Publications by authors named "Sybille Reder"

29 Publications

  • Page 1 of 1

Effective rational humanization of a PASylated anti-galectin-3 Fab for the sensitive PET imaging of thyroid cancer in vivo.

Sci Rep 2021 Apr 1;11(1):7358. Epub 2021 Apr 1.

Lehrstuhl für Biologische Chemie, Technische Universität München, 85354, Freising (Weihenstephan), Germany.

The lack of a non-invasive test for malignant thyroid nodules makes the diagnosis of thyroid cancer (TC) challenging. Human galectin-3 (hGal3) has emerged as a promising target for medical TC imaging and diagnosis because of its exclusive overexpression in malignant thyroid tissues. We previously developed a human-chimeric αhGal3 Fab fragment derived from the rat monoclonal antibody (mAb) M3/38 with optimized clearance characteristics using PASylation technology. Here, we describe the elucidation of the hGal3 epitope recognized by mAb M3/38, X-ray crystallographic analysis of its complex with the chimeric Fab and, based on the three-dimensional structure, the rational humanization of the Fab by CDR grafting. Four CDR-grafted versions were designed using structurally most closely related fully human immunoglobulin V/V regions of which one-employing the acceptor framework regions of the HIV-1 neutralizing human antibody m66-showed the highest antigen affinity. By introducing two additional back-mutations to the rodent donor sequence, an affinity toward hGal3 indistinguishable from the chimeric Fab was achieved (K = 0.34 ± 0.02 nM in SPR). The PASylated humanized Fab was site-specifically labelled with the fluorescent dye Cy7 and applied for the immuno-histochemical staining of human tissue sections representative for different TCs. The same protein was conjugated with the metal chelator Dfo, followed by radiolabelling with Zr(IV). The resulting protein tracer allowed the highly sensitive and specific PET/CT imaging of orthotopic tumors in mice, which was confirmed by quantitative analysis of radiotracer accumulation. Thus, the PASylated humanized αhGal3 Fab offers clinical potential for the diagnostic imaging of TC.
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http://dx.doi.org/10.1038/s41598-021-86641-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016950PMC
April 2021

Development of a Chimeric Antigen-Binding Fragment Directed Against Human Galectin-3 and Validation as an Immuno-Positron Emission Tomography Tracer for the Sensitive Imaging of Thyroid Cancer.

Thyroid 2020 09 30;30(9):1314-1326. Epub 2020 Apr 30.

Klinikum rechts der Isar, Department of Nuclear Medicine, Technical University Munich, Munich, Germany.

The lack of facile methods for the specific characterization of malignant thyroid nodules makes the diagnosis of thyroid cancer (TC) challenging. Due to its restricted expression in such nodules, the cell-associated lectin galectin-3 (Gal3) has emerged as a marker for TC with growing interest for imaging as well as targeted radionuclide therapy. To accelerate translation into clinical application, we have developed a cognate chimeric human antigen-binding fragment (Fab) derived from the rat anti-Gal3 monoclonal antibody M3/38. The variable immunoglobulin (Ig) light and heavy chain sequences were cloned from the hybridoma cell line, and the corresponding Fab carrying human IgG1/κ constant genes was functionally produced in the periplasm of and purified to homogeneity. To moderately prolong its plasma half-life and, thus, increase tumor uptake, the recombinant Fab was fused with a long disordered amino acid chain comprising in total 200 Pro, Ala, and Ser residues (PASylation). This novel tracer was subjected to characterization and validation by using two thyroid cancer orthotopic murine models. To this end, the αGal3-Fab-PAS was conjugated with deferoxamine (Dfo), labeled with Zr under mild conditions and tested for binding on TC cell lines. Athymic nude mice were inoculated either with FRO82-1 or with CAL62 tumor cells into the left thyroid lobe. After intravenous injection with ∼3.0 MBq of Zr-Dfo-PAS-Fab, these mice were subjected to positron emission tomography (PET)/computed tomography imaging followed by quantification of tumor accumulation and immunohistochemical analysis. The αGal3-Fab-PAS revealed high affinity toward the recombinant Gal3 antigen, with a dissociation constant ≤1 nM as measured via enzyme-linked immunosorbent assay, surface plasmon resonance spectroscopy, and radioactive cell binding assay. The Gal3-targeting by the Zr(IV)-labeled protein tracer, as investigated by immuno-PET, demonstrated highly selective and fast accumulation in orthotopically implanted tumors, with strong contrast images achieved 24 hours postinjection, and no uptake in the tumor-free thyroid lobe, as also confirmed by biodistribution studies. The chimeric αGal3 Zr-Dfo-PAS-Fab tracer exhibits selective accumulation in the tumor-bearing thyroid lobe of xenograft mice. Thus, this novel radioactive probe offers potential to change TC management, in addition to current diagnostic procedures, and to reduce unnecessary thyroidectomies.
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http://dx.doi.org/10.1089/thy.2019.0670DOI Listing
September 2020

Development of a high affinity Anticalin directed against human CD98hc for theranostic applications.

Theranostics 2020 12;10(5):2172-2187. Epub 2020 Jan 12.

Lehrstuhl für Biologische Chemie, Technische Universität München, 85354 Freising, Germany.

Enhanced amino acid supply and dysregulated integrin signaling constitute two hallmarks of cancer and are pivotal for metastatic transformation of cells. In line with its function at the crossroads of both processes, overexpression of CD98hc is clinically observed in various cancer malignancies, thus rendering it a promising tumor target. : We describe the development of Anticalin proteins based on the lipocalin 2 (Lcn2) scaffold against the human CD98hc ectodomain (hCD98hcED) using directed evolution and protein design. X-ray structural analysis was performed to identify the epitope recognized by the lead Anticalin candidate. The Anticalin - with a tuned plasma half-life using PASylation technology - was labeled with Zr and investigated by positron emission tomography (PET) of CD98-positive tumor xenograft mice. : The Anticalin P3D11 binds CD98hc with picomolar affinity and recognizes a protruding loop structure surrounded by several glycosylation sites within the solvent exposed membrane-distal part of the hCD98hcED. studies revealed specific binding activity of the Anticalin towards various CD98hc-expressing human tumor cell lines, suggesting broader applicability in cancer research. PET/CT imaging of mice bearing human prostate carcinoma xenografts using the optimized and Zr-labeled Anticalin demonstrated strong and specific tracer accumulation (8.6 ± 1.1 %ID/g) as well as a favorable tumor-to-blood ratio of 11.8. : Our findings provide a first proof of concept to exploit CD98hc for non-invasive biomedical imaging. The novel Anticalin-based αhCD98hc radiopharmaceutical constitutes a promising tool for preclinical and, potentially, clinical applications in oncology.
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http://dx.doi.org/10.7150/thno.38968DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019167PMC
April 2021

Room Temperature Al F Labeling of 2-Aminomethylpiperidine-Based Chelators for PET Imaging.

ChemMedChem 2020 02 7;15(3):284-292. Epub 2020 Jan 7.

Department of Nuclear Medicine, Klinikum rechts der Isar TU München, Ismaningerstraße 22, 81675, Munich, Germany.

