First Biodistribution Study of [68Ga]Ga-NOTA-Insulin Following Intranasal Administration in Adult Vervet Monkeys.

Background: Intranasal insulin (INI) is being explored as a treatment for Alzheimer's disease (AD). Improved memory, functional ability, and cerebrospinal fluid (CSF) AD biomarker profiles have been observed following INI administration. However, the method of intranasal delivery may significantly affect outcomes. Objective: To show reliable delivery of insulin to the brain using the Aptar Cartridge Pump System (CPS) intranasal delivery system. Methods: To visualize INI biodistribution, we developed a novel PET radiotracer, Gallium 68-radiolabeled (NOTA-conjugated) insulin, [68Ga]Ga-NOTA-insulin. We used the Aptar CPS to administer [68Ga]Ga-NOTA-insulin to anesthetized healthy adult vervet monkeys and measured brain regional activity and whole-body dosimetry following PET/CT scans. Results: We observed brain penetration of [68Ga]Ga-NOTA-insulin following intranasal administration with the Aptar CPS. Radioactive uptake was seen in multiple regions, including the amygdala, putamen, hypothalamus, hippocampus, and choroid plexus. A safety profile and whole-body dosimetry were also established in a second cohort of vervets. Safety was confirmed: vitals remained stable, blood glucose levels were unchanged, and no organ was exposed to more than 2.5 mSv of radioactivity. Extrapolations from vervet organ distribution allowed for estimation of the [68Ga]Ga-NOTA-insulin absorbed dose in humans, and the maximum dose of [68Ga]Ga-NOTA-insulin that can be safely administered to humans was determined to be 185 MBq. Conclusions: The use of [68Ga]Ga-NOTA-insulin as a PET radiotracer is safe and effective for observing brain uptake in vervet monkeys. Further, the Aptar CPS successfully targets [68Ga]Ga-NOTA-insulin to the brain. The data will be essential in guiding future studies of intranasal [68Ga]Ga-NOTA-insulin administration in humans.

Development and Evaluation of DOTA-FAPI-Maleimide as a Novel Radiotracer for Tumor Theranostic with Extended Circulation.

This study aimed to evaluate a novel albumin-binding strategy for addressing the challenge of insufficient tumor retention of fibroblast activation protein inhibitors (FAPIs). Maleimide, a molecule capable of covalent binding to free thiol groups, was modified to conjugate with FAPI-04 in order to enhance its binding to endogenous albumin, resulting in an extended blood circulation half-life and increased tumor uptake. DOTA-FAPI-maleimide was prepared and radiolabeled with Ga-68 and Lu-177, followed by cellular assays, pharmacokinetic analysis, PET/CT, and SPECT/CT imaging to assess the probe distribution in various tumor-bearing models. Radiolabeling of the modified probe was successfully achieved with a radiochemical yield of over 99% and remained stable for 144 h. Cellular assays showed that the ligand concentration required for 50% inhibition of the probe was 1.20 ± 0.31 nM, and the Kd was 0.70 ± 0.07 nM with a Bmax of 7.94 ± 0.16 fmol/cell, indicative of higher specificity and affinity of DOTA-FAPI-maleimide compared to other FAPI-04 variants. In addition, DOTA-FAPI-maleimide exhibited a persistent blood clearance half-life of 7.11 ± 0.34 h. PET/CT images showed a tumor uptake of 2.20 ± 0.44%ID/g at 0.5 h p.i., with a tumor/muscle ratio of 5.64 in HT-1080-FAP tumor-bearing models. SPECT/CT images demonstrated long-lasting tumor retention. At 24 h p.i., the tumor uptake of [177Lu]Lu-DOTA-FAPI-maleimide reached 5.04 ± 1.67%ID/g, with stable tumor retention of 3.40 ± 1.95%ID/g after 4 days p.i. In conclusion, we developed and evaluated the thiol group-attaching strategy, which significantly extended the circulation and tumor retention of the adapted FAPI tracer. We envision its potential application for clinical cancer theranostics.

Development and first-in-human study of PSMA-targeted PET tracers with improved pharmacokinetic properties.

