Design, Synthesis, In Vitro and In Vivo Evaluation of Heterobivalent SiFAlin-Modified Peptidic Radioligands Targeting Both Integrin αvβ3 and the MC1 Receptor—Suitable for the Specific Visualization of Melanomas?

Abstract

Combining two peptides addressing two different receptors to a heterobivalent peptidic ligand (HBPL) is thought to enable an improved tumor-targeting sensitivity and thus tumor visualization, compared to monovalent peptide ligands. In the case of melanoma, the Melanocortin-1 receptor (MC1R), which is stably overexpressed in the majority of primary malignant melanomas, and integrin αvβ3, which is involved in lymph node metastasis and therefore has an important role in the transition from local to metastatic disease, are important target receptors. Thus, if a radiolabeled HBPL could be developed that was able to bind to both receptor types, the early diagnosis and correct staging of the disease would be significantly increased. Here, we report on the design, synthesis, radiolabeling and in vitro and in vivo testing of different SiFAlin-modified HBPLs (SiFA = silicon fluoride acceptor), consisting of an MC1R-targeting (GG-Nle-c(DHfRWK)) and an integrin αvβ3-affine peptide (c(RGDfK)), being connected by a symmetrically branching framework including linkers of differing length and composition. Kit-like 18F-radiolabeling of the HBPLs 1–6 provided the labeled products [18F]1–[18F]6 in radiochemical yields of 27–50%, radiochemical purities of ≥95% and non-optimized molar activities of 17–51 GBq/µmol within short preparation times of 25 min. Besides the evaluation of radiotracers regarding logD(7.4) and stability in human serum, the receptor affinities of the HBPLs were investigated in vitro on cell lines overexpressing integrin αvβ3 (U87MG cells) or the MC1R (B16F10). Based on these results, the most promising compounds [18F]2, showing the highest affinity to both target receptors (IC50 (B16F10) = 0.99 ± 0.11 nM, IC50 (U87MG) = 1300 ± 288 nM), and [18F]4, exhibiting the highest hydrophilicity (logD(7.4) = −1.39 ± 0.03), were further investigated in vivo and ex vivo in a xenograft mouse model bearing both tumors. For both HBPLs, clear visualization of B16F10, as well as U87MG tumors, was feasible. Blocking studies using the respective monospecific peptides demonstrated both peptide binders of the HBPLs contributing to tumor uptake. Despite the somewhat lower target receptor affinities (IC50 (B16F10) = 6.00 ± 0.47 nM and IC50 (U87MG) = 2034 ± 323 nM) of [18F]4, the tracer showed higher absolute tumor uptakes ([18F]4: 2.58 ± 0.86% ID/g in B16F10 tumors and 3.92 ± 1.31% ID/g in U87MG tumors; [18F]2: 2.32 ± 0.49% ID/g in B16F10 tumors and 2.33 ± 0.46% ID/g in U87MG tumors) as well as higher tumor-to-background ratios than [18F]2. Thus, [18F]4 demonstrates to be a highly potent radiotracer for the sensitive and bispecific imaging of malignant melanoma by PET/CT imaging and impressively illustrates the suitability of the underlying concept to develop heterobivalent integrin αvβ3- and MC1R-bispecific radioligands for the sensitive and specific imaging of malignant melanoma by PET/CT.

Synthesis, Characterization and In Vitro Evaluation of Hybrid Monomeric Peptides Suited for Multimodal Imaging by PET/OI: Extending the Concept of Charge—Cell Binding Correlation

“…For this purpose, the peptides were synthesized on solid support (Scheme 1A, B and C) and in the following further modified using a PEG3 linker unit followed by 4-maleimidobutyric acid to obtain peptides analogs amenable to efficient chemoselective functionalization with the MIUs (Scheme 1D). In detail, the three peptides 2 a-c were completely built on solid support using standard Fmoc-based solid phase-assisted synthesis protocols, as reported in previous procedures [27][28][29]. Cyclization of peptides was performed while being still attached to the resin and after removal of the respective protecting groups (Mtt-and O-2-Ph i Pr-PG (2 b) were removed under mildly acidic conditions, and the All-PG (2 c) by treatment with phenylsilane (PhSiH3) and tetrakis(triphenylphosphine) palladium(0) (Pd(PPH3)4)).…”

“…The same procedure was repeated for the following amino acids. Detailed syntheses of the three peptide units can be found in the corresponding references [27][28][29]. The PEG spacer (4 equiv.)…”

Synthesis, Radiolabeling, and In Vitro and In Vivo Characterization of Heterobivalent Peptidic Agents for Bispecific EGFR and Integrin αvβ3 Targeting

“…The data show that the internal reference peptides hEGF, c(RGDyK), and c(RGDfK) showed the expected affinity to their respective targets with IC 50 values of 19.3 ± 2.7 nM for hEGF to the EGFR, 630 ± 144 nM for c(RGDyK) and 872 ± 198 nM for c(RGDfK) to integrin α v β 3 , being in the range of published values. 44,45 In comparison, the HBPLs showed an only slightly decreased affinity to integrin α v β 3 , presenting IC 50 values to this receptor type of 1.39 ± 0.12 μM for 1 and 1.48 ± 0.29 μM for 2. In contrast, both heterobivalent agents failed to demonstrate any affinity toward the EGFR up to a concentration of 1 mM.…”

Peptide‐based diagnostic and therapeutic agents: Where we are and where we are heading?

“…Although many peptide receptor targets are available for exploration today, optimization of peptide ligands that are relevant to a disease state requires further investigation. Alternative strategies such as multi‐receptor targeting by utilizing combinations of peptides and heterodimer (hybrid) peptides labelled with various radionuclides can be employed to advance selectivity, stability, binding affinity, and related imaging quality (Cheng et al, 2021; Judmann et al, 2020; Liu et al, 2009).…”