Chemistry and radiochemistry of As, Re and Rh isotopes relevant to radiopharmaceutical applications: high specific activity radionuclides for imaging and treatment.

The chemistry and radiochemistry of high specific activity radioisotopes of arsenic, rhenium and rhodium are reviewed with emphasis on University of Missouri activities over the past several decades, and includes recent results. The nuclear facilities at the University of Missouri (10 MW research reactor and 16.5 MeV GE PETtrace cyclotron) allow research and development into novel theranostic radionuclides. The production, separation, enriched target recovery, radiochemistry, and chelation chemistry of 72,77As, 186,188Re and 105Rh are discussed.

Synthesis and biological evaluation of copper-64 radiolabeled [DUPA-6-Ahx-(NODAGA)-5-Ava-BBN(7-14)NH2], a novel bivalent targeting vector having affinity for two distinct biomarkers (GRPr/PSMA) of prostate cancer.

UNLABELLED: Gastrin-releasing peptide receptors (GRPr) and prostate-specific membrane antigen (PSMA) are two identifying biomarkers expressed in very high numbers on prostate cancer cells and could serve as a useful tool for molecular targeting and diagnosis of disease via positron-emission tomography (PET). The aim of this study was to produce the multipurpose, bivalent [DUPA-6-Ahx-((64)Cu-NODAGA)-5-Ava-BBN(7-14)NH2] radioligand for prostate cancer imaging, where DUPA = (2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid), a small-molecule, PSMA-targeting probe, 6Ahx = 6-aminohexanoic acid, 5-Ava = 5-aminovaleric acid, NODAGA = [2-(4,7-biscarboxymethyl)-1,4,7-(triazonan-1-yl)pentanedioic acid] (a derivative of NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid)), and BBN(7-14)NH2 = bombesin, a GRPr-specific peptide targeting probe. METHODS: The PSMA/GRPr dual targeting ligand precursor [DUPA-6-Ahx-K-5-Ava-BBN(7-14)NH2], was synthesized by solid-phase and manual peptide synthesis, after which NODAGA was added via manual conjugation to the ε-amine of lysine (K). The new bivalent GRPr/PSMA targeting vector was purified by reversed-phase high performance liquid chromatography (RP-HPLC), characterized by electrospray-ionization mass spectrometry (ESI-MS), and metallated with (64)CuCl2 and (nat)CuCl2. The receptor binding affinity was evaluated in human, prostate, PC-3 (GRPr-positive) and LNCaP (PSMA-positive) cells and the tumor-targeting efficacy determined in severe combined immunodeficient (SCID) and athymic nude mice bearing PC-3 and LNCaP tumors. Whole-body maximum intensity microPET/CT images of PC-3/LNCaP tumor-bearing mice were obtained 18 h post-injection (p.i.). RESULTS: Competitive binding assays in PC-3 and LNCaP cells indicated high receptor binding affinity for the [DUPA-6-Ahx-((nat)Cu-NODAGA)-5-Ava-BBN(7-14)NH2] conjugate. MicroPET scintigraphy in PC-3/LNCaP tumor-bearing mice indicated that xenografted tumors were visible at 18h p.i. with collateral, background radiation also being observed in non-target tissue. CONCLUSIONS: DUPA-6-Ahx-((64)Cu-NODAGA)-5-Ava-BBN(7-14)NH2] targeting vector, as described herein, is the first example of a dual GRPr-/PSMA-targeting radioligand for molecular of imaging prostate tumors. Detailed in vitro studies and microPET molecular imaging investigations of [DUPA-6-Ahx-((64)Cu-NODAGA)-5-Ava-BBN(7-14)NH2 in tumor-bearing mice indicate that further studies are necessary to optimize uptake and retention of tracer in GRPr- and PSMA-positive tissues.

A heterodimeric [RGD-Glu-[(64)Cu-NO2A]-6-Ahx-RM2] αvß3/GRPr-targeting antagonist radiotracer for PET imaging of prostate tumors.

