FZD Inhalt Life Science - HZDR · Small animal positron-emission tomography in radiation therapy response monitoring ... As for the Life Sciences research program scientists at OncoRay

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LIFE SCIENCES

TRIENNIAL SCIENTIFIC REPORT 2004 -2007 I Volume 3

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PREFACE

FOCUS

FACILITIES FOR EUROPE

The Radiation Source ELBE

The PET Center

Laser-Particle Acceleration

RESEARCH

PET-tracer development: from basic research to clinical applications

Small animal positron-emission tomography in radiation therapyresponse monitoring

Neuroendocrine hormone receptors as molecular targets for cancer diagnosis and therapy

Protein oxidation and disease

Radioactive metals for tumor therapy

Medical impact and beauty of cluster compounds

Moving targets - correcting patient movement in positron-emission tomography

In-beam PET for radiotherapy monitoring

Cell damage after X-ray irradiation

Biomolecular Switches: Molecular evolution conserves function, but allows diversification

FACTS & FIGURES

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Content

Cover picture: 3D scheme of the molecular structure of a multimeric neurotensin derivative(Holger Stephan, graphic: Sander Münster)

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Preface

This volume of the Triennial Scientific Report highlights the scientific output of the FZDresearch program “Life Sciences”, covering the years 2004 to 2006 and the first sixmonths of 2007. It is one out of three volumes that are published this year for the firsttime. The first part of this report introduces the “Life Sciences” program as well as thelarge-scale facilities that are used for research within this program. The second partconsists of ten articles on research projects that were conducted by scientists of theInstitute of Radiopharmacy and the Institute of Radiation Physics addressing currentresearch into cancer-related biomolecular function, imaging, and therapy.

The last eighteen months were characterized by an intense discussion about the future ofthe Forschungszentrum Dresden-Rossendorf (FZD). In meetings and seminars we debatedabout our topical status and our future, asking questions like: “What are our futurescientific objectives?” “Which research methods and facilities are required in order toreach those goals?” “How can new research activities be funded and who are our futurecooperation partners?”

As for the Life Sciences research program scientists at OncoRay - Center for RadiationResearch in Oncology - have become the most important cooperation partners. The FZDis one out of three members of this German Federal Ministry of Education and Research(BMBF) funded “Center for Innovation Competence”. It is actively engaged in promotingan ion-therapy center in Dresden, which would present a very promising location giventhe scientific background of the OncoRay center that is supported by the UniversityHospital Dresden and the Technische Universität Dresden on the one hand and theexpertise of FZD scientists on the other hand. For example, scientists from the FZDInstitute of Radiation Physics have developed an effective system of online-monitoring forthe ion-beam radiotherapy facility at the Gesellschaft für Schwerionenforschung (GSI) inDarmstadt. Another example: the Institute of Radiopharmacy performs tumor research onsmall animal models in close collaboration with OncoRay using a variety of tools andmethods at the molecular-imaging center for small animals of the FZD.

The newly established collaboration of the OncoRay center with the ultraoptics center inJena, which started at the beginning of 2007, promises new impulses for radiotherapy tocombat cancer in the future. This exciting “onCOOPtics” project, also funded by theBMBF, with laser physicists from Jena, radiotherapy experts from the University HospitalDresden, and scientists from the FZD, is dedicated to the fundamental understanding ofhigh-energy laser processes for acceleration of ions, which is required to explore their yet

Roland Sauerbrey I Scientific Director

PREFACE

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Prof. Dr. Roland Sauerbrey

unexploited potential for novel therapeutic approaches in the next decade. A high-intensity laser laboratory is currently under construction at the FZD and will be put intooperation in the beginning of 2008. The Laser-Particle Acceleration Group that isresponsible for this laser was founded in November 2006 and comprises six scientists andlaser engineers now. All these steps undertaken by us and our new cooperation partnersin Dresden and Jena are building blocks of our common vision to turn Dresden into anexcellent tumor research location designed to produce first-class scientific results in itsown right.

The new high-power laser facility at the FZD will at the same time strengthen theinterdisciplinary cooperation of scientists from different institutes of the FZD. For example,the Institute of Radiation Physics will investigate the effect of particle radiation producedby high-intensity lasers on living cells. Ph.D. work on dosimetry with respect to futureradiotherapy using high-intensity lasers has already started under the supervision ofscientists from OncoRay and the FZD. Scientific groups of the Institute of Ion-BeamPhysics and Materials Research as well as the Institute of Radiation Physics are interestedin basic questions of particle-matter interaction. Alternative concepts for the accelerationof particles like plasma wakefield will be the common focus of physicists from theRadiation Source ELBE and the Laser-Particle Acceleration Group. Especially laser drivenwakefield for the post-acceleration of ELBE electron bunches are to be mentioned here.

Finally, I would like to thank our partners in both the state and the federal government fortheir continued support, our national and international scientific cooperation partners formany successful joint research endeavours and, last but not least, the entire staff of theFZD for their multidisciplinary contributions to the improvement of human health usingstate-of-the-art physical approaches.

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Jörg Steinbach

The scientific activities in the Life Sciencesresearch program focus on molecularapproaches to tumor diagnostics andtherapy. This is motivated by the fact thatin Germany, malignant tumors account for25% of mortality. To be more specific,every 6th person between the ages of 20and 65 years dies from cancer. Today,cancer is treated with three fundamentaltherapies: surgery, radiation therapy, andchemotherapy. Curative treatment,however, fails for about half of tumorpatients. Radiation therapy may beimproved considerably by combiningtechnologically-advanced external

radiotherapy, radionuclide therapy, andmolecular targeting methods. The LifeSciences research program focuses oninvestigations related to the disease-relevant behavior of cells and tissues aswell as biomolecular structure anddynamics using in vitro studies, in vivoexperiments, and clinical studies inpatients. Additional research aims toimprove diagnostics and therapy. The Institutes of Radiopharmacy andRadiation Physics contribute to thisresearch program. They bring togetherexpertise from radiopharmaceuticalscience, radiation physics, and spectros-copy using a unique combination ofresources, such as the Radiation Source

ELBE and the PET facility in combinationwith Radiopharmaceutical Chemistry andRadiopharmaceutical Biology at the FZD.

(i) Radiopharmaceutical tumor research A detailed understanding of cancer-relatedmolecular and cellular processes is requiredto develop new methods for sensitive andspecific diagnostics and therapy. Therefore,a major goal is to improve MolecularImaging and Therapy of Tumorscomplemented by metabolic researchbased on radiopharmaceutical methods. In so doing, we aim to identify new bio-molecules which may be used as targetsfor radioactively-labeled tracers in

Life Sciences program at the Forschungszentrum Dresden-Rossendorf

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FOCUS

diagnostics or therapy of solid tumors,metastases, or inflammatory processes.Eventually, we will develop new targetingconcepts for drugs labeled with particle-emitting radionuclides. Their radiation iscapable of destroying tumors, but spareshealthy tissue. Also, we are investigatingdiagnostically and therapeutically relevantradionuclides, in particular particleemitters.

Preclinical studies for pharmacologicalcharacterization of appropriate substancesand predrugs are part of these researchactivities. The studies include differentkinds of molecular imaging in animalmodels of disease in order to investigateprocesses in vivo and to enable thecharacterization and preclinical applicationof newly developed substances. Yet,molecular imaging generates largeamounts of data. Data handling and imagecorrection, i.e., for patient movement, aretherefore both important parts of ourwork. In order to translate our researchresults to a clinical environment, the FZDand the Technische Universität Dresden are collaborating in a common PET centerlocated on the grounds of the FZD.

(ii) Radiotherapy monitoring and radiation damageToday, more than 50% of tumor patientsreceive radiation treatment—and thatnumber is rising. The crucial challenge ofradiotherapy is to destroy the tumorcompletely while saving the surroundinghealthy tissue. Yet, in many cases radio-therapy, which is based on photon orelectron beams delivered by compactelectron linear accelerators, fails.Therefore, new technologies for gener-ating and monitoring radiotherapy beamsneed to be developed and transferred toclinical application, which is the subject of our research. Due to their favorablephysical and radiobiological properties, ionbeams have recently started being used inradiotherapy. A unique method for image-guided radiotherapy, in-beam PET, isapplied to clinical ion beam treatments. Weare also investigating whether other kindsof radiation like ultra-hard photons can beapplied to cancer treatment. Moreover, the

radiobiological properties of new radiationqualities which are interesting for medicalapplication are tested by in vitro experi-ments with both tumor and normal tissuecells. Currently, the main experimentalfacility used for these experiments is theELBE radiation source delivering novelunconventional beams, such as veryintense ultra-short pulsed electron beamsand monochromatic tunable X-rays. In thefuture, we also plan to investigate laser-accelerated particle beams with respect totheir potential for cancer treatment.

(iii) Dynamics of membrane receptor structureStructural transitions within biopolymers,such as DNA and proteins, play key roles in many physiological disorders includingcancer. Detailed knowledge of thesemolecular switching processes isparticularly required for membraneproteins. Here, structural information isvery limited and especially desired for G-protein-coupled receptors (GPCRs).They are targeted by 50% of the currentpharmacotherapeutics and are involved inthe genesis of various cancerous diseases.Using infrared spectroscopy, functionally-relevant structural transitions are studied in real time to systematically understandthese molecular switching mechanisms inmembrane proteins. New strategies forGPCR-based diagnostics and therapies are being explored in collaboration withindustrial partners.

All these activities are primarily embeddedin a scientific network with the Faculty ofMedicine and the Faculty of Mathematicsand Natural Sciences of TechnischeUniversität Dresden. The scientific programis coordinated in close cooperation withthe Center of Radiation Research inOncology (OncoRay®) which is jointlyoperated by Technische UniversitätDresden, FZD, and the University HospitalCarl Gustav Carus Dresden and hosted atthe Faculty of Medicine of TU Dresden.We are also a member of the Center forRegenerative Therapies Dresden which isincreasingly important for our futurescientific cooperation.

Photo: Jürgen Lösel

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Jörg Pawelke, Karim Fahmy

The basic component of the RadiationSource ELBE (Electron Linear acceleratorwith high Brilliance and low Emittance) is asuperconducting electron linear acceleratorwhich provides an electron beam with anaverage current of up to 1 mA and beamenergy of between 5 and 40 MeV. Theparticular properties of this primary beamare its low transverse emittance (i.e., anearly parallel beam of small spot size),short (~ps) pulses, low energy spread, and flexible temporal structure. Theseoutstanding properties allow a variety ofsecondary radiations for experiments inbasic as well as applied research. In the LifeSciences program, experiments are focusedon the Biostructures and Radiation

research area utilizing (i) intense infraredlight beams delivered by a free-electronlaser (FEL) for biophysics research and (ii)novel unconventional beams of ionizingradiation which are of potential medicalinterest for tumor diagnostics and therapy.

Monitoring and manipulatingbiomolecular structures by FEL infrared light The high IR-pulse repetition (13 MHz) and,correspondingly, the high energy which isemitted with low divergence from theELBE-FEL allow biophysical studies wherethese parameters are critical. For example,it has been shown that the high energy ofthe ELBE-FEL even enables the chemicalcharacterization of single layers ofbiomolecules on reflecting surfaces using

IR reflection absorption spectroscopy. Dueto the narrow spot size of the IR beam andits well-defined incident angle, suchmeasurements can be performed with aspatial resolution close to the diffractionlimit. In combination with the intrinsicallyhigh time-resolution of the FEL, this willallow sensitive experiments on faststructural transitions in monomolecularlayers on solid supports, as they areapplied in many areas of biotechnology. Inaddition, it has been shown that disease-related biomolecules, such as DNA, can bestructurally manipulated using FEL light(Fig. 1). Current research utilizes variousFEL-pulse regimes to generate layers ofDNA with spatially defined structuralalterations which are of great interest forbiotechnological applications.

The Radiation Source ELBE

Jörg Pawelke and Anna Lehnert at the X-ray beamline at ELBE.

Facilities for Europe


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New radiation qualities for radiooncologyIn some cases, state of the art radiotherapy fails to destroy the tumor completely whilesaving the surrounding healthy tissue. This requires new technologies in generating,forming, and monitoring radiotherapy beams which are developed with the help ofexperiments at ELBE. For this purpose, new radiation qualities are utilized. Theirradiobiological properties must be investigated for both tumor and normal tissue cells.Both the new radiation qualities and ELBE experiments have proven advantageous inseveral ways: (i) beams of ultra-hard photons can potentially be better focused on thetumor; they are monitored by the in-beam PET method at ELBE. (ii) The high-currentelectron beam of ELBE allows unique experiments of split-dose cell irradiation. This meansthat a priming radiation exposure is followed by re-irradiation after a delay lasting for anydesired time period from milliseconds up to a few minutes. Priming radiation exposure isfollowed by the early steps of DNA damage recognition and repair, which brings aboutenhanced cell killing after re-irradiation. But the radiobiological consequences of shortpulse irradiation can also be investigated. This is interesting for future compact particleaccelerators which are developed on the basis of high-intensity laser systems. Such anaccelerator will deliver ultra-short and intense bunches of particles comparable with thoseof the ELBE electron beam. (iii) The low emittance of the electron beam allows generatingchanneling radiation, i.e., monochromatic X-rays, which is tunable in the 10 to 100 keVrange (Fig. 2). X-rays in this energy range are widely used in medical diagnostics, andexperiments at ELBE can provide photon-energy resolved information on basicradiobiological effects and mechanisms following an exposure of living cells to X-rays.

Fig. 2: Beamline of thechanneling X-ray facility at ELBE.The electron beam coming fromthe ELBE accelerator (from theleft) and entering the vacuumchamber (in the middle) hits asingle diamond crystal.Channeling radiation is emittedby the relativistic electrons whentraveling through the crystalalong a lattice plane.

Fig. 1: The Brewster-angle-microscopic image ofa DNA film of 1 µm thickness. The central area of 40 µm diameter was exposed to IR-FELmacropulses at a wavelength of 9 µm which areabsorbed by the chemical groups of the DNAbackbone. Structural transitions induced bypicosecond micropulses at a repetition rate of 13 MHz accumulate during the 400 µs longmacropulse causing an almost linear increase inreflectivity (bottom). After exposure to themacropulse, the relaxation of the DNA structureproceeds on a ms time scale (top).

