Radiolabeled high affinity peptidomimetic antagonist selectively targets αvβ3 receptor-positive tumor in mice

https://doi.org/10.1016/j.nucmedbio.2007.02.002Get rights and content

Abstract

Objectives

The aim of this research was to synthesize radiolabeled peptidomimetic integrin αvβ3 antagonists that selectively target integrin αvβ3 receptor and clear rapidly from the whole body.

Methods

Integrin αvβ3 antagonists, 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-(S)-aminoethylsulfonyl-amino-β-alanine (IA) and 4-[2-(3,4,5,6-tetrahydro-pyrimidin-2-ylamino)-ethyloxy]benzoyl-2-(S)-[N-(3-amino-neopenta-1-carbamyl)]-aminoethylsulfonylamino-β-alanine hydrochloride (IAC), a hydrophobic carbamate derivative of IA, were conjugated with 2-p-isothiocyanatobenzyl-DOTA at the amino terminus and labeled with 111In. The 111In labeled IA and IAC were subjected to in vitro receptor binding, biodistribution and imaging studies using nude mice bearing the receptor-positive M21 human melanoma xenografts.

Results

The 111In-labeled IA (40%) and -IAC (72%) specifically bound in vitro to αvβ3 (0.8 μM) at a molar excess. This receptor binding was completely blocked by a molar excess of cold IA to αvβ3. The higher receptor-binding affinity of the 111In-labeled IAC was reflected in higher tumor uptake and retention: 5.6±1.4 and 4.5±0.7 %ID/g vs. 3.8±0.9 and 2.0±0.3 %ID/g for the 111In-labeled IA at 0.33 and 2 h. The tumor uptakes were inhibited by the co-injection of 200 μg of IA, indicating that the uptake was receptor mediated. These antagonists were excreted primarily via the renal system. The 111In activity retained in the whole body was quite comparable between the 111In-labeled IA (24% ID) and the 111In-labeled IAC (33% ID) at 2 h. The higher peak tumor uptake and longer retention resulted in higher tumor-to-background ratios for the 111In-labeled IAC at 2 h with 9.7, 2.3, 0.8, 1.9, 7.1, 2.2, 0.9, 3.7 and 9.9 for blood, liver, kidney, lung, heart, stomach, intestine, bone and muscle, respectively. The imaging studies with the 111In-labeled IAC also clearly visualized the receptor-positive tumor at 4 h.

Conclusions

The 111In-labeled IAC showed an improve tumor targeting kinetics with rapid accumulation and prolonged retention in the αvβ3 receptor-positive tumor. This together with the rapid whole-body clearance pharmacokinetics warrants further studies on this IAC analog for molecular imaging of tumor-induced angiogenic vessels and various malignant human tumors expressing the receptor.

Introduction

Integrin αvβ3 is a heterodimeric transmembrane glycoprotein [1] and is overexpressed in tumor-induced angiogenic vessels and in various malignant human tumors [2], [3]. Selective targeting of this αvβ3 receptor with radioligands may enable the assessment of angiogenesis and receptor status in tumors.

Integrin αvβ3 is a receptor for extracellular proteins including vitronectin, fibronectin and fibrinogen that contain an arginine-glycine-aspartic acid (RGD) sequence [4], [5]. Recently, RGD peptides and peptidomimetic antagonists specific for αvβ3 receptor have been labeled with various gamma and positron emitters for scintigraphic detection [6], [7], [8], [9], [10], and gamma and beta emitters for radiotherapy of tumors [11], [12], [13], [14], [15]. Steady progress has been reported in optimizing the labeling methodologies to increase tumor-to-nontumor tissue ratios, especially the tumor-to-liver and tumor-to-kidney ratios by increasing the hydrophilicity of the product via glycosylation and PEGylation of radiolabeled RGD peptides [16], [17], [18], [19]. Recently, dimeric and tetrameric RDG peptides labeled with 18F and 64Cu were synthesized [20], [21], [22]. It was reported that these oligomeric RDG peptides enhanced the receptor-binding affinity, thereby improving the tumor targeting and slowing the wash-out of radioactivity from tumor.

