Research ArticleBasic Science Investigation

Effects of the Amino Acid Linkers on the Melanoma-Targeting and Pharmacokinetic Properties of 111In-Labeled Lactam Bridge–Cyclized α-MSH Peptides

Haixun Guo, Jianquan Yang, Fabio Gallazzi and Yubin Miao
Journal of Nuclear Medicine April 2011, 52 (4) 608-616; DOI: https://doi.org/10.2967/jnumed.110.086009

Abstract

The purpose of this study was to examine the profound effects of the amino acid linkers on the melanoma-targeting and pharmacokinetic properties of 111In-labeled lactam bridge–cyclized DOTA-[X]-CycMSHhex {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-[X]-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2; X = GGNle, GENle, or NleGE; GG = -Gly-Gly- and GE = -Gly-Glu-} peptides. Methods: Three novel peptides (DOTA-GGNle-CycMSHhex, DOTA-GENle-CycMSHhex, and DOTA-NleGE-CycMSHhex) were designed and synthesized. The melanocortin-1 (MC1) receptor–binding affinities of the peptides were determined in B16/F1 melanoma cells. The melanoma-targeting and pharmacokinetic properties of 111In-DOTA-GGNle-CycMSHhex and 111In-DOTA-GENle-CycMSHhex were determined in B16/F1 melanoma–bearing C57 mice. Results: DOTA-GGNle-CycMSHhex and DOTA-GENle-CycMSHhex displayed 2.1 and 11.5 nM MC1 receptor–binding affinities, whereas DOTA-NleGE-CycMSHhex showed 873.4 nM MC1 receptor–binding affinity. The introduction of the -GG- linker maintained high melanoma uptake while decreasing kidney and liver uptake of 111In-DOTA-GGNle-CycMSHhex. The tumor uptake of 111In-DOTA-GGNle-CycMSHhex was 19.05 ± 5.04 and 18.6 ± 3.56 percentage injected dose per gram at 2 and 4 h after injection, respectively. 111In-DOTA-GGNle-CycMSHhex exhibited 28%, 32%, and 42% less kidney uptake than 111In-DOTA-Nle-CycMSHhex we reported previously, and 61%, 65%, and 68% less liver uptake than 111In-DOTA-Nle-CycMSHhex at 2, 4, and 24 h after injection, respectively. Conclusion: The amino acid linkers exhibited profound effects on the melanoma-targeting and pharmacokinetic properties of the 111In-labeled lactam bridge–cyclized α-melanocyte–stimulating hormone peptides. Introduction of the -GG- linker maintained high melanoma uptake while reducing kidney and liver uptake of 111In-DOTA-GGNle-CycMSHhex, highlighting its potential as an effective imaging probe for melanoma detection, as well as a therapeutic peptide for melanoma treatment when labeled with a therapeutic radionuclide.

Over the last decade, both radiolabeled linear and cyclized α-melanocyte–stimulating hormone (α-MSH) peptides have been designed to target G protein–coupled melanocortin-1 (MC1) receptors (1–5) for melanoma radioimaging and radiotherapy (6–20). Because of the stabilization of secondary structures (i.e., β-turns), the cyclic peptides possess less conformational freedom and higher stability than the linear peptides. Furthermore, the stabilization of secondary structures makes the cyclic peptides better fit the receptor-binding pocket, thus enhancing their receptor-binding affinities. Presently, disulfide bond, metal, and lactam bridge have been successfully used to cyclize the radiolabeled α-MSH peptides (9–13,15–20). Among these cyclization strategies, metal and lactam bridge cyclization resulted in greater tumor uptake and lower kidney uptake of the radiolabeled α-MSH peptides than the disulfide bridge cyclization (12,13,15–20).

We have successfully developed a class of 111In-labeled lactam bridge–cyclized DOTA-conjugated α-MSH peptides for primary and metastatic melanoma detection (15–19). Initially, a Lys-Asp lactam bridge was used to cyclize the MC1 receptor–binding motif (His-dPhe-Arg-Trp) to yield a 12–amino acid cyclic α-MSH peptide {CycMSH: c[Lys-Nle-Glu-His-dPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]}. DOTA was conjugated to the N terminus of the CycMSH with or without an amino acid linker (-Gly-Glu- [GE]) for radiolabeling. 111In-DOTA-GlyGlu-CycMSH displayed high melanoma uptake (10.40 ± 1.40 percentage injected dose [%ID]/g at 2 h after injection) in B16/F1 melanoma–bearing C57 mice (15). When 111In-DOTA-GlyGlu-CycMSH was used as an imaging probe, both flank primary and pulmonary metastatic melanoma lesions could be clearly visualized by small-animal SPECT/CT (15,16).

Recently, we identified another DOTA-conjugated lactam bridge–cyclized α-MSH peptide with a 6–amino acid peptide ring {DOTA-Nle-CycMSHhex: DOTA-Nle-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2} for melanoma targeting. The receptor-binding motif of His-dPhe-Arg-Trp was directly cyclized by an Asp-Lys lactam bridge. Interestingly, reduction of the ring size dramatically enhanced melanoma uptake (19.39 ± 1.65 %ID/g at 2 h after injection) and reduced kidney uptake (9.52 ± 0.44 %ID/g at 2 h after injection) of 111In-DOTA-Nle-CycMSHhex, compared with 111In-DOTA-GlyGlu-CycMSH, in B16/F1 melanoma–bearing C57 mice (15,19).