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Jörg van den Hoff

The Positron Emission Tomography (PET)Center is jointly operated by the FZD andthe Technische Universität Dresden. Theinfrastructure comprises a dedicatedcyclotron, GMP (Good ManufacturingPractice) approved radiopharmaceuticallaboratories, and several tomographs: ahuman PET tomograph and, for smalllaboratory animals, dedicated tomographsfor PET, magnetic resonance imaging andspectroscopy (7 Tesla MRI/MRS), andcomputed tomography (CT).

As its central resource, the PET Centerprovides molecular imaging capabilities fornoninvasive in vivo investigation ofphysiological and biochemical processesrelated to different pathophysiologicalstates, notably in tumor diseases. The PETtechnique is able to noninvasively deliverquantitative information on cellulartransport processes and metabolism (tissueperfusion, distribution volumes, turnoverrates, and so on) in an unparalleled way.PET utilizes radioactive tracers at the nano-and picomolar concentration level, thus

excluding any interference of the measure-ment with the systemic metabolism. PETaccurately provides the three-dimensionaldistribution of the tracer concentrationwith a spatial resolution of better than2 mm in the case of dedicated small animaltomographs. Performing these measure-ments allows assessing the time depend-ence of the tracer distribution and, thus,quantification of relevant pharmacologicalparameters.

Research at the PET Center is focused oninvestigations of transport, metabolism andsignal transduction in normal tissue as wellas damaged and tumor tissues with thePET method. Our multi-disciplinaryresearch group of radiochemists, bio-chemists, biologists, physicists, softwareengineers, and physicians addresses thistask by the development of new PETtracers, biological characterization of theirproperties, multi-modal imaging of smallanimal tumor models with new andestablished tracers, development of newimage data acquisition and evaluationtechniques, and, finally, clinical studies inhumans.

An important part of this work is toadvance biologically individualized,technically optimized radiotherapy, whichis also the main goal of the Center forRadiation Research in Oncology“OncoRay” recently founded in Dresden(the FZD being a founding member). Inthis context, our efforts are focused on thesystematic evaluation of various tracers forassessing tumor vitality and radiationsensitivity and the subsequent seamlessintegration of the quantitative informationderived from PET into radiation treatmentplanning and therapy response control.Secondly, research at the PET Centerfocuses on the investigation of patho-mechanisms of metabolic and inflamma-tory diseases such as the metabolicsyndrome. Apart from these researchprojects, our imaging techniques are alsoutilized in pre-phase I clinical trials for drugdevelopment in collaboration with thepharmaceutical industry.

The PET Center

Chemistry student Katrin Müller in a radiopharmaceutical laboratory.


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Ulrich Schramm

Over the past several decades, particleaccelerators have developed into versatiletools for many aspects of science. Theyprovide beams for such diverse areas asbasic research, brilliant light sources, aswell as diagnosis and therapy in lifesciences. However, acceleration andespecially transport of energetic particlebeams requires large installations, whichhas always fostered great interest in noveltechniques which could circumvent theserestrictions. Within just the last few years,laser-particle acceleration—a techniquewhich uses relativistic laser plasmainteraction to provide accelerating fieldswhich exceed conventional ones by at leastthree orders of magnitude—has maturedin a way that allows us to envision the firstapplications. High-energy (GeV) electronbeams of only a few millimeters as well asintense high-quality ion bunches havebeen generated.

Therefore, FZD decided to establish a high-power laser laboratory planned to deliverthe first 100 Terawatt laser pulses totargets by early 2008. The laboratory hasbeen installed in close proximity to theELBE accelerator in order to bundle theexpertise in state-of-the-art supercon-ducting accelerator and gun and the laseraccelerator technology.

The most ambitious goal of this group is todevelop a compact and reliable laser-ionaccelerator which can be positioned closeto an irradiated object, thus eliminating theneed of costly ion beam lines. Developmentof dedicated laser targets as well as ofhigh-power laser technology will beperformed in-house. Within the frameworkof onCOOPtics—a cooperation projectbetween laser experts from Jena andoncologists from Dresden funded by theFederal Ministry of Education and Research(BMBF)—irradiation of tissue probes withlaser accelerated particles will be studied sothat at last a compact machine for cancertherapy could be envisioned.

In addition to the scattering of laserphotons from ELBE, accelerated electronswill be exploited as a variable and fullysynchronized source of soft X-rayradiation. Unprecedented photon yield canbe expected from combining the ultra-brilliant ELBE photo-gun, which is presentlybeing developed with a laser beam of amatched repetition rate. A source like thiswill pave the way for time-resolved X-rayimaging and future diagnosis techniques,such as phase contrast imaging. It will be developed in collaboration with theFraunhofer Institute for Applied Optics and Precision Engineering (IOF) in Jena.

Stefan Bock preparing laser experiments.

Laser-Particle Acceleration (Laboratory)

RESEARCH INSTITUTE OF RADIOPHARMACY

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Cathleen Haase, Ralf Bergmann, Jens Pietzsch

In recent years, positron-emissiontomography (PET) has evolved as avaluable imaging modality in oncology,neurology, cardiology, rheumatology, andother medical branches. In oncology, forinstance, PET has become an essential toolfor displaying cancer, monitoring treatmentresponse, investigating tumor recurrence,and for objective assessment of overalltherapeutic efficiency. Apart from theseclinical applications, PET plays an ever-increasing role in basic and applied tumorresearch due to its inherent property ofdepicturing and functional characterizationof physiologic, metabolic, and molecularpathways in the living organism in aquantitative manner. Several radiopharma-ceutical compounds have been developedfor PET which have proven suitable forimaging and functional characterization oftumors. In this regard, amino acid-basedPET tracers are especially important. It iswell known that the uptake of amino acidsis increased in tumor tissue compared tonormal “healthy” tissue. Among variousavailable amino acid-based radiopharma-ceuticals, the fluorine-18-labeled aminoacid “3-O-methyl-[18F]fluoro-L-DOPA”([18F]OMFD; Fig. 1), which has been

developed and evaluated by the Instituteof Radiopharmacy at FZD, is a verypromising PET tracer and has already beensuccessfully applied in brain tumor imagingin close cooperation with the Clinic andPoliclinic of Nuclear Medicine at theUniversity Hospital Carl Gustav CarusDresden (Fig. 2) [1, 2].

The aim of our current research is todetermine whether [18F]OMFD also haspotential in the diagnosis of clinicallyrelevant tumor entities other than braintumors. Moreover, we evaluated thepotential of [18F]OMFD for differentiationof tumorigenic and inflammatory

processes, which is an important challengein nuclear medicine. Furthermore, we focuson cellular and molecular characterizationof different tumor entities in vitro and invivo, thereby gaining new insights into themechanisms of proliferation and geneexpression. We investigated the mainamino acid transport systems for theuptake of [18F]OMFD in various cells, e.g.,in human head and neck squamous cellcarcinoma cells (FaDu) and in the phorbolester stimulated human monocyte/macrophage cell line (THP-1). We alsostudied [18F]OMFD uptake in thecorresponding tumor (FaDu) xenograftmodels in nude mice in vivo. For molecular

PET-tracer development: from basic research to clinical applications

Research

Fig. 1: Chemical structure of the fluorine-18-labeled amino acid derivative 3-O-methyl-6-[18F]fluoro-L-DOPA ([18F]OMFD).

Fig. 2: Positron emission tomography (frontal, transversal and sagittal projection) of the brain with[18F]OMFD: brain tumor recurrent (astrocytoma) 4 years after surgical resection and postoperativeradiotherapy. The figure shows the distribution of 18F radioactivity, the red color indicating the highestradioactivity.

Fig.3: Time course of [18F]OMFD(2 MBq/mL) uptake into tumorFaDu cells and THP-1 macro-phages.*P < 0.05.

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characterization of the amino acidtransport systems, detailed quantitativegene expression analyses of the differenttransporter subtypes within the so called L-amino acid system (L-system; LAT1,LAT2, LAT3, LAT4 and 4F2hc subtype) and

the alanine-serine-cysteine system (ASCsystem; ASC1 and ASC2) were performed.

It was demonstrated that the uptake of[18F]OMFD in all cell lines tested wasmediated mainly by the sodium-independent high-capacity L-system.Kinetic analyses in cells demonstrated anincreased uptake in the human head andneck squamous cell carcinoma (FaDu) cellsin comparison to the THP-1 cells (Fig. 3).The latter were used as a model for tumor-associated (proinflammatory) macro-phages. The accumulated radioactivity in the FaDu cells was three times higher than in THP-1 cells (Fig. 3). The relativeexpression level of the L-amino acidtransporter subtypes LAT1 and LAT4 inFaDu tumor cells and the correspondingFaDu xenografts was significantly higherwhen compared to THP-1 macrophages(Fig. 4) [3]. These data gave rise to theassumption that [18F]OMFD is not only agood tracer for brain tumors but also forother, especially poorly-differentiatedtumor entities, e.g., head and necksquamous cell carcinoma. To substantiatethis assumption, small animal PET studieswere performed in tumor-bearing micexenotransplanted with human head andneck carcinoma cells (FaDu), also revealinga high [18F]OMFD uptake in tumor tissue(Fig. 5) [3].

In conclusion, an important finding is that[18F]OMFD offers the opportunity to studythe specific L-amino acid transport systemin certain tumor entities overexpressingthis transport system with PET in vivo. The use of [18F]OMFD may allow

quantification of important cellularprocesses related to tumor proliferation in these tumor entities. However, thecontribution of endothelial cells andproinflammatory cells like macrophagesand the role of different LAT subtypes onthe overall uptake of [18F]OMFD in tumorsand inflammatory lesions must beresearched further.

Rossendorf Beamline

Environment and Safety

Structure of Matter

Life Sciences

TOPFLOW FacilityHigh Magnetic Field Lab.

PET Center

Ion Beam Center

Radiation Source ELBE

Fig. 4: LAT1 and LAT4 geneexpression studies in FaDutumor cells and the corres-ponding xenografts, and THP-1macrophages using quantitativereal-time Polymerase ChainReaction. Arithmetic chart ofthe threshold cycle (Ct) forLAT1 and LAT4 was normalizedto house-keeping-gene 18S-rRNA expression. *P < 0.05.

Fig. 5: Representative small animal positronemission tomography image (maximumintensity projection) of a mouse human-headand neck squamous cell carcinoma xenograftmodel (FaDu) after intravenous administrationof 10 MBq of [18F]OMFD (60 min p.i.). Thehighest radioactivity concentration can beobserved in the tumor and in the pancreas. Thelatter is of particular interest and is hypothesizedto indicate high amino acid transport activity inthis organ due to increased amino acidrequirement for the synthesis of severalhormones and enzymes.

References*[1] Radiotherapy Treatment Planning in

Brain Tumors with 18F-3-O-methyl-fluordopa (18F-OMFD) and Positron Emission Tomography (PET), H. Alheit1, L. Oehme1, C. Winkler1, F. Füchtner, A. Hoepping2, J. Kotzerke1, B. Beuthien-Baumann1, European Journal of Nuclear Medicine and Molecular Imaging (2007), submitted

[2] Diagnostic impact of PET with (18)F-FDG, (18)F-DOPA and 3-O-methyl-6-[18F]fluoro-DOPA in recurrent or metastatic medullary thyroid carcinoma, B. Beuthien-Baumann1, A. Strumpf1, J. Zessin, J. Bredow1, J. Kotzerke1, European Journal of Nuclear Medicine and Molecular Imaging 34 (10), 1604 – 1609 (2007)

[3] L-Type Amino Acid Transporters LAT1 and LAT4 in Cancer: Uptake of 3-O-Methyl-6-[18F]fluoro-L-Dopa (18F-OMFD) in Human Adenocarcinoma and Squamous Cell Carcinoma in vitro and in vivo, C. Haase, R. Bergmann, F. Fuechtner, A. Hoepping2, J. Pietzsch, Journal of Nuclear Medicine (2007), in press

Project partners1Clinic of Nuclear Medicine, University Hospital Carl Gustav Carus Dresden, Germany

2ABX Advanced Biochemical Compounds GmbH, Radeberg, Germany

*In this report we quote mainly the most important papers that were published by FZD scientists and their partners.


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Ralf Bergmann, Jens Pietzsch, Bettina Beuthien-Baumann1,2

Tumors are abnormal masses of tissue that result from unusually high cell divisionor retarded cell death.A fundamental prop-erty of neoplasia is the Warburg effect,which means that tumors have a highmetabolic rate and accumulate glucose at a higher rate than normal tissue. 2-[18F]fluoro-2-deoxy-D-glucose([18F]FDG) Positron-Emission Tomography(PET) assesses this tumor property.[18F]FDG is an analog of deoxyglucose that has been employed for tumor imagingusing the radioactive label 18F (110 minhalf-life) (Fig. 1). The molecular

[18F]FDG-PET imaging technique offers acomplementary approach to anatomicimaging, such as computer tomographyand magnetic resonance imaging, and ismore sensitive and specific in detectingcertain cancers. [18F]FDG-PET has beenwidely applied in oncology, primarily as astaging and restaging tool that can guidepatient care. But as it accurately detectsrecurrent or residual diseases, [18F]FDG-PET also has significant potential forassessing therapy response. In this regard,it can improve patient management byidentifying responders at an early stagebefore the tumor size is reduced; incontrast, nonresponders could discontinuefutile therapy. Moreover, the reduction of

the [18F]FDG-PET signal within days orweeks after initiating therapy (e.g., inlymphoma, non-small cell lung, andesophageal cancer) significantly correlateswith prolonged survival and other clinicalendpoints. Applications of [18F]FDGinclude assessing response to therapy alsofor tumor models. [18F]FDG can thereforebe used to test the efficacy of radiationtherapy, including preclinical animalstudies. Serial studies are potentially usefulto determine if the therapy inhibits tumormetabolism and growth.

A tumor starts to grow from a singletransformed cell. This is also the case fortumors used in our preclinical research that

Small animal positron-emission tomographyin radiation therapy response monitoring

Ralf Bergmann at the MICRO-PET device of the FZD.

