Apoptosis-induced concomitant release of cytosolic proteins and factors which prevent cell death.

In the course of the apoptotic cell death, cells fragment into apoptotic bodies, the elimination of which by phagocytosis is thought to avoid the release of cytosolic constituents whose occurrence is indicative for necrotic cell death. Confluent cultures of chicken embryo fibroblasts, however, show a different behaviour. After serum deprivation, they transiently released with the same time course mitogenic activity, lactate dehydrogenase and cytosolic peptidyl prolyl cis-trans isomerases into the serum-free culture medium. The release correlated in time with a decrease of the cell number which started approximately 3 h after serum removal and ceased within approximately 10 h at about half of the initial cell density. Morphological features like cell shrinkage, membrane blebbing and cell fragmentation as well as internucleosomal DNA fragmentation indicated apoptotic cell death whereas necrotic cell death could be excluded. Conditioned medium (M(r) > or = 30 kDa) from serum-deprived cultures of chicken embryo fibroblasts completely prevented chicken embryo fibroblasts to undergo apoptosis as did phorbol 12-myristate, 13-acetate and, to -60%, L-cysteine. Cycloheximide had no effect on serum deprivation-induced apoptosis. From the present results it can be concluded that chicken embryo fibroblasts and possibly other cells undergoing apoptosis release cytosolic components and endogenous survival factor(s) which prevent apoptosis

Neuroimaging with Radiopharmaceuticals Targeting the Glutamatergic System

Radiopharmacy at ETH has worked on the development of novel PET tracers for neuro-, cardiac- and tumor imaging for many years. In this paper, our efforts on targeting the glutamatergic system of the metabotropic glutamate receptor subtype 5 (mGluR5) and the ionotropic N-methyl-D-aspartate (NMDA) receptor are summarized. We briefly described the principles of positron emission tomography (PET) tracer development for the central nervous system (CNS) and the radiolabeling methods used in our laboratory. To assess the radioligands, results of in vitro autoradiography, biodistribution, and metabolite studies as well as PET imaging data are discussed. Furthermore, key PET parameters for kinetic modeling and quantification methods are provided. Two mGluR5 PET tracers, [11C]ABP688 and [18F]PSS232, were translated in our GMP labs and evaluated in human subjects. The newly developed GluN2B PET tracer [11C]Me-NB1 is currently being investigated in a first-in-human PET study and several F-18 labeled tracers are being evaluated in non-human primates in which the first-in-class will be translated for human studies

Neuroimaging with Radiopharmaceuticals Targeting the Glutamatergic System

Radiopharmacy at ETH has worked on the development of novel PET tracers for neuro-, cardiac- and tumor imaging for many years. In this paper, our efforts on targeting the glutamatergic system of the metabotropic glutamate receptor subtype 5 (mGluR5) and the ionotropic N-methyl-D-aspartate (NMDA) receptor are summarized. We briefly described the principles of positron emission tomography (PET) tracer development for the central nervous system (CNS) and the radiolabeling methods used in our laboratory. To assess the radioligands, results of in vitro autoradiography, biodistribution, and metabolite studies as well as PET imaging data are discussed. Furthermore, key PET parameters for kinetic modeling and quantification methods are provided. Two mGluR5 PET tracers, [11C]ABP688 and [18F]PSS232, were translated in our GMP labs and evaluated in human subjects. The newly developed GluN2B PET tracer [11C]Me-NB1 is currently being investigated in a first-in-human PET study and several F-18 labeled tracers are being evaluated in non-human primates in which the first-in-class will be translated for human studies.ISSN:0009-429