PURPOSE: A series of new 68Ga-labeled tracers based on [68Ga]Ga-PSMA-617 were developed to augment the tumor-to-kidney ratio and reduce the activity accumulation in bladder, ultimately minimize radiation toxicity to the urinary system. METHODS: We introduced quinoline group, phenylalanine and decanoic acid into different tracers to enhance their lipophilicity, strategically limiting their metabolic pathway through the urinary system. Their binding affinity onto LNCaP cells was determined through in vitro saturation assays and competition binding assays. In vivo metabolic study, PET imaging and biodistribution experiment were performed in LNCaP tumor-bearing B-NSG male mice. The most promising tracer was selected for first-in-human study. RESULTS: Four radiotracers were synthesized with radiochemical purity (RCP) > 95% and molar activity in a range of 20.0-25.5 GBq/µmol. The binding affinities (Ki) of TWS01, TWS02 to PSMA were in the low nanomolar range (< 10 nM), while TWS03 and TWS04 exhibited binding affinities with Ki > 20 nM (59.42 nM for TWS03 and 37.14 nM for TWS04). All radiotracers exhibited high stability in vivo except [68Ga]Ga-TWS03. Micro PET/CT imaging and biodistribution analysis revealed that [68Ga]Ga-TWS02 enabled clear tumor visualization in PET images at 1.5 h post-injection, with higher tumor-to-kidney ratio (T/K, 0.93) and tumor-to-muscle ratio (T/M, 107.62) compared with [68Ga]Ga-PSMA-617 (T/K: 0.39, T/M: 15.01) and [68Ga]Ga-PSMA-11 (T/K: 0.15, T/M: 24.00). In first-in-human study, [68Ga]Ga-TWS02 effectively detected PCa-associated lesions including primary and metastatic lesions, with lower accumulation in urinary system, suggesting that [68Ga]Ga-TWS02 might be applied in the detection of bladder invasion, with minimized radiation toxicity to the urinary system. CONCLUSION: Introduction of quinoline group, phenylalanine and decanoic acid into different tracers can modulate the binding affinity and pharmacokinetics of PSMA in vivo. [68Ga]Ga-TWS02 showed high binding affinity to PSMA, excellent pharmacokinetic properties and clear imaging of PCa-associated lesions, making it a promising radiotracer for the clinical diagnosis of PCa. Moreover, TWS02 with a chelator DOTA could also label 177Lu and 225Ac, which could be used for PCa treatment without significant side effects. TRIAL REGISTRATION: The clinical evaluation of this study was registered On October 30, 2021 at https://www.chictr.org.cn/ (No: ChiCTR2100052545).

Synthesis and Evaluation of [18F]AlF-NOTA-c-DVAP: A Novel PET Probe for Imaging GRP78 in Cancer.

GRP78, a member of the HSP70 superfamily, is an endoplasmic reticulum chaperone protein overexpressed in various cancers, making it a promising target for cancer imaging and therapy. Positron emission tomography (PET) imaging offers unique advantages in real time, noninvasive tumor imaging, rendering it a suitable tool for targeting GRP78 in tumor imaging to guide targeted therapy. Several studies have reported successful tumor imaging using PET probes targeting GRP78. However, existing PET probes face challenges such as low tumor uptake, inadequate in vivo distribution, and high abdominal background signal. Therefore, this study introduces a novel peptide PET probe, [18F]AlF-NOTA-c-DVAP, for targeted tumor imaging of GRP78. [18F]AlF-NOTA-c-DVAP was radiolabeled with fluoride-18 using the aluminum-[18F]fluoride ([18F]AlF) method. The study assessed the partition coefficients, stability in vitro, and metabolic stability of [18F]AlF-NOTA-c-DVAP. Micro-PET imaging, pharmacokinetic analysis, and biodistribution studies were carried out in tumor-bearing mice to evaluate the probe's performance. Docking studies and pharmacokinetic analyses of [18F]AlF-NOTA-c-DVAP were also performed. Immunohistochemical and immunofluorescence analyses were conducted to confirm GRP78 expression in tumor tissues. The probe's binding affinity to GRP78 was analyzed by molecular docking simulation. [18F]AlF-NOTA-c-DVAP was radiolabeled in just 25 min with a high yield of 51 ± 16%, a radiochemical purity of 99%, and molar activity within the range of 20-50 GBq/µmol. [18F]AlF-NOTA-c-DVAP demonstrated high stability in vitro and in vivo, with a logD value of -3.41 ± 0.03. Dynamic PET imaging of [18F]AlF-NOTA-c-DVAP in tumors showed rapid uptake and sustained retention, with minimal background uptake. Biodistribution studies revealed rapid blood clearance and excretion through the kidneys following a single-compartment reversible metabolic model. In PET imaging, the T/M ratios for A549 tumors (high GRP78 expression), MDA-MB-231 tumors (medium expression), and HepG2 tumors (low expression) at 60 min postintravenous injection were 10.48 ± 1.39, 6.25 ± 0.47, and 3.15 ± 1.15% ID/g, respectively, indicating a positive correlation with GRP78 expression. This study demonstrates the feasibility of using [18F]AlF-NOTA-c-DVAP as a PET tracer for imaging GRP78 in tumors. The probe shows promising results in terms of stability, specificity, and tumor targeting. Further research may explore the clinical utility and potential therapeutic applications of this PET tracer for cancer diagnosis.

Preparation of 177Lu-PSMA-617 in Hospital Radiopharmacy: Convenient Formulation of a Clinical Dose Using a Single-Vial Freeze-Dried PSMA-617 Kit Developed In-House.

