Synthesis and biological evaluation of potent α_vβ₃-integrin receptor antagonists

Abstract

Introduction

α_vβ₃ Integrin is expressed in sprouting endothelial cells in growing tumors, whereas it is absent in quiescent blood vessels. In addition, various tumor cell types express α_vβ₃ integrin. α_vβ₃ Integrin, a transmembrane heterodimeric protein, binds to the arginine–glycine–aspartic acid (RGD) amino acid sequence of extracellular matrix proteins such as vitronectin and plays a pivotal role in invasion, proliferation and metastasis. Due to the selective expression of α_vβ₃ integrin in tumors, radiolabeled RGD peptides and peptidomimetics are attractive candidates for tumor targeting.

Methods

A cyclic RGD peptide, a peptoid–peptide hybrid, an all-peptoid and a peptidomimetic compound were synthesized, conjugated with 1,4,7,10-tetraazadodecane-N,N′,N″,N‴-tetraacetic acid (DOTA) and radiolabeled with ¹¹¹In. Their in vitro and in vivo α_vβ₃-binding characteristics were determined.

Results

IC₅₀ values were 236 nM for DOTA-E-c(RGDfK), 219 nM for DOTA-peptidomimetic, >10 mM for DOTA-all-peptoid and 9.25 mM for the peptoid–peptide hybrid DOTA-E-c(nRGDfK). ¹¹¹In-labeled compounds, except for [¹¹¹In]DOTA-all-peptoid, showed specific uptake in human α_vβ₃-expressing tumors xenografted in athymic mice. Tumor uptake for [¹¹¹In]DOTA-E-c(RGDfK) was 1.73±0.4% ID/g (2 h postinjection) and that of [¹¹¹In]DOTA-peptidomimetic was 2.04±0.3% ID/g. Tumor uptake for the peptoid–peptide hybrid [¹¹¹In]DOTA-E-c(nRGDfK) was markedly lower (0.45±0.07% ID/g). The all-peptoid [¹¹¹In]DOTA-E-c(nRGnDnFnK) did not show specific uptake in tumors (0.11±0.04% ID/g).

Conclusions

The peptidomimetic compound and the cyclic RGD peptide have a high affinity for α_vβ₃ integrin, and these compounds have better tumor-targeting characteristics than the peptoid–peptide hybrid and the all-peptoid.

Introduction

Integrins are an important class of receptors involved in cell–cell and cell–matrix interactions [1]. Integrins are heterodimeric transmembrane glycoproteins consisting of an α-subunit and a β-subunit. One of the most prominent members of this class of adhesion molecules is α_vβ₃ integrin, which plays a role in a series of pathological processes such as osteoporosis [2], restenosis after balloon angioplasty [3], inflammation [4], rheumatoid arthritis [5], metastasis [6] and tumor-induced angiogenesis [7]. α_vβ₃ Integrin is expressed in sprouting endothelial cells during angiogenesis but not in quiescent endothelial cells. In addition, α_vβ₃ is expressed in various tumor cell types, such as melanoma, ovarian carcinoma and gliomas. This integrin interacts with the arginine–glycine–aspartic acid (RGD) amino acid sequence of extracellular matrix proteins such as vitronectin [8]. Based on this RGD tripeptide sequence, a series of small peptides has been designed to antagonize the function of α_vβ₃ integrin [9]. Some of these peptides, particularly the cyclic ones, have a relatively high and specific affinity for α_vβ₃ integrin [10], [11].

Potentially, radiolabeled RGD peptides and analogs could be used to visualize angiogenesis in tumors. Therefore, several radiolabeled ligands for α_vβ₃ integrin have been synthesized. The first radiolabeled α_vβ₃ antagonist for the investigation of angiogenesis and metastasis in vivo was [¹²⁵I]-3-iodo-d-Tyr⁴-cyclo(–Arg–Gly–Asp–d-Tyr–Val–) [12]. To develop a clinically useful radiolabeled α_vβ₃-integrin antagonist, a variety of factors, such as receptor affinity, α_vβ₃ specificity, hydrophilicity and metabolic stability, must be considered [13]. After a systematic search, Haubner et al. [14] developed a stable ¹⁸F-labeled galactosylated cyclic pentapeptide, with a high affinity and selectivity for α_vβ₃ that accumulated specifically in α_vβ₃-positive tumors and cleared rapidly via the kidneys. In a recent study, they showed that this positron emission tomography tracer can be used to visualize α_vβ₃ expression in tumors [15].

In peptidomimetic compounds, amino acids are replaced by nonpeptidic building blocks [16]. These modifications can be divided into backbone modifications, such as N-methylation and side-chain modification, introducing unnatural amino acids. N-methylation of peptide bonds could improve the binding characteristics of a peptide. For example, N-methylation of the valine residue of the α_vβ₃ antagonist cyclo(RGDfV) resulted in a more active compound, cyclo(RGDf-N(Me)V-). The antiangiogenic activity of this compound, cilengitide, is currently in clinical Phase II studies [17]. Peptoids are a class of peptidomimetics built from N-substituted glycine residues. In these so-called peptoid residues, the position of the amino acid side chain is shifted from the α-carbon atom to the nitrogen atom [18]. Evidently, these modifications have consequences for the biological activity of a peptoid or a peptoid–peptide hybrid compared to the parent peptide.

The aim of this study was to develop RGD analogs with improved in vivo α_vβ₃-targeting characteristics. Here, we describe the synthesis and the radiolabeling of four 1,4,7,10-tetraazadodecane-N,N′,N″,N‴-tetraacetic acid (DOTA)-conjugated compounds: a cyclic pentapeptide RGDfK (Fig. 1A), a cyclic peptoid–peptide hybrid in which arginine residue is replaced by the corresponding peptoid residue (Fig. 1B), an all-peptoid in which all amino acid residues are replaced by their corresponding peptoid residues (Fig. 1C) and a peptidomimetic α_vβ₃ receptor antagonist recently described by Hood et al. [19] (Fig. 1D). Stability and in vitro and in vivo α_vβ₃-binding characteristics are systematically studied.