Positron emission tomography (PET) is a non-invasive molecular imaging technology that is constantly expanding, with a high demand for specific antibody-derived imaging probes. The use of tracers based on temperature-sensitive molecules (i. e. Fab, svFab, nanobodies) is increasing and has led us to design a class of chelators based on the structure of 2-aminomethylpiperidine (AMP) with acetic and/or hydroxybenzyl pendant arms (2-AMPTA, NHB-2-AMPDA, and 2-AMPDA-HB), which were investigated as such for {Al F} -core chelation efficiency. All the compounds were characterized by HPLC-MS analysis and NMR spectroscopy. The AlF-18 labeling reactions were performed under various conditions (pH/temperature), and the radiolabeled chelates were purified and characterized by radio-TLC and radio-HPLC. The stability of labeled chelates was investigated up to 240 min in human serum (HS), EDTA 5 mM, PBS and 0.9 % NaCl solutions. The in vivo stability of [Al F(2-AMPDA-HB)] was assessed in healthy nude mice (n=6). Radiochemical yields between 55 % and 81 % were obtained at pH 5 and room temperature. High stability in HS was measured for [Al F(2-AMPDA-HB)] , with 90 % of F-18 complexed after 120 min. High stability in vivo, rapid hepatobiliary and renal excretion, with low accumulation of free F-18 in bones were measured. Thus, this new Al F-chelator may have a great impact on immuno-PET radiopharmacy, by facilitating the development of new fluorine-18-labeled heat-sensitive biomolecules.
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http://dx.doi.org/10.1002/cmdc.201900652DOI Listing
February 2020

Reproducibility and Comparability of Preclinical PET Imaging Data: A Multicenter Small-Animal PET Study.

J Nucl Med 2019 10 8;60(10):1483-1491. Epub 2019 Mar 8.

Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Eberhard-Karls University Tübingen, Tübingen, Germany.

The standardization of preclinical imaging is a key factor to ensure the reliability, reproducibility, validity, and translatability of preclinical data. Preclinical standardization has been slowly progressing in recent years and has mainly been performed within a single institution, whereas little has been done in regards to multicenter standardization between facilities. This study aimed to investigate the comparability among preclinical imaging facilities in terms of PET data acquisition and analysis. In the first step, basic PET scans were obtained in 4 different preclinical imaging facilities to compare their standard imaging protocol for F-FDG. In the second step, the influence of the personnel performing the experiments and the experimental equipment used in the experiment were compared. In the third step, the influence of the image analysis on the reproducibility and comparability of the acquired data was determined. Distinct differences in the uptake behavior of the 4 standard imaging protocols were determined for the investigated organs (brain, left ventricle, liver, and muscle) due to different animal handling procedures before and during the scans (e.g., fasting vs. nonfasting, glucose levels, temperature regulation vs. constant temperature warming). Significant differences in the uptake behavior in the brain were detected when the same imaging protocol was used but executed by different personnel and using different experimental animal handling equipment. An influence of the person analyzing the data was detected for most of the organs, when the volumes of interest were manually drawn by the investigators. Coregistration of the PET to an MR image and drawing the volume of interest based on anatomic information yielded reproducible results among investigators. It has been demonstrated that there is a huge demand for standardization among multiple institutions.
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http://dx.doi.org/10.2967/jnumed.118.221994DOI Listing
October 2019

Galectin-3 Targeting in Thyroid Orthotopic Tumors Opens New Ways to Characterize Thyroid Cancer.

J Nucl Med 2019 06 25;60(6):770-776. Epub 2018 Oct 25.

Nuklearmedizinische Klinik und Poliklinik, Klinikum rechts der Isar, Technische Universität München, München, Germany

Preoperative characterization of thyroid nodules is challenging since thyroid scintigraphy fails to distinguish between benign and malignant lesions. Galectin-3 (gal-3) is expressed in well-differentiated and in undifferentiated thyroid cancer types but not in normal thyrocytes and benign thyroid lesions. Herein, we aimed to validate gal-3 targeting as a specific method to detect non-radioiodine-avid thyroid cancer in thyroid orthotopic tumor models. Papillary (BcPAP) and anaplastic (CAL62 and FRO82-1) thyroid carcinoma cell lines were characterized via Western blot and polymerase chain reaction for gal-3 and sodium-iodide symporter (NIS) expression. An Zr-labeled F(ab') antigal-3 was generated and characterized for binding versus I on 2- and 3-dimensional cell cultures. The thyroid carcinoma cells were inoculated into the left thyroid lobe of athymic nude mice, and the orthotopic tumor growth was monitored via ultrasound and fluorescence molecular tomography. Head-to-head PET/CT comparison of I versus Zr-deferoxamine (DFO)-F(ab') antigal-3 was performed, followed by biodistribution studies and immunohistochemical analysis for gal-3 and NIS expression. The thyroid carcinoma cells investigated were invariably gal-3-positive while presenting low or lost NIS expression. Zr-DFO-F(ab') antigal-3 tracer showed high affinity to gal-3 (dissociation constant, ∼3.9 nM) and retained immunoreactivity (>75%) on 2-dimensional cell cultures and on tumor spheroids. I internalization in FRO82-1, BcPAP, and CAL62 was directly dependent on NIS expression, both in 2-dimensional and tumor spheroids. PET/CT imaging showed Zr-DFO-F(ab') antigal-3 signal associated with the orthotopically implanted tumors only; no signal was detected in the tumor-free thyroid lobe. Conversely, PET imaging using I showed background accumulation in tumor-infiltrated lobe, a condition simulating the presence of non-radioiodine-avid thyroid cancer nodules, and high accumulation in normal thyroid lobe. Imaging data were confirmed by tracer biodistribution studies and immunohistochemistry. A specific and selective visualization of thyroid tumor by targeting gal-3 was demonstrated in the absence of radioiodine uptake. Translation of this method into the clinical setting promises to improve the management of patients by avoiding the use of unspecific imaging methodologies and reducing unnecessary thyroid surgery.
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http://dx.doi.org/10.2967/jnumed.118.219105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581231PMC
June 2019

Preclinical Evaluation of the Hsp70 Peptide Tracer TPP-PEG-DFO[Zr] for Tumor-Specific PET/CT Imaging.

Cancer Res 2018 11 18;78(21):6268-6281. Epub 2018 Sep 18.

Radiation Immuno Oncology Group, Center for Translational Cancer Research (TranslaTUM), Campus Klinikum rechts der Isar, Technische Universität München (TUM), Munich, Germany.

High precision PET/CT imaging of solid tumors improves diagnostic credibility and clinical outcome of patients. An epitope of the oligomerization domain of Hsp70 is exclusively exposed on the membrane of a large variety of tumor types, but not on normal cells, and thus provides a universal tumor-specific target. Here we developed a novel PET tracer TPP-PEG-DFO[Zr] based on the tumor cell-penetrating peptide probe TPP, which specifically recognizes membrane Hsp70 (mHsp70) on tumor cells. The implemented PEG moiety supported tracer stability and improved biodistribution characteristics The of the tracer ranged in the low nanomolar range (18.9 ± 11.3 nmol/L). Fluorescein isothiocyanate (FITC)-labeled derivatives TPP-[FITC] and TPP-PEG-[FITC] revealed comparable and specific binding to mHsp70-positive 4T1, 4T1, a derivative of the 4T1 cell line sorted for high Hsp70 expression, and CT26 tumor cells, but not to mHsp70-negative normal fibroblasts. The rapid internalization kinetics of mHsp70 into the cytosol and the favorable biodistribution of the peptide-based tracer TPP-PEG-DFO[Zr] enabled a tumor-specific accumulation with a high tumor-to-background contrast and renal body clearance. The tumor-specific enrichment of the tracer in 4T1 (6.2 ± 1.1%ID/g), 4T1 (4.3 ± 0.7%ID/g), and CT26 (2.6 ± 0.6%ID/g) mouse tumors with very high, high, and intermediate mHsp70 densities, respectively, reflected mHsp70 expression profiles of the different tumor types, whereas benign mHsp70-negative fibroblastic hyperplasia showed no tracer accumulation (0.2 ± 0.03%ID/g). The ability of our chemically optimized peptide-based tracer TPP-PEG-DFO[Zr] to detect mHsp70 suggests its broad applicability in targeting and imaging with high specificity for any tumor type that exhibits surface expression of Hsp70. A novel peptide-based PET tracer against the oligomerization domain of Hsp70 has potential for universal tumor-specific imaging across many tumor type. .
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http://dx.doi.org/10.1158/0008-5472.CAN-18-0707DOI Listing
November 2018

In-depth Characterization of a TCR-specific Tracer for Sensitive Detection of Tumor-directed Transgenic T Cells by Immuno-PET.

