Elevating theranostics: The emergence and promise of radiopharmaceutical cell-targeting heterodimers in human cancers.

Optimal therapeutic and diagnostic efficacy is essential for healthcare's global mission of advancing oncologic drug development. Accurate diagnosis and detection are crucial prerequisites for effective risk stratification and personalized patient care in clinical oncology. A paradigm shift is emerging with the promise of multi-receptor-targeting compounds. While existing detection and staging methods have demonstrated some success, the traditional approach of monotherapy is being reevaluated to enhance therapeutic effectiveness. Heterodimeric site-specific agents are a versatile solution by targeting two distinct biomarkers with a single theranostic agent. This review describes the innovation of dual-targeting compounds, examining their design strategies, therapeutic implications, and the promising path they present for addressing complex diseases.

Development of Heterobivalent Theranostic Probes Having High Affinity/Selectivity for the GRPR/PSMA.

In this study, we describe the development of heterobivalent [DUPA-6-Ahx-([111In]In-DO3A)-8-Aoc-BBN ANT] and [DUPA-6-Ahx-([177Lu]Lu-DO3A)-8-Aoc-BBN ANT] radiotracers that display very high selectivity/specificity for gastrin-releasing peptide receptor (GRPR)-/prostate-specific membrane antigen (PSMA)-expressing cells. These studies include metallation, purification, characterization, and in vitro and in vivo evaluation of the new small-molecule-/peptide-based radiopharmaceuticals having utility for imaging and potentially therapy. Competitive displacement binding assays using PC-3 cells and LNCaP cell membranes showed high binding affinity for the GRPR or the PSMA. Biodistribution studies showed favorable excretion pharmacokinetics with high tumor uptake in PC-3 or PC-3 prostatic inhibin peptide (PIP) tumor-bearing mice. For example, tumor accumulation at the 1 h time point ranged from (4.74 ± 0.90) to (7.51 ± 2.61)%ID/g. Micro-single-photon emission computed tomography (microSPECT) molecular imaging investigations showed very high uptake in tumors with minimal accumulation of tracers in the surrounding collateral tissues in xenografted mice at 4 h postintravenous injection. In conclusion, [DUPA-6-Ahx-([111In]In-DO3A)-8-Aoc-BBN ANT] and [DUPA-6-Ahx-([177Lu]Lu-DO3A)-8-Aoc-BBN ANT] tracers displayed favorable pharmacokinetic and excretion profiles with high uptake and retention in tumors.

Preclinical Evaluation of Novel 64Cu-Labeled Gastrin-Releasing Peptide Receptor Bioconjugates for PET Imaging of Prostate Cancer.

We report herein the preclinical evaluation of new [64Cu]Cu-gastrin-releasing peptide receptor (GRPR)-targeting tracers, employing the potent peptide antagonist DPhe-Gln-Trp-Ala-VaI-Gly-His-Sta-Leu-NH2 conjugated to NOTA (in 1) or NODAGA (in 2) chelators via a 6-aminohexanoic acid linker. The Cu-1/2 metalated peptides were synthesized by reacting 1/2 with CuCl2 and were characterized by LC-ESI-MS and HR-ESI-MS. Cu-1/2 exhibited high GRPR-binding affinities with IC50 values <3 nM, as measured in a competition assay using the GRPR-expressing human PC-3 prostate cancer cell line and [125I]I-Tyr4-BBN as the competing ligand. Tracers [64Cu]Cu-1/2 were prepared in quantitative radiochemical yield (by radio-HPLC), and their identities were confirmed by coelution with their Cu-1/2 standards via comparative HPLC studies. Lipophilicity was measured in 1-octanol/PBS (pH 7.4), and the negative log D7.4 values (≤-1) confirmed the anticipated hydrophilic character for [64Cu]Cu-1/2. Both tracers demonstrated excellent in vitro stability, with ≥98% remaining intact through 24 h at physiological conditions (PBS, pH 7.4, 37 °C). Biodistribution in PC-3 tumor-bearing mice demonstrated good tumor uptake (%ID/g at 4 h: 4.34 ± 0.71 for [64Cu]Cu-1, 3.92 ± 1.03 for [64Cu]Cu-2) and rapid renal clearance (≥87% ID at 4 h). Tumor uptake was receptor-mediated, as verified by parallel GRPR-blocking studies. Small-animal PET/CT imaging studies validated the biodistribution data. These preclinical data support that the [64Cu]Cu-1/2 tracers show promise for further development as diagnostic PET imaging agents of GRPR-expressing tumors.

