle-CycMSH_hex. All three peptides displayed greater than 90% purity after HPLC purification. The identities of MAG₃-GGNle-CycMSH_hex, AcCG₃-GGNle-CycMSH_hex and HYNIC-GGNle-CycMSH_hex were confirmed by electrospray ionization mass spectrometry. The calculated and measured molecular weights of MAG₃-GGNle-CycMSH_hex, AcCG₃-GGNle-CycMSH_hex and HYNIC-GGNle-CycMSH_hex are presented in Table 1. The measured molecular weights matched the calculated molecular weights. The schematic structures of MAG₃-GGNle-CycMSH_hex, AcCG₃-GGNle-CycMSH_hex and HYNIC-GGNle-CycMSH_hex are shown in Figure 1. The IC₅₀ values of MAG₃-GGNle-CycMSH_hex, AcCG₃-GGNle-CycMSH_hex and HYNIC-GGNle-CycMSH_hex were 1.0 ± 0.05, 1.2 ± 0.19 and 0.6 ± 0.04 nM in B16/F1 melanoma cells, respectively (Table 1).

Table 1.

IC₅₀ values and molecular weights (MWs) of MAG₃-GGNle-CycMSH_hex, AcCG₃-GGNle-CycMSH_hex and HYNIC-GGNle-CycMSH_hex.

Peptide	IC50 (nM)	Calculated MW	Measured MW
MAG3-GGNle-CycMSHhex	1.0 ± 0.05	1340.6	1341.0
AcCG3-GGNle-CycMSHhex	1.2 ± 0.19	1411.6	1412.1
HYNIC-GGNle-CycMSHhex	0.6 ± 0.04	1231.0	1230.8

Figure 1.

Schematic structures of MAG₃-GGNle-CycMSH_hex (A), AcCG₃-GGNle-CycMSH_hex (B) and HYNIC-GGNle-CycMSH_hex (C).

Both MAG₃-GGNle-CycMSH_hex and AcCG₃-GGNle-CycMSH_hex were readily labeled with ^99mTc with greater than 95% radiolabeling yields in tartrate buffer using SnCl₂ as a reducing agent. ^99mTc-MAG₃-GGNle-CycMSH_hex and ^99mTc-AcCG₃-GGNle-CycMSH_hex were completely separated from their excess non-labeled peptides by RP-HPLC. The retention times of ^99mTc-MAG₃-GGNle-CycMSH_hex and ^99mTc-AcCG₃-GGNle-CycMSH_hex were 14.9 and 13.0 min, whereas the retention times of MAG₃-GGNle-CycMSH_hex and AcCG₃-GGNle-CycMSH_hex were 12.0 and 12.0 min, respectively. Both ^99mTc-MAG₃-GGNle-CycMSH_hex and ^99mTc-AcCG₃-GGNle-CycMSH_hex showed greater than 98% radiochemical purities after HPLC purification. HYNIC-GGNle-CycMSH_hex was radiolabeled with ^99mTc either via an IsoLink^™ carbonyl labeling agent or EDDA/Tricine solution with greater than 95% radiolabeling yields. ^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex and ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex were completely separated from their excess non-labeled peptides by RP-HPLC. The retention times of ^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex and ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex were 18.0 and 17.9 min, whereas the retention time of HYNIC-GGNle-CycMSH_hex was 15.5 min. Both ^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex and ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex displayed greater than 98% radiochemical purities after HPLC purification.