Theranostics 2017 15;7(9):2402-2416. Epub 2017 Jun 15.

Department of Nuclear Medicine, Klinikum rechts der Isar, Technische Universität München, Germany.

A number of different technologies have been developed to monitor the distribution of gene-modified T cells used in immunotherapy. Nevertheless, in-depth characterization of novel approaches with respect to sensitivity and clinical applicability are so far missing. We have previously described a novel method to track engineered human T cells in tumors using Zr-Df-aTCRmu-F(ab') targeting the murinized part of the TCR beta domain (TCRmu) of a transgenic TCR. Here, we performed an in-depth characterization of the tracer in terms of antigen affinity, immunoreactivity, influence on T-cell functionality and stability and . Of particular interest, we have developed diverse experimental settings to quantify TCR-transgenic T cells . Local application of Zr-Df-aTCRmu-F(ab')-labeled T cells in a spot-assay revealed signal detection down to approximately 1.8x10 cells. In a more clinically relevant model, NSG mice were intravenously injected with different numbers of transgenic T cells, followed by injection of the Zr-Df-aTCRmu-F(ab') tracer, PET/CT imaging and subsequent T-cell quantification in the tumor. Using this setting, we defined a comparable detection limit of 1.0x10 T cells. PET signals correlated well to total numbers of transgenic T cells detected independently of the engraftment rates observed in different individual experiments. Thus, these findings confirm the high sensitivity of our novel PET/CT T-cell tracking method and provide critical information about the quantity of transgenic T cells in the tumor environment suggesting our technology being highly suitable for further clinical translation.
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http://dx.doi.org/10.7150/thno.17994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5525745PMC
March 2018

Noninvasive In Vivo Imaging and Biologic Characterization of Thyroid Tumors by ImmunoPET Targeting of Galectin-3.

Cancer Res 2016 06 23;76(12):3583-92. Epub 2016 May 23.

Pathology Research Laboratory, Sant'Andrea University Hospital, Rome, Italy. Pathology Research Laboratory, Cancer Center Karolinska, Karolinska Hospital, Stockholm, Sweden.

The high prevalence of thyroid nodules in the adult population and the relatively low incidence of thyroid cancer make the preoperative identification of malignant lesions challenging. The β-galactoside-binding protein galectin-3 is widely expressed in well-differentiated thyroid carcinomas, but not in normal thyrocytes and benign thyroid nodules. This molecule offers a candidate biomarker to improve thyroid cancer diagnosis. Here we report the development of an immunoPET approach for noninvasive imaging of thyroid cancer. The method employs a (89)Zr-labeled mAb to galectin-3, which shows high specificity and binding affinity in vitro Reliable and specific immunoPET imaging was obtained of thyroid cancer in vivo in murine xenograft models of human thyroid cancer. Our findings provide a method to improve the clinical management of patients with thyroid nodules while reducing unnecessary surgery and social costs. Cancer Res; 76(12); 3583-92. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-3046DOI Listing
June 2016

Comparison of 18F-Labeled Fluoroalkylphosphonium Cations with 13N-NH3 for PET Myocardial Perfusion Imaging.

J Nucl Med 2015 Oct 11;56(10):1581-6. Epub 2015 Jun 11.

Department of Nuclear Medicine, Chonnam National University Hwasun Hospital, Hwasun, Korea; and

Unlabelled: Despite substantial advances in the diagnosis of cardiovascular disease, there is a need for 18F-labeled myocardial perfusion agents for the diagnosis of ischemic heart disease because current PET tracers for myocardial perfusion imaging have a short half-life that limits their widespread clinical use in PET. Thus, 18F-labeled fluoroalkylphosphonium derivatives (18F-FATPs), including (5-18F-fluoropentyl)triphenylphosphonium cation (18F-FPTP), (6-18F-fluorohexyl)triphenylphosphonium cation (18F-FHTP), and (2-(2-18F-fluoroethoxy)ethyl)triphenylphosphonium cation (18F-FETP), were synthesized. The myocardial extraction and image quality of the 18F-FATPs were compared with those of 13N-NH3 in rat models.

Methods: The first-pass extraction fraction (EF) values of the 18F-FATPs (18F-FPTP, 18F-FHTP, 18F-FETP) and 13N-NH3 were measured in isolated rat hearts perfused with the Langendorff method (flow velocities, 0.5, 4.0, 8.0, and 16.0 mL/min). Normal and myocardial infarction rats were imaged with small-animal PET after intravenous injection of 37 MBq of 18F-FATPs and 13N-NH3. To determine pharmacokinetics, a region of interest was drawn around the heart, and time-activity curves of the 18F-FATPs and 13N-NH3 were generated to obtain the counts per pixel per second. Defect size was analyzed on the basis of polar map images of 18F-FATPs and 13N-NH3.

Results: The EF values of 18F-FATPs and 13N-NH3 were comparable at low flow velocity (0.5 mL/min), whereas at higher flows EF values of 18F-FATPs were significantly higher than those of 13N-NH3 (4.0, 8.0, and 16.0 mL/min, P<0.05). Myocardium-to-liver ratios of 18F-FPTP, 18F-FHTP, 18F-FETP, and 13N-NH3 were 2.10±0.30, 4.36±0.20, 3.88±1.03, and 0.70±0.09, respectively, 10 min after injection, whereas myocardium-to-lung ratios were 5.00±0.25, 4.33±0.20, 7.98±1.23, and 2.26±0.14, respectively. Although 18F-FATPs and 13N-NH3 sharply delineated myocardial perfusion defects, defect size on the 13N-NH3 images was significantly smaller than on the 18F-FATP images soon after tracer injection (0-10 min, P=0.027).

Conclusion: 18F-FATPs exhibit higher EF values and more rapid clearance from the liver and lung than 13N-NH3 in normal rats, which led to excellent image quality in a rat model of coronary occlusion. Therefore, 18F-FATPs are promising new PET radiopharmaceuticals for myocardial perfusion imaging.
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http://dx.doi.org/10.2967/jnumed.115.156794DOI Listing
October 2015

Assessment of the 18F-labeled PET tracer LMI1195 for imaging norepinephrine handling in rat hearts.

J Nucl Med 2013 Jul 13;54(7):1142-6. Epub 2013 May 13.

Nuklearmedizinische Klinik und Poliklinik, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.

Unlabelled: A novel (18)F-labeled tracer, LMI1195 (N-[3-bromo-4-(3-(18)F-fluoro-propoxy)-benzyl]-guanidine), is being developed for sympathetic nerve imaging; its high specificity for neural uptake-1 mechanism has previously been demonstrated in cell associative studies and in rabbit and nonhuman primate studies assessing heart uptake. The aim of this study was to investigate the mechanisms of (18)F-LMI1195 cardiac uptake in the rat, which is known to contain norepinephrine uptake mechanisms beyond uptake-1.

