Synthesis and Biological Evaluation of a Novel 177 Lu-DOTA-[Gly 3 -Cyclized(Dap 4,d -Phe 7,Asp 10 )-Arg 11 ]α-MSH 3–13 Analogue for Melanocortin-1 Receptor-Positive Tumor Targeting

Abstract

In this study, a novel α-melanocyte stimulating hormone (α-MSH) analogue 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) coupled [Gly³-cyclized(Dap⁴, _d-Phe⁷, Asp¹⁰)-Arg¹¹]α-MSH_3–13 (DOTA-GMSH) for melanocortin-1 receptor (MC-1R) targeting was newly synthesized, radiolabeled with ¹⁷⁷Lu, and in vitro and in vivo characterized. ¹⁷⁷Lu-labeled peptides were prepared with a high radiolabeling yield (>98%), and its Log p value was −2.89. No degradation was observed not only by serum incubation at 37°C for 7 days but also by an HPLC analysis of radioactive metabolites in urine. A cell binding assay revealed that an inhibitory concentration of 50% (IC₅₀) of the peptide was 3.80 nM. The tumor-to-blood ratio, which was 14.27 at 2 hours p.i., was increased to 56.37 at 24 hours p.i., which means that the radiolabeled peptide was highly accumulated in a tumor and was rapidly cleared from the blood pool. We, therefore, conclude that ¹⁷⁷Lu-DOTA-GMSH has promising characteristics for application in nuclear medicine, namely for the diagnosis of MC-1R over-expressing tumors.

Introduction

Melanoma is a type of skin cancer and is well known by its malignancy.¹ Since it is important to detect melanoma in its early stages for enhancing the survival rates, there is a great need to develop accurate diagnostic molecules for the early detection of both primary and metastatic melanoma.² In the field of cancer diagnosis and therapy, radiopharmaceuticals stand out clearly, as they can accurately detect cancerous sites and effectively deliver therapeutic doses of ionizing radiation to the targeted region.³ Since the melanocortin-1 receptor (MC-1R) is overexpressed in 80% of human melanoma tumors, radiopharmaceuticals for targeting MC-1R have been investigated for the diagnosis and treatment of melanoma over the past several years.

The MC-1R is a seven-transmembrane G-protein coupled receptor whose natural ligand as an agonist is melanocortin peptides, adrenocorticotropic hormone, and α-, β-, and γ-melanocyte stimulating hormone (MSH).⁴ α-MSH analogues have been studied as diagnostic and therapeutic tools for metastatic melanoma due to their high receptor affinity.⁵ Various radio-labeled α-MSH derivatives with various radionuclides such as ^99mTc, ¹⁸⁸Re, ¹¹¹In, ⁶⁸Ga, ⁹⁰Y, and ¹⁷⁷Lu have been investigated for SPECT imaging and therapy in metastatic melanoma.^6

–9 To improve its binding affinity and in vivo stability, α-MSH was modified chemically via peptide cyclization and the replacement of a certain amino acid in the structure.^10,11

In general, bifunctional chelating agents (BFCA) are used for the preparation of radiolabeled compounds, in order to introduce radionuclides to the targeting molecules without any distortion of their structure and not reduce their binding affinity and stability.

Among them, the DOTA is able to strongly chelate many radionuclides such as ⁶⁸Ga, ¹¹¹In, ¹⁴⁹Pm, ²¹²Pb, ⁹⁰Y, and ¹⁷⁷Lu.^12

–16 In particular, ¹⁷⁷Lu emits medium and lower-energy β-rays (497 keV), and, thus, ¹⁷⁷Lu is considered a suitable radionuclide for treating small tumors or metastatic deposits. In addition, ¹⁷⁷Lu emits γ-rays (113 and 208 keV, 6% and 11%) that allow scintigraphic imaging and dosimetry.⁶

In this study, we designed a novel α-MSH analogue, DOTA-GMSH for melanoma targeted therapy using ¹⁷⁷Lu, and DOTA BFCAs were introduced into the peptide using conventional solid-phase peptide synthesis. The 7th and 11th amino acids of the α-MSH sequence were, respectively, changed to _d-form Phe and Arg to enhance its binding affinity, and the analogue was cyclized to increase the in vivo stability. After radiolabeling this synthesized peptide, we conducted in vitro and in vivo melanoma-targeting studies.

