Formation of Size-Controllable Tetragonal Nanoprisms by Crystallization-Directed Ionic Self-Assembly of Anionic Porphyrin and PEO-Containing Triblock Cationic Copolymer.

The creation of anisotropic nanostructures with precise size control is desirable for new properties and functions, but it is challenging for ionic self-assembly (ISA) because of the non-directional electrostatic interactions. Herein, the formation of size-controllable tetragonal nanoprisms is reported via crystallization-directed ionic self-assembly (CDISA) through evaporating a micellar solution on solid substrates. First, ISA is designed with a crystalline polyethylene oxide (PEO) containing cationic polymer poly(2-(2-guanidinoethoxy)ethyl methacrylate)-b-poly(ethyleneoxide)-b-poly(2-(2-guanidinoethoxy)-ethylmethacrylate) (PGn -PEO230 -PGn ) and an anionic 5,10,15,20-Tetrakis(4-sulfonatophenyl) porphyrin (TPPS) to form micelles in aqueous solution. The PG segments binds excessive TPPS with amplenet chargeto form hydrophilic corona, while the PEO segments are unprecedentedly dehydrated and tightly packed into cores. Upon naturally drying the micellar solution on a silicon wafer, PEO crystallizationdirects the micelles to aggregate into square nanoplates, which are further connected to nanoprisms. Length and width of the nanoprisms can be facilely tuned by varying the initial concentration. In this hierarchical process, the aqueous self-assembly is prerequisite and the water evaporation rate is crucial for the formation of nanostructures, which provides multiple factors for morphology regulating. Such precise size-control strategy is highly expected to provide a new vision for the design of advanced materials with size controllable anisotropic nanostructures.

Ankylosing spondylitis PET imaging and quantifications via P2X7 receptor-targeting radioligand [18F]GSK1482160.

PURPOSE: Ankylosing spondylitis (AS) is a chronic inflammatory disease of the axial spine; however, the quantitative detection of inflammation in AS remains a challenge in clinical settings. We aimed to investigate the feasibility of using a specific P2X7R-targeting 18F-labeled tracer [18F]GSK1482160 for positron emission tomography (PET) imaging and the quantification of AS. METHODS: The radioligand [18F]GSK1482160 was obtained based on nucleophilic aliphatic substitution. Dynamic [18F]GSK1482160 and [18F]FDG micro-PET/CT imaging were performed on AS mice (n = 8) and age-matched controls (n = 8). Tracer kinetics modeling was performed using Logan's graphical arterial input function analysis to quantify the in vivo expression of P2X7R. The post-PET tissues were collected for hematoxylin-eosin (H&E), immunohistochemical (IHC), and immunofluorescence (IF) staining. RESULTS: [18F]GSK1482160 PET/CT imaging revealed that the specific binding in the ankle joint and sacroiliac joint (SIJ) of the AS at 8 weeks group (BPNDankle-AS-8W (non-displaceable binding potential of the ankle) 3.931 ± 0.74; BPND SIJ-AS-8W (BPBD of the SIJ) 4.225 ± 0.84) were significantly higher than the controls at 8 weeks group (BPNDankle-Ctr-8W 0.325 ± 0.15, BPNDSJJ-Ctr-8W 0.319 ± 0.17) respectively, and the AS at 14 weeks group (BPNDankle-AS-14W 12.212 ± 2.25; BPNDSJJ-AS-14W 13.389 ± 3.60) were significantly higher than the controls at 14 weeks group (BPNDankle-Ctr-14W 0.204 ± 0.16, BPNDSJJ-Ctr-14W 0.655 ± 0.35) respectively. The four groups had no significant difference in the [18F]FDG uptake of ankle and SIJ. IHC and IF staining revealed that the overexpression of P2X7R was colocalized with activated macrophages from the ankle synovium and spinal endplate in mice with AS, indicating that quantification of P2X7R may contribute to the understanding of the pathogenesis of inflammation in human AS. CONCLUSION: This study developed a novel P2X7R-targeting PET tracer [18F]GSK1482160 to detect the expression of P2X7R in AS mouse models and provided powerful non-invasive PET imaging and quantification for AS.

Application of Dynamic 18F-FDG PET/CT for Distinguishing Intrapulmonary Metastases from Synchronous Multiple Primary Lung Cancer.