INTRODUCTION: In the present study, we describe a (64)Cu-radiolabeled heterodimeric peptide conjugate for dual αvß3/GRPr (αvß3 integrin/gastrin releasing peptide receptor) targeting of the form [RGD-Glu-[(64)Cu-NO2A]-6-Ahx-RM2] (RGD: the amino acid sequence [Arg-Gly-Asp], a nonregulatory peptide used for αvß3 integrin receptor targeting; Glu: glutamic acid; NO2A: 1,4,7-triazacyclononane-1,4-diacetic acid; 6-Ahx: 6-amino hexanoic acid; and RM2: (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2), an antagonist analogue of bombesin (BBN) peptide used for GRPr targeting). METHODS: RGD-Glu-6Ahx-RM2] was conjugated to a NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) complexing agent to produce [RGD-Glu-[NO2A]-6-Ahx-RM2], which was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and characterized by electrospray ionization-mass spectrometry (ESI-MS). Radiolabeling of the conjugate with (64)Cu produced [RGD-Glu-[(64)Cu-NO2A]-6-Ahx-RM2 in high radiochemical yield (≥95%). In vivo behavior of the radiolabeled peptide conjugate was investigated in normal CF-1 mice and in the PC-3 human prostate cancer experimental model. RESULTS: A competitive displacement receptor binding assay in human prostate PC-3 cells using (125)I-[Tyr(4)]BBN as the radioligand showed high binding affinity of [RGD-Glu-[(nat)Cu-NO2A]-6-Ahx-RM2] conjugate for the GRPr (3.09±0.34 nM). A similar assay in human, glioblastoma U87-MG cells using (125)I-Echistatin as the radioligand indicated a moderate receptor-binding affinity for the αvß3 integrin (518±37.5 nM). In vivo studies of [RGD-Glu-[(64)Cu-NO2A]-6-Ahx-RM2] showed high accumulation (4.86±1.01 %ID/g, 1h post-intravenous injection (p.i.)) and prolonged retention (4.26±1.23 %ID/g, 24h p.i.) of tracer in PC-3 tumor-bearing mice. Micro-positron emission tomography (microPET) molecular imaging studies produced high-quality, high contrast images in PC-3 tumor-bearing mice at 4h p.i. CONCLUSIONS: The favorable pharmacokinetics and enhanced tumor uptake of (64)Cu-NOTA-RGD-Glu-6Ahx-RM2 warrant further investigations for dual integrin and GRPr-positive tumor imaging and possible radiotherapy.

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

BACKGROUND: 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. METHODS: PC-3 cells were orthotopically implanted in severe combined immunedeficient or thymic nude (nu/nu) male mice. Tumor growth was monitored using magnetic resonance imaging. Alexa Fluor 680 conjugated BBN[7-14]NH2 (AF680-BBN) peptides were administered intravenously at 4-7 weeks post-tumor-implantation. Near-infrared fluorescence (NIRF) imaging was performed for up to 6 hr post-injection. The imaging sensitivity and specificity were assessed by co-registration of AF680-BBN NIRF imaging and luciferase bioluminescence imaging of the PC-3/Luc+ orthotopic mouse model. RESULTS: AF680-BBN showed a high binding affinity and selectivity to GRPR-positive cancer in vitro and in vivo. LN and peritoneal metastases were detected by NIRF imaging, and confirmed by histopathology. Tumor-to-muscle (T/M) ratio was the highest at 2-hr post-injection (4.12 ± 1.77). Blocking experiments, using unlabeled BBN as the inhibiting agent, significantly reduced the T/M ratio (1.64 ± 0.21, P = 0.02). AF680-BBN NIRF imaging had a sensitivity of 89.4%, specificity of 92.9%, and accuracy of 90.2% for the detection of metastases in mice. CONCLUSIONS: [corrected] The studies suggest the potential of use and development of NIR-fluorescent BBN probes as site-directed agents to help improve the current detection and LN staging accuracy in prostate cancer.

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.