Hydrocarbon, amino acid, and polyethylene glycol linkers displayed profound favorable effects on the receptor-binding affinities and pharmacokinetics of radiolabeled bombesin (21–25), RGD (26–29), and α-MSH peptides (15,16). To examine the effects of the amino acid linkers on melanoma-targeting and pharmacokinetic properties, we designed 3 novel DOTA-conjugated lactam bridge–cyclized CycMSHhex peptides with different amino acid linkers in this study based on the unique structure of DOTA-Nle-CycMSHhex we previously reported (19). A neutral -Gly-Gly- (GG) linker and a negatively charged -GE- linker were inserted between the DOTA and Nle to generate DOTA-GGNle-CycMSHhex and DOTA-GENle-CycMSHhex. Furthermore, the negatively charged -GE- linker was introduced between Nle and CycMSHhex to yield DOTA-NleGE-CycMSHhex. The MC1 receptor–binding affinities of these 3 peptides were determined in B16/F1 melanoma cells. Only DOTA-GGNle-CycMSHhex and DOTA-GENle-CycMSHhex displayed low-nanomolar MC1 receptor–binding affinities. Hence, we further determined the melanoma-targeting and pharmacokinetic properties of 111In-DOTA-GGNle-CycMSHhex and 111In-DOTA-GENle-CycMSHhex in B16/F1 melanoma–bearing C57 mice.

MATERIALS AND METHODS

Chemicals and Reagents

Amino acids and resin were purchased from Advanced ChemTech Inc. and Novabiochem. DOTA-tri-t-butyl ester was purchased from Macrocyclics Inc. for peptide synthesis. 125I-Tyr2-[Nle4, dPhe7]-α-MSH (NDP-MSH) was obtained from PerkinElmer, Inc. for in vitro receptor-binding assay. 111InCl3 was purchased from MDS Nordion, Inc., for radiolabeling. All other chemicals used in this study were purchased from Thermo Fischer Scientific and used without further purification. B16/F1 murine melanoma cells were obtained from American Type Culture Collection.

Peptide Synthesis

New DOTA-GGNle-CycMSHhex, DOTA-GENle-CycMSHhex, and DOTA-NleGE-CycMSHhex peptides were synthesized using standard fluorenylmethyloxycarbonyl chemistry according to our published procedure (19) with modifications. Briefly, linear peptide backbones of (tBu)3DOTA-GGNle-Asp(O-2-PhiPr)-His(Trt)-dPhe-Arg(Pbf)-Trp(Boc)-Lys(Dde), (tBu)3DOTA-GE(OtBu)-Nle-Asp(O-2-PhiPr)-His(Trt)-dPhe-Arg(Pbf)-Trp(Boc)-Lys(Dde), and (tBu)3DOTA-NleGE(OtBu)-Asp(O-2-PhiPr)-His(Trt)-dPhe-Arg(Pbf)-Trp(Boc)-Lys(Dde) were synthesized on Sieber Amide resin by an Advanced ChemTech multiple-peptide synthesizer. Seventy micromoles of resin, 210 μmol of each fluorenylmethyloxycarbonyl-protected amino acid, and 210 μmol of (tBu)3DOTA were used for the synthesis. 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 through treatment with a mixture of 2.5% of trifluoroacetic acid and 5% of triisopropylsilane. After the precipitation with ice-cold ether and characterization by liquid chromatography–mass spectroscopy, each protected peptide was dissolved in H2O/CH3CN (50:50) and lyophilized to remove the reagents. Then, each protected peptide was further cyclized by coupling the carboxylic group from the Asp with the ε-amino group from the Lys. The cyclization reaction was achieved by an overnight reaction in dimethylformamide using benzotriazole-1-yl-oxy-tris-pyrrolidinophosphonium-hexafluorophosphate as a coupling agent in the presence of N,N-diisopropylethylamine. After the characterization by liquid chromatography–mass spectroscopy, each cyclized protected peptide was dissolved in H2O/CH3CN (50:50) and lyophilized to remove the reagents. The protecting groups were totally removed by treatment with a mixture of trifluoroacetic acid, 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 4 times, purified by reverse-phase high-performance liquid chromatography (RP-HPLC), and characterized by liquid chromatography–mass spectroscopy.

In Vitro Receptor-Binding Assay

The receptor-binding affinities (inhibitory concentration of 50% [IC50]) of DOTA-GGNle-CycMSHhex, DOTA-GENle-CycMSHhex, and DOTA-NleGE-CycMSHhex were determined by an in vitro competitive binding assay according to our published procedure (19), with modifications. B16/F1 cells in 24-well cell culture plates (5 × 105 cells per well) were incubated at room temperature (25°C) for 2 h with approximately 60,000 cpm of 125I-Tyr2-NDP-MSH in the presence of a 10−12 to 10−5 M concentration of each peptide in 0.3 mL of binding medium {Dulbecco's modified Eagle's medium with 25 mM N-(2-hydroxyethyl)-piperazine-N′-(2-ethanesulfonic acid), pH 7.4, 0.2% bovine serum albumin, 0.3 mM 1,10-phenathroline}. The medium was aspirated after the incubation. The cells were rinsed twice with 0.5 mL of ice-cold 0.2% bovine serum albumin (pH 7.4)/0.01 M phosphate-buffered saline and lysed in 0.5 mL of 1N NaOH for 5 min. The activities associated with cells were measured in a Wallac 1480 automated γ-counter (PerkinElmer). The IC50 of each peptide was calculated using Prism software (GraphPad Software).

