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4. Direct labeling of peptides with [18F]FDG

Author

Hultsch, C., Bergmann, R., Berndt, M., and Wüst, F.

Abstract

Objectives: The routine 18F labeling of biomacromolecules like peptides and proteins mainly exploits the use of bifunctional labeling precursors, also referred to as prosthetic groups. 2-[18F]Fluoro-2-deoxy-D-glucose ([18F]FDG) is the most important clinical PET radiotracer, but only very few examples using readily available [18F]FDG as a building block for the radiosynthesis of 18F-labeled compounds. The present study describes the use of [18F]FDG as a 18F building block for the direct labeling of various aminooxy-functionalized peptides via chemoselective oxime formation. The potential of this novel peptide labeling reaction was expemplified by means of various neurotensin NT(8-13) derivatives. Methods: The labeling reaction was performed using a 0.9% NaCl solution of [18F]FDG and aminooxy-functionalized peptide at different concentrations in a mixture of MeOH/H2O at 80 °C for 30 min. The reaction mixture was analyzed by radio-HPLC to determine the radiochemical yield of the conjugation reaction (Fig. 1). Results: Direct labeling of aminooxy-functionalized peptides with [18F]FDG was strongly dependent on the amount of used peptide. Monomeric NT(8-13) derivative gave best radiochemical yields of up to 80% based upon [18F]FDG. More complex dimeric and tetrameric neurotensin derivatives gave lower radiochemical yields at comparable peptide concentrations. Increase of [18F]FDG activity also lowered radiochemical yield due to an increasing competitive reaction with glucose originating from the [18F]FDG solution. Depending on the size of the used peptide, separation of [18F]FDG-labeled peptide from glucose-labeled peptide is possible by semi-preparative HPLC. The formation of isomers during the aminooxy-aldehyde conjugation reaction between [18F]FDG and aminooxy-functionalized peptides in aqueous media leads to the formation of isomers according to literature reports. Conclusions: For the first time, readily available PET radiotracer [18F]FDG was shown to be a suitable prosthetic group for direct labeling of aminooxy-functionalized peptides with fluorine-18 under mild conditions. The reaction is especially suitable for small peptides. However, application of larger peptides seems to be limited by increasing separation difficulties of the corresponding glucose-peptide conjugate.

Published
2009

6. Radiolabeling of multimeric neurotensin(8-13) analougues with the short-lived positron emitter fluorine-18

Author

Hultsch, C., Berndt, M., Bergmann, R., and Wüst, F.

Subjects
Tumor Targeting, Neurotensin, Fluorine-18, Multimer
Abstract

Introduction: Neurotensin receptors are expressed with high incidence in several human tumour entities. Thus, radiolabeled neurotensin derivatives might be used for tumour targeting. However, its application is limited by insufficient metabolic stability. Metabolic stability might be improved by the synthesis of multimeric peptides. Experimental: Three methods for 18F-labeling of dimeric and tetrameric neurotensin(8-13) derivatives were evaluated with respect to the labeling yield and the required peptide amounts. Results and Discussion: Labeling using N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) gave low radiochemical yield for the dimeric peptides. Coupling of the tetramer with [18F]SFB was not successful. Furthermore, labeling of aminooxy-functionalized neurotensin(8-13) derivatives using 4-[18F]fluorobenzaldehyde ([18F]FBA) was investigated. High yields of up to 96% were obtained for the dimer whilst coupling of the tetramer only gave low yields of up to 10%. In contrast to these findings, labelling of sulfhydryl-functionalized neurotensin(8-13) derivatives using the maleinimide 4-[18F]fluorobenzaldehyde O-[6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexyl]-oxime ([18F]FBAM) resulted in high radiochemical yields for both, the dimer and the tetramer, being 94% and 40%, respectively. Therefore, [18F]FBAM seems to be the most suitable 18F-labeling agent for multimeric neurotensin(8-13) derivatives. The synthesized 18F-labeled multimeric neurotensin derivatives are depicted in Fig. 1. Conclusion: In summary, 18F-radiolabeling of dimeric and tetrameric NT(8-13) derivates was investigated. Suitable results were obtained by labeling of the sulfhydryl-functionalized peptides using [18F]FBAM. Using [18F]SFB or [18F]FBA as the labeling agent gave only low radiochemical yields. Furthermore, labeling of the dimer gave better radiochemical yields than labeling of the tetramer – independent from the labeling method which was used. Therefore, the nature of the peptide exhibits a strong influence on the coupling reaction.

