Univ., Diss., 2009--Marburg.

Ulm, Univ. Diss., 2006.

Introduction [11C]choline is a very effective PET radiopharma-ceutical for the study of prostate cancer. To support the increasing demand for [11C]choline, several different synthetic approaches have been described in the literature, including different automated production methods using remote-controlled synthesis modules [1–4]. The most popular method uses a C18 Sep-Pak as solid support for methylation and, subsequently, a CM Sep-Pak for purification [2]. We report an optimized method for producing [11C]choline using only one CM Sep-Pak for both reaction and purification as was shown in the literature [4]. For synthesis of [11C]choline we used two modules Tracerlab FXC for preparation of methylation reagent [11C]CH3I and GPF-101 for [11C]choline synthesis. Material and Methods TracerlabFXC GE, GPF-101 Veenstra Instrument, 2-(dimethylamino)-ethanol (DMAE) ABX, Sep-Pak Light Accell Plus CM cation-exchange cartridges Waters used without conditioning, precursor 50 µL of DMAE dissolved in 25 µL of ethanol and loaded on a CM Sep-Pak. Schematic diagram of the automated system for the production of [11C]choline is given below. [11C]CH4 was produced in two standard Nitra target IBA irradiation of mixture 90 % N2/10 % H2 with 15 MeV protons using dual beam. Results and Conclusion [11C]CH4 was prepared in the targets and connected with Tracerlab FXC. [11C]CH3I was pre-pared in a loop in which allowed to react of elemental iodine at a temperature 720 oC. Con-version to [11C]CH3I usually is around 50% uncorrected activity. Activity is within the range 15–18 GBq of [11C]CH3I and time of production 10 min. Synthesis of [11C]choline is based on the reaction DMAE with [11C]CH3I on a Accell Plus CM cation-exchange column which serves both as a support for reaction and for separation of choline from DMAE by ethanol washing. The basic parameters are shown in TABLE 1. Beam current 2X 20 µA Irradiation time 30 min DMAE 50 µl Synthesis time from EOB 25 min Absolute yield without correction 6.6 GBq Radiochemical purity > 99 % Residual DMAE in product < 5 ppm Ethanol < 1000 mg/L pH 4.5–8.5 TABLE 1. Reaction parameters and result of production of [11C]choline syntheses Conclusion We have applied a simple synthesis method for [11C]choline preparation using automated commercial equipments with one column used both for reaction and separation purpose. The main advantage of using one column is lower contamination of the product [11C]choline with DMAE. When for synthesis of [11C]choline two columns C18 for synthesis and CM for separation is used, higher contamination of DMAE can be found in the product due to a release of DMAE from C18 column.

: Choline is being used in all mammalian cells as a precursor for synthesis of a major phospholipide phosphatidylcholine and as a donor of acetyl residues. Cholinergic neurons in addition require choline to synthesize neuromediator acetylcholine. The ability of cells to create choline via de novo synthesis is limited and therefore they need to transport choline from extracellular space. Limited availability of choline in brain leads specifically to diminished function of cholinergic neurons and in general to impaired reparation of biological membranes. Dysfunctions of cholinergic signaling in brain is characteristic for Alzheimer's disease. Aim of this work was to investigate whether gene and protein expression of high- affinity cholinergic transporters is altered in 5-6 months old APPswe/PS1dE9 mouse model of Alzheimer's disease. Expression of specific high-affinity cholinergic transporter CHT1 (responsible for transport of choline to be used for acetylcholine synthesis) and putative high-afinity choline transporter CTL1 (generally present in all cells and related to high affinity choline transport for phospholipide synthesis) in cerebral cortex was measured. Compared to non-trangenic littermates, no changes in the expression of both genes were detected at either mRNA (quantitative PCR) or protein...

