Background

[PubMed]

N-[2-(4-[¹⁸F]Fluorobenzamido)ethyl]maleimide-sulfhydryl-cyclic-arginine-glycine-aspartic acid peptide ([¹⁸F]FBEM-SRGD) is an integrin-targeted molecular imaging agent developed for positron emission tomography (PET) of tumor vasculature and angiogenesis (1). ¹⁸F is a positron emitter with a physical half-life (t_½) of 110 min.

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. 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). Chen et al. (9) evaluated ⁶⁴Cu-labeled and 18F-labeled cyclic RGD peptide [c(RGDyK)] monomers in nude mice bearing breast tumors. [¹⁸F]FB-c(RGDyK) showed high tumor accumulation but also rapid tumor washout with unfavorable biliary excretion (10). To improve the pharmacokinetics and tumor retention of the radiolabeled peptide, a dimer analog was synthesized as [¹⁸F]FB-[c(RGDyK)]₂, which showed improved tumor localization and predominant renal excretion (11). Radiofluorination can generally be achieved through the functional groups of amino, carboxylic acid, and sulfhydryl. Labeling of RGD peptide with ¹⁸F in most fluorinated peptide studies used ¹⁸F-synthons such as N-succinimidyl 4-[¹⁸F]fluorobenzoate to form a stable amide bond by reacting with primary amino groups of RGD peptides at the N terminus or the lysine side chain (1). One of the main concerns with this approach is the potential interference with the biological activities of these peptides. Cai et al. (1) suggested that ¹⁸F-labeling of RGD peptides via the carboxylic acid group at the C terminus or internal glutamic/aspartic acid side chain might minimize the potential interference. They reported the use of a thiol-reactive ¹⁸F-synthon, ¹⁸F-FBEM, as the prosthetic group for labeling a sulfhydryl-functionalized c(RGDyK) monomeric peptide (SRGD) and a E[c(RGDyK)]₂ dimeric peptide (SRGD2). The maleimide group in ¹⁸F-FBEM allows for a thiol-specific Michael addition reaction.

Synthesis

[PubMed]

The c(RGDyK) peptide was first prepared via solution cyclization of the fully protected linear pentapeptide H-Gly-Asp(OtBu)-D-Tyr(OtBu)-Lys(Boc)-Arg(Pbf)-OH, followed by deprotection with trifluoroacetic acid (TFA) in the presence of the free radical scavenger triisopropylsilane (1, 12). The c(RGDyK) was conjugated to N-succinimidyl S-acetylthioacetate (SATA) by mixing c(RGDyK) in sodium borate (Na₂B₄O₇) buffer (pH 8.5) with SATA in dimethyl sulfoxide (1). The yield was 95%. The crude SATA-c(RGDyK) was then dissolved in water and mixed with l-hydroxylamine (pH 6.0) for 2 h to yield the SRGD. The final product was purified with semi-preparative high-performance liquid chromatography (HPLC). The overall yield was 80%. The identity of the SRGD peptide (C₂₉H₄₃N₉O₉S) was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy. FBEM was obtained by reacting N-(2-aminoethyl)maleimide with N-succinimidyl 4-fluorobenzoate in Na₂B₄O₇ (pH 8.5) at 50ºC for 20 min. The reaction was quenched with 2% TFA. The yield was 85% after HPLC purification.

Cai et al. (1) reported the radiolabeling of [¹⁸F]FBEM-SRGD with the use of [¹⁸F]FBEM, which was first prepared from N-succinimidyl 4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB) (10, 13). Then [¹⁸F]SFB in acetonitrile was mixed with N-(2-aminoethyl)maleimide and N,N-diisopropylethylamine. The reaction mixture was heated at 40ºC for 20 min and then quenched with TFA. The HPLC-purified [¹⁸F]FBEM was obtained with a total reaction time of 150 ± 20 min. The non–decay-corrected radiochemical yield was 5 ± 2% (on the basis of ¹⁸F-F−) with a specific activity of 150–200 TBq/mmol (4,050–5,400 Ci/mmol). Purified [¹⁸F]FBEM in phosphate-buffered saline was mixed with the SRGD peptide in dimethyl sulfoxide and tris(2-carboxyethyl)phosphine hydrochloride in water. The pH of the mixture was adjusted to 7.0–7.5 and incubated at room temperature for 20 min. The resulting [¹⁸F]FBEM-SRGD was purified with HPLC. The radiochemical yield was 85 ± 5% (non–decay-corrected) on the basis of [¹⁸F]FBEM. The total reaction time, including final HPLC purification, was ~200 ± 25 min. The overall decay-corrected radiochemical yield was 20 ± 4% (n = 5), and the specific activity was 100–150 TBq/mmol (2,700–4,050 Ci/mmol). The radiochemical purity was >98%.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Cai et al. (1) determined the octanol/water partition coefficient (Log P) of [¹⁸F]FBEM-SRGD to be 0.93 ± 0.02. In vitro cell-binding affinity studies of the unlabeled FBEM-SRGD was conducted in U87MG glioblastoma cells with ¹²⁵I-echistatin as the integrin α_vβ₃–specific radioligand. The inhibition concentration (IC₅₀) for FBEM-SRGD was determined to be 66.8 ± 5.1 nM. In comparison, the IC₅₀ for c(RGDyK) was determined to be 51.3 ± 4.2 nM.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

NIH Support

NIH CA93862, NCI P50 CA114747.

References

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