Materials and Methods

Materials

All chemicals were of an analytical grade, were purchased from a chemical company, and were used without further purification. Automated solid-phase synthesis was accomplished by the use of a Multiple Biomolecular Synthesizer (Peptron). Analytical and preparative RP-HPLC was performed on a Shimazu prominence HPLC using a Shiseido capcell pak C-18 column. A wavelength of 220 nm was used for UV detection for analytical RP-HPLC. The LC/MS was performed using an HP 1100 series. The radioactivity was determined with a Wallac 1470 automated gamma counter (PerkinElmer Life Science). ¹⁷⁷Lu was purchased from Perkin-Elmer, and the radioactivity was measured using an ionizing chamber (Atomlab 200; Bio-dex). The radiolabeling yield and radiochemical purity (RCP) were determined using a gamma detector-equipped HPLC analyzer (Waters).

Preparation of chelator conjugated peptide: DOTA-GMSH

The peptide was prepared by the use of an automated Multiple Biomolecular Synthesizer (Peptron). The DOTA-GMSH was synthesized by applying a standard Fmoc (fluorenylmethyloxycarbonyl) strategy as detailed in Figure 1A. Briefly, Fmoc-Val-OH conjugated 4-methylbenzhydrylamine resin was used as an anchor polymeric support for a solid-phase synthesis. After removing the Fmoc protecting group from resin-bounded Fmoc-Val-OH under a standard cleavage condition (20% piperidine in N,N-dimethylformamide), the linear sequence peptide was prepared by the sequential coupling of Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(Allyl)-OH, Fmoc-Trp(tBoc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-_d-Phe-OH, Fmoc-His(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Dap(Alloc)-OH, Fmoc-Gly-OH, and DOTA(OtBu)₃ introduced into the peptide. HBTU and HOBt were applied as an activating reagent to ensure efficient coupling. Lactam cyclization of DOTA(OtBu)₃-Gly-Dap-Glu-His-_d-Phe-Arg-Trp-Asp-Arg-Pro-Val-NH₂ was accomplished by the reaction between an amine of DAP (diaminopropionic acid) and a carboxylic acid of Asp (aspartic acid) under a conventional amide coupling procedure after the deprotection of allyl and alloc groups using tetrakis(triphenylphosphine)(0)palladium and 1, 3-dimethyl-barbituric acid in dichloromethane. After the lactam cyclization, the resulting peptide was cleaved from the polymeric support by treatment with a mixture solvent of 90% trifluroacetic acid (TFA) containing 2.5% triisopropylsilane (TIS), 2.5% ethanedithiol (EDT), 2.5% thioanisole, and 2.5% deionized water (TFA:TIS:EDT:Thioanisole:H₂O=90:2.5:2.5:2.5:2.5). The crude product was purified by Shimadzu HPLC equipped with a Capcell pak C18 column on a binary gradient system at a flow rate of 1.0 mL/min using an elution solvent of 0.1% TFA in water (A) and 0.1% TFA in acetonitrile (B) with a gradient elution profile of (B): 0%–10% in 2 minutes; 10%–40% in 10 minutes; and 40%–70% in 2 minutes. The molecular mass was analyzed on LC-MS.

FIG. 1.

Solid-phase synthesis route for DOTA-GMSH (A) and formula of DOTA-GMSH (B). The peptide sequence was DOTA-Gly-Dap-Glu-His-_d-Phe-Arg-Trp-Asp-Arg-Pro-Val-NH₂ (Cyclized between Dap-Asp). DOTA-GMSH, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-coupled [Gly³-cyclized(Dap⁴, _d-Phe⁷, Asp¹⁰)-Arg¹¹]α-MSH_3–13.