It has been a big challenge to distinguish synchronous multiple primary lung cancer (sMPLC) from primary lung cancer with intrapulmonary metastases (IPM). We aimed to assess the clinical application of dynamic 18F-FDG PET/CT in patients with multiple lung cancer nodules. We enrolled patients with multiple pulmonary nodules who had undergone dynamic 18F-FDG PET/CT and divided them into sMPLC and IPM groups based on comprehensive features. The SUVmax, fitted K i value based on dynamic scanning, and corresponding maximum diameter (D max) from the two largest tumors were determined in each patient. We determined the absolute between-tumor difference of SUVmax/D max and K i /D max (ΔSUVmax/D max; ΔK i /D max) and assessed the between-group differences. Further, the diagnostic accuracy was evaluated by ROC analysis and the correlation between ΔSUVmax/D max and ΔK i /Dmax from all groups was determined. There was no significant difference for ΔSUVmax/D max between the IPM and sMPLC groups, while the IPM group had a significantly higher ΔK i /Dmax than the sMPLC group. The AUC of ΔK i /D max for differentiating sMPLC from IPM was 0.80 (cut-off value of K i = 0.0059, sensitivity 79%, specificity 75%, p < 0.001). There was a good correlation (Pearson r = 0.91, 95% CI: 0.79-0.96, p < 0.0001) between ΔSUVmax/D max and ΔK i /D max in the IPM group but not in the sMPLC group (Pearson r = 0.45, p > 0.05). Dynamic 18F-FDG PET/CT could be a useful tool for distinguishing sMPLC from IPM. K i calculation based on Patlak graphic analysis could be more sensitive than SUVmax in discriminating IPM from sMPLC in patients with multiple lung cancer nodules.

A Positron Emission Tomography Tracer Targeting the S2 Subunit of SARS-CoV-2 in Extrapulmonary Infections.

Tracking the pathogen of coronavirus disease 2019 (COVID-19) in live subjects may help estimate the spatiotemporal distribution of SARS-CoV-2 infection in vivo. This study developed a positron emission tomography (PET) tracer of the S2 subunit of spike (S) protein for imaging SARS-CoV-2. A pan-coronavirus inhibitor, EK1 peptide, was synthesized and radiolabeled with copper-64 after being conjugated with 1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid (NOTA). The in vitro stability tests indicated that [64Cu]Cu-NOTA-EK1 was stable up to 24 h both in saline and in human serum. The binding assay showed that [64Cu]Cu-NOTA-EK1 has a nanomolar affinity (Ki = 3.94 ± 0.51 nM) with the S-protein of SARS-CoV-2. The cell uptake evaluation used HEK293T/S+ and HEK293T/S- cell lines that showed that the tracer has a high affinity with the S-protein on the cellular level. For the in vivo study, we tested [64Cu]Cu-NOTA-EK1 in HEK293T/S+ cell xenograft-bearing mice (n = 3) and pseudovirus of SARS-CoV-2-infected HEK293T/ACE2 cell bearing mice (n = 3). The best radioactive xenograft-to-muscle ratio (X/Nxenograft 8.04 ± 0.99, X/Npseudovirus 6.47 ± 0.71) was most evident 4 h postinjection. Finally, PET imaging in the surrogate mouse model of beta-coronavirus, mouse hepatic virus-A59 infection in C57BL/6 J mice showed significantly enhanced accumulation in the liver than in the uninfected mice (1.626 ± 0.136 vs 0.871 ± 0.086 %ID/g, n = 3, P < 0.05) at 4 h postinjection. In conclusion, our experimental results demonstrate that [64Cu]Cu-NOTA-EK1 is a potential molecular imaging probe for tracking SARS-CoV-2 in extrapulmonary infections in living subjects.

Hetero-aryl bromide precursor fluorine-18 radiosynthesis and preclinical evaluation of a novel positron emission tomography (PET) tracer [18F]GSK1482160.

The purinergic P2X7 receptor (P2X7R), an ATP gated ion channel, is an important therapeutic target for various inflammatory immune and neurodegenerative diseases. A novel P2X7R targeting radiotracer GSK1482160 was radiosynthesized by hetero-aryl bromides precursor 10 with [18F]Et4NF, 20-30 % radiochemical yield, > 68 GBq/µmol specific activity, >98 % radiochemical purity. Evaluation in healthy male Sprague-Dawley rats revealed that [18F]GSK1482160 ([18F]11) was stably retained 87.81 %, 72.45 %, and 56.32 % in brain, blood and liver respectively 60-min post-injection. Ex-vivo biodistribution of [18F]11 proved that it was able to target the P2X7R in vivo and there was no defluorination in the major organs. PET/MRI imaging and autoradiography revealed that [18F]11 was able to penetrate the blood-brain barrier (BBB) and to be a promising P2X7R PET radioligand for clinical translation.