OBJECTIVE: In the recent time, endoradionuclide therapy for metastatic castration-resistant prostate carcinoma employing 177Lu-PSMA-617 has yielded encouraging results and several clinical trials with the agent are currently ongoing. Routine preparation of 177Lu-PSMA-617 patient doses can be made simpler and convenient, if the ingredients essential for radiolabeling are made available in a ready-to-use lyophilized form. METHODS: PSMA-617 freeze-dried kit was formulated and used for the preparation of 177Lu-PSMA-617 clinical dose with high radiochemical purity using low/medium specific activity 177Lu. Detailed radiochemical studies were performed to determine the maximum activity and volume of 177LuCl3, which can be added in the kit for the formulation of 177Lu-PSMA-617. Studies were also performed to determine the shelf life of the kit to ensure its long-term usage. Studies were performed in buffer as well as human serum medium to determine the stability of the 177Lu-PSMA-617 complex after storing in respective media up to 7 days postpreparation. About ten patient doses of 177Lu-PSMA-617 were administered, and posttherapy scans were acquired. RESULTS: The formulated freeze-dried kit of PSMA-617 could be radiolabeled with an average percentage radiochemical purity > 98.53 ± 0.38. The freeze-dried kit was found suitable for tolerating up to 0.5 mL of 177LuCl3 (in 0.01 N HCl) and specific activity of 555 MBq/µg (15 mCi/µg) for the preparation of the patient dose of 177Lu-PSMA-617. The 177Lu-PSMA-617 complex prepared using the freeze-dried kit of PSMA-617 was observed to maintain % radiochemical purity (RCP) of 96.74 ± 0.87 and 94.81 ± 2.66, respectively, even after storing up to 7 days in buffer and human serum, respectively. 177Lu-PSMA-617 prepared using the in-house formulated freeze-dried kit of PSMA-617 exhibited accumulation in metastatic lesions picked up in a pretherapy PET scan. Reduction in number as well as size of lesions was observed in posttherapy scans acquired after two months of administering the first therapeutic dose of 177Lu-PSMA-617. CONCLUSIONS: The freeze-dried kit of PSMA-617 could be used for the preparation of 177Lu-PSMA-617 with high radiochemical purity (>98%) in a reproducible manner. 177Lu-PSMA-617 prepared using the developed kit was successfully evaluated in patients suffering from metastatic prostate cancer.

Evaluation of 177Lu and 47Sc Picaga-Linked, Prostate-Specific Membrane Antigen-Targeting Constructs for Their Radiotherapeutic Efficacy and Dosimetry.

Lu-177-based, targeted radiotherapeutics/endoradiotherapies are an emerging clinical tool for the management of various cancers. The chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) remains the workhorse for such applications but can limit apparent molar activity or efficient charge modulation, which can impact target binding and, as a consequence, target efficacy. Previously, our lab had developed the small, rare earth selective bifunctional chelator, picaga, as an efficient bifunctional chelator for scandium and lutetium isotopes. Here, we assess the performance of these constructs for therapy in prostate-specific membrane antigen (PSMA)-expressing tumor xenografts. To assess the viability of picaga conjugates in conjunction with long in vivo circulation, a picaga conjugate functionalized with a serum albumin binding moiety, 177Lu-picaga-Alb53-PSMA, was also synthesized. A directly comparative, low, single 3.7 MBq dose treatment study with Lu-PSMA-617 was conducted. Treatment with 177Lu-picaga-Alb53-PSMA resulted in tumor regression and lengthened median survival (54 days) when compared with the vehicle (16 days), 47Sc-picaga-DUPA-, 177Lu-picaga-DUPA-, and 177Lu-PSMA-617-treated cohorts (21, 23, and 21 days, respectively).

Comparison of phosphonate, hydroxypropyl and carboxylate pendants in Fe(III) macrocyclic complexes as MRI contrast agents.

Fe(III) macrocyclic complexes containing a macrocycle and three pendant groups including phosphonate (NOTP =1,4,7-triazacyclononane-1,4,7-triyl-tris(methylenephosphonic acid), carboxylate (NOTA = 1,4,7 - triazacyclononane - N,N',N″ - triacetate) or hydroxypropyl (NOHP =(2S,2'S,2"S)-1,1',1″-(1,4,7-triazonane-1,4,7-triyl)tris(propan-2-ol)) were studied in order to compare the effect of these donor groups on solution chemistry and water proton relaxivity. All three complexes, Fe(NOTP), Fe(NOHP) and Fe(NOTA), display a large degree of kinetic inertness to dissociation in the presence of phosphate and carbonate, under acidic conditions of 100 mM HCl or 1 M HCl or to trans-metalation with Zn(II). The r1 proton relaxivity of the complexes at 1.4 T, 33 °C is compared over the pH range of 1 to 10. At pH 7.4, 33 °C, 1.4 T, Fe(NOHP) has the largest relaxivity (1.5 mM-1 s-1), Fe(NOTP) is second at 1.0 mM-1 s-1, whereas Fe(NOTA) is the lowest at 0.61 mM-1 s-1. Fe(NOTP), Fe(NOHP) and Fe(NOTA) all show an increase in relaxivity at very acidic pH values (< 3) that is consistent with an acid-catalyzed process. Variable temperature 17O NMR studies at near neutral pH are consistent with the absence of an inner-sphere water molecule for Fe(NOTP) and Fe(NOHP), supporting second-sphere or outer-sphere water contributions to proton relaxation. Fe(NOTP) shows contrast enhancement in T1 weighted MRI studies in mice and clears through a renal pathway.