Non-invasive microCT imaging characterization and in vivo targeting of BB2 receptor expression of a PC-3 bone metastasis model.

PURPOSE: A devastating progression of human prostate cancer is the development of bone metastasis. Animal models of bone metastasis induced by inoculating human prostate cell lines into mice are well established. Here, we report the characterization of a mouse model of prostatic bone metastasis using non-invasive microCT and targeted microSPECT imaging of bone tumors using the bombesin receptor (BB2r)-avid radiolabeled peptide, (111)In-DOTA-8-Aoc-BBN[7­14]NH(2). PROCEDURES: Immunocompromised mice were inoculated with human prostate cancer cells by intracardiac injection. Metastatic lesion development was monitored by serially imaging mice weekly with microCT. Mice with CT imaging-confirmed bone lesions were administered (111)In-DOTA-8-Aoc-BBN[7­14]NH(2) for microSPECT imaging of BB2r expressing lesions. RESULTS: Metastatic bone lesions as small as 0.3 mm in diameter were detected by microCT image analysis as early as 21 days after tumor cell inoculation and had wide anatomical distribution. MicroSPECT imaging using (111)In-DOTA-8-Aoc-BBN[7­14]NH(2) successfully targeted BB2r expressing metastatic bone lesions of the tibia at day 29. CONCLUSIONS: MicroCT imaging can accurately and non-invasively follow the onset and progression of metastatic bone lesions in mouse models of prostate cancer. Micro-CT coupled with BB2r Micro-SPECT imaging affords the opportunity to obtain a combined receptor/anatomic map of metastatic bone lesion status in this mouse model.

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.

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.

Evaluation of [99mTc-(CO)3-X-Y-Bombesin(7-14)NH2] conjugates for targeting gastrin-releasing peptide receptors overexpressed on breast carcinoma.

BACKGROUND: Gastrin-releasing peptide (GRP) receptors are overexpressed on a variety of human carcinomas, including those of the breast. These receptors may be targeted with bombesin (BBN), which binds to GRP receptors with high affinity and specificity. The aim of this study was to develop a (99m)Tc(I)-BBN analog with favorable pharmacokinetic properties in order to improve the visualization of breast cancer tissue. MATERIALS AND METHODS: Solid-phase peptide synthesis was used to produce a series of X-Y-BBN-NH2 conjugates, where X is pyrazolyl (PZ1) or 2,3-diaminopropionic acid (DPR) and Y is a spacer sequence. Their metallated counterparts were prepared by reacting [(99m)Tc-(H(2)O)(3)(CO)(3)](+) with the corresponding ligand. RESULTS: While the PZ1 conjugates exhibited higher GRP receptor binding affinities in vitro, the DPR analogs demonstrated superior target tissue accumulation and pharmacokinetic properties in vivo. CONCLUSION: These results demonstrate the ability of the DPR derivatives (Y=glycylserylglycine, triserine) to clearly identify the T47-D tumor tissue in xenografted SCID mice.

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.

Design, synthesis, and biological evaluation of an antagonist-bombesin analogue as targeting vector.

The gastrin releasing peptide receptor (GRP-R) is overexpressed on a number of tumors and cancer cell lines including pancreas, prostate, breast, gastrointestinal, and small cell lung cancer (SCLC). Radiolabeled bombesin (BBN) analogues have exhibited high binding affinity and specificity to the GRP-R. A bombesin analogue with an antagonist targeting vector at the C-terminus, DOTA-aminohexanoyl-[D-Phe(6), Leu-NHCH 2CH 2CH3(13), des Met(14)] BBN[6-14] (1, "Bomproamide"), has been synthesized and displays high binding affinity (IC50 = 1.36 +/- 0.09 nM) against (125)I-Tyr (4)-BBN in in vitro competitive assays using PC-3 cells. Maximum internalization of (111)In-1 reached 14% in PC-3 cells after 45 min of incubation. Rapid (0.25 h PI) and high (12.21 +/- 3.2%ID/g) pancreatic uptake of (111)In-1 was observed in healthy CF-1 mice, and 90% of the activity was blocked by coinjection of 100 mug of BBN. Rapid (0.25 h PI) and high uptake (6.90 +/- 1.06%ID/g) was observed in PC-3 prostate cancer xenografts in SCID mice, as well as visualized clearly in a SPECT/CT study. These results support the use of a bombesin construct with an antagonist C-terminal vector as a candidate of choice for specific in vivo imaging of tumors overexpressing GRP-receptors.