Methods: Tracer accumulation in the heart was studied over time after intravenous administration of (18)F-LMI1195 in healthy male Wistar rats by quantitative in vivo PET imaging. The uptake mechanism was assessed by pretreatment with the nonselective norepinephrine uptake-1 and norepinephrine uptake-2 inhibitor phenoxybenzamine (50 mg/kg intravenously; n = 4), the selective norepinephrine uptake-1 inhibitor desipramine (2 mg/kg intravenously; n = 4), or saline control (intravenously; n = 4).

Results: (18)F-LMI1195 produced high and sustained heart uptake allowing clear delineation of the left ventricular wall over 60 min after tracer administration. Pretreatment with phenoxybenzamine markedly reduced the (18)F-LMI1195 cardiac uptake when compared with controls. In contrast, there was preserved (18)F-LMI1195 uptake after desipramine pretreatment.

Conclusion: In rats, cardiac uptake of (18)F-LMI1195 was significantly inhibited by phenoxybenzamine but not desipramine, suggesting (18)F-LMI1195 is a substrate for the uptake-2 mechanism and is consistent with the rat heart having a dominant level of the mechanism.
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http://dx.doi.org/10.2967/jnumed.112.104232DOI Listing
July 2013

Small-animal PET imaging of amyloid-beta plaques with [11C]PiB and its multi-modal validation in an APP/PS1 mouse model of Alzheimer's disease.

PLoS One 2012 9;7(3):e31310. Epub 2012 Mar 9.

Nuklearmedizinische Klinik und Poliklinik, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.

In vivo imaging and quantification of amyloid-β plaque (Aβ) burden in small-animal models of Alzheimer's disease (AD) is a valuable tool for translational research such as developing specific imaging markers and monitoring new therapy approaches. Methodological constraints such as image resolution of positron emission tomography (PET) and lack of suitable AD models have limited the feasibility of PET in mice. In this study, we evaluated a feasible protocol for PET imaging of Aβ in mouse brain with [(11)C]PiB and specific activities commonly used in human studies. In vivo mouse brain MRI for anatomical reference was acquired with a clinical 1.5 T system. A recently characterized APP/PS1 mouse was employed to measure Aβ at different disease stages in homozygous and hemizygous animals. We performed multi-modal cross-validations for the PET results with ex vivo and in vitro methodologies, including regional brain biodistribution, multi-label digital autoradiography, protein quantification with ELISA, fluorescence microscopy, semi-automated histological quantification and radioligand binding assays. Specific [(11)C]PiB uptake in individual brain regions with Aβ deposition was demonstrated and validated in all animals of the study cohort including homozygous AD animals as young as nine months. Corresponding to the extent of Aβ pathology, old homozygous AD animals (21 months) showed the highest uptake followed by old hemizygous (23 months) and young homozygous mice (9 months). In all AD age groups the cerebellum was shown to be suitable as an intracerebral reference region. PET results were cross-validated and consistent with all applied ex vivo and in vitro methodologies. The results confirm that the experimental setup for non-invasive [(11)C]PiB imaging of Aβ in the APP/PS1 mice provides a feasible, reproducible and robust protocol for small-animal Aβ imaging. It allows longitudinal imaging studies with follow-up periods of approximately one and a half years and provides a foundation for translational Alzheimer neuroimaging in transgenic mice.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0031310PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3302888PMC
August 2012

Molecular imaging of early αvβ3 integrin expression predicts long-term left-ventricle remodeling after myocardial infarction in rats.

J Nucl Med 2012 Feb;53(2):318-23

Nuklearmedizinische Klinik, Technischen Universität München, Munich, Germany.

Unlabelled: (18)F-galacto-RGD ((18)F-RGD) is a PET tracer binding to α(v)β(3) integrin receptors that are upregulated after myocardial infarction (MI) as part of the healing process. We studied whether myocardial (18)F-RGD uptake early after MI is associated with long-term left-ventricle (LV) remodeling in a rat model.

Methods: Wistar rats underwent sham operation (n = 9) or permanent coronary ligation (n = 25). One week after MI, rats were injected with (18)F-RGD to evaluate α(v)β(3) integrin expression using a preclinical PET system. In the same rats, LV volumes and defect size were measured 1 and 12 wk after MI by (13)N-ammonia PET and MRI, respectively.

Results: One week after MI, (18)F-RGD uptake was increased in the defect area as compared with the remote myocardium of MI rats or sham-operated controls (percentage injected dose per cubic centimeter, 0.20 ± 0.05 vs. 0.06 ± 0.03 and 0.07 ± 0.04, P < 0.001). At this time, (18)F-RGD uptake was associated with capillary density in histologic sections. Average (18)F-RGD uptake in the defect area was lowest in the rats demonstrating greater than 20% relative increase in the LV end-diastolic volume from 1 to 12 wk (percentage injected dose per centimeter cubed, 0.15 ± 0.07 vs. 0.21 ± 0.05, P < 0.05). In a multivariable logistic regression analysis, low (18)F-RGD uptake was a significant predictor of increase in end-diastolic volume (r = 0.51, P < 0.05).

Conclusion: High levels of (18)F-RGD uptake in the perfusion defect area early after MI were associated with the absence of significant LV remodeling after 12 wk of follow-up. These results suggest that α(v)β(3) integrin expression is a potential biomarker of myocardial repair processes after MI and enables the monitoring of these processes by molecular imaging to derive possible prognostic information.
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http://dx.doi.org/10.2967/jnumed.111.091652DOI Listing
February 2012

Functional Imaging of Pheochromocytoma with Ga-DOTATOC and C-HED in a Genetically Defined Rat Model of Multiple Endocrine Neoplasia.

Int J Mol Imaging 2011 8;2011:175352. Epub 2011 Jun 8.

Department of Nuclear Medicine, Johannes Gutenberg University of Mainz, Langenbeck Strasse 1, 55131 Mainz, Germany.

Rats affected by the MENX multitumor syndrome develop pheochromocytoma (100%). Pheochromocytomas are uncommon tumors and animal models are scarce, hence the interest in MENX rats to identify and preclinically evaluate novel targeted therapies. A prerequisite for such studies is a sensitive and noninvasive detection of MENXassociated pheochromocytoma. We performed positron emission tomography (PET) to determine whether rat pheochromocytomas are detected by tracers used in clinical practice, such as 68Ga-DOTATOC (somatostatin analogue) or (11)C-Hydroxyephedrine (HED), a norepinephrine analogue. We analyzed four affected and three unaffected rats. The PET scan findings were correlated to histopathology and immunophenotype of the tumors, their proliferative index, and the expression of genes coding for somatostatin receptors or the norepinephrine transporter. We observed that mean 68Ga-DOTATOC standard uptake value (SUV) in adrenals of affected animals was 23.3 ± 3.9, significantly higher than in control rats (15.4 ± 7.9; P = .03). The increase in mean tumor-to-liver ratio of (11)C-HED in the MENX-affected animals (1.6 ± 0.5) compared to controls (0.7 ± 0.1) was even more significant (P = .0016). In a unique animal model, functional imaging depicting two pathways important in pheochromocytoma biology discriminated affected animals from controls, thus providing the basis for future preclinical work with MENX rats.
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http://dx.doi.org/10.1155/2011/175352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3132633PMC
July 2011

Simplified quantification of myocardial flow reserve with flurpiridaz F 18: validation with microspheres in a pig model.

J Nucl Med 2011 Apr;52(4):617-24

Nuklearmedizinische Klinik und Poliklinik der Technischen Universität München, Munich, Germany.

Unlabelled: The novel PET flow tracer flurpiridaz F 18 shows high myocardial extraction and slow washout. flurpiridaz F 18 PET data analysis with tracer kinetic modeling provides accurate absolute myocardial blood flow (MBF) measurements but requires in-scanner injection and complex processing. We evaluated the hypothesis that myocardial retention and standardized uptake values (SUVs) based on late uptake provide accurate estimates of myocardial flow reserve (MFR) and, thus, might allow simplified quantification after tracer injection outside the scanner.