Radiochemistry of ¹⁷⁷Lu-labeled DOTA-GMSH

Preparation of ¹⁷⁷Lu-labeled DOTA-GMSH

DOTA-GMSH was dissolved in a 50 mM sodium acetate buffer (pH=5.5) to give a concentration of 10⁻⁶ M/mL. 74 MBq of ¹⁷⁷Lu solution diluted in a 50 mM acetate buffer (pH=5.5) was injected into 2×10⁻⁸ M of DOTA-GMSH solution vial to give a final volume of 1 mL, and heated at 90°C for 15 minutes. The radiolabeling yield and radiochemical RCP/stability of the radiolabeled compound were analyzed by a Waters Chromatograph equipped with an X-Terra C-18 column. The column was eluted with a binary gradient system with a flow rate of 1.0 mL/min using an elution solvent of 0.1% TFA in water and 0.1% TFA in ACN. The gradient elution profile based on the solution of 0.1% TFA in ACN is as follows: 0%, 5 minutes; 0%–25%, 1 minutes; 25%–34%, 3 minutes; 34%–100%, 11 minutes; 100%, 10 minutes; 100%–0%, 1 minutes; and 0%, 5 minutes.

Serum stability assay

Serum stability was evaluated as described by Nguyen and coworkers with some modification.¹⁷ ¹⁷⁷Lu-DOTA-GMSH was added to 200 μL of 25% human serum in phosphate-buffered saline (PBS), and incubated at 37°C for 7 days. Two hundred microliter aliquots of the incubations were taken for the following time periods: 0, 1, and 7 days. The aliquots were mixed with 40 mL of 15% trichloroacetic acid (TCA) and incubated at 4°C for at least 15°minutes to precipitate the serum proteins. Five microliter of 1°M NaOH was supplemented to the TCA to prevent peptide precipitation. The supernatant was collected for each sample after centrifugation at 13,000°rpm for 10°minutes and analyzed by HPLC analysis as just described.

Determination of log p value

37 KBq of ¹⁷⁷Lu-DOTA-GMSH was dissolved in an equal volume mixture of 1-octanol and a PBS buffer (1°mL:1°mL). After stirring vigorously for ∼20°minutes, the mixture was centrifuged at a speed of 8,000°rpm for 5°minutes. About 100°μL of samples from both 1-octanol and PBS layers were transferred, and the radioactivity was measured using a Wallac 1470 Wizard automated gamma counter (PerkinElmer Life Science). Partition coefficients were measured at three different times. The log p values were reported as the average of three independent measurements.

Biological evaluation of ¹⁷⁷Lu-DOTA-GMSH

Cell culture

B16-F10 mouse melanoma cells were obtained from the American Type Culture Collection and grown in 100-mm culture dishes (Corning). The cells were cultured in Dulbecco's Modified Eagle's medium (DMEM; Lonza), supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 g/mL streptomycin (Sigma) in an atmosphere of 5% CO₂ in air at 37°C for up to an approximately 90% confluence.

Competitive binding assay

The inhibitory concentration of 50% (IC₅₀) values of DOTA-GMSH were determined by using methods previously described with some modifications.¹⁸ 1×10⁶ B16-F10 cells were placed in 12-well plates, and grown for 24 hours at 37 C. After being washed once with a binding buffer (serum-free DMEM media, Ph 7.4, 25 mM HEPES, 0.2% bovine serum albumin, 1,10-phenathroline), the cells were incubated at 37 C for 2 hours with 50,000 cpm of [¹²⁵I]-[Nle⁴, D-Phe⁷]-α-MSH (Perkin-Elmer) in the presence of increasing concentrations of the DOTA-GMSH (10⁻⁶–10⁻¹² M) in a 1 mL binding buffer. The reaction media was collected. Cells were then washed twice with a cold binding buffer and solubilized with 1 N NaOH for 5 minutes. The activity was then determined in a gamma-counter. IC₅₀ values for the peptides were calculated through a nonlinear regression analysis using the GraphPad Prism5 computer fitting program.

