In VitroMouse and Human Serum Stability of a Heterobivalent Dual-Target Probe That Has Strong Affinity to Gastrin-Releasing Peptide and Neuropeptide Y1 Receptors on Tumor Cells

Abstract

Receptor-targeting radiolabeled molecular probes with high affinity and specificity are useful in studying and monitoring biological processes and responses. Dual- or multiple-targeting probes, using radiolabeled metal chelates conjugated to peptides, have potential advantages over single-targeting probes as they can recognize multiple targets leading to better sensitivity for imaging and radiotherapy when target heterogeneity is present. Two natural hormone peptide receptors, gastrin-releasing peptide (GRP) and Y1, are specifically interesting as their expression is upregulated in most breast and prostate cancers. One of our goals has been to develop a dual-target probe that can bind both GRP and Y1 receptors. Consequently, a heterobivalent dual-target probe, t-BBN/BVD15-DO3A (where a GRP targeting ligand J-G-Abz4-QWAVGHLM-NH and Y1 targeting ligand INP-K [ɛ-J-(α-DO3A-ɛ-DGa)-K] YRLRY-NH were coupled), that recognizes both GRP and Y1 receptors was synthesized, purified, and characterized in the past. Competitive displacement cell binding assay studies with the probe demonstrated strong affinity (IC values given in parentheses) for GRP receptors in T-47D cells (18 ± 0.7 nM) and for Y1 receptors in MCF7 cells (80 ± 11 nM). As a further evaluation of the heterobivalent dual-target probe t-BBN/BVD15-DO3A, the objective of this study was to determine its mouse and human serum stability at 37°C. The in vitro metabolic degradation of the dual-target probe in mouse and human serum was studied by using a Gd-labeled t-BBN/BVD15-DO3A and a high-performance liquid chromatography/radioisotope detector analytical method. The half-life (t) of degradation of the dual-target probe in mouse serum was calculated as 7 hours and only ∼20% degradation was seen after 6 hours incubation in human serum. The slow in vitro metabolic degradation of the dual-target probe can be compared with the degradation t of the corresponding monomeric probes, BVD15-DO3A and AMBA: 15, and ∼40 minutes for BVD15-DO3A and 3.1 and 38.8 hours for AMBA in mouse and human serum, respectively. A possible pathway for in vitro metabolic degradation of the t-BBN/BVD15-DO3A in mouse serum is proposed based on the chromatographic retention times of the intact probe and its degradants.

Design, Synthesis, In Vitro, and Initial In Vivo Evaluation of Heterobivalent Peptidic Ligands Targeting Both NPY(Y1)- and GRP-Receptors—An Improvement for Breast Cancer Imaging?

“…So far, only one example of a heterobivalent NPY(Y 1 )R- and GRPR-targeting ligand has been described [ 9 , 10 ] and for this substance, no descriptions of radiolabeling with a PET isotope, in vitro cell uptake or in vivo imaging data are available. Thus, the general feasibility of the approach has not been demonstrated so far.…”

Identification of a Suitable Peptidic Molecular Platform for the Development of NPY(Y1)R‐Specific Imaging Agents

“…Taking the results of the serum stability and the liver microsomal stability assays together, [ 68 Ga]3 possesses the highest resistance against proteolytic degradation towards human serum as well as human liver peptidases. A further comparative testing of the agents in mice does not seem to be reasonable due to the different proteolytic activities towards peptides of human origin in both species, [33] not allowing inferences on the stability of the agents in humans by studying it in rodents. Although these findings do not allow to predict a high in vivo stability of [ 68 Ga]3, thus potentially requiring further stabilization of the peptide sequence, the presented results are a good indication of the relative stabilities of the tested agents.…”

“…[12,14,15,17,18,19,20,21,29,30] Among these, several showed low affinities, NPY(Y 1 ) receptor subtype selectivities or stabilities. Of those remaining, we chose to directly compare five different peptide analogues regarding their metabolic stability (Figure 1), representing the most promising agents in terms of in vitro properties (NPY(Y 1 )R affinity, Table 1, and receptor subtype selectivity) within their respective group of linear, cyclic and dimeric truncated NPY analogues: i) linear [Pro 30 ,Lys(DOTA) 31 DOTA = (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid), serving as reference compound with known low metabolic stability but high NPY(Y 1 )R affinity and receptor subtype selectivity, [18] ii) [Pro 30 ,Lys(DOTA) 31 ,Bip 32 ,Leu 34 ]NPY [28][29][30][31][32][33][34][35][36] (2), a variant of 1 comprising an artificial Bip (biphenylalanine) amino acid in position 32 instead of tyrosine, [12] and Lys(DOTA) in position 31 for radiolabeling, [15] iii) [Lys(lauroyl) 27 ,Pro 30 ,Lys (DOTA) 31 ,Bip 32 ,Leu 34 ]NPY [27][28][29][30][31][32][33][34][35][36] (3), a variant of 2 modified with an additional N ɛ -Lys-lauroyl in position 27 for full receptor agonism and high NPY(Y 1 )R af...…”

“…Hence, the vast majority of approaches aimed at the development of truncated versions of NPY, ideally showing a strong receptor subtype preference as well as a high affinity to the NPY(Y 1 )R. One challenge is that the peptidereceptor interaction was demonstrated to be highly sensitive to structural changes of the peptide. The C-terminal part of the peptide, NPY [28][29][30][31][32][33][34][35][36] , is the minimal core sequence for receptor binding and Arg33, Arg35 as well as the terminal Tyr36-amide are essential for NPY(Y 1 )R binding. [2,7,8] Furthermore, Thr32 and Gln34 were found to be crucial for receptor subtype preference.…”

First in vivo evaluation of a potential SPECT brain radiotracer for the gonadotropin releasing hormone receptor

“…Serum stability analysis is an initial step in the in vitro evaluation [ 18 ]. Compound- 1 revealed sufficient stability for imaging following incubations in saline, rat and human serum.…”