The melanoma targeting and pharmacokinetic properties of ^99mTc-MAG₃-GGNle-CycMSH_hex, ^99mTc-AcCG₃-GGNle-CycMSH_hex, ^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex and ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex were determined in B16/F1 melanoma-bearing mice at 2 h post-injection to select a lead radiolabeled peptide for further evaluation. The biodistribution results of these four ^99mTc-conjugates are shown in Table 2. Although ^99mTc-MAG₃-GGNle-CycMSH_hex and ^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex displayed substantial tumor uptake of 4.64 ± 1.06% ID/g and 5.84 ± 1.26% ID/g at 2 h post-injection, ^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex showed higher liver uptake (38.11 ± 2.31 vs. 1.18 ± 0.15% ID/g) and renal uptake (17.69 ± 4.06 vs. 1.20 ± 0.51% ID/g) than ^99mTc-MAG₃-GGNle-CycMSH_hex at 2 h post-injection. Meanwhile, ^99mTc-AcCG₃-GGNle-CycMSH_hex displayed improved melanoma uptake of 9.76 ± 0.51% ID/g coupled with low liver and renal uptake (1.69 ± 0.35 and 2.62 ± 0.45% ID/g, respectively) at 2 h post-injection. ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex exhibited the highest tumor uptake (14.14 ± 4.90% ID/g) among these four ^99mTc-conjugates at 2 h post-injection in B16/F1 melanoma-bearing C57 mice. Interestingly, ^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex showed the highest renal and liver uptake, as well as the highest accumulation in blood, heart, lung, spleen, stomach, bone and skin at 2 h post-injection among these four ^99mTc-conjugates. Compared to ^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex, both ^99mTc-MAG₃-GGNle-CycMSH_hex and ^99mTc-AcCG₃-GGNle-CycMSH_hex displayed much lower normal organ accumulation at 2 h post-injection. The uptake of ^99mTc-MAG₃-GGNle-CycMSH_hex or ^99mTc-AcCG₃-GGNle-CycMSH_hex was lower than 3.4% ID/g in stomach, liver and kidneys at 2 h post-injection. However, only 27.53 ± 6.99% ID of ^99mTc-MAG₃-GGNle-CycMSH_hex and 38.20 ± 3.98% ID of ^99mTc-AcCG₃-GGNle-CycMSH_hex were excreted through the urinary system, while 55.02 ± 10.44% ID of ^99mTc-MAG₃-GGNle-CycMSH_hex and 50.91 ± 2.72% ID of ^99mTc-AcCG₃-GGNle-CycMSH_hex were excreted through the GI system. On the other hand, ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex exhibited the highest tumor uptake (14.14 ± 4.90% ID/g) and the fastest urinary clearance (91.26 ± 1.96% ID) at 2 h post-injection. Thus, we selected ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex as a lead peptide to further examine its full biodistribution and melanoma imaging properties.

Table 2.

Biodistribution comparison among ^99mTc-MAG₃-GGNle-CycMSH_hex {MAG₃}, ^99mTc-AcCG₃-GGNle-CycMSH_hex {AcCG₃}, ^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex {(CO)₃} and ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex {EDDA} in B16/F1 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=5)

Tissues	MAG3	AcCG3	(CO)3	EDDA
	Percent injected dose/gram (%ID/g)
Tumor	4.64±1.06	9.76±0.51	5.84±1.26	14.14±4.90
Brain	0.05±0.05	0.04±0.03	0.12±0.02	0.05±0.04
Blood	0.25±0.13	0.27±0.13	2.17±1.91	0.17±0.05
Heart	0.33±0.13	0.27±0.14	1.94±0.22	0.09±0.03
Lung	0.46±0.10	0.47±0.09	4.42±1.62	0.41±0.06
Liver	1.18±0.15	1.69±0.35	38.11±2.31	0.52±0.05
Spleen	0.30±0.12	0.35±0.10	4.70±1.13	0.18±0.08
Stomach	3.38±0.78	2.86±1.80	4.01±1.78	1.02±0.31
Kidneys	1.20±0.51	2.62±0.45	17.69±4.06	7.52±0.96
Muscle	0.08±0.02	0.12±0.09	0.17±0.07	0.04±0.02
Pancreas	0.41±0.54	0.32±0.16	1.02±0.55	0.04±0.04
Bone	0.29±0.13	0.50±0.28	1.91±0.52	0.12±0.05
Skin	0.28±0.10	0.20±0.07	1.75±0.62	0.33±0.10
	Percent injected dose (%ID)
Intestines	55.02±10.44	50.91±2.72	18.35±5.49	1.09±0.06
Urine	27.53±6.99	38.20±3.98	27.65±2.53	91.26±1.96

