18F-4V for PET–CT Imaging of VCAM-1 Expression in Atherosclerosis

ObjectivesThe aim of this study was to iteratively develop and validate an 18F-labeled small vascular cell adhesion molecule (VCAM)-1 affinity ligand and demonstrate the feasibility of imaging VCAM-1 expression by positron emission tomography–computed tomography (PET-CT) in murine atherosclerotic arteries.BackgroundHybrid PET-CT imaging allows simultaneous assessment of atherosclerotic lesion morphology (CT) and may facilitate early risk assessment in individual patients. The early induction, confinement of expression to atherosclerotic lesions, and accessible position in proximity to the blood pool render the adhesion molecule VCAM-1 an attractive imaging biomarker for inflamed atheroma prone to complication.MethodsA cyclic, a linear, and an oligomer affinity peptide, internalized into endothelial cells by VCAM-1–mediated binding, were initially derivatized with DOTA to determine their binding profiles and pharmacokinetics. The lead compound was then 18F-labeled and tested in atherosclerotic apoE−/− mice receiving a high-cholesterol diet as well as wild type murine models of myocardial infarction and heart transplant rejection.ResultsThe tetrameric peptide had the highest affinity and specificity for VCAM-1 (97% inhibition with soluble VCAM-1 in vitro). In vivo PET-CT imaging using 18F-4V showed 0.31 ± 0.02 SUV in murine atheroma (ex vivo %IDGT 5.9 ± 1.5). 18F-4V uptake colocalized with atherosclerotic plaques on Oil Red O staining and correlated to mRNA levels of VCAM-1 measured by quantitative reverse transcription polymerase chain reaction (R = 0.79, p = 0.03). Atherosclerotic mice receiving an atorvastatin-enriched diet had significantly lower lesional uptake (p < 0.05). Furthermore, 18F-4V imaging in myocardial ischemia after coronary ligation and in transplanted cardiac allografts undergoing rejection showed high in vivo PET signal in inflamed myocardium and good correlation with ex vivo measurement of VCAM-1 mRNA by quantitative polymerase chain reaction.Conclusions 18F-4V allows noninvasive PET-CT imaging of VCAM-1 in inflammatory atherosclerosis, has the dynamic range to quantify treatment effects, and correlates with inflammatory gene expression

Polymeric Nanoparticle PET/MR Imaging Allows Macrophage Detection in Atherosclerotic Plaques

Author Manuscript 2013 March 02.Rationale: Myeloid cell content in atherosclerotic plaques associates with rupture and thrombosis. Thus, imaging of lesional monocytes and macrophages could serve as a biomarker of disease progression and therapeutic intervention. Objective: To noninvasively assess plaque inflammation with dextran nanoparticle (DNP)-facilitated hybrid positron emission tomography/magnetic resonance imaging (PET/MRI). Methods and Results: Using clinically approved building blocks, we systematically developed 13-nm polymeric nanoparticles consisting of cross-linked short chain dextrans, which were modified with desferoxamine for zirconium-89 radiolabeling ([superscript 89]Zr-DNP) and a near-infrared fluorochrome (VT680) for microscopic and cellular validation. Flow cytometry of cells isolated from excised aortas showed DNP uptake predominantly in monocytes and macrophages (76.7%) and lower signal originating from other leukocytes, such as neutrophils and lymphocytes (11.8% and 0.7%, P<0.05 versus monocytes and macrophages). DNP colocalized with the myeloid cell marker CD11b on immunohistochemistry. PET/MRI revealed high uptake of [superscript 89]Zr-DNP in the aortic root of apolipoprotein E knock out (ApoE[superscript −/−]) mice (standard uptake value, ApoE[superscript −/−] mice versus wild-type controls, 1.9±0.28 versus 1.3±0.03; P<0.05), corroborated by ex vivo scintillation counting and autoradiography. Therapeutic silencing of the monocyte-recruiting receptor C-C chemokine receptor type 2 with short-interfering RNA decreased [superscript 89]Zr-DNP plaque signal (P<0.05) and inflammatory gene expression (P<0.05). Conclusions: Hybrid PET/MRI with a 13-nm DNP enables noninvasive assessment of inflammation in experimental atherosclerotic plaques and reports on therapeutic efficacy of anti-inflammatory therapy.National Heart, Lung, and Blood InstituteNational Institutes of Health (U.S.). Dept. of Health and Human Services (HHSN268201000044C)National Institutes of Health (U.S.). Dept. of Health and Human Services (R01-HL096576)National Institutes of Health (U.S.). Dept. of Health and Human Services (R01-HL095629)National Institutes of Health (U.S.). Dept. of Health and Human Services (T32-HL094301