Development and Preclinical Evaluation of 99mTc- and 186Re-Labeled NOTA and NODAGA Bioconjugates Demonstrating Matched Pair Targeting of GRPR-Expressing Tumors.

PURPOSE: The goal of this work was to develop hydrophilic gastrin-releasing peptide receptor (GRPR)-targeting complexes of the general formula fac-[M(CO)3(L)]+ [M = natRe, 99mTc, 186Re; L: NOTA for 1, NODAGA for 2] conjugated to a powerful GRPR peptide antagonist (DPhe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) via a 6-aminohexanoic acid linker. PROCEDURES: Metallated-peptides were prepared employing the [M(OH2)3(CO)3]+ [M = Re, 99mTc, 186Re] precursors. Re-1/2 complexes were characterized with HR-MS. IC50 studies were performed for peptides 1/2 and their respective Re-1/2 complexes in a binding assay utilizing GRPR-expressing human PC-3 prostate cancer cells and [125I]I-Tyr4-BBN as the competing ligand. The 99mTc/186Re-complexes were identified by HPLC co-injection with their Re-analogues. All tracers were challenged in vitro at 37 °C against cysteine/histidine (phosphate-buffered saline 10 mM, pH 7.4) and rat serum. Biodistribution and micro-SPECT/CT imaging of [99mTc]Tc-1/2 and [186Re]Re-2 were performed in PC-3 tumor-bearing ICR SCID mice. RESULTS: High in vitro receptor affinity (IC50 2-3 nM) was demonstrated for all compounds. The 99mTc/186Re-tracers were found to be hydrophilic (log D7.4 ≤ - 1.35) and highly stable. Biodistribution in PC-3 xenografted mice revealed good tumor uptake (%ID/g at 1 h: 4.3 ± 0.7 for [99mTc]Tc-1, 8.3 ± 0.9 for [99mTc]Tc-2 and 4.2 ± 0.8 for [186Re]Re-2) with moderate retention over 24 h. Rapid renal clearance was observed for [99mTc]Tc-2 and [186Re]Re-2 (> 84 % at 4 h), indicating favorable pharmacokinetics. Micro-SPECT/CT images for the 99mTc-tracers clearly visualized PC-3 tumors in agreement with the biodistribution data and with superior imaging properties found for [99mTc]Tc-2. CONCLUSIONS: [99mTc]Tc-2 shows promise for further development as a GRPR-imaging agent. [186Re]Re-2 demonstrated very similar in vivo behavior to [99mTc]Tc-2, and further studies are therefore justified to explore the theranostic potential of our approach for targeting of GRPR-positive cancers.

PD-L1 Expression with Epithelial Mesenchymal Transition of Circulating Tumor Cells Is Associated with Poor Survival in Curatively Resected Non-Small Cell Lung Cancer.

In addition to the FDA-approved definition of a circulating tumor cell (CTC), various CTC phenotypes have been discovered. Epithelial-mesenchymal transition (EMT) of cancer cells is directly linked to PD-L1 upregulation. The goal of the study was to investigate PD-L1 expression and EMT in CTCs of non-small cell lung cancer (NSCLC) patients, and perform an outcome analysis. Prospectively, 7.5 mL peripheral blood was collected from 30 NSCLC patients that underwent surgery and 15 healthy controls. CTCs were enriched by size-based microfilter and immunofluorescence stainings performed (cytokeratin (CK) 8/18/19, EpCAM, CD45, PD-L1, EMT markers vimentin, and N-Cadherin, DAPI). Patient-matched NSCLC tissues were also stained. CTC staining intensity was quantified with a software and correlated with patient-matched NSCLC tissues and survival. PD-L1 and EMT markers were expressed at significantly higher proportions in CTCs than patient-matched NSCLC tissues (p < 0.05); ≥3 PD-L1pos/EMTposCTCs were associated with significantly poorer survival after curative surgery (p < 0.05). No CTCs were detected in 15 healthy controls. This study shows that PD-L1 expression and EMT of CTCs is a negative survival predictor for NSCLC patients. The therapeutic role of the molecular linkage of PD-L1 and EMT will need to be further investigated, as linked pathways could be targeted to improve NSCLC outcome.