The imaging quantification of multiple organs by dynamic 18F-FDG PET/CT in discharged COVID-19 patients: A prospective pilot study.

Purpose: To early identify abnormal lesions by applying the 18F-FDG PET dynamic modeling approach for discharged patients recovering from COVID-19. Methods: Seven discharged COVID-19 patients (COVID-19 group), twelve healthy volunteers (control group 1), and eight cancer patients with normal pulmonary function (control group 2) were prospectively enrolled. Control group 1 completed static 18F-FDG PET/CT only; COVID-19 group and control group 2 completed 60-min dynamic 18F-FDG PET/CT. Among COVID-19 group and control group 2, the uptake of FDG on the last frame (at 55-60 min) of dynamic scans was used for static analysis. Prior to performing scans, COVID-19 patients provided negative real-time Reverse Transcription-Polymerase Chain Reaction (rRT-PCR) of SARS-CoV-2, normal lung functions test, and normal laboratory test. Organ-to-liver standard uptake ratio (OLR, i.e. SUVmax evaluated organ/ SUVmax liver) from conventional static data and Patlak analysis based on the dynamic modeling to calculate the 18F-FDG net uptake rate constant (Ki) were performed. Results: Compared to the control groups, COVID-19 patients at two to three months after discharge still maintained significantly higher Ki values in multiple organs (including lung, bone marrow, lymph nodes, myocardium and liver), although results for regular OLR measurements were normal for all discharged COVID-19 patients. Taking the image of lung as an example, the differences of SUVmax images between COVID-19 group and control group were hard to distinguish. In contrast, a high 18F-FDG signal of the lung among the COVID-19 group was observed for Ki images. Conclusion: The Ki from 18F-FDG PET/CT dynamic imaging quantification might contribute to identifying residual lesions for COVID-19 survivors. Trial registration: The trial is registered with ClinicalTrials.gov, number NCT04519255 (IRB-approved number, K52-1).

Loss-of-function Mutations K11E or E271K Lead to Novel Tumor Suppression, Implicate Nucleolar Helicase DDX24 Oncogenicity.

Purpose: Mutations (K11E or E271K) of DEAD-box RNA helicase 24 (DDX24) were related to multi-organ venous lymphatic malformation syndrome (MOVLD). However, the relationship between these mutations and DDX24-function still remains unknown. Understanding whether K11E and E271K cause "loss-of-function" or "gain-of-function" for DDX24 is significant for related diseases. DDX24 was reported to be related to tumors closely, thus this study aims to explore how K11E and E271K affect DDX24-function in tumor proliferation. Methods: Cell lines stably expressing wild-type DDX24, K11E-DDX24, E271K-DDX24, along with vector only based on Chinese hamster ovary cells (CHO) and Balb/c tumor-bearing mice models were constructed. Then immunofluorescence staining, proliferation assay and colony formation assay in vitro and 18F-FDG PET/CT-scan were performed. Finally, the tumor tissues were collected to perform transcriptome sequencing to predict the potential mechanism. Results: Contrasted with CHO-WT-DDX24, CHO-K11E-DDX24 or CHO-E271K-DDX24 showed a decreased number of nucleoli, a slower proliferation rate and a lower colony formation rate significantly. Moreover, mice, inoculated with CHO-K11E-DDX24 or CHO-E271K-DDX24 cells, showed lower tumor formation rate, slower tumor growth rate, better prognosis, reduced standard uptake value and Ki of glucose in subcutaneous tumors. Sequencing indicated CHO-K11E-DDX24 or CHO-E271K-DDX24 caused increasing expression of TNF or chemokines and alteration in immune-related signal pathways. Conclusion: K11E or E271K mutation could lead to "loss-of-function" of DDX24 in cell proliferation and tumor bearing mice, which may be acted by non-specific immune killing to inhibit tumor growth.

[Effect of Notch1 on extracellular matrix deposition in the renal tubulointerstitium of diabetes].