Peptide Radiolabeling with 111In

Since DOTA-NleGE-CycMSHhex exhibited at least 78-fold lower receptor-binding affinity than DOTA-GGNle-CycMSHhex and DOTA-GENle-CycMSHhex, we only further evaluated DOTA-GGNle-CycMSHhex and DOTA-GENle-CycMSHhex. 111In-DOTA-GGNle-CycMSHhex and 111In-DOTA-GENle-CycMSHhex were prepared in a 0.5 M NH4OAc-buffered solution at pH 4.5 according to our published procedure (19). Briefly, 50 μL of 111InCl3 (37–74 MBq in 0.05 M HCl aqueous solution), 10 μL of a 1 mg/mL peptide aqueous solution, and 400 μL of 0.5 M NH4OAc (pH 4.5) were added to a reaction vial and incubated at 75°C for 45 min. After the incubation, 10 μL of 0.5% ethylenediaminetetraacetic acid aqueous solution were added to the reaction vial to scavenge potential unbound 111In3+ ions. The radiolabeled complexes were purified to a single species by Waters RP-HPLC on a Grace Vydac C-18 reverse-phase analytic column using the following gradient at a 1 mL/min flow rate. The mobile phase consisted of solvent A (20 mM HCl aqueous solution) and solvent B (100% CH3CN). The gradient was initiated and kept at 82:18 A/B for 3 min followed by a linear gradient of 82:18 A/B to 72:28 A/B over 20 min. Then, the gradient was changed from 72:28 A/B to 10:90 A/B over 3 min followed by an additional 5 min at 10:90 A/B. Thereafter, the gradient was changed from 10:90 A/B to 82:18 A/B over 3 min. Each purified peptide sample was purged with N2 gas for 20 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. The in vitro serum stability of 111In-DOTA-GGNle-CycMSHhex and 111In-DOTA-GENle-CycMSHhex was determined by incubation in mouse serum at 37°C for 24 h and monitored for degradation by RP-HPLC.

Biodistribution Studies

All animal studies were conducted with the approval of the Institutional Animal Care and Use Committee. The pharmacokinetics of 111In-DOTA-GGNle-CycMSHhex and 111In-DOTA-GENle-CycMSHhex were determined in B16/F1 melanoma–bearing C57 female mice (Harlan). Each C57 mouse was subcutaneously inoculated with 1 × 106 B16/F1 cells in the right flank to generate B16/F1 melanoma. Ten days after inoculation, the tumor weights reached approximately 0.2 g. Each melanoma-bearing mouse was injected with 0.037 MBq of 111In-DOTA-GGNle-CycMSHhex or 111In-DOTA-GENle-CycMSHhex via the tail vein. Groups of 5 mice were sacrificed at 0.5, 2, 4, and 24 h after 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 specificities of tumor uptake of 111In-DOTA-GGNle-CycMSHhex and 111In-DOTA-GENle-CycMSHhex were determined by coinjecting 10 μg (6.07 nmol) of unlabeled NDP-MSH, which is a linear α-MSH peptide analog with picomolar MC1 receptor–binding affinity.

Melanoma Imaging

Since 111In-DOTA-GGNle-CycMSHhex displayed more favorable tumor targeting and pharmacokinetic properties than 111In-DOTA-GENle-CycMSHhex, we only further evaluated the melanoma imaging property of 111In-DOTA-GGNle-CycMSHhex. One B16/F1 melanoma–bearing C57 mouse (10 d after cell inoculation) was injected with 14.8 MBq of 111In-DOTA-GGNle-CycMSHhex via the tail vein. The mouse was sacrificed for small-animal SPECT/CT (Nano-SPECT/CT; BioScan) imaging at 2 h after injection. The CT was immediately followed by the whole-body SPECT. SPECT scans of 24 projections were acquired. Reconstructed SPECT and CT data were visualized and coregistered using InVivoScope (BioScan).

Metabolites of 111In-DOTA-GGNle-CycMSHhex in Melanoma and Urine

From the mouse used for SPECT/CT, both melanoma and urine were collected for analyses of 111In-DOTA-GGNle-CycMSHhex metabolites. The tumor was homogenized for 5 min. An equal volume of ethanol was added to the tumor sample. The tumor sample was stirred in a vortex mixer and then centrifuged at 16,000g for 5 min. The supernatant was transferred to a glass test tube and purged with N2 gas for 20 min to remove the ethanol. Aliquots of the supernatant were injected into the HPLC. The urinary sample was directly centrifuged at 16,000g for 5 min before the HPLC analysis. Thereafter, aliquots of the urine were injected into the HPLC. The HPLC gradient already described was used for the analyses of metabolites.

Statistical Analysis

Statistical analysis was performed using the Student t test for unpaired data. A 95% confidence level was chosen to determine the significance of differences in tumor and kidney uptake between 111In-DOTA-GGNle-CycMSHhex and 111In-DOTA-GENle-CycMSHhex, as well as the significance of differences in tumor and kidney uptake in 111In-DOTA-GGNle-CycMSHhex or 111In-DOTA-GENle-CycMSHhex with and without NDP-MSH coinjection. Differences at the 95% confidence level (P < 0.05) were considered significant.

RESULTS

Three α-MSH peptides—DOTA-GGNle-CycMSHhex, DOTA-GENle-CycMSHhex, and DOTA-NleGE-CycMSHhex—were synthesized and purified by HPLC. All 3 peptides displayed greater than 95% purity after HPLC purification. The schematic structures of the peptides are shown in Figure 1. The identities of the peptides were confirmed by electrospray ionization mass spectrometry. The calculated and found molecular weights of the peptides are presented in Table 1. The receptor-binding affinities of the peptides were determined in B16/F1 melanoma cells. The IC50 values of DOTA-GGNle-CycMSHhex, DOTA-GENle-CycMSHhex, and DOTA-NleGE-CycMSHhex were 2.1, 11.5, and 873.4 nM in B16/F1 cells, respectively (Table 1; Fig. 2).

FIGURE 1.
FIGURE 1.

Structures of DOTA-Nle-CycMSHhex, DOTA-GGNle-CycMSHhex, DOTA-GENle-CycMSHhex, and DOTA-NleGE-CycMSHhex. Structure of DOTA-Nle-CycMSHhex was cited from reference 19 for comparison.

FIGURE 2.
FIGURE 2.

