Early Prediction of Radiation-Induced Pulmonary Fibrosis Using Gastrin-Releasing Peptide Receptor-Targeted PET Imaging.

Early diagnosis of radiation-induced pulmonary fibrosis (RIPF) in lung cancer patients after radiation therapy is important. A gastrin-releasing peptide receptor (GRPR) mediates the inflammation and fibrosis after irradiation in mice lungs. Previously, our group synthesized a GRPR-targeted positron emission tomography (PET) imaging probe, [64Cu]Cu-NODAGA-galacto-bombesin (BBN), an analogue peptide of GRP. In this study, we evaluated the usefulness of [64Cu]Cu-NODAGA-galacto-BBN for the early prediction of RIPF. We prepared RIPF mice and acquired PET/CT images of [18F]F-FDG and [64Cu]Cu-NODAGA-galacto-BBN at 0, 2, 5, and 11 weeks after irradiation (n = 3-10). We confirmed that [64Cu]Cu-NODAGA-galacto-BBN targets GRPR in irradiated RAW 264.7 cells. In addition, we examined whether [64Cu]Cu-NODAGA-galacto-BBN monitors the therapeutic efficacy in RIPF mice (n = 4). As a result, the lung uptake ratio (irradiated-to-normal) of [64Cu]Cu-NODAGA-galacto-BBN was the highest at 2 weeks, followed by its decrease at 5 and 11 weeks after irradiation, which matched with the expression of GRPR and was more accurately predicted than [18F]F-FDG. These uptake results were also confirmed by the cell uptake assay. Furthermore, [64Cu]Cu-NODAGA-galacto-BBN could monitor the therapeutic efficacy of pirfenidone in RIPF mice. We conclude that [64Cu]Cu-NODAGA-galacto-BBN is a novel PET imaging probe for the early prediction of RIPF-targeting GRPR expressed during the inflammatory response.

18 F-labeled 1,2,3-triazole-linked Glu-urea-Lys-based PSMA ligands have good pharmacokinetic properties for positron emission tomography imaging of prostate cancer.

BACKGROUND: Prostate-specific membrane antigen (PSMA) is increasingly recognized as an excellent target for prostate cancer imaging and therapy. Finding compounds with a high target-to-nontarget ratio are an important challenge in the development of positron emission tomography (PET) imaging agents. In this study, we attempted to find a suitable compound from a simply-synthesized compound library. METHOD: 18 F-labeling was achieved in a two-step synthesis consisting of [18 F]fluorination of azido sulfonates followed by copper(I)-catalyzed click ligation. In vitro binding experiment and in vivo studies were carried out using isogenic PSMA+ PC3-PIP and PSMA- PC3-flu cells and 22RV1 cells. [125 I]MIP-1095 was used to measure the binding affinities of compounds through a competitive binding assay, and [18 F]DCFPyL was used for a comparative assessment of compounds. Radiation dosimetry data were obtained using OLINDA/EXM software. RESULTS: Nine novel PSMA ligands were synthesized by the combination of three azido compounds and three terminal acetylene-containing Glu-urea-Lys compounds. Among them, compound 6f having a pyridine moiety showed a high binding affinity of 6.51 ± 0.19 nM (Ki ). 18 F-labeled compounds were obtained at moderate yields within 70 to 75 minutes (including high-performance liquid chromatography purification). Compound [18 F]6c had the lowest log P of -2.693. MicroPET/computed tomography (CT) images were acquired from 22RV1 cell xenograft mice after injecting [18 F]6c, [18 F]6f, and [18 F]6i. Additional microPET/CT experiments of [18 F]6c and [18 F]6f were performed using PSMA+ PC3-PIP and PSMA- PC3-flu cell-bearing mice. [18 F]6c was selected for further studies because it was found to have high uptake in tumors and rapid renal clearance, resulting in great tumor-to-nontumor ratios and distinct tumor images with very low background activity. Human dosimetry estimation of [18 F]6c using OLINDA/EXM software was calculated, resulting in an effective dose of 4.35 × 10-3 mSv/MBq. CONCLUSIONS: [18 F]6c showed significant tumor uptake, a high tumor-to-nontumor ratio, and good radiation dosimetry results, suggesting further development as a potential diagnostic PET agent for prostate cancer.

PEGylated liposome encapsulating nido-carborane showed significant tumor suppression in boron neutron capture therapy (BNCT).