The aim of the present study was to observe the effects of Notch1 and autophagy on extracellular matrix deposition in renal tubulointerstitium of diabetes and to explore the mechanism. The mice were randomly divided into normal control group (db/m mice) and diabetes group (db/db mice). After 12 weeks of feeding, the mice were sacrificed and the corresponding biochemical indexes were measured. Rat renal tubular epithelial cells NRK52E were cultured under normal glucose (NG) and high glucose (HG) respectively, and the expression of Notch1 and LC3 proteins were detected by Western blotting. Autophagosomes in NRK52E cells with overexpressed and knockdown Notch1 under NG and HG conditions were observed by confocal microscope, and the expression changes of Notch1, Collagen-I and III protein were detected by immunofluorescence. The results showed that the Notch1 and Collagen-III expressions were increased (P < 0.01) and the LC3 expression was decreased (P < 0.05) in db/db mice compared with db/m mice. In vitro, the Notch1 was increased (P < 0.01) and the LC3 expression was decreased significantly (P < 0.01) in NRK52E cells of HG group compared with NG group. There was no significant change of Notch1 and LC3 expression between the mannitol (MA) group and the NG group. Autophagy was decreased and extracellular matrix deposition was aggravated when Notch1 was overexpressed. In contrast, autophagy was increased and extracellular matrix deposition was relieved by knockdown of Notch1 under HG conditions. In conclusion, Notch1 protein expression was increased and autophagy was reduced in renal tissue of diabetes and renal tubular epithelial cells under HG. The extracellular matrix deposition in the renal tubulointerstitium was relieved by regulating autophagy after the knockdown of Notch1.

Preclinical Evaluation of [64Cu]NOTA-CP01 as a PET Imaging Agent for Metastatic Esophageal Squamous Cell Carcinoma.

Targeting metastatic esophageal squamous cell carcinoma (ESCC) has been a challenge in clinical practice. Emerging evidence demonstrates that C-X-C chemokine receptor 4 (CXCR4) highly expresses in ESCC and plays a pivotal role in the process of tumor metastasis. We developed a copper-64 (t1/2 = 12.7 h, 19% beta+) labeling route of NOTA-CP01 derived from LY2510924, a cyclopeptide-based CXCR4 potent antagonist, in an attempt to noninvasively visualize CXCR4 expression in metastatic ESCC. Precursor NOTA-CP01 was designed by modifying the C-terminus of LY2510925 with bis-t-butyl NOTA via a butane-1,4-diamine linker. The radiolabeling process was finished within 15 min with high radiochemical yield (>95%), radiochemical purity (>99%), and specific activity (10.5-21 GBq/µmol) (non-decay-corrected). The in vitro solubility and stability tests revealed that [64Cu]NOTA-CP01 had a high water solubility (log P = -3.44 ± 0.12, n = 5) and high stability in saline and fetal bovine serum. [64Cu]NOTA-CP01 exhibited CXCR4-specific binding with a nanomolar affinity (IC50 = 1.61 ± 0.96 nM, Kd = 0.272 ± 0.14 nM) similar to that of the parental LY2510924. The in vitro cell uptake assay indicated that the [64Cu]NOTA-CP01-selective accumulation in EC109 cells was CXCR4-specific. Molecular docking of the CXCR4/NOTA-CP01 complex suggested that the Lys, Arg, and NOTA of this ligand have a strong polar interaction with the key residues of CXCR4, which explains the tight affinity of [64Cu]NOTA-CP01 for CXCR4. To test the target engagement in vivo, prolonged-time positron emission computed tomography (PET) imaging was performed at 0.5, 4, 6, 8, 12, 16, and 24 h postinjection of [64Cu]NOTA-CP01 to the EC109 tumor-bearing mice. The EC109 tumors were most visible with high contrast to the contralateral background at 6 h postinjection. The tracer revealed receptor-specific tumor accumulation, which was illustrated by effective blocking via coinjection with a blocking dose of LY2510924. Quantification analysis of the prolonged-time images showed that there was obvious radioactivity accumulation in the tumor (1.27 ± 0.19%ID/g) with the best tumor-to-blood ratio (4.79 ± 0.06) and tumor-to-muscle ratio (15.44 ± 2.94) at 6 h postinjection of the probe. The immunofluorescence and immunohistochemistry confirmed the positive expression of CXCR4 in the EC109 tumor and ESCC and metastatic lymph nodes of patients, respectively. We concluded that [64Cu]NOTA-CP01 possessed a very high target engagement for CXCR4-positive ESCC and could be a potential candidate in the clinical detection of metastatic ESCC.

Influx rate constant of 18F-FDG increases in metastatic lymph nodes of non-small cell lung cancer patients.