Methods: Nine pigs had dynamic PET scans after repeated injections of flurpiridaz F 18 at rest and combined adenosine and dobutamine stress. flurpiridaz F 18 PET with a 3-compartment model and coinjected radioactive microspheres were used to delineate MBF. These quantitative measurements were compared with myocardial retention (%/min) and SUV of flurpiridaz F 18 after summing data over 5-10, 5-12, 5-15, 10-15, and 10-20 min after tracer injection.

Results: MBF ranged from 0.5 to 2.8 mL/min/g. There was a good correlation between both flurpiridaz F 18 retention and SUVs from 5 to 12 min after injection and MBF measured using 3-compartment model- or microsphere-derived MBF (r = 0.73, P < 0.05, and r = 0.68, P < 0.05, respectively, for retention; r = 0.88, P < 0.001, and r = 0.92, P < 0.001, respectively, for SUV). At later time points, retention and SUV underestimated stress microsphere flow (at 10-20 min: r = 0.41, P = not significant, and r = 0.46, P = not significant, respectively, for retention; r = 0.41, P = not significant, and r = 0.65, P < 0.05, respectively, for SUV). When measured 5-12 min after injection, there was a close agreement between MFR measured with either flurpiridaz F 18 retention or SUV and MFR measured using microspheres (mean difference, -0.08 ± 0.36 and -0.18 ± 0.25, respectively).

Conclusion: Myocardial retention and SUVs of the (18)F-labeled flow tracer flurpiridaz F 18 accurately reflect the MFR. These simplified analysis methods may facilitate the combination of quantitative assessment of perfusion reserve and rapid clinical imaging protocols.
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http://dx.doi.org/10.2967/jnumed.110.083196DOI Listing
April 2011

Evaluation of a novel (18)F-labeled positron-emission tomography perfusion tracer for the assessment of myocardial infarct size in rats.

Circ Cardiovasc Imaging 2009 Mar 26;2(2):77-84. Epub 2009 Jan 26.

Department of Nuclear Medicine, Klinikum Rechts der Isar, Technische Universität München, Munich, Germany.

Background: The goal of this study was to evaluate a new (18)F-labeled positron-emission tomography (PET) perfusion tracer, (18)F BMS747158-02, for the assessment of myocardial infarct (MI) size.

Methods And Results: Wistar rats were studied 24 hours after ligation of the left coronary artery either permanently (n=15) or transiently (n=16) for 30 minutes. Seven nonoperated rats were studied as controls. The rats were injected with 37 MBq of (18)F BMS747158-02 and imaged with a small animal PET scanner for 20 minutes. Polar maps were generated for measurement of PET defect size, and left ventricular systolic and diastolic volumes were assessed in gated images. As a reference, MI size was determined by 2,3,5-triphenyltetrazolium chloride staining of left ventricular tissue samples. Permanent or transient ligation of the left coronary artery produced transmural or subendocardial MI of variable sizes, respectively. In normal rats, PET imaging demonstrated intense and homogeneous uptake of (18)F BMS747158-02 throughout the myocardium. After ligation, sharply defined perfusion defects were present. Throughout the imaging period, the defect size correlated closely with the MI size either after permanent (r=0.88; P<0.01; mean difference, 1.86%) or transient (r=0.92; P<0.01; mean difference, 2.16%) ligation of the left coronary artery. Moreover, reduction of left ventricular systolic function measured with PET correlated with the MI size (r=-0.81; P<0.01; n=23).

Conclusions: Myocardial (18)F BMS747158-02 PET imaging provides excellent image quality and uptake properties, enabling accurate evaluation of MI size and left ventricular function in rats. It is a promising technique for evaluation of MI size in clinical trials.
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http://dx.doi.org/10.1161/CIRCIMAGING.108.815423DOI Listing
March 2009

A new 18F-labeled myocardial PET tracer: myocardial uptake after permanent and transient coronary occlusion in rats.

J Nucl Med 2008 Oct 15;49(10):1715-22. Epub 2008 Sep 15.

Department of Nuclear Medicine, Klinikum Rechts der Isar, Technische Universität München, Munich, Germany.

Unlabelled: Conventional myocardial perfusion PET tracers require onsite tracer production because of their short radioactive half-lives. To investigate the potential of a new (18)F-labeled pyridazinone analog ((18)F-BMS-747158-02), we characterized this tracer in a rat model of permanent and transient coronary occlusion using small-animal PET.

Methods: Myocardial (18)F-BMS-747158-02 distribution in healthy rats (n = 7), rats with transient (3-min) left coronary artery occlusion (n = 11), and rats with permanent left coronary occlusion (n = 11) was analyzed with a dedicated small-animal PET scanner.

Results: Normal hearts demonstrated intense and almost homogeneous tracer uptake throughout the left ventricle for more than 2 h. During permanent coronary occlusion, PET demonstrated perfusion defects, which remained unchanged (37.6% +/- 8.8%, 37.4% +/- 10.2%, and 36.2% +/- 9.8% left ventricle at 15, 45, and 115 min, respectively, after tracer injection). After transient ischemia, the induced defect size decreased significantly after reperfusion (16.2% +/- 9.3%, 6.0% +/- 6.5%, and 1.4% +/- 1.3% left ventricle). Tracer reinjection after transient ischemia resulted in normalization of the induced defect.

Conclusion: Coronary occlusion yielded distinct myocardial (18)F-BMS-747158-02 uptake defects in the area of ischemia, which demonstrated normalization of activity after reperfusion and reinjection. These promising kinetic parameters may allow for assessment of flow using exercise-rest protocols similar to those used in combination with exercise and rest perfusion SPECT.
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http://dx.doi.org/10.2967/jnumed.108.053967DOI Listing
October 2008

Initial characterization of an 18F-labeled myocardial perfusion tracer.

J Nucl Med 2008 Apr 14;49(4):630-6. Epub 2008 Mar 14.

Department of Nuclear Medicine, Klinikum Rechts der Isar, Technische Universität München, Munich, Germany.

Unlabelled: PET allows for quantitative, regional myocardial perfusion imaging. The short half-lives of the perfusion tracers currently in use limit their clinical applicability. Here, the biodistribution and imaging quality of a new 18F-labeled myocardial perfusion agent (18F-BMS-747158-02) in an animal model are described.

Methods: The biodistribution of 18F-BMS-747158-02 was determined at 10 and 60 min after injection. The first-pass extraction fraction of the tracer was measured in isolated rat hearts perfused with the Langendorff method. Small-animal PET imaging was used to study tracer retention.

Results: The biodistribution at 10 min after injection demonstrated high myocardial uptake (3.1 percentage injected dose per gram [%ID/g]) accompanied by little activity in the lungs (0.3 %ID/g) and liver (1.0 %ID/g). The tracer showed a high and flow-independent myocardial first-pass extraction fraction, averaging 0.94 (SD = 0.04). PET imaging provided excellent delineation of myocardial structures. The heart-to-lung activity ratio increased from 4.7 to 10.2 between 1 and 15 min after tracer injection (at rest). Adenosine infusion (140 microg/kg/min) led to a significant increase in myocardial tracer retention (from 1.68 [SD = 0.23]) s(-1) to 3.21 [SD = 0.92] s(-1); P = 0.03).