Evaluation of the pharmacokinetic effects of various linking group using the 111In-DOTA-X-BBN(7-14)NH2 structural paradigm in a prostate cancer model.

The high incidence of BB2 receptor (BB2r) expression in various cancers has prompted investigators to pursue the development of BB2r-targeted agents for diagnostic imaging, chemotherapy, and radiotherapy. Development of BB2r-targeted agents, based on the bombesin (BBN) peptide, has largely involved the use of the bifunctional chelate approach in which the linking group serves several key roles including pharmacokinetic modification. Understanding the in vivo properties of the various pharmacokinetic modifying linking groups is crucial for developing BB2r-targeted agents with improved targeting and clearance characteristics. The goal of this study was to systematically evaluate the pharmacokinetic profile of aliphatic hydrocarbon, aromatic, and poly(ethylene glycol) (ether) functional groups in order to obtain a better understanding of the in vivo properties of these pharmacokinetic modifiers. Specifically, we synthesized six radioconjugates with the structure 111In-DOTA- X-BBN(7-14)NH2, where X = 8-aminooctanoic acid (8-AOC), 5-amino-3-oxapentyl-succinamic acid (5-ADS), 8-amino-3,6-dioxaoctyl-succinamic acid (8-AOS), p-aminobenzoic acid (AMBA), Gly-AMBA, and Gly- p-aminomethylbenzoic acid (Gly-AM2BA). All of the (nat)In-conjugates demonstrated nanomolar binding affinities to the BB2r. In CF-1 mice, the BB2r uptake in the pancreas of radioconjugates containing aromatic linking groups was found to be significantly higher at 1 h postinjection than the radioconjugates with ether linker moieties. For PC-3 tumor-bearing SCID mice, the tumor uptake was found to be 6.66 +/- 2.00, 6.21 +/- 1.57, 6.36 +/- 1.60, 4.46 +/- 0.81, and 7.76 +/- 1.19 %ID/g for the 8-AOC, 8-ADS, AMBA, Gly-AMBA, and Gly-AM2BA radioconjugates, respectively, at 15 min postinjection. By 24 h postinjection, the radioconjugates containing aromatic groups exhibited the highest percentage tumor retention with 11.4%, 19.8%, 26.6%, 25.8%, and 25.5% relative to the 15 min values remaining in the tumor tissue for the 8-AOC, 8-ADS, AMBA, Gly-AMBA, and Gly-AM2BA radioconjugates, respectively. Fused Micro-SPECT/CT imaging studies performed at 24 h postinjection revealed substantial accumulation of radioactivity in the tumor tissue for all radioconjugates. In both biodistribution and Micro-SPECT/CT imaging studies, the radioconjugates containing aromatic linking groups typically exhibited significantly higher G.I. tract retention than the hydrocarbon or ether linking moieties. In conclusion, our studies indicate that radioconjugates incorporating aromatic linking groups, of the type investigated, generally demonstrated enhanced retention in BB2r expressing tissues in comparison to either the hydrocarbon or ether linking moieties. Furthermore, this investigation clearly demonstrates the significance of the linking group upon not only the in vivo clearance of the radiopharmaceutical, but also on the in vivo uptake and retention of the BB2r-targeted agent in tumor tissue. Future designs of BB2r-targeted agents should include a careful consideration of the effect linking group functionality has upon tumor targeting and retention.

99mTc(CO)3-DTMA bombesin conjugates having high affinity for the GRP receptor.