Published
2007

9. Biodistribution and catabolism of 18F-labeled N-epsilon-fructoselysine as a model of Amadori products

Author

Hultsch, C., Hellwig, M., Pawelke, B., Bergmann, R., Rode, K., Pietzsch, J., Krause, R., and Henle, T.

Subjects
Maillard reaction, Fructosamine 3-kinase, N-epsilon-fructoselysine, Positron emission tomography, Amadori products
Abstract

Amadori products are formed in the early stage of the so-called Maillard reaction between reducing sugars and amino acids or proteins. Such nonenzymatic glycosylation may occur during the heating or storage of foods, but also under physiological conditions. N-epslion-fructoselysine is formed via this reaction between the epsilon-amino group of peptide-bound lysine and glucose. Despite the fact that, in certain heated foods, up to 50% of lysyl moieties may be modified to such lysine derivatives, up to now, very little is known about the metabolic fate of alimentary administered Amadori compounds. In the present study, N-succinimidyl-4-[18F]fluorobenzoate was used to modify N-epsilon-fructoselysine at the a-amino group of the lysyl moiety. The in vitro stability of the resulting 4-[18F]fluorobenzoylated derivative was tested in different tissue homogenates. Furthermore, the 4-[18F]fluorobenzoylated N-epsilon-fructoselysine was used in positron emission tomography studies, as well as in studies concerning biodistribution and catabolism. The results show that the 4-[18F]fluorobenzoylated N-epsilon-fructoselysine is phosphorylated in vitro, as well as in vivo. This phosphorylation is caused by fructosamine 3-kinases and occurs in vivo, particularly in the kidneys. Despite the action of these enzymes, it was shown that a large part of the intravenously applied radiolabeled N-epsilon-fructoselysine was excreted nearly unchanged in the urine. Therefore, it was concluded that the predominant part of peptide-bound lysine that was fructosylated during food processing is not available for nutrition.

Published
2006

10. Biodistribution and catabolism of 18F-labelled isopeptide N (varepsilon)-(gamma-glutamyl)-L-lysine

Author

Hultsch, C., Bergmann, R., Pawelke, B., Pietzsch, J., Wüst, F., Johannsen, B., and Henle, T.

Abstract

Isopeptide bonds between the varepsilon-amino group of lysine and the gamma-carboxamide group of glutamine are formed during strong heating of pure proteins or, more important, by enzymatic reaction mediated by transglutaminases. Despite the wide use of a microbial transglutaminase in food biotechnology, up to now little is known about the metabolic fate of the isopeptide N(varepsilon)-(gamma-glutamyl)-L-lysine. In the present study, N-succinimidyl-4-[18F]fluorobenzoate was used to modify N(varepsilon)-(gamma-glutamyl)-L-lysine at each of its two alpha-amino groups, resulting in the 4-[18F]fluorobenzoylated derivatives, for which biodistribution, catabolism, and elimination were investigated in male Wistar rats. A significant different biochemical behavior of the two labelled isopeptides was observed in terms of in vitro stability, in vivo metabolism as well as biodistribution. The results suggest that the metabolic fate of isopeptides is likely to be dependent on how they are reabsorbed - free or peptide bound.

Published
2005

11. Evaluation of F-18 labelled annexin V: apoptosis imaging in mice

Author

Bergmann, R., Hultsch, C., Bergmann, S., Pietzsch, J., Gunawan, J., Burchert, W., and Hoff, J.