The full biodistribution results of ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex are presented in Table 3. ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex displayed high tumor uptake and prolonged tumor retention in B16/F1 melanoma-bearing C57 mice. The tumor uptake was 13.75 ± 3.49% ID/g at 30 min post-injection and reached its peak value of 14.14 ± 4.90% ID/g at 2 h post-injection. Compared with the tumor uptake at 2 h post-injection, 93.6% of the radioactivity remained in the tumor at 4 h post-injection. In the melanoma uptake blocking study, co-injection of NDP-MSH blocked 96.8% of the tumor uptake (p<0.05) at 2 h post-injection, demonstrating that the tumor uptake was MC1 receptor-medicated. Normal organ uptake of ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex was lower than 1.02% ID/g in normal tissues except for kidneys at 2, 4 and 24 h post-injection. High tumor/blood and high tumor/normal organ uptake ratios were achieved as early as 0.5 h post-injection. As the major excretion pathway of ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex, the kidney uptake was 11.21 ± 1.68% ID/g at 0.5 h post-injection and decreased to 1.28 ± 0.28% ID/g at 24 h post-injection. The tumor/kidney uptake ratios of ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex were 1.88, 2.50 and 3.55 at 2, 4 and 24 h post-injection. Co-injection of NDP-MSH didn’t reduce the renal uptake of the ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex activity at 2 h post-injection, indicating that the renal uptake was not MC1 receptor-mediated. Meanwhile, L-lysine co-injection decreased 42% of the renal uptake (p<0.05) without affecting the tumor uptake. Moreover, ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex exhibited rapid urinary excretion. Approximately 91% of the activity cleared out of the body at 2 h post-injection. The representative whole-body, coronal and transversal SPECT/CT images are presented in Figure 2. The melanoma lesions were clearly visualized by SPECT/CT using ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex as an imaging probe at 2 h post-injection. ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex exhibited high tumor to normal organ uptake ratios except for the kidneys, which was consistent with the biodistribution results. Urinary metabolites of ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex were analyzed by RP-HPLC 2 h post-injection. The radioactive HPLC profile of urine is illustrated in Figure 3. The urine analysis suggested that ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex remained intact in urine at 2 h post-injection.

Table 3.

Biodistribution of ^99mTc(EDDA)-HYNIC-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)

Tissues	0.5 h	2 h#	4 h	24 h	2-h NDP blockade	2-h L-Lys co-injection
	Percent injected dose/gram (%ID/g)
Tumor	13.75±3.49	14.14±4.90	13.23±2.35	4.54±0.70	0.45±0.29*	14.10±2.90
Brain	0.10±0.02	0.05±0.04	0.03±0.04	0.01±0.01	0.01±0.01	0.03±0.02
Blood	2.62±0.09	0.17±0.05	0.12±0.08	0.03±0.01	0.24±0.07	0.09±0.05
Heart	0.84±0.31	0.09±0.03	0.05±0.04	0.02±0.02	0.10±0.05	0.1±0.05
Lung	2.73±0.76	0.41±0.06	0.24±0.04	0.09±0.01	0.34±0.11	0.17±0.02
Liver	1.15±0.08	0.52±0.05	0.60±0.19	0.16±0.02	0.52±0.07	0.35±0.05
Spleen	0.99±0.10	0.18±0.08	0.20±0.18	0.05±0.03	0.13±0.09	0.09±0.06
Stomach	2.23±0.52	1.02±0.31	0.52±0.24	0.06±0.03	0.49±0.22	0.63±0.44
Kidneys	11.21±1.68	7.52±0.96	5.29±1.84	1.28±0.28	7.67±1.99	4.37±0.36*
Muscle	0.27±0.05	0.04±0.02	0.04±0.02	0.03±0.01	0.03±0.02	0.06±0.05
Pancreas	0.50±0.21	0.04±0.04	0.07±0.03	0.03±0.02	0.05±0.04	0.13±0.04
Bone	0.76±0.10	0.12±0.05	0.09±0.01	0.09±0.03	0.09±0.10	0.13±0.08
Skin	2.94±0.14	0.33±0.10	0.33±0.12	0.13±0.02	0.34±0.13	0.23±0.03
	Percent injected dose (%ID)
Intestines	1.41±0.14	1.09±0.06	1.88±1.06	0.24±0.17	0.73±0.21	0.51±0.15
Urine	73.09±2.72	91.26±1.96	92.29±1.73	97.93±0.28	94.90±1.39	94.96±1.54
	Uptake ratio of tumor/normal tissue
Tumor/liver	11.96	27.19	22.05	28.38	0.87	40.29
Tumor/kidney	1.23	1.88	2.50	3.55	0.06	3.23
Tumor/lung	5.03	34.49	55.13	50.44	1.32	82.94
Tumor/muscle	50.93	353.50	330.75	151.33	15.0	235.0
Tumor/blood	5.26	83.18	110.25	151.33	1.88	156.67
Tumor/skin	4.67	42.85	40.09	34.92	1.32	61.30

^#2 h Data were cited from Table 2 for comparison;

^*p<0.05 for determining the significance of differences in tumor and kidney uptake between ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex with or without NDP-MSH peptide blockade, as well as with or without L-lysine co-injection at 2 h post-injection.