Somatostatin receptor targeting with hydrophilic [99mTc/186Re]Tc/Re-tricarbonyl NODAGA and NOTA complexes.

INTRODUCTION: The aim of this work was to develop diagnostic (99mTc) and therapeutic (186Re) agents for targeting somatostatin receptor (SSTR)-positive neuroendocrine tumors (NETs). In this regard, we evaluated in vitro complexes of the general formula [M(CO)3(L-sst2-ANT)] (M = 99mTc, 186Re), where L denotes NODAGA or NOTA and sst2-ANT denotes the potent SSTR2 antagonist 4-NO2-Phe-c(DCys-Tyr-DTrp-Lys-Thr-Cys)-DTyr-NH2. Moreover, we assessed the in vivo properties of the 99mTc-complexes in an animal SSTR-tumor model. METHODS: The [99mTc]/[186Re][Tc/Re(OH2)3(CO)3]+ precursors were utilized to prepare the 99mTc/186Re-complexes, which were identified by HPLC co-injection with their natRe analogues. The tracers were challenged in vitro at 37 °C against cysteine and histidine in phosphate-buffered saline (pH 7.4) and in rat serum. Biodistribution and micro-SPECT/CT imaging studies of the 99mTc-tracers were performed in AR42J tumor-bearing female ICR SCID mice. RESULTS: The 99mTc-complexes were prepared in high radiochemical yield (RCY > 90%, by HPLC), with lower RCY (≤30%) obtained for 186Re-complexes. Tracers remained intact in vitro and displayed low non-specific binding (10-25%) to rat serum proteins. Biodistribution of [99mTc]Tc-NODAGA-sst2-ANT revealed low tumor uptake (2.78 ±â€¯0.27 %ID/g) at 1 h, while high tumor uptake (16.70 ±â€¯3.32 %ID/g) was found for [99mTc]Tc-NOTA-sst2-ANT. Moderate to low tumor retention was observed for both tracers after 4 and 24 h. Tumor uptake for [99mTc]Tc-NOTA-sst2-ANT was receptor-mediated, as demonstrated by parallel SSTR blocking studies. Rapid renal clearance was observed for both tracers, and SPECT/CT images clearly delineated the tumors, in agreement with the biodistribution data. CONCLUSIONS: The [99mTc]Tc-NOTA-sst2-ANT complex demonstrated high tumor uptake and rapid clearance in a SSTR-tumor mouse model, showing potential for further development. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Preclinical data support the feasibility of the [99mTc]Tc/[186Re]Re-NOTA/NODAGA labeling strategy for use in the development of theranostic radiopharmaceuticals for translation into the human clinic for targeting of SSTR-expressing NETs.

NOTA and NODAGA [99mTc]Tc- and [186Re]Re-Tricarbonyl Complexes: Radiochemistry and First Example of a [99mTc]Tc-NODAGA Somatostatin Receptor-Targeting Bioconjugate.