PURPOSE: Primary tumor (PT) and metastatic lymph node (MLN) status have a great influence on diagnosis and treatment of lung cancer. Our main purpose was to investigate the imaging characteristics of PT or MLN by applying the 18F-FDG PET dynamic modeling approach for non-small cell lung cancer (NSCLC). METHODS: Dynamic 18F-FDG PET scans were performed for 76 lung cancer patients, and 62 NSCLC cases were finally included in this study: 37 with newly diagnosed early and locally advanced lung cancer without distant metastases (group M0) and 25 metastatic lung cancer (group M1). Patlak graphic analysis (Ki calculation) based on the dynamic modeling and SUV analysis from conventional static data were performed. RESULTS: For PT, both KiPT (0.050 ± 0.005 vs 0.026 ± 0.004 min-1, p < 0.001) and SUVPT (8.41 ± 0.64 vs 5.23 ± 0.73, p < 0.01) showed significant higher values in group M1 than M0. For MLN, KiMLN showed significant higher values in M1 than M0 (0.033 ± 0.005 vs 0.016 ± 0.003 min-1, p < 0.01), while no significant differences were found for SUVMLN between M0 and M1 (4.22 ± 0.49 vs 5.57 ± 0.59, p > 0.05). Both SUV PT and KiPT showed significant high values in squamous cell carcinoma than adenocarcinoma, but neither SUVPT nor KiPT showed significant differences between EGFR mutants versus wild types. The overall Spearman analysis for SUV and Ki from different groups showed variable correlation (r = 0.46-0.94). CONCLUSION: The dynamic modeling for MLN (KiMLN) showed more sensitive than the static analysis (SUV) to detect metastatic lymph nodes in NSCLC, although both methods were sensitive for PT. This methodology of non-invasive imaging may become an important tool to evaluate MLN and PT status for patients who cannot undergo histological examination. CLINICAL TRIAL REGISTRATION: The clinical trial registration number is NCT03679936 (http://www.clinicaltrials.gov/).

Hydration-Facilitated Fine-Tuning of the AIE Amphiphile Color and Application as Erasable Materials with Hot/Cold Dual Writing Modes.

Hydration water greatly impacts the color of inorganic crystals, but it is still unknown whether hydration water can be utilized to systematically manipulate the emission color of organic luminescent groups. Now, metal ions with different hydration ability allow fine-tuning the emission color of a fluorescent group displaying aggregation induced emission (AIE). Because the hydration water can be removed easily by gentle heating or mechanical grinding and re-gained by solvent fuming, rewritable materials can be fabricated both in the hot-writing and cold-writing modes. This hydration-facilitated strategy will open up a new vista in fine-tuning the emission color of AIE molecules based on one synthesis and in the design of smart luminescent devices.

Early Therapeutic Vaccination Prediction of Hepatocellular Carcinoma via Imaging OX40-Mediated Tumor Infiltrating Lymphocytes.

The overall prognosis for hepatocellular carcinoma (HCC) patients is poor but immunotherapeutic strategies may represent a novel and effective tool for HCC. However, the prediction of the early response for the immunotherapeutic effect of HCC remains a big challenge. We developed a novel near-infrared fluorescence (NIRF) probe (IRDye800-AbOX40) for OX40-targeted imaging. The H22 dual-tumor-bearing mice models were established and treated with CpG ODN intratumoral vaccination. Sixteen hours after vaccination, the mice were injected with the probe via the tail vein and conducted with NIRF imaging. The uptake of this probe in HCC tumors was greatly increased as early as 40 h post vaccination and reached a plateau between 54 and 112 h, while the untreated tumors showed a lower uptake, which was further confirmed by ex vivo imaging and flow cytometry. Immunofluorescence staining identified the colocalization of CD3 and OX40 in the tumor microenvironment. Moreover, immunohistochemistry analysis showed that OX40 expression level on tumor infiltrating lymphocytes (TILs) was associated with the fluorescence signal of the H22 tumors. IRDye800-AbOX40 could be used as a specific NIRF probe for noninvasive imaging of OX40 expression on TILs, which may aid in predicting the early response to immunotherapy of HCC.

Kinetic modeling of [18 F]VAT, a novel radioligand for positron emission tomography imaging vesicular acetylcholine transporter in non-human primate brain.

Molecular imaging of vesicular acetylcholine transporter (VAChT) in the brain provides an important cholinergic biomarker for the pathophysiology and treatment of dementias including Alzheimer's disease. In this study, kinetics modeling methods were applied and compared for quantifying regional brain uptake of the VAChT-specific positron emission tomography radiotracer, ((-)-(1-(-8-(2-fluoroethoxy)-3-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)piperidin-4-yl)(4-fluorophenyl)-methanone) ([18 F]VAT) in macaques. Total volume distribution (VT ) estimates were compared for one-tissue compartment model (1TCM), two-tissue compartment model (2TCM), Logan graphic analysis (LoganAIF) and multiple linear analysis (MA1) with arterial blood input function using data from three macaques. Using the cerebellum-hemispheres as the reference region with data from seven macaques, three additional models were compared: reference tissue model (RTM), simplified RTM (SRTM), and Logan graphic analysis (LoganREF). Model selection criterion indicated that a) 2TCM and SRTM were the most appropriate kinetics models for [18 F]VAT; and b) SRTM was strongly correlated with 2TCM (Pearson's coefficients r > 0.93, p < 0.05). Test-retest studies demonstrated that [18 F]VAT has good reproducibility and reliability (TRV < 10%, ICC > 0.72). These studies demonstrate [18 F]VAT is a promising VAChT positron emission tomography tracer for quantitative assessment of VAChT levels in the brain of living subjects.

