This study aimed to examine how negatively charged amino acid linkers influence the tumor-targeting and biodistribution properties of [177Lu]-Lu-labeled 4-p-(tolyl)-butyric acid (p-TBA)-conjugated alpha-melanocyte-stimulating hormone peptides. The p-TBA moiety functions as an albumin binder (ALB), either directly attached to the peptide or through a negatively charged aspartic acid (Asp) or glutamic acid (Glu) linker. DOTA-Lys-(p-TBA)-GGNle-CycMSHhex {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-Lys-(4-p-(tolyl)-butyric acid)-GlyGly-Nle-c-[Asp-His-DPhe-Arg-Trp-Lys]-CONH2}, DOTA-Lys-(Asp-p-TBA)-GGNle-CycMSHhex, and DOTA-Lys-(Glu-p-TBA)-GGNle-CycMSHhex were synthesized using fluorenylmethoxycarbonyl (Fmoc) chemistry. Their melanocortin-1 receptor (MC1R) binding affinities were measured in B16/F10 melanoma cells, and the biodistribution of [177Lu]-Lu-labeled peptides was examined in B16/F10 melanoma-bearing mice at 0.5, 2, 4, and 24 h postinjection. Additionally, the melanoma imaging capability of [177Lu]-Lu-DOTA-Lys-(Asp-p-TBA)-GGNle-CycMSHhex was examined in B16/F10 melanoma-bearing mice. The IC50 values were 0.5 ± 0.09 (p-TBA), 0.03 ± 0.03 (Asp-p-TBA), and 7.2 ± 0.07 nM (Glu-p-TBA). Among the three, [177Lu]-Lu-DOTA-Lys-(Asp-p-TBA)-GGNle-CycMSHhex displayed the highest tumor/kidney and tumor/liver uptake ratios at 2 and 4 h postinjection. The B16/F10 melanoma uptake of [177Lu]-Lu-DOTA-Lys-(Asp-p-TBA)-GGNle-CycMSHhex was 20.40 ± 1.91, 45.24 ± 1.72, 31.30 ± 2.85, and 11.16 ± 1.79%ID/g at 0.5, 2, 4, and 24 h postinjection, respectively. The B16/F10 melanoma lesions were clearly visualized by SPECT/CT using [177Lu]-Lu-DOTA-Lys-(Asp-p-TBA)-GGNle-CycMSHhex as an imaging probe at 2 h postinjection. Overall, the presence and charge of the linker significantly affected MC1R binding and tumor uptake. The linker charge played a key role in the liver and kidney uptake of [177Lu]-Lu-DOTA-Lys-(Asp-p-TBA)-GGNle-CycMSHhex and [177Lu]-Lu-DOTA-Lys-(Glu-p-TBA)-GGNle-CycMSHhex. [177Lu]-Lu-DOTA-Lys-(Asp-p-TBA)-GGNle-CycMSHhex exhibited the highest tumor/liver and tumor/kidney uptake ratios among the three [177Lu]-Lu-peptides, highlighting its potential for future melanoma therapy.

Cancer remains one of the most challenging medical conditions. Nanotechnology is revolutionizing the approach to cancer management as an efficient tool for early diagnosis, therapy and monitoring. Nanoparticles (NPs) have shown promising results in advancing precision medicine by integrating diagnostic imaging and targeted therapy within a single platform, a concept of theranostics. Nanocarriers are synthesized via the use of biocompatible materials that are functionalized with specific ligands that bind to receptors that are overexpressed on the surface of cancer cells. For theranostic purposes, these NPs are loaded with suitable radioisotopes that emit gamma, positron, alpha or beta radiation for imaging and/or therapy. Despite the significant successes, most of the work is still at the preclinical stages awaiting clinical translation. In this review, a comprehensive examination of the current state of receptor-targeted radiotheranostic NPs is presented, with a focus on their design, functionalization, receptor-specific targeting, and clinical translational potential. The role of key tumor markers is highlighted in guiding NP delivery and improving precision and therapy. The exploration of these cutting-edge advancements and associated challenges will provide insights into the revolution of radiotheranostic nanomedicine and its impact on the future of precision oncology.