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were transplanted to immunocompromisedmice. However, shortly after the tumorshad been transplanted to the host animals,they differed considerably in size, in thelocalization of the growing tissue inside thetumors, in the amount of blood vessels inthe tumors, in oxygen consumption, andother functional and molecular parameters.Increased glucose accumulation in thetumor is associated with the rate oftransport across the cell membrane, theactivity of hexokinase, and the rate ofdephosphorylation in the tissue. Thetransport of [18F]FDG across cellmembranes is mediated by structurally-related proteins constituting a family ofglucose transporters, Glut-1 to Glut-5.Significantly elevated expression levels of Glut-1 and Glut-3 are believed tocontribute to the accumulation of [18F]FDGin malignant tumors. It has also beensuggested that the activity level ofhexokinase-II (HKII) influences [18F]FDGaccumulation in various malignant tumors.Tumor tissues also show intratumoralheterogeneity in their various propertieswhich may originate from the diversephenotypic properties of tumor cells ormay be induced by their metabolicmicroenvironment. In this regard,intratumoral heterogeneity of [18F]FDGdistribution has been well demonstrated.However, there has been little informationon the biologic mechanisms involved in theintratumoral heterogeneous distribution of [18F]FDG. Moreover, the relationshipsbetween the intratumoral distribution of[18F]FDG and the response to X-raytherapy remain to be investigated. Thesedata provide the biological basis fordiagnosing, staging, and prognosticatingmalignant tumors and monitoring therapyresponse by [18F]FDG.

Our objective was to investigate theradiation response of tumors with identicalgenetic background in relation to theamount and heterogeneity of [18F]FDGuptake (Fig. 2). Research has been partlysupported by the EU BioCare project(“Molecular Imaging for BiologicallyOptimized Cancer Therapy”). Human headand neck squamous cell carcinoma (hSCC;origin FaDu cells) with a diameter of 7 mmxenotransplanted to nude mice wereincluded in the investigations. The tumor

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Structure of Matter

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PET Center

Ion Beam Center


Fig. 1: Schema of the [18F]FDG-uptake in the cells. The glucosetransporters transport the neutral[18F]FDG through the cellmembrane, like glucose. Thehexokinase phosphorylate the[18F]FDG using adenosinetriphosphate (ATP) to adenosinediphosphate and [18F]FDG-6-phosphate, which is an anionthat remains inside the cells. Theaccumulation of [18F]FDG in thecells is mainly correlated to thefunctional expression of theglucose transporters and thehexokinase.

Fig. 2: [18F]FDG distribution in HT-29 and FaDu tumors one hour after injection.

Fig. 3: Actuarial local control rates for FaDu-tumors after irradiation with single doses under ambientconditions stratified according to FDG uptake. Tumors with SUVmax (a) below median value, (b) above median. Solid lines: 35 Gy, dotted lines: 25 Gy.


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uptake of [18F]FDG was measured withoutanesthesia immediately prior to irradiationas well as one, four, and eleven days afterirradiation. The radiotracer uptake wasdetermined as a maximum standardizeduptake value (SUVmax), i.e., the con-centration of [18F]FDG in the tumornormalized to the injected dose and to thebody weight. This allows a comparison ofthe data of animals with different bodyweights. Single-dose irradiations with lowand high amounts were applied undernormal blood-flow conditions using X-rays.The mice were observed for 120 days afterirradiation. This long time period guaran-tees identification of the reaction of thetumor to therapy, for instance whether thetumors have shrunk, which is quantified as tumor control. This is an authentic“clinical” end point and not a surrogate. In analyzing the animals, the tumor controlprobability after irradiation with the lowerdose (25Gy) was significantly lower thanafter irradiation with the higher dose(35Gy). The animals were divided into twogroups, i.e. animals with pretreatment[18F]FDG uptake in the tumor higher thanthe median SUVmax (1.59) and the groupwith [18F]FDG uptake in the tumor lowerthan this. In tumors with [18F]FDG uptakeless than the median SUV(max), localcontrol was 37 % after 25Gy whereas itamounted to 47 % after 35Gy. In contrast,substantial differences in local tumorcontrol were found in tumors with FDGuptake above the median SUV(max)(Fig. 3). Multivariate Cox analysis revealeda significant decrease of the recurrencehazard with an increasing dose andSUV(max).

To characterize the functional differencesand relations in the tumor tissue, the[18F]FDG uptake was also compared on the tissue level by coregistration of quan-titative autoradiography and functionalhistological images. The [18F]FDG uptakein xenotransplanted squamous cellcarcinoma was higher than in human

adenocarcinoma (HT-29 cells) (Fig. 2). InhSCC tumors, the [18F]FDG uptake wasincreased in hypoxic and proliferatingtumor regions compared to vital regionswithout signs of hypoxia and proliferation.However, the [18F]FDG uptake in humanadenocarcinoma did not show any de-pendence on the microenvironment in the vital tumor regions.

The characterization of the tumor hetero-geneity by animal PET in xenotransplantedmice has been extended to the imaging ofhypoxia using [18F]Fluoromisonidazole([18F]FMISO). These investigations are stillunderway. In summary, tumors are veryheterogeneous, not only with regard totheir various origins, but also in terms ofintratumoral differentiation. We were ableto show that hSCC and adenocarcinomadiffer in the [18F]FDG uptake and thedevelopment of hypoxic regions (Fig. 4).Due to this, tumors also respondeddifferently to radiation therapy. Moreover,we could show that in our model systems,the [18F]FDG uptake is related to thetherapy response of the tumors. This sup-ports the hypothesis that pre-treatment

[18F]FDG-PET may provide usefulinformation for individual treatment;specific radiation doses can be prescribedto single tumor areas.

References[1] Effect of increase of radiation dose on

local control relates to pre-treatment FDG uptake in FaDu tumours in nude mice, C. Schütze1, R. Bergmann, A. Yaromina1, F. Hessel1, J. Kotzerke2, J. Steinbach, M. Baumann1, B. Beuthien-Baumann1,2, Radiotherapy and Oncology 83 (3), 311 – 315 (2007)

Project partners1OncoRay – Center for Radiation Research in Oncology, Department of Radiation Oncology, Faculty of Medicine, Technische Universität Dresden, Germany

2Department of Nuclear Medicine, Faculty of Medicine, Technische Universität Dresden, Germany

Fig. 4: Autoradiographic andfunctional histological images ofhSCC tumor sections. The imagesrepresent the [18F]FDG-uptake(autoradiography), the histology,and the marked regions as masksfor the vital, necrotic, and hypoxicregions (left). The right side showsthe overlaid masks with theautoradiographic image.

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Ralf Bergmann, Frank Wuest, Jens Pietzsch

Neurotensin and its receptor subtypesExperimental and clinical data indicate thatG-protein-coupled peptide hormonereceptors such as neurotensin (NT) play acrucial, but often not fully-appreciated rolein the genesis of tumors and metastases.The imaging of these G-protein-coupledpeptide hormone receptors and thefunctional information derived from it will assist in tumor localization, define or predict tumor histology, identifymetastases, and plan, guide, and monitortherapy [1]. A particularly devastatingdisease with very poor prognosis is thepancreatic ductal carcinoma. This tumorurgently requires novel approaches forprecise and more effective molecularly-targeted diagnosis and therapy. As thistumor expresses NT receptors, welaunched a research project aimed at

developing new radiopharmaceuticals formolecular imaging of these neurotensinreceptors [2 – 7]. This long-standingproject was initiated within the EUBIOMED 2 framework and has created ascientific network of fruitful collaborationwith various European partners, particu-larly with the Department of OrganicChemistry of the Vrije Universiteit Brussels.

Neurotensin is a tridecapeptide (pGlu1-Leu2-Tyr3-Gly4-Asn5-Lys6-Pro7-Arg8-Arg9-Pro10-Tyr11-Ile12-Leu13-OH) produced inthe central nervous system. It is mainlyfound in the gastrointestinal tract inperipheral tissues. The pharmacologicaleffects of neurotensin [2] result from thespecific interaction of the peptide with cell-surface neurotensin receptors (NTR).Neurotensin receptors are described as twoG-protein-coupled receptors NTR1 andNTR2, and the non-G-protein-coupledreceptor NTR3 which is identical with gp95

sortilin. Neurotensin is rapidly degraded inblood plasma by endogenous peptidasesand proteases. Several proteolytic enzymeshave been reported to cleave intactneurotensin.

Initial studies were aimed at the metabolicfate of neurotensin. For this purpose, theN-terminus of the NTR-binding NT(8-13)fragment was successfully radiolabeledwith N-succimidyl-4-[18F]fluorobenzoate([18F]SFB). Several metabolites could beidentified, whereas expected catabolites ordecomposition products like [18F]fluoro-benzoic acid and [18F]fluoride were notdetected in plasma or urine. As a conse-quence, we proposed a new metabolismscheme of [18F]FB-NT(8-13) based onthese data (Fig. 1) [2].

Neurotensin receptors in cancerNeurotensin receptors 1 and 3 areexpressed in various tumor cell linesincluding small cell lung cancer,neuroblastoma, pancreatic, and coloniccancer (HT-29) (Fig. 2). Binding anduptake studies on human adenocarcinomatumor cell lines showed that afterinteraction with the peptide, substantialamounts of the receptors were internalized(Fig. 3). Visualization of binding andinternalization of a fluorescent probe of a neurotensin analogue were carried outby confocal microscopy (Fig. 4). Theinvestigations were complementedadditionally by two-dimensional massspectrometry of tumor sections (Fig. 5). As is generally known, neurotensinreceptors occur abnormally often in avariety of primary human tumors, such asmeningioma, Ewing’s sarcoma, and ductalpancreatic carcinoma. But neurotensinreceptors are also found, though less

Neuroendocrine hormone receptorsas molecular targets for cancer diagnosis and therapy

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Fig. 1: Metabolism of [18F]FB-NT(8-13).


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often, in astrocytoma, medulloblastoma,medullary thyroid cancers, and small-celllung cancers. These neoplasms displayNTR1 receptor proteins, as well as NTR1mRNA. However, NTR1 has rarely beenfound in a number of other cancer entities[1]. The neurotensin peptide itself appearsto be expressed by numerous tumors ortumor cell lines. For additional biochemicalstudies, we cloned the NTR1 gene,expressed it in an E. coli system, andisolated the protein. The NTR1 protein willbe used as target for the production ofspecific antibodies. Later, we plan toperform targeting studies.

New radiolabeled neurotensinanaloguesFor the quantitative characterization of NT analogues, we established chemical,radiochemical, biochemical, as well asradiopharmacological and imagingmethods. The animal facility of theInstitute of Radiopharmacy at FZD is fullyequipped for maintaining immunodeficientand genetically-engineered small animals.The necessary tumor models (Fig. 6) wereestablished and produced in closecooperation with the Centre for RadiationResearch in Oncology “OncoRay” and the Department of Radiooncology, bothlocated at the Faculty of Medicine ofTechnische Universität Dresden.

To increase the biological half-life ofneurotensin analogues as one prerequisiteof higher tumor accumulation, wefollowed two strategies: (1) synthesis ofstabilized peptides by insertion of non-natural amino acids and formation ofpseudo-peptide bonds, and (2) develop-ment of multivalent NT(8-13) derivatives[4-6]. Moreover, the increased molecularweight will reduce the glomerular filtrationin the kidneys. Two of the new multimericNT(8-13) tetramers [5] showed an in vitrobinding affinity towards the NTR1 com-parable to that of the parent compoundNT(8-13) (Fig. 7). In the current project,new labeling approaches based onprosthetic group chemistry and metalcomplexes for positron and single-photonemission tomography will be used toperform extensive radiopharmacologicalstudies.

Fig. 2: Expression ofNTR1 in cancer celllines on (A) mRNAlevel in FaDu, HT-29, WiDr and(B) protein levelusing 2D-electro-phoresis of HT-29cell proteins.

Fig. 3: Internalization kinetics of adouble stabilized 18F-labeled NT-analogue.

Fig. 5: Image fusion of theautoradiogram of 3H-labeled NT(green) and the distribution ofNT(8-13) measured by 2D massspectrometry (MALDI-TOF; red)on a HT-29 tumor section (left)and the corresponding histologicalimage (right), cooperation withGerald Steiner and Rainer Salzer(Institute of Analytical Chemistry,Technische Universität Dresden).

Fig. 4: Localization of afluorescence-labeled NT analogue(green) on HT-29 cells(autofluorescence, blue) (bycourtesy of Thomas Hanke,Institute of Materials Science,Technische Universität Dresden).

A) B)

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Future research on neurotensinreceptors for diagnosis and therapyThe aim of our future research is to testthe following working hypotheses: (i) functional expression of the NT receptoris characteristic of tumor stem cells, (ii) newly developed multimeric neu-rotensin congeners have an increasedaffinity to the receptors, and (iii) they alsoshow an increased metabolic stability bypreserving at least one intact peptide chain for receptor binding if proteolyticdegradation in the organism occurs.

The internalization of the agents will allowtargeting of intracellular structures with ß-emitting radionuclides. The metalcomplex analogues are potential agents for therapeutic applications when labeledwith 90Y, 117Lu, and 188Re.

The progress made so far with in vitroexperiments in the model of pancreastumor (HT-29) and the well-establishedscientific network are the basis forsuccessful preclinical in vivo studies on the role and functional expression of

neurotensin receptors in tumors bymolecular imaging. This will also lead toadditional therapeutic opportunities,especially for small and heterogeneoustumors. Research on the role of theneurotensin receptors in cancer and thedevelopment of radiolabeled diagnosticsand therapeutics represent an excitingchallenge and should thus be extended toother neuroendocrine human receptorssuch as the neuropeptide y receptorsystem.