Neuroimaging with Radiopharmaceuticals Targeting the Glutamatergic System

Radiopharmacy at ETH has worked on the development of novel PET tracers for neuro-, cardiac- and tumor imaging for many years. In this paper, our efforts on targeting the glutamatergic system of the metabotropic glutamate receptor subtype 5 (mGluR5) and the ionotropic N-methyl-D-aspartate (NMDA) receptor are summarized. We briefly described the principles of positron emission tomography (PET) tracer development for the central nervous system (CNS) and the radiolabeling methods used in our laboratory. To assess the radioligands, results of in vitro autoradiography, biodistribution, and metabolite studies as well as PET imaging data are discussed. Furthermore, key PET parameters for kinetic modeling and quantification methods are provided. Two mGluR5 PET tracers, [11C]ABP688 and [18F]PSS232, were translated in our GMP labs and evaluated in human subjects. The newly developed GluN2B PET tracer [11C]Me-NB1 is currently being investigated in a first-in-human PET study and several F-18 labeled tracers are being evaluated in non-human primates in which the first-in-class will be translated for human studies

A first-in-man PET study of [18F]PSS232, a fluorinated ABP688 derivative for imaging metabotropic glutamate receptor subtype 5

PURPOSE: Non-invasive imaging of metabotropic glutamate receptor 5 (mGlu5) in the brain using PET is of interest in e.g., anxiety, depression, and Parkinson's disease. Widespread application of the most widely used mGlu5 tracer, [11C]ABP688, is limited by the short physical half-life of carbon-11. [18F]PSS232 is a fluorinated analog with promising preclinical properties and high selectivity and specificity for mGlu5. In this first-in-man study, we evaluated the brain uptake pattern and kinetics of [18F]PSS232 in healthy volunteers. METHODS: [18F]PSS232 PET was performed with ten healthy male volunteers aged 20-40 years. Seven of the subjects received a bolus injection and the remainder a bolus/infusion protocol. Cerebral blood flow was determined in seven subjects using [15O]water PET. Arterial blood activity was measured using an online blood counter. Tracer kinetics were evaluated by compartment modeling and parametric maps were generated for both tracers. RESULTS: At 90 min post-injection, 59.2 ± 11.1% of total radioactivity in plasma corresponded to intact tracer. The regional first pass extraction fraction of [18F]PSS232 ranged from 0.41 ± 0.06 to 0.55 ± 0.03 and brain distribution pattern matched that of [11C]ABP688. Uptake kinetics followed a simple two-tissue compartment model. The volume of distribution of total tracer (V T, ml/cm3) ranged from 1.18 ± 0.20 for white matter to 2.91 ± 0.51 for putamen. The respective mean distribution volume ratios (DVR) with cerebellum as the reference tissue were 0.88 ± 0.06 and 2.12 ± 0.10, respectively. The tissue/cerebellum ratios of a bolus/infusion protocol (30/70 dose ratio) were close to the DVR values. CONCLUSIONS: Brain uptake of [18F]PSS232 matched the distribution of mGlu5 and followed a two-tissue compartment model. The well-defined kinetics and the possibility to use reference tissue models, obviating the need for arterial blood sampling, make [18F]PSS232 a promising fluorine-18 labeled radioligand for measuring mGlu5 density in humans

Radiation dosimetry of [F]-PSS232-a PET radioligand for imaging mGlu5 receptors in humans

PURPOSE (E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone O-(3-(2-[F]-fluoroethoxy)propyl) oxime ([F]-PSS232) is a new PET tracer for imaging of metabotropic glutamate receptor subtype 5 (mGlu5), and has shown promising results in rodents and humans. The aim of this study was to estimate the radiation dosimetry and biodistribution in humans, to assess dose-limiting organs, and to demonstrate safety and tolerability of [F]-PSS232 in healthy volunteers. METHODS PET/CT scans of six healthy male volunteers (mean age 23.5 ± 1.7; 21-26 years) were obtained after intravenous administration of 243 ± 3 MBq of [F]-PSS232. Serial whole-body (vertex to mid-thigh) PET scans were assessed at ten time points, up to 90 min after tracer injection. Calculation of tracer kinetics and cumulated organ activities were performed using PMOD 3.7 software. Dosimetry estimates were calculated using the OLINDA/EXM software. RESULTS Injection of [F]-PSS232 was safe and well tolerated. Organs with highest absorbed doses were the gallbladder wall (0.2295 mGy/MBq), liver (0.0547 mGy/MBq), and the small intestine (0.0643 mGy/MBq). Mean effective dose was 3.72 ± 0.12 mSv/volunteer (range 3.61-3.96 mSv; 0.0153 mSv/MBq). CONCLUSION [F]-PSS232, a novel [F]-labeled mGlu5 tracer, showed favorable dosimetry values. Additionally, the tracer was safe and well tolerated