A peptidomimetic integrin αvβ3 antagonist, 4-[2-(3,4,5,6-tetrahydropyrimidine-2-ylamino)ethyloxy]benzoyl-2-(S)-aminoethylsulfonyl-amino-β-alanine (IA), has been exploited as an integrin receptor-mediated tumor targeting agent. IA was coupled to a cationic polymerized lipid-based nanoparticle and was successfully used to deliver a mutant Raf gene [23] and 90Y [24] to αvβ3 receptor on the neovasculature for tumor regression. Recently, Burnett et al. [25] modified the ethylamine terminus of IA to a series of the corresponding carbamate derivatives and reported that the hydrophobic carbamate-linked appendages improved the αvβ3 receptor-binding affinity of the parent compound IA by 2 to 20 times. They conjugated the highest affinity antagonist 4-[2-(3,4,5,6-tetrahydro-pyrimidin-2-ylamino)-ethyloxy]benzoyl-2-(S)-[N-(3-amino-neopenta-1-carbamyl)]-aminoethylsulfonylamino-β-alanine hydrochloride (IAC) to fluorescein isothiocyanate and demonstrated that this IAC with the optical probe was avidly taken up by the receptor-expressing M21 melanoma cells in vitro and in vivo. We extended this research by labeling IA and IAC with 111In using 2-(p-isothiocyanatobenzyl)-DOTA, as a bifunctional chelator, to synthesize αvβ3 receptor-targeting radiopharmaceuticals that rapidly target the receptor expressing tumor while being excreted via the renal system to produce low nontarget radioactivity in the abdomen. In this paper, we report the 111In labeling of IA and IAC, and their in vitro αvβ3 receptor-binding study, biodistribution and imaging studies using nude mice implanted with the receptor-positive M21 melanoma cells.

Section snippets

Chemistry of conjugation and radiolabeling

The synthesis and chemical identification of IA and IAC were previously reported [25]. To label with 111In, the amino terminus of IA or IAC (10-20 mM) was reacted with 2-(p-isothiocyanatobenzyl)-DOTA (Macrocyclics, Inc., Dallas, TX; SCN-Bz-DOTA; 1-2 mM) in 40 μl of 0.1 M sodium bicarbonate at pH 8.4 for 2 days at room temperature. The product formation was monitored by reverse-phase HPLC. It was determined that the reaction of 2 mM SCN-Bz-DOTA with 20 mM IA or IAC produced a quantitative yield

Chemistry of conjugation and radiolabeling

The conjugation yield of SCN-Bz-DOTA to the amino end of IA or IAC increased proportionally as the concentration of IA or IAC was increased when monitored by reverse-phase HPLC. A quantitative conjugation yield was obtained, based on reverse-phase HPLC analysis, when the IA concentration was >20 mM and the reaction was performed at an IA or IAC to SCN-Bz-DOTA molar ratio of 10 at pH 8.4 for 2 days. Under this condition, the product formed is believed to be a 1:1 conjugate of the DOTA at the

Discussion

The goal of this research was to synthesize 111In-labeled peptidomimetic antagonists that would target αVβ3 receptor-positive tumor and produce a high target to nontarget radioactivity ratio at an early time after injection. In this research, we have investigated the effect of the receptor-binding affinity of the 111In-labeled peptidomimetic antagonists on the in vivo tumor-targeting kinetics. We used SCN-Bz-DOTA as a bifunctional chelator because DOTA forms inert complexes with a variety of

Acknowledgments

We thank Monica Bur for her assistance with the animal studies and Colleen Henrichsen for editorial assistance.

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    1

    Current address: Department of Radiology, The Methodist Hospital, 6565 Fannin Street, MB1-066 Houston, TX 77030, USA.

    2

    Current address: Nuclear Medicine Service, Memorial Sloan-Kettering Cancer Center, Mailbox 77, 1275 York Avenue, New York, NY 10021, USA.

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