(Radio)pharmacological characterization of novel α-MSH derivatives

Objectives: Melanocortin-1 receptor (MC1R) is well known to be overexpressed in melanoma. Thus, it has been a great interest in targeting this receptor for diagnosis of human metastasized melanoma. We aimed at investigating
(radio)pharmacological properties of novel derivatives of the α-melanocyte-stimulating hormone (α-MSH) and selecting most promising candidates for further studies in melanoma models in vivo.
Methods: Linear and cyclic α-MSH derivatives (NAP-NS1(1), NOTA-NAP-NS1(2), ^natCu-NOTA-NAP-NS1(3), NAP-NS2(4), NOTA-NAP-NS2(5), ^natCu-NOTA-NAP-NS2(6), DPA-NAP-NS1(7) and Re-tricarbonyl-DPA-NAPNS1(8)) were investigated in competition assays in both murine B16F10 and human MeWo cells. In vitro stabilities of [⁶⁴Cu]Cu-2, [⁶⁴Cu]Cu-5 and ^99mTc-tricarbonyl-7 were tested in phosphate buffer (pH=7.4) and human serum at 37°C for 1h and 24h. Transchelation and octanol/water partition coefficients of radiolabeled peptides were also investigated. Additionally, [⁶⁴Cu]Cu-2, [⁶⁴Cu]Cu-5 and ^99mTc-tricarbonyl-7 with high radiochemical purities and specific activities were applied in saturation assays and kinetic studies.
Results: Linear α-MSH derivatives (1, 2, 3, 7 and 8) showed higher affinities on both murine and human cells than cyclic α-MSH derivatives (4, 5, 6). Linking the chelator to the peptide and coordinating the chelator-peptide with
^natCu or Re were accompanied by some loss of affinity. [⁶⁴Cu]Cu-2, [⁶⁴Cu]Cu-5 and ^99mTc-tricarbonyl-7 were stable in phosphate buffer and serum at 37°C after incubation for 1h and 24h. No transchelation of radiolabeled peptides was observed in cysteine and histidine challenge experiments. LogD values suggested that [⁶⁴Cu]Cu-2 (-2.30±0.01) and [⁶⁴Cu]Cu-5 (-3.39±0.04) had higher hydrophilicity than ^99mTc-tricarbonyl-7 (-0.43±0.01). Saturation studies in both cell lines resulted in K_d values (nM) in the lower nanomolar ranges for [⁶⁴Cu]Cu-2 (B16F10: 1.7±0.2; MeWo: 2.6±0.5) and ^99mTc-tricarbonyl-7 (B16F10: 6.0±0.5; MeWo: 4.5±0.8). But B_max (fmol/mg protein) of [⁶⁴Cu]Cu-2 on murine and human cells (B16F10: 46.6±3.9; MeWo: 16.6±1.6) was notably lower than that of ^99mTc-tricarbonyl-7 (B16F10: 403.5±46.1; MeWo: 50.3±6.4). Kinetic study of [⁶⁴Cu]Cu-2 in murine cells showed rapid cellular association and dissociation in vitro.
Conclusions: [⁶⁴Cu]Cu-2 showed high stability, hydrophilicity, binding affinities and rapid cellular association and dissociation in vitro, which made it promising for further investigations in melanoma models.