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References[1] Peptide receptors as molecular targets

for cancer diagnosis and therapy, J.C. Reubi3, Endocrine Reviews 24, 389 – 427 (2003)

[2] Biodistribution and catabolism of (18)F-labeled neurotensin(8-13) analogs, R. Bergmann, M. Scheunemann, C. Heichert, P. Mading, H. Wittrisch, M. Kretzschmar, H. Rodig, D. Tourwe1, K. Iterbeke1, K. Chavatte1, D. Zips2, J.C. Reubi3, B. Johannsen, Nuclear Medicine and Biology 29, 61 – 72 (2002)

[3] Neurotensin receptors in adeno- and squamous cell carcinoma, C. Haase, R. Bergmann, J. Oswald, D. Zips2, J. Pietzsch, Anticancer Research 26, 3527 – 3533 (2006)

[4] Radiolabeling of multimeric neu-rotensin(8-13) analogs with the short-lived positron emitter fluorine-18, C. Hultsch, M. Berndt, R. Bergmann, F. Wuest, Applied Radiation and Isotopes (2007), accepted, electronically published

[5] Synthesis and evaluation of novel multimeric neurotensin(8-13) analogs, C. Hultsch, B. Pawelke, R. Bergmann, F. Wuest, Bioorganic and Medical Chemistry 14, 5913 – 5920 (2006)

[6] Fluorescein-labeled stable neurotensin derivatives, V. Maes1, C. Hultsch, S. Kohl, R. Bergmann, T. Hanke4, D. Tourwe1, Journal of Peptide Science 12, 505 – 508 (2006)

Project partners1Department of Organic Chemistry, Vrije Universiteit Brussels, Belgium

2OncoRay – Center for Radiation Research in Oncology, Clinic of Radiotherapy, Faculty of Medicine, Technische Universität Dresden, Germany

3Institute of Pathology, University of Berne, Switzerland

4Institute of Materials Science, Technische Universität Dresden, Germany

Fig. 6: Whole body autoradiogram of [18F]FB-NT(8-13) analogue distribution andhistological section of a HT-29 tumor-bearing mouse.

Fig. 7: IC50 of tetrameric NT(8-13) molecules [5]. Synthesis and characterization of the complexcompound (left; unpublished result) is part of Anne Kretzschmanns's diploma thesis at the TechnischeUniversität Dresden.


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Jens Pietzsch, Frank Wuest

Controlling the damaging effects ofreactive oxygen species (ROS) on proteinsis a major unsolved problem in currentbiology and medicine [1]. In the 1960s,Garrison and others carried out pioneeringstudies on oxidative modification of pro-teins exposed to ionizing radiation. Recentprogress in life sciences has led to thedevelopment of excellent methods andapproaches enabling an increasinglydetailed investigation of the phenomenon

of protein oxidation at both a micro andmacrostructural level under various physio-logical and pathophysiological conditions.Recent experimental and clinical evidenceunderpins the argument that proteinoxidation is a significant causative orassociated factor in aging as well as in theetiology, progression, and manifestation ofa panoply of human diseases and disorders(Table 1) [1]. Protein oxidation is definedhere as covalent modification of proteinsinduced either directly by reactive oxygenspecies or indirectly by reactions with

secondary by-products of oxidative stress(Fig. 1). Proteins are the major target ofreactive oxygen species due to their abun-dance in biological systems as well as theirhigh rate constants for reaction [1]. Proteinoxidation may be important in vivo fortwo reasons: Firstly, it affects the functionof receptors, enzymes, transport proteins,and cell signaling mechanisms. Secondly,protein damage can lead to secondarydamage to other biomolecules, e.g., byinactivating DNA repair enzymes.

Most protein damage is non-repairableand has deleterious consequences on thestructure and function of proteins. Butsome oxidation processes can be reversedin some circumstances. This is particularlyimportant because the generation ofreactive oxygen species cannot solely beconsidered as a purely pathophysiologicalphenomenon. When nitric oxide (NO•)was identified as an endothelium-derivedrelaxing factor, it became incontrovertiblethat reactive oxygen species are essentialentities in mammalian biochemistry.Reversible protein oxidation is thus aroutine, purposeful aspect of the cell’snormal function [1]. In order to understandhow protein oxidation causes disease, it isimportant to find out which proteins areaffected by reactive oxygen species and towhat degree they are modified in vivo. Offurther interest are the specific functionalconsequences of protein modification andthe critical ‘hits’ of reactive oxygen speciesthat are relevant in the etiopathology ofdiseases.

Protein oxidation and disease

Fig. 1: Generation of reactive oxygen species(ROS) leading to protein oxidation in the humanorganism. ROS is a collective term includingoxygen, nitrogen, or halogen radicals, (e.g., OH•,O2

•-, NO•, Cl•) and certain nonradicals (e.g., 1O2,H2O2, HOCl, HOBr, ONOO-, Maillard reactionproducts, homocysteine thiolactone) that areoxidizing, nitrating, halogenating, glycating, or

homocysteinylating agents or are easilyconverted into radicals. ROS and otherprooxidative reactive species i) are present aspollutants in the normal atmosphere, ii) aregenerated as by-products of normal metabolicprocesses, iii) are formed by bioactivation ofxenobiotics, and iv) are formed during exposureto X, γ, or UV radiation.

· Acute respiratory distress syndrome · Alcoholic liver disease*· Alzheimer’s disease· Amyotrophic lateral sclerosis· Atherosclerosis*

The asterisk denotes diseases that show a relevant involvement of modified lipoproteins in their pathogenesis.

· Cancer· Diabetes mellitus (and complications)*· Glomerulonephritis*· Hypertension*· Impaired glucose tolerance (Prediabetes)*

· Infectious diseases · Inflammatory bowel diseases*· Injury· Nephrotic syndrome*· Obesity*

· Osteoarthritis· Parkinson’s disease· Progeria· Rheumatoid arthritis*· Sepsis

Table 1: Human disorders and diseases associated with increased protein oxidation:

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These issues are in the heart of research atthe FZD Institute of Radiopharmacy, whichcollaborates closely with the Departmentof Internal Medicine at the Faculty ofMedicine Carl Gustav Carus of TechnischeUniversität Dresden. The aim is to providenovel targets for further research into thedisease process as well as sites of potentialtherapeutic interest. In particular, we haveintensively studied the oxidative modifi-cation of apolipoprotein B-100, the majorprotein of human low-density lipoproteins(LDL). Due to the “oxidative modificationhypothesis of atherosclerosis” [1], oxi-dative modification of apolipoprotein B-100 by reactive oxygen species is widely regarded as a crucial event in theatherogenic process. Only recently couldwe provide new evidence supporting therole of oxidative damage of apolipoprotein

B-100 in the pathogenesis of impairedglucose tolerance, a prediabetic statemarking an important facet of the Meta-bolic Syndrome [2]. The major finding ofthis study is that low density lipoproteinsobtained from subjects with impairedglucose tolerance exhibited an increasedapolipoprotein B-100 glycoxidative statuswhen compared with low-density lipo-proteins from normoglycemic subjects.Low-density lipoprotein modifications wereclosely correlated with sustained baselinehyperglycemia observed in subjects withimpaired glucose tolerance as well as withtemporary hyperglycemia after an oralglucose challenge. As a consequence ofapolipoprotein B-100 modification, low-density lipoproteins from subjects withimpaired glucose tolerance evoked asignificantly altered expression of certain

scavenger receptors and peroxisome-prolif-erator activated receptors, respectively, inmacrophages. In conclusion, impairedglucose tolerance is causally related toglycoxidative modification of circulatinglow-density lipoproteins. Also, it is associa-ted with early events in the pathogenesisof cardiovascular complications in predia-betic and diabetic patients. These datafurther underline the importance of earlypreventive treatment and strategies [2].

Despite the evidence from clinical studies,however, the role of circulating modifiedlow-density lipoprotein particles in the invivo development of atherosclerosis is stilla matter of debate. It could be argued thatperhaps an increased level of circulatingoxidized low-density lipoproteins is anepiphenomenon that merely showscorrelation with the basic pathological ordegenerative processes or with impairmentof antioxidant barriers. On the other hand,circulating oxidized low-density lipopro-teins, e.g., descending from an inflam-mation site as the inflamed joints of sub-jects suffering from rheumatoid arthritiscould be a causative substrate for thedevelopment of a pathological situation in another compartment, such as lesionformation in the arterial wall [1].Therefore, we developed highly sensitive

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Fig. 2: Reaction scheme forfluorine-18 radiolabeling ofnative and modified lowdensity lipoproteins (LDL)with the amino group reactiveagent N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB)under mild reaction con-ditions. ApoB-100 denotesthe apolipoprotein B-100, themajor structural protein ofhuman LDL.

Fig. 3: Study design and principleof small animal positron emissiontomography studies aiming atcharacterization and differentiationof the metabolic fate of native andmodified low density lipoproteins inanimal (rodent) models of diseaseand controls.


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and specific methods for radiolabeling ofnative and modified proteins with thepositron emitter fluorine-18. This allowsinitial direct assessment of intravasculartransfer and catabolism of modified low-density lipoproteins using dynamic smallanimal positron emission tomography inrodent models in vivo (Fig. 2 – 3) [1, 3, 4].In this regard, our experiments showed anextremely fast and complete bloodclearance of glycoxidized low-densitylipoproteins in vivo. This has beenexplained by the concerted action ofvarious scavenger receptors on residentmacrophages and endothelial cells in liver,spleen, and kidney (Fig. 4). These cells

form an efficient scavenger apparatusprotecting the organism from the athero-genic action of glycoxidized/ oxidized low-density lipoproteins in the circulating bloodcompartment.

Other circulating modified low-densitylipoprotein species, e.g., glycated LDL,avoid this scavenger pathway and arererouted to various tissue-specificproinflammatory pathways [1 – 4]. The in vivo distribution and kinetics ofboth native low density lipoproteins and modified low density lipoproteinscorrelated well with the anatomicallocalization and functional expression of low density lipoprotein receptors,scavenger receptors, and receptors foradvanced glycation end products.Interestingly, the vasculature in rodentsshows a substantial temporary retention ofglycated low-density lipoproteins whencompared with native or glycoxidized low-density lipoproteins. This retention cannotbe explained by the circulating blood poolor perfusion effects alone. Therefore, it isindicative of a tissue-specific interactionwith glycated low-density lipoproteins thatis potentially proatherogenic (Fig. 4 – 5)[5]. Given the intrinsic properties of smallanimal positron emission tomography, ourapproach enables first-time quantitativecharacterization and discrimination of thekinetics and the metabolic fate of nativeand oxidatively modified proteins, and,vice versa, of functional expression of their pathophysiologically relevant tissue-specific binding sites in vivo [5]. Ongoingstudies in animal models of disease fundedby the German Research Foundation(DGF) will thus provide detailed informa-tion on the importance of circulating

modified lipoproteins in the etiology,progression, and manifestation ofprediabetes, the Metabolic Syndrome, and other pathologies.

Fig. 5: Representative whole body smallanimal positron emission tomography scans(maximum intensity projection) showing themetabolic fate of fluorine-18 radiolabeled([18F]fluorobenzoylated, [18F]FB-) native andcertain modified low density lipoproteinspecies (high radioactivity concentration isindicated by black color) in a rat model at180 min post injection.

Fig. 4: Representative time-activity-curves showing kinetics of the fluorine-18 radioactivity concentration during 120 min after injection of radiolabeled([18F]fluorobenzoylated, [18F]FB-) nativelow density lipoproteins (LDL), glycatedLDL, and glycoxidized LDL as calculatedfrom region-of-interest analysis ofdynamic small animal positron emissiontomography scans of the heart (mainly

representing the cardiac blood pool).Results are expressed as means ± SD offive independent experiments. Dynamicpositron emission tomography datademonstrated a significantly delayedcatabolism of glycated LDL comparedwith native LDL. In contrast, glycoxidizedLDL showed an enhanced catabolismwhen compared with native LDL [1].

References[1] Protein Oxidation and Disease,

J. Pietzsch (ed.), Research Signpost, Trivandrum/ India (2006)

[2] Oxidized and glycated LDL isolated from subjects with impaired glucose tolerance increases CD36 and PPARg gene expression in macrophages, J. Graessler1, J. Pietzsch, T. Westendorf1, U. Julius1, S.R. Bornstein1, S. Kopprasch1, Diabetologia 50, 1080 – 1088 (2007)

[3] Catabolism of native and oxidized low density lipoproteins (LDL): in vivo insights from small animal positron emission tomography studies, J. Pietzsch, R. Bergmann, F. Wuest, B. Pawelke, C. Hultsch, J. van den Hoff, Amino Acids 29, 389 – 404 (2005)

[4] Labeling of low-density lipoproteins using the 18F-labeled thiol-reactive reagent N-[6-(4-[18F]fluorobenzy-lidene)aminooxyhexyl]maleimide [18F]FBAM, M. Bernd, J. Pietzsch, F. Wuest, Nuclear Medicine and Biology 34, 5 – 15 (2007)

[5] Imaging and functional characterization of metabolic pathways of modified lipoproteins in vivo using small animal positron emission tomography (PET), J. Pietzsch, Vascular Disease Prevention(2008), in press

Project partner1Department of Internal Medicine, Faculty of Medicine, Technische Universität Dresden, Germany

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Hans-Jürgen Pietzsch, Holger Stephan,Jörg Steinbach

Ionizing rays, after the surgeon’s knife, arethe most successful and most frequently-applied weapon against cancer. In externalradiotherapy, high radiation doses areconcentrated onto a small area in the body.This aims at destroying the pathologicallymodified body cells in the tumor whereas,at the same time, the adjacent healthytissue is spared. However, externalradiation is limited by metastasing condi-tions. In these cases, the treatment methodmust be systemic, i.e., therapeutic tumoragents must reach the (partly invisible)

metastases and solid tumors via thebloodstream. This is the field ofchemotherapy and targeted internalradionuclide therapy, which make use ofparticle radiation emitted by radionuclides(usually beta radiation) as a therapeuticallyeffective dose. In the course of treatment,the radiolabeled substance is transportedto the tumor and the radiation energyreleased there causes the tumor cells to dieoff. The main challenges of this therapyare targeting the tumor and the timedistribution of the radioactivity.Radioimmunotherapy (RIT) as a specialform of endoradionuclide therapy hasgained in clinical importance. With it,

antibodies specifically targeting tumor cellsare labeled radioactively. Together with theantibodies, the radionuclides arrive at thetumor where their radioactive radiation can deploy its cytocidal effect. In doing so, healthy tissue is largely spared (Fig. 1).Radiotherapeutical applications requireradionuclide-emitting particles (beta andalpha particles), the half-life periods ofwhich range from several hours up to afew days. Still, a high local radiation dose is generated. As a consequence, thereshould be sufficient time to prepare themedication, transport it to the tumor and,above all, release the radiation dose intothe tumor cells. However, in order to

Radioactive metals for tumor therapy

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Preparation of radiopharmaceuticals in hot cells. Fig. 1: Principle of radioimmunotherapy: a radio-therapeutical clings to a tumor cell where radio-active radiation unfolds its cell-destructive effect.