Animal models

Female C57BL/6 mice were purchased at 6 weeks of age from Nara Biotec, Inc. and were allowed 1 week for quarantine and acclimatization. For the induction of tumor xenografts, B16-F10 cells were subcutaneously injected in the right upper flank at a concentration of 1×10⁶ cells/mouse with 200 μL of a 1:1 mixture of a DMEM culture medium and Matrigel. The tumors were allowed to grow for 2 weeks, and their size were calculated using the following formula: width×length×depth×π/6. The animals were housed in a room maintained at 23±2°C with 50±5% relative humidity, under artificial lighting from 08:00 to 20:00 with 13–18 air changes per hour. They were housed 4 animals per cage and given tap water and commercial rodent chow (Samyang Feed) ad libitum. The Institutional Animal Care and Use Committee at KAERI approved the protocols used in all of these experiments, and the animals were cared for in accordance with the Guidelines for Animal Experiments.

Biodistribution assay

A biodistribution assay was carried out using a C57BL/6 black mice-bearing B16-F10 mouse melanoma xenograft. B16-F10 xenograft-bearing mice with 1.5–2 g tumors were randomly divided into three groups (n=4). Saline was added to the ¹⁷⁷Lu-labeled GMSH solution to give a radiotracer concentration of 370 KBq/10⁻⁹ mol/200 (¹⁷⁷Lu/Ligand/Volume), and 0.2 mL of saline was administered into a tail vein. After 2 and 24 hours p.i., all animals were sacrificed using a 60%–70% CO₂ chamber, and each sample was collected respectively. Blood samples were withdrawn from the heart. The tumor and normal organs (liver, kidney, spleen, heart, intestine, lung, and stomach) were excised, weighed, and counted on a Wallac 1470 Wizard automated gamma counter. The organ uptake was calculated as a percentage of the injected dose per organ (%ID/organ) and a percentage of the injected dose per gram of organ tissue (%ID/g). For the blocking experiment, 4 animals were preadministered 10⁻⁷ M of α-MSH (Sigma-Aldrich) in 0.2 mL of saline through the tail vein 30 minutes before the injection of ¹⁷⁷Lu-labeled GMSH, and after 2 hours p.i., a biodistribution experiment was performed.

Urinary metabolites of ¹⁷⁷Lu-DOTA-GMSH

7.4 MBq of ¹⁷⁷Lu-DOTA-GMSH was injected into ICR mice through the tail vein (n=4). After administration, the urinary samples were collected for 3 hours using metabolic cages. The 200 μL of radioactive metabolites in urine were mixed with 40 μL of 15% TCA and incubated at 4°C for 15°minutes to precipitate serum proteins. Five microliters of 1 M NaOH was supplemented to the TCA to prevent peptide precipitation. The supernatant was collected for each sample after centrifugation at 13,000 rpm for 10°minutes and analyzed using HPLC analysis as just described.

Results

The DOTA-GMSH was easily prepared using a solid-phase synthetic method as described in Figure 1A, and the final structural formula is shown in Figure 1B. As shown in Figure 2, the retention time of the analytical HPLC for DOTA-GMSH was found to be 6.57°minutes. The peptide sequence was DOTA-Gly-Dap-Glu-His-_d-Phe-Arg-Trp-Asp-Arg-Pro-Val-NH₂ (Cyclized between Dap-Asp). In addition, the MS data was consistent with the proposed formula. The measured ion peak [M+1 (m/z)] was found at 1752 (Calculated°=°1752). The key sequence for MC-1R targeting, His-Phe-Arg-Trp, was placed in the middle of the peptide.

FIG. 2.

HPLC analysis (A) and LC/MS profiles (B) of DOTA-GMSH. The crude product was purified by Shimadzu HPLC equipped with a Capcell pak C-18 column, and the molecular mass was analyzed on LC-MS.

For ¹⁷⁷Lu radiolabeling, DOTA-GMSH was mixed with an ¹⁷⁷Lu solution and heated at 90°C for 15°minutes. A gamma detector-equipped HPLC analyzer was used to evaluate the labeling yield of the peptides. A high labeling yield (>98%) was achieved, and directly used without further purification (Fig. 3).

FIG. 3.

Typical profiles of ¹⁷⁷Lu-DOTA-GMSH determined by HPLC analysis using a C-18 column. Retention time: ¹⁷⁷Lu at 3.5 minutes and ¹⁷⁷Lu-DOTA-GMSH at 10.8 minutes.