 In vitro competitive binding curves of DOTA-Nle-CycMSHhex, DOTA-GGNle-CycMSHhex, DOTA-GENle-CycMSHhex, and DOTA-NleGE-CycMSHhex in B16/F1 melanoma cells. IC50 values of DOTA-Nle-CycMSHhex, DOTA-GGNle-CycMSHhex, DOTA-GENle-CycMSHhex, and DOTA-NleGE-CycMSHhex were 1.8, 2.1, 11.5, and 873.4 nM, respectively. Data of DOTA-Nle-CycMSHhex were cited from reference 19 for comparison.

View this table:
TABLE 1

DOTA-Conjugated Lactam Bridge–Cyclized α-MSH Peptides

We further evaluated only DOTA-GGNle-CycMSHhex and DOTA-GENle-CycMSHhex because both peptides displayed low-nanomolar MC1 receptor–binding affinities. The peptides were readily labeled with 111In in 0.5 M ammonium acetate solution at pH 4.5 with greater than 95% radiolabeling yield. Each 111In-labeled peptide was completely separated from its excess nonlabeled peptide by RP-HPLC. The retention times of the peptides and their 111In-labeled conjugates are shown in Table 1. The retention times of 111In-DOTA-GGNle-CycMSHhex and 111In-DOTA-GENle-CycMSHhex were 17.7 and 21.7 min, respectively. They showed greater than 98% radiochemical purity after HPLC purification and were stable in mouse serum at 37°C for 24 h. Only intact 111In-labeled conjugates were detected by RP-HPLC after 24 h of incubation in mouse serum.

We further evaluated the melanoma-targeting and pharmacokinetic properties of 111In-DOTA-GGNle-CycMSHhex and 111In-DOTA-GENle-CycMSHhex in B16/F1 melanoma–bearing C57 mice. The biodistribution results are shown in Table 2. 111In-DOTA-GGNle-CycMSHhex exhibited rapid, high melanoma uptake and prolonged tumor retention. The tumor uptake of 111In-DOTA-GGNle-CycMSHhex was 18.39 ± 2.22 %ID/g at 0.5 h after injection and peaked at 19.05 ± 5.04 %ID/g at 2 h after injection. 111In-DOTA-GGNle-CycMSHhex displayed similar high tumor uptake (18.6 ± 3.56 %ID/g) at 4 h after injection. Even at 24 h after injection, 6.77 ± 0.84 %ID/g of 111In-DOTA-GGNle-CycMSHhex activity remained in the tumor. Approximately 98% of the tumor uptake of 111In-DOTA-GGNle-CycMSHhex was blocked by 10 μg (6.07 nmol) of nonradiolabeled NDP-MSH (P < 0.05), demonstrating that the tumor uptake was specific and MC1 receptor–mediated. Whole-body clearance of 111In-DOTA-GGNle-CycMSHhex was rapid, with approximately 88.4% of the injected radioactivity cleared through the urinary system by 2 h after injection. Normal-organ uptake of 111In-DOTA-GGNle-CycMSHhex was low (<1.31 %ID/g), except for the kidneys at 2, 4, and 24 h after injection. Liver uptake of 111In-DOTA-GGNle-CycMSHhex was less than 0.61 %ID/g at 2 h after injection. Kidney uptake was 15.19 ± 2.75 %ID/g at 0.5 h after injection and decreased to 6.84 ± 0.92 %ID/g at 2 h after injection. Coinjection of NDP-MSH did not significantly reduce kidney uptake of the 111In-DOTA-GGNle-CycMSHhex activity at 2 h after injection, indicating that kidney uptake was not MC1 receptor–mediated. High tumor uptake and prolonged tumor retention coupled with rapid whole-body clearance resulted in high ratios of tumor-to-blood uptake and tumor–to–normal-organ uptake as early as 0.5 h after injection. The ratios of tumor-to-liver uptake for 111In-DOTA-GGNle-CycMSHhex were 33.42 and 31.0 at 2 and 4 h, after injection, respectively, whereas the ratios of tumor-to-kidney uptake for 111In-DOTA-GGNle-CycMSHhex were 2.79 and 2.73 at 2 and 4 h after injection, respectively.

View this table:
TABLE 2

Biodistribution of 111In-DOTA-GGNle-CycMSHhex and 111In-DOTA-GENle-CycMSHhex in B16/F1 Melanoma–Bearing C57 Mice

As we anticipated, 111In-DOTA-GENle-CycMSHhex showed lower tumor uptake than 111In-DOTA-GGNle-CycMSHhex at 0.5, 2, and 4 h after injection. Tumor uptake of 111In-DOTA-GGNle-CycMSHhex was 2, 2.5, and 3 times that of 111In-DOTA-GENle-CycMSHhex at 0.5, 2, and 4 h after injection, respectively (Table 2). Coinjection of nonradioactive NDP-MSH blocked 95.6% of tumor uptake at 2 h after injection (P < 0.05), indicating that tumor uptake of 111In-DOTA-GENle-CycMSHhex was MC1 receptor–specific. Despite the fact that kidney uptake of 111In-DOTA-GENle-CycMSHhex was similar to that of 111In-DOTA-GGNle-CycMSHhex at 2, 4, and 24 h after injection, 111In-DOTA-GENle-CycMSHhex showed 40% lower kidney uptake than 111In-DOTA-GGNle-CycMSHhex at 0.5 h after injection (P < 0.05). Kidney uptake of 111In-DOTA-GENle-CycMSHhex was as low as 9.06 ± 2.20 %ID/g at 0.5 h after injection and decreased to 5.54 ± 0.63 %ID/g at 2 h after injection.