Photo: Jürgen Lösel


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minimize the exposure of healthy tissue to radiation, the radionuclides must decaywithin a relatively short time into non-radioactive, i.e., stable daughter nuclides.Fig. 2 presents a survey of elements theradioisotopes of which are potent therapynuclides.

We started our research using the radio-nuclides copper-64 (Cu-64), copper-67(Cu-67), and rhenium-188 (Re-188) withhalf-life periods of approximately 13, 62,and 17 hours, respectively. Apart from thedesired particle radiation, these radio-nuclides simultaneously emit positron orgamma radiation, which by means ofspecial detection techniques allowsregistering the distribution of radioactivesubstances and visualizing them. Fortu-nately, these radionuclides are easilyavailable. For instance, we can produceCu-64 with the help of our own cyclotron.For obtaining Re-188, we use a commer-cial radionuclide generator which can alsobe installed in hospitals. As the maximumrange of radiation in tissue varies betweenabout 1 millimeter for both Cu-64 and Cu-67, and 11 millimeters for Re-188, tumorsof various sizes can be targeted.

Therapeutical application of radionuclidesrequires radiopharmaceuticals to have ahigh metabolic and radiolytic stability. Thismeans that the compound applied muststay intact until it has reached the desiredplace where it should remain until itsradiation has abated. Possible metabolitesof these radiopharmaceuticals must leavethe body without damaging healthy tissue.Thus, we are searching for compoundswhich incorporate a complex-formingsection for metallic radionuclides to fulfillseveral tasks (Fig. 3). Due to the differingcoordination chemistry of rhenium andcopper compounds, both radiometals

require a differentiated approach.Currently, we are pursuing two approachesto develop radiolabeled rhenium andcopper compounds:

Radiolytically resistant Re-188-S4

chelates for labelling biomoleculesChemically very robust radioactive rheniumcomplexes can be generated on the basisof bridged dimercaptosuccinic acid chelateunits (Fig. 4), permitting wide structuralvariety as well. Furthermore, solubility-mediating units can be tied to the carboxylgroups of the dimercaptosuccinic acid. Yetsynthesizing such rhenium complexes is achallenge to chemists as stereoisomers aregenerated during production, i.e., com-pounds with the same constitution, butwith a different spatial, three-dimensionalarrangement of their atoms and atomgroups [1, 2].

The Re-188 labeling procedure runsquickly in good yields and under mildconditions, such as aqueous solution,neutral pH, and room temperature. Theappropriate Re-188-S4 complexes werefound to be very stable regarding re-oxidation and ligand exchange in vitro

Fig. 2: Survey of elements the radioisotopes of which are potent therapeutical nuclides.

Fig. 3: Principle structure of a radiometalpharmaceuticalBasically, a radiometal pharmaceutical consistsof four components: a bifunctional chelategroup, a spacer unit, a radionuclide, and abiomolecule. The bifunctional chelate groupmust bind the radionuclide in a most stablemanner and contain a couplable group to tiebiomolecules. Biomolecules, such as specificpeptides, proteines, antibodies, or aptameres,are linked via a spacer to the bifunctionalchelate unit and are expected to enablecontrollable biodistribution. Labeling thechelate unit equipped with the biomolecule bymeans of the radionuclide shall preferably becarried out at the last stage in order to facilitatethe user to apply a simple single-step approach.

Fig. 4: Rhenium complex on the basis ofbridged dimercaptosuccinic acid chelate units.

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and in vivo. All in all, the new Re-S4

complexes offer the possibility of stableand highly-specific activity labeling ofbiomolecules for therapeutic applications[3, 4].

Highly stable complexes of radioactive copper nuclides with bispidinesWe are developing a ligand system withcolleagues from the Institute of InorganicChemistry at Heidelberg University whichis suitable for generating extremely stablecopper(II)-complexes. Fig. 5 shows onerepresentative. Here, the Cu(II) ion isbound by a total of six donor atoms (twoamine nitrogen and four pyridine nitrogenatoms) and is practically completelyshielded from its environment, whichexplains its high stability. Studies duringwhich these ligands were labeled with

copper radionuclides indicate a rapidformation of stable complexes under mildconditions. Furthermore, the bispidinestructure opens suitable chemical ap-proaches to introduce biomolecules, whichare important in view of the targeting ofsuch complexes. Due to these promisingfeatures, bispidines are predestined asattractive candidates for developing newcopper-based radiopharmaceuticals [5].

Principally, the concepts presented heremay also be applied to the design of metalcomplexes of other therapeutically relevantradionuclides. For this purpose, a multitudeof subtasks, particularly with regard toresearch on ligand synthesis, coordinationchemistry, as well as tumor-biological andradiopharmacological aspects, must besolved.

Radionuclide therapy is challenging, butstill evolving in regards to the investigationof new molecular constructs, new radio-nuclides and radiochemistry, improveddosimetry, prediction of tumor responseand host toxicities, and better targetingstrategies to prevent or overcome hosttoxicities, particularly liver and kidneytoxicity and myelosuppression. Hopefully,the advances in radioimmunotherapyregarding hematologic malignancies willtranslate to progress in the therapy ofradioresistant solid tumors.

In the field of developing radiometal com-pounds for tumor therapy, we are enjoyingexternal collaboration with experts at thePaul Scherrer Institut (Switzerland), and at the universities in Dresden, Heidelberg,and Padua (Italy). In the future, theseefforts will be intensified strongly in thecontext of “OncoRay”—Centre forRadiation Research in Oncology, which is jointly run by Technische UniversitätDresden, FZD, and University Hospital Carl Gustav Carus in Dresden.

References[1] A novel rhenium chelate system derived

from dimercaptosuccinic acid for the selective labeling of biomolecules, T.K. Heinrich, W. Kraus1, H.-J. Pietzsch, C. Smuda, H. Spies, Inorganic Chemistry 44 (26), 9930 – 9937 (2005)

[2] A new molecular mechanics force field for the design of oxotechnetium(V) and oxorhenium(V) radiopharmaceuticals, P. Comba2, A. Daubinet2, B. Martin2, H.-J. Pietzsch, H. Stephan, Journal of Organometallic Chemistry 691, 2495 – 2502 (2006)

[3] Preparation and Biological Characterization of Isomeric 188Re(V) Oxocomplexes with Tetradentate S4

Ligands Derived from DMSA for Labeling of Biomolecules, S. Seifert, T. Heinrich, C. Jentschel, C. Smuda, R. Bergmann, H.-J. Pietzsch, Bioconjugate Chemistry 17, 1601 – 1606 (2006)

[4] Stability of 188Re complexes prepared with highly concentrated [188Re]perrhenate eluates from 188W/188Re generators, S. Seifert, C. Jentschel, R. Bergmann, H.-J. Pietzsch,G. Wunderlich3, J. Kotzerke3, J. Stein-bach, Nuklearmedizin/Nuclear Medicine 46, 181 (2007)

[5] Synthesis, characterization and evaluation of novel chelating agents for copper radionuclides, H. Stephan, S. Juran, M. Walther, J. Steinbach, K. Born2, P. Comba2, in: Technetium, Rhenium and other Metals in Chemistry and Nuclear Medicine, vol. 7, U. Mazzi (ed.), SGE Editoriali, Padova, Italy, 2006, 219 – 222

Project partners1Federal Institute for Materials Research and Testing (BAM), Berlin, Germany

2Institute of Inorganic Chemistry, Universität Heidelberg, Germany

3Clinic of Nuclear Medicine, University Hospital Dresden, Germany

Institute of Inorganic Chemistry andSurfaces, Consiglio Nazionale delle Ricerche,Padova, Italy

Fig. 5: Bispidine ligand of four pyridine groupsand the appropriate copper(II)-complex.

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Holger Stephan, Gerhard Geipel

Polynuclear metal compounds may haveconsiderable potential as metallic drugs.The most prominent representatives arepolyoxometalates, which have been underinvestigation since the last third of the 19th

century. They contain transition metal ionssuch as tungsten, molybdenum, vanadium,and so on which are bridged by oxygenatoms (Fig. 1) [1]. In addition toapplications in catalysis, separation,analysis, and as electron-dense imagingagents, some of these substances havebeen shown to exhibit biological activity invitro as well as in vivo ranging from anti-cancerous, antibiotic, and antiviral up toanti-diabetic effects. Yet fundamentalquestions regarding the mechanism ofmany of the observed medical effects haveremained essentially unanswered. Modernexperimental techniques developed in thelast decades may help to explore this

exciting area. This requires interdisciplinarycollaboration between chemists, crystallo-graphers, physicists, biochemists, pharma-cists, and physicians.

Polymetalates represent a diverseensemble of nanostructures with almostinfinite variability in their chemical,physical, and biological properties. Typicalcovalent-bridged cluster compounds aresized between 1 and 3 nm, creatingfascinating and beautiful molecules (Fig.1–5). Attaching special surface groups onthe periphery of cluster compounds mayresult in self-assembled non-covalentorganized structures larger than 5 nmwhich are characteristic of bio-moleculessuch as enzymes. Cells of mammalianorganisms are typically between 10 and30 µm; however, sub-cellular organelledimensions are smaller—in the sub-µmrange. This comparison of size dimensionsillustrates that polymetalates are smallenough to allow the cell membrane to bepenetrated without excessive interference.Evidently, some types of polymetalates canbe transported into cells, particularly intomitochondria. Our aim is to develop novelcluster compounds with improved chemicaland metabolic stability. Furthermore,increased recognition of target biomol-ecules such as enzymes is a goal as well.Regarding this, the conjugation of organicgroups to the inorganic metal clustercompounds could be used to enhance

biological targeting. The development ofsuch inorganic-organic hybrids is particu-larly challenging.

During exploration of biological activity of polynuclear cluster compounds, werecently recognized polyoxometalates as anew class of potent enzyme inhibitors [2].E-NTPDases (ecto-nucleoside triphosphatediphosphohydrolases) are surface-locatednucleotide-hydrolyzing enzymes involvedin the regulation of signaling cascades byactivating G protein-coupled P2 receptors.Currently, there are significant effortsunderway to find efficient and selectiveinhibitors for these enzymes in order tomodulate receptor activity and to influencethe pharmacological effects as a conse-quence. The most potent compounddescribed to date is K6H2[TiW11CoO40]exhibiting Ki values which are significantlylower than those of known standardinhibitors (Fig. 2). In future jointexperiments with the PharmaceuticalInstitute of the Universität Bonn, thenature of the enzyme inhibition mech-anism caused by polyoxometalates will be researched. Most importantly, we wishto determine the method of action for the biological effects including their anti-cancer activity.

A promising new class of clustercompounds are hexanuclear rheniumcomplexes with bridging sulfur, selenium,

Medical impact and beauty of cluster compounds

Fig. 2: Concentration-inhibition curves for the enzymes E-NTPDase 1 – 3 with K6H2[TiW11CoO40].

Fig. 1: a) Molecular structure of a typical Keggintype polyanion [Ti2W10PO40]

7- b) Crystalpacking of K4H3[Ti2W10PO40] · 15H2O, with the[W/TiO6] octahedral coordination polyeder(green), potassium cations (blue), and watermolecules as light gray spheres.

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and/or tellurium atoms (Fig. 3) [3 – 5].Increased attention to these complexes isdue to their structural, redox, andphotoluminescent properties as well astheir rich chemistry which derives from asimple modification of their coordinationenvironment. Attaching organic ligands tothe cluster environment seems to be veryattractive in view of developingbiocompatible and bio-available hybridcompounds for therapeutic purposes. Forinstance, octahedral rhenium complexeswith grafted pyrazole ligands possessbright red luminescence (Fig. 4) whichmakes them attractive for medicaltreatment of cancer by means ofphotodynamic therapy. Furthermore, theinherent potential of this novel class ofcluster compounds for anti-tumor activity,photosensitizing, and radiation-sensitizingproperties can provide synergetic medicalefficacy by simultaneously applying variousmethodologies. Thus, chemotherapy couldbe combined with photodynamic therapyor radiation therapy.

The enormous versatility and variety ofthese cluster structures offer considerableopportunity in these areas. In the future,

the know-how of our Russian colleagues at the Nikolaev Institute of InorganicChemistry in Novosibirsk, Russia insynthesizing appropriate rhenium clustercompounds and our expertise indeveloping dendritic (tree-like) ligandsshall be allied in order to create intelligentvehicles with tunable transport properties.The dendritic modification of polynuclearmetal compounds allows the intendedstructures to have versatile chemical,physical, and biological properties (Fig. 5).Most importantly, both the biodistributionand the biological targeting may beinfluenced by grafting a multitude ofsuitable bio-molecules onto the surface ofthe metal cluster compounds. To achievethis ambitious goal, the Nikolaev Instituteof Inorganic Chemistry, the LeibnizInstitute of Polymer Research Dresden, and the FZD will collaborate on a projectfunded by the International Bureau of theFederal Ministry of Education and Research(BMBF). This will focus on the synthesis ofnovel cluster compounds and thecharacterization of the luminescenceproperties by laser spectroscopy. Structuralcharacterization will be performed incollaboration with colleagues from the

Federal Institute for Materials Researchand Testing (BAM) in Berlin and biologicalactivity will be tested at the PharmaceuticalInstitute in Bonn.

