Charles J. Smith

[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, [64Cu-NOTA-8-Aoc-BBN(7-14)NH2] (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)NH2] conjugates were synthesized, where X = a specific pharmacokinetic modifier. The IC50 of [NOTA-8-Aoc-BBN(7-14)NH2] was determined by a competitive displacement cell-binding assay in PC-3 human prostate cancer cells using 125I-[Tyr4]-BBN as the displacement ligand. An IC50 of 3.1 ± 0.5 nM was obtained, demonstrating high binding affinity of [NOTA-8-Aoc-BBN] for the GRPr. [64Cu-NOTA-X-BBN] conjugates were prepared by the reaction of 64CuCl2 with peptides in buffered aqueous solution. In vivo studies of [64Cu-NOTA-8-Aoc-BBN(7-14)NH2] 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 [64Cu-NOTA-8-Aoc-BBN(7-14)NH2] with little or no in vivo dissociation of 64Cu2+ 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.

Laminin receptor specific therapeutic gold nanoparticles (198AuNP-EGCg) show efficacy in treating prostate cancer

Systemic delivery of therapeutic agents to solid tumors is hindered by vascular and interstitial barriers. We hypothesized that prostate tumor specific epigallocatechin-gallate (EGCg) functionalized radioactive gold nanoparticles, when delivered intratumorally (IT), would circumvent transport barriers, resulting in targeted delivery of therapeutic payloads. The results described herein support our hypothesis. We report the development of inherently therapeutic gold nanoparticles derived from the Au-198 isotope; the range of the 198Au β-particle (approximately 11 mm in tissue or approximately 1100 cell diameters) is sufficiently long to provide cross-fire effects of a radiation dose delivered to cells within the prostate gland and short enough to minimize the radiation dose to critical tissues near the periphery of the capsule. The formulation of biocompatible 198AuNPs utilizes the redox chemistry of prostate tumor specific phytochemical EGCg as it converts gold salt into gold nanoparticles and also selectively binds with excellent affinity to Laminin67R receptors, which are over expressed in prostate tumor cells. Pharmacokinetic studies in PC-3 xenograft SCID mice showed approximately 72% retention of 198AuNP-EGCg in tumors 24 h after intratumoral administration. Therapeutic studies showed 80% reduction of tumor volumes after 28 d demonstrating significant inhibition of tumor growth compared to controls. This innovative nanotechnological approach serves as a basis for designing biocompatible target specific antineoplastic agents. This novel intratumorally injectable 198AuNP-EGCg nanotherapeutic agent may provide significant advances in oncology for use as an effective treatment for prostate and other solid tumors.

Gastrin releasing peptide (GRP) receptor targeted radiopharmaceuticals: a concise update.

The gastrin releasing peptide (GRP) receptor is becoming an increasingly attractive target for development of new radiolabeled peptides with diagnostic and therapeutic potential. The attractiveness of the GRP receptor as a target is based upon the functional expression of GRP receptors in several tumors of neuroendocrine origin including prostate, breast, and small cell lung cancer. This concise review outlines some of the efforts currently underway to develop new GRP receptor specific radiopharmaceuticals by employing a variety of radiometal chelation systems.

Optimization, biological evaluation and microPET imaging of copper-64-labeled bombesin agonists, [64Cu-NO2A-(X)-BBN(7–14)NH2], in a prostate tumor xenografted mouse model☆