We further evaluated the melanoma-imaging properties of 111In-DOTA-GGNle-CycMSHhex, because it showed more favorable biodistribution properties than 111In-DOTA-GENle-CycMSHhex. The whole-body SPECT/CT images are presented in Figure 3. Flank melanoma tumors were clearly visualized by SPECT/CT using 111In-DOTA-GGNle-CycMSHhex as an imaging probe. The whole-body images showed high ratios of tumor–to–normal-organ uptake except for the kidneys, as was consistent with the biodistribution results. Melanoma and urinary metabolites of 111In-DOTA-GGNle-CycMSHhex were analyzed by RP-HPLC at 2 h after injection. Figure 4 illustrates the HPLC profiles for both melanoma and urine samples. 111In-DOTA-GGNle-CycMSHhex remained intact in both tumor and urine 2 h after injection (Fig. 4).

FIGURE 3.
FIGURE 3.

Representative whole-body SPECT/CT image of B16/F1 melanoma–bearing mouse (10 d after cell inoculation) 2 h after injection of 14.8 MBq of 111In-DOTA-GGNle-CycMSHhex.

FIGURE 4.
FIGURE 4.

Radioactive HPLC profiles of 111In-DOTA-GGNle-CycMSHhex (injected conjugate) and its metabolites in urine and tumor 2 h after injection.

DISCUSSION

We have been interested in developing lactam bridge–cyclized α-MSH peptides to target the MC1 receptors for melanoma detection (15–19). Unique lactam bridge cyclization makes the cyclic α-MSH peptides resistant to proteolytic degradation in vivo and provides the flexibility for fine structural modification (15,17,19). Recently, we identified 111In-DOTA-Nle-CycMSHhex with a 6–amino acid ring targeting the MC1 receptors for melanoma imaging (19). Among these reported 111In-labeled lactam bridge–cyclized α-MSH peptides (15,17,19), 111In-DOTA-Nle-CycMSHhex displayed the highest melanoma uptake (24.94 ± 4.58 %ID/g at 0.5 h after injection and 19.39 ± 1.65 %ID/g at 2 h after injection) in B16/F1 melanoma–bearing mice (19). Reduction of the ring size improved tumor uptake and reduced kidney uptake of 111In-DOTA-Nle-CycMSHhex, providing new insight into the design of lactam bridge–cyclized α-MSH peptides for melanoma targeting.

Hydrocarbon, amino acid, and polyethylene glycol linkers have been used to optimize the receptor-binding affinities and modify the pharmacokinetic properties of radiolabeled bombesin (21–25), RGD (26–29), and α-MSH peptides (15,16). For instance, Hoffman et al. reported that the hydrocarbon linkers ranging from 5-carbon to 8-carbon between the DOTA and bombesin peptide resulted in 0.6–1.7 nM receptor-binding affinities for the DOTA-conjugated bombesin peptides. Either shorter or longer hydrocarbon linkers dramatically reduce the receptor-binding affinity by 100-fold (21). Parry et al. reported the profound effects of amino acid linkers (-Gly-Gly-Gly-, -Gly-Ser-Gly-, -Gly-Ser-Ser-, and -Gly-Glu-Gly-) between the DOTA and bombesin peptide on tumor and normal-organ uptake of the radiolabeled peptides (25). 64Cu-labeled DOTA-conjugated bombesin peptide with the -Gly-Gly-Gly- linker displayed higher PC-3 tumor uptake, whereas the -Gly-Ser-Gly- linker resulted in lower kidney uptake (25). Recently, the group led by Liu reported an improvement in the tumor uptake and pharmacokinetics of 64Cu- and 99mTc-labeled cyclic RGD peptides using the -Gly-Gly-Gly- and polyethylene glycol 4 (PEG4) linkers (26–29). We also demonstrated that the introduction of a negatively charged -GE- linker enhanced melanoma uptake and reduced kidney uptake of 111In-DOTA-GlyGlu-CycMSH, compared with 111In-DOTA-CycMSH (15). Hence, we evaluated the effects of -GG- and -GE- linkers on the melanoma-targeting and pharmacokinetic properties of 111In-DOTA-[X]-CycMSHhex peptide constructs in this study.

DOTA-Nle-CycMSHhex displayed 1.8 nM MC1 receptor–binding affinity in B16/F1 melanoma cells in our previous report (19). The MC1 receptor–binding sequence of His-dPhe-Arg-Trp was directly cyclized by an Asp-Lys lactam bridge to generate the CycMSHhex moiety. The radiometal chelator DOTA was conjugated to the CycMSHhex moiety via an Nle to form DOTA-Nle-CycMSHhex peptide. Based on the unique structure of DOTA-Nle-CycMSHhex, we initially introduced the amino acid linker (-GE-) between the DOTA and Nle or between the Nle and CycMSHhex moiety to determine which position was suitable for an amino acid linker. We found that the moiety of Nle-CycMSHhex was critical for maintaining the low-nanomolar MC1 receptor–binding affinity of the peptide. The introduction of the -GE- linker between the Nle and CycMSHhex moiety dramatically reduced the MC1 receptor–binding affinity to 873.4 nM, whereas the introduction of the -GE- linker between the DOTA and Nle decreased the MC1 receptor–binding affinity only to 11.5 nM. Interestingly, the -GG- linker between the DOTA and Nle maintained the MC1 receptor–binding affinity at 2.1 nM, further indicating that the moiety of Nle-CycMSHhex played a crucial role in maintaining the low-nanomolar MC1 receptor–binding affinity of the peptide. Furthermore, the amino acid between the DOTA and the moiety of Nle-CycMSHhex also showed a significant impact on the MC1 receptor–binding affinity of the peptide. The neutral -GG- linker was better than the negatively charged -GE- linker in maintaining the low-nanomolar MC1 receptor–binding affinity of the peptide. The IC50 value of DOTA-GENle-CycMSHhex was 5.5 times that of DOTA-GGNle-CycMSHhex. It was likely that the electrostatic interaction between the negatively charged Glu in the -GE- linker and the positively charged Arg in the moiety of Nle-CycMSHhex affected the configuration of the MC1 receptor–binding region (His-dPhe-Arg-Trp). The difference in MC1 receptor–binding affinity between DOTA-GGNle-CycMSHhex and DOTA-GENle-CycMSHhex (2.1 nM vs. 11.5 nM) was also observed in the difference in melanoma uptake between 111In-DOTA-GGNle-CycMSHhex and 111In-DOTA-GENle-CycMSHhex in B16/F1 melanoma–bearing C57 mice. Tumor uptake of 111In-DOTA-GGNle-CycMSHhex was 2, 2.5, and 3 times that of 111In-DOTA-GENle-CycMSHhex at 0.5, 2, and 4 h after injection, respectively (Table 2). In our previous report, the introduction of a negatively charged -GE- linker resulted in 44% reduced kidney uptake of 111In-DOTA-GE-CycMSH at 4 h after injection, compared with 111In-DOTA-CycMSH (15). In this study, kidney uptake for 111In-DOTA-GENle-CycMSHhex was 40% lower than that for 111In-DOTA-GGNle-CycMSHhex at 0.5 h after injection (P < 0.05) (Table 2).

