Synthesis and Characterization of a Melanoma-Targeted Fluorescence Imaging Probe by Conjugation of a Melanocortin 1 Receptor (MC1R) Specific Ligand

Abstract

The incidence of malignant melanoma is rising faster than that of any other cancer in the United States. The melanocortin 1 receptor (MC1R) is overexpressed in most human melanoma metastases, thus making it a promising target for imaging and therapy of melanomas. We have previously reported the development of a peptidomimetic ligand with high specificity and affinity for MC1R. Here, we have conjugated near-infrared fluorescent dyes to the C-terminus of this ligand via lysine-mercaptopropionic acid linkers to generate MC1R specific optical probes (MC1RL-800, 0.4 nM Ki and MC1RL-Cy5, 0.3 nM Ki). Internalization of the imaging probe was studied in vitro by fluorescence microscopy using engineered A375/MC1R cells and B16F10 cells with endogenous MC1R expression. The in vivo tumor targeting of MC1RL-800 was evaluated by intravenous injection of probe into nude mice bearing bilateral subcutaneous A375 xenograft tumors with low MC1R expression and engineered A375/MC1R tumors with high receptor expression. Melanotic B16F10 xenofgrafts were also studied. Fluorescence imaging showed that the agent has higher uptake values in tumors with high expression compared to low (p<0.05), demonstrating the effect of expression levels on image contrast-to-noise. In addition, tumor uptake was significantly blocked by co-injection of excess NDP-α-MSH peptide (p<0.05). In conclusion, the MC1R-specific imaging probe developed in this study displays excellent potential for the intraoperative detection of regional node involvement and for margin detection during melanoma metastasis resection.

Trigonal scaffolds for multivalent targeting of melanocortin receptors

“…41,42 In the present study with HEK293 cells, these assays exhibited relatively poor signal-to-noise ratios due to cell detachment during the multiple wash and reagent addition steps. The use of more adherent A375 cells bearing melanocortin 1 receptors 43 also gave poor signal-to-noise ratios due to the lower receptor expression levels of these cells. While the engineered HEK293 cells express about 640,000 MC4 receptors per cell, 9 the A375 cells express about 75,000 MC1 receptors per cell.…”

“…While the engineered HEK293 cells express about 640,000 MC4 receptors per cell, 9 the A375 cells express about 75,000 MC1 receptors per cell. 43 …”

In Vivo and in Silico Pharmacokinetics and Biodistribution of a Melanocortin Receptor 1 Targeted Agent in Preclinical Models of Melanoma

“…The MC1RL-Cy5 probe has high affinity for MC1R, with a K i of 0.3 ± 0.05 nM. 24 The binding affinity is comparable to that of unlabeled ligand, 0.24 nM K i . 23 …”

“…24 After tumor growth to approximately 500–800 mm 3 , the previously reported IRDye800CW conjugate (MC1RL-800) 24 was injected intravenously and fluorescence accumulation was monitored over time. The MC1RL-800 probe has high affinity for MC1R, with a 0.4 ± 0.1 nM K i 24 which is comparable to that of unlabeled ligand, 0.24 nM K i . 23 At 2h post-injection, A375 tumors with low MC1R expression had significantly lower normalized fluorescence signal compared to A375/MC1R tumors (P≤0.05, n=3) (Figure 3A).…”

“…23 More recently, we have conjugated near-infrared fluorescent (NIRF) dyes to this ligand and demonstrated in vivo specificity for tumors with MC1R expression. 24 …”