Effective molecular imaging and targeted cancer therapy rely on receptor-specific targeted delivery systems that are both metabolically stable and kinetically inert for optimal in vivo performance. Until now, no single metal complexing agent has demonstrated the versatility to coordinate metals across the periodic table while maintaining the kinetic inertness required for clinical theranostic applications. Therefore, enhancing the in vivo kinetic stability of radiolabeled, cell-targeting, biologically active compounds remains a critical goal to minimize unintended accumulation of radioactivity in collateral tissues. This review describes the usage of NOTA [NOTA = 1,4,7-triazacyclononane-1,4,7-triacetic acid] and derivatives of NOTA, a metal complexing agent that has been found to have the ability to effectively coordinate with a wide range of radiometals, including metal-radiohalogens, to form stable complexes. This enables the development of new cell-targeting small molecule and peptide conjugates with the potential to resist demetallation in vivo, thereby reducing radionuclide uptake in non-target tissues. Herein, we discuss the design and development of NOTA-based, cell-targeting, small molecules having very high affinity and selectivity for the GRPR (Gastrin-Releasing Peptide Receptor), the SSTR2 (Somatostatin Receptor Subtype 2), and the MC1R (Melanocortin-1) receptors that are present on the surfaces of numerous solid primary human tumors and their metastatic counterparts.

Alpha therapy (TAT) relies on combining alpha-emitting radionuclides with specific cell-targeting vectors to deliver a high payload of cytotoxic radiation capable of destroying tumor tissues. TAT efficacy comes from the tissue selectivity of the targeting vector, the high linear energy transfer (LET) of the radionuclide, and the short range of alpha particles in tissues. Recent research studies have been directed to evaluate TAT on a preclinical and clinical scale, including evaluating damage to tumor tissues with minimal toxic radiation effects on surrounding healthy tissues. This review highlights the use of Actinium-225/Bismuth-213 radionuclides as promising candidates for TAT. Herein, we begin with a discussion on the production and supply of [225Ac]Ac/[213Bi]Bi followed by the formulation of [225Ac]Ac/[213Bi]Bi-radiopharmaceuticals using different radiolabeling techniques. Finally, we have summarized the preclinical and clinical evaluation of these potential radiotheranostic agents.

Background In the United States, breast cancer is the second leading cause of cancer-related death. Triple-negative breast cancer (TNBC) is of substantial concern, as it lacks the receptors usually targeted by conventional treatments. Triple-negative breast tumors have a high degree of copper metabolism for the synthesis of transporters, enzymes, and chaperones. Tetrathiomolybdate (TM) is a well-tolerated oral therapy that has been investigated for chelating copper from tumors in TNBC patients, resulting in extended remission. The overall goal of this research was to evaluate [64Cu]CuCl2 PET/CT imaging of copper utilization in this disease, in the presence and absence of TM. Methods Uptake, internalization, and efflux studies were performed in TNBC cells versus normal cells. Biodistribution experiments were then conducted in TNBC xenograft-bearing mice that were administered TM versus controls. PET/CT imaging of mice carrying TNBC tumors was also performed in the presence and absence of TM. Finally, imaging was performed in a healthy cat and in a cat with mammary carcinoma. Results SUM149 TNBC cells selectively took up, internalized, and retained [64Cu]CuCl2 more avidly than normal fibroblasts. When SUM149-bearing mice were given TM, tumor uptake decreased and tracer accumulation shifted predominantly to the liver and kidneys, compared to control mice, in which large quantities of 64Cu were excreted into the intestines. These results were supported by PET/CT imaging of the mice. PET/CT of companion cats gave results similar to those obtained in mice, with high accumulation of radioactivity observed in the liver and gallbladder and moderate intestinal and renal clearance. In a cat with mammary carcinoma, tumor uptake of [64Cu]CuCl2 was highly conspicuous, even in close proximity to the liver. Conclusions Utilization of [64Cu]CuCl2 in triple-negative breast cancer can be detected efficiently in cell and animal models of this disease. The tracer was also used successfully to evaluate TM administration in the SUM149 TNBC mouse model. Furthermore, PET/CT imaging of both mice and cats with breast cancer shows the potential to monitor treatment with TM in a facile, noninvasive manner. We are currently conducting a clinical trial of [64Cu]CuCl2 PET/CT in companion cats with mammary carcinoma, with the future goal of evaluating the efficacy of TM in feline patients.