Figure 2.

Representative whole-body (A), coronal (B) and transversal (C) SPECT/CT images of ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex in a B16/F1 melanoma-bearing C57 mouse at 2 h post-injection. The tumor lesions (T) were highlighted with arrows on the images.

Figure 3.

Radioactive HPLC profile of urine sample of a B16/F1 melanoma-bearing C57 mouse at 2 h post-injection of ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex. The arrow indicates the retention time (17.9 min) of the original compound of ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex prior to the tail vein injection.

DISCUSSION

It has been of interest to develop ^99mTc-labeled α-MSH peptides targeting the MC1 receptors for melanoma imaging. Both linear and cyclic ^99mTc-labeled α-MSH peptides were reported for melanoma targeting. Specifically, bifunctional chelator Ac-Cys-Gly-Cys-Gly- (for ^99mTc radiolabeling) was coupled to the N-terminus of the linear NDP-MSH peptide to generate ^99mTc-CGCG-NDPMSH.^{32 99m}Tc-CGCG-NDPMSH displayed 6.52 ± 1.11% ID/g of tumor uptake in B16/F1 melanoma-bearing C57 mice at 30 min post-injection. However, ^99mTc-CGCG-NDPMSH showed a rapid tumor washout. The tumor uptake of ^99mTc-CGCG-NDPMSH dramatically decreased to 0.56 ± 0.14% ID/g at 4 h post-injection. Meanwhile, ^99mTc-CGCG-NDPMSH exhibited high liver uptake (6.80 ± 1.22 and 4.58 ± 1.02% ID/g) at 30 min and 1 h post-injection. Another bifunctional chelator MAG₂ (tetrafluorophenyl mercaptoacetylglycylglycyl-gamma-aminobutyrate) was coupled to the epsilon amino group of Lys¹¹ to yield ^99mTc-MAG₂-NDPMSH. Compared to ^99mTc-CGCG-NDPMSH, ^99mTc-MAG₂-NDPMSH showed lower tumor uptake (4.17 ± 1.34 and 2.39 ± 1.21% ID/g at 30 min and 1 h post-injection) in B16/F1 melanoma-bearing C57 mice.³²

It was an interesting approach to introduce three cycteines at the 3^rd, 4^th and 10^th position of CCMSH peptide for preparing ^99mTc-CCMSH. Three Cys^3,4,10 sulfhydryls and one Cys⁴ amide nitrogen in the CCMSH peptide provided a site-specific chelator for complexing ^99mTc.^2,3,33 Importantly, ^99mTc-CCMSH exhibited dramatically enhanced tumor uptake and prolonged tumor retention compared to ^99mTc-CGCG-NDPMSH in B16/F1 melanoma-bearing C57 mice. The tumor uptake of ^99mTc-CCMSH was 12.97 ± 1.38 and 11.64 ± 1.54% ID/g at 30 min and 1 h post-injection, respectively. The tumor uptake of ^99mTc-CCMSH was 9.51 ± 1.97% ID/g at 4 h post-injection, which was 16.98 times the tumor uptake of ^99mTc-CGCG-NDPMSH at 4 h post-injection.^3,32 Furthermore, the substation of Lys¹¹ with Arg¹¹ generated ^99mTc-(Arg¹¹)CCMSH, which exhibited enhanced tumor uptake and dramatically reduced renal uptake in B16/F1 melanoma-bearing C57 mice.¹⁸ The tumor uptake of ^99mTc-(Arg¹¹)CCMSH was 1.21 and 1.17 times the tumor uptake of ^99mTc-CCMSH at 1 and 4 h post-injection, whereas the renal uptake of ^99mTc-(Arg¹¹)CCMSH was only 59.2% and 37.9% of the renal uptake of ^99mTc-CCMSH at 1 and 4 h post-injection.^3,18 High tumor uptake and reduced renal uptake highlighted ^99mTc-(Arg¹¹)CCMSH as a lead metal-cyclized imaging probe for melanoma detection.