Abstract

Apoptosis imaging with PET plays an increasing role in various medical fields like oncology, cardiology, transplant rejection, and inflammation, but the radiotracer distribution in the tis-sues is influenced by various mechanisms. In this study a recombinant annexin-V (courtesy N. Budisa, Max Planck Institute of Biochemistry) derivative and human serum albumin (HSA) were radiolabelled using N-succinimidyl-4-F-18-fluorobenzoate (SFB), and characterized. Mechanism and specificity of both F-18-annexin-V and F-18-HSA biodistribution and accu-mulation were examined in rodents. Recombinant annexin-V derivative and HSA were radiolabelled using SFB and the products were confirmed by size exclusion chromatography and SDS-PAGE. The radiotracer distribu-tions in animals were studied in rats and mice ex vivo by organ extraction, autoradiography, and in vivo with animal PET. For apoptosis imaging, F-18-annexin-V or F-18-HSA were in-travenously applied in 4 groups of mice that received either intraperitoneal 100 µg Anti-Fas antibody in 200 µL isotonic NaCl or 200 µL isotonic NaCl 2 hours before the radiotracer. The degree of liver apoptosis was characterized by plasma ALAT and ASAT activity measure-ments. The radiochemical yield was in the range of 10 to 30% (corrected for decay) with a specific activity of more than 20 GBq/µmol. The accumulation of F-18-annexin-V and F-18-HSA, respectively in the apoptotic livers (4 – 16%ID/g) were correlated to the ASAT and increased up to 4 times in comparison to control. The biodistribution of the tracers were comparable except for the renal elimination of F-18-annexin-V, which was up to 3 times higher than of F-18-HSA.

Published
2005

12. Assessment of metabolism of native and oxidized low density lipoprotein in vivo: insights from animal positron emission tomography (PET) studies

Author

Pietzsch, J., Bergmann, R., Wüst, F., Grote, M., Hultsch, C., Pawelke, B., and Hoff, J.

Subjects
lipids (amino acids, peptides, and proteins)
Abstract

Oxidative modification of low density lipoprotein (LDL) is regarded as a crucial event in athergenesis. Data concerning the role of circulating oxidized LDL (oxLDL) in the development of atherosclerosis are scarce. One reason for this is the shortage of methaods for direct assessment of metabolism of oxLDL in vivo. We reprot an improved methodology for labelling of both native LDL (nLDL) and oxLDL with fluorine-18 (18F) by N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) and the use of LDL-[18]FB-conjugates in dynamic PET studies in Wistar rats. For labelling experiments, pools of chemically well characterized human nLDL and oxLDL, respectively, were used. Preparation of [18F]SFB was achieved within 40 min with radiochemical yields of 50±5% and purity of >95% using O-(N-succinimidyl)-N-N,N´,N´-tetramethyluronium tetrafluoroborate (TSTU) as activating reagent. LDL labelling with [18F]SFB resulted in radiochemical yields of 30±10%. The method was evaluated with respect to uptake of FB-conjugated nLDL in HepG2 cells and of FB-conjugated oxLDL in primary human macrophages, respectively. Biodistribution studies revealed high in vivo stability for the LDL-[18F]FB conjugates. The metabolic fate of LDL-[18F]FB conjugates in vivo was delineated by PET using a dedicated small animal tomograph (microPET; spatial resolution of 2 mm). In conclusion, [18F]SFB-labelling of LDL and the use of PET provide a valuable tool for assessment of metabolism of nLDL and oxLDL in vivo.

Published
2003

14. Radiolabelling of isopeptide NEpsilon-(Gamma-glutamyl)-L-lysine by conjugation with N-succinimidyl-4-[18F]fluorobenzoate

Author

Wüst, F., Hultsch, C., Bergmann, R., Johannsen, B., and Henle, T.

Subjects
positron emission tomography, 18F-labelling, isopeptide, [18F]SFB
Abstract

The isopeptide NEpsilon-(Gamma-glutamyl)-L-lysine 4 was labelled with 18F via N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB). A modified approach for the convenient synthesis of [18F]SFB was used, and [18F]SFB could be obtained in decay-corrected radiochemical yields of 44-53% (n=20) and radiochemical purity >95% within 40 min after EOB. For labelling NEpsilon-(Gamma-glutamyl)-L-lysine with [18F]SFB the effects of isopeptide concentration, temperature, and pH were studied to determine the optimum reaction conditions. The coupling reaction was shown to be temperature and pH independent while being strongly affected by the isopeptide concentration. Using the optimized labelling conditions, in a typical experiment 1.3 GBq of [18F]SFB could be converted into 447 MBq (46%, decay-corrected) of [18F]fluorobenzoylated isopeptide within 45 min, including HPLC purification.