Monocyte-Directed RNAi Targeting CCR2 Improves Infarct Healing in Atherosclerosis-Prone Mice

Background—Exaggerated and prolonged inflammation after myocardial infarction (MI) accelerates left ventricular remodeling. Inflammatory pathways may present a therapeutic target to prevent post-MI heart failure. However, the appropriate magnitude and timing of interventions are largely unknown, in part because noninvasive monitoring tools are lacking. Here, we used nanoparticle-facilitated silencing of CCR2, the chemokine receptor that governs inflammatory Ly-6Chigh monocyte subset traffic, to reduce infarct inflammation in apolipoprotein E–deficient (apoE−/−) mice after MI. We used dual-target positron emission tomography/magnetic resonance imaging of transglutaminase factor XIII (FXIII) and myeloperoxidase (MPO) activity to monitor how monocyte subset–targeted RNAi altered infarct inflammation and healing. Methods and Results—Flow cytometry, gene expression analysis, and histology revealed reduced monocyte numbers and enhanced resolution of inflammation in infarcted hearts of apoE−/− mice that were treated with nanoparticle-encapsulated siRNA. To follow extracellular matrix cross-linking noninvasively, we developed a fluorine-18–labeled positron emission tomography agent (18F-FXIII). Recruitment of MPO-rich inflammatory leukocytes was imaged with a molecular magnetic resonance imaging sensor of MPO activity (MPO-Gd). Positron emission tomography/magnetic resonance imaging detected anti-inflammatory effects of intravenous nanoparticle-facilitated siRNA therapy (75% decrease of MPO-Gd signal; P<0.05), whereas 18F-FXIII positron emission tomography reflected unimpeded matrix cross-linking in the infarct. Silencing of CCR2 during the first week after MI improved ejection fraction on day 21 after MI from 29% to 35% (P<0.05). Conclusion—CCR2-targeted RNAi reduced recruitment of Ly-6Chigh monocytes, attenuated infarct inflammation, and curbed post-MI left ventricular remodeling.National Heart, Lung, and Blood InstituteUnited States. Dept. of Health and Human Services (contract No. HHSN268201000044C)National Institutes of Health (U.S.) (grant R01-HL096576)National Institutes of Health (U.S.) (grant R01-HL095629)National Institutes of Health (U.S.) (grant T32-HL094301)Deutsche Forschungsgemeinschaft (HE-6382/1-1

Use of 18F-2-Fluorodeoxyglucose to Label Antibody Fragments for Immuno-Positron Emission Tomography of Pancreatic Cancer

We generated 18F-labeled antibody fragments for positron emission tomography (PET) imaging using a sortase-mediated reaction to install a trans-cyclooctene-functionalized short peptide onto proteins of interest, followed by reaction with a tetrazine-labeled-18F-2-deoxyfluoroglucose (FDG). The method is rapid, robust, and site-specific (radiochemical yields > 25%, not decay corrected). The availability of 18F-2-deoxyfluoroglucose avoids the need for more complicated chemistries used to generate carbon–fluorine bonds. We demonstrate the utility of the method by detecting heterotopic pancreatic tumors in mice by PET, using anti-Class II MHC single domain antibodies. We correlate macroscopic PET images with microscopic two-photon visualization of the tumor. Our approach provides easy access to 18F-labeled antibodies and their fragments at a level of molecular specificity that complements conventional 18F-FDG imaging

Synthesis and thermal automerizations of 4- and 6-exo-deuteriobicyclo[3.1.0]hex-2-enes