Presently, the lactam bridge–cyclized 111In-DOTA-Nle-CycMSHhex and the metal-cyclized 111In-DOTA-Re(Arg11)CCMSH have displayed the highest comparable melanoma uptake among all reported 111In-labeled linear and cyclic α-MSH peptides (13,19). The respective values for melanoma uptake were 17.29 ± 2.49 and 17.41 ± 5.63 %ID/g at 2 and 4 h after injection for 111In-DOTA-Re(Arg11)CCMSH (13) and 19.39 ± 1.65 and 17.01 ± 2.54 %ID/g at 2 and 4 h after injection for 111In-DOTA-Nle-CycMSHhex (19). Meanwhile, 111In-DOTA-Nle-CycMSHhex showed ratios of tumor-to-kidney uptake that were similar to those for 111In-DOTA-Re(Arg11)CCMSH at 2 and 24 h after injection (19). In this study, the introduction of the -GG- linker maintained high melanoma uptake of 111In-DOTA-GGNle-CycMSHhex (19.05 ± 5.04 and 18.6 ± 3.56 %ID/g at 2 and 4 h after injection, respectively), compared with 111In-DOTA-Nle-CycMSHhex, as was consistent with their similar MC1 receptor–binding affinities (2.1 nM vs. 1.8 nM). Interestingly, the introduction of the -GG- linker reduced liver and kidney uptake of 111In-DOTA-GGNle-CycMSHhex, compared with 111In-DOTA-Nle-CycMSHhex (19). The reduction in liver and kidney uptake might be attributed to the relatively faster whole-body clearance of 111In-DOTA-GGNle-CycMSHhex. Approximately 88% of 111In-DOTA-GGNle-CycMSHhex activity cleared from the body via the urinary system at 2 h after injection, whereas 82% of 111In-DOTA-Nle-CycMSHhex activity washed out of the body via the urinary tract at 2 h after injection (19). 111In-DOTA-GGNle-CycMSHhex exhibited 61%, 65%, and 68% less liver uptake than 111In-DOTA-Nle-CycMSHhex (Fig. 5) and 28%, 32%, and 42% less kidney uptake than 111In-DOTA-Nle-CycMSHhex at 2, 4, and 24 h after injection, respectively (Fig. 5). The maintained high melanoma uptake coupled with the decreased liver and kidney uptake resulted in enhanced ratios of tumor-to-liver and tumor-to-kidney uptake for 111In-DOTA-GGNle-CycMSHhex, compared with 111In-DOTA-Nle-CycMSHhex, at 2 and 4 h after injection (Fig. 6). The tumor-to-liver ratios of 111In-DOTA-GGNle-CycMSHhex were 2.5 and 3.1 times those of 111In-DOTA-Nle-CycMSHhex at 2 and 4 h after injection, whereas the tumor-to-kidney ratios of 111In-DOTA-GGNle-CycMSHhex were 1.4 and 1.6 times those of 111In-DOTA-Nle-CycMSHhex at 2 and 4 h after injection.

FIGURE 5.
FIGURE 5.

Kidney (A) and liver (B) uptake of 111In-DOTA-Nle-CycMSHhex and 111In-DOTA-GGNle-CycMSHhex. Data of 111In-DOTA-Nle-CycMSHhex were cited from reference 19 for comparison.

FIGURE 6.
FIGURE 6.

Tumor-to-kidney (A) and tumor-to-liver (B) ratios of 111In-DOTA-Nle-CycMSHhex and 111In-DOTA-GGNle-CycMSHhex 2 and 4 h after injection. Data of 111In-DOTA-Nle-CycMSHhex were cited from reference 19 for comparison.

As shown in Figure 3, the enhanced tumor-to-liver and tumor-to-kidney ratios of 111In-DOTA-GGNle-CycMSHhex generated high contrast between tumor and background. The flank melanoma lesions were clearly visualized by SPECT/CT using 111In-DOTA-GGNle-CycMSHhex as an imaging probe, highlighting its potential as an effective imaging agent for melanoma detection. 111In-DOTA-GGNle-CycMSHhex maintained intact in melanoma and urine at 2 h after injection (Fig. 4). From the therapeutic point of view, the enhanced tumor-to-liver and tumor-to-kidney ratios of 111In-DOTA-GGNle-CycMSHhex would decrease the absorbed doses to the liver and kidneys when the therapeutic radionuclide–labeled DOTA-GGNle-CycMSHhex is used for melanoma treatment. In other words, the improvement in tumor-to-liver and tumor-to-kidney ratios would potentially increase the absorbed dose to the tumor while keeping the liver and kidneys safe when melanoma is treated with therapeutic radionuclide–labeled DOTA-GGNle-CycMSHhex.