Introduction [11C]choline is a very effective PET radiopharma-ceutical for the study of prostate cancer. To support the increasing demand for [11C]choline, several different synthetic approaches have been described in the literature, including different automated production methods using remote-controlled synthesis modules [1–4]. The most popular method uses a C18 Sep-Pak as solid support for methylation and, subsequently, a CM Sep-Pak for purification [2]. We report an optimized method for producing [11C]choline using only one CM Sep-Pak for both reaction and purification as was shown in the literature [4]. For synthesis of [11C]choline we used two modules Tracerlab FXC for preparation of methylation reagent [11C]CH3I and GPF-101 for [11C]choline synthesis. Material and Methods TracerlabFXC GE, GPF-101 Veenstra Instrument, 2-(dimethylamino)-ethanol (DMAE) ABX, Sep-Pak Light Accell Plus CM cation-exchange cartridges Waters used without conditioning, precursor 50 µL of DMAE dissolved in 25 µL of ethanol and loaded on a CM Sep-Pak. Schematic diagram of the automated system for the production of [11C]choline is given below. [11C]CH4 was produced in two standard Nitra target IBA irradiation of mixture 90 % N2/10 % H2 with 15 MeV protons using dual beam. Results and Conclusion [11C]CH4 was prepared in the targets and connected with Tracerlab FXC. [11C]CH3I was pre-pared in a loop in which allowed to react of elemental iodine at a temperature 720 oC. Con-version to [11C]CH3I usually is around 50% uncorrected activity. Activity is within the range 15–18 GBq of [11C]CH3I and time of production 10 min. Synthesis of [11C]choline is based on the reaction DMAE with [11C]CH3I on a Accell Plus CM cation-exchange column which serves both as a support for reaction and for separation of choline from DMAE by ethanol washing. The basic parameters are shown in TABLE 1. Beam current 2X 20 µA Irradiation time 30 min DMAE 50 µl Synthesis time from EOB 25 min Absolute yield without correction 6.6 GBq Radiochemical purity > 99 % Residual DMAE in product < 5 ppm Ethanol < 1000 mg/L pH 4.5–8.5 TABLE 1. Reaction parameters and result of production of [11C]choline syntheses Conclusion We have applied a simple synthesis method for [11C]choline preparation using automated commercial equipments with one column used both for reaction and separation purpose. The main advantage of using one column is lower contamination of the product [11C]choline with DMAE. When for synthesis of [11C]choline two columns C18 for synthesis and CM for separation is used, higher contamination of DMAE can be found in the product due to a release of DMAE from C18 column.