UNLABELLED Gastrin-releasing peptide receptors (GRPr) are a member of the bombesin (BBN) receptor family. GRPr are expressed in high numbers on specific human cancers, including human prostate cancer. Therefore, copper-64 ((64)Cu) radiolabeled BBN(7-14)NH(2) conjugates could have potential for diagnosis of human prostate cancer via positron-emission tomography (PET). The aim of this study was to produce [(64)Cu-NO2A-(X)-BBN(7-14)NH(2)] conjugates for prostate cancer imaging, where X=pharmacokinetic modifier (beta-alanine, 5-aminovaleric acid, 6-aminohexanoic acid, 8-aminooctanoic acid, 9-aminonanoic acid or para-aminobenzoic acid) and NO2A=1,4,7-triazacyclononane-1,4-diacetic acid [a derivative of NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid)]. METHODS [(X)-BBN(7-14)NH(2)] Conjugates were synthesized by solid-phase peptide synthesis (SPPS), after which NOTA was added via manual conjugation. The new peptide conjugates were radiolabeled with (64)Cu radionuclide. The receptor-binding affinity was determined in human prostate PC-3 cells, and tumor-targeting efficacy was determined in PC-3 tumor-bearing severely combined immunodeficient (SCID) mice. Whole-body maximum intensity microPET/CT images of PC-3 tumor-bearing SCID mice were obtained 18 h postinjection (pi). RESULTS Competitive binding assays in PC-3 cells indicated high receptor-binding affinity for the [NO2A-(X)-BBN(7-14)NH(2)] and [(nat)Cu-NO2A-(X)-BBN(7-14)NH(2)] conjugates. In vivo biodistribution studies of the [(64)Cu-NO2A-(X)-BBN(7-14)NH(2)] conjugates at 1, 4 and 24 h pi showed very high uptake of the tracer in GRPr-positive tissue with little accumulation and retention in nontarget tissues. High-quality, high-contrast microPET images were obtained, with xenografted tumors being clearly visible at 18 h pi. CONCLUSIONS NO2A chelator sufficiently stabilizes copper(II) radiometal under in vivo conditions, producing conjugates with very high uptake and retention in targeted GRPr. Preclinical evaluation of these new peptide conjugates in tumor-bearing mice provides some impetus for clinical evaluation in human patients.

Radiolabeled bombesin analogs for prostate cancer diagnosis: preclinical studies

INTRODUCTION Radionuclide imaging can be a useful tool for the diagnosis of prostate cancer. Bombesin (BBN) is a molecule with high affinity for gastrin releasing peptide (GRP) receptors which are over-expressed in that tumor. This report compares (99m)Tc-HYNIC-betaAla-BBN(7-14)NH2 [(99m)Tc-HYNIC-BBN] and (99m)Tc identical withN(PNP6)-Cys-betaAla-BBN(7-14)NH2 [(99m)TcN(PNP6)-Cys-BBN] with regard to labeling procedures as well as in vitro and in vivo evaluation (biodistribution and scintigraphic imaging). METHODS Peptide synthesis was performed in an automated peptide synthesizer. HYNIC-BBN was radiolabeled with pertechnetate using tricine and ethylenediamine diacetic acid (EDDA) as coligands. Cys- BBN was radiolabeled in a two-step procedure with the preparation of the precursor (99m)Tc-Nitrido first and then introducing diphosphine (PNP6). Radiochemical evaluation of conjugates, as well as studies of stability, transchelation toward cysteine, and partition coefficient were done. Biological studies included internalization, biodistribution in healthy animals and in animals bearing PC3 cancer cells with acquisition of images from the tumor-bearing animals. RESULTS Both complexes showed a high radiochemical yield along with good stability. Biodistribution studies pointed out strong renal excretion for the former complex due to its hydrophilic profile and marked hepatobiliary excretion for the latter, corresponding to observed lipophilicity. Tumor uptake was higher for (99m)Tc-HYNIC-BBN and the same occurred with internalization findings, which exceeded those of (99m)TcN(PNP6)-BBN. Blocking studies in mice bearing PC-3 tumor cells revealed significantly reduced pancreas and tumor uptake, demonstrating receptor specificity of the conjugates. CONCLUSION The best radiotracer was (99m)Tc-HYNIC-BBN on the basis of high radiochemical yield, fast radiolabeling procedure without need for a purification step, and more consistent tumor uptake.

In vitro and in vivo analysis of [64Cu-NO2A-8-Aoc-BBN(7–14)NH2]: 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.