INTRODUCTION: Targeted diagnosis of specific human cancer types continues to be of significant interest in nuclear medicine. 99mTc is ideally suited as a diagnostic radiometal for in vivo tumor targeting due to its ideal physical characteristics and diverse labeling chemistries in numerous oxidation states. METHODS: In this study, we report a synthetic approach toward design of a new tridentate amine ligand for the organometallic aqua-ion [99mTc(H2O)3(CO)3]+. The new chelating ligand framework, 2-(N,N'-Bis(tert-butoxycarbonyl)diethylenetriamine) acetic acid (DTMA), was synthesized from a diethylenetriamine precursor and fully characterized by mass spectrometry and nuclear magnetic resonance spectroscopy (1H and 13C). DTMA was conjugated to H2N-(X)-BBN(7-14)NH2, where X=an amino acid or aliphatic pharmacokinetic modifier and BBN=bombesin peptide, by means of solid phase peptide synthesis. DTMA-(X)-BBN(7-14)NH2 conjugates were purified by reversed-phase high-performance chromatography and characterized by electrospray-ionization mass spectrometry. RESULTS: The new conjugates were radiolabeled with [99mTc(H2O)3(CO)3]+ produced via Isolink radiolabeling kits to produce [99mTc(CO)3-DTMA-(X)-BBN(7-14)NH2]. Radiolabeled conjugates were purified by reversed-phase high-performance chromatography. Effective receptor binding behavior was evaluated in vitro and in vivo. CONCLUSIONS: [99mTc(CO)3-DTMA-(X)-BBN(7-14)NH2] conjugates displayed very high affinity for the gastrin releasing peptide receptor in vitro and in vivo. Therefore, these conjugates hold some propensity to be investigated as molecular imaging agents that specifically target human cancers uniquely expressing the gastrin releasing peptide receptor subtypes.

Organometallic 99mTc(III) '4 + 1' bombesin(7-14) conjugates: synthesis, radiolabeling, and in vitro/in vivo studies.

Bombesin (BBN) peptide exhibits high selectivity and affinity for the gastrin-releasing peptide receptor (GRPr). The GRPr is overexpressed on many human cancer cell types, thus making BBN a potent delivery vehicle for radionuclide targeting. In this study, the biologically active minimal sequence BBN(7-14) was labeled using the novel Tc '4 + 1' mixed-ligand system, [Tc(NS3)(CN-R)], in which Tc(III) is coordinated by a monodentate isocyanide linker bearing the peptide and the tetradentate, tripodal chelator, 2,2',2''-nitrilotriethanethiol (NS3). BBN(7-14) was N-terminally modified with Gly-Gly-Gly, betaAla, and Ser-Ser-Ser spacer groups (X) and functionalized with 4-(isocyanomethyl)benzoic acid (L1) or 4-isocyanobutanoic acid (L2), resulting in a series of [M(NS3)(L-X-BBN(7-14))] conjugates (M = 99mTc, Re). The isocyanide ligand frameworks were introduced using novel bifunctional coupling agents. The spacer groups (X), the monodentate isocyanide units, and a tetradentate NS3 chelator bearing a pendant carboxylic acid (NS3COOH) were proposed as pharmacological modifiers. 99mTc-labeling was performed in a two-step procedure by first preparing 99mTc-EDTA/mannitol followed by reactions with the isocyanides and NS3 or NS3COOH ligand frameworks. The 99mTc complexes were obtained with a radiochemical yield of 30-80% depending on the amount of the isocyanide (20-100 nmol) used. These new conjugates were purified by reversed-phased high-performance liquid chromatography (RP-HPLC) to give a radiochemical purity of >or=95%. The 99mTc conjugates exhibited high in vitro stability (>90%, 24 h). Analogous nonradioactive Re conjugates were synthesized and characterized by electrospray ionization mass spectrometry (ESI-MS). RP-HPLC analyses of the Re conjugates indicated that they exhibited identical retention times to the corresponding 99mTc conjugates under identical HPLC conditions, demonstrating structural similarity between the two metalated species. The [Re(NS3)(L-X-BBN(7-14))] conjugates exhibited GRPr affinity in the nanomolar range as demonstrated by in vitro competitive binding assays using PC-3 human prostate cancer cells. In vitro internalization/externalization assays indicated that approximately 65% of [99mTc(NS3)(L2-betaAla-BBN(7-14))] conjugate was either surface-bound or internalized in PC-3 cells. Cell-associated activity for all other 99mTc conjugates was below 20%. Biodistribution studies of [99mTc(NS3)(L-betaAla-BBN(7-14))], L = L1 or L2, in normal, CF-1 mice showed minimal accumulation in normal pancreas (a tissue expressing the GRPr in high density in rodent models) and rapid hepatobiliary elimination. Introduction of a carboxyl group onto the NS3 ligand framework had only minimal effects to increase renal excretion. Activity distribution and accumulation was highly dominated by the relatively lipophilic '4 + 1' complex unit.