Conclusion: The observation of a high and flow-independent first-pass extraction fraction promises linearity between tracer uptake and myocardial blood flow. Sustained myocardial tracer uptake, combined with high image contrast, will allow for imaging protocols with tracer injection at peak exercise followed by delayed imaging. Thus, 18F-BMS-747158-02 is a promising new tracer for the quantitative imaging of myocardial perfusion and can be distributed to imaging laboratories without a cyclotron.
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http://dx.doi.org/10.2967/jnumed.107.044727DOI Listing
April 2008

Assessment of alphavbeta3 integrin expression after myocardial infarction by positron emission tomography.

Cardiovasc Res 2008 May 6;78(2):395-403. Epub 2008 Feb 6.

Nuklearmedizinische Klinik und Poliklinik der Technischen Universität München, Klinikum rechts der Isar, Ismaninger Strasse 22, 81675 Munich, Germany.

Aims: The purpose of this study was to determine the feasibility of a new positron emission tomography (PET) imaging approach using an (18)F-labelled alpha(v)beta(3) integrin antagonist ((18)F-Galacto-RGD) to monitor the integrin expression after myocardial infarction.

Methods And Results: Male Wister rats were subjected to 20 min transient left coronary artery occlusion followed by reperfusion. Autoradiographic analysis and in vivo PET imaging were used to determine myocardial (18)F-Galacto-RGD uptake at different time points following reperfusion.

Results: PET imaging and autoradiography demonstrated no significant focal myocardial (18)F-Galacto-RGD uptake in non-operated control rats and at day 1 after reperfusion. However, focal accumulation in the infarct area started at day 3 (uptake ratio = 1.91 +/- 0.22 vs. remote myocardium), peaked between 1 (3.43 +/- 0.57) and 3 weeks (3.43 +/- 0.95), and decreased to 1.96 +/- 0.40 at 6 months after reperfusion. Pretreatment with alpha(v)beta(3) integrin antagonist c(-RGDfV-) significantly decreased tracer uptake, indicating the specificity of tracer uptake. The time course of focal tracer uptake paralleled vascular density as measured by CD31 immunohistochemical analysis.

Conclusion: Regional (18)F-Galacto-RGD accumulation suggests up-regulation of alpha(v)beta(3) integrin expression after myocardial infarction, which peaks between 1 and 3 weeks and remains detectable until 6 months after reperfusion. This new PET tracer is promising for the monitoring of myocardial repair processes.
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http://dx.doi.org/10.1093/cvr/cvn033DOI Listing
May 2008

Characterization of normal and infarcted rat myocardium using a combination of small-animal PET and clinical MRI.

J Nucl Med 2007 Feb;48(2):288-94

Nuklearmedizinische Klinik und Poliklinik der Technischen Universität München, Klinikum rechts der Isar, Munich, Germany.

Unlabelled: The combination of small-animal PET and MRI data provides quantitative in vivo insights into cardiac pathophysiology, integrating information on biology and morphology. We sought to determine the feasibility of PET and MRI for the quantification of ischemic injury in the rat model.

Methods: Fourteen healthy male Wistar rats were studied with 18F-FDG PET and cine MRI. Myocardial viability was determined in a transmural myocardial infarction model in 12 additional rats, using 18F-FDG PET and delayed-enhancement MRI with gadolinium-diethylenetriaminepentaacetic acid. All PET was acquired with a dedicated small-animal PET system. MRI was performed on a 1.5-T clinical tomograph with a dedicated small-animal electrocardiographic triggering device and a small surface coil.

Results: In normal rats, 18F-FDG uptake was homogeneous throughout the left ventricle. The lowest mean uptake of the 18F-FDG was found in the apical regions (79% +/- 6.0% of maximum) and the highest uptake was in the anterior wall (93% +/- 4.3 % of maximum). Myocardial infarct size as determined by histology correlated well with defects of glucose metabolism obtained with 18F-FDG PET (r = 0.89) and also with delayed-enhancement MRI (r = 0.91). Left ventricular ejection fraction in normal rats measured by cine MRI was 57% +/- 5.4% and decreased to 38% +/- 12.9% (P < 0.001) in the myocardial infarction model.

Conclusion: Integrating information from small-animal PET and clinical MRI instrumentation allows for the quantitative assessment of cardiac function and infarct size in the rat model. The MRI measurements of scar can be complemented by metabolic imaging, addressing the extent and severity of ischemic injury and providing endpoints for therapeutic interventions.
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February 2007

Performance evaluation of the Philips MOSAIC small animal PET scanner.

Eur J Nucl Med Mol Imaging 2007 Apr 22;34(4):532-40. Epub 2006 Nov 22.

Nuklearmedizinische Klinik und Poliklinik, Klinikum rechts der Isar der Technischen Universitaet Muenchen, Ismaninger Strasse 22, 81675, Munich, Germany.

Purpose: In this study an evaluation of the performance of the Philips MOSAIC small animal PET scanner is presented, with special emphasis on the ability of the system to provide quantitatively accurate PET images.

Methods: The performance evaluation was structured according to NEMA-like procedures.

Results: The transaxial spatial resolution of the system (radial component) ranged between 2.7 mm FWHM at the centre and 3.2 mm FWHM at a radial offset of 45 mm from the centre. The axial spatial resolution of the system ranged between 3.4 mm FWHM at the centre and 5.8 mm FWHM at a radial offset of 45 mm from the centre. The scatter fraction was determined for a mouse- as well as for a rat-sized phantom, and the values obtained were 9.6% and 16.8%, respectively. For the mouse phantom, the maximum count rate measured was 560 kcps at 93 MBq; the maximum NEC rate equalled 308 kcps at 1.7 MBq/ml. For the rat phantom, these values were 400 kcps at 100 MBq and 129 kcps at 0.24 MBq/ml, respectively. The sensitivity of the system was derived to be 0.65%. An energy window between 410 and 665 keV was used in all experiments.

Conclusion: The MOSAIC system exhibits moderate spatial resolution and sensitivity values, but good NEC performance. In combination with its relatively large field of view, the system allows for high-throughput whole-body imaging of mice and rats. The accurate measurement of relative changes in radiotracer distributions is feasible.
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http://dx.doi.org/10.1007/s00259-006-0271-7DOI Listing
April 2007

Noninvasive characterization of myocardial molecular interventions by integrated positron emission tomography and computed tomography.

J Am Coll Cardiol 2006 Nov 31;48(10):2107-15. Epub 2006 Oct 31.

Nuklearmedizinische Klinik und Poliklinik, Technische Universität München, Germany.

Objectives: We sought to investigate the usefulness of integrated positron emission tomography (PET) and computed tomography (CT) for in vivo characterization of an angiogenesis-directed molecular intervention.

Background: Controversies about the effectiveness of molecular therapies for cardiovascular disease have prompted the need for more powerful noninvasive imaging techniques.

Methods: In a model of regional adenoviral transfer of the VEGF(121) gene to myocardium of healthy pigs, PET-CT using multiple molecular-directed radiotracers was employed.

Results: Two days after gene transfer, successful transgene expression was noninvasively confirmed by a reporter probe targeting co-expressed HSV1-sr39tk reporter gene. The CT-derived ventricular function and morphology remained unaltered (left ventricular ejection fraction 57 +/- 5% in adenovirus-injected animals vs. 53 +/- 5% in controls; p = 0.36). Increased regional perfusion was identified in areas overexpressing VEGF (myocardial blood flow during adenosine-induced vasodilation 1.47 +/- 0.49 vs. 1.14 +/- 0.27 ml/g/min in remote areas; p = 0.01), corroborating in vivo effects on microvascular tone and permeability. Finally, regional angiogenesis-associated alpha(v)beta3 integrin expression was not enhanced, suggesting little contribution to the perfusion increase. Fusion of CT morphology and tracer-derived molecular signals allowed for accurate regional localization of biologic signals. Findings were validated by control vectors, sham-operated animals, and ex vivo tissue analysis.