Published
2003

16. Synthesis and evaluation of novel multimeric neurotensin(8-13) analogs

Author

Hultsch, C., Pawelke, B., Bergmann, R., Wüst, F., Hultsch, C., Pawelke, B., Bergmann, R., and Wüst, F.

Abstract

Neurotensin(8–13) is a hexapeptide with subnanomolar affinity to the neurotensin receptor 1 which is expressed with high incidence in several human tumor entities. Thus, radiolabeled neurotensin(8–13) might be used for tumor targeting. However, its application is limited by insufficient metabolic stability. The present study aims at improving metabolic stability by the synthesis of multimeric neurotensin(8–13) derivatives rather than commonly employed chemical modifications of the peptide itself. Thus, different dimeric and tetrameric peptides carrying C- or N-terminal attached neurotensin(8–13) moieties have been synthesized and their binding affinity toward the neurotensin receptor has been determined. The results demonstrate that branched compounds containing neurotensin(8–13) attached via its C-terminus only show low receptor affinities, whilst derivatives with neurotensin(8–13) attached via the N-terminus show IC50 values in the nanomolar range. Moreover, within the multimeric neurotensin(8–13) derivatives with neurotensin(8–13) attached via the N-terminus an increasing number of branching units lead to higher binding affinities toward the neurotensin receptor.

Published
2006

17. 4-[18F]Fluorobenzaldehyde-O-(2-{2-[2-(pyrol-2,5-dion-1-yl)ethoxy] ethoxy}ethyl)oxim ([18F]FBOM): A novel prosthetic group for mild labelling of SH-group bearing biomacromolecules

Author

Vogler, L., Berndt, M., Pietzsch, J., Hultsch, C., Wüst, F., Vogler, L., Berndt, M., Pietzsch, J., Hultsch, C., and Wüst, F.

Abstract

The synthesis and application of 4-[18F]fluorobenzaldehyde-O-(2-{2-[2-(pyrol-2,5-dion-1-yl)ethoxy]ethoxy}ethyl)oxim ([18F]FBOM) as a novel prosthetic group for 18F labelling of SH-group bearing biomacromolecules is described. The aminooxy-functionalised labelling precursor for the radiosynthesis of [18F]FBOM was synthesised in a four-step synthesis sequence with a total yield of 14%. [18F]FBOM could be obtained in a condensation reaction between the labelling precursor and [18F]fluorobenzaldehyde through formation of an oxim. The radiochemical yield ranged between 14 and 19% (decay-corrected). In a typical experiment, starting from 6.5 GBq of [18F]fluoride 730MBq of [18F]FBOM could be obtained within 80 minutes (including HPLC purification). The specific activity was determined to be 51 GBq/µmol. The lipophilicity of [18F]FBOM was determined to be logP = 0.84. The use of [18F]FBOM as SH-reactive group prosthetic group was demonstrated by the reaction with glutathion, low densitiy lipoproteins (LDL) and modified neurotensin derivatives. [18F]FBOM can easily be synthesised, and [18F]FBOM represents an interesting novel prosthetic group for the labelling of SH-group containing biomacromolecules with 18F under mild conditions.

Published
2006

18. Fluorescein-labeled stable neurotensin derivatives

Author

Maes, V., Hultsch, C., Kohl, S., Bergmann, R., Hanke, T., Tourwé, D., Maes, V., Hultsch, C., Kohl, S., Bergmann, R., Hanke, T., and Tourwé, D.

Abstract

Neurotensin(8-13) analogs containing a glycine or 5-aminovaleroyl spacer were labeled with fluorescein through formation of a N-terminal thiourea function. The receptor binding was measured in HT-29 cell cultures and showed a substantial decrease in affinity, especially for the metabolically stabilized[MeArg9, Tle11] analog. Using fluorescence microscopy, the internalization of the fluorescent neurotensin analogs into HT-29 cells was observed.