¹⁷⁷Lu-DOTA-GMSH showed excellent in vitro stability in serum at 37°C for 7 days (>98%) (Table 1). To evaluate whether ¹⁷⁷Lu is decomposed from the peptide in serum, free ¹⁷⁷Lu both in a stock solution and in serum was analyzed by HPLC to confirm its typical profiles. The free ¹⁷⁷Lu was observed at 3.5 minutes in the stock solution, but was not at 5.8 minutes in the serum incubation (Fig. 4). The radiolabeled compound was stable in serum for 7 days.

FIG. 4.

Typical profiles of free ¹⁷⁷Lu at 3.5 minutes in the stock solution (A) and at 5.8 minutes in the serum incubation (B) for 7 days.

Table 1.

Stability of ¹⁷⁷ Lu-DOTA-GMSH for 7 Days in Human Serum

	0 Day (%)	4 Day (%)	7 Day (%)
¹⁷⁷Lu (3.2 minutes)	0	0	0
¹⁷⁷Lu in serum (5.7 minutes)	0	0	0
¹⁷⁷Lu-DOTA-GMSH (10.8 minutes)	>98	>98	>98

DOTA-GMSH, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-coupled [Gly³-cyclized(Dap⁴, _d-Phe⁷, Asp¹⁰)-Arg¹¹]α-MSH_3–13.

The log p value of ¹⁷⁷Lu-DOTA-GMSH was determined to be −2.89, indicating a relatively high hydrophilicity of the radiolabeled compound.

In an attempt to evaluate the binding affinity of the synthesized α-MSH analogue, it was tested on B16-F10 mouse melanoma cells (Fig. 5). DOTA-GMSH inhibited the binding of ¹²⁵I-α-MSH, showing a typical sigmoid curve. An IC₅₀ was obtained for DOTA-GMSH (3.80 nM), indicating a highly nanomolar affinity for MC-1R in this cell line.

FIG. 5.

Binding of [¹²⁵I]-[Nle⁴, D-Phe⁷]-α-MSH on B16-F10 cells by the treatment of DOTA-GMSH (●). Results expressed as a percentage of binding are mean±standard deviation in triplicate. 1×106 B16-F10 cells were incubated at 37°C for 2 hours with 50,000 cpm of [¹²⁵I]-[Nle⁴, D-Phe⁷]-α-MSH (Perkin-Elmer) in the presence of increasing concentrations of the DOTA-GMSH (10⁻⁶–10⁻¹² M) in a 1 mL binding buffer. IC₅₀ values were calculated through a nonlinear regression analysis using the GraphPad Prism5 computer fitting program.

Figure 6 shows the biodistribution of the ¹⁷⁷Lu-DOTA-GMSH in B16-F10 xenografted mice. As shown in Figure 6A, the biggest proportion of the radiolabeled peptide was in the small intestine at 2 hours p.i. and in the large intestine at 24 hours p.i. The %ID of the tumor (1.70±0.35 g) was 4.61±1.89 (2 hours p.i.) and 3.38±1.23 (24 hours p.i.). The %ID/g showed a fast blood clearance and high tumor uptake. The %ID/g of blood was quickly decreased for 24 hours (0.19±0.12 at 2 hours p.i. and 0.04±0.04 at 24 hours), and the %ID/g of the tumor was 2.70±1.06 at 2 hours p.i. and 2.06±0.28 at 24 hours p.i. As a result, the tumor/blood ratio was increased from 14.27 at 2 hours p.i. to 56.37 at 24 hours p.i., which means a high tumor uptake. The radiolabeled peptide was highly retained in the kidneys, at 4.92±0.86%ID/g and 4.85±0.50%ID/g. The tumor/liver ratio was relatively low (6.83 at 2 hours p.i. and 5.42 at 24 hours p.i., respectively). Through a blocking study, there was no significance from the pretreatment of α-MSH peptide by a 100 times molar ratio 30 minutes before the treatment of ¹⁷⁷Lu-DOTA-GMSH.

FIG. 6.

