α-Melanocyte-stimulating hormone was a gift from the Novartis Institutes of Biomedical Research (Basel, Switzerland). [Nle⁴,D-Phe⁷]-α-MSH (NDP-MSH) was obtained from Bachem (Bubendorf, Switzerland). DOTA-tris(tert-butyl ester) (1,4,7,10-tetraazacyclododecane-1,4,7-tris-tert-butyl acetate-10-acetic acid) was purchased from Macrocyclics (Dallas, TX, USA), Fmoc-PAL-PEG-PS resin from Applied Biosystems (Rotkreuz, Switzerland), Fmoc-d-Asp(tBu)-TentaGel S AC resin from Rapp-Polymere (Tübingen, Germany), Fmoc-amino acids from Novabiochem (Läufelfingen, Switzerland), and Kaiser test kits from Sigma-Aldrich (Buchs, Switzerland). N-Succinimidyl iodoacetate and iodogen tubes were from Pierce Biotechnology (Rockford, IL, USA), Na¹²⁵I (3.7 GBq/mL) from Perkin Elmer (Waltham, MA, USA), and ¹¹¹InCl₃ (370 MBq/mL) from Mallinckrodt (Petten, The Netherlands). 1,10-Phenanthroline was bought from Merck (Darmstadt, Germany), and all other organic reagents were obtained from Sigma-Aldrich. All reagents were of highest purity available. Cell culture media were from Biochrom AG (Berlin, Germany) and Sigma-Aldrich. Penicillin, streptomycin, vitamins, and non-essential amino acids were bought from Gibco/Invitrogen (Carlsbad, CA, USA) or Sigma-Aldrich.

Continuous-flow peptide synthesis was carried out on a Pioneer peptide synthesizer from PerSeptive Biosystems (Framingham, MA, USA). Analytical reverse-phase high-pressure liquid chromatography (RP-HPLC) was performed on a PU-980 system from Jasco (Easton, MD, USA) using a Vydac 218TP54 C18 (5 µm, 4.6 mm × 250 mm) or a Phenomenex Jupiter C18 300 Å (5 µm, 4.6 mm × 250 mm) analytical column. DOTA-NAP-amide-d-Asp-d-Asp was chromatographed with a gradient between solvent A [0.1% trifluoroacetic acid (TFA) in H₂O] and solvent B (0.1% TFA in 70:30 acetonitrile/H₂O). The 40-min gradient cycle consisted of the following parts: 95% A (0–2 min), 95–70% A (2–10 min), 70–30% A (10–30 min), 30–5% A (30–34 min), 5% A (34–36 min), 5–95% A (36–38 min), and 95% A (38–40 min); the flow rate was 1 mL/min. UV absorption was recorded at 280 nm using a Jasco UV-1570 detector. DOTA-Phospho-MSH_2-9 was chromatographed on a Phenomenex Jupiter column using the same gradient, except that solvent A was replaced by 0.02 M ammonium acetate. Mass spectra were determined on a Finnigan LCQ Deca electrospray ion trap mass spectrometry (MS) system.

The purity of the radioligands was assessed by RP-HPLC using a dedicated Jasco PU-980 chromatography system equipped with a Spherisorb ODS2/5-μm column and a Radiomatic 500TR LB506C1 γ-detector (Packard, Meriden, CT, USA). Solvent A was 0.1% TFA in H₂O; solvent B was 0.1% TFA in acetonitrile; the gradient consisted of 96% A (0–2 min), 96–45% A (2–22 min), 45–25% A (22–30 min), 25% A (30–32 min), and 25–96% A (32–34 min); the flow rate was 1.0 mL/min. A cell harvester (Packard) was used to collect cell-bound radioactivity from binding assays on filters. Their radioactivity was measured on a TopCount microplate scintillation counter (Packard). Radioactivity in internalization and biodistribution assays was measured on a Cobra II Auto-Gamma γ-counter (Packard). For bioassays, melanin content in cell culture media of each well was quantified on a Spectra Max 190 microplate reader (Molecular Devices, Menlo Park, CA, USA) using a wavelength of 310 nm.