CONCLUSION

The amino acid linkers exhibited profound effects on the melanoma-targeting and pharmacokinetic properties of the 111In-labeled lactam bridge–cyclized α-MSH peptides. Introduction of the -GG- linker maintained high melanoma uptake while reducing kidney and liver uptake of 111In-DOTA-GGNle-CycMSHhex, highlighting its potential as an effective imaging probe for melanoma detection and as a therapeutic peptide for melanoma treatment when labeled with a therapeutic radionuclide.

DISCLOSURE STATEMENT

The costs of publication of this article were defrayed in part by the payment of page charges. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Acknowledgments

This work was supported in part by NIH grant NM-INBRE P20RR016480 and the University of New Mexico STC Gap Fund. Figure 3 was generated by the Keck-UNM Small Animal Imaging Resource established with funding from the W.M. Keck Foundation and the University of New Mexico Cancer Research and Treatment Center (NIH P30 CA118100).

REFERENCES

  1. 1.↵
    1. Siegrist W,
    2. Solca F,
    3. Stutz S,
    4. et al
    . Characterization of receptors for alpha-melanocyte-stimulating hormone on human melanoma cells. Cancer Res. 1989;49:6352–6358.
    OpenUrlAbstract/FREE Full Text
  2. 2.
    1. Tatro JB,
    2. Reichlin S
    . Specific receptors for alpha-melanocyte-stimulating hormone are widely distributed in tissues of rodents. Endocrinology. 1987;121:1900–1907.
    OpenUrlCrossRefPubMed
  3. 3.
    1. Miao Y,
    2. Whitener D,
    3. Feng W,
    4. Owen NK,
    5. Chen J,
    6. Quinn TP
    . Evaluation of the human melanoma targeting properties of radiolabeled alpha-melanocyte stimulating hormone peptide analogues. Bioconjug Chem. 2003;14:1177–1184.
    OpenUrlCrossRefPubMed
  4. 4.
    1. Miao Y,
    2. Owen NK,
    3. Whitener D,
    4. Gallazzi F,
    5. Hoffman TJ,
    6. Quinn TP
    . In vivo evaluation of 188Re-labeled alpha-melanocyte stimulating hormone peptide analogs for melanoma therapy. Int J Cancer. 2002;101:480–487.
    OpenUrlCrossRefPubMed
  5. 5.↵
    1. Chen J,
    2. Cheng Z,
    3. Hoffman TJ,
    4. Jurisson SS,
    5. Quinn TP
    . Melanoma-targeting properties of 99mtechnetium-labeled cyclic alpha-melanocyte-stimulating hormone peptide analogues. Cancer Res. 2000;60:5649–5658.
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    1. Froidevaux S,
    2. Calame-Christe M,
    3. Tanner H,
    4. Eberle AN
    . Melanoma targeting with DOTA-alpha-melanocyte-stimulating hormone analogs: structural parameters affecting tumor uptake and kidney uptake. J Nucl Med. 2005;46:887–895.
    OpenUrlAbstract/FREE Full Text
  7. 7.
    1. Froidevaux S,
    2. Calame-Christe M,
    3. Schuhmacher J,
    4. et al
    . A gallium-labeled DOTA-alpha-melanocyte-stimulating hormone analog for PET imaging of melanoma metastases. J Nucl Med. 2004;45:116–123.
    OpenUrlAbstract/FREE Full Text
  8. 8.
    1. Froidevaux S,
    2. Calame-Christe M,
    3. Tanner H,
    4. Sumanovski L,
    5. Eberle AN
    . A novel DOTA-alpha-melanocyte-stimulating hormone analog for metastatic melanoma diagnosis. J Nucl Med. 2002;43:1699–1706.
    OpenUrlAbstract/FREE Full Text
  9. 9.↵
    1. Miao Y,
    2. Owen NK,
    3. Fisher DR,
    4. Hoffman TJ,
    5. Quinn TP
    . Therapeutic efficacy of a 188Re-labeled alpha-melanocyte-stimulating hormone peptide analog in murine and human melanoma-bearing mouse models. J Nucl Med. 2005;46:121–129.
    OpenUrlAbstract/FREE Full Text
  10. 10.
    1. Miao Y,
    2. Hylarides M,
    3. Fisher DR,
    4. et al
    . Melanoma therapy via peptide-targeted alpha-radiation. Clin Cancer Res. 2005;11:5616–5621.
    OpenUrlAbstract/FREE Full Text
  11. 11.
    1. Wei L,
    2. Butcher C,
    3. Miao Y,
    4. et al
    . Synthesis and biologic evaluation of 64Cu-labeled rhenium-cyclized alpha-MSH peptide analog using a cross-bridged cyclam chelator. J Nucl Med. 2007;48:64–72.
    OpenUrlAbstract/FREE Full Text
  12. 12.↵
    1. Miao Y,
    2. Benwell K,
    3. Quinn TP
    . 99mTc- and 111In-labeled alpha-melanocyte-stimulating hormone peptides as imaging probes for primary and pulmonary metastatic melanoma detection. J Nucl Med. 2007;48:73–80.
    OpenUrlAbstract/FREE Full Text
  13. 13.↵
    1. Cheng Z,
    2. Chen J,
    3. Miao Y,
    4. Owen NK,
    5. Quinn TP,
    6. Jurisson SS
    . Modification of the structure of a metallopeptide: synthesis and biological evaluation of 111In-labeled DOTA-conjugated rhenium-cyclized alpha-MSH analogues. J Med Chem. 2002;45:3048–3056.
    OpenUrlCrossRefPubMed
  14. 14.
    1. Cheng Z,