[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.

In vivo evaluation and small-animal PET/CT of a prostate cancer mouse model using 64Cu bombesin analogs: side-by-side comparison of the CB-TE2A and DOTA chelation systems.

UNLABELLED: The BB2 receptor subtype, of the bombesin family of receptors, has been shown to be highly overexpressed in a variety of human tumors, including prostate cancer. Bombesin (BBN), a 14-amino acid peptide, has been shown to target the BB2 receptor with high affinity. 64Cu (half-life = 12.7 h, beta+: 18%, E(beta+ max) = 653 keV; beta-: 37%, E(beta- max) = 578 keV) is a radioisotope that has clinical potential for application in both diagnostic imaging and radionuclide therapy. Recently, new chelation systems such as 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A) have been reported to significantly stabilize the 64Cu radiometal in vivo. The increased stability of the 64Cu-CB-TE2A chelate complex has been shown to significantly reduce nontarget retention compared with tetraazamacrocycles such as 1,4,7,10-tetraazacyclodoadecane-N,N',N'',N'''-tetraacetic acid (DOTA). The aim of this study was to determine whether the CB-TE2A chelation system could significantly improve the in vivo stability of 64Cu bombesin analogs. The study directly compares 64Cu bombesin analogs using the CB-TE2A and DOTA chelation systems in a prostate cancer xenograft SCID (severely compromised immunodeficient) mouse model. METHODS: The CB-TE2A-8-AOC-BBN(7-14)NH2 and DOTA-8-AOC-BBN(7-14)NH2 conjugates were synthesized and radiolabeled with 64Cu. The receptor-binding affinity and internalization profile of each metallated conjugate was evaluated using PC-3 cells. Pharmacokinetic and small-animal PET/CT studies were performed using female SCID mice bearing PC-3 xenografts. RESULTS: In vivo BB2 receptor targeting was confirmed by tumor uptake values of 6.95 +/- 2.27 and 4.95 +/- 0.91 %ID/g (percentage injected dose per gram) at the 15-min time point for the 64Cu-CB-TE2A and 64Cu-DOTA radioconjugates, respectively. At the 24-h time point, liver uptake was substantially reduced for the 64Cu-CB-TE2A radioconjugate (0.21 +/- 0.06 %ID/g) compared with the 64Cu-DOTA radioconjugate (7.80 +/- 1.51 %ID/g). The 64Cu-CB-TE2A-8-AOC-BBN(7-14)NH2 radioconjugate demonstrated significant clearance, 98.60 +/- 0.28 %ID, from the mouse at 24 h after injection. In contrast, only 67.84 +/- 5.43 %ID of the 64Cu activity was excreted using the 64Cu-DOTA-8-AOC-BBN(7-14)NH2 radioconjugate because of nontarget retention. CONCLUSION: The pharmacokinetic and small-animal PET/CT studies demonstrate significantly improved nontarget tissue clearance for the 64Cu-CB-TE2A8-AOC-BBN(7-14)NH2. This is attributed to the improved in vivo stability of the 64Cu-CB-TE2A chelate complex as compared with the 64Cu-DOTA chelate complex.

