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