Prostate cancer remains a major global health concern demanding innovative therapeutic strategies. This study introduces a novel green nanotechnology approach for the development of a nanomedicine agent, also referred to as a nano-radiopharmaceutical, which integrates the antitumor and high antioxidant capacity of resveratrol phytochemical (RESV) with radioactive gold nanoparticles (198AuNPs) for targeted prostate cancer therapy and diagnostics. The radioactive formulation, RESV-198AuNP, was developed at the University of Missouri Research Reactor (MURR) using neutron-activated gold-198, which exhibited high radionuclidic and radiochemical purity. Stability testing in rat serum and saline demonstrated durability of up to 15 days. In vivo biodistribution studies in CF-1 mice and PC-3 tumor-bearing SCID mice have provided insights into pharmacokinetics and optimum tumor retention. Intratumoral administration of RESV-198AuNP in SCID mice demonstrated strong retention within prostate cancer xenografts, suggesting tumor-specific uptake and retention. This study underscores the potential of RESV-198AuNP as a dual-functional nano-radiopharmaceutical for prostate cancer theranostics. By combining resveratrol's anticancer properties with the therapeutic and imaging benefits of 198AuNPs, this platform offers a promising avenue for improving treatment efficacy and enabling real-time therapeutic response monitoring. This research reveals the potential of RESV as a tumor-targeting agent and introduces a new perspective of green nanotechnology for dual anti-inflammatory radiosynovectomy as well as for use in cancer treatment. In-depth in vivo studies on the therapeutic efficacy of intratumorally administered RESV-198AuNP revealed that over 85% of the injected dose (ID) remained within prostate tumors for up to 24 h. By the fourth week post-treatment, the treated group exhibited a greater than tenfold reduction in tumor volumes than the control group receiving saline. This study highlights emerging opportunities in green nanotechnology and introduces a clinically feasible approach to utilize resveratrol as a tumor-targeting agent in oncology, particularly for the application of RESV-198AuNP in cancer treatments.

We developed a novel site-specific bimodal MRI/fluorescence nanoparticle contrast agent targeting gastrin-releasing peptide receptors (GRPrs), which are overexpressed in aggressive prostate cancers. Biocompatible ultra-small superparamagnetic iron oxide (USPIO) nanoparticles were synthesized using glucose and casein coatings, followed by conjugation with a Cy7.5-K-8AOC-BBN [7-14] peptide conjugate. The resulting USPIO(Cy7.5)-BBN nanoparticles were purified by 100 kDa membrane dialysis and fully characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared (FTIR) spectroscopy, and magnetic resonance imaging (MRI) relaxivity, as well as evaluated for in vitro and in vivo binding specificity and imaging efficacy in PC-3 prostate cancer cells and xenografted tumor-bearing mice. The USPIO(Cy7.5)-BBN nanoparticles had a core diameter of 4.93 ± 0.31 nm and a hydrodynamic diameter of 35.56 ± 0.58 nm. The r2 relaxivity was measured to be 70.2 ± 2.5 s−1 mM−1 at 7T MRI. The Cy7.5-K-8AOC-BBN [7-14] peptide-to-nanoparticle ratio was determined to be 21:1. The in vitro GRPr inhibitory binding (IC50) value was 2.5 ± 0.7 nM, indicating a very high binding affinity of USPIO(Cy7.5)-BBN to the GRPr on PC-3 cells. In vivo MRI showed significant tumor-to-muscle contrast enhancement in the uptake group at 4 h (31.1 ± 3.4%) and 24 h (25.7 ± 2.1%) post-injection compared to the blocking group (4 h: 15.3 ± 2.0% and 24 h: −2.8 ± 6.8%; p < 0.005). In vivo and ex vivo near-infrared fluorescence (NIRF) imaging revealed significantly increased fluorescence in tumors in the uptake group compared to the blocking group. These findings demonstrate the high specificity of bimodal USPIO(Cy7.5)-BBN nanoparticles towards GRPr-expressing PC-3 cells, suggesting their potential for targeted imaging in aggressive prostate cancer.

