The pharmacological properties of 3-arm or 4-arm DOTA constructs for conjugation to α-melanocyte-stimulating hormone analogues for melanoma imaging

Background Although a 3-arm DOTA construct, which has three carboxylic acids, h has been applied for conjugation to many peptides, we investigated if a 4-arm DOTA construct conjugated to peptides improves chemical properties for melanoma imaging of the melanocortin 1 receptor compared to 3-arm DOTA-conjugated peptides. Methods Specific activities, radiolabeling efficiencies, and partition coefficients were evaluated using 111In-labeled 3-arm and 4-arm DOTA-α-melanocyte-stimulating hormone (MSH). For assessment of MC1-R affinity and accumulation in tumor cells in vitro, B16-F1 melanoma and/or 4T1 breast cancer cells were incubated with 111In-labeled 3-arm and 4-arm DOTA-α-MSH with and without α-MSH as a substrate. The stability was evaluated using mouse liver homogenates and plasma. Biological distribution and whole-body single photon emission computed tomography imaging of 111In-labeled 3-arm and 4-arm DOTA-α-MSH were obtained using B16-F1 melanoma-bearing mice. Results Specific activities and radiolabeling efficiencies of both radiotracers were about 1.2 MBq/nM and 90–95%, respectively. The partition coefficients were −0.28 ± 0.03 for 111In-labeled 3-arm DOTA-α-MSH and −0.13 ± 0.04 for 111In-labeled 4-arm DOTA-α-MSH. Although accumulation was significantly inhibited by α-MSH in B16-F1 cells, the inhibition rate of 111In-labeled 4-arm DOTA-α-MSH was lower than that of 111In-labeled 3-arm DOTA-α-MSH. 111In-labeled 4-arm DOTA-α-MSH was taken up early into B16-F1 cells and showed higher accumulation than 111In-labeled 3-arm DOTA-α-MSH after 10 min of incubation. Although these stabilities were relatively high, the stability of 111In-labeled 4-arm DOTA-α-MSH was higher than that of 111In-labeled 3-arm DOTA-α-MSH. Regarding biological distribution, 111In-labeled 4-arm DOTA-α-MSH showed significantly lower average renal accumulation (1.38-fold) and significantly higher average melanoma accumulation (1.32-fold) than 111In-labeled 3-arm DOTA-α-MSH at all acquisition times. 111In-labeled 4-arm DOTA-α-MSH showed significantly higher melanoma-to-kidney, melanoma-to-blood, and melanoma-to-muscle ratios than 111In-labeled 3-arm DOTA-α-MSH. Conclusions The 4-arm DOTA construct has better chemical properties for peptide radiotracers than the 3-arm DOTA construct.


Introduction
The incidence rate of malignant melanoma has been steadily increasing over the past 40 years. The 5-year survival rate with stage IV metastatic melanoma is currently less than 20% because few effective treatments have been established [1,2]. Because survival is associated with an earlier stage at detection and treatment, specific and highly detectable imaging of melanoma tumors is strongly desired.
The melanocortin 1 receptor (MC1-R) is one of the most targeted melanoma antigens and belongs to the melanocortin family of G protein-coupled receptors, which consists of five receptor subtypes, MC1-R to MC5-R. MC1-R is expressed in nearly all primary and metastatic melanomas [3] and 95% of uveal melanomas [4]. MC1-R is an attractive receptor for molecular-targeted imaging and radionuclide therapy of melanoma. α-melanocyte-stimulating hormone (α-MSH), a tridecapeptide, is an endogenous ligand for the melanocortin family of receptors, with subnanomolar binding affinity to MC1-R [5].
Radionuclide-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)chelate has been conjugated to many peptides and provides better specific activity, MC1-R affinity, and stability for melanoma imaging of MC1-R [12]. Although the 3-arm DOTA construct, which has three carboxylic acids, has been applied for conjugation to many peptides, the 4-arm DOTA construct, which has four carboxylic acids, improves radiochemical yield, specific activity, and stability of therapeutically active conjugates to antibodies for radioimmunotherapy of cancer [13]. In this study, we investigated if the 4-arm DOTA construct conjugated to peptides improves the chemical properties for melanoma imaging of MC1-R compared to 3-arm DOTA-conjugated peptides. Numerous α-MSH conjugated peptide analogues have been developed with high affinities and specificities for α-MSH receptors [14]. Native α-MSH, as an example of a peptide, was selected to evaluate improvement of chemical properties using the 4-arm DOTA construct because 3-arm DOTA-conjugated native α-MSH (3-arm DOTA-α-MSH, Fig 1A) yielded relatively low MC1-R affinity and stability compared to other 3-arm DOTA-conjugated peptides [14].