A lactam bridge-cyclized α-MSH peptide was radiolabeled with the ^99mTc(CO)₃ core to generate ^99mTc(CO)₃-pz-βAla-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH₂.²⁶ Not surprisingly, ^99mTc(CO)₃-pz-βAla-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH₂ exhibited high tumor uptake and prolonged tumor retention. The tumor uptake of ^99mTc(CO)₃-pz-βAla-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH₂ was 9.26 ± 0.83 and 11.31 ± 1.83% ID/g at 1 h and 4 post-injection, respectively. Unfortunately, ^99mTc(CO)₃-pz-βAla-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH₂ showed extremely high renal and liver uptake at 1 and 4 h post-injection. The renal uptake of ^99mTc(CO)₃-pz-βAla-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH₂ was 71.06 ± 6.44% and 32.12 ± 1.57% ID/g at 1 and 4 h post-injection, whereas the liver uptake of ^99mTc(CO)₃-pz-βAla-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH₂ was 42.19 ± 5.05 and 22.86 ± 1.17% ID/g at 1 and 4 h post-injection.²⁶

In this study, we developed new ^99mTc-labeled lactam bridge-cyclized α-MSH peptides building upon the GGNle-CycMSH_hex peptide construct we identified.²⁵ Specifically, we coupled three bifunctional chelating agents, namely MAG₃, AcCG₃ and HYNIC, to GGNle-CycMSH_hex to generate new MAG₃-GGNle-CycMSH_hex, AcCG₃-GGNle-CycMSH_hex and HYNIC-GGNle-CycMSH_hex peptides. The coupling of MAG₃, AcCG₃ and HYNIC to GGNle-CycMSH_hex retained low nanomolar MC1 receptor binding affinities of the peptides. The IC₅₀ was 1.0 ± 0.05 nM for MAG₃-GGNle-CycMSH_hex, 1.2 ± 0.19 nM for AcCG₃-GGNle-CycMSH_hex and 0.6 ± 0.04 nM HYNIC-GGNle-CycMSH_hex (Table 1), respectively. It is worthwhile to note that both MAG₃ and AcCG₃ provide N₃S chelators for ^99mTc conjugation, whereas the HYNIC chelator allows ^99mTc conjugation via either the ^99mTc(CO)₃ or ^99mTc(EDDA) core. Therefore, we prepared ^99mTc-MAG₃-GGNle-CycMSH_hex, ^99mTc-AcCG₃-GGNle-CycMSH_hex, ^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex and ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex conjugates in this study. Furthermore, we determined their biodistribution properties in B16/F1 melanoma-bearing C57 mice at 2 h post-injection to examine which bifunctional chelator was the best in retaining favorable melanoma targeting and pharmacokinetic properties. Despite the slight difference in receptor binding affinity among MAG₃-GGNle-CycMSH_hex, AcCG₃-GGNle-CycMSH_hex and HYNIC-GGNle-CycMSH_hex, we observed dramatic differences in melanoma uptake, accumulation in non-target tissues, and excretion pathways among these four ^99mTc-conjugates. As shown in Table 2, ^99mTc-MAG₃-GGNle-CycMSH_hex and ^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex showed substantial tumor uptake, whereas ^99mTc-AcCG₃-GGNle-CycMSH_hex displayed enhanced melanoma uptake. ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex exhibited the highest tumor uptake among these four ^99mTc-peptides at 2 h post-injection in B16/F1 melanoma-bearing C57 mice. It was worthwhile to note that ^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex exhibited high liver and renal uptake, as well as higher accumulation in the normal organs than the other three ^99mTc-peptides at 2 h post-injection. It was likely that ^99mTc was demetallated from the peptide in vivo. Obviously, the disadvantage associated with ^99mTc-MAG₃-GGNle-CycMSH_hex and ^99mTc-AcCG₃-GGNle-CycMSH_hex was the accumulation in the GI system. Approximately 55.02 ± 10.44% ID of ^99mTc-MAG₃-GGNle-CycMSH_hex and 50.91 ± 2.72% ID of ^99mTc-AcCG₃-GGNle-CycMSH_hex were excreted through the GI system. On the other hand, ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex exhibited the fastest urinary clearance (91.26 ± 1.96% ID) at 2 h post-injection. Thus, we further examined the full biodistribution and melanoma imaging properties of ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex.

