Synthesis, In Vitro Receptor Binding, and In Vivo Evaluation of Fluorescein and Carbocyanine Peptide-Based Optical Contrast Agents

Abstract

Site-specific delivery of drugs and contrast agents to tumors protects normal tissues from the cytotoxic effects of drugs and enhances the contrast between normal and pathologic tissues. One approach to achieve selectivity is to target overexpressed receptors on the membranes of tumor cells and to visualize the tumors by a noninvasive optical imaging method. Accordingly, we conjugated fluorescein and carbocyanine dyes to somatostatin and bombesin receptor-avid peptides and examined their receptor binding affinities. We also prepared potential dual imaging probes consisting of a bioactive peptide for tumor targeting, a biocompatible dye for optical imaging, and a radioactive or paramagnetic metal chelator for scintigraphic or magnetic resonance imaging of tumors. Using these approaches, the resulting carbocyanine derivatives of somatostatin and bombesin analogues retained high binding for their respective receptors. Further evaluation of representative molecules in rats bearing somatostatin- and bombesin-positive tumors showed selective uptake of the agents by the tumor cells. Unlike carbocyanine derivatives, the receptor binding of fluorescein-somatostatin peptide conjugates was highly sensitive to the type of linker and the site of fluorescein attachment on the nonreceptor binding region of the peptide. In general, the presence of flexible linkers disrupted binding affinity, possibly due to the interaction of the linker's thiourea group with the peptide's cyclic disulfide bond. While the receptor binding affinity of the dual probes was not dependent on the type of chelating group examined, it was affected by the relative positions of fluorescein and chelator on the lysine linker. For somatostatin compounds, best results were obtained when the chelator was on the alpha-amino lysine linker and fluorescein was on the epsilon-amino group. In contrast, conjugation of the chelator to epsilon- and fluorescein to the alpha-amino lysine linker of bombesin peptides resulted in high receptor binding. These findings indicate that despite their small size, conjugation of dyes to truncated somatostatin and bombesin peptide analogues results in promising diagnostic agents that retain high receptor binding activity in vitro. The results further show that these contrast agents can selectively and specifically localize in receptor-positive tumors in rat models.

Evaluation of a Fluorescent and Radiolabeled Hybrid Somatostatin Analog In Vitro and in Mice Bearing H69 Neuroendocrine Xenografts

“…One of the main drawbacks of dual-labeled tracers is the potential loss of receptor affinity and in vivo targeting capability, which has previously been reported for various dual-labeled tracers (16,17,26,27). Cy5-111 In-DTPA-Tyr 3 -octreotate and 111 In-DTPA-Tyr 3 -octreotate showed comparable profiles when applied in our in vivo model, especially with respect to tumor uptake and limited off-target uptake; the only major difference between 111 In-DTPA-Tyr 3 -octreotate and Cy5-111 In-DTPA-Tyr 3 -octreotate was the renal uptake, which was higher with the dual-labeled tracer than with the nuclear-only tracer.…”

Agonist−Antagonist Dilemma in Molecular Imaging: Evaluation of a Monomolecular Multimodal Imaging Agent for the Somatostatin Receptor

“…Cytate was included as a reference because of its high SSTr2 specific accumulation in a xenografted tumor (CA20948) tumor in as little as 90 minutes (14,19). The internalization of LS172 and nat Cu-LS172 by A427-7 cells was directly observed by NIR fluorescence microscopy because they are both conjugated to cypate ( Figure 4 and Figure 5).…”

“…To design radionuclear-optical MOMIA, it would be easier to incorporate a lysine residue at the N-terminus of the peptide to provide two amino groups for conjugating DOTA and cypate. However, in a previous study, we showed that this molecular construct can compromise the peptide's binding affinity for SSTr2 (14). In developing macrocyclic scaffold for molecular optical imaging, we found that adding lysine to the C-terminus of Y3-TATE (Y3-K9-TATE) retained the binding affinity of Y3-TATE (17) and provided a second reactive group for coupling cypate at a distal position to DOTA.…”

Synergistic effects of light-emitting probes and peptides for targeting and monitoring integrin expression

“…ABIbinding cyclo [GRGDfV], was purchased from Peptide International (Louisville, KY) and used for blocking studies. Octreotate, an octapeptide that binds somatostatin receptors but not ABI, was prepared as described (23). Purification and analysis of the new peptides were performed on an HPLC system equipped with a tunable UV-visible detector.…”

“…The linear peptides were prepared by standard fluorenylmethyl (Fmoc) protocol (25), as described (21,23). Conjugation of Cypate with peptides was performed on solid support.…”