Boron neutron capture therapy (BNCT) is a binary radiotherapy based on nuclear reactions that occur when boron-10 is irradiated with neutrons, which result in the ejection of high-energy alpha particles. Successful BNCT requires the efficient delivery of a boron-containing compound to effect high concentrations in tumor cells while minimizing uptake in normal tissues. In this study, PEGylated liposomes were employed as boron carriers to maximize delivery to tumors and minimize uptake in the reticuloendothelial system (RES). The water-soluble potassium salt of nido-7,8-carborane, nido-carborane, was chosen as the boron source due to its high boron content per molecule. Nido-carborane was encapsulated in the aqueous cores of PEGylated liposomes by hydrating thin lipid films. Repeated freezing and thawing increased nido-carborane loading by up to 47.5 ± 3.1%. The average hydrodynamic diameter of the prepared boronated liposomes was determined to be 114.5 ± 28 nm through dynamic light scattering (DLS) measurement. Globular liposomes approximately 100 nm in diameter were clearly visible in transmission electron microscope (TEM) images. The viability of tumor cells following BNCT with 70 µM nido-carborane was reduced to 17.1% compared to irradiated control cells, which did not contain boronated liposomes. Confocal microscopy revealed that fluorescently labeled liposomes injected into the tail veins of mice were deeply and evenly distributed in tumor tissues and localized in the cytoplasm of tumor cells. When mice were properly shielded with a 12 mm-thick polyethylene board during in-vivo irradiation at a thermal neutron flux of 1.94 × 104/cm2·sec, almost complete tumor suppression was achieved in tumor models injected with boronated liposomes (21.0 mg 10B/kg). Two BNCT cycles spaced 10 days apart further enhanced the therapeutic anti-tumor effect, even when the dose was lowered to 10.5 mg 10B/kg. No notable weight loss was observed in the tumor models during the BNCT study.

Immobilization of the Gas Signaling Molecule H2 S by Radioisotopes: Detection, Quantification, and In Vivo Imaging.

Hydrogen sulfide (H2 S) has multifunctional roles as a gas signaling molecule in living systems. However, the efficient detection and imaging of H2 S in live animals is very challenging. Herein, we report the first radioisotope-based immobilization technique for the detection, quantification, and in vivo imaging of endogenous H2 S. Macrocyclic (64) Cu complexes that instantly reacted with gaseous H2 S to form insoluble (64) CuS in a highly sensitive and selective manner were prepared. The H2 S concentration in biological samples was measured by a thin-layer radiochromatography method. When (64) Cu-cyclen was injected into mice, an elevated H2 S concentration in the inflamed paw was clearly visualized and quantified by Cerenkov luminescence and positron emission tomography (PET) imaging. PET imaging was also able to pinpoint increased H2 S levels in a millimeter-sized infarcted lesion of the rat heart.

Apoptosis imaging studies in various animal models using radio-iodinated peptide.

Apoptosis has a role in many medical disorders and treatments; hence, its non-invasive evaluation is one of the most riveting research topics. Currently annexin V is used as gold standard for imaging apoptosis. However, several drawbacks, including high background, slow body clearance, make it a suboptimum marker for apoptosis imaging. In this study, we radiolabeled the recently identified histone H1 targeting peptide (ApoPep-1) and evaluated its potential as a new apoptosis imaging agent in various animal models. ApoPep-1 (CQRPPR) was synthesized, and an extra tyrosine residue was added to its N-terminal end for radiolabeling. This peptide was radiolabeled with (124)I and (131)I and was tested for its serum stability. Surgery- and drug-induced apoptotic rat models were prepared for apoptosis evaluation, and PET imaging was performed. Doxorubicin was used for xenograft tumor treatment in mice, and the induced apoptosis was studied. Tumor metabolism and proliferation were assessed by [(18)F]FDG and [(18)F]FLT PET imaging and compared with ApoPep-1 after doxorubicin treatment. The peptide was radiolabeled at high purity, and it showed reasonably good stability in serum. Cell death was easily imaged by radiolabeled ApoPep-1 in an ischemia surgery model. And, liver apoptosis was more clearly identified by ApoPep-1 rather than [(124)I]annexin V in cycloheximide-treated models. Three doxorubicin doses inhibited tumor growth, which was evaluated by 30-40% decreases of [(18)F]FDG and [(18)F]FLT PET uptake in the tumor area. However, ApoPep-1 demonstrated more than 200% increase in tumor uptake after chemotherapy, while annexin V did not show any meaningful uptake in the tumor compared with the background. Biodistribution data were also in good agreement with the microPET imaging results. All of the experimental data clearly demonstrated high potential of the radiolabeled ApoPep-1 for in vivo apoptosis imaging.

Synthesis and Evaluation of New Generation Cross-Bridged Bifunctional Chelator for (64)Cu Radiotracers.