As shown in Table 3, ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex displayed high tumor uptake and prolonged tumor retention in B16/F1 melanoma-bearing C57 mice. The tumor uptake reached its peak value of 14.14 ± 4.90% ID/g at 2 h post-injection, with 93.6% of the radioactivity remaining in tumor at 4 h post-injection. Co-injection of NDP-MSH blocked 96.8% of tumor uptake (p<0.05) without affecting the renal uptake at 2 h post-injection, demonstrating that the tumor uptake was MC1 receptor-medicated and the renal uptake was non-specific. Meanwhile, L-lysine co-injection decreased 42% of the renal uptake (p<0.05) without affecting the tumor uptake, indicating that the overall positive charge of the ^99mTc-peptide contributed to the non-specific renal uptake. It was worthwhile to note that there was a positively-charged side chain in Arg⁸, which is critical for MC1 receptor binding. ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex exhibited low accumulation in normal organs and a rapid urinary excretion, resulting in high tumor to normal organ uptake ratios. As we anticipated, the B16/F1 melanoma lesions were clearly visualized by SPECT/CT using ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex as an imaging probe.

At the present time, ^99mTc-(Arg¹¹)CCMSH displayed more favorable melanoma targeting and pharmacokinetic properties than ^99mTc(CO)₃-pz-βAla-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH₂ and ^99mTc-CGCG-NDPMSH. Although ^99mTc(CO)₃-pz-βAla-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH₂ showed similar high tumor uptake (11.31 ± 1.81% ID/g) with ^99mTc-(Arg¹¹)CCMSH, ^99mTc(CO)₃-pz-βAla-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH₂ displayed high accumulation and prolonged retention in both liver (22.86 ± 1.17% ID/g) and kidneys (32.12 ± 1.57% ID/g) at 4 h post-injection. High liver and renal uptake of ^99mTc(CO)₃-pz-βAla-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH₂ would limit its potential application in metastatic melanoma imaging. Remarkably, ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex exhibited comparably high melanoma uptake (13.23 ± 2.35% ID/g) to ^99mTc-(Arg¹¹)CCMSH at 4 h post-injection. Importantly, as shown in Figure 4, ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex displayed faster urinary clearance than ^99mTc-(Arg¹¹)CCMSH (92.3% ID vs. 83.8% ID) and much lower intestinal accumulation than ^99mTc-(Arg¹¹)CCMSH (1.9% ID vs. 11.5% ID) at 4 h post-injection. The differences in intestinal accumulation and urinary clearance were likely due to the structural differences between ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex and ^99mTc-(Arg¹¹)CCMSH.

Figure 4.

Comparisons in tumor uptake (A), urine excretion (B) and intestine accumulation (C) at 4 h post-injection among ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex (blue), ^99mTc-(Arg¹¹)CCMSH (pink), ^99mTc(CO)₃-Pz-βAla-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]NH₂ (green) and ^99mTc-CGCG-NDPMSH (red). Data of ^99mTc-(Arg¹¹)CCMSH, ^99mTc(CO)₃-Pz-βAla-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]NH₂ and ^99mTc-CGCG-NDPMSH were cited from references 18, 26 and 32 for comparison.

In conclusion, the biodistribution of ^99mTc-MAG₃-GGNle-CycMSH_hex, ^99mTc-AcCG₃-GGNle-CycMSH_hex, ^99mTc(CO)₃-HYNIC-GGNle-CycMSH_hex and ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex were determined in B16/F1 melanoma-bearing C57 mice in this study. Among these four ^99mTc-peptides, ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex exhibited the highest melanoma uptake and fastest urinary clearance at 2 h post-injection. Overall, the properties of high melanoma uptake, low accumulation in intestines and fast urinary clearance of ^99mTc(EDDA)-HYNIC-GGNle-CycMSH_hex highlighted its potential as an imaging probe for metastatic melanoma detection in the future.