Noninvasive Detection of HER2 Expression in Gastric Cancer by 64Cu-NOTA-Trastuzumab in PDX Mouse Model and in Patients.

The purpose of this study was to establish the quality control and quantify the novel 64Cu-NOTA-Trastuzumab in gastric cancer patient-derived xenografts (PDX) mice models and patients by applying the molecular imaging technique. Trastuzumab was labeled with 64Cu using NCS-Bz-NOTA as bifunctional chelator, and hIgG1 was labeled with the same procedures as a negative control agent. HER2-positive (case 176, n = 12) and HER2-negative (case 168, n = 3) PDX models were established and validated by Western blot, DNA amplification, and immunohistochemistry (IHC). Both models were conducted for micro-PET imaging by tail injection of 18.5 MBq of 64Cu-NOTA-Trastuzumab or 64Cu-NOTA-hIgG1. Radioprobe uptake in tumor and main organs was quantified by region of interested (ROI) analysis of the micro-PET images and autoradiography. Finally, gastric cancer patients were enrolled in preliminary 64Cu-NOTA-Trastuzumab PET/CT scans. NOTA-Trastuzumab was efficiently radiolabeled with 64Cu over a 99% radiochemical purity and 17.5 GBq/µmol specific activity. The immune activity was preserved as the nonmodified antibody, and the radiopharmaceutical proved to be stable for up to 5 half-decay lives of 64Cu both in vitro and in vivo. Two serials of PDX gastric cancer models were successfully established: case 176 for HER2 positive and case 168 for HER2 negative. In micro-PET imaging studies, 64Cu-NOTA-Trastuzumab exhibits a significant higher tumor uptake (11.45 ± 0.42 ID%/g) compared with 64Cu-NOTA-IgG1 (3.25 ± 0.28 ID%/g, n = 5, p = 0.0004) at 36 h after intravenous injection. Lower level uptake of 64Cu-NOTA-Trastuzumab (6.35 ± 0.48 ID%/g) in HER2-negative PDX tumor models further confirmed specific binding of the radioprobe. Interestingly, the coinjection of 2.0 mg of Trastuzumab (15.52 ± 1.97 ID%/g) or 2.0 mg of hIgG1 (15.64 ± 3.54 ID%/g) increased the 64Cu-NOTA-Trastuzumab tumor uptake in PDX tumor (HER2+) models compared with 64Cu-NOTA-Trastuzumab alone ( p < 0.05) at 36 h postinjection. There were good correlations between micro-PET images and IHC ( n = 4) and autoradiography in PDX (HER2+) tumor tissues. Therefore, 64Cu-NOTA-Trastuzumab successfully translated to clinical PET imaging, and 64Cu-NOTA-Trastuzumab PET/CT scan in gastric cancer patients showed good detection ability. In conclusion, we reported quality control and application of novel 64Cu-NOTA-Trastuzumab for HER2 expression in PDX gastric cancer mice models and gastric cancer patients. Moreover, 64Cu-NOTA-Trastuzumab holds great potential for noninvasive PET detection, staging, and follow-up of HER2 expression in gastric cancer.

PET Study of Sphingosine-1-Phosphate Receptor 1 Expression in Response to Vascular Inflammation in a Rat Model of Carotid Injury.

Sphingosine-1-phosphate receptor (S1PR) activation plays a key role in vascular inflammatory response. Here, we report in vivo validation of [11C]TZ3321, a potent S1PR1 radioligand, for imaging vascular inflammation in a rat model of carotid injury. The right common carotid artery of male adult Sprague-Dawley rats was injured by balloon overinflation that denuded the endothelium and distended the vessel wall. Animals received a 60-minute micro-positron emission tomography (micro PET) scan with [11C]TZ3321 at 72 hours after injury. Ex vivo autoradiography was also conducted. The expression and cellular location of S1PR1 were examined by immunohistological analysis. Three-dimensional (3D) reconstruction of the first 100-second microPET/computed tomography (CT) image indicated the location of bilateral common carotid arteries. [11C]TZ3321 displayed significantly higher accumulation (standardized uptake values: 0.93 ± 0.07 vs 0.78 ± 0.09, n = 6, P = .001) in the injured carotid artery than in the contralateral side. Increased tracer uptake in the injured artery was confirmed by autoradiography (photostimulated luminescence measures: 85.5 ± 0.93 vs 71.48 ± 6.22, n = 2). Concordantly, high S1PR1expression was observed in infiltrated inflammatory cells in the injured artery. Our studies demonstrate [11C]TZ3321 microPET is able to detect the acute upregulation of S1PR1 expression in inflamed carotid artery. Therefore, [11C]TZ3321 has potential to be a PET radiotracer for detecting early inflammatory response and monitoring therapeutic efficacy of vascular inflammation.

