Date Published: March 22, 2019
Publisher: Public Library of Science
Author(s): Masato Kobayashi, Toshitaka Kato, Kohshin Washiyama, Masaaki Ihara, Asuka Mizutani, Kodai Nishi, Leo G. Flores, Ryuichi Nishii, Keiichi Kawai, Gayle E. Woloschak.
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.
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.
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.
The 4-arm DOTA construct has better chemical properties for peptide radiotracers than the 3-arm DOTA construct.
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.
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 111In-labeled 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 111In-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 111In-labeled 3-arm and 4-arm DOTA-α-MSH (Fig 4). However, the lipophilicity of 111In-labeled 4-arm DOTA-α-MSH was slightly closer to zero than that of 111In-labeled 3-arm DOTA-α-MSH according to the partition coefficients. We estimated that the lipophilicity resulted in a slightly different distribution between 111In-labeled 3-arm DOTA-α-MSH and 111In-labeled 4-arm DOTA-α-MSH (Tables 2 and 3).
111In-labeled 4-arm DOTA-α-MSH yields higher tumor accumulation and lower renal accumulation than 111In-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.