References[1] Chitosan-encapsulated Keggin anion

[Ti2W10PO40]7-: synthesis, character-

ization and cell uptake studies, T. Meißner, R. Bergmann, J. Oswald, K. Rode, H. Stephan, W. Richter, H. Zänker, W. Kraus1, F. Emmerling1, G. Reck1, Transition Metal Chemistry 31, 603 – 610 (2006)

[2] Polyoxometalates – a new class of potent ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) inhibitors, C.E. Müller2, J. Iqbal2, Y. Baqi2, H. Zimmermann2, A. Röllich, H. Stephan, Bioorganic and Medical Chemistry Letters 16, 5943 – 5947 (2006)

[3] [Re6Q7O(3,5-Me2PzH)6]Br2·3,5-Me2PzH (Q = S, Se) – new octahedral rhenium cluster complexes with organic ligands: original synthetic approach and unexpected ligand exchange in cluster core, Y.V. Mironov3, M.A. Shestopalov3, K.A. Brylev3, A.S. Yarovoi3, G.V. Roma-nenko3, V.E. Fedorov3, H. Spies, H.-J. Pietzsch, H. Stephan, G. Geipel, G. Bernhard, W. Kraus1, European Journal of Inorganic Chemistry, 657 – 661 (2005)

[4] Octahedral rhenium cluster complexes with inner organic ligands: synthesis, structure and properties of [Re6Q8(3,5-Me2PzH)6]Br2·2(3,5-Me2PzH) (Q = S, Se), Y.V. Mironov3, K.A. Brylev3, M.A. Shestopalov3, S.S. Yarovoi3, V.E. Fedorov3, H. Spies, H.-J. Pietzsch, H. Stephan, G. Geipel, G. Bernhard, W. Kraus1, Inorganica Chimica Acta 359, 1129 – 1134 (2006)

[5] Cluster core controlled reaction of substitution of terminal bromide ligands by triphenylphosphine in octahedral rhenium chalcobromide complexes, M.A. Shestopalov3, Y.V. Mironov3, K.A. Brylev3, D.Y. Naumov3, V.E. Fedorov3, H. Spies, H.-J. Pietzsch, H. Stephan, G. Geipel, G. Bernhard, Journal of the American Chemical Society 129, 3714 – 3721 (2007)

Project partners1Federal Institute for Materials Research and Testing (BAM), Berlin, Germany

2Pharmaceutical Institute, Universität Bonn, Germany

3Nikolaev Institute of Inorganic Chemistry, Novosibirsk, Russia

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Fig. 3: Molecular structure of the clusterpolyanion [Re6S8Br6]

3- (rhenium – green,sulfur – yellow, bromine – red).

Fig. 4: Luminescence spectra of the cluster compound [Re6Se8L6]Br2L2 (L = 3,5-dimethylpyrazole).

Fig. 5: Cluster compound with pendantdendritic ligands.


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Jörg van den Hoff

Positron Emission Tomography (PET) is atomographic imaging technique whichenables quantitative investigation ofcellular and molecular processes, such as blood flow, transport across diffusionbarriers, substrate metabolism, proteinsynthesis, enzyme activity, receptor affinity,and so on. Today, PET is firmly establishedin preclinical and clinical research as well asfor clinical applications. The PET techniqueassesses in vivo the three-dimensionaldistribution of special substances (tracers)labeled with short-lived positron emittingisotopes. An important example is thesugar 18F-Deoxyglucose (18F-FDG),currently the most widely used tracer for clinical oncological investigations.

The measurement of the regional tracerdistribution depends upon detecting theradioactive decay of individual atomicnuclei. Upon the decay of the radioactivelabel, a positron is emitted from the atomicnucleus and is rapidly stopped in thesurrounding tissue (typical mean range

in water < 1 mm). The positron thenannihilates with an electron, which isaccompanied by collinear emission of two511 keV photons in opposite directions.Thereupon, these photons are detectedwith several thousand scintillationdetectors arranged in a ring coaxial to the patient’s body axis. The coincidentdetection of two photons within a timewindow of a few nanoseconds defines theso called Line of Response (LOR) alongwhich the primary decay of the radioactivelabel has occurred. In other words, thiscoincidence measurement provides thedirectional information for the subsequenttomographic reconstruction of the tracerdistribution (Fig. 1).

The resulting tomographic images quanti-tatively provide the regional concentrationof the applied tracer substance. Yet PETrequires long-term data acquisition—fromseveral minutes up to more than one hour.During this “exposure time”, patientmovements are unavoidable and deteri-orate the image quality, reducing thespatial resolution and creating image

artifacts (“ghost images”). As the intrinsicspatial resolution of current PET scannersconstantly improves, patient movementincreasingly limits the image quality whichis practically achievable. As far as the PET’scontribution to biologically individualizedand technically optimized radiotherapy isconcerned—a subject which we areworking on with the recently establishedOncoRay research center in Dresden—reliable elimination of motion artifactsappears highly desirable. Therefore, ourgroup is developing methods for accuratecorrection of two different types of patientmovement: first, cyclic organ and bodymotion correlated to the heart andbreathing cycle and secondly, erraticinvoluntary movement of the patient.

As far as the first item is concerned, we usea system of infrared video cameras fortracking the motion of targets attached tothe patient (Fig. 2). Every 50 milliseconds,the system reports the patient’s positionwith sub-millimeter accuracy. The crosscalibration to the tomograph is performedat the same level of accuracy by imagingthe motion tracking target with thetomograph using an external radioactivesource.Substantially more effort is necessary withregard to the remaining issues of the

Moving targets – correcting patient movement in positron-emission tomography

In order to perform accurate motion cor-rection, several problems must be solved:1. Monitoring of the patient with external

motion tracking devices providing high temporal and spatial resolution,

2. Spatial and temporal synchronization of the motion tracking data with the reference frame of the PET scanner (cross calibration),

3. Modifying the PET data acquisition technique,

4. Development of algorithms for performing the actual motion correction within the PET data.

Fig. 1: Schematic overview ofmeasurement process in PET.

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above list. During routine operation, thecoincident events are fed into the dataprocessing electronics of the tomographand stored in a so-called histogrammicmemory. Each Line of Response (LOR)corresponds to a single memory cellcontaining the cumulative number ofevents which occurred along this LORduring data acquisition. (This can becompared to the way photographs arestored on the memory cards of digitalcameras.) Only after measurement are the histogram data transferred to thecomputer hard disc. In this way, significantdata compression is achieved, but at theprice of losing the timing information ofthe specific events. Since it is exactly thistiming information which is needed formotion correction, we use the list mode inwhich each coincident event is directlystored on the computer’s hard disc

together with its timing information.(Regarding the example from digitalphotography, this corresponds toseparately storing location and time of thearrival of each individual photon hitting theCCD chip of a digital camera.) Since listmode measurements are not adequatelysupported by the tomograph’s software,we have developed tools from scratch tomanage large list mode data sets, notablywith regard to integrating multiple triggersignals from breathing monitors andelectrocardiograms (ECG). Fig. 3 shows an example of the benefits of using abreathing trigger and list mode data.

The main challenge is to correct theprimary list mode data prior totomographic reconstruction. If this can beaccomplished, tomographic images whichare completely free of any motion blurring

or other motion artifacts could beobtained. For each coincident event, ouralgorithm computes a reorientation whichrepositions the LOR to its originalorientation within the detector ring. Whilethis reorientation is straightforward, severalcomplications exist. The most obvious aredifferences in detector efficiencies,discretization and interpolation errors, and,most notably, LORs drifting out of thescanner’s field of view and/or reenteringthe field of view during measurement. Lastbut not least, efficient implementation ofthe algorithms on multi-processor systemsis necessary so the methods can be usedfor clinical studies. Fig. 4 shows the rewardof using such elaborate methods. Thistechnique is already operational and hasbeen used, for instance, in pre-phase oneclinical trials in the context of drugdevelopment in collaboration with thepharmaceutical industry. Currently, we areimproving the algorithms and are aiming atextending the technique to whole bodyinvestigations.

References[1] An accurate method for correction of

head movement in PET, P. Bühler, U. Just, E. Will, J. Kotzerke1, J. van den Hoff, IEEE Transactions on Medical Imaging 23 (9), 1176 – 1185 (2004)

[2] Optimized list-mode acquisition and data processing procedures for ACS2 based PET systems, J. Langner, P. Bühler, U. Just, C. Pötzsch, E. Will, J. van den Hoff, Zeitschrift für medizinische Physik 16, 75 – 82 (2006)

Project partner1Clinic of Nuclear Medicine, University Hospitel Dresden, Germany

Fig. 2: The motion tracking system for brain applications.

Fig. 4: Images acquired during rest (left) show significantly higher quality than those influencedby patient motion (center). Motion correction (right) essentially restores the full image quality.

Fig. 3: Correction of breathing cycle-relatedmotion. In the corrected image the tumor ismuch better delineated and significant errorsin assessing the size and metabolic state areavoided.

at rest not corrected corrected

RESEARCH INSTITUTE OF RADIATION PHYSICS

Wolfgang Enghardt1, Fine Fiedler, Daniela Möckel, Katia Parodi2

Radiation therapy is one of the corner-stones of modern cancer treatment. Withincreasing tendency, more than 50% oftumor patients are irradiated, either as theexclusive form of treatment or in com-bination with other modalities such assurgery or chemotherapy. The centralchallenge of radiotherapy is to destroy the tumor completely while saving thesurrounding healthy tissue. In some cases,for example, for compact, deep-seated,radioresistant tumors growing in closevicinity to organs at risk, these objectivescannot be reached by state-of-the-artradiotherapy technology which is basedupon hard photon or electron beamsdelivered by compact electron linearaccelerators. Therefore, proton and lightion (e.g., carbon) beams become more and more important due to their favorablephysical and radiobiological properties. Totranslate this potential into clinical results,new technologies in generating, forming,

and monitoring ion beams are required. In-beam positron-emission tomography(PET), one of these innovative technol-ogies, has been developed and transferredto clinical application by FZD at theexperimental carbon ion therapy facilitylocated at the Gesellschaft für Schwer-ionenforschung (GSI) Darmstadt in collab-oration with the Radiological Clinic of the University Hospital and the GermanCancer Research Center Heidelberg.

The unique knowledge of the FZD on in-beam PET is subject of a collaboration withSiemens AG Medical Solutions, with thegoal of transferring this into thedevelopment of a clinical in-beam PETscanner for ion therapy facilities that areplanned or already under constructionworldwide. This know-how comprisesdetector, signal processing, and dataacquisition technology for the criticaloperation of PET detectors at high-energyion beams [1], tomographic reconstructionalgorithms optimized for in-beam PET, andexperience in clinical application [2].

Making invisible beams visibleThe technological basis of in-beam PET is a double head positron camera [1]integrated into the therapy unit (Fig. 1).During therapeutic irradiation, this devicedetects the annihilation γ-rays followingthe decay of minor amounts of positronemitting nuclei (predominantly 11C and15O) which are produced via nuclearreactions between the impinging ions andthe atomic nuclei of the tissue (Fig. 2).Sophisticated algorithms of tomographicreconstruction deliver the spatialdistribution of positron emitters in vivo.They are related to the dose distribution bymeans of a precise Monte Carlo simulationof the production of positron emitters and

In-beam PETfor radiotherapy monitoring

Fig. 1: Double head positron camera developed byFZD at the treatment site of GSI Darmstadt. Thehorizontal carbon ion beam leaves the beam pipethrough a 20 x 20 cm2 window visible in the centreof the picture. To provide sufficient space for patientpositioning, the PET scanner can be moved on railsparallel to the beam between the measuring pos-ition displayed and the parking position upbeam.(Photos: GSI)

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the detection of annihilation γ-rays (Fig. 3).When these are compared with themeasured PET images, deviations betweenthe planned and actual dose distributionscan be revealed, quantified [2], andcompensated for in the course of furtherfractionated treatment. Deviations arecaused by ion range modifications due tominor patient positioning errors in com-bination with large tissue density gradientsor due to changes of the tissue densitydistribution within the irradiated volume(e.g., radiation induced tumor shrinking)during the three weeks of fractionatedradiotherapy. At the carbon ion therapyfacility of GSI, more than 350 cancerpatients, most with tumors in the head andneck region, have been treated since 1997(134 in the years between 2004 and2006). All of these treatments weremonitored by means of in-beam PET forquality assurance. In-beam PET phantomstudies with protons at FZD/GSI [3]triggered research on post-radiationPET/CT imaging at Massachusetts GeneralHospital, Boston (Fig. 4). Initial clinicalresults [4] confirm the predicted positiveimpact.

For radiobiological reasons, not onlyproton and carbon beams are highlydesirable for therapy, but also a largevariety of ion species with atomic numbersbetween 1 (hydrogen) and 8 (oxygen).Novel ion therapy accelerators (such as theone at the Heidelberger Ionenstrahl-Therapiezentrum) are capable of deliveringbeams of all these ions with therapeuticallyrelevant energy values. Since in-beam PEToffers the unique possibility to measureparticle ranges in-vivo, it allows sensitivetesting of the physical beam modelunderlying the dose calculation algorithmsfor treatment planning. This is highlyrelevant for commissioning new ion speciesfor therapy. Therefore, the FZD closelycollaborating with the HeidelbergerIonenstrahl-Therapiezentrum and theEuropean Organization for NuclearResearch/CERN uses its in-beam PETscanner at the GSI therapy facility tomeasure physical data which are necessaryfor extending in-beam PET to other ionbeams like helium and oxygen with veryhigh precision (Fig. 2).

Fig. 2: Depth distributions of calculated dose (blue, dashed) and measured β+-activity (red, solid) inducedby beams of protons as well as 3He, 12C and 16O ions in thick targets of polymethyl methacrylate. Theprominent maxima in the cases of 12C and 16O are formed by positron radioactive projectile fragments,whereas the pedestals as well as the distributions generated by 1H and 3He are due to target fragments.

Fig. 3: Clinical application of in-beam PET at the carbon ion therapy facility at GSI Darmstadt. As anexample, the irradiation of a chondrosarcoma of the skull base with a lateral portal coming from theleft side of the patient, i.e., right side in the picture (maximal dose: 0.63 Gy), is displayed. As indicatedby the dose distribution superimposed onto the computed tomogram (left), the carbon ions must notpenetrate the brain stem as an organ at risk. The comparison of the predicted (middle) with themeasured (right) β+-activity distributions shows that this was fulfilled during the treatment. The isodoseand isoactivity lines are decoded in rainbow colours and denote 5, 15, … 95 % of the maxima.

