Melanoma targeting with [^99mTcN(PNP3)]-labeled α-MSH peptide analogs: Preliminary studies

Objectives: The purpose of this study was to examine the effect of cyclization on the biological profiles of [^99mTcN(PNP3)]-labeled α-MSH analogs.
Methods: The linear peptide H-Cys-Ahx-bAla-Nle-Asp-His-D-Phe-Arg-Trp-Gly-NH₂ (NAP-NS1) (1) and a corresponding lactam bridge-cyclized peptide, H-Cys-Ahx-bAla³-c[Lys⁴-Glu-His-D-Phe-Arg-Trp-Glu¹⁰]-Arg¹¹-Pro-Val-NH₂ (NAP-NS2) (2), were synthesized, characterized by ESI-MS, and their melanocortin-1 receptor (MC1R) binding affinity was determined in B16F10 melanoma cells. In vitro stability and pharmacological parameters of [^99mTc(N)(NAP-NS1)(PNP3)]⁺ (1a) and [^99mTc(N)(NAP-NS2)(PNP3)]⁺ (2a) were assessed. Challenges with an excess of glutathione and cysteine and LogD values were also investigated. Furthermore, 1a and 2a were applied to study in vivo stability and the pharmacokinetic profiles on healthy rats.
Results: 1a and 2a were obtained in high yield (RCY > 90%). LogD values demonstrated the hydrophilic nature of the radiolabeled peptides: -1.43 for 1a; - 2.09 for 2a. No significant variations in RCPs of both the complexes were observed. Both complexes showed high stability after incubation in human and rat sera as well as in rat liver homogenate. A fast degradation of 2a was detected in kidneys homogenate. 1a retained a high receptor affinity (K_d: 7.1±0.5 nM). Biodistribution of 1a displayed a favorable pharmacokinetic profile with fast blood clearance and elimination from normal tissues. Rapid renal excretion of 1a was observed due to the high hydrophilic character. The pharmacokinetic profile of 2a was reflected in reduction of the blood clearance and the elimination from the other organs; especially the kidneys showed restraint elimination.
Conclusions: Compared with the linear peptide 1, cyclization affected the pharmacological properties of 2 negatively by reducing its stability, its binding affinity to MC1Rs (Ki: 0.9±0.3 nM for 1; 7.1±2.4 nM for 2) and decreasing the overall excretion rate of the corresponding [^99mTcN(PNP3)]-labeled peptide from the body. Thus, only the linear labeled peptide 1a will be considered for further investigations in tumor bearing mice.