In Vitro Mouse 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

摘要

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-NH2 and Y1 targeting ligand INP-K [ɛ-J-(α-DO3A-ɛ-DGa)-K] YRLRY-NH2 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 (IC50 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 ¹⁵³Gd-labeled t-BBN/BVD15-DO3A and a high-performance liquid chromatography/radioisotope detector analytical method. The half-life (t1/2) 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 t1/2 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.