Research article
Development of a 111In-labeled peptide derivative targeting a chemokine receptor, CXCR4, for imaging tumors

https://doi.org/10.1016/j.nucmedbio.2006.01.006Get rights and content

Abstract

The chemokine receptor CXCR4 is highly expressed in tumor cells and plays an important role in tumor metastasis. The aim of this study was to develop a radiopharmaceutical for the imaging of CXCR4-expressing tumors in vivo. Based on structure–activity relationships, we designed a 14-residue peptidic CXCR4 inhibitor, Ac-TZ14011, as a precursor for radiolabeled peptides. For 111In-labeling, diethylenetriaminepentaacetic acid (DTPA) was attached to the side chain of d-Lys8 which is distant from the residues indispensable for the antagonistic activity. In-DTPA-Ac-TZ14011 inhibited the binding of a natural ligand, stromal cell-derived factor-1α, to CXCR4 in a concentration-dependent manner with an IC50 of 7.9 nM (Ac-TZ14011: 1.2 nM). In biodistribution experiments, more 111In-DTPA-Ac-TZ14011 accumulated in the CXCR4-expressing tumor than in blood or muscle. Furthermore, the tumor-to-blood and tumor-to-muscle ratios were significantly reduced by coinjection of Ac-TZ14011, indicating a CXCR4-mediated accumulation in tumor. These findings suggested that 111In-DTPA-Ac-TZ14011 would be a potential agent for the imaging of CXCR4 expression in metastatic tumors in vivo.

Introduction

Chemokines are a family of small proteins (8–14 kDa) that chemoattract leukocytes by binding to cell surface receptors, chemokine receptors [1]. The chemokine receptor family, which belongs to a superfamily of seven transmembrane domain G-protein-coupled receptors, comprises 18 members [2]. In 1996, one member, CXCR4, was identified as a coreceptor for the entry of T-cell line-tropic HIV-1 [3]. Since then, this receptor has attracted considerable attention as a pathogenic factor or a therapeutic target for HIV infection. Recent studies indicated that CXCR4 and its ligand, stromal cell-derived factor-1 (SDF-1), play an important role also in tumor metastasis [4], [5], [6], [7], [8]. Müller et al. [4] reported that CXCR4 was highly expressed in breast cancer and SDF-1 was highly expressed in organs representing the first destinations of metastasis. Moreover, they demonstrated that neutralization with anti-CXCR4 monoclonal antibody significantly inhibited the metastasis of breast cancer cells in mice. Similar results were obtained in other types of cancer [5], [6], [7], [8]. These findings suggest that CXCR4 is a potential target for the in vivo imaging of metastatic tumors.

We have previously demonstrated that a peptide with anti-HIV-1 activity, T22 ([Tyr5,12, Lys7]-polyphemusin II), is an inhibitor of CXCR4 that blocks the entry of T-cell line-tropic HIV-1 mediated by this receptor. T22 is an 18-residue peptide amide, which was previously found by us based on an analysis of the structure–activity relationships of self-defense peptides of horseshoe crabs, tachyplesin and polyphemusin [9], [10]. On the basis of the structure of T22, we designed and synthesized several downsized analogs, 14-residue peptides [11], [12]. Among them, T140 showed the greatest inhibitory effect on the binding of an anti-CXCR4 monoclonal antibody to CXCR4 and the strongest inhibitory activity against HIV-1 entry [12]. The aim of this study was to develop a radiolabeled T140 derivative as an imaging agent for metastatic tumors. Considering that the three residues on the restricted backbone (l-3-(2-naphthyl)alanine (Nal)3, Tyr5 and Arg14) and the single residue in the flexible region (Arg2) form the intrinsic pharmacophore of T140 [13], [14], [15], we designed a 14-residue peptidic inhibitor, Ac-TZ14011, as the precursor for radiolabeled peptides (Fig. 1). This precursor contains the above four residues which are necessary for the inhibitory activity against CXCR4. Furthermore, for site-selective conjugation of radiolabels, Ac-TZ14011 has a single amino group (d-Lys8), which is distant from the pharmacophore, and the carboxyl group of Arg14 of Ac-TZ14011 is protected via amidation for stability in vivo [16], [17].

111In constitutes one of the most useful radionuclides for the radiolabeling of peptides for diagnostic applications in nuclear medicine. Diethylenetriaminepentaacetic acid (DTPA) is still an attractive chelating agent with which to prepare 111In-labeled peptides since it provides 111In-labeled peptides with highly specific activity. In addition, the development of a monoreactive DTPA derivative has provided an easy and efficient way to prepare DTPA-conjugated peptides [18], [19]. In this study, DTPA-Ac-TZ14011 was prepared using a monoreactive DTPA derivative and coordinated with nonradioactive In or radioactive 111In. Furthermore, the antagonistic activity of In-DTPA-Ac-TZ14011 and in vivo behavior of 111In-DTPA-Ac-TZ14011 were investigated and the applicability of 111In-DTPA-Ac-TZ14011 as a radiopharmaceutical for imaging tumors was evaluated.

Section snippets

Reagents and chemicals

111InCl3 (74 MBq/ml in 0.02N HCl) was kindly supplied by Nihon Medi-Physics (Nishinomiya, Japan). 9-Fluorenylmethoxycarbonyl (Fmoc)-protected amino acids and 4-(2′,4′-dimethoxyphenylaminomethyl)phenoxy (SAL) resin were purchased from Watanabe Chemical Industries (Hiroshima, Japan) or Calbiochem-Novabiochem Japan (Tokyo, Japan). 1-tert-Butyl hydrogen 3,6,9-tris((tert-butoxycarbonyl)methyl)-3,6,9-triazaundecanedioic acid (mDTPA) was synthesized as reported previously [18]. All the other chemicals

Results and discussion

T140 and its analogs have one disulfide bond and maintain an antiparallel β-sheet structure connected by a type II′ β-turn with d-Lys8-Pro9 at the (i+1) and (i+2) positions, and the side chain of d-Lys8 is distant from the pharmacophore for the antagonistic activity [14], [15]. Therefore, we designed Ac-TZ14011 as a mother compound that contains the residues indispensable for the antagonistic activity and has a single amino group of d-Lys8 for site-selective conjugation of DTPA (Fig. 1). In

Acknowledgments

We are grateful to Nihon Medi-Physics (Nishinomiya, Japan) for the gift of 111InCl3. This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Health, Labour and Welfare.

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