Streptococcus pneumoniae ist der häufigste Erreger bakterieller Meningitiden. Eine Pneumokokken-Meningitis führt trotz Ausschöpfung aller heute verfügbaren Behandlungsmöglichkeiten in 25 % der Fälle zum Tod. Steigende Antibiotikaresistenzen und die Limitation verfügbarer Vakzine auf einige Serotypen von S. pneumoniae erfordern neue Ansätze in der antimikrobiellen Therapie. Cholin-bindende Proteine (CBPs) sind gemeinsames Merkmal aller Pneumokokkenstämme und für die Virulenz dieses Bakteriums essenziell. Durch Zugabe potenter Anticholinergika können die CBPs von der Bakterienoberfläche abgelöst und damit inhibiert werden. In dieser Arbeit wurde untersucht, ob durch Inhibition von CBPs während des Wachstums der Pneumokokken deren Phagozytose durch Mikrogliazellen in vitro gesteigert werden kann. Während ihres Wachstums wurden die Bakterien dazu mit potenten Anticholinergika inkubiert und am Ende ihrer exponentiellen Wachstumsphase auf murine Mikrogliazellen gegeben. Nicht alle eingesetzten Anticholinergika konnten die Inhibition der CBPs – angezeigt durch die Bildung langer Kokkenketten – bewirken, obwohl für sie alle eine hohe Affinität zu den CBPs in früheren Arbeiten nachgewiesen worden war. Ipratropium, das in höheren Konzentrationen das Pneumokokkenwachstum inhibiert, induzierte in den von uns eingesetzten niedrigen Konzentrationen weder die Bildung von Ketten noch führte es zu einer erhöhten Phagozytoseleistung. Mit DMAE funktionalisiert zeigten PAMAM-Dendrimere der 1. Generation ebenfalls keine Inhibition der CBPs: Es bildeten sich weder Kokkenketten noch zeigte sich eine erhöhte Bakterienaufnahme der Mikroglia. Im Gegensatz dazu stellte sich unter Einfluss von PPI-g2-DMAE neben ausbleibender Kettenbildung ein dosisabhängiger phagozytosehemmender Effekt dar. Einzig durch Co-Inkubation mit dem mit Cholin funktionalisierten PPI-Dendrimer der 2. Generation gelang die Inhibition der CBPs mit resultierender Bildung langer Ketten. Die Phagozytoseleistung zeigte eine dosisabhängige Steigerung sowohl für eine CoInkubation während der gesamten exponentiellen Wachstumsphase als auch nach Co-Inkubation während ihrer letzen 2 Stunden. Dennoch konnte im Sepsismodell der Maus durch intraperitoneale Injektion dieses Dendrimers 15 min vor Infektion mit S. pneumoniae kein protektiver Effekt erzielt werden: Zwischen den mit Dendrimeren behandelten Tieren und denen der Kontrollgruppe zeigten sich keine Unterschiede in Überlebenszeit und Sterblichkeit, dem krankheitsbedingten Gewichtsverlust, dem klinischen Score und der durch Ausplattieren von Milzhomogenaten ermittelten Keimkonzentration im Blut infektionsbedingt verstorbener Tiere. Die von uns eingesetzten Konzentrationen von Ipratropium scheinen für eine Inhibition der CBPs nicht ausgereicht zu haben. Der bislang nicht genau geklärte wachstumsinhibitorische Effekt, der sich in unseren Versuchen bereits ab 5 mM bemerkbar machte, könnte jedoch durch Inhalation von Ipratropium gezielt zur Prophylaxe von Pneumokokken-Pneumonien genutzt werden. Bei an Dendrimere gekoppelten, eigentlich potenten Liganden der CBPs konnte beobachtet werden, dass sie als Teil des Dendrimers ihre Affinität gegenüber den CBPs nicht nur deutlich verändern, sondern auch unerwartete Effekte (Verminderung der Phagozytose) hervorrufen können. Wegen der raschen Elimination scheint die einmalige Gabe eines potenten Dendrimers zur Inhibition der CBPs in vivo nicht auszureichen und erklärt das Versagen im Sepsismodell. Neuere Untersuchungen zur Distribution im Hirnparenchym nach intraventrikulärer oder subarachnoidaler Injektion lassen hoffen, dass durch Gabe subtoxischer Dosen die von uns beobachtete Phagozytosesteigerung in vivo reproduzierbar ist. Durch Inhibition der CBPs ist es möglich, die Virulenz aller Serotypen des Pneumokokkus stark zu reduzieren. Potente Inhibitoren könnten sowohl als Therapeutikum als auch zur Infektionsprophylaxe eingesetzt werden, ohne dass es dabei zur Ausbildung von Resistenzen kommt, da zeitgleich mehrere Virulenzfaktoren inhibiert werden. Es ist daher von großem medizinischen Interesse, Inhibitoren der CBPs zu entwickeln, die in subtoxischen Dosen eine hohe Affinität zu den CBPs aufweisen und diese inhibieren.