Association between [68Ga]NODAGA-RGDyK uptake and dynamics of angiogenesis in a human cell-based 3D model.

Radiolabeled RGD peptides targeting expression of αvß3 integrin have been applied to in vivo imaging of angiogenesis. However, there is a need for more information on the quantitative relationships between RGD peptide uptake and the dynamics of angiogenesis. In this study, we sought to measure the binding of [68Ga]NODAGA-RGDyK to αvß3 integrin in a human cell-based three-dimensional (3D) in vitro model of angiogenesis, and to compare the level of binding with the amount of angiogenesis. Experiments were conducted using a human cell-based 3D model of angiogenesis consisting of co-culture of human adipose stem cells (hASCs) and of human umbilical vein endothelial cells (HUVECs). Angiogenesis was induced with four concentrations (25%, 50%, 75%, and 100%) of growth factor cocktail resulting in a gradual increase in the density of the tubule network. Cultures were incubated with [68Ga]NODAGA-RGDyK for 90 min at 37 °C, and binding of radioactivity was measured by gamma counting and digital autoradiography. The results revealed that tracer binding increased gradually with neovasculature density. In comparison with vessels induced with a growth factor concentration of 25%, the uptake of [68Ga]NODAGA-RGDyK was higher at concentrations of 75% and 100%, and correlated with the amount of neovasculature, as determined by visual evaluation of histological staining. Uptake of [68Ga]NODAGA-RGDyK closely reflected the amount of angiogenesis in an in vitro 3D model of angiogenesis. These results support further evaluation of RGD-based approaches for targeted imaging of angiogenesis.

Preclinical Assessment Addressing Intravenous Administration of a [68Ga]Ga-PSMA-617 Microemulsion: Acute In Vivo Toxicity, Tolerability, PET Imaging, and Biodistribution.

It has been herein presented that a microemulsion, known to be an effective and safe drug delivery system following intravenous administration, can be loaded with traces of [68Ga]Ga-PSMA-617 without losing its properties or causing toxicity. Following tolerated IV injections the capability of the microemulsion in altering [68Ga]Ga-PSMA-617 distribution was presented at 120 min post injection based on its ex vivo biodistribution results.

Evaluation of glucagon-like peptide-1 receptor expression in nondiabetic and diabetic atherosclerotic mice using PET tracer 68Ga-NODAGA-exendin-4.

Cardiovascular effects of glucagon-like peptide-1 receptor (GLP-1R) agonist therapies are potentially mediated by anti-inflammatory effects on atherosclerosis. Our study demonstrates that 68Ga-NODAGA-exendin-4, a radioligand specifically targeting GLP-1R, detects GLP-1R expression in inflamed atherosclerotic lesions in nondiabetic and diabetic hypercholesterolemic mice. Immunofluorescence staining suggests that GLP-1R is primarily localized in M2 macrophages in lesions. This study describes a new potential tool that may have translational relevance for studies of pharmacological modification of GLP-1R signaling in atherosclerosis.

In Vivo Molecular Imaging of the Efficacy of Aminopeptidase N (APN/CD13) Receptor Inhibitor Treatment on Experimental Tumors Using 68Ga-NODAGA-c(NGR) Peptide.