Bifunctional chelators have been successfully used to construct (64)Cu-labeled radiopharmaceuticals. Previously reported chelators with cross-bridged cyclam backbones have various essential features such as high stability of the copper(II) complex, high efficiency of radiolabeling at room temperature, and good biological inertness of the radiolabeled complex, along with rapid body clearance. Here, we report a new generation propylene-cross-bridged chelator with hybrid acetate/phosphonate pendant groups (PCB-TE1A1P) developed with the aim of combining these key properties in a single chelator. The PCB-TE1A1P was synthesized from cyclam with good overall yield. The Cu(II) complex of our chelator showed good robustness in kinetic stability evaluation experiments, such as acidic decomplexation and cyclic voltammetry studies. The Cu(II) complex of PCB-TE1A1P remained intact under highly acidic conditions (12 M HCl, 90 °C) for 8 d and showed quasi-reversible reduction/oxidation peaks at -0.77 V in electrochemical studies. PCB-TE1A1P was successfully radiolabeled with (64)Cu ions in an acetate buffer at 60 °C within 60 min. The electrophoresis study revealed that the (64)Cu-PCB-TE1A1P complex has net negative charge in aqueous solution. The biodistribution and in vivo stability study profiles of (64)Cu-PCB-TE1A1P indicated that the radioactive complex was stable under physiological conditions and cleared rapidly from the body. A whole body positron emission tomography (PET) imaging study further confirmed high in vivo stability and fast clearance of the complex in mouse models. In conclusion, PCB-TE1A1P has good potential as a bifunctional chelator for (64)Cu-based radiopharmaceuticals, especially those involving peptides.

In Vivo Fluorescence Molecular Imaging Using Covalent Organic Nanosheets Without Labeling.

Organic nanomaterials, as nanocarrier platforms, have tremendous potential for biomedical applications. The authors successfully prepared novel two-dimensional covalent organic nanosheets (CONs) that can be used as efficient in vivo bioimaging probes by condensing 1,3,5-triformylglucinol (Tp) and 2,7-diaminopyrene (Py) to produce TpPy covalent organic frameworks (COFs). TpPy COFs are then subjected to a liquid exfoliation process to obtain TpPy CONs (< 200 nm in size and < 1.7 nm in thickness). TpPy CONs disperse well in water to provide a stable, homogeneous colloidal suspension, which shows favorable photoluminescence properties. Cell viability tests using MDA-MB-231 and RAW 264.7 cells reveal that TpPy CONs are low in cytotoxicity. Confocal microscopy reveals clear fluorescent cell images after incubation with TpPy CONs for 24 h, without reduction in cell activity or cytosolic aggregation. To investigate the biological behavior of the TpPy CONs, the authors perform an in vivo fluorescence imaging study using MDA-MB-231 tumor-bearing mice. After intravenous injection of TpPy CONs disperse in phosphate-buffered saline (PBS), persistent and strong fluorescence signals are observed in the tumor region, with low background signals from normal tissues at 1, 3, 12, and 24 h after injection. Furthermore, these in vivo imaging results concurred with ex vivo biodistribution and histological results.

Cancer therapy by antibody-targeted Cerenkov light and metabolism-selective photosensitization.

Photodynamic therapy (PDT) is an effective cancer treatment option, but it suffers from penetration limit of light, making it available only for superficial and endoscopically accessible cancers. Recently, there have been reports that Cerenkov luminescence originated from radioisotopes can be utilized as an excitation source for PDT without external light illumination. Here, cancer-selective agents, i.e., (1) clinically available 5-aminolevulinic acid (5-ALA), which promotes cancer metabolism-specific accumulation of protoporphyrin IX (PpIX), and (2) 64Cu-DOTA-trastuzumab, which has HER2-expressing cancer selective uptake, are separately applied as a photosensitizer and an in situ radiator, respectively, to potentiate tumor-specific Cerenkov luminescence energy transfer (CLET) from 64Cu to PpIX for high-precision PDT of cancer. It is shown that the combinational administration and tumor colocalization of 5-ALA and 64Cu-DOTA-trastuzumab exert significant in vitro cytotoxicity (cell viability <9%) as well as in vivo antitumor effects (tumor volume ratio of 0.50 on 14 days post-injection) on HER2-expressing breast and gastric cancer models. This study proves that high-precision treatment regimen using dual-targeted CLET-based PDT is feasible for HER2-expressing cancers. Furthermore, the results offer great potential for clinical translation to the dual-targeted CLET-based PDT because the treatment regimen uses components, 5-ALA and 64Cu-DOTA-trastuzumab, which are already in clinical uses.

Validation of Image Qualities of a Novel Four-Mice Bed PET System as an Oncological and Neurological Analysis Tool.