The purpose of this study was the investigation of several transport systems expressed at the BBB. The identification and functional characterization of such transport systems is essential to provide a basis for strategies to regulate drug disposition into the brain. Immortalized rat brain endothelial cells (RBE4 cells) have been used in this study as an in vitro model of the BBB. The present study has shown that the RBE4 cells are a suitable model of the BBB for transporter studies. These cells do express the amino acid transport systems L and y+, which are known to be present at the BBB. The uptake of L-tryptophan, a neutral amino acid transported by system L, exhibited a half saturation constant (Kt) of 31 µM and a maximal velocity rate (Vmax) of about 1 nmol/mg/min in RBE4 cells. The kinetic constants of the L-arginine uptake, representing system y+ transport activity, into RBE4 cells were determined with a Kt value of about 55 µM and a Vmax of 0.56 nmol/mg/min. Furthermore the expression of two sodium dependent transporters, the 5-HT transporter (SERT) and the organic cation/carnitine transporter OCTN2, was shown at the RBE4 cells. Uptake studies with radiolabeled 5-HT exhibited a saturable, sodium dependent transport at RBE4 cells with a Kt value of about 0.40 µM and a Vmax of about 52 fmol/mg/min. L-carnitine and TEA (tetraethylammonium) are known to be transported by the OCTN2 transporter. The uptake of L-carnitine into RBE4 cells was shown to be sodium dependent and saturable with a Kt value of 54 µM and a maximal velocity of about 3.6 pmol/mg/min. In contrast, the organic cation TEA follows a sodium independent uptake mechanism at RBE4 cells. Also a sodium independent choline uptake into the cells was discovered but the molecular identity remained unknown. This saturable choline transport exhibited a Kt value of about 22 µM and a maximal velocity of about 52 pmol/mg/min.

The purpose of this study was the investigation of several transport systems expressed at the BBB. The identification and functional characterization of such transport systems is essential to provide a basis for strategies to regulate drug disposition into the brain. Immortalized rat brain endothelial cells (RBE4 cells) have been used in this study as an in vitro model of the BBB. The present study has shown that the RBE4 cells are a suitable model of the BBB for transporter studies. These cells do express the amino acid transport systems L and y+, which are known to be present at the BBB. The uptake of L-tryptophan, a neutral amino acid transported by system L, exhibited a half saturation constant (Kt) of 31 µM and a maximal velocity rate (Vmax) of about 1 nmol/mg/min in RBE4 cells. The kinetic constants of the L-arginine uptake, representing system y+ transport activity, into RBE4 cells were determined with a Kt value of about 55 µM and a Vmax of 0.56 nmol/mg/min. Furthermore the expression of two sodium dependent transporters, the 5-HT transporter (SERT) and the organic cation/carnitine transporter OCTN2, was shown at the RBE4 cells. Uptake studies with radiolabeled 5-HT exhibited a saturable, sodium dependent transport at RBE4 cells with a Kt value of about 0.40 µM and a Vmax of about 52 fmol/mg/min. L-carnitine and TEA (tetraethylammonium) are known to be transported by the OCTN2 transporter. The uptake of L-carnitine into RBE4 cells was shown to be sodium dependent and saturable with a Kt value of 54 µM and a maximal velocity of about 3.6 pmol/mg/min. In contrast, the organic cation TEA follows a sodium independent uptake mechanism at RBE4 cells. Also a sodium independent choline uptake into the cells was discovered but the molecular identity remained unknown. This saturable choline transport exhibited a Kt value of about 22 µM and a maximal velocity of about 52 pmol/mg/min.