A promising carbon-11-labeled sphingosine-1-phosphate receptor 1-specific PET tracer for imaging vascular injury.

BACKGROUND: Sphingosine-1-phosphate receptor 1 (S1PR1) is highly expressed in vascular smooth muscle cells from intimal lesions. PET imaging using S1PR1 as a biomarker would increase our understanding of its role in vascular pathologies including in-stent restenosis. METHODS: The S1PR1 compound TZ3321 was synthesized for in vitro characterization and labeled with Carbon-11 for in vivo studies. The biodistribution of [11C]TZ3321 was evaluated in normal mice; microPET and immunohistochemistry (IHC) studies were performed using a murine femoral artery wire-injury model of restenosis. RESULTS: The high potency of TZ3321 for S1PR1 (IC 50 = 2.13 ± 1.63 nM), and high selectivity (>1000 nM) for S1PR1 over S1PR2 and S1PR3 were confirmed. Biodistribution data revealed prolonged retention of [11C]TZ3321 in S1PR1-enriched tissues. MicroPET imaging of [11C]TZ3321 showed higher uptake in the wire-injured arteries of ApoE-/- mice than in injured arteries of wild-type mice (SUV 0.40 ± 0.06 vs 0.28 ± 0.04, n = 6, P < .001); FDG-PET showed no difference (SUV 0.98 ± 0.04 vs 0.94 ± 0.01, n = 6, P > .05). Post-PET autoradiography showed >4-fold higher [11C]TZ3321 retention in the injured artery of ApoE-/- mice than in wild-type mice. Subsequent IHC staining confirmed higher expression of S1PR1 in the neointima of the injured artery of ApoE-/- mice than in wild-type mice. CONCLUSIONS: This preliminary study supports the potential use of PET for quantification of the S1PR1 expression as a biomarker of neointimal hyperplasia.

Synthesis, resolution, and in vitro evaluation of three vesicular acetylcholine transporter ligands and evaluation of the lead fluorine-18 radioligand in a nonhuman primate.

The vesicular acetylcholine transporter (VAChT) is a reliable biomarker for assessing cholinergic dysfunction associated with dementia. We recently reported three new potent and selective carbon-11 labeled VAChT radiotracers. Herein, we report the resolution with a Chiralcel OD column of three additional fluorine containing VAChT ligands in which a fluoroethoxy or fluoroethylamino moiety was substituted for the methoxy group. An in vitro competitive binding assay showed that (-)-7 had high potency for VAChT (Ki-VAChT = 0.31 ± 0.03 nM) and excellent selectivity for VAChT versus σ receptors (Ki-σ1 = 1870 ± 250 nM, Ki-σ2 = 5480 ± 140 nM). Three different radiolabeling approaches were explored; the radiosynthesis of (-)-[18F]7 was successfully accomplished via a stepwise two-pot, three-step method with moderate yield (11 ± 2%) and high radiochemical purity (>98%). PET imaging studies in a nonhuman primate indicated that (-)-[18F]7 rapidly entered the brain and accumulated in the VAChT-enriched striatum. The uptake of (-)-[18F]7 in the target striatal area peaked at 10 min and displayed improved clearance kinetics compared to the VAChT tracer [18F]VAT, which has been approved by the Food and Drug Administration (FDA) for first-in-man studies. These studies justify further investigation of (-)-[18F]7 and exploration of the structure-activity relationships of these fluoroethoxy and fluoroethylamino analogs.

Chiral resolution of serial potent and selective σ1 ligands and biological evaluation of (-)-[18F]TZ3108 in rodent and the nonhuman primate brain.

