Receptor-Mediated Melanoma Targeting with Radiolabeled α-Melanocyte-Stimulating Hormone: Relevance of the Net Charge of the Ligand

Abstract

A majority of melanotic and amelanotic melanomas overexpress melanocortin type 1 receptors (MC1Rs) for α-melanocyte-stimulating hormone. Radiolabeled linear or cyclic analogs of α-MSH have a great potential as diagnostic or therapeutic tools for the management of malignant melanoma. Compounds such as [¹¹¹In]DOTA-NAP-amide exhibit high affinity for the MC1R in vitro, good tumor uptake in vivo, but they may suffer from relatively high kidney uptake and retention in vivo. We have shown previously that the introduction of negative charges into radiolabeled DOTA-NAP-amide peptide analogs may enhance their excretion and reduce kidney retention. To address the question of where to place negative charges within the ligand, we have extended these studies by designing two novel peptides, Ac-Nle-Asp-His-d-Phe-Arg-Trp-Gly-Lys(DOTA)-d-Asp-d-Asp-OH (DOTA-NAP-d-Asp-d-Asp) with three negative charges at the C-terminal end (overall net charge of the molecule -2) and DOTA-Gly-Tyr(P)-Nle-Asp-His-d-Phe-Arg-Trp-NH₂ (DOTA-Phospho-MSH_2-9) with two negative charges in the N-terminal region (net charge -1). The former peptide showed markedly reduced receptor affinity and biological activity by >10-fold compared to DOTA-NAP-amide as reference compound, and the latter peptide displayed similar bioactivity and receptor affinity as the reference compound. The uptake by melanoma tumor tissue of [¹¹¹In]DOTA-Phospho-MSH_2-9 was 7.33 ± 0.47 %ID/g 4 h after injection, i.e., almost equally high as with [¹¹¹In]DOTA-NAP-amide. The kidney retention was 2.68 ± 0.18 %ID/g 4 h after injection and hence 44% lower than that of [¹¹¹In]DOTA-NAP-amide. Over an observation period from 4 to 48 h, the tumor-to-kidney ratio of [¹¹¹In]DOTA-Phospho-MSH_2-9 was 35% more favorable than that of the reference compound. In a comparison of DOTA-NAP-d-Asp-d-Asp, DOTA-Phospho-MSH_2-9 and DOTA-NAP-amide with five previously published analogs of DOTA-NAP-amide that altogether cover a range of peptides with an overall net charge between +2 and -2, we now demonstrate that a net charge of -1, with the extra negative charges preferably placed in the N-terminal region, has led to the lowest kidney uptake and retention. Charges of +2 or -2 markedly increased kidney uptake and retention. In conclusion, the novel DOTA-Phospho-MSH_2-9 may represent a new lead compound for negatively charged linear MC1R ligands that can be further developed into a clinically relevant melanoma targeting radiopeptide.

Keywords: kidney toxicity; melanoma; net charge; phosphopeptide; radiolabeled peptide; tissue distribution; tumor targeting; α-melanocyte-stimulating hormone.

Figure 1

Amino acid sequence of α-MSH, DOTA-NAP-amide, DOTA-NAP-d-Asp-d-Asp, and DOTA-Phospho-MSH_2-9.

Figure 2

Chemical structure of DOTA-NAP-d-Asp-d-Asp (A) and DOTA-Phospho-MSH_2-9 (B).

Figure 3

Determination of internalization of [¹¹¹In] DOTA-NAP-d-Asp-d-Asp (A) and [¹¹¹In]DOTA-Phospho-MSH_2-9 (B) by cultured B16-F1 cells exposed to the peptides at 37°C for 0.5, 2, and 3.5 h. Surface-bound radioligand was released by an acid buffer wash, and internalized radioligand was determined by lysing cells with detergent. Results are expressed in percent of the added dose per million cells.

Figure 4

Tissue distribution of [¹¹¹In]DOTA-Phospho-MSH_2-9 in B16-F1 melanoma tumor-bearing mice at 4, 24, and 48 h postinjection. Results are expressed as percent of injected dose per g of tissue (%ID/g; means ± SEM; n = 3 experiments).

Figure 5

Uptake by the kidneys of eight different ¹¹¹In-labeled DOTA-MSH peptides with net charges ranging from −2 to +2. For structures of peptides A–H, see Table 4. Results are expressed as percent of injected dose per gram of tissue (%ID/g; means ± SEM; n = 3).

Figure 6

Uptake by the liver of eight different ¹¹¹In-labeled DOTA-MSH peptides with net charges ranging from −2 to +2. For structures of peptides A–H, see Table 4. Results are expressed as percent of injected dose per gram of tissue (%ID/g; means ± SEM; n = 3).