Biodistribution results for ¹⁷⁷Lu-DOTA-GMSH in B16-F10 melanoma xenografted mice. 370 KBq/10^–9 M of the ¹⁷⁷Lu-DOTA-GMSH was administered into a tail vein of 1.70±0.35 g B16-F10 tumor-bearing mice. After 2 and 24 hours p.i., each group was sacrificed, and radioactivities of the organs were counted. The blocking group was administered 10^–7 M of α-MSH 30 minutes before the treatment of ¹⁷⁷Lu-DOTA-GMSH. Data were expressed as a percentage of the administered activity per organ (%ID/organ) (A) and of the administered activity per gram of organ (%ID/g) (B).

In view of high renal uptake proportion, we analyzed the urinary metabolites of ¹⁷⁷Lu-DOTA-GMSH at 3 hours p.i. The HPLC elution profiles of ¹⁷⁷Lu-DOTA-GMSH are shown in Figure 7. The single peak was observed at the same position of ¹⁷⁷Lu-DOTA-GMSH.

FIG. 7.

HPLC profiles of radioactive urine samples. Urine samples were collected for 3 hours after the administration of ¹⁷⁷Lu-DOTA-GMSH using a metabolic cage, and analyzed by HPLC using a C-18 column.

Discussion

The current treatment options for metastatic melanoma are unsatisfactory due to its resistance to conventional chemotherapy and external beam radiation therapy.⁶ Therefore, the great need to develop novel treatment approaches for metastatic melanoma is increasing. In this study, we developed a novel ¹⁷⁷Lu-DOTA-GMSH for melanoma imaging and therapy. In addition, its in vitro and in vivo characteristics were evaluated.

Many anti-melanoma peptides have been developed for the identification of melanoma-associated antigens and receptors.¹⁹ The highly characterized class among them belongs to the α-MSH family, and wild-type α-MSH is a linear tricapeptide (Ac-Ser¹-Tyr²-Ser³-Met⁴-Glu⁵-His⁶-Phe⁷-Arg⁸-Trp⁹-Gly¹⁰-Lys¹¹-Pro¹²-Val¹³-NH₂).²⁰ Since the native α-MSH is rapidly metabolized by proteolytic enzymes, many studies have improved their stability and biological activity in vivo. In an effort to improve it, peptide cyclization enhanced the molecules resistant to in vivo degradation while retaining high bioactivities,^21

–24 and cyclization using lactam bond formation, which was also used in this study, demonstrated an improvement of in vivo stability.¹⁸ In addition, the strategies of substitution of Phe⁷ with D-form Phe enhanced their binding affinity.⁶

To prepare a α-MSH analogue that is more stable and has a higher binding affinity than native α-MSH, we designed a α-MSH analogue, which substituted Phe⁷ and Lys¹¹ with D-form Phe and Arg, respectively. Furthermore, cyclization by a lactam formation between Dap and Asp, and a reduction of the peptide size, except for the sequence of the binding site, were designed, and DOTA BFCAs were conjugated to the compound apart from the receptor-binding site by a solid-phase synthesis to introduce ¹⁷⁷Lu. As a result of modifying the sequence in this study, it still had a nanomolar binding affinity (IC₅₀=3.80 nM) and showed high stability. It was not degraded by serum incubation at 37°C for 7 days (>98%). In addition, the urinary metabolite analysis revealed that ¹⁷⁷Lu-DOA-GMSH was not degraded at 3 hours postinjection, which means a high in vivo stability. It is planned to be compared with the previous α-MSH peptide analogues, which were degraded in a urine sample.^6,18

Interestingly, the HPLC-peak of free ¹⁷⁷Lu was moved by serum incubation. It is assumed that free ¹⁷⁷Lu was bound with an unidentified protein in serum, and its charge and molecular weight were changed. In addition, it might be caused by the sample preparation. Therefore, we have the need to demonstrate ¹⁷⁷Lu in both a stock solution and serum.