    2. Xiong Z,
    3. Subbarayan M,
    4. Chen X,
    5. Gambhir SS
    . 64Cu-labeled alpha-melanocyte-stimulating hormone analog for MicroPET imaging of melanocortin 1 receptor expression. Bioconjug Chem. 2007;18:765–772.
    OpenUrlCrossRefPubMed
  15. 15.↵
    1. Miao Y,
    2. Gallazzi F,
    3. Guo H,
    4. Quinn TP
    . 111In-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone peptide analogues for melanoma imaging. Bioconjug Chem. 2008;19:539–547.
    OpenUrlCrossRefPubMed
  16. 16.↵
    1. Guo H,
    2. Shenoy N,
    3. Gershman BM,
    4. Yang J,
    5. Sklar LA,
    6. Miao Y
    . Metastatic melanoma imaging with an 111In-labeled lactam bridge-cyclized alpha-melanocyte-stimulating hormone peptide. Nucl Med Biol. 2009;36:267–276.
    OpenUrlCrossRefPubMed
  17. 17.↵
    1. Guo H,
    2. Yang J,
    3. Gallazzi F,
    4. Prossnitz ER,
    5. Sklar LA,
    6. Miao Y
    . Effect of DOTA position on melanoma targeting and pharmacokinetic properties of 111In-labeled lactam bridge-cyclized α-melanocyte stimulating hormone peptide. Bioconjug Chem. 2009;20:2162–2168.
    OpenUrlCrossRefPubMed
  18. 18.
    1. Guo H,
    2. Yang J,
    3. Shenoy N,
    4. Miao Y
    . Gallium-67-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone peptide for primary and metastatic melanoma imaging. Bioconjug Chem. 2009;20:2356–2363.
    OpenUrlCrossRefPubMed
  19. 19.↵
    1. Guo H,
    2. Yang J,
    3. Gallazzi F,
    4. Miao Y
    . Reduction of the ring size of radiolabeled lactam bridge-cyclized alpha-MSH peptide resulting in enhanced melanoma uptake. J Nucl Med. 2010;51:418–426.
    OpenUrlAbstract/FREE Full Text
  20. 20.↵
    1. Chen J,
    2. Cheng Z,
    3. Owen NK,
    4. et al
    . Evaluation of an 111In-DOTA-rhenium cyclized α-MSH analog: a novel cyclic-peptide analog with improved tumor-targeting properties. J Nucl Med. 2001;42:1847–1855.
    OpenUrlAbstract/FREE Full Text
  21. 21.↵
    1. Hoffman TJ,
    2. Gali H,
    3. Smith CJ,
    4. et al
    . Novel series of 111In-labeled bombesin analogs as potential radiopharmaceuticals for specific targeting of gastrin-releasing peptide receptors expressed on human prostate cancer cells. J Nucl Med. 2003;44:823–831.
    OpenUrlAbstract/FREE Full Text
  22. 22.
    1. Garcia Garayoa E,
    2. Schweinsberg C,
    3. Maes V,
    4. et al
    . Influence of the molecular charge on the biodistribution of bombesin analogues labeled with the [99mTc(CO)3]-core. Bioconjug Chem. 2008;19:2409–2416.
    OpenUrlCrossRefPubMed
  23. 23.
    1. Fragogeorgi EA,
    2. Zikos C,
    3. Gourni E,
    4. et al
    . Spacer site modifications for the improvement of the in vitro and in vivo binding properties of 99mTc-N3S-X-bombesin[2-14] derivatives. Bioconjug Chem. 2009;20:856–867.
    OpenUrlCrossRefPubMed
  24. 24.
    1. Garrison JC,
    2. Rold TL,
    3. Sieckman GL,
    4. et al
    . Evaluation of the pharmacokinetic effects of various linking group using the 111In-DOTA-X-BBN(7-14)NH2 structural paradigm in a prostate cancer model. Bioconjug Chem. 2008;19:1803–1812.
    OpenUrlCrossRefPubMed
  25. 25.↵
    1. Parry JJ,
    2. Kelly TS,
    3. Andrews R,
    4. Rogers BE
    . In vitro and in vivo evaluation of 64Cu-labeled DOTA-linker-bombesin(7-14) analogues containing different amino acid linker moieties. Bioconjug Chem. 2007;18:1110–1117.
    OpenUrlCrossRefPubMed
  26. 26.↵
    1. Liu S,
    2. He Z,
    3. Hsieh WY,
    4. Kim YS,
    5. Jiang Y
    . Impact of PKM linkers on biodistribution characteristics of the 99mTc-labeled cyclic RGDfK dimer. Bioconjug Chem. 2006;17:1499–1507.
    OpenUrlCrossRefPubMed
  27. 27.
    1. Shi J,
    2. Wang L,
    3. Kim YS,
    4. et al
    . Improving tumor uptake and excretion kinetics of 99mTc-labeled cyclic arginine-glycine-aspartic (RGD) dimers with triglycine linkers. J Med Chem. 2008;51:7980–7990.
    OpenUrlCrossRefPubMed
  28. 28.
    1. Wang L,
    2. Shi J,
    3. Kim YS,
    4. et al
    . Improving tumor-targeting capability and pharmacokinetics of 99mTc-labeled cyclic RGD dimers with PEG4 linkers. Mol Pharm. 2009;6:231–245.
    OpenUrlCrossRefPubMed
  29. 29.↵
    1. Shi J,
    2. Kim YS,
    3. Zhai S,
    4. Liu Z,
    5. Chen X,
    6. Liu S
    . Improving tumor uptake and pharmacokinetics of 64Cu-labeled cyclic RGD peptide dimers with Gly3 and PEG4 linkers. Bioconjug Chem. 2009;20:750–759.
    OpenUrlCrossRefPubMed
  • Received for publication November 30, 2010.
  • Accepted for publication January 12, 2011.
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Bookmark this article

Jump to section