Published
2006

20. Catabolism of native and oxidized low density lipoproteins (LDL): in vivo insights from small animal positron emission tomography studies

Author

Pietzsch, J., Bergmann, R., Wüst, F., Pawelke, B., Hultsch, C., Hoff, J., Pietzsch, J., Bergmann, R., Wüst, F., Pawelke, B., Hultsch, C., and Hoff, J.

Abstract

The human organism is exposed to numerous processes that generate reactive oxygen species (ROS). ROS may directly or indirectly cause oxidative modification and damage of proteins. Protein oxidation is regarded as a crucial event in the pathogenesis of various diseases ranging from rheumatoid arthritis to Alzheimer's disease and atherosclerosis. As a representative example, oxidation of low density lipoprotein (LDL) is regarded as a crucial event in atherogenesis. Data concerning the role of circulating oxidized LDL (oxLDL) in the development and outcome of diseases are scarce. One reason for this is the shortage of methods for direct assessment of the metabolic fate of circulating oxLDL in vivo. We present an improved methodology based on the radiolabelling of apoB-100 of native LDL (nLDL) and oxLDL, respectively, with the positron emitter fluorine-18 (18F) by conjugation with N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB). Radiolabelling of both nLDL and oxLDL using [18F]SFB causes neither additional oxidative structural modifications of LDL lipids and proteins nor alteration of their biological activity and functionality, respectively, in vitro. The method was further evaluated with respect to the radiopharmacological properties of both [18F]fluorobenzoylated nLDL and oxLDL by biodistribution studies in male Wistar rats. The metabolic fate of [18F]fluorobenzoylated nLDL and oxLDL in rats in vivo was further delineated by dynamic positron emission tomography (PET) using a dedicated small animal tomograph (spatial resolution of 2 mm). From this study we conclude that the use of [18F]FB-labelled LDL particles is an attractive alternative to, e.g., LDL iodination methods, and is of value to characterize and to discriminate the kinetics and the metabolic fate of nLDL and oxLDL in small animals in vivo.

Published
2005

21. Bioverteilung und Metabolisierung des 18F-markierten Amadori-Produktes Fructoselysin

Author

Hultsch, C., Hellwig, M., Bergmann, R., Henle, T., Hultsch, C., Hellwig, M., Bergmann, R., and Henle, T.

Abstract

Ziel: Amadore-Produkte (1-Amino-1-desoxy-2-ketosen) werden in der Frühphase der sogenannten Maillard-Reaktion zwischen reduzierenden Zuckern und primären Aminen in Lebensmitteln bei der zubereitung oder Lagerung sowie in vivo gebildet. Täglich werden etwa 1 g Amadori-Produkte mit der nahrung aufgenommem [1]. Jedoch ist nur der Verbleib von ca. 5% dieser Verbindungen bislang bekannt. Die ernährungsphysiologischen Konsequenzen, die sich aus der Aufnahme dieser Verbindungen ergeben, sind weitestgehend ungeklärt. Ziel dieser Arbeit war es daher, Bioverteilung und eventuelle Metabolisierungsreaktionen des Amadori-Produktes Fructoselysin auch vor dem Hintergrund der kürzlich entdeckten Fructosamin-3-Kinasen [2] zu untersuchen. Methodik: Fructoselysin wurde mit N-Succinimidyl-4-[18F]-Fluorbenzonat zum [18F]fluorbenzoylierten Fructoselysin (FB-FL) umgesetzt. Dieses wurde mit Organhomogenaten (Herz, Hirn, Leber, Milz, Niere, Pankreas), Vollblut, Plasma und Vollblutlysat von männlichen Wistar-Ratten inkubiert. Dabei wurde der Einfluss von ATP und 1-Desoxy-1-morpholino-fructose (DMF), einem spezifischen Inhibitor der Fructosamin-3-Kinase, auf die Umsetzung des FB-FL untersucht. Weiterhin erfolgte die Messung der Bioverteilung un der in vivo-Stabilität. Ergebnisse: Weder in den untersuchten Organhomogenaten noch im Vollblut oder Plasma fand eine Umsetzung des FB-FL statt. Nur im Vollblutlysat konnte die Bildung eines Umwandlungsproduktes m1 beobachtet werden. Diese Umsetzung konnte durch die Zugabe von ATP beschleunigt und durch Zugabe von DMF gehemmt werden.Die Inkubation von m1 mit alkalischer Phosphatase führte zu einem vollständigen Abbau. Bei m1 handelt es sich damit voraussichtlich um ein Phosphatester, gebildet durch eine Fructosamin-3-Kinase. Bei der Messung der Bioverteilung nach intravenöser Applikation kam es zu einer schnellen Aufnahme des Präparates in die Niere. 30% der injizierten Dosis waren auch 60 min nach