Fig. 4: Monte Carlo calculated (left) and measured (right) activity distribution after proton irradiation ofa clivus chordoma patient at Massachusetts General Hospital, Boston. Images by courtesy of K. Parodiand T. Bortfeld.


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Back to the photonsThe successful application of PET formonitoring radiation therapy with ionsmotivated investigations about thefeasibility of the method for hard photonbeams. This is studied within the EC FP6integrated project “BioCare – MolecularImaging for Biologically Optimized CancerTherapy” in collaboration with theKarolinska Institute, Stockholm, theMathematics-Physics Department atStockholm University, the Soltan Institutefor Nuclear Studies, Otwock-Swierk,Poland, and CERN in Geneva. The aim ofthis research is to prove the feasibility ofcombining an in-beam PET scanner withan extremely compact 50 MeV electronaccelerator delivering a pencil-like beam ofultrahard bremsstrahlung photons. In thiscase, positron emitters are generated by (γ,n) photonuclear reactions in the tissue atphoton energy values above 20 MeV. Thein-beam PET-related experimental researchof the BioCare project is carried out at 20until 40 MV bremsstrahlung beams

delivered by the Radiation Source ELBE. Bymeans of Monte Carlo calculations, it hasalready been shown that for electron beamenergies beyond 30 MeV, the induceddose-related activity density is comparablewith that obtained during irradiation withcarbon ions. This has been successfullyconfirmed in the second step [5] in whichthe 11C activity generated in plasticphantoms during photon irradiation atELBE was measured and quantified bymeans of the human PET scanner of theInstitute of Radiopharmacy at FZD (Fig. 5).Just recently, bremsstrahlung-inducedpositron emitters (11C and 15O) have beenimaged in-beam for the first time world-wide by means of a small limited anglepositron camera installed at the ELBEbeam. The encouraging result was thatdosimetry (a collaboration with theInstitute of Nuclear and Particle Physics ofTechnische Universität Dresden) as well asthe control of patient positioning on thebasis of in-beam PET appears feasible(Fig. 5).

Spin-off for educationBased on the expertise on PET instru-mentation available at the Institute ofRadiation Physics, a PET scanner has beeninstalled for education (Fig. 6). This deviceis aimed at practical training in the generalprinciples of tomography, in physics andmathematics of PET imaging and,additionally, in the fundamentals of multi-parameter measurements in nuclear,radiation, and particle physics. The scanneris used at the Institute of Nuclear andParticle Physics of TU Dresden for hands-on training of students of medical radiationsciences at the Postgraduate School of theCenter for Radiation Research in Oncology“OncoRay”—located at the Faculty ofMedicine of Technische UniversitätDresden—as well as of undergraduatephysics students.

References[1] Charged hadron tumour therapy moni-

toring by means of PET, W. Enghardt1, P. Crespo, F. Fiedler, R. Hinz, K. Parodi2, J. Pawelke, F. Pönisch, Nuclear Instruments and Methods in Physics Research A525, 284 (2004)

[2] Dose quantification from in-beam positron emission tomography, W. Enghardt1, K. Parodi2, P. Crespo, F. Fiedler, J. Pawelke, F. Pönisch, Radio-therapy and Oncology 73, S96 (2004)

[3] Experimental study on the feasibility of in-beam PET for accurate monitoring of proton therapy, K. Parodi2, F. Pönisch, W. Enghardt1, IEEE Transactions on Nuclear Science 52, 778 (2005)

[4] Patient study on in-vivo verification of beam delivery and range using PET/CT imaging after proton therapy, K. Parodi2

et al., International Journal of Radiation Oncology Biology Physics 68, 920 (2007)

[5] Quantification of β+ activity generated by hard photons by means of PET, D. Möckel, H. Müller, J. Pawelke, M. Sommer, E. Will, W. Enghardt, Physics in Medicine and Biology 52, 2515 (2007)

Project partners1OncoRay – Center for Radiation Research in Oncology, Faculty of Medicine, Technische Universität Dresden, Germany

2Heidelberger Ionenstrahl-Therapiezentrum, Heidelberg, Germany

Fig. 6: Left: photograph of the PET-scanner for education. Right: detailed picture with (1) detectorsystem, (2) object table, (3) crate with components for signal processing, (4) step motor controller,(5) standard PC for data acquisition and tomographic reconstruction.

Fig. 5: Two-dimensional β+-activity distributionsgenerated by ultrahard bremsstrahlung (coming fromthe left) in an inhomogeneous phantom consisting of 2 cm thick slabs of polymethyl methacrylate(PMMA), polyethylene (PE) and different tissue-equivalent materials (lung, adipose and bone). Upperimage: in-beam PET result measured during 34 MVphoton irradiation showing the distribution of 11C(half-life: 20 min) and 15O (half-life: 2 min) from thephotonuclear reactions 12C(γ, n) and 16O(γ, n),

respectively. Lower image: result of the PET measurement at the human scanner of the Institute ofRadiopharmacy. This measurement started 30 min after finishing the 30 MV photon irradiation.Therefore, it only shows the 11C distribution (15O has nearly completely decayed during 30 min) leadingto a reduced contrast between PE, PMMA, adipose tissue, and bone compared to the in-beam image.

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Jörg Pawelke, Elke Beyreuther, Wolfgang Wagner

In 1895, Wilhelm Conrad Röntgendiscovered X-rays and they wereimmediately and enthusiastically applied tomedical diagnostics. The side effects whichwere observed, such as skin damage orloss of hair, triggered interest in both theradiobiological properties of X-rays andtheir use for therapeutic irradiation—thefirst treatments being performed as earlyas 1896. More than 100 years after theirdiscovery, X-rays are still the subject ofradiobiological research.

In vitro cell experiments allow the study of basic radiobiological effects andmechanisms following an exposure ofliving cells to ionizing radiation. Suchexperiments revealed that biological effectsin human cells are caused by the fact thatthe DNA is the main target of radiation.Furthermore, these effects are influenced,among other elements, by the dose andquality of the radiation. The latter isexpressed by the quantity of the relativebiological effectiveness (RBE), i.e., the ratioof the absorbed doses of two types ofradiation producing the same specificeffect. While γ-rays were declared to bethe reference radiation, an RBE value of 1was assigned to photon radiations of allenergies. However, in vitro studies havealready shown that, especially at lowdoses, low-energy X-rays possess anincreased biological effectiveness (RBE > 1)compared to high-energy photons. Thesedifferences are interesting in and ofthemselves, but they must also be takeninto account when various photonradiations are used as reference radiationor applied in radiation therapy anddiagnostics.

The study of the RBE dependence onphoton energy by means of in vitro cellirradiations is part of the radiobiologicalresearch at the FZD and subject ofcollaboration with the TechnischeUniversität Dresden. This interdisciplinarytopic necessarily combines the expertisefrom several fields: (i) operation of anappropriate radiation source, (ii) physicalcharacterization of the radiation field, (iii) irradiation of probes of living cellsincluding an accurate determination of the dose absorbed by the cells, and (iv) suitable methods of determining theradiation-induced biological effects in cells on a cellular and molecular level.

Effectiveness of mammography X-rays The studies were triggered by aninsufficient knowledge of the RBE of low-energy X-rays (below about 30 keV) whichare mandatory for mammography becauseof the necessary tissue contrast. Beforeestablishing nationwide screening pro-grams, the risk-benefit ratio has to be

reliably estimated. The relative biologicaleffectiveness determined for variousmammalian cell lines ranges from less thanone up to about eight and depends on cellline, biological endpoint, and appliedreference photon radiation. So far, noexperimental studies have been performedon human mammary cells, although thesecells require investigation as they aresupposed to induce breast cancer.

Therefore, two human mammary epithelialcell lines have been chosen to determinethe RBE of 10 kV and 25 kV soft X-raysrelative to 200 kV X-rays, all generated byconventional X-ray tubes. Photon energiesbelow 10 keV are less relevant to exposureto the human body due to their strongattenuation in tissue. But they areparticularly interesting for biophysicalconsiderations and to show the trend ofenergy dependence. Various endpointshave been investigated includingclonogenic cell survival and several typesof DNA damage including the repairkinetics of DNA double strand breaks(Fig. 1). The results [1 – 2] confirm a

Cell damage after X-ray irradiation

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Fig. 1: Mammary epithelial celldamaged by X-ray irradiationwith a dose of 2 Gy:chromosomal damage shownby the induced micronuclei (topleft), chromosomal aberration(tricentric) determined byfluorescent plus Giemsastaining (top right), chromo-some specific aberration (twotranslocations involvingchromosome 1, colored pink)observed by the means offluorescence in situhybridization (bottom left) andDNA double strand breaksindicated by the formation of γ-H2AX plus 53BP1 foci(colored green, bottom right).

moderately increased relative biologicaleffectiveness of mammography X-raysranging between 1 and 2; however, theydo not support the high values of a recentclaim based on an experiment on celltransformations in a human hybrid cell lineand on re-interpretation of various earlierRBE data. Comparable RBE values havebeen determined for both of the mammarycell lines, displaying the same photonenergy dependence (Fig. 2). They are alsoconsistent with experimental data achievedfor human lymphocytes as well as with theresults of several theoretical calculations ofrelative biological effectiveness.

Since any material included in the beamcan substantially modify the dose absorbedinto the cells at these low photon energyvalues, implementation of an accuratedosimetry [3, 4] considering the exactirradiation conditions was important forthe reliability of the data. Thanks to thedetermination of the X-ray spectra, thecontribution of the different energy rangesto the dose could be calculated andcompared. Low-energy components arepresent in the broad X-ray spectrum(Fig. 3), and the biological effects of thedifferent photon energy ranges aresuperimposed.

Intense, monochromatic channeling X-rays at ELBE Experiments with an X-ray tube cannotprovide complete photon-energy resolvedinformation on biological effectiveness.Channeling radiation, which is emitted byrelativistic electrons during their passagethrough a diamond crystal parallel to acrystal plane (Fig. 4), was announced as a bright and tunable monochromatic X-ray source in the 1990s. Yet such anunconventional X-ray source, dedicated topractical application, has been realized atthe Radiation Source ELBE for the firsttime.

The yield and spectral distribution of planarchanneling radiation have been measured,depending on the crystal properties as wellas the parameters of the ELBE electronbeam at low beam current (several nA)[5 – 7]. The results show that by variationof the electron beam energy, the photon


32

Fig. 2: Yields of chromosomalexchange aberrations per cellinduced by different X-rayqualities depending on dose.

Fig. 3: Energy distribution ofan X-ray tube with tungstenanode operated at 25 kV.

Fig. 4: Scheme of channelingX-ray generation.

Fig. 5: Spectra of quasi-monochromatic channelingradiation with the associatedbremsstrahlung (blue line)measured for 14.6 MeVelectrons channeled in the(110) plane of a 42 µm thickdiamond crystal. The narrowpeak (red line) representsmonochromatic X-rays afterBragg reflection at the (002)plane of a planar large-areaHOPG crystal.

33

Rossendorf Beamline


Structure of Matter

Life Sciences


PET Center

Ion Beam Center


energy can be tuned from 10 keV up toabout 100 keV, requiring thicker diamondcrystals if large channeling radiationintensities are required. An intense sourceof channeling radiation dedicated toradiobiological application was designedand implemented at the Radiation SourceELBE. After commissioning, stableoperation was proven over hours with anaverage electron beam current of up to100 µA, which allows reaching photonrates of quasi-monochromatic channelingradiation of the order of 1011 s-1. For this,various technological challenges had to besolved: (i) precise crystal alignment withrespect to the beam axis by a newlyconstructed goniometer, (ii) water-coolingof the crystal while guaranteeing that theelectron beam passes through the 150 µmthick diamond crystal of 4 x 4 mm2 size,(iii) on-line monitoring of the photon-energy spectra at high beam current bymeans of a Compton spectrometer, and

(iv) X-ray monochromatization by applyingBragg diffraction at a highly-orderedpyrolytic graphite (HOPG) crystal. Here,monochromatization means to filter outsome part of the spectral distribution at those photon energies where thechanneling radiation line superimposes the broad polychromatic bremsstrahlungbackground naturally associated with theproduction of channeling radiation (Fig. 5).

Cooperation with industrial partners wasessential for the success. The uniqueHOPG technology of Bourevestnik Inc.,St. Petersburg, Russia, allowed designingand building a special toroidal HOPG X-rayreflector which focuses the X-rays on theprobe for irradiation of living cells.Radiobiological experiments with quasi-monochromatic channeling radiation X-rays are planned at ELBE at the end of 2007.

References[1] RBE of 25 kV X-rays for the survival and

induction of micronuclei in the human mammary epithelial cell line MCF-12A, A. Lehnert, E. Leßmann, J. Pawelke, W. Dörr1,2, Radiation and Environmental Biophysics 45, 253 (2006)

[2] RBE of 10 kV X-rays determined for the human mammary epithelial cell line MCF-12A, A. Lehnert, E. Leßmann, J. Pawelke, W. Dörr1,2, Radiation Research (2007), accepted

[3] Application of advanced Monte Carlo methods in numerical dosimetry, U. Reichelt, J. Henniger3, C. Lange3, Radiation Protection Dosimetry 119, 479 (2006)

[4] Investigation of a TSEE dosimetry system for determination of dose in a cell monolayer, A. Lehnert, E. Beyreuther, E. Lessmann, J. Pawelke, Radiation Measurements, doi: 10.1016/j.radmeas. 2007.03.007 (2007)

[5] Electron beam monitoring for channeling radiation measurements, W. Neubert, B. Azadegan, W. Enghardt2, K. Heidel, J. Pawelke, W. Wagner, Nuclear Instru-ments and Methods B 245, 319 (2007)

[6] Dependence of the linewidth of planar electron channeling radiation on the thickness of the diamond crystal, B. Azadegan, W. Wagner, J. Pawelke, Physical Review B 74, 045209-1 (2006)

[7] Planar channeling radiation from electrons in quartz, W. Wagner, B. Azadegan, L.Sh. Grigoryan4, J. Pawelke, Europhysics Letters 78, 56004 (2007)

Project partners1Clinic of Radiotherapy, Faculty of Medicine, Technische Universität Dresden, Germany

2OncoRay – Center for Radiation Research in Oncology, Faculty of Medicine, Technische Universität Dresden, Germany

3Institute of Nuclear and Particle Physics, Technische Universität Dresden, Germany

4Institute of Applied Problems of Physics, National Academy of Sciences, Yerevan, Armenia Accelerator module of the Radiation Source ELBE. Photo: Sven Claus


34

Biomolecular Switches: Molecular evolutionconserves function, but allows diversification

Karim Fahmy, Sineej Madathil

The integration of the multitude ofbiochemical processes occurring simul-taneously in a cell critically depends onmolecular switching events. They rely onmolecules which can adopt differentstructures, allowing them to interactspecifically with other components of acell. Linking molecular switches into cross-talking signaling chains generates anintricate network of information flowwithin a cell. Such networks guarantee the proper differentiation, function, andeven the initiation of the death of a cell.