INTRODUCTION: The aminopeptidase N (APN/CD13) receptor plays an important role in the neoangiogenic process and metastatic tumor cell invasion. Clinical and preclinical studies reported that bestatin and actinonin are cytotoxic to APN/CD13-positive tumors and metastases due to their APN/CD13-specific inhibitor properties. Our previous studies have already shown that 68Ga-labeled NGR peptides bind specifically to APN/CD13 expressing tumor cells. The APN/CD13 specificity of 68Ga-NGR radiopharmaceuticals enables the following of the efficacy of antiangiogenic therapy with APN/CD13-specific inhibitors using positron emission tomography (PET). The aim of this in vivo study was to assess the antitumor effect of bestatin and actinonin treatment in subcutaneous transplanted HT1080 and B16-F10 tumor-bearing animal models using 68Ga-NODAGA-c(NGR). MATERIALS AND METHODS: Three days after the inoculation of HT1080 and B16-F10 cells, mice were treated with intraperitoneal injection of bestatin (15 mg/kg) or actinonin (5 mg/kg) for 7 days. On the 5th and 10th day, in vivo PET scans and ex vivo biodistribution studies were performed 90 min after intravenous injection of 5.5 ± 0.2 MBq68Ga-NODAGA-c(NGR). RESULTS: Control-untreated HT1080 and B16-F10 tumors were clearly visualized by the APN/CD13-specific 68Ga-NODAGA-c(NGR) radiopharmaceutical. The western blot analysis also confirmed the strong APN/CD13 positivity in the investigated tumors. We found significantly (p ≤ 0.05) lower radiopharmaceutical uptake after bestatin treatment and higher radiotracer accumulation in the actinonin-treated HT1080 tumors. In contrast, significantly lower (p ≤ 0.01) 68Ga-NODAGA-c(NGR) accumulation was observed in both bestatin- and actinonin-treated B16-F10 melanoma tumors compared to the untreated-control tumors. Bestatin inhibited tumor growth and 68Ga-NODAGA-c(NGR) uptake in both tumor models. CONCLUSION: The bestatin treatment is suitable for suppressing the neoangiogenic process and APN/CD13 expression of experimental HT1080 and B16-F10 tumors; furthermore, 68Ga-NODAGA-c(NGR) is an applicable radiotracer for the in vivo monitoring of the efficacy of the APN/CD13 inhibition-based anticancer therapies.

Novel Peptide-Based PET Probe for Non-invasive Imaging of C-X-C Chemokine Receptor Type 4 (CXCR4) in Tumors.

The recently reported CXCR4 antagonist 3 (Ac-Arg-Ala-[DCys-Arg-2Nal-His-Pen]-CO2H) was investigated as a molecular scaffold for a CXCR4-targeted positron emission tomography (PET) tracer. Toward this end, 3 was functionalized with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-triazacyclononanetriacetic acid (NOTA). On the basis of convincing affinity data, both tracers, [68Ga]NOTA analogue ([68Ga]-5) and [68Ga]DOTA analogue ([68Ga]-4), were evaluated for PET imaging in "in vivo" models of CHO-hCXCR4 and Daudi lymphoma cells. PET imaging and biodistribution studies revealed higher CXCR4-specific tumor uptake and high tumor/background ratios for the [68Ga]NOTA analogue ([68Ga]-5) than for the [68Ga]DOTA analogue ([68Ga]-4) in both in vivo models. Moreover, [68Ga]-4 and [68Ga]-5 displayed rapid clearance and very low levels of accumulation in all nontarget tissues but the kidney. Although the high tumor/background ratios observed in the mouse xenograft model could partially derive from the hCXCR4 selectivity of [68Ga]-5, our results encourage its translation into a clinical context as a novel peptide-based tracer for imaging of CXCR4-overexpressing tumors.

Imaging Tumor-Infiltrating Lymphocytes in Brain Tumors with [64Cu]Cu-NOTA-anti-CD8 PET.

PURPOSE: Glioblastoma (GBM) is the most common malignant brain tumor in adults. Various immunotherapeutic approaches to improve patient survival are being developed, but the molecular mechanisms of immunotherapy resistance are currently unknown. Here, we explored the ability of a humanized radiolabeled CD8-targeted minibody to noninvasively quantify tumor-infiltrating CD8-positive (CD8+) T cells using PET. EXPERIMENTAL DESIGN: We generated a peripheral blood mononuclear cell (PBMC) humanized immune system (HIS) mouse model and quantified the absolute number of CD8+ T cells by flow cytometry relative to the [64Cu]Cu-NOTA-anti-CD8 PET signal. To evaluate a patient-derived orthotopic GBM HIS model, we intracranially injected cells into NOG mice, humanized cohorts with multiple HLA-matched PBMC donors, and quantified CD8+ tumor-infiltrating lymphocytes by IHC. To determine whether [64Cu]Cu-NOTA-anti-CD8 images brain parenchymal T-cell infiltrate in GBM tumors, we performed PET and autoradiography and subsequently stained serial sections of brain tumor tissue by IHC for CD8+ T cells. RESULTS: Nontumor-bearing NOG mice injected with human PBMCs showed prominent [64Cu]Cu-NOTA-anti-CD8 uptake in the spleen and minimal radiotracer localization to the normal brain. NOG mice harboring intracranial human GBMs yielded high-resolution PET images of tumor-infiltrating CD8+ T cells. Radiotracer retention correlated with CD8+ T-cell numbers in spleen and tumor tissue. Our study demonstrates the ability of [64Cu]Cu-NOTA-anti-CD8 PET to quantify peripheral and tumor-infiltrating CD8+ T cells in brain tumors. CONCLUSIONS: Human CD8+ T cells infiltrate an orthotopic GBM in a donor-dependent manner. Furthermore, [64Cu]Cu-NOTA-anti-CD8 quantitatively images both peripheral and brain parenchymal human CD8+ T cells.

Stabilization Strategies for Linear Minigastrin Analogues: Further Improvements via the Inclusion of Proline into the Peptide Sequence.