Published
2005

23. Fluorine-18 radiolabeling of low-density lipoproteins: a potential approach for characterization and differentiation of metabolism of native and oxidized low-density lipoproteins in vivo.

Author

Pietzsch, J., Bergmann, R., Rode, K., Hultsch, C., Pawelke, B., Wüst, F., Hoff, J., Pietzsch, J., Bergmann, R., Rode, K., Hultsch, C., Pawelke, B., Wüst, F., and Hoff, J.

Abstract

Oxidative modification of low-density lipoprotein (LDL) is regarded as a crucial event in atherogenesis. Assessing the metabolic fate of oxidized LDL (oxLDL) in vivo with radiotracer techniques is hindered by the lack of suitable sensitive and specific radiolabeling methods. We evaluated an improved methodology based on the radiolabeling of native LDL (nLDL) and oxLDL with the positron emitter fluorine-18 ((18)F) by conjugation with N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). We investigated whether radiolabeling of LDL induces adverse structural modifications. Results suggest that radiolabeling of both nLDL and oxLDL using [(18)F]SFB causes neither additional oxidative structural modifications of LDL lipids and proteins nor alteration of their biological activity and functionality, respectively. Thus, radiolabeling of LDL using [(18)F]SFB could prove to be a promising approach for studying the kinetics of oxLDL in vivo.

Published
2004

24. 13th Workshop of the Central European Division e.V. of the International Isotope Society

Author

Derdau, Volker, primary, Atzrodt, Jens, additional, Markó, István E., additional, Harwood, Simon J., additional, Moenius, Th., additional, Salter, R., additional, Wietfeld, B., additional, Burtscher, P., additional, Zueger, C., additional, Clayden, Jonathan, additional, Bordeaux, K., additional, Metz, Y., additional, Rodriguez, I., additional, Ruetsch, R., additional, Voges, R., additional, Gardiner, John M., additional, Stimpson, William, additional, Panchal, Nitesh, additional, Herbert, John, additional, Ellames, George J., additional, Beller, Matthias, additional, Kozempel, Ján, additional, Kadeřávek, Jan, additional, Lešetický, Ladislav, additional, Lebeda, Ondřej, additional, Wähälä, Kristiina, additional, Kiuru, Paula, additional, Leppälä, Eija, additional, Pohjoispää, Monika, additional, Parikka, Kirsti, additional, Raffaelli, Barbara, additional, Herbert, John M., additional, Janssen, Cor G. M., additional, Verluyten, Willy L. M., additional, Vliegen, Maarten, additional, Mäding, P., additional, Füchtner, F., additional, Bergmann, R., additional, Pietzsch, J., additional, Hultsch, C., additional, Wüst, F., additional, Scheunemann, M., additional, Vercouillie, J., additional, Fischer, St., additional, Sorger, D., additional, Großmann, U., additional, Schliebs, R., additional, Sabri, O., additional, and Steinbach, J., additional

Published
2006
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25. Radiolabelling of isopeptide Nε-(γ-glutamyl)-l-lysine by conjugation with N-succinimidyl-4-[18F]fluorobenzoate.

Author

Wüst, F., Hultsch, C., Bergmann, R., Johannsen, B., and Henle, T.