Mouse B16-F1 melanoma cells (39) were cultured in modified Eagle’s medium (MEM) containing 10% heat-inactivated fetal calf serum, 2 mmol/L l-glutamine, 1% non-essential amino acids, 1% vitamin solution, 50 IU/mL penicillin, and 50 µg/mL streptomycin, in a humidified atmosphere consisting of 95% air and 5% CO₂ at 37°C. For cell expansion or experiments with isolated cells, the B16-F1 cells were detached with 0.02% EDTA in phosphate-buffered saline (PBS) (150 mM, pH 7.2–7.4). The human HBL melanoma cell line (3, 4) was cultured in modified RPMI medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM l-glutamine, 50 IU/mL penicillin, and 50 µg/mL streptomycin in the same conditions as for B16-F1 cells.

Triplicates of 100-µL volumes of B16-F1 or HBL cell suspensions adjusted to 4 × 10⁶/mL were incubated in 96-well U-bottom microplates (Falcon 3077). The binding medium consisted of MEM with Earle’s salts, 0.2% BSA, and 0.3 mM 1,10-phenanthroline. This binding medium was called mouse binding medium (MBM). Triplicates of competitor peptide solution (50 µL) yielding final concentrations ranging from 1 × 10⁻⁶ to 1 × 10⁻¹² M were added. [¹²⁵I]NDP-MSH (50,000 cpm) in 50 µL was finally added to each well. The plates were incubated at 15°C for 3 h for B16-F1 cells and at 37°C for 2 h for HBL cells. The incubation was stopped by covering the plates with ice for 10 min. A cell harvester was used to collect cell-bound radioactivity on filters (Packard Unifilter-96 GF/B). The collected radioactivity was counted on a TopCount scintillation counter (Packard) after addition of 50 µL Microscint-20 scintillation cocktail (Perkin Elmer). The IC₅₀ values were calculated with the Prism 6 software (GraphPad Software Inc., San Diego CA, USA).

The biological activity of the α-MSH derivatives was assessed with an in situ melanin assay (40). Briefly, B16-F1 cells (2,500 cells per well in 100 µL) were distributed into 96-well flat-bottom cell culture plates. MEM without phenol red, supplemented with 10% heat-inactivated fetal calf serum, 2 mM l-glutamine, 0.31 mmol/L l-tyrosine, 1% non-essential amino acids, 1% vitamin solution, 50 IU/mL penicillin, and 50 µg/mL streptomycin, was used as culture medium. After overnight incubation at cell culture conditions mentioned above, concentrations of α-MSH derivatives ranging from 1 × 10⁻⁸ to 1 × 10⁻¹² in 100-µL volumes were added (in threefold dilution steps), and the incubation was continued for an additional 72 h. Melanin production was quantified by determining the absorbance at 310 nm in a microplate reader.

B16-F1 cells were seeded in six-well plates and incubated overnight in MEM at 37°C. For the internalization experiments, MEM was replaced by 1 mL MBM (see above) as internalization buffer. After a 1-h incubation at 37°C, 74 kBq of radioligand (¹¹¹In-labeled peptides) was added, and the plates were incubated for different time periods. Non-specific internalization was determined by addition of 50 µL of a 1 µM α-MSH solution to the incubation mixture. After the desired incubation times, the cells were extensively washed with MBM kept at 37°C to remove excess radioligand. Incubation in 2 mL ice-cold acid buffer (acetate-buffered Hank’s balanced salt solution, pH 5) for 10 min allowed dissociation of surface-bound ligand. After collection of the acid buffer fraction, the cells were rinsed once with cold MBM, and the washings were pooled with the acid buffer fraction. The cells were washed again with MBM kept at 37°C, lysed in a 1% Triton X-100 solution, and finally transferred to tubes for quantification. The radioactivity of all collected fractions was measured in a γ-counter. A counting plate underwent the same treatment as the plate incubated for the longest time, but its cells were detached with 0.02% EDTA in PBS instead of being lysed with the Triton X-100 solution. Cells from three wells were collected, counted, and thus allowed for normalization of the results obtained. Results were expressed as percent of the added dose per million cells.