Synthesis and purification of 3-arm and 4-arm DOTA constructs
The conjugation procedure was performed using the methods of Maguire and Greg et al., with some modifications [13,15]. Briefly, to form the 3-arm DOTA-α-MSH, α-MSH free acid (1 eq) was dissolved in purified water (10 mg/mL solution) and conjugated to DOTA-NHS-ester (40 eq), which was dissolved in N,N-dimethylformamide (40 mg/mL solution), using 0.1 M NaHCO 3 buffer (pH 7-8) for 6 hours at room temperature.
These resulting products were purified using high performance liquid chromatography (HPLC, Hitachi, Ibaraki, Japan) using a 5C18-AR-II column (Cosmosil, Nakalai Tesque, Kyoto, Japan), a combination gamma counter, and 210-nm ultraviolet light. For the 3-arm DOTA construct, the initial solvent was 0.05% trifluoroacetic acid in water (pH 2.2): acetonitrile at 78:22 after injection, and was gradually changed to 74:26 after 20 min as the eluent at a flow rate of 1.0 mL/min. For the 4-arm DOTA construct, the initial solvent was 0.05% trifluoroacetic acid in water (pH 2.2): acetonitrile at 78:22 after injection, and was gradually changed to 75:25 after 40 min as the eluent at a flow rate of 1.0 mL/min.
The partition coefficients of 111 In-labeled 3-arm and 4-arm DOTA-α-MSH were measured using 2.0 mL n-octanol as the organic phase and 2.0 mL 0.l M phosphate buffer as the aqueous phase (pH 7.4 for plasma) [18]. N-octanol and the buffer were pre-mixed twice using a mechanical mixer for 1 min at room temperature. Then, 20 υL radioactive sample were added and mixed twice using a mechanical mixer for 1 min at room temperature. The radioactivity of 200 uL of each phase was measured after centrifugation. Calculation of log (n-octanol/0.l M phosphate buffer) was performed.

Tumor cells
Mouse skin melanoma B16-F1 cells with high MC1-R expression and mouse breast cancer 4T1 cells with low MC1-R expression were obtained from American Type Culture Collection. B16-F1 cells and 4T1 cells were cultured in Dulbecco's Modified Eagle's Medium (Wako, Osaka, Japan) and Roswell Park Memorial Institute-1640 (Wako), respectively, supplemented with 10% fetal bovine serum (Dainippon Sumitomo, Osaka, Japan) at 37˚C in a 5% CO 2 incubator.

In vitro assays with B16-F1 and 4T1 cells
In vitro assays were performed using our methods, with some modifications [19]. Briefly, for the inhibition study, B16-F1 and 4T1 cells were seeded into 24-well cell culture multiwell plates at a density of 5×10 5 cells/well. Assays were conducted 24 hours after seeding. B16-F1 and 4T1 cells were pre-incubated with each type of medium including 1.0 υM α-MSH for 180 min at 37˚C. B16-F1 and 4T1 cells were incubated for 180 min with 111 In-labeled 3-arm or 4-arm DOTA-α-MSH.
For the accumulation study with 111 In-labeled 3-arm and 4-arm DOTA-α-MSH, each well was incubated with each type of medium for 10 min at 37˚C. Then, 20 kBq/well 111 In-labeled 3-arm or 4-arm DOTA-α-MSH was added and incubated for 10, 30, 60, 120, and 180 min at 37˚C as the control condition. At the end of the incubation in the inhibition and accumulation studies, each well was rapidly washed twice with 500 μL 0.5 M HEPES buffer. Cells were then solubilized in 500 μL 0.1 M NaOH. The radioactivity that accumulated in B16-F1 and 4T1 cells was measured with a gamma counter (ARC-380; Hitachi-Aloka Medical, Tokyo, Japan). B16-F1 and 4T1 cells were detached with trypsin, and the protein in the cells was measured using a protein assay. All experimental conditions were examined with quadruplicate assays.