Im Rahmen dieser Arbeit wurde der Einfluss des GPIIb/IIIa- Antagonisten Tirofiban auf die Vollblut-Konzentration des Phospholipase D Metaboliten Cholin (2- hydroxyethyltrimethylammonium, "whole blood cholin", WBCHO) bei Patienten mit akutem Koronarsyndrom untersucht. Die Phospholipase D hat eine Schlüsselfunktion bei der Destabilisierung atherosklerostischer Plaques, Aktivierung von Thrombozyten und Sekretion von Matrixmetalloproteinasen durch Makrophagen. Als Analyseverfahren für Cholin wurde die Hochleistungsflüssigkeits-Chromatographie (HPLC) in Verbindung mit der Massenspektrometrie (MS) eingesetzt. Die Klassifikation der Patienten erfolgte nach den aktuellen Richtlinien der European Society of Cardiology (ESC) und des American College of Cardiology (ACC) für das akute Koronarsyndrom. Aus einem Kollektiv von 342 Patienten wurden 32 Patienten mit akutem Koronarsyndrom in diese Studie aufgenommen, in zwei Gruppen mit jeweils 16 Patienten mittels matched pairs Technik unterteilt und analysiert. Eine Gruppe erhielt zusätzlich zur Standard- Therapie Tirofiban. Es wurden Blutabnahmen bei Aufnahme, nach 3-6 Stunden und nach 12-24 Stunden gewonnen. Hieraus wurden Troponin I und T, Myoglobin, Kreatinkinase Isoenzym MB, sowie Vollblut-Cholin bestimmt. Es gab einen signifikanten Verlauf der WBCHO- Konzentration (p = 0,006) in der mit Tirofiban behandelten Gruppe im Gegensatz zur Gruppe die nur die Standardtherapie erhielt (p = 0,174). Für den Verlauf der Standardmarker (Myoglobin, Kreatinkinase, Troponin I und T), wurde keine signifikante Beeinflussung durch die Therapie mit Tirofiban nachgewiesen. Im Vergleich zu Troponin I und T, Myoglobin und Kreatinkinase hatte WBCHO das zeitlich früheste Maximum. WBCHO könnte als Markersubstanz der Phospholipase D Aktivität zusätzliche Informationen über die Möglichkeit einer Destabilisierung einer atherosklerotischen Plaque bei Patienten mit akutem Koronarsyndrom geben. Dies könnte in Kombination mit anderen Markern eine verbesserte Risikostratifizierung in der Frühphase des akuten Koronarsyndroms ermöglichen. Zusätzlich scheint ein Monitoring der Tirofiban Therapie durch die WBCHO Konzentration möglich zu sein. / This research work deals with the measurement of the phospholipase D metabolite choline in patients with acute coronary syndrome (ACS) undergoing GPIIb/IIIa antagonist therapy. The influence of GPIIb/IIIa antagonists on concentration levels of the PLD metabolite 2- hydroxyethyltrimethylammonium in blood (whole blood choline, WBCHO) was studied. The activation of phospholipase D (PLD) has a key function in plaque destabilisation, activation of platelets and secretion of matrixmetalloproteinases by macrophages. For the detection of the PLD metabolite WBCHO high pressure liquid chromatography (HPLC) with a mass spectrometer (MS) was used. The classification of patients was performed according to the current guidelines of the European Society of Cardiology (ESC) and the American College of Cardiology (ACC). 32 patients with ACS out of a 342 patient study were included and analysed by matched pairs technique as two groups with 16 patients. One group was treated with Tirofiban (aggrastrat) in addition to standard therapy. Blood samples were taken at admission, after 3-6 hours and after 12-24 hours and in addition to the troponines, myoglobin and creatinkinase Isoenzyme MB, whole blood choline was analyzed. There was a significant (p = 0,006) decrease of WBCHO level in the group treated with Tirofiban in contrast to the reference group with no significant decrease (p = 0,174). The levels of conventional markers as troponin I and T, CK-MB mass and myoglobin had no significant changes in relationship to the Tirofiban therapy. WBCHO had the earliest maximum in contrast to all other markers. We concluded that WBCHO can be used as an additional early risk marker in ACS. Since GPIIb/IIIa- antagonist- therapy may influence WBCHO level, WBCHO has potential to be used for monitoring of therapy.

Proton MR spectroscopy is a non-invasive tool for measuring in vivo concentrations of several metabolites. The aim of this thesis was to test its applicability and reliability in neurosurgical praxis. In the first part of the study multiple MR spectroscopy methods were applied in a group of patients after surgery and oncologic treatment for high-grade glioma to test which method performed best in discriminating recurrent tumor from radionecrosis in the presence of a new enhancing lesion. The best diagnostic yield was achieved by comparison of choline, creatine and lactate between lesion and contralateral side (sensitivity 93.3%, specificity 78.6%). Creatine was significantly decreased in patients compared to controls. The inhibiting effect of ongoing oncologic treatment on cerebral and tumoral metabolism makes differential diagnosis trickier. Therefore, a diagnosis of radionecrosis assessed during ongoing radio- and chemotherapy should be confirmed after its completion. In the second part of the study MR spectroscopy data was compared with MR hippocampal volumetry and transcranial doppler examination in a cohort of patients with unilateral occlusion of the internal carotid artery. The N-acetylaspartate/choline ratio and hippocampal volume were significantly lower in both hemispheres of patients...