With the long-term goal of developing theranostic agents for applications in nuclear medicine, in this work we evaluated the well-known NOTA and NODAGA chelators as bifunctional chelators (BFCs) for the [99mTc/186Re]Tc/Re-tricarbonyl core. In particular, we report model complexes of the general formula fac-[M(L)(CO)3]+ (M = Re, 99mTc, 186Re) where L denotes NOTA-Pyr (1) or NODAGA-Pyr (2), which are derived from conjugation of NOTA/NODAGA with pyrrolidine (Pyr). Further, as proof-of-principle, we synthesized the peptide bioconjugate NODAGA-sst2-ANT (3) and explored its complexation with the fac-[Re(CO)3]+ and fac-[99mTc][Tc(CO)3]+ cores; sst2-ANT denotes the somatostatin receptor (SSTR) antagonist 4-NO2-Phe-c(DCys-Tyr-DTrp-Lys-Thr-Cys)-DTyr-NH2. Rhenium complexes Re-1 through Re-3 were synthesized and characterized spectroscopically, and receptor binding affinity was demonstrated for Re-3 in SSTR-expressing cells (AR42J, IC50 = 91 nM). Radiolabeled complexes [99mTc]Tc/[186Re]Re-1/2 and [99mTc]Tc-3 were prepared in high radiochemical yield (>90%, determined by radio-HPLC) by reacting [99mTc]/[186Re][Tc/Re(OH2)3(CO)3]+ with 1-3 and correlated well with the respective Re-1 through Re-3 standards in comparative HPLC studies. All radiotracers remained intact through 24 h (99mTc-labeled complexes) or 48 h (186Re-labeled complexes) against 1 mM l-histidine and 1 mM l-cysteine (pH 7.4, 37 °C). Similarly, rat serum stability studies displayed no decomposition and low nonspecific binding of 9-24% through 4 h. Biodistribution of [99mTc]Tc-3 in healthy CF-1 mice demonstrated a favorable pharmacokinetic profile. Rapid clearance was observed within 1 h post-injection, predominantly via the renal system (82% of the injected dose was excreted in urine by 1 h), with low kidney retention (% ID/g: 11 at 1 h, 5 at 4 h, and 1 at 24 h) and low nonspecific uptake in other organs/tissues. Our findings establish NOTA and NODAGA as outstanding BFCs for the fac-[M(CO)3]+ core in the design and development of organometallic radiopharmaceuticals. Future in vivo studies of [99mTc]Tc- and [186Re]Re-tricarbonyl complexes of NODAGA/NOTA-biomolecule conjugates will further probe the potential of these chelates for nuclear medicine applications in diagnostic imaging and targeted radiotherapy, respectively.

Peptide-Based Radiopharmaceuticals for Molecular Imaging of Prostate Cancer.

Given the high incidence of prostate cancer, there is a continuing need for advances in early detection and in effective treatments. Over the last several years, radiolabeled peptides have been developed, which can target receptors on prostate tumors with high affinity and specificity. These peptides are eliminated from normal tissues rapidly, producing high contrast for PET and SPECT imaging. Receptors of interest for tumor imaging include prostate specific membrane antigen (PSMA), gastrin-releasing peptide receptor (GRPR), and αvß3 integrin. Because radiolabeled peptides afford high tumor-to-normal tissue uptake ratios, the potential of peptide-based targeted radiotherapy of prostate cancer is being explored. In addition, targeting either of two receptors with one peptide may allow more tumors to be detected and aid in the delineation of early versus advanced disease. Taken together, all these developments in peptide-based imaging and therapy of prostate cancer offer the promise of personalized, molecular medicine for individual patients.

Design, Synthesis, and in Vitro and in Vivo Evaluation of High Affinity and Specificity Near-Infrared Fluorescent Bombesin Antagonists for Tumor Imaging.

The bombesin (BBN) antagonist binds with high affinity to the gastrin releasing peptide receptor (GRPr), a receptor overexpressed on many human cancers. We present an investigation employing BBN antagonist for highly specific near-infrared fluorescence (NIRF) imaging of GRPr-positive tumors. Nine NIRF-dye labeled BBN antagonists with differing linkers and dyes were synthesized and characterized to screen for the optimal agent. Three novel agents, AF750-G-pip-Sta-BBN (1), AF750-GSG-Sta-BBN (2), and AF750-6Ahx-Sta-BBN (3), exhibited an excellent binding-specificity and affinity to human PC-3 prostate cancer cells in vitro, and a remarkable in vivo tumor-selectivity and NIRF imaging sensitivity in PC-3 tumor-bearing mice. Compound 1 showed the fastest, and 3, the slowest, pharmacokinetics on the tumor sites. Despite of high tumor uptake, 2 had a low pancreas uptake distinct from 1 and 3 at 0.44 nmol dose. This difference was attributed to the inherent linker properties such as the hydrophilicity, polarity, and charge.

Matched-pair, 86Y/90Y-labeled, bivalent RGD/bombesin antagonist, [RGD-Glu-[DO3A]-6-Ahx-RM2], as a potential theranostic agent for prostate cancer.