Background: Micro-positron emission tomography (micro-PET), a small-animal dedicated PET system, is used in biomedical studies and has the quantitative imaging capabilities of radiotracers. A single-bed system, commonly used in micro-PET, is laborious to use in large-scale studies. Here, we evaluated the image qualities of a multi-bed system. Methods: Phantom imaging studies were performed to assess the recovery coefficients (RCs), uniformity, and spill-over ratios (SORs) in water- and air-filled chambers. 18F-FDG and 18F-FPEB PET images of xenograft and normal mice from the multi-bed and single-bed systems were compared. Results: For small diameters (< 3 mm), the RC values between the two systems differed significantly. However, for large diameters (> 4 mm), there were no differences in RC values between the two systems. Uniformity and SORs of both systems were within the tolerance limit of 15%. In the oncological study, the estimation of 18F-FDG uptake in the tumor was significantly lower in the multi-bed system than that in the single-bed system. However, 18F-FDG PET in xenograft mice with tumor size > 4 mm revealed the variation between subjects within the multi-bed system group to be less than 12%. In the neurological study, SUV for the multi-bed group was 25-26% lower than that for the single-bed group; however, inter-object variations within the multi-bed system were below 7%. Conclusions: Although the multi-bed system showed lower estimation of radiotracer uptake than that of the single-bed system, the inter-subject variations were within acceptable limits. Our results indicate that the multi-bed system can be used in oncological and neurological studies.

Imaging Strategy that Achieves Ultrahigh Contrast by Utilizing Differential Esterase Activity in Organs: Application in Early Detection of Pancreatic Cancer.

Most nanoparticles show much higher uptake in mononuclear phagocyte system (MPS) organs than in tumors, which has been a long-lasting dilemma in nanomedicine. Here, we report an imaging strategy that selectively decreases MPS organ uptakes by utilizing the differential esterase activity in tumors and other organs. When an esterase-labile radiotracer loaded liposome was injected into the body, radioactivity was rapidly excreted from the liver and spleen after breakage of the ester bond by esterase. However, the lipophilic radiotracer delivered to the tumor remained in the tumor with minimal bond cleavage. The underlying mechanism was fully characterized in vitro and in vivo in colon tumor models. As a proof of concept, the liposomal radiotracer was further optimized for the early detection of pancreatic cancer. The folate-coated liposomal radiotracer showed highly selective tumor uptake. At 4 h postinjection, a pancreatic tumor a few millimeters in size was unambiguously visualized in orthotopic tumor models by PET imaging. At 24 h, an exceptionally high tumor-to-background ratio was achieved, enabling the visualization of tumors alone with minimal background noise. More than 9% of the total radioactivity was found in the tumor. Utilizing our imaging strategy, various tumor imaging agents can be developed for sensitive detection with ultrahigh contrast.

Evaluation of the Neuroprotective Effect of Microglial Depletion by CSF-1R Inhibition in a Parkinson's Animal Model.

PURPOSE: Neuroinflammation in Parkinson's disease (PD) is known to play a pivotal role in progression to neuronal degeneration. It has been reported that colony-stimulation factor 1 receptor (CSF-1R) inhibition can effectively deplete microglia. However, its therapeutic efficacy in PD is unclear still now. PROCEDURES: To elucidate this issue, we examined the contribution of microglial depletion to PD by behavioral testing, positron emission tomography (PET) imaging, and immunoassays in sham, PD, and microglial depletion PD model (PLX3397 was administered to PD groups, with n = 6 in each group). RESULTS: The microglial depletion in PD model showed improved sensory motor function and depressive-like behavior. NeuroPET revealed that PLX3397 treatment resulted in partial recovery of striatal neuro-inflammatory functions (binding values of [18F]DPA-174 for PD, 1.47 ± 0.12, p < 0.01 vs. for PLX3397 in PD: 1.33 ± 0.26) and the dopaminergic (binding values of 18F-FP-CIT for PD, 1.32 ± 0.07 vs. for PLX3397 in PD: 1.54 ± 0.10, p < 0.01) and glutamatergic systems (binding values of [18F]FPEB for PD: 9.22 ± 0.54 vs. for PLX3397 Tx in PD: 9.83 ± 0.96, p > 0.05). Western blotting for microglia showed similar changes. CONCLUSION: Microglial depletion has inflammation-related therapeutic effects, which have beneficial effects on motor and nonmotor symptoms of PD.

Propylene cross-bridged macrocyclic bifunctional chelator: a new design for facile bioconjugation and robust (64)Cu complex stability.

The first macrocyclic bifunctional chelator incorporating propylene cross-bridge was efficiently synthesized from cyclam in seven steps. After the introduction of an extra functional group for facile conjugation onto the propylene cross-bridge, the two carboxylic acid pendants could contribute to strong coordination of Cu(II) ions, leading to a robust Cu complex. The cyclic RGD peptide conjugate of PCB-TE2A-NCS was prepared and successfully radiolabeled with (64)Cu ion. The radiolabeled peptide conjugate was evaluated in vivo through a biodistribution study and animal PET imaging to demonstrate high tumor uptake with low background.