A Modular Dual-Labeling Scaffold That Retains Agonistic Properties for Somatostatin Receptor Targeting

Abstract

Fluorescence-guided surgery is an emerging imaging technique that can enhance the ability of surgeons to detect tumors when compared with visual observation. To facilitate characterization, fluorescently labeled probes have been dual-labeled with a radionuclide to enable cross-validation with nuclear imaging. In this study, we selected the somatostatin receptor imaging agent DOTATOC as the foundation for developing a dual-labeled analog. We hypothesized that a customized dual-labeling approach with a multimodality chelation (MMC) scaffold would minimize steric effects of dye conjugation and retain agonist properties. An MMC conjugate (MMC-TOC) was synthesized on solid-phase and compared with an analog prepared using conventional methods (DA-TOC). Both analogs were conjugated to IRDye 800 using copper-free click chemistry. The resulting compounds, MMC(IR800)-TOC and DA(IR800)-TOC, were labeled with Cu andCu and tested in vitro in somatostatin receptor subtype 2-overexpressing HEK-293 cells to assess agonist properties, and in AR42J rat pancreatic cancer cells to determine receptor binding characteristics. Multimodality imaging was performed in AR42J xenografts. Cu-MMC(IR800)-TOC demonstrated higher potency for cyclic adenosine monophosphate inhibition (half maximal effective concentration [EC]: 0.21 ± 0.18 vs. 1.38 ± 0.54 nM) and receptor internalization (EC: 41.9 ± 29.8 vs. 455 ± 299 nM) than Cu-DA(IR800)-TOC. Radioactive uptake studies showed that blocking with octreotide caused a dose-dependent reduction in Cu-MMC(IR800)-TOC uptake whereasCu-DA(IR800)-TOC was not affected. In vivo studies revealed higher tumor uptake for Cu-MMC(IR800)-TOC thanCu-DA(IR800)-TOC (5.2 ± 0.2 vs. 3.6 ± 0.4 percentage injected dose per gram). In vivo blocking studies with octreotide reduced tumor uptake of Cu-MMC(IR800)-TOC by 66%. Excretion ofCu-MMC(IR800)-TOC was primarily through the liver and spleen whereas Cu-DA(IR800)-TOC was cleared through the kidneys. Ex vivo analysis at 24 h confirmed PET/CT data by showing near-infrared fluorescence signal in tumors and a tumor-to-muscle ratio of 5.3 ± 0.8 as determined by γ-counting. The findings demonstrate that drug design affected receptor pharmacology and suggest that the MMC scaffold is a useful tool for the development of dual-labeled imaging agents.

Specific Targeting of Somatostatin Receptor Subtype-2 for Fluorescence-Guided Surgery

“…Therefore, efficacy is dependent upon the specificity of the agent for its target and its overexpression in tumor tissue. We previously showed SSTR2-mediated uptake of 64 Cu-MMC(IR800)-TOC in vitro and in vivo, where competition with octreotide strongly reduced uptake in SSTR2-expressing tumors (36). In a subsequent study with 68 Ga-MMC(IR800)-TOC, we demonstrated selective in vitro uptake by SSTR2-expressing cells that was 20-fold higher than cells that lacked the receptor, as well as a 16-fold reduction in uptake when coincubated with octreotide (12).…”

A Cyanine‐Bridged Somatostatin Hybrid Probe for Multimodal SSTR2 Imaging in Vitro and in Vivo: Synthesis and Evaluation

“…The tumor‐to‐background ratios 1 h after injection were moderate for the muscles and low for blood. Other studies with SSTR2‐targeting hybrid probes observed similar values 3–4 h after injection, [14b,18a] which could be improved when imaging at later time points, such as 24 or 48 h after injection with 67 Ga [18c] …”

“…According to the PET/MRI and biodistribution data, the clearance route is predominantly renal with a high uptake in the kidneys (∼30 % IA g −1 ). Attaching a fluorophore to 68 Ga‐DOTATATE usually causes higher renal uptake compared to the dye‐free 68 Ga‐DOTATATE, [21,23] leading to values of up to 70 % IA g −1 [14b,18a] . The same applies, usually to a smaller extent, for the uptake in the liver, which is also influenced by nonpolar structural moieties as typically given by fluorophores.…”

Overview of Radiolabeled Somatostatin Analogs for Cancer Imaging and Therapy

“…The development of bivalent radiotracers to target several receptors concomitantly expressed could be of interest to improve targeting [ 237 ]. Similarly, improved detection and sensitivity could be achieved using bimodal agents [ 238 ]. Besides, the clinical success for radiolabeled somatostatin analogs both with diagnostic and therapeutic radionuclides paved the way for new promising peptide derivatives, such as bombesin, neurotensin, or CXCR4 ligands, and, in a similar way, PSMA ligands, for cancer theranostics [ 49 , 233 , 239 , 240 ].…”