INTRODUCTION: In this study, we describe development of a true matched-pair theranostic agent that is able to target the αVß3 integrin and the gastrin releasing peptide receptor (GRPR). We herein describe methods to metallate and characterize the new conjugate and to validate its biological efficacy by in vitro and in vivo methods. METHODS: We have previously described the development of [RGD-Glu-6Ahx-RM2] (where RGD: Arg-Gly-Asp; Glu: glutamic acid; 6-Ahx: 6-amino hexanoic acid; RM2: (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2)) that has been conjugated to a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) bifunctional chelating agent (BFCA) to afford [RGD-Glu-[DO3A]-6-Ahx-RM2] peptide. In this study, we have radiolabeled [RGD-Glu-[DO3A]-6-Ahx-RM2] peptide with 86Y or 90Y. Natural-metallated (natY) conjugates were assessed for binding affinity for the αVß3 integrin or GRPR in human glioblastoma U87-MG and prostate PC-3 cell lines, respectively. The effective stability of the new tracers was also evaluated prior to in vivo evaluation in normal CF-1 mice and SCID mice bearing xenografted tumors. RESULTS: Competitive displacement binding assays in PC-3 cells showed high binding affinity for the GRPR (IC50, 5.65 ±â€¯0.00 nM). On the other hand, competitive displacement binding assays in U87-MG cells revealed only moderate binding to the αVß3 integrin (IC50, 346 ±â€¯5.30 nM). Biodistribution studies in PC-3 tumor-bearing mice [RGD-Glu-[[90Y]Y-DO3A]-6-Ahx-RM2] showed high tumor uptake (8.70 ±â€¯0.35%ID/g at 1 h post-intravenous injection) and retention of tracer (5.28 ±â€¯0.12%ID/g) at 24 h post-intravenous injection. Micro-positron emission tomography (microPET) in PC-3 tumor-bearing mice using [RGD-Glu-[[86Y]Y-DO3A]-6-Ahx-RM2] correlated well with biodistribution investigations over the various time points that were studied. CONCLUSIONS: The [RGD-Glu-[[86Y]Y-DO3A]-6-Ahx-RM2] and [RGD-Glu-[[90Y]Y-DO3A]-6-Ahx-RM2] matched-pair conjugates described herein exhibit favorable microPET and pharmacokinetic profiles and merit further investigations for molecular imaging and/or therapeutic evaluation in larger animal models and potentially humans. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: The theranostic, heterobivalent, agents described herein perform comparably with other mono- and multivalent conjugates we have reported and offer the potential of improved sensitivity for detecting prostate cancer cells that might exhibit differing profiles of receptor expression on tumor cells in human patients.

Lutetium-177 Labeled Bombesin Peptides for Radionuclide Therapy.

The rare-earth radionuclides that decay by beta particle (ß-) emission are considered to be ideal in the context of targeted radiotherapy. The rare-earth isotopes exist primarily in the 3+ oxidation state and are considered to be hard metal centers, requiring multidentate, hard donor ligands such as the poly(aminocarboxylates) for in vivo kinetic inertness. 177Lu is a rare-earth radionuclide that is produced in moderate specific activity (740 GBq/mg) by direct neutron capture of enriched 176Lu via the 176Lu(n,γ)177Lu nuclear reaction. 177Lu has a half-life of 6.71 d, decays by beta emission (Ebmax = 0.497 MeV), and emits two imagable photons (113keV, 3% and 208kev, 11%). High specific activity, no-carrier-added 177Lu can also be prepared by an indirect neutron capture nuclear reaction on a 176Yb target. Herein, we report upon bombesin (BBN) peptides radiolabeled with 177Lu. The impetus driving many of the research studies that we have described in this review is that the high-affinity gastrin releasing peptide receptor (GRPR, BBN receptor subtype 2, BB2) has been identified in tissue biopsy samples and immortalized cell lines of many human cancers and is an ideal biomarker for targeting early-stage disease. Early on, the ability of GRPR agonists to be rapidly internalized coupled with a high incidence of GRPR expression on various neoplasias was a driving force for the design and development of new diagnostic and therapeutic agents targeting GRP receptor-positive tumors. Recent reports, however, show compelling evidence that radiopharmaceutical design and development based upon antagonist-type ligand frameworks clearly bears reexamination. Last of all, the ability to target multiple biomarkers simultaneously via a heterodimeric targeting ligand has also provided a new avenue to investigate the dual targeting capacity of bivalent radioligands for improved in vivo molecular imaging and treatment of specific human cancers. In this report, we describe recent advances in 177Lu-labeled bombesin peptides for targeted radiotherapy that includes agonist, antagonist, and multivalent cell-targeting agents. In vitro, in vivo translational, and in vivo human clinical investigations are described.