Subjects
*RADIOLABELING, *RADIOCHEMICAL analysis
Abstract

The isopeptide Nε-(γ-glutamyl)-l-lysine 4 was labelled with 18F via N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB). A modified approach for the convenient synthesis of [18F]SFB was used, and [18F]SFB could be obtained in decay-corrected radiochemical yields of 44–53% (n=20) and radiochemical purity >95% within 40 min after EOB. For labelling Nε-(γ-glutamyl)-l-lysine with [18F]SFB the effects of isopeptide concentration, temperature, and pH were studied to determine the optimum reaction conditions. The coupling reaction was shown to be temperature and pH independent while being strongly affected by the isopeptide concentration. Using the optimized labelling conditions, in a typical experiment 1.3 GBq of [18F]SFB could be converted into 447 MBq (46%, decay-corrected) of [18F]fluorobenzoylated isopeptide within 45 min, including HPLC purification. [Copyright &y& Elsevier]

Published
2003
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26. Biodistribution and catabolism of 18F-labelled isopeptide Nɛ-(γ-glutamyl)-L-lysine

Author

Hultsch, C., Bergmann, R., Pawelke, B., Pietzsch, J., Wuest, F., Johannsen, B., and Henle, T.

Abstract

Summary.: Isopeptide bonds between the ɛ-amino group of lysine and the γ-carboxamide group of glutamine are formed during strong heating of pure proteins or, more important, by enzymatic reaction mediated by transglutaminases. Despite the wide use of a microbial transglutaminase in food biotechnology, up to now little is known about the metabolic fate of the isopeptide Nɛ-(γ-glutamyl)-L-lysine. In the present study, N-succinimidyl-4-[18F]fluorobenzoate was used to modify Nɛ-(γ-glutamyl)-L-lysine at each of its two α-amino groups, resulting in the 4-[18F]fluorobenzoylated derivatives, for which biodistribution, catabolism, and elimination were investigated in male Wistar rats. A significant different biochemical behavior of the two labelled isopeptides was observed in terms of in vitro stability, in vivo metabolism as well as biodistribution. The results suggest that the metabolic fate of isopeptides is likely to be dependent on how they are reabsorbed – free or peptide bound.

Published
2005
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27. (S)-4-(3-18F-fluoropropyl)-L-glutamic acid: an 18F-labeled tumor-specific probe for PET/CT imaging--dosimetry.

Author

Smolarz K, Krause BJ, Graner FP, Wagner FM, Hultsch C, Bacher-Stier C, Sparks RB, Ramsay S, Fels LM, Dinkelborg LM, and Schwaiger M

Subjects
Female, Humans, Isotope Labeling, Male, Middle Aged, Radiometry, Safety, Glutamates adverse effects, Multimodal Imaging methods, Neoplasms diagnostic imaging, Positron-Emission Tomography, Tomography, X-Ray Computed
Abstract

Unlabelled: The glutamic acid derivative (S)-4-(3-(18)F-Fluoropropyl)-l-glutamic acid ((18)F-FSPG, alias BAY 94-9392), a new PET tracer for the detection of malignant diseases, displayed promising results in non-small cell lung cancer patients. The aim of this study was to provide dosimetry estimates for (18)F-FSPG based on human whole-body PET/CT measurements., Methods: (18)F-FSPG was prepared by a fully automated 2-step procedure and purified by a solid-phase extraction method. PET/CT scans were obtained for 5 healthy volunteers (mean age, 59 y; age range, 51-64 y; 2 men, 3 women). Human subjects were imaged for up to 240 min using a PET/CT scanner after intravenous injection of 299 ± 22.5 MBq of (18)F-FSPG. Image quantification, time-activity data modeling, estimation of normalized number of disintegrations, and production of dosimetry estimates were performed using the RADAR (RAdiation Dose Assessment Resource) method for internal dosimetry and in general concordance with the methodology and principles as presented in the MIRD 16 document., Results: Because of the renal excretion of the tracer, the absorbed dose was highest in the urinary bladder wall and kidneys, followed by the pancreas and uterus. The individual organ doses (mSv/MBq) were 0.40 ± 0.058 for the urinary bladder wall, 0.11 ± 0.011 for the kidneys, 0.077 ± 0.020 for the pancreas, and 0.030 ± 0.0034 for the uterus. The calculated effective dose was 0.032 ± 0.0034 mSv/MBq. Absorbed dose to the bladder and the effective dose can be reduced significantly by frequent bladder-voiding intervals. For a 0.75-h voiding interval, the bladder dose was reduced to 0.10 ± 0.012