Background Prostate cancer affects 1 in 6 men, and it is the second‑leading cause of cancer-related death in American men. Surgery is one of the main treatment modalities for prostate cancer, but it often results in incomplete resection margins or complete resection that leads to nerve damage and undesirable side effects. In the present work, we have developed a new bimodal tracer, NODAGA-sCy7.5 PSMAi (prostate-specific membrane antigen inhibitor), labeled with the true matched theranostic pair 64Cu/67Cu and a near-infrared fluorescent dye. This agent could potentially be used for concomitant PET imaging, optical surgical navigation, and targeted radiopharmaceutical therapy. Methods A prostate-specific membrane antigen (PSMA)-targeting urea derivative was conjugated to NODAGA for copper radiolabeling and to the near-infrared fluorophore sulfo-Cy7.5 (sCy7.5). Binding studies were performed in PSMA-positive PC-3 PIP cells, as well as uptake and internalization assays in PC-3 PIP cells and PSMA-negative PC-3 wild type cells. Biodistribution studies of the 64Cu-labeled compound were performed in PC-3 PIP- and PC-3 tumor-bearing mice, and 67Cu biodistributions of the agent were obtained in PC-3 PIP tumor-carrying mice. PET imaging and fluorescence imaging were also performed, using the same molar doses, in the two mouse models. Results The PSMA conjugate bound with high affinity to PSMA-positive prostate cancer cells, as opposed to cells that were PSMA-negative. Uptake and internalization were rapid and PSMA-mediated in PC-3 PIP cells, while only minimal non-specific uptake was observed in PC-3 cells. Biodistribution studies showed specific uptake in PC-3 PIP tumors, while accumulation in PC-3 tumor-bearing mice was low. Furthermore, tumor uptake of the 67Cu-labeled agent in the PC-3 PIP model was statistically equivalent to that of 64Cu. PET and fluorescence imaging at 0.5 nmol per mouse also demonstrated that PC-3 PIP tumors could be clearly detected, while PC-3 tumors showed no tumor accumulation. Conclusions NODAGA-sCy7.5-PSMAi was specific and selective in detecting PSMA-positive, as opposed to PSMA-negative, tumors in mouse models of prostate cancer. This bioconjugate could potentially be used for PET staging with 64Cu, targeted radiopharmaceutical therapy with 67Cu, and/or image-guided surgery with sCy7.5.

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.

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.

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.

INTRODUCTION: The aim of this work was to develop diagnostic (Tc-99m) 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 sst(2)-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 Tc-99m-complexes in an animal SSTR-tumor model. METHODS: The [99mTc]/[186Re][Tc/Re(OH2)3(CO)3](+) precursors were utilized to prepare the Tc-99m/186Re-complexes, which were identified by HPLC co-injection with their natRe analogues. The tracers were challenged in vitro at 37 degrees C against cysteine and histidine in phosphate-buffered saline (pH7.4) 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-sst(2)-ANT revealed low tumor uptake (2.78?+/-?0.27 %ID/g) at 1?h, while high tumor uptake (16.70?+/-?3.32 %ID/g) was found for [Tc-99m]Tc-NOTA-sst(2)-ANT. Moderate to low tumor retention was observed for both tracers after 4 and 24?h. Tumor uptake for [Tc-99m]Tc-NOTA-sst(2)-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 [Tc-99m]Tc-NOTA-sst(2)-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 [Tc-99m]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. Copyright (C) 2019 Elsevier Inc. All rights reserved.

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.

INTRODUCTION: The aim of this work was to develop diagnostic (Tc-99m) 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 sst(2)-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 Tc-99m-complexes in an animal SSTR-tumor model.METHODS: The [99mTc]/[186Re][Tc/Re(OH2)3(CO)3](+) precursors were utilized to prepare the Tc-99m/186Re-complexes, which were identified by HPLC co-injection with their natRe analogues. The tracers were challenged in vitro at 37 degrees C against cysteine and histidine in phosphate-buffered saline (pH7.4) 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-sst(2)-ANT revealed low tumor uptake (2.78?+/-?0.27 %ID/g) at 1?h, while high tumor uptake (16.70?+/-?3.32 %ID/g) was found for [Tc-99m]Tc-NOTA-sst(2)-ANT. Moderate to low tumor retention was observed for both tracers after 4 and 24?h. Tumor uptake for [Tc-99m]Tc-NOTA-sst(2)-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 [Tc-99m]Tc-NOTA-sst(2)-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 [Tc-99m]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. Copyright (C) 2019 Elsevier Inc. All rights reserved.

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.

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 αvβ3 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.

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.

INTRODUCTION:In this study, we describe development of a true matched-pair theranostic agent that is able to target the αVβ3 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 αVβ3 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 ± 0.00 nM). On the other hand, competitive displacement binding assays in U87-MG cells revealed only moderate binding to the αVβ3 integrin (IC50, 346 ± 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 ± 0.35%ID/g at 1 h post-intravenous injection) and retention of tracer (5.28 ± 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.

In this study, we have prepared a novel, dual-biomarker, targeting ligand having high affinity and specificity for PSMA/GRPr receptors that are expressed on most prostate cancers. [DUPA-6-Ahx-Lys(DOTA)-6-Ahx-RM2] was synthesized and the new conjugate was metallated macroscopically with GaCl 3 , InCl 3 , and LuCl 3 to form [DUPA-6-Ahx-Lys(M-DOTA)-6-Ahx-RM2] (where M = Ga, In, or Lu). These new agents, when radiolabeled with Ga-68, In-111, or Lu-177 hold theranostic potential for patients presenting with prostate cancer disease.

The rare-earth radionuclides that decay by beta particle (β-) 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.