Positron-emission tomography (PET) imaging agents for diagnosis of human prostate cancer: agonist vs. antagonist ligands.

AIM: The present study adds scientific support to the growing debate regarding the superiority of radiolabeled bombesin-based antagonist peptides over agonists for molecular imaging and therapy of human tumors overexpressing the gastrin-releasing peptide receptor (GRPR) and describes a detailed in vitro and in vivo comparison of 64Cu-NODAGA-6-Ahx-BBN(7-14)NH2 agonist and 64Cu-NODAGA-6-Ahx-DPhe6-BBN(6-13)NHEt antagonist ligands. MATERIALS AND METHODS: Conjugates were synthesized by solid-phase peptide synthesis, purified by reversed-phase high-performance liquid chromatography, and characterized by electrospray ionization-mass spectroscopy. The conjugates were radiolabeled with 64Cu. RESULTS: In vitro and in vivo data support the hypothesis for targeting of the GRPR by these tracer molecules. Maximum-intensity micro Positron Emission Tomography (microPET) imaging studies show the agonist ligand to provide high-quality, high-contrast images with very impressive tumor uptake and background clearance, with virtually no residual gastrointestinal or renal-urinary radioactivity. CONCLUSION: Based on microPET imaging experiments, we conclude the agonist peptide ligand to be a superior molecular imaging agent for targeting GRPR.

Bombesin analogues for gastrin-releasing peptide receptor imaging.

OBJECTIVES: The present study describes the design and development of a series of new bombesin (BBN) antagonist peptide ligands of the form [(64)Cu-(NO2A-X-D-Phe(6)-BBN(6-13)NHEt)], where Cu-64=a positron emitting radiometal; NO2A=1,4,7-triazacyclononane-1,4-diacetic acid; X=6-amino hexanoic acid, 8-amino octanoic acid or 9-Aminononanoic acid; and BBN(6-13)NHEt=Gln-Trp-Ala-Val-Gly-His-Leu-NHEt, an antagonist analogue of bombesin peptide for specific targeting of the gastrin-releasing peptide receptor (GRPR). METHODS: [NO2A-X-D-Phe(6)-BBN(6-13)NHEt] conjugates were manually conjugated with NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), and the resulting conjugates were labeled with (64)Cu to yield [(64)Cu-(NO2A-X-D-Phe(6)-BBN(6-13)NHEt)]. The metallated and nonmetallated conjugates were purified via reversed-phase high-performance liquid chromatography and characterized by electrospray ionization-mass spectrometry. RESULTS: Competitive displacement binding assays displayed nanomolar binding affinities toward human GRPR for all of the newly formed peptide analogues. Biodistribution studies showed very high uptake and retention of tumor-associated radioactivity in PC-3 (a prostate tumor model known to express the GRPR) tumor-bearing rodent models. The radiolabeled conjugates also exhibited rapid urinary excretion and very high tumor to background ratios. Micro-positron emission tomography (PET) molecular imaging investigations showed clear visualization of tumors in female PC-3 tumor-bearing mice 15 h postinjection. CONCLUSION: The biodistribution and molecular imaging study suggests that these conjugates can be considered as potential PET tracer candidates for the diagnosis of GRPR-positive tumors in human patients.

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

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 (64)Cu-NO2A-RGD-Glu-6-Ahx-BBN(7-14)NH(2) 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 α(v)ß(3) receptors up-regulated on tumor cells and neovasculature; and BBN(7-14)NH(2)=Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH(2), an agonist analogue of bombesin peptide for specific targeting of the gastrin-releasing peptide receptor (GRPr). METHODS: RGD-Glu-6-Ahx-BBN(7-14)NH(2) was manually coupled with NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), and the resulting conjugate was labeled with (64)Cu to yield (64)Cu-NO2A-RGD-Glu-6-Ahx-BBN(7-14)NH(2). 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 IC(50) 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.

Radiotracers for different angiogenesis receptors in a melanoma model.

Early and reliable diagnosis of melanoma, a skin tumor with a poor prognosis, is extremely important. Phage display peptide libraries are a convenient screening resource for identifying bioactive peptides that interact with cancer targets. The aim of this study was to evaluate two technetium-99m tracers for angiogenesis detection in a melanoma model, using cyclic pegylated pentapeptide with RGD and NGR motifs conjugated with the bifunctional chelator mercaptoacetyltriglycine (MAG(3)). The conjugated peptides (10 µl of a µg/µl solution) were labeled with technetium-99m using a sodium tartrate buffer. Radiochemical evaluation was carried out by instant thin-layer chromatography and confirmed by high-performance liquid chromatography. The partition coefficient was determined and internalization assays were performed in two melanoma cell lines (B16F10 and SKMEL28). Biodistribution evaluation of the tracers was carried out in healthy animals at different time points and also in tumor-bearing mice, 120 min post injection. Blocking studies were also conducted by coinjection of cold peptides. The conjugates displayed a rather similar pharmacokinetic profile. They were radiolabeled with high radiochemical purity (>97%) and both were hydrophilic with preferential renal excretion. Yet, tumor uptake was higher for human than for murine melanoma cells, especially for [(99m)Tc]-MAG(3)-PEG(8)-c(RGDyk) (7.85±2.34%injected dose/g 120 min post injection). The performance of [(99m)Tc]-MAG(3)-PEG(8)-c(RGDyk) was better than the NGR tracer with regard to human melanoma uptake. In this sense, it should be considered for future radiotracer studies of tumor diagnosis.

Radiolabeled regulatory peptides for imaging and therapy.