Biallelic RFX6 mutations can cause childhood as well as neonatal onset diabetes mellitus.

Neonatal diabetes is a highly genetically heterogeneous disorder. There are over 20 distinct syndromic and non-syndromic forms, including dominant, recessive and X-linked subtypes. Biallelic truncating or mis-sense mutations in the DNA-binding domain of the RFX6 transcription factor cause an autosomal recessive, syndromic form of neonatal diabetes previously described as Mitchell-Riley syndrome. In all, eight cases have been reported, with the age at onset of diabetes in the first 2 weeks of life. Here we report two individuals born to double first cousins in whom intestinal atresias consistent with a diagnosis of Mitchell-Riley syndrome were diagnosed at birth, but in whom diabetes did not present until the ages of 3 and 6 years. Novel compound heterozygous RFX6 nonsense mutations (p.Arg726X/p.Arg866X) were identified at the 3' end of the gene. The later onset of diabetes in these patients may be due to incomplete inactivation of RFX6. Genetic testing for RFX6 mutations should be considered in patients presenting with intestinal atresias in the absence of neonatal diabetes.

Characterization and evaluation of DOTA-conjugated Bombesin/RGD-antagonists for prostate cancer tumor imaging and therapy.

INTRODUCTION: Here we present the metallation, characterization, in vivo and in vitro evaluations of dual-targeting, peptide-based radiopharmaceuticals with utility for imaging and potentially treating prostate tumors by virtue of their ability to target the αVß3 integrin or the gastrin releasing peptide receptor (GRPr). METHODS: [RGD-Glu-6Ahx-RM2] (RGD: Arg-Gly-Asp; Glu: glutamic acid; 6-Ahx: 6-amino hexanoic acid; RM2: (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2)) was conjugated to a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) bifunctional chelator (BFCA) purified via reversed-phase high-performance liquid chromatography (RP-HPLC), characterized by electrospray ionization-mass spectrometry (ESI-MS), and radiolabeled with (111)In or (177)Lu. Natural-metallated compounds were assessed for binding affinity for the αVß3 integrin or GRPr in human glioblastoma U87-MG and prostate PC-3 cell lines and stability prior to in vivo evaluation in normal CF-1 mice and SCID mice xenografted with PC-3 cells. RESULTS: Competitive displacement binding assays with PC-3 and U87-MG cells revealed high to moderate binding affinity for the GRPr or the αVß3 integrin (IC50 range of 5.39±1.37 nM to 9.26±0.00 nM in PC-3 cells, and a range of 255±47 nM to 321±85 nM in U87-MG cells). Biodistribution studies indicated high tumor uptake in PC-3 tumor-bearing mice (average of 7.40±0.53% ID/g at 1h post-intravenous injection) and prolonged retention of tracer (mean of 4.41±0.91% ID/g at 24h post-intravenous injection). Blocking assays corroborated the specificity of radioconjugates for each target. Micro-single photon emission computed tomography (microSPECT) confirmed favorable radiouptake profiles in xenografted mice at 20h post-injection. CONCLUSIONS: [RGD-Glu-[(111)In-DO3A]-6-Ahx-RM2] and [RGD-Glu-[(177)Lu- DO3A]-6-Ahx-RM2] show favorable pharmacokinetic and radiouptake profiles, meriting continued evaluation for molecular imaging in murine U87-MG/PC-3 xenograft models and radiotherapy studies with (177)Lu and (90)Y conjugates. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: These heterovalent, peptide-targeting ligands perform comparably with many mono- and multivalent conjugates with the potential benefit of increased sensitivity for detecting cancer cells exhibiting differential expression of target receptors.

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.

PET of tumors expressing gastrin-releasing peptide receptor with an 18F-labeled bombesin analog.