In vitro and in vivo evaluation of Alexa Fluor 680-bombesin[7-14]NH2 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 H(2)N-glycylglycylglycine-BBN[7-14]NH(2) peptide with the following general sequence: H(2)N-G-G-G-Q-W-A-V-G-H-L-M-(NH(2)). 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]NH(2) conjugate was prepared by reaction of Alexa Fluor 680 succinimidyl ester to H(2)N-G-G-G-BBN[7-14]NH(2) in dimethylformamide (DMF). In vitro competitive binding assays, using (125)I-Tyr(4)-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]NH(2) 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.

The effects of linking substituents on the in vivo behavior of site-directed, peptide-based, diagnostic radiopharmaceuticals.

A number of human cancers are known to over-express the gastrin-releasing peptide receptor (GRPr) on cell surfaces. The high specificity and affinity of bombesin (BBN), an amphibian analogue of mammalian gastrin-releasing peptide, for the GRPr makes it an ideal candidate for delivery of diagnostic probes, such as 99mTc radiometal, to tumor sites. An optimized targeting agent possesses high tumor uptake with minimal uptake in normal tissues. In this study, 99mTc-targeting vectors of bombesin using various amino acid/aliphatic pharmacokinetic modifiers or linking groups were evaluated to determine the effect of the spacer on receptor binding affinity, internalization/externalization and biodistribution. Conjugates of the general type [DPR-X-BBN] (X = amino acid/aliphatic pharmacokinetic modifier) were synthesized by solid phase peptide synthesis (SPPS) and metallated with either low-valent, radioactive Tc-99m(I) or non-radioactive Re(I)-tricarbonyl precursors. All of the new non-metallated and metallated conjugates were characterized by electrospray ionization mass spectrometry (ESI-MS). Receptor binding affinity, internalization/externalization and biodistribution studies in normal (CF-1) and tumor (human prostate PC-3-bearing mice) are reported. The effectiveness of targeting xenografted PC-3 tumors in rodents for two of the new 99mTc-BBN conjugates is demonstrated herein using small animal single photon emission computed tomography (SPECT).

Selective targeting of E. coli heat-stable enterotoxin analogs to human colon cancer cells.

BACKGROUND: Radiolabeled analogs of the E. coli heat-stable enterotoxin (ST(h)) are currently under study as imaging and therapeutic agents for colorectal cancer. The aim of these studies is to compare in vitro and in vivo characteristics of two novel ST(h) analogs with appended DOTA chelating moieties. MATERIALS AND METHODS: ST(h) analogs were synthesized with pendant N-terminal DOTA moieties and radiolabeled with indium-111. In vitro cell binding was studied using cultured T-84 human colorectal cancer cells, and in vivo biodistribution studies were carried out using T-84 human colorectal tumor xenografts in SCID mice. RESULTS: Competitive radioligand binding assays employing T-84 human colon cancer cells demonstrated similar IC50 values for the F19-ST(h)(2-19) and F9-ST(h)(6-19) analogs. Addition of DOTA to the N-terminus of these peptides elicited distinctly different effects on binding affinities in vitro, effects that were largely unchanged by metallation with nonradioactive (nat)In. In vivo pharmacokinetic studies in SCID mice bearing T-84 human colon cancer-derived tumor xenografts demonstrated tumor uptake of 0.74 +/- 0.1% ID/g at 4 h post-injection (p.i.) for the 111In-DOTA-F19-ST(h)(2-19) analog, and significantly reduced tumor localization (0.27 + 0.08 % ID/g) for the 111In-DOTA-F9-ST(h)(6-19) analog. CONCLUSION: These results demonstrate that placement of a DOTA moiety immediately adjacent to Cys 6 in ST(h) significantly inhibits receptor binding in vitro and in vivo, highlighting the need for intervening spacer residues between the pharmacophore and the DOTA chelating moiety in effective ST(h)-based radiopharmaceutical constructs.