The labeling yield was greater than 98% by 15 minutes heating at 90°C. It is well known that a conjugated DOTA chelator can be radiolabeled with a lot of radionuclides such as ⁹⁰Y, ¹⁷⁷Lu, and ¹¹¹In, in various applications for cancer imaging and treatment, which is encouraging.²⁵

Miao et al. has reported melanoma targeting studies using lactam bridge-cyclized α-MSH peptide analogues that are similar with the peptide used in this study. Tumor uptake of ¹¹¹In-DOTA-CycMSH was 9.53±1.41%ID/g at 2 hours p.i. and 2.22±0.51%ID/g at 24 hours p.i., and ¹¹¹In-DOTA-GlyGlu-CycMSH was 10.40±1.40%ID/g at 2 hours and 2.37±0.28%ID/g at 24 hours.¹⁸ In this study, tumor uptake of the radiolabeled peptide was much lower, which was 2.71±1.06%ID/g and 2.05±0.28%ID/g, respectively. However, it is remarkable that the decreased rate of tumor %ID/g was much lower than that of ¹¹¹In-DOTA-CycMSH and ¹¹¹In-DOTA-GlyGlu-CycMSH, at 24 hours p.i., and all peptides in a tumor had a similar %ID/g. Therefore, we can assume that ¹⁷⁷Lu-DOTA-GMSH sustains the binding power more than other peptides. In addition, the tumor-to-blood ratio increased significantly from 14.27 at 2 hours p.i. to 56.37 at 24 hours p.i., which indicates a fast blood clearance and a high tumor uptake. These results are encouraging compared with the results showing that the tumor-to-blood ratio of other peptides were decreased from 2 hours p.i. to 24 hours p.i. However, further investigations that enhance in vivo tumor uptake are still needed.

Kidney retention was observed, and the tumor-to-kidney ratio was 0.55 at 2 hours p.i. and 0.42 at 24 hours p.i., which were somewhat higher than that of other peptides. At 2 hours, most of the radiolabeled peptide was observed in the small intestine, and at 24 hours, it shifted to the large intestine. This demonstrates that it was excreted mainly through the intestine pathway. The administered-radiolabeled peptide was predominantly retained in the tumor except for the excretory pathway, and, therefore, it is desirable to use this peptide for MC-1R mediated-melanoma targeting.

In a blocking study, although pretreated α-MSH peptide was 1000-times greater than administered ¹⁷⁷Lu-DOTA-GMSH, there was no significant difference in the tumor uptake. Since native α-MSH is quickly decomposed in vivo, this might be the cause for a lack of significance in the results of the blocking study. Otherwise, the affinity of DOTA-GMSH is 2.36-times higher than the native α-MSH, and, thus, the uptake of α-MSH might be displaced by ¹⁷⁷Lu-DOTA-GMSH.

In conclusion, a novel ¹⁷⁷Lu-labeled α-MSH analogue, ¹⁷⁷Lu-DOTA-[Gly³-Cyclized(Dap⁴, _d-Phe⁷, Asp¹⁰)-Arg¹¹]α-MSH_3–13 analogue exhibited high stability and binding affinity in vitro, and MC-1R-mediated tumor uptake and retention with fast blood clearance in B16-F10 murine melanoma mouse model in vivo.

Footnotes

Acknowledgments

This study was supported by the Nuclear R&D program through the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology (57141-11).

Disclosure Statement

There are no existing financial conflicts.

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Synthesis and Biological Evaluation of a Novel 177 Lu-DOTA-[Gly 3 -Cyclized(Dap 4,d -Phe 7,Asp 10 )-Arg 11 ]α-MSH 3–13 Analogue for Melanocortin-1 Receptor-Positive Tumor Targeting

Abstract

Introduction

Materials and Methods

Materials

Preparation of chelator conjugated peptide: DOTA-GMSH

Radiochemistry of 177Lu-labeled DOTA-GMSH

Preparation of 177Lu-labeled DOTA-GMSH

Serum stability assay

Determination of log p value

Biological evaluation of 177Lu-DOTA-GMSH

Cell culture

Competitive binding assay

Animal models

Biodistribution assay

Urinary metabolites of 177Lu-DOTA-GMSH

Results

Discussion

Footnotes

Acknowledgments

Disclosure Statement

References

Radiochemistry of ¹⁷⁷Lu-labeled DOTA-GMSH

Preparation of ¹⁷⁷Lu-labeled DOTA-GMSH

Biological evaluation of ¹⁷⁷Lu-DOTA-GMSH

Urinary metabolites of ¹⁷⁷Lu-DOTA-GMSH