Stability analysis of 111 In-labeled 3-arm and 4-arm DOTA-α-MSH in mice
All animal studies were conducted following approval by the Animal Care Committee of Kanazawa University (AP-122339). Fasted B16-F1-bearing C57BL6 male mice (5 weeks old) were administered 111 In-labeled 3-arm or 4-arm DOTA-α-MSH (3.7 MBq/mouse) via the tail vein. At 10, 30, 60, 120, and 180 min after injection, mice (n = 3 per time point) were euthanized with isoflurane, and blood and liver were collected and analyzed with thin layer chromatography (TLC). Briefly, 300 uL blood in a heparinized tube was centrifuged at 18,000 ×g at 4˚C for 5 min. A total of 30 uL perchloric acid was added to the supernatant, which was centrifuged again, after which the final supernatant was spotted onto the TLC plate as plasma. Krebs-Ringer phosphate buffer (pH 7.4) was added to the liver samples, followed by homogenization with an ultrasonic homogenizer (SONIFIER250, Branson, MO, USA). Then, ethanol was added to the homogenate to remove proteins, and the sample was centrifuged at 18,000 ×g at 4˚C for 5 min. The final supernatant was spotted onto the reverse-phase TLC plate, and the TLC spots were developed using 50 mM EDTA (pH 4-5) solvents. After development and complete drying, the TLC plates were cut into 21 fractions, and the radioactivity associated with each fraction was quantified using a γ-ray counter. The fraction ratios of 111 In 3+ , 111 Inlabeled 3-arm or 4-arm DOTA-α-MSH, and all metabolites were calculated by dividing the radioactive counts for each fraction by the total radioactivity counts. A stability study showed that the fraction of 111 In-labeled 3-arm or 4-arm DOTA-α-MSH remained the same at all injection times.

Biological distribution and whole-body single photon emission computed tomography (SPECT) imaging of 111 In-labeled 3-arm and 4-arm DOTA-α-MSH in B16-F1-bearing mice
C57BL6 male mice (5 weeks old) were transplanted with B16-F1 cells (5 × 10 5 cells/100 μL) into the thigh of the mice [19]. The tumors appeared and gradually increased in size after about 10 days. The mice were housed and continuously monitored each day for 2 weeks in a 12-hour light/12-hour dark cycle with free access to food and water. The tumor size reached 8.7 ± 2.8 mm at 2 weeks after transplant of cells. Two mice with tumor sizes of 13 and 15 mm were excluded from our experiments. Mice with a tumor size <10 mm were fasted with no food overnight with water supplied ad libitum before experiments.
For SPECT imaging, 111 In-labeled 3-arm or 4-arm DOTA-α-MSH was injected into the tail vein of four B16-F1-bearing mice in total (37 MBq/mouse). For SPECT imaging, acquisition was started at 10 min after injection and continued every 10 min for 180 min. The data were reconstructed using the ordered subset expectation maximization method with 16 subsets and six iterations including no scatter and attenuation correction. The voxel size was set to 0.8×0.8×0.8 mm. Post-reconstruction smoothing filtering was applied using a 1.0-mm 4-arm DOTA construct affects the biological distribution Gaussian filter. Image displays were obtained using medical image data analysis software, AMIDE (ver. 1.04). The coronal images were displayed as maximum intensity projections. In these images, three to five regions of interest were placed over the kidney, muscle, and B16-F1 melanoma in which accumulation could be seen, and the time activity curve of each was obtained as the %ID/g.

Statistical analysis
Data are presented as the mean and standard deviation (SD). P values were calculated using a two-tailed Student's t-test. Results were considered to be significant at P < 0.01.

Results
Specific activity, radiolabeling efficiency, and partition coefficients of