UNLABELLED: The gastrin-releasing peptide receptor (GRPR) is overexpressed in human prostate cancer. Bombesin (BBN) is a neurotransmitter of 14 amino acids and binds with selectivity and with high affinity to GRPRs. We have synthesized a NOTA-conjugated bombesin derivative, NOTA-8-Aoc-BBN(7-14)NH(2), to label this analog with (18)F using the new Al(18)F method. In this study, the GRPR-targeting potential of (18)F-labeled NOTA-8-Aoc-BBN(7-14)NH(2) was studied using (68)Ga-NOTA-8-Aoc-BBN(7-14)NH(2) as a reference. METHODS: The NOTA-conjugated bombesin analog was synthesized and radiolabeled with (68)Ga or (18)F. For (18)F labeling, we used our new 1-pot, 1-step method. The labeled product was purified by reversed-phase high-performance liquid chromatography. The log P values of the radiotracers were determined. The tumor-targeting characteristics of the compounds were assessed in mice with subcutaneously growing PC-3 xenografts. GRPR-binding specificity was studied by coinjection of an excess of unlabeled NOTA-8-Aoc-BBN(7-14)NH(2). Small-animal PET/CT images were acquired. RESULTS: NOTA-8-Aoc-BBN(7-14)NH(2) could be efficiently labeled with (18)F or with (68)Ga. NOTA-8-Aoc-BBN(7-14)NH(2) was labeled with (18)F in a single step, with 50%-90% yield. Radiolabeling, including purification, was performed in 45 min and resulted in a specific activity of greater than 10 GBq/µmol. The log P values of (18)F- and (68)Ga-labeled NOTA-8-Aoc-BBN(7-14)NH(2) were -1.47 ± 0.05 and -1.98 ± 0.03, respectively. In mice, both radiolabeled compounds cleared rapidly from the blood (<0.07 percentage injected dose per gram at 1 h after injection), mainly via the kidneys. At 1 h after injection, the uptake of (18)F- and (68)Ga-labeled NOTA-8-Aoc-BBN(7-14)NH(2) in the PC-3 tumors was 2.15 ± 0.55 and 1.24 ± 0.26 percentage injected dose per gram, respectively. GRPR-binding specificity was demonstrated by reduced tumor uptake of radiolabeled NOTA-8-Aoc-BBN(7-14)NH(2) after coinjection of a 100-fold excess of unlabeled NOTA-8-Aoc-BBN(7-14)NH(2) peptide. The accumulation of (18)F-NOTA-8-Aoc-BBN(7-14)NH(2) in the subcutaneous PC-3 tumors could be visualized via small-animal PET. CONCLUSION: NOTA-8-Aoc-BBN(7-14)NH(2) could be labeled rapidly and efficiently with (18)F using a 1-pot, 1-step method. Radiolabeled NOTA-8-Aoc-BBN(7-14)NH(2) specifically accumulated in the GRPR-expressing PC-3 tumors and should be evaluated clinically.

Comparison of two peptide radiotracers for prostate carcinoma targeting.

OBJECTIVES: Scintigraphy is generally not the first choice treatment for prostate cancer, although successful studies using bombesin analog radiopeptides have been performed. Recently, a novel peptide obtained using a phage display library demonstrated an affinity for prostate tumor cells. The aim of this study was to compare the use of a bombesin analog to that of a phage display library peptide (DUP-1) radiolabeled with technetium-99m for the treatment of prostate carcinoma. The peptides were first conjugated to S-acetyl-MAG3 with a 6-carbon spacer, namely aminohexanoic acid. METHODS: The technetium-99m labeling required a sodium tartrate buffer. Radiochemical evaluation was performed using ITLC and was confirmed by high-performance liquid chromatography. The coefficient partition was determined, and in vitro studies were performed using human prostate tumor cells. Biodistribution was evaluated in healthy animals at various time points and also in mice bearing tumors. RESULTS: The radiochemical purity of both radiotracers was greater than 95%. The DUP-1 tracer was more hydrophilic (log P = -2.41) than the bombesin tracer (log P = -0.39). The biodistribution evaluation confirmed this hydrophilicity by revealing the greater kidney uptake of DUP-1. The bombesin concentration in the pancreas was greater than that of DUP-1 due to specific gastrin-releasing peptide receptors. Bombesin internalization occurred for 78.32% of the total binding in tumor cells. The DUP-1 tracer showed very low binding to tumor cells during the in vitro evaluation, although tumor uptake for both tracers was similar. The tumors were primarily blocked by DUP1 and the bombesin radiotracer primarily targeted the pancreas. CONCLUSION: Further studies with the radiolabeled DUP-1 peptide are recommended. With further structural changes, this molecule could become an efficient alternative tracer for prostate tumor diagnosis.