PURPOSE OF REVIEW: The purpose of the present review is to describe new, innovative strategies of diagnosing and treating specific human cancers using a cadre of radiolabeled regulatory peptides. RECENT FINDINGS: Peptide receptor-targeted radionuclide therapy is a method of site-directed radiotherapy that specifically targets human cancers expressing a cognate receptor-subtype in very high numbers. Ideally, the procedure targets only the primary or metastatic disease and is minimally invasive, with little radiation damage to normal, collateral tissues. For treatment strategies of this type to be effective, it is critical to evaluate the toxicity of the treatment protocol, the radiation dosimetry of the therapeutic regimen, and the biological profile of the radiopharmaceutical, including biodistribution and pharmacokinetics of the drug. Site-directed molecular imaging procedures via gamma-scintigraphy can address many of the critical issues associated with peptide receptor-targeted radionuclide therapy and it is, therefore, necessary to describe the effective balance between the clinical benefits and risks of this treatment strategy. SUMMARY: Continued development in the design or chemical structure of radiolabeled, biologically active peptides could do much to improve the targeting ability of these drugs, thereby creating new and innovative strategies for diagnosis or treatment of human cancers.

In vitro and in vivo analysis of [(64)Cu-NO2A-8-Aoc-BBN(7-14)NH(2)]: a site-directed radiopharmaceutical for positron-emission tomography imaging of T-47D human breast cancer tumors.

INTRODUCTION: Human breast cancer, from which the T-47D cell line was derived, is known to overexpress the gastrin-releasing peptide receptor (GRPR) in some cases. Bombesin (BBN), an agonist for the GRPR, has been appended with a radionuclide capable of positron-emission tomography (PET) imaging and therapy. (64)Cu-NO2A-8-Aoc-BBN(7-14)NH(2) (NO2A=1,4,7-triazacyclononane-1,4-diacetate) has produced high-quality microPET images of GRPR-positive breast cancer xenografted tumors in mice. METHODS: The imaging probe was synthesized by solid-phase peptide synthesis followed by manual conjugation of the 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) bifunctional chelator and radiolabeling in aqueous solution. The radiolabeled conjugate was subjected to in vitro and in vivo studies to determine its specificity for the GRPR and its pharmacokinetic profile. A T-47D tumor-bearing mouse was imaged with microPET/CT and microMRI imaging. RESULTS: The (64)Cu-NO2A-8-Aoc-BBN(7-14)NH(2) targeting vector was determined to specifically localize in GRPR-positive tissue. Accumulation was observed in the tumor in sufficient quantities to allow for identification of tumors in microPET imaging procedures. For example, uptake and retention in T-47D xenografts at 1, 4 and 24 h were determined to be 2.27+/-0.08, 1.35+/-0.14 and 0.28+/-0.07 % ID/g, respectively. CONCLUSIONS: The (64)Cu-NO2A-8-Aoc-BBN(7-14)NH(2) produced high-quality microPET images. The pharmacokinetic profile justifies investigation of this bioconjugate as a potentially useful diagnostic/therapeutic agent. Additionally, the bioconjugate would serve as a good starting point for modification and optimization of similar agents to maximize tumor uptake and minimize nontarget accumulation.

[64Cu-NOTA-8-Aoc-BBN(7-14)NH2] targeting vector for positron-emission tomography imaging of gastrin-releasing peptide receptor-expressing tissues.

Radiolabeled peptides hold promise as diagnostic/therapeutic targeting vectors for specific human cancers. We report the design and development of a targeting vector, [(64)Cu-NOTA-8-Aoc-BBN(7-14)NH(2)] (NOTA = 1,4,7-triazacyclononane-1,4,7-triacetic acid, 8-Aoc = 8-aminooctanoic acid, and BBN = bombesin), having very high selectivity and affinity for the gastrin-releasing peptide receptor (GRPr). GRPrs are expressed on a variety of human cancers, including breast, lung, pancreatic, and prostate, making this a viable approach toward site-directed localization or therapy of these human diseases. In this study, [NOTA-X-BBN(7-14)NH(2)] conjugates were synthesized, where X = a specific pharmacokinetic modifier. The IC(50) of [NOTA-8-Aoc-BBN(7-14)NH(2)] was determined by a competitive displacement cell-binding assay in PC-3 human prostate cancer cells using (125)I-[Tyr(4)]-BBN as the displacement ligand. An IC(50) of 3.1 +/- 0.5 nM was obtained, demonstrating high binding affinity of [NOTA-8-Aoc-BBN] for the GRPr. [(64)Cu-NOTA-X-BBN] conjugates were prepared by the reaction of (64)CuCl(2) with peptides in buffered aqueous solution. In vivo studies of [(64)Cu-NOTA-8-Aoc-BBN(7-14)NH(2)] in tumor-bearing PC-3 mouse models indicated very high affinity of conjugate for the GRPr. Uptake of conjugate in tumor was 3.58 +/- 0.70% injected dose (ID) per g at 1 h postintravenous injection (p.i.). Minimal accumulation of radioactivity in liver tissue (1.58 +/- 0.40% ID per g, 1 h p.i.) is indicative of rapid renal-urinary excretion and suggests very high in vivo kinetic stability of [(64)Cu-NOTA-8-Aoc-BBN(7-14)NH(2)] with little or no in vivo dissociation of (64)Cu(2+) from the NOTA chelator. Kidney accumulation at 1 h p.i. was 3.79 +/- 1.09% ID per g. Molecular imaging studies in GRPr-expressing tumor models produced high-contrast, high-quality micro-positron-emission tomography images.