About 5 × 10⁵ B16-F1 cells were implanted subcutaneously to female B6D2F1 mice (C57BL/6 × DBA/2F1 hybrids; breeding pairs obtained from IFFA-CREDO, France). Seven days later, 185 kBq of ¹¹¹In-labeled ligand in 200 µL PBS/0.1% BSA were injected intravenously into the lateral tail vein of each mouse. Control mice were injected with a mixture of the tracer and 50 µg α-MSH to determine the non-specific uptake of radioligand. The animals were sacrificed 4, 24, and 48 h postinjection, dissected, and the tissues of interest collected, rinsed of excess blood, and weighed. The radioactivity emitted by each organ was measured in a γ-counter to determine the tissue uptake as percentage of the injected dose (ID) per gram of tissue. The total of injected radioactivity per animal was determined by extrapolation from the counts of a standard collected from the injection solution for each animal. Urine samples were collected at 10, 15, and 20 min and 4 h after injection and kept frozen at −80°C until use. Urine (1 vol) was mixed with methanol (2 vol) to precipitate proteins, and the supernatant was analyzed by RP-HPLC/γ-detection, as described in Ref. (12).

Results are expressed as means ± SEM, unless otherwise stated. The statistical evaluation of data was performed using the one- or two-way ANOVA test. When significant overall effects were obtained by ANOVA, multiple comparisons were made with the Bonferroni correction. P < 0.05 was considered statistically significant. The area under the curve (AUC) was calculated with the GraphPad Prism 6 software for the indicated period of time, using the mean tissue uptake value at each time point.

All DOTA-peptides were obtained in >99% purity. The synthesis of DOTA-NAP-amide had an overall yield (after RP-HPLC purification) of 15%, DOTA-NAP-d-Asp-d-Asp 8.3%, and DOTA-Phospho-MSH_2-9 5.3%. The expected molecular weights were confirmed by MS. The net charges of DOTA-NAP-d-Asp-d-Asp and DOTA-Phospho-MSH_2-9 at physiological pH are −2 and −1, respectively, calculated on the basis of known pK_a values for amino acid residues and functional groups.

The binding affinity of the peptides to MC1R was assessed by competition binding assays using [¹²⁵I]-NDP-MSH as radioligand and both murine B16-F1 and human HBL cells. Table 1 summarizes the IC₅₀ values obtained for the tested peptide compared to the values of the native ligand α-MSH and the reference peptide DOTA-NAP-amide. DOTA-Phospho-MSH_2-9 displayed affinities in the nanomolar range on both cell lines. Although the IC₅₀ obtained for DOTA-Phospho-MSH_2-9 with B16-F1 cell line was slightly lower than that of DOTA-NAP-amide (2.32 ± 0.80 vs. 1.38 ± 0.35 nmol/L), the binding affinity with HBL cells was comparable (3.03 ± 0.59 vs. 3.09 ± 1.11 nmol/L). DOTA-Phospho-MSH_2-9 displayed good α-MSH agonist activity (Table 1), as demonstrated by the induction of melanin synthesis by B16-F1 cells at a dose matching its IC₅₀. By contrast, C-terminal extension of DOTA-NAP by d-Asp-d-Asp led to a ~10-fold lower MC1R affinity compared to that of DOTA-NAP-amide with both B16-F1 and HBL cells. The biological activity in the melanin assay was also around 11-fold lower than the reference peptide.

[¹¹¹In]DOTA-NAP-d-Asp-d-Asp and [¹¹¹In]DOTA-Phospho-MSH_2-9 exhibited excellent internalization profiles when studied in vitro with cultured B16-F1 cells (Figure 3). The plateau phase was not quite reached by the first peptide after 3.5 h, probably because of the lower receptor affinity; this may predict lower in vivo tumor uptake. By contrast, the plateau phase for [¹¹¹In]DOTA-Phospho-MSH_2-9 was almost complete after 3.5 h, indicating that maximal internalization of the peptide was reached after 4 h, the first test point with in vivo biodistribution studies. It appears that MC1R internalization was not altered by the negatively charged ligands.

