Background

[PubMed]

4-[2-(3,4,5,6-Tetrahydropyrimidin-2-ylamino)ethyloxy]benzoyl-2-(S)-[N-(3-amino-neopenta-1-carbamyl)]-aminoethylsulfonylamino-β-alanine fluorescein thiourea (FITC-IAC) is an integrin-targeted molecular imaging agent conjugated with fluorescein isothiocyanate (FITC) that was developed for fluorescence imaging of tumor vasculature and tumor angiogenesis (1). FITC is a fluorochrome with a peak excitation wavelength of 495 nm and a peak emission wavelength of 519 nm.

Cellular survival, invasion, and migration control embryonic development, angiogenesis, tumor metastasis, and other physiological processes (2, 3). Among the molecules that regulate angiogenesis are integrins, which comprise a superfamily of cell adhesion proteins that form heterodimeric receptors for extracellular matrix (ECM) molecules (4, 5). These transmembrane glycoproteins consist of two noncovalently associated subunits, α and β (18 α- and 8 β-subunits in mammals), which are assembled into at least 24 α/β pairs. Several integrins, such as integrin α_vβ₃, have affinity for the arginine-glycine-aspartic acid (RGD) tripeptide motif, which is found in many ECM proteins. Expression of integrin α_vβ₃ receptors on endothelial cells is stimulated by angiogenic factors and environments. The integrin α_vβ₃ receptor is generally not found in normal tissue, but it is strongly expressed in vessels with increased angiogenesis, such as tumor vasculature. It is significantly upregulated in certain types of tumor cells and in almost all tumor vasculature. Molecular imaging probes carrying the RGD motif that binds to the integrin α_vβ₃ can be used to image tumor vasculature and evaluate angiogenic response to tumor therapy (6, 7).

Various RGD peptides in both linear and cyclic forms have been developed for in vivo binding to integrin α_vβ₃ (8). Non-peptide integrin α_vβ₃–targeted molecular probes have also been investigated (1, 9, 10). Duggan et al. (9) modified a potent fibrinogen receptor antagonist from the sulfonamide exo-site class to generate non-peptide compounds with high affinity for the integrin α_vβ₃ receptor. The centrally constrained benzoylamino-3-propionic acid scaffold appeared to provide optimum spacing between the acidic and basic portions of these ligands for binding. Optical imaging is an imaging method that utilizes light photons emitted from bioluminescence and fluorescence probes (7). Depth penetration is a major limiting factor for in vivo optical imaging. Currently, in vivo optical imaging has wide applications in small animal imaging but only limited applications in large animal and human studies (11). Fluorescent dyes conjugated RGD peptides such as Cy5.5-c(RGDyK),Cy7-E{E(c(RGDyK)]₂}₂, and Cyp-GRD have been shown to visualize subcutaneously implanted integrin α_vβ₃−positive tumors (12-15). Based on the potent α_vβ₃ inhibitor 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-(S)-aminoethylsulfonyl-amino-β-alanine (IA) which has an inhibition concentration (IC₅₀) of 0.04 μM and was used by Hood et al. (10) to covalently couple to a cationic nanoparticle for targeted gene therapy, Burnett et al. (1) designed and synthesized a series of aliphatic carbamate derivatives with enhanced binding affinity. The peptidomimetic IAC ligand was successfully conjugated to FITC as an integrin α_vβ₃ molecular probe for optical imaging of α_vβ₃-expressing tumors.

Synthesis

[PubMed]

Burnett et al. (1) reported the synthesis of FITC-IAC from commercially available 3-amino neopentanol. The amine was first protected as the t-butoxycarbonyl (t-Boc) derivative and then activated as 3-(Boc-amino)neopentyl-1-O-carbonylimidazole. The activated alcohol was then coupled to the free amine of IA to provide the Boc carbamate. The Boc carbamate was deprotected with anhydrous hydrochloric acid in dioxane to yield 4-[2-(3,4,5,6-tetrahydropyrimidin-2-ylamino)ethyloxy]benzoyl-2-(S)-[N-(3-amino-neopenta-1-carbamyl)]-aminoethylsulfonylamino-β-alanine hydrochloride. N,N-diisopropylethylamine was added to this ligand in anhydrous dimethyl sulfoxide. One molar equivalent of FITC was then added, and the reaction mixture was stirred under argon at 40ºC for 18 h. The final FITC-IAC was purified by high-performance liquid chromatography with a yield of 24%. The identity of FITC-IAC was confirmed by (¹H and ¹³C) nuclear magnetic resonance and high-resolution mass spectrometry.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

An in vitro binding affinity assay of FITC-IAC was performed with purified human α_vβ₃ integrin in ELISA assays and α_vβ₃-expressing M21 melanoma cells in cell adhesion assays (1). The natural ligand vitronectin was used as the competing ligand. The IC₅₀ of FITC-IAC was determined to be 3.4 ± 0.33 nM (n = 3). In comparison, the IA and IAC had IC₅₀ values of 14.1 ± 1.4 nM and 2.94 ± 0.19 nM, respectively. Inhibition of cell adhesion of FITC-IAC was similar to that of IA. Comparing results with other neopentyl derivatives in the same study, the authors (1) suggested that the geminal methyl groups favored interaction with the receptor. The cellular distribution of FITC-IAC in the M21 cells was also examined by fluorescence microscopy. The study suggested that the fluorescence signal came from the cell membrane surface after 5 min of incubation.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

NIH Support

NIH intramural support.

References

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This MICAD chapter is not included in the Open Access Subset, because it was authored / co-authored by one or more investigators who was not a member of the MICAD staff.