Structural requirements for novel coenzyme-substrate derivatives to inhibit intracellular ornithine decarboxylase and cell proliferation

Creating transition-state mimics has proven to be a powerful strategy in developing inhibitors to treat malignant diseases in several cases. In the present study, structurally diverse coenzyme-substrate derivatives mimicking this type for pyridoxal 5;-phosphate-dependent human ornithine decarboxylase (hODC), a potential anticancer target, were designed, synthesized, and tested to elucidate the structural requirements for optimal inhibition of intracellular ODC as well as of tumor cell proliferation. Of 23 conjugates, phosphopyridoxyl- and pyridoxyl-L-tryptophan methyl ester (pPTME, PTME) proved significantly more potent in suppression proliferation (IC50 up to 25 microM) of glioma cells (LN229) than alpha-DL-difluoromethylornithine (DFMO), a medically used irreversible inhibitor of ODC. In agreement with molecular modeling predictions, the inhibitory action of pPTME and PTME toward intracellular ODC of LN229 cells exceeded that of the previous designed lead compound POB. The inhibitory active compounds feature hydrophobic side chain fragments and a kind of polyamine motif (-NH-(CHX)4-NH-). In addition, they induce, as polyamine analogs often do, the activity of the polyamine catabolic enzymes polyamine oxidase and spermine/spermidine N(1)-acetyltransferase up to 250 and 780%, respectively. The dual-action mode of these compounds in LN229 cells affects the intracellular polyamine metabolism and might underlie the more favorable cell proliferation inhibition in comparison with DFMO.-Wu, F., Gehring, H. Structural requirements for novel coenzyme-substrate derivatives to inhibit intracellular ornithine decarboxylase and cell proliferation

Antibody-guided Molecular Imaging of Aspergillus Lung Infections in Leukemia Patients

This is the author accepted manuscript.Werner Siemens FoundationEuropean Union FP

Characterization and Analysis of the PikD Regulatory Factor in the Pikromycin Biosynthetic Pathway of Streptomyces venezuelae

The Streptomyces venezuelae pikD gene from the pikromycin biosynthetic cluster was analyzed, and its deduced product (PikD) was found to have amino acid sequence homology with a small family of bacterial regulatory proteins. Database comparisons revealed two hypothetical domains, including an N-terminal triphosphate-binding domain and a C-terminal helix-turn-helix DNA-binding motif. Analysis of PikD was initiated by deletion of the corresponding gene (pikD) from the chromosome of S. venezuelae, resulting in complete loss of antibiotic production. Complementation by a plasmid carrying pikD restored macrolide biosynthesis, demonstrating that PikD is a positive regulator. Mutations were made in the predicted nucleotide triphosphate-binding domain, confirming the active-site amino acid residues of the Walker A and B motifs. Feeding of macrolide intermediates was carried out to gauge the points of operon control by PikD. Although the pikD mutant strain was unable to convert macrolactones (10-deoxymethynolide and narbonolide) to glycosylated products, macrolide intermediates (YC-17 and narbomycin) were hydroxylated with high efficiency. To study further the control of biosynthesis, presumed promoter regions from pik cluster loci were linked to the xylE reporter and placed in S. venezuelae wild-type and pikD mutant strains. This analysis demonstrated that PikD-mediated transcriptional regulation occurs at promoters controlling expression of pikRII, pikAI, and desI but not those controlling pikRI or pikC