Discussion
In this study, the 4-arm DOTA construct was newly applied for conjugation to α-MSH to evaluate specific activity, radiolabeling efficiency, MC1-R affinity, stability, and tumor accumulation in melanoma imaging. Although specific activity and radiolabeling efficiency of 111 Inlabeled 4-arm DOTA-α-MSH were not much different from those of 3-arm DOTA-α-MSH, which is the generally used DOTA construct, specific activity was quite low compared to labeling with other MC1-R analogues [14,20]. Because the partition coefficients of 111 In-labeled 3-arm and 4-arm DOTA-α-MSH were negative, these radiotracers are water soluble and are usually renally excreted. We observed high renal accumulation and excretion of 111 In-labeled 3-arm and 4-arm DOTA-α-MSH (Fig 4). However, the lipophilicity of 111 In-labeled 4-arm DOTA-α-MSH was slightly closer to zero than that of 111 In-labeled 3-arm DOTA-α-MSH according to the partition coefficients. We estimated that the lipophilicity resulted in a slightly different distribution between 111 In-labeled 3-arm DOTA-α-MSH and 111 In-labeled 4-arm DOTA-α-MSH (Tables 2 and 3).
In the inhibition study (Fig 2), because accumulation of both 111 In-labeled 3-arm and 4-arm DOTA-α-MSH was significantly inhibited by the substrate, α-MSH, in B16-F1 cells with high MC1-R expression, and not inhibited in 4T1 cells with low MC1-R expression, both   Table 1). The stability of 111 In-labeled 4-arm DOTA-α-MSH was higher than that of 111 In-labeled 3-arm DOTA-α-MSH. In addition, these stabilities were relatively higher than another peptide radiotracer conjugated to 3-arm DOTA [21]. One reason may be that eightcoordinate complexes of 111 In-labeled 4-arm DOTA conjugation are generally more stable than seven-coordinate complexes of 111 In-labeled 3-arm DOTA conjugation [22,23].
In the biological distribution of 111 In-labeled 3-arm (Table 2) and 4-arm DOTA-α-MSH (Table 3) and whole-body SPECT imaging (Fig 4), accumulation of 111 In-labeled 4-arm DOTA-α-MSH was significantly higher than that of 111 In-labeled 3-arm DOTA-α-MSH in B16-F1 melanoma. This also supports the similar results from the in vitro study in Fig 3. 111 Inlabeled 4-arm DOTA-α-MSH showed significantly lower renal accumulation than 111 Inlabeled 3-arm DOTA-α-MSH at all acquisition times. The number of carboxy groups affects electric charges in the whole body and changes the whole-body distribution of peptide radiotracers [24]. Thus, the effect of electric charges may yield lower renal accumulation. For peptide radiotracers, reducing renal accumulation is important. Behr et al. showed that systemic administration of cationic amino acids reduces renal reabsorption and accumulation of peptide radiotracers [25]. However, amino acid infusion may change the whole-body distribution and increase background accumulation in muscle, blood, and kidney etc. On the other hand, 111 In-labeled 4-arm DOTA-α-MSH showed significantly higher melanoma accumulation than 111 In-labeled 3-arm DOTA-α-MSH. This may be because of the slightly higher lipophilicity, significantly higher MC1-R affinity, and relatively higher stability of 4-arm DOTA-α-MSH compared to 3-arm DOTA-α-MSH. If renal accumulation is reduced and tumor accumulation is increased using other peptides conjugated to the 4-arm DOTA construct compared with the 3-arm DOTA construct, the 4-arm construct will be a simple and useful DOTA construct for labeling of peptide radiotracers.

Time (min)
Melanoma-to-kidney Melanoma-to-blood Melanoma-to-muscle In-labeled DOTA peptides [12] because we selected the native α-MSH peptide hormone to evaluate the chemical properties between the 3-arm DOTA and 4-arm DOTA constructs. We selected native α-MSH as a good peptide to evaluate improvement of chemical properties using the 4-arm DOTA construct because 3-arm DOTA-α-MSH has relatively low yield, MC1-R affinity, and stability compared to other 3-arm DOTA-conjugated peptides [14]. The IC 50 values of 111 In-labeled 3-arm and 4-arm DOTA-α-MSH were 4.8 ± 0.5 nM and 5.3 ± 0.4 nM, respectively. These are similar to or better than those of numerous 111 In-labeled α-MSH analogues (0.9-78.6 nM) [12]. Therefore, utilization of α-MSH for comparison of 111 In-labeled 3-arm and 4-arm DOTA peptides was considered appropriate. However, when other peptides are applied for 111 In labeling of the 4-arm DOTA construct, melanoma-to-tissue ratios will improve more than other 111 In-labeled DOTA peptides with the 3-arm DOTA construct. Thus, 4-arm DOTA peptides will be effective for tumor diagnosis because of their slightly higher lipophilicity, higher MC1-R affinity, and relatively higher stability compared to 3-arm DOTA peptides. https://doi.org/10.6084/m9.figshare.7698239

111
In-labeled 4-arm DOTA-α-MSH yields higher tumor accumulation and lower renal accumulation than 111 In-labeled 3-arm DOTA-α-MSH because the chemical properties of the 4-arm DOTA construct include slightly higher lipophilicity, significantly higher MC1-R affinity, and relatively higher stability. Therefore, 4-arm DOTA constructs provide better chemical properties for peptide radiotracers than 3-arm DOTA constructs.