Tissue distributions in B16-F1 melanoma-bearing mice of [¹¹¹In]DOTA-NAP-d-Asp-d-Asp and [¹¹¹In]DOTA-Phospho-MSH_2-9 were compared with the reference peptide [¹¹¹In]DOTA-NAP-amide (Table 2). Tissues, including melanoma tumors, were collected 4, 24, and 48 after injection of the radiopeptides. Clearance from the blood was faster for the two new radiopeptides compared with the reference peptide as almost no radioactivity could be detected after 4 h. Kidney uptake of [¹¹¹In]DOTA-NAP-d-Asp-d-Asp was 5.95 ± 0.85 %ID/g 4 h after injection, i.e., the uptake was 25% higher than that of [¹¹¹In]DOTA-NAP-amide (4.77 ± 0.26 %ID/g), which was not expected. After 24 h, the radioactive load was even 70% higher than with the reference peptide. On the other hand, owing to the lower receptor affinity of [¹¹¹In]DOTA-NAP-d-Asp-d-Asp, it was anticipated that tumor uptake (1.93 ± 0.11 %ID/g after 4 h) was lower than that of [¹¹¹In]DOTA-NAP-amide (7.77 ± 0.35 %ID/g). Other organs did not substantially accumulate the radiopeptide.

[¹¹¹In]DOTA-Phospho-MSH_2-9 displayed more promising data: after 4 h, tumor uptake was 7.33 ± 0.47 %ID/g, i.e., almost identical with that of [¹¹¹In]DOTA-NAP-amide, and kidney uptake was 2.68 ± 0.18 %ID/g, which corresponds to 56% of that of the reference peptide (Table 2). Non-specific uptake by other organs was very low and did not exceed the values found with [¹¹¹In]DOTA-NAP-amide (Figure 4), thus excluding an altered excretion pathway. Coinjection of 50 µg of α-MSH together with the radiopeptide significantly blocked melanoma uptake and confirmed MC1R-mediated internalization (data not shown). The retention of radioactivity by the tumor was decreased to 40% at 24 h postinjection and to 15% at 48 h postinjection. These data are similar to those found with other MSH radiopeptides. On the other hand, clearance from the kidney appeared to be slightly slower. The tumor-to-kidney ratios calculated for 4, 24, and 48 h were 2.75, 1.55, and 1.16, respectively (Table 3). This results in an AUC (4–48 h) of 1.81 compared with 1.17 for [¹¹¹In]DOTA-NAP-amide.

Kidney and liver uptake of [¹¹¹In]DOTA-NAP-d-Asp-d-Asp, [¹¹¹In]DOTA-Phospho-MSH_2-9, and [¹¹¹In]DOTA-NAP-amide (reference peptide) was compared with that of five previously published peptides (14) that altogether cover an overall net charge ranging between +2 and −2 (Table 4). As shown in Figure 5, kidney uptake is lowest for net charge −1; it is considerably increased for −2 and +2 and moderately for +1 or 0. Of the two peptides with a net charge of −1, [¹¹¹In]DOTA-Phospho-MSH_2-9 showed a slightly lower kidney uptake than [¹¹¹In-DOTA-Nle⁴,Asp⁵,d-Phe⁷,Lys¹¹(Suc)]-α-MSH_4-11-carboxylate, indicating that incorporating the negative charges in the N-terminal region is more advantageous than in the C-terminal region. In addition, the AUC (4–48 h) for [¹¹¹In]DOTA-Phospho-MSH_2-9 was 1.81, i.e., the tumor-to-kidney ratio was threefold higher than that of [¹¹¹In-DOTA-Nle⁴,Asp⁵,d-Phe⁷,Lys¹¹(Suc)]-α-MSH_4-11-carboxylate exhibiting an AUC (4–48 h) of 0.62 (14). With respect to the liver, non-specific uptake does not follow the same pattern as outlined for the kidneys (Figure 6). Other factors such as differences in lipophilicity may play a more important role. It should be stressed, however, that liver uptake is about 20-fold lower than that reported for the kidneys and therefore of minor importance for these peptides.