64Cu-NO2A-RGD-Glu-6-Ahx-BBN(7-14)NH2: a heterodimeric targeting vector for positron emission tomography imaging of prostate cancer

Abstract

Introduction The present study describes the design and development of a new heterodimeric RGD-bombesin (BBN) agonist peptide ligand for dual receptor targeting of the form 64Cu-NO2A-RGD-Glu-6-Ahx-BBN(7-14)NH2 in which Cu-64=a positron emitting radiometal; NO2A=1,4,7-triazacyclononane-1,4-diacetic acid; Glu=glutamic acid; 6-Ahx=6-aminohexanoic acid; RGD=the amino acid sequence [Arg-Gly-Asp], a nonregulatory peptide that has been used extensively to target αvβ3 receptors up-regulated on tumor cells and neovasculature; and BBN(7-14)NH2=Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2, an agonist analogue of bombesin peptide for specific targeting of the gastrin-releasing peptide receptor (GRPr). Methods RGD-Glu-6-Ahx-BBN(7-14)NH2 was manually coupled with NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), and the resulting conjugate was labeled with 64Cu to yield 64Cu-NO2A-RGD-Glu-6-Ahx-BBN(7-14)NH2. Purification was achieved via reversed-phase high-performance liquid chromatography and characterization confirmed by electrospray ionization–mass spectrometry. Results Competitive displacement binding assays displayed single-digit nanomolar IC50 values showing very high binding affinities toward the GRPr for the new heterodimeric peptide analogues. In vivo biodistribution studies showed high uptake and retention of tumor-associated radioactivity in PC-3 tumor-bearing rodent models with little accumulation and retention in nontarget tissues. The radiolabeled conjugate also exhibited rapid urinary excretion and high tumor-to-background ratios. Micro-positron emission tomography (microPET) molecular imaging investigations produced high-quality, high-contrast images in PC-3 tumor-bearing mice 15 h postinjection. Conclusions Based on microPET imaging experiments that show high-quality, high-contrast images with virtually no residual gastrointestinal radioactivity, this new heterodimeric RGD-BBN conjugate can be considered as a promising PET tracer candidate for the diagnosis of GRPr-positive tumors in human patients.

N-Terminal Modifications Improve the Receptor Affinity and Pharmacokinetics of Radiolabeled Peptidic Gastrin-Releasing Peptide Receptor Antagonists: Examples of 68Ga- and 64Cu-Labeled Peptides for PET Imaging

“…Various radiolabeled GRPr agonists have been synthesized, evaluated, and applied in nuclear oncology (4,(16)(17)(18)(19)(20)(21)(22)(23). Innovative developments using agonists hybridized with other targeting units such as a v b 3 integrins or hypoxia-targeting agents such as 2-nitroimidazoles (25). The successful pharmacokinetic performance of somatostatin-based antagonistic radiotracers preclinically (6,26,27) and clinically (28) led several research groups to develop GRPr antagonists, which indeed exhibit pharmacokinetics superior to agonists (7)(8)(9)(10)(29)(30)(31)(32).…”

A heterodimeric [RGD-Glu-[64Cu-NO2A]-6-Ahx-RM2] αvβ3/GRPr-targeting antagonist radiotracer for PET imaging of prostate tumors

“…NOTA bifunctional chelating agent was manually conjugated to the [RGD-Glu-6Ahx-RM2] dimeric peptide analogue via an active ester per a previously described procedure [28] to yield [RGDGlu-[NO2A]-6-Ahx-RM2]. All other reagents/solvents were purchased from Fisher Scientific (Pittsburgh, PA, USA) or Sigma-Aldrich Chemical Company (St. Louis, MO, USA) and used without further purification.…”

“…Female CF-1 and Institute of Cancer Research severe combined immunodeficient (SCID) female mice (4–5 weeks of age) were supplied from Taconic Farms (Germantown, NY, USA). PC-3 cells were injected into the subcutaneous flanks of female SCID mice according to published procedure [28]. Tumors were allowed to grow two to three weeks postinoculation, developing tumors ranging in mass from 0.05 g to 0.34 g. Biodistribution studies in CF-1 and SCID mice were performed by injection of ~20 μCi (~0.75 MBq, 8.08x10 -14 -2.02x10 -13 mol, 9.16x10 18 Bq/mol) of [RGD-Glu-[ 64 Cu-NO2A]-6-Ahx-RM2] (100 μL, 0.9% NaCl) into the tail vein.…”

“…according to published procedure [28]. Micro-computed tomography (microCT) coronal images were also obtained on the Siemens INVEON small-animal CT unit (Nashville, TN, USA) immediately following microPET imaging for the purpose of fusing the anatomic and molecular data.…”

“…These compounds have traditionally been modeled after a single, endogenous peptide hormone, small peptide, or antibody, which begets the tendency for them to bind only to those cancer cells which adequately express the target molecule. However, results of new heterodimeric peptide conjugates have continued to show improved molecular imaging properties when compared to monomeric peptide conjugates [28-31]. This is presumably due to an effective increase in the total number of receptors that are available for binding the heterodimer.…”