Conclusions: Integrated PET-CT has the potential to dissect cardiovascular biologic mechanisms from gene expression to physiologic function and morphology. The VEGF overexpression in healthy myocardium increases myocardial perfusion without significant up-regulation of alpha(v)beta3 integrin adhesion molecules early after the intervention.
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http://dx.doi.org/10.1016/j.jacc.2006.08.029DOI Listing
November 2006

Myocardial kinetics of reporter probe 124I-FIAU in isolated perfused rat hearts after in vivo adenoviral transfer of herpes simplex virus type 1 thymidine kinase reporter gene.

J Nucl Med 2005 Jan;46(1):98-105

Nuklearmedizinische Klinik und Poliklinik, Technische Universität München, Ismaninger Strasse 22, 81675 Münich, Germany.

Unlabelled: Reporter gene imaging holds promise for noninvasive monitoring of cardiac gene therapy. We recently demonstrated that (124)I-labeled 2'-fluoro-2'-deoxy-5'-iodo-1beta-d-arabinofuranosyluracil ((124)I-FIAU) is suitable for PET of myocardial expression of herpes simplex virus type 1 thymidine kinase reporter gene (HSV1-tk). In contrast to previous studies in tumors, early specific uptake was followed by rapid washout. Myocardial kinetics of (124)I-FIAU are still poorly understood. This study aimed at a further investigation under controlled conditions using an isolated heart perfusion model.

Methods: Male Wistar rats underwent transthoracic regional injection of replication-defective adenovirus (2.5 x 10(9) plaque-forming units) containing either HSV1-tk (n = 16) or LacZ reporter gene (n = 15) into the inferior wall. Nonmanipulated rats (n = 5) served as further controls. Hearts were excised 2 d later and perfused according to the Langendorff technique with (124)I-FIAU-containing buffer (15 min, followed by 30 min of nonradioactive perfusion). Experiments were performed under baseline conditions and in the presence of thymidine (competitive substrate) or fludarabine (in vitro inhibitor of 5'-nucleotidase). Time-activity curves were acquired by external coincident detectors. The myocardial rate of (124)I-FIAU uptake (K(i)), clearance rate (K(o)), and volume of distribution (V(d) = K(i)/K(o)) were calculated. Subsequently, hearts were subjected to gamma-counting, followed by microtome slicing and autoradiography.

Results: The V(d) from Langendorff perfusion significantly correlated with final whole-heart tracer retention (r = 0.88, P = 0.019) and the autoradiographic area of regional myocardial activity (r = 0.89, P = 0.016). HSV1-tk hearts showed higher K(i) and V(d) of (124)I-FIAU compared with that of controls (P < 0.001) and detectable but slower washout compared with that of the LacZ group (P < 0.01). Addition of thymidine to the perfusate inhibited myocardial uptake of (124)I-FIAU by reducing V(d) and K(i) in HSV1-tk and LacZ hearts compared with the baseline. Addition of fludarabine did not result in the expected reduction of washout in HSV1-tk hearts due to inhibition of 5'-nucleotidases (which may dephosphorylate (124)I-FIAU monophosphate). It acted as an uptake inhibitor similar to thymidine, reducing V(d) in HSV1-tk hearts.

Conclusion: Assessment of specific reporter probe kinetics after regional in vivo reporter gene transfer is feasible using the isolated perfused rat heart preparation. This model allows one to study the effects of pharmacologic interventions and may refine understanding of the reporter probe signal for in vivo imaging. Different nucleoside analogs significantly inhibit (124)I-FIAU uptake, emphasizing the importance of transporter mechanisms for reporter probe kinetics.
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January 2005

PET of cardiac transgene expression: comparison of 2 approaches based on herpesviral thymidine kinase reporter gene.

J Nucl Med 2004 Nov;45(11):1917-23

Nuklearmedizinische Klinik und Poliklinik, Technische Universität München, 81675 Munich, Germany.

Unlabelled: PET of reporter gene expression holds promise for noninvasive monitoring of gene therapy. Previously, 2 approaches based on the herpes simplex virus type 1 thymidine kinase gene (HSV1-tk) have been successfully applied to the heart. Wild-type HSV1-tk was imaged with (124)I-labeled 2'-fluoro-2'-deoxy-5-iodo-1-beta-D-arabinofuranosyl-5-iodouracil (FIAU), and a mutant HSV1-tk (HSV1-sr39tk) was imaged with (18)F-labeled 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG). The aim of this study was to compare these 2 combinations with regard to specificity, imaging contrast, and reporter probe kinetics using dynamic PET in small and large animals.

Methods: Similar titers of adenovirus-expressing wild-type HSV1-tk (Ad(tk)), mutant HSV1-sr39tk (Ad(sr39tk)), or control genes were directly injected into the myocardium of 24 rats and 8 pigs. Two days later, dynamic PET was performed with a clinical scanner during the 120 min after injection of (124)I-FIAU (Ad(tk) animals and controls) or (18)F-FHBG (Ad(sr39tk) animals and controls). Imaging with (13)N-ammonia was performed to identify cardiac regions of interest.

Results: In rats, significant cardiac (124)I-FIAU accumulation occurred in images obtained early (10-30 min) after Ad(tk) injection. Because of tracer washout, however, no difference between Ad(tk)-injected animals and controls was seen in the images obtained later. For (18)F-FHBG, specific myocardial accumulation greater than background levels was detected in Ad(sr39tk)-injected animals at early imaging and, in contrast to (124)I-FIAU accumulation, increased over time until the latest imaging (105-120 min). At maximum, cardiac (18)F-FHBG concentration showed a 4.15 +/- 1.65-fold increase compared with controls (105-120 min), and cardiac (124)I-FIAU concentration reached a maximal increase of 1.34 +/- 0.38-fold compared with controls (10-30 min, P = 0.0014). Global cardiac reporter probe kinetics in rats were confirmed by regional myocardial analysis in pig hearts. Transgene expression was specifically visualized by both approaches. The highest target-to-background ratio of (124)I-FIAU in Ad(tk)-infected pig myocardium was 1.50 +/- 0.20, versus 2.64 +/- 0.49 for (18)F-FHBG in Ad(sr39tk)-infected areas (P = 0.01). In vivo results were confirmed by ex vivo counting and autoradiography.

Conclusion: Both reporter gene/probe combinations were feasible for noninvasive imaging of cardiac transgene expression in different species. Specific probe kinetics suggest different myocardial handling of pyrimidine (FIAU) and acycloguanosine (FHBG) derivatives. The results favor (18)F-FHBG with mutant HSV1-sr39tk because of continuous accumulation over time and higher imaging contrast.
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November 2004

Coexpression of herpesviral thymidine kinase reporter gene and VEGF gene for noninvasive monitoring of therapeutic gene transfer: an in vitro evaluation.

J Nucl Med 2004 Oct;45(10):1743-6

Institut für Experimentelle Onkologie und Therapieforschung, Technische Universität München, Munich, Germany.

Unlabelled: Coexpression of a reporter gene and a therapeutic gene may allow for noninvasive monitoring of cardiac gene therapy. We sought to evaluate the usefulness of an adenoviral vector expressing mutant herpesviral thymidine kinase reporter gene (HSV1-sr39tk) and vascular endothelial growth factor (VEGF) 121 in independent expression cassettes (Ad4tk).