Samples of racemic 4- exo -deuteriobicyclo[3.1.0]hex-2-ene (4 x - 1 ) and racemic 6- exo -deuteriobicyclo[3.1.0]hex-2-ene (6 x - 1 ) were synthesized with high incorporation and stereoselectivity of deuterium label. Mole percent concentrations as functions of time profiles for the four isomers were secured from the thermolysis of 4 x - 1 at 224.9, 240.1, and 255.0°C. From this data rate constants for the three isomerization paths ( k r , k i , and k 1,5 ) at the three temperatures were calculated. Relative participation of the three reaction paths was determined; the k r :k i :k 1,5 proportions were 48:11:36. The calculated activation parameters for k r , k i , and k 1,5 were Ea 43.8, 44.8, 44.3 kcal/mole and log A 14.1, 14.1, 14.2, respectively. Thermolysis of 6 x - 1 at 224.9, 240.1, and 255.0°C provided mole percent concentration as functions of time profiles that were compared with the profiles obtained with 4 x - 1 . This comparison demonstrates that one-centered epimerization, k 6e , plays no kinetically important role at the temperatures and time domains studied

Imaging Therapeutic PARP Inhibition In Vivo through Bioorthogonally Developed Companion Imaging Agents12

A number of small-molecule poly (ADP-ribose) polymerase (PARP) inhibitors are currently undergoing advanced clinical trials. Determining the distribution and target inhibitory activity of these drugs in individual subjects, however, has proven problematic. Here, we used a PARP agent for positron emission tomography-computed tomography (PET-CT) imaging (18F-BO), which we developed based on the Olaparib scaffold using rapid bioorthogonal conjugation chemistries. We show that the bioorthogonal 18F modification of the parent molecule is simple, highly efficient, and well tolerated, resulting in a half maximal inhibitory concentration (IC50) of 17.9 ± 1.1 nM. Intravital imaging showed ubiquitous distribution of the drug and uptake into cancer cells, with ultimate localization within the nucleus, all of which were inhibitable. Whole-body PET-CT imaging showed tumoral uptake of the drug, which decreased significantly, after a daily dose of Olaparib. Standard 18F-fludeoxyglucose imaging, however, failed to detect such therapy-induced changes. This research represents a step toward developing a more generic approach for the rapid codevelopment of companion imaging agents based on small-molecule therapeutic inhibitors

Characterizing the Interactions of Organic Nanoparticles with Renal Epithelial Cells <i>in Vivo</i>

Nanotechnology approaches are actively being pursued for drug delivery, novel diagnostics, implantable devices, and consumer products. While considerable research has been performed on the effects of these materials on targeted tumor or phagocytic cells, relatively little is known about their effects on renal cells. This becomes critical for supersmall nanoparticles (<10 nm), designed to be renally excreted. The active endocytic machinery of kidney proximal tubules avidly internalizes filtered proteins, which may also be the case for filtered nanoparticles. To test whether such interactions affect kidney function, we injected mice with either 5 nm dextran-based nanoparticles (DNP) that are similar in composition to FDA-approved materials or poly(amido amine) dendrimer nanoparticles (PNP) of comparable size. These fluorescently tagged nanoparticles were both filtered and internalized by renal tubular epithelial cells in a dose- and time-dependent fashion. The biological effects were quantitated by immunocytochemistry, measuring kidney injury markers and performing functional tests. DNP administration resulted in a dose-dependent increase in urinary output, while cellular albumin endocytosis was increased. The expression of megalin, a receptor involved in albumin uptake, was also increased, but AQP1 expression was unaffected. The effects after PNP administration were similar but additionally resulted in increased clathrin expression and increased endocytosis of dextran. We conclude that there are no major detrimental renal effects of DNP on overall kidney function, but changes in endocytosis-mediating protein expression do occur. These studies provide a framework for the testing of additional nanoparticle preparations as they become available

Hybrid PET-optical imaging using targeted probes

Fusion imaging of radionuclide-based molecular (PET) and structural data [x-ray computed tomography (CT)] has been firmly established. Here we show that optical measurements [fluorescence-mediated tomography (FMT)] show exquisite congruence to radionuclide measurements and that information can be seamlessly integrated and visualized. Using biocompatible nanoparticles as a generic platform (containing a 18F isotope and a far red fluorochrome), we show good correlations between FMT and PET in probe concentration (r2 > 0.99) and spatial signal distribution (r2 > 0.85). Using a mouse model of cancer and different imaging probes to measure tumoral proteases, macrophage content and integrin expression simultaneously, we demonstrate the distinct tumoral locations of probes in multiple channels in vivo. The findings also suggest that FMT can serve as a surrogate modality for the screening and development of radionuclide-based imaging agents