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Introduction: To probe the interplay between radiotracer stability and somatostatin receptor affinity, Tyr(3)-octreotate and six variations of its peptide sequence, for which the Re-cyclized products were previously reported, were radiolabeled with (99m)Tc and investigated for their in vitro stability., Methods: Radiolabeling of the peptides was effected by ligand exchange from (99m)Tc-glucoheptonate, and the desired products were purified by radio-RP-HPLC. The in vitro stability in phosphate buffered saline, mouse serum and cysteine solutions at physiological temperature and pH for all seven (99m)Tc-cyclized peptides was determined by radio-RP-HPLC and radio-TLC. Normal CF-1 mouse biodistribution studies were performed for three of the (99m)Tc-cyclized peptides., Results: Based on the fully characterized Re-cyclized peptide analogues, four (99m)Tc-coordination motifs were proposed for the (99m)Tc-cyclized peptides. Technetium-99m-cyclized Tyr(3)-octreotate derivatives with N(2)S(2) metal coordination modes and large metal ring sizes were susceptible to oxidation and loss of (99m)Tc in the form of (99m)TcO(4)(-), as evidenced by their instability in the various solutions under physiological conditions (15-58% intact at 24 h). As anticipated, the addition of a third cysteine to the sequence stabilized the (99m)Tc metal coordination, and peptides with NS(3) coordination modes remained >85% intact out to 24 h. No significant differences were observed in the biodistribution studies performed with three peptides of varying stabilities., Conclusions: Improvements in stability were not sufficient to outweigh the low somatostatin receptor affinity for the peptides in this study. Further improvements in the peptide sequence and/or metal coordination are needed to result in a radiodiagnostic/radiotherapeutic pair for targeting the somatostatin receptor., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Introduction: Development of radiolabeled octreotide analogues is of interest for targeting somatostatin receptor (SSTR)-positive tumors for diagnostic and therapeutic purposes. We are investigating a direct labeling approach for incorporation of a Re ion into octreotide analogues, where the peptide sequences are cyclized via coordination to Re rather than through a disulfide bridge., Methods: Various octreotide analogue sequences and coordination systems (e.g., S(2)N(2) and S(3)N) were synthesized and cyclized with nonradioactive Re. In vitro competitive binding assays with (111)In-DOTA-Tyr(3)-octreotide in AR42J rat pancreatic tumor cells yielded IC(50) values as a measure of SSTR affinity of the Re-cyclized analogues. Three-dimensional structures of Re-cyclized Tyr(3)-octreotate and its disulfide-bridged analogue were calculated from two-dimensional NMR experiments to visualize the effect of metal cyclization on the analogue's pharmacophore., Results: Only two of the 11 Re-cyclized analogues investigated showed moderate in vitro binding affinity toward somatostatin subtype 2 receptors. Three-dimensional molecular structures of Re- and disulfide-cyclized Tyr(3)-octreotate were calculated, and both of their pharmacophore turns appear to be very similar with minor differences due to metal coordination to the amide nitrogen of one of the pharmacophore amino acids., Conclusions: Various Re-cyclized analogues were developed and analogue 4 had moderate affinity toward somatostatin subtype 2 receptors. In vitro stable studies that are in progress showed stable radiometal cyclization of octreotide analogues via NS(3) and N(2)S(2) coordination forming five- and six-membered chelate rings. In vivo biodistribution studies are underway of (99m)Tc-cyclized analogue 4., (Copyright 2010 Elsevier Inc. All rights reserved.)

The development of receptor-targeting radiopharmaceuticals commonly involves the use of peptides, antibodies or small molecules. Resulting from the numerous modifications that can be made to these basic targeting agents, research in this field often generates a series of compounds as potential ligands for in vivo investigation. However, measuring each variant of a series in vivo can be both costly and time-consuming. Therefore, a number of in vitro assays, to study interactions between the targeted receptor and the ligands of interest, are frequently used to quickly and inexpensively narrow the field and identify a lead compound(s) for further investigation. For example, in saturation binding studies, the amount of radioligand required to saturate the receptors is measured and analyzed to determine the radioligand equilibrium dissociation constant (Kd), a useful gauge of the receptor binding affinity of a radioligand. In competitive binding experiments, a ligand of interest competes for available receptor sites with a standard radioligand of known high receptor affinity. Competition data are analyzed to yield another indicator of receptor affinity, called an IC50 value, which can be used to rank the relative receptor binding affinities for a series of ligands. In internalization and efflux studies, the rate and extent of receptor-mediated radioligand taken into and subsequently released from cells is measured, providing insight into cellular uptake and retention of the radioligand. Individually or taken together, these in vitro receptor binding assays are useful tools in radiopharmaceutical development.

The structure-activity relationships of a series of rhenium (Re)-cyclized octreotide derivatives are described. The effects of changes in the peptide sequence, N-terminus, and C-terminus on metal cyclization, as well as binding to the somatostatin receptor, were investigated. Each peptide complex was found to have an integrated Re(V) core with a single metal oxo group, two coordination sites filled by the cysteine sulfhydryls, and another by the amide nitrogen of Phe (3)/Tyr (3). The final coordination site was determined by the peptide N-terminus: the N-terminal amine coordinated for N-NH 2 peptides and the amide nitrogen of Thr (6) for peptides with acetylated N-termini. Re-cyclization of the octreotide derivatives led to structural perturbations of the somatostatin receptor-binding sequence relative to the Re-free disulfide analogues, resulting in reduced binding affinities. The findings presented herein demonstrate the importance of understanding the consequences of structural modifications when designing metal-peptide complexes for somatostatin receptor targeting.