G-protein-coupled receptors (GPCRs) forma particularly important class of moleculeswhich switch between functionally activeand inactive states after receiving a specificsignal. GPCRs couple a large variety ofextracellular stimuli to cell-specific answers.Key representatives of GPCRs are neuro-transmitters and hormone receptors. Uponligand binding at the extracellular side ofthe receptors, these membrane proteinsalter their structure at the intracellular side.This allows interaction with guanosine-binding proteins (G-proteins) which initiatesignal amplification and transductionwithin the cell. The human genome con-tains about 1000 GPCRs. Each member ofthis receptor superfamily is a potential drugtarget and approximately 50% of today’spharmacotherapeutics target at GPCRs.Intensive structural and functional studiestry to exploit their pharmacologicalpotential.

Within evolutionary related GPCRs,functional mechanisms are believed to beconserved; however, it is not understoodhow an extremely large variety ofSet-up for biophysical experiments with the free-electron laser at ELBE.

35

Rossendorf Beamline


Structure of Matter

Life Sciences


PET Center

Ion Beam Center


structurally different ligands can operateessentially the same mechanism. We havestudied the physical basis of switching inthe bovine photoreceptor rhodopsin, theonly GPCR for which X-ray structures havebeen determined, so that structural modelsof receptor activation can be generated[1]. Rhodopsin provides the molecularbasis of vision. It is a prototypical GPCRwhich allows transducing the absorption of light into a nerve pulse in the retina ofthe eye. The process of vision is primarilybased on the photoisomerization of thecovalently bound ligand 11-cis retinal,which evokes structural changes inrhodopsin very similar to the changesinduced by the binding of a hormone to

a hormone receptor. Today, most studies of rhodopsin aim at an understanding ofgeneral switching mechanisms in GPCRs.

The plasmamembrane: A specificreaction environment involved inconformational switching of GPCRsOur work focuses on the role of specificlipid protein interactions within the plasmamembrane which affect the switchingprocess of GPCRs. The function of the lipidbilayer into which GPCRs are integrated isnot deducible from the crystal structures,where lipids are absent. Using spectro-scopic techniques, we have investigatedtwo regions of rhodopsin which are

involved in functionally important lipidprotein interactions. One region is the C-terminal extramembraneousamphipathic helix 8. In photoactivatedrhodopsin, it binds to the G-protein. It has been suggested to be a “local” switchregion because it can adopt differentstructures even in the absence of the restof the receptor molecule, depending onthe surrounding lipids [2].

To understand the long-range coupling ofthe cytosolic helix 8 to the transmembraneregion where the light-dependentstructural alteration occurs, we havesimultaneously observed two independentspectroscopic properties by which

Fig. 2: Fluorescence spectroscopy of an isolatedsynthetic hydrophobic peptide corresponding tohelix 3 of rhodopsin exhibits proton-sensitivefluorescence energy transfer from the nativetyrosin in the conserved ERY motif to anengineered tryptophan. The unusually high pK of ~6 is assigned to the neighboring strictlyconserved glutamic acid as the only titratablegroup in this pH range [5]. The high pK indicatesthat the protonated (neutral) state of the Glu134

side chain becomes stabilized by hydrophobicinteractions in a surrounding lipophilic phase aspresented in the bilayer model above. The rightpanel shows the position of the ERY motif in thecontext of the full-length receptor, where itlocalizes close to the membrane water interface.

Fig. 1: a) Sketch of the rhodopsin structure (pdbid 1L9H) emphasizing the location of thecovalently bound fluorescent dye fluorescein athelix 8 and the position of protonated Asp83, forwhich the C=O stretching mode has beenassigned and used here to monitor changes in the intramembrane H-bond network above the11-cis-retinal. TM1 – 3 refers to theintramembraneous helices 1 – 3, whereas helix 8 runs roughly parallel to the cytosolic membranesurface. b) Fluorescence-infrared-cross-correlation spectroscopy evidences the kineticcoupling between the physical environment ofhelix 8 and the intramolecular H-bond networkconnecting Asp83 to the conserved NPxxY motifthrough two water molecules (green). Thisprovides a signaling pathway from the interior ofthe GPCR to its cytosolic surface as indicated bythe red arrow.

a) b)


36

structural transitions can be monitored:fluorescence emission and IR-absorption.In cooperation with Ulrike Alexiev fromFreie Universität Berlin, this experiment hasallowed us to identify highly synchronizedchanges around the lipid-interacting helix8 (where a fluorescent label was attachedto Cys316) and the transmembrane H-bondnetwork around Asp83 in the interior of theprotein. The latter was monitored byinfrared spectroscopy. The data reveal astructural connectivity that couplesphotoisomerization to a lipid-dependentswitching motif at the extramembraneousG-protein recognition site [3]. In contrast,another internal domain around Glu122

communicates with an H-bond networkwhich is not coupled to the helix 8environment, but appears to be importantfor retinal binding.

The kinetic analysis of distantconformational changes within rhodopsinclearly shows that ligand specificity canevolve independently from evolutionaryconservation of function. This is stronglysupported by the fact that Asp83 and thetransmembrane motif to which it is linkedthrough water molecules have beenstrongly conserved throughout evolution,comprising many hormone receptors,whereas Glu122 has only been conserved in the subfamily of photoreceptors.

Hydrophobicity and side chainprotonation in GPCR function:Searching for artificial mimics of natural switchesWe have extended our studies to anothersurface region of rhodopsin which alsointeracts with the lipidic matrix and havestarted to systematically explore the role of side chain hydrophobicity in membraneproteins. Hydrophobicity describes theease by which a chemical group can be

transferred from an aqueous to a nonpolarenvironment such as the interior of aplasma membrane. Recently, the biologicalimportance of hydrophobicity for theinsertion of membrane proteins into theplasma membrane has been accuratelyquantified. This led to a scale of freeenergy of the transfer of any of the 24naturally-occurring amino acids in proteinsfrom a hydrophobic to an aqueousenvironment [4]. Importantly, the influenceof the charges of a protonatable group inthese amino acids has been addressed. Wehave investigated whether the modulationof side chain hydrophobicity byprotonation may provide a link betweenproton exchange reactions occurring inGPCRs and other membrane proteins andthe transition between their functionallydistinct states. Surprisingly, a specific singletransmembrane-spanning domain ofrhodopsin, i.e., one helical rod-likestructural element out of seven whichmake up a GPCR, exhibits proton-dependent structural changes in anartificial membrane [5].

In cooperation with the Max PlanckInstitute of Molecular Physiology,Dortmund, and M-fold GmbH, Tübingenin a project funded by the Federal Ministryof Education and Research (BMBF), we areapplying our approach to a large scale ofsynthetic peptides with charged residuesstrategically located near the water lipidphase boundary, where proton-dependentstructural reorganizations can be expected.These model systems will not only verifyimportant hypotheses on GPCR functionbut the knowledge gained from this classof membrane receptors will also allow thesynthesis of artificial transmembraneswitching devices such as the trans-membrane proton sensor which we arecurrently developing in this project.

References[1] Rhodopsin: a structural primer for

G-protein coupled receptors,R.E. Stenkamp, D.C. Teller, K. Palczewski, Archiv der Pharmazie (Weinheim) 338, 209 – 216 (2005)

[2] Evidence that helix 8 of rhodopsin acts as a membrane-dependent conform-ational switch, A.G. Krishna1, S.T. Menon1, T.J. Terry1, T.P. Sakmar1, Biochemistry 41, 8298 – 8309 (2002)

[3] Linkage between the intramembrane H-bond network around aspartic acid 83 and the cytosolic environment of helix 8 in photoactivated rhodopsin, N. Lehmann2, U. Alexiev2, K. Fahmy, Journal of Molecular Biology 366, 1129 – 1141 (2007)

[4] Do protein-lipid interactions determine the recognition of transmembrane helices at the ER translocon?,S.H. White, G. von Heijne, Biochemical Society Transactions 33, 1012 – 1015 (2005)

[5] Structure and pH sensitivity of the transmembrane segment 3 of rhodopsin, S. Madathil, G. Furlinski, K. Fahmy, Biopolymers 82, 329 – 833 (2006)

Project partners1 Indian Institute of Technology Madras (IITM, Chennai)

2Freie Universität Berlin, Germany

37

FACTS & FIGURES

The Forschungszentrum Dresden-Rossendorf (FZD) is a multi-disciplinary research centerfor natural sciences and technology. It is the largest institute of the Leibniz Associationand is equally funded by the Federal Republic of Germany and the Federal States, inparticular by the Free State of Saxony. At the FZD, around 225 scientists are engaged inthree different research programs of basic and application-oriented research. Scientistsworking in the Structure of Matter program investigate the reactions of matter wheninfluenced by high fields and minuscule dimensions. Research and development in theLife Sciences program is focused on the imaging of tumors and the effective radiationtreatment of cancer. How can humankind and the environment be protected fromtechnical risks? – This question is in the center of research in the Environment and Safetyprogram of the FZD.

In the following Facts & Figures section data presenting the scientific output in the Life Sciences research program are given as well as information on staff and funding at the FZD.

Facts & Figures

3838

Structure of Matter Life Sciences Environment and Safety

10

8

6

4

2

02004 2005 2006

50

40

30

20

10

02004 2005 2006

Patents

Publications

staff scientists third-party funded scientistsguest scientists postdocs

30

20

10

02004 2005 2006

Scientific staff

This figure shows the number of applications for a patent filed in eachresearch program of the FZD during the last years. National andinternational applications for a patent of one and the same inventionare only counted once.

This chart displays the evolution of peer-reviewed articles by scientistsfrom the FZD’s Life Sciences program.

This chart shows the evolution of posts occupied by scientificpersonnel in the Life Sciences program of the FZD. Third-party funded scientists, guest scientists, and postdocsrepresented by the corresponding figures are given in units ofpaid full-time posts.

2004 2005 2006

Research Programs

Structure of Matter

Life Sciences


Facilities

Sum

Public FundingT€

13.858

8.241

13.005

20.721

55.825

Project FundingT€

1.190

801

3.180

911

6.082

Public FundingT€

14.209

6.266

12.950

20.428

53.853

Project FundingT€

1.259

645

3.656

756

6.316

Public FundingT€

19.575

8.191

11.866

17.988

57.620

Project FundingT€

1.405

784

3.990

1.643

7.822

Budget

This table displays the share of each research program as well as the experimental facilities located at the FZD of both public and projectfunding during the last three years.

39

Latvia

India

Hungary

Algeria

Japan

Israel

Spain

USA

Romania

Australia

other

5

5

3

3

36

13

10

9

8

6

5

Russia

Ukraine

Bulgaria

Czech Republic

Poland

China

99

29

25

22

19

16

80

60

40

20

02004 2005 2006

Doctoral students

International guest scientists

This figure shows the evolution of the doctoral students atthe FZD from 2004 until 2006.

Here, the distribution of the international guest scientistswho visited the FZD for the purpose of research between2004 and 2006 is shown according to their countries oforigin.

FACTS & FIGURES

40

Structure of Matter

Life Sciences

Research Technology

Dr. Frank Herbrand

Administration

Andrea Runow

Technical Service

Dr. Wolfgang Matz

Institute of Ion Beam Physics andMaterials Research

Prof. W. Möller and Prof. M. Helm

Institute of Radiopharmacy

Prof. Jörg Steinbach

Dresden High Magnetic FieldLaboratory

Prof. Joachim Wosnitza

Institute of Radiation Physics

N.N.

Laser-Particle Acceleration

Dr. Ulrich Schramm


Institute of Safety Research

Prof. Frank-Peter Weiß

Institute of Radiochemistry

Prof. Gert Bernhard

General Assembly

Scientific Advisory Board Chair: Prof. August Schubiger

Supervisory BoardChairman: Dr. Gerd UhlmannDeputy: Dr. Jan Grapentin

Scientific Technical Council Chair: Prof. Frank-Peter Weiß

Works Committee Chair: Siegfried Dienel

Support Staff

Board of Directors Scientific Director Administrative DirectorProf. Roland Sauerbrey Dr. Dr. h. c. Peter Joehnk

Organizational Chart

IMPRINT

FZD Triennial Scientific Report 2004-2007 I Volume 3Volume 1 Structure of MatterVolume 2 Environment and SafetyVolume 3 Life Sciences

Published by Forschungszentrum Dresden-RossendorfConcept and editorial work Dr. Christine Bohnet & Anja Bartho, FZDDesign and layout WA Preußel, CoswigPhotos C. Preußel & FZD employeesAvailable from Forschungszentrum Dresden-Rossendorf

Public RelationsBautzner Landstr. 12801328 Dresden / GermanyPhone: +49 351 260 2450Email: [email protected]://www.fzd.de

ISSN 1437-322XWissenschaftlich-Technische BerichteFZD-472, October 2007

Copying is welcomed, provided the source is quoted.

Forschungszentrum Dresden-RossendorfP.O. Box 51 01 19 I 01314 Dresden/GermanyScientific Director I Prof. Dr. Roland SauerbreyPhone +49 351 260 2625Fax +49 351 260 2700Email [email protected]

Member of the Leibniz Association

Documents

FZD Inhalt Life Science - HZDR · Small animal positron-emission tomography in radiation therapy response monitoring ... As for the Life Sciences research program scientists at OncoRay