Minigastrin (MG) analogues, known for their high potential to target cholecystokinin-2 receptor (CCK2R) expressing tumors, have limited clinical applicability due to low enzymatic stability. By introducing site-specific substitutions within the C-terminal receptor-binding sequence, reduced metabolization and improved tumor targeting can be achieved. In this work, the influence of additional modification within the N-terminal sequence has been explored. Three novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated CCK2R ligands with proline substitution at different positions were synthesized. Substitution did not affect CCK2R affinity, and the conjugates labeled with indium-111 and lutetium-177 showed a high enzymatic stability in different incubation media as well as in vivo (57-79% intact radiopeptide in blood of BALB/c mice at 1 h p.i.) combined with enhanced tumor uptake (29-46% IA/g at 4 h in xenografted BALB/c nude mice). The inclusion of Pro contributes significantly to the development of CCK2R ligands with optimal targeting properties for application in targeted radiotherapy.

Facile preparation of a novel Ga-67-labeled NODAGA-conjugated lactam-cyclized alpha-MSH peptide at room temperature for melanoma targeting.

In this study, the melanoma targeting property of 67Ga-NODAGA-GGNle-CycMSHhex {1,4,7-triazacyclononane,1-gluteric acid-4,7-acetic acid-GlyGlyNle-c[Asp-His-D-Phe-Arg-Trp-Lys]-CONH2} was determined on B16/F10 melanoma-bearing C57 mice to demonstrate the feasibility of NODAGA as a radiometal chelator for facile room temperature radiolabeling of NODAGA-GGNle-CycMSHhex. The IC50 value of NODAGA-GGNle-CycMSHhex was 0.87 ± 0.12 nM on B16/F10 melanoma cells. 67Ga-NODAGA-GGNle-CycMSHhex was readily prepared at room temperature with greater than 98% radiolabeling yield and displayed MC1R-specific binding on B16/F10 melanoma cells. The B16/F10 melanoma uptake of 67Ga-NODAGA-GGNle-CycMSHhex was 10.31 ± 0.78, 14.96 ± 1.34, 13.7 ± 3.33 and 10.4 ± 2.2% ID/g at 0.5, 2, 4 and 24 h post-injection, respectively. Approximately 85% of the injected dose was cleared out the body via urinary system at 2 h post-injection. 67Ga-NODAGA-GGNle-CycMSHhex showed high tumor/blood, tumor/muscle and tumor/skin uptake ratios after 2 h post-injection. Overall, 67Ga-NODAGA-GGNle-CycMSHhex could be easily prepared at room temperature and exhibited favorable melanoma targeting property, suggesting the potential use of NODAGA as a radiometal chelator for facile room temperature radiolabeling of α-MSH peptides.

Alpha radioimmunotherapy using 225Ac-proteus-DOTA for solid tumors - safety at curative doses.

This is the initial report of an α-based pre-targeted radioimmunotherapy (PRIT) using 225Ac and its theranostic pair, 111In. We call our novel tumor-targeting DOTA-hapten PRIT system "proteus-DOTA" or "Pr." Herein we report the first results of radiochemistry development, radiopharmacology, and stoichiometry of tumor antigen binding, including the role of specific activity, anti-tumor efficacy, and normal tissue toxicity with the Pr-PRIT approach (as α-DOTA-PRIT). A series of α-DOTA-PRIT therapy studies were performed in three solid human cancer xenograft models of colorectal cancer (GPA33), breast cancer (HER2), and neuroblastoma (GD2), including evaluation of chronic toxicity at ~20 weeks of select survivors. Methods: Preliminary biodistribution experiments in SW1222 tumor-bearing mice revealed that 225Ac could not be efficiently pretargeted with current DOTA-Bn hapten utilized for 177Lu or 90Y, leading to poor tumor uptake in vivo. Therefore, we synthesized Pr consisting of an empty DOTA-chelate for 225Ac, tethered via a short polyethylene glycol linker to a lutetium-complexed DOTA for picomolar anti-DOTA chelate single-chain variable fragment (scFv) binding. Pr was radiolabeled with 225Ac and its imaging surrogate, 111In. In vitro studies verified anti-DOTA scFv recognition of [225Ac]Pr, and in vivo biodistribution and clearance studies were performed to evaluate hapten suitability and in vivo targeting efficiency. Results: Intravenously (i.v.) administered 225Ac- or 111In-radiolabeled Pr in mice showed rapid renal clearance and minimal normal tissue retention. In vivo pretargeting studies show high tumor accumulation of Pr (16.71 ± 5.11 %IA/g or 13.19 ± 3.88 %IA/g at 24 h p.i. for [225Ac]Pr and [111In]Pr, respectively) and relatively low uptake in normal tissues (all average ≤ 1.4 %IA/g at 24 h p.i.). Maximum tolerated dose (MTD) was not reached for either [225Ac]Pr alone or pretargeted [225Ac]Pr at administered activities up to 296 kBq/mouse. Single-cycle treatment consisting of α-DOTA-PRIT with either huA33-C825 bispecific anti-tumor/anti-DOTA-hapten antibody (BsAb), anti-HER2-C825 BsAb, or hu3F8-C825 BsAb for targeting GPA33, HER2, or GD2, respectively, was highly effective. In the GPA33 model, no complete responses (CRs) were observed but prolonged overall survival of treated animals was 42 d for α-DOTA-PRIT vs. 25 d for [225Ac]Pr only (P < 0.0001); for GD2, CRs (7/7, 100%) and histologic cures (4/7, 57%); and for HER2, CRs (7/19, 37%) and histologic cures (10/19, 56%) with no acute or chronic toxicity. Conclusions: [225Ac]Pr and its imaging biomarker [111In]Pr demonstrate optimal radiopharmacologic behavior for theranostic applications of α-DOTA-PRIT. For this initial evaluation of efficacy and toxicity, single-cycle treatment regimens were performed in all three systems. Histologic toxicity was not observed, so MTD was not observed. Prolonged overall survival, CRs, and histologic cures were observed in treated animals. In comparison to RIT with anti-tumor IgG antibodies, [225Ac]Pr has a much improved safety profile. Ultimately, these data will be used to guide clinical development of toxicity and efficacy studies of [225Ac]Pr, with the goal of delivering massive lethal doses of radiation to achieve a high probability of cure without toxicity.