Twelve optically pure enantiomers were obtained using either crystallization or chiral high performance liquid chromatography (HPLC) separation methodologies to resolve six racemic sigma-1 (σ1) receptor ligands. The in vitro binding affinities of each enantiomer for σ1, σ2 receptors and vesicular acetylcholine transporter (VAChT) were determined. Out of the 12 optically pure enantiomers, five displayed very high affinities for σ1 (Ki<2nM) and high selectivity for σ1 versus σ2 and VAChT (>100-fold). The minus enantiomer, (-)-14a ((-)-TZ3108) (Ki-σ1=1.8±0.4nM, Ki-σ2=6960±810nM, Ki-VAChT=980±87nM), was chosen for radiolabeling and further in vivo evaluation in rodents and nonhuman primates (NHPs). A biodistribution study in Sprague Dawley rats showed brain uptake (%ID/gram) of (-)-[18F]TZ3108 reached 1.285±0.062 at 5min and 0.802±0.129 at 120min. NHP microPET imaging studies revealed higher brain uptake of (-)-[18F]TZ3108 and more favorable pharmacokinetics compared to its racemic counterpart. Pretreatment of the animal using two structurally different σ1 ligands significantly decreased accumulation of (-)-[18F]TZ3108 in the brain. Together, our in vivo evaluation results suggest that (-)-[18F]TZ3108 is a promising positron emission tomography (PET) tracer for quantifying σ1 receptor in the brain.

Preclinical evaluation of a promising C-11 labeled PET tracer for imaging phosphodiesterase 10A in the brain of living subject.

Phosphodiesterase 10A (PDE10A) plays a key role in the regulation of brain striatal signaling. A PET tracer for PDE10A may serve as a tool to evaluate PDE10A expression in vivo in central nervous system disorders with striatal pathology. Here, we further characterized the binding properties of a previously reported radioligand we developed for PDE10A, [(11)C]TZ1964B, in rodents and nonhuman primates (NHPs). The tritiated counterpart [(3)H]TZ1964B was used for in vitro binding characterizations in rat striatum homogenates and in vitro autoradiographic studies in rat brain slices. The carbon-11 labeled [(11)C]TZ1964B was utilized in the ex vivo autoradiography studies for the brain of rats and microPET imaging studies for the brain of NHPs. MicroPET scans of [(11)C]TZ1964B in NHPs were conducted at baseline, as well as with using a selective PDE10A inhibitor MP-10 for either pretreatment or displacement. The in vivo regional target occupancy (Occ) was obtained by pretreating with different doses of MP-10 (0.05-2.00 mg/kg). Both in vitro binding assays and in vitro autoradiographic studies revealed a nanomolar binding affinity of [(3)H]TZ1964B to the rat striatum. The striatal binding of [(3)H]TZ1964B and [(11)C]TZ1964B was either displaced or blocked by MP-10 in rats and NHPs. Autoradiography and microPET imaging confirmed that the specific binding of the radioligand was found in the striatum but not in the cerebellum. Blocking studies also confirmed the suitability of the cerebellum as an appropriate reference region. The binding potentials (BPND) of [(11)C]TZ1964B in the NHP striatum that were calculated using either the Logan reference model (LoganREF, 3.96 ± 0.17) or the simplified reference tissue model (SRTM, 4.64 ± 0.47), with the cerebellum as the reference region, was high and had good reproducibility. The occupancy studies indicated a MP-10 dose of 0.31 ± 0.09 mg/kg (LoganREF)/0.45 ± 0.17mg/kg (SRTM) occupies 50% striatal PDE10A binding sites. Studies in rats and NHPs demonstrated radiolabeled TZ1964B has a high binding affinity and good specificity for PDE10A, as well as favorable in vivo pharmacokinetic properties and binding profiles. Our data suggests that [(11)C]TZ1964B is a promising radioligand for in vivo imaging PDE10A in the brain of living subject.

Synthesis and in vitro characterization of cinnoline and benzimidazole analogues as phosphodiesterase 10A inhibitors.

Fifteen cinnoline analogues and six benzimidazole phosphodiesterase 10A (PDE10A) inhibitors were synthesized as potential PET radiopharmaceuticals and their in vitro activity as PDE10A inhibitors was determined. Nine out of twenty-one compounds were potent inhibitors of PDE10A with IC50 values ranging from 1.5 to 18.6nM. Notably, the IC50 values of compounds 26a, 26b, and 33c were 1.52±0.18, 2.86±0.10, and 3.73±0.60nM, respectively; these three compounds also showed high in vitro selectivity (>1000-fold) for PDE10A over PDE 3A/3B, PDE4A/4B. The high potency and selectivity of these three compounds suggests that they could be radiolabeled with PET radionuclides for further evaluation of their in vivo pharmacological behavior and ability to quantify PDE10A in the brain.