Methods: Accumulation of 14C-2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (FIAU) and 9-(4-18F-fluoro-3-hydroxymethylbutyl)guanine (FHBG) as reporter probes, and secretion of VEGF into medium, were determined for Ad4tk-infected H9c2 rat cardiac cells in vitro.

Results: In vitro tracer uptake increased with increasing vector concentration and over time. It was comparable to cells infected with adenovirus expressing only wild-type HSV1-tk (reporter probe: 14C-FIAU) or mutant HSV1-sr39tk (reporter probe: 18F-FHBG). No significant uptake was observed in cells infected with adenovirus expressing VEGF alone. With increasing vector concentration, Ad4tk-infected cells increasingly released VEGF into medium. VEGF production correlated significantly with cellular reporter probe uptake (r = 0.93; P = 0.0003).

Conclusion: The usefulness of a vector coexpressing HSV1-tk and VEGF for noninvasive imaging of expression of a therapeutic transgene has been demonstrated in vitro. This approach may allow for future in vivo monitoring of cardiac angiogenesis gene therapy.
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October 2004

Delayed response of insulin-stimulated fluorine-18 deoxyglucose uptake in glucose transporter-4-null mice hearts.

J Am Coll Cardiol 2004 May;43(9):1690-7

Nuklearmedizinische Klinik und Poliklinik, Klinikum rechts der Isar der Technischen Universität München, Munich, Germany.

Objectives: We sought to evaluate the time course of insulin-stimulated myocardial glucose uptake (MGU) in mice that had undergone ablation of glucose transporter-4 (GLUT4).

Background: The relative importance of GLUT4, the most abundant insulin-responsive glucose transporter, to modulate myocardial glucose metabolism is not well defined.

Methods: Myocardial glucose uptake was assessed at various time points after glucose (1 mg/g) and insulin (8 mU/g) injection in GLUT4-null (G4N) (n = 48) and wild-type (WT) (n = 48) mice with (18)F-2-deoxy-2-fluoro-d-glucose (FDG) using in vivo positron emission tomography (PET), in vitro gamma-counter biodistribution, and isolated, perfused hearts.

Results: Baseline assessment with PET imaging showed comparable MGU in G4N (0.66 +/- 0.12) and WT (0.67 +/- 0.11, p = 0.70) mice. Early after insulin injection, WT mice demonstrated a 3.5-fold increase in MGU (2.45 +/- 0.45, p = 0.03), whereas G4N mice presented no increase (1.11 +/- 0.24, p = 0.28). At 60 min, MGU was comparable in G4N (3.19 +/- 0.60) and WT (2.66 +/- 0.47, p = 0.28) mice. In vitro gamma-counter biodistribution evaluation confirmed in G4N mice a lack of MGU increase early after insulin, but a slow response over 120 min. The isolated, perfused hearts of G4N mice during short-term (15 min) insulin stimulation displayed no increase in MGU (0.08 +/- 0.01 ml/g/min), whereas WT mice presented a threefold increase (0.22 +/- 0.01 ml/g/min, p < 0.01). With long-term (60 min) insulin stimulation, similar MGU was found in G4N (0.31 +/- 0.02 ml/g/min) and WT (0.33 +/- 0.04 ml/g per min, p = 0.04) mice.

Conclusions: The G4N mice displayed an increase of MGU in response to insulin similar to that of controls, but with a markedly delayed time response. Our findings underscore the important role of GLUT4 in the rapid adaptive response of myocardial glucose metabolism.
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http://dx.doi.org/10.1016/j.jacc.2003.12.038DOI Listing
May 2004

Evaluation of cardiac beta-adrenoreceptors in the isolated perfused rat heart using (S)-11C-CGP12388.

J Nucl Med 2004 Mar;45(3):471-7

Nuklearmedizinische Klinik und Poliklinik der Technischen Universität München, Munich, Germany.

Unlabelled: (S)-(11)C-CGP12388 ((11)C-CGP12388) was recently developed as an in vivo PET tracer for the evaluation of cardiac beta-adrenergic receptors. The purpose of this study was to evaluate the myocardial kinetics of (11)C-CGP12388 using the perfused rat heart model.

Methods: Normal rat hearts were cannulated for retrograde perfusion according to the Langendorff method. Studies were performed using constant coronary flow rates of 12 mL/min (high flow: n = 6) and 6 mL/min (low flow: n = 6). Beta-adrenergic-blocking studies were also done using propranolol (blocking: n = 6). Two bolus injections of (11)C-CGP12388 were administered at a 25-min interval, and time-activity curves were measured using bismuth germanate detectors. The beta-adrenergic receptor density (B(max)) and total distribution volume (DV(tot)) were estimated using compartmental modeling. After the experiment, B(max) in vitro was measured for all hearts using (3)H-CGP12177, and the values were compared with the B(max) estimated in isolated hearts.

Results: DV(tot) was significantly lower in the blocking group than in the high-flow group (P < 0.01), and there was no significant difference in DV(tot) between the high- and the low-flow groups. B(max) values estimated from (11)C-CGP12388 kinetics were 5.05 +/- 0.90 pmol/g under the high-flow model and 5.20 +/- 0.63 pmol/g under the low-flow model. The B(max) results in isolated hearts correlated significantly with the measured in vitro B(max) values (r(2) = 0.69; P < 0.001).

Conclusion: Beta-adrenoreceptor density in the isolated rat heart can be quantified using (11)C-CGP12388 and a 2-injection protocol. The binding of the tracer was flow independent, with low nonspecific binding. These results suggest that (11)C-CGP12388 is a promising PET tracer that may be applicable to human studies.
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March 2004

Noninvasive imaging of transgene expression by use of positron emission tomography in a pig model of myocardial gene transfer.

Circulation 2003 Oct 6;108(17):2127-33. Epub 2003 Oct 6.

Nuklearmedizinische Klinik und Poliklinik, Technische Universität München, Klinikum rechts der Isar, Ismaninger Strasse 22, 81675 München, Germany.

Background: Radionuclide imaging of reporter gene expression may be useful for noninvasive monitoring of clinical cardiac gene therapy. Experience until now, however, has been limited to small animals.

Methods And Results: To evaluate feasibility in a clinically applicable setting, pigs were studied by conventional positron emission tomography (PET) 2 days after regional intramyocardial injection of control adenovirus or adenovirus carrying herpesviral thymidine kinase reporter gene (HSV1-tk). Myocardial blood flow was quantified by use of [13N]ammonia. Subsequently, kinetics of the reporter substrate [124I]-2'-fluoro-2'-deoxy-5-iodo-1-beta-d-arabino-furanosyluracil (FIAU) were assessed over a period of 2 hours. Areas infected with adenovirus expressing HSV1-tk showed significantly elevated FIAU retention during the first 30 minutes after injection. At later times, washout was observed, and retention was not different from that in areas infected with control virus or remote myocardium. Early in vivo FIAU uptake correlated with ex vivo images, autoradiography, and immunohistochemistry for reporter gene product after euthanasia. After intramyocardial injection of both adenoviruses, myocardial blood flow was mildly elevated compared with that in remote areas, consistent with histological signs of regional inflammation.

Conclusions: In vivo quantification of regional myocardial transgene expression is feasible with clinical PET methodology, the radioiodinated reporter probe FIAU, and the HSV1-tk reporter gene. Radioactivity efflux after specific initial uptake was not observed previously in tumor studies, suggesting that tissue-specific differences in nucleoside metabolism influence reporter probe kinetics. By coregistering reporter gene expression with additional biological parameters such as myocardial blood flow, PET allows for noninvasive characterization of the success of cardiac gene transfer along with its functional correlates.
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http://dx.doi.org/10.1161/01.CIR.0000091401.26280.A0DOI Listing
October 2003
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