In Vitro and In Vivo Evaluation of Alexa Fluor 680- Bombesin(7-14)NH 2 Peptide Conjugate, a High-Affinity Fluorescent Probe with High Selectivity for the Gastrin- Releasing Peptide Receptor

Gastrin-releasing peptide (GRP) receptors are overexpressed on several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. Bombesin (BBN), a 14–amino acid peptide that is an analogue of human GRP, binds to GRP receptors with very high affinity and specificity. The aim of this study was to develop a new fluorescent probe based on BBN having high tumor uptake and optimal pharmacokinetics for specific targeting and optical imaging of human breast cancer tissue. In this study, solid-phase peptide synthesis was used to produce H2N-glycylglycylglycine-BBN[7–14]NH2 peptide with the following general sequence: H2N-G-G-G-Q-W-A-V-G-H-L-M-(NH2). This conjugate was purified by reversed-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectra. The fluorescent probe Alexa Fluor 680-G-G-G-BBN[7–14]NH2 conjugate was prepared by reaction of Alexa Fluor 680 succinimidyl ester to H2N-G-G-G-BBN[7–14]NH2 in dimethylformamide (DMF). In vitro competitive binding assays, using 125I-Tyr4-BBN as the radiolabeling gold standard, demonstrated an inhibitory concentration 50% value of 7.7 ± 1.4 nM in human T-47D breast cancer cells. Confocal fluorescence microscopy images of Alexa Fluor 680-G-G-G-BBN[7–14]NH2 in human T-47D breast cancer cells indicated specific uptake, internalization, and receptor blocking of the fluorescent bioprobe in vitro. In vivo investigations in SCID mice bearing xenografted T-47D breast cancer lesions demonstrated the ability of this new conjugate to specifically target tumor tissue with high selectivity and affinity.

99mTc‐HYNIC‐Bombesin (7‐14)NH2: Radiochemical Evaluation with Co‐ligands EDDA (EDDA = Ethylenediamine‐N,N′‐diacetic Acid), Tricine, and Nicotinic Acid

Bombesin (BBN) is a peptide exhibiting high affinity for the gastrin releasing peptide receptor (GRPr), which is over‐expressed on a variety of human tumors including breast, prostate, lung and pancreatic cancers. The specific aim of this study was to identify a new 99mTc‐radiolabeled BBN analogue based upon the bifunctional chelating ligand HYNIC (2‐hydrazinonicotinamide) that might be used as a noninvasive tool for diagnosis of GRP receptor‐positive tumors. In this study, HYNIC‐β‐Ala‐BBN(7‐14)NH2 and HYNIC‐5‐Ava‐BBN(7‐14)NH2 were synthesized using traditional solid phase peptide synthetic techniques. The newly‐formed conjugates were radiolabeled using 99mTc in the presence of different coligands including tricine, ethylenediamine diacetic acid (EDDA), tricine/EDDA, and tricine/nicotinic acid. Radiolabeling conditions (i.e., pH, temperature, and reaction time) were optimized and evaluated by ITLC and reversed‐phase HPLC. This material is the result of work supported with resources and the use of facilities at the Radiopharmacy Center, Instituto de Pesquisas Energéticas e Nucleares (IPEN/CNEN), São Paulo, SP, Brazil, the Harry S. Truman Memorial Veterans Hospital, Columbia, MO, the University of Missouri-Columbia School of Medicine Departments of Radiology and Internal Medicine, and the Missouri University Research Reactor, Columbia, MO.

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.

Near-infrared fluorescence imaging of gastrin releasing peptide receptor targeting in prostate cancer lymph node metastases

Development of high affinity and specificity molecular imaging probes that increase accuracy for early detection of lymph node (LN) metastases is important for improving survivorship in prostate cancer. We evaluated the specificity, sensitivity, and accuracy of fluorescence‐labeled bombesin (BBN) peptides to detect LN and systematic metastases in orthotopic mouse models bearing gastrin releasing peptide receptor (GRPR)‐positive human prostate cancer.