Preparation, Characterization, and Radiolabeling of [68Ga]Ga-NODAGA-Pamidronic Acid: A Potential PET Bone Imaging Agent.

Early diagnosis of bone metastases is crucial to prevent skeletal-related events, and for that, the non-invasive techniques to diagnose bone metastases that make use of image-guided radiopharmaceuticals are being employed as an alternative to traditional biopsies. Hence, in the present work, we tested the efficacy of a gallium-68 (68Ga)-based compound as a radiopharmaceutical agent towards the bone imaging in positron emitting tomography (PET). For that, we prepared, thoroughly characterized, and radiolabeled [68Ga]Ga-NODAGA-pamidronic acid radiopharmaceutical, a 68Ga precursor for PET bone cancer imaging applications. The preparation of NODAGA-pamidronic acid was performed via the N-Hydroxysuccinimide (NHS) ester strategy and was characterized using liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MSn). The unreacted NODAGA chelator was separated using the ion-suppression reverse phase-high performance liquid chromatography (RP-HPLC) method, and the freeze-dried NODAGA-pamidronic acid was radiolabeled with 68Ga. The radiolabeling condition was found to be most optimum at a pH ranging from 4 to 4.5 and a temperature of above 60 °C. From previous work, we found that the pamidronic acid itself has a good bone binding affinity. Moreover, from the analysis of the results, the ionic structure of radiolabeled [68Ga]Ga-NODAGA-pamidronic acid has the ability to improve the blood clearance and may exert good renal excretion, enhance the bone-to-background ratio, and consequently the final image quality. This was reflected by both the in vitro bone binding assay and in vivo animal biodistribution presented in this research.

Evaluation of 111In-DOTA-F56 peptide targeting VEGFR1 for potential non-invasive gastric cancer xenografted tumor mice Micro-SPECT imaging.

Non-invasive imaging of vascular endothelial growth factor receptor 1 (VEGFR1) remains a great challenge in the early diagnosis of tumors, especially in gastric cancer. Here, we designed and evaluated a novel 111In-DOTA-F56 peptide as a radioactive analogue of F56 (peptide WHSDMEWWYLLG) to bind VEGFR1. It was obtained by radiolabeling DOTA-F56 with 111InCl3 with 98% radiochemical purity and 1.4 ± 0.4 GBq/µmol specific activity. 111In-DOTA-F56 was obtained by the reaction of DOTA-F56 (10 µg) with 111InCl3 in pH 4.0 sodium acetate buffer at 85 °C for 20 min. 111In-DOTA-F56 shows good stability in 0.01 M Phosphate Buffered Saline (PBS) and 5% Human Serum Albumin (HSA). 111In-DOTA-F56 has a high binding affinity for human gastric cancer BGC-823 cells. Bio-distribution studies of 111In-DOTA-F56 were performed in nude mice xenografted with human gastric cancer BGC-823 cells and the results revealed tumor uptake accumulation. A blocking dose of DOTA-F56 significantly reduced the tumor uptake of 111In-DOTA-F56. Tumors were observed with Micro-SPECT images, and the uptake in the tumor increased with time from 4 h to 24 h. The MIP of the Micro-SPECT also showed that the excess DOTA-F56 can specifically block 111In-DOTA-F56 in a mouse tumor model. We successfully synthesized the 111In-DOTA-F56 VEGFR1-targeted peptide as a non-invasive molecule with fine radiochemical properties. Micro-SPECT indicates tumor uptake, which can be further blocked by excess of the F56 peptide, indicating that 111In-DOTA-F56 peptide has potential for early detection of VEGFR1 positive gastric cancer and is worthy of further clinical investigations.

Comparison of Various Aryl-Dithiolethiones and Aryl-Dithiolones As Hydrogen Sulfide Donors in the Presence of Rat Liver Microsomes.

It has been reported that microsomal metabolism of ADT (5-(p-methoxyphenyl)-3H-1,2-dithiole-3-thione, anetholedithiolethione, Sulfarlem) and ADO (5-(p-methoxyphenyl)-3H-1,2-dithiole-3-one, anetholedithiolone) led to formation of H2S mainly derived from oxidations catalyzed by cytochrome P450-dependent monooxygenases and that ADO was a better H2S donor than ADT under these conditions. This article compares the H2S donor abilities of 18 dithiolethione and dithiolone analogs of ADT and ADO upon incubation with rat liver microsomes. It shows that, for all the studied compounds, maximal H2S formation was obtained after incubation with microsomes and NADPH and that this formation greatly decreased in the presence of N-benzylimidazole, a known inhibitor of cytochrome P450. This indicates that H2S formation from all the studied compounds requires, as previously observed in the case of ADT and ADO, oxidations catalyzed by cytochrome P450-dependent monooxygenases. Under these conditions, the studied dithiolones were almost always better H2S donors than the corresponding dithiolethiones. Interestingly, the best H2S yields (up to 75%) were observed in microsomal oxidation of ADO and its close analogs, pCl-Ph-DO and Ph-DO, in the presence of glutathione (GSH), whereas only small amounts of H2S were formed in microsomal incubations of those compounds with GSH but in the absence of NADPH. A possible mechanism for this effect of GSH is proposed on the basis of results obtained from reactions of GSH with 5-(p-methoxyphenyl)-3H-1,2-dithiole-3-one-1-sulfoxide, the ADO metabolite involved in H2S formation in microsomal oxidation of ADO. SIGNIFICANCE STATEMENT: A series of 18 dithiolethiones and dithiolones were compared for their ability to form hydrogen sulfide (H2S) in oxidations catalyzed by microsomal monooxygenases. The studied dithiolones were better H2S donors than the corresponding dithiolethiones, and the addition of glutathione to the incubations strongly increased H2S formation. A possible mechanism for this effect of GSH is proposed on the basis of results obtained from reactions of GSH with 5-(p-methoxyphenyl)-3H-1,2-dithiole-3-one-1-sulfoxide, a metabolite of the choleretic and sialologic drug Sulfarlem.

Folate Receptor ß-Targeted PET Imaging of Macrophages in Autoimmune Myocarditis.

Currently available imaging techniques have limited specificity for the detection of active myocardial inflammation. Aluminum 18F-labeled 1,4,7-triazacyclononane-N,N',N″-triacetic acid conjugated folate (18F-FOL) is a PET tracer targeting folate receptor ß (FR-ß), which is expressed on activated macrophages at sites of inflammation. We evaluated 18F-FOL PET for the detection of myocardial inflammation in rats with autoimmune myocarditis and studied the expression of FR-ß in human cardiac sarcoidosis specimens. Methods: Myocarditis was induced by immunizing rats (n = 18) with porcine cardiac myosin in complete Freund adjuvant. Control rats (n = 6) were injected with Freund adjuvant alone. 18F-FOL was intravenously injected, followed by imaging with a small-animal PET/CT scanner and autoradiography. Contrast-enhanced high-resolution CT or 18F-FDG PET images were used for coregistration. Rat tissue sections and myocardial autopsy samples from 6 patients with cardiac sarcoidosis were studied for macrophages and FR-ß. Results: The myocardium of 10 of 18 immunized rats showed focal macrophage-rich inflammatory lesions, with FR-ß expression occurring mainly in M1-polarized macrophages. PET images showed focal myocardial 18F-FOL uptake colocalizing with inflammatory lesions (SUVmean, 2.1 ± 1.1), whereas uptake in the remote myocardium of immunized rats and controls was low (SUVmean, 0.4 ± 0.2 and 0.4 ± 0.1, respectively; P < 0.01). Ex vivo autoradiography of tissue sections confirmed uptake of 18F-FOL in myocardial inflammatory lesions. Uptake of 18F-FOL in inflamed myocardium was efficiently blocked by a nonlabeled FR-ß ligand folate glucosamine in vivo. The myocardium of patients with cardiac sarcoidosis showed many FR-ß-positive macrophages in inflammatory lesions. Conclusion: In a rat model of autoimmune myocarditis, 18F-FOL shows specific uptake in inflamed myocardium containing macrophages expressing FR-ß, which were also present in human cardiac sarcoid lesions. Imaging of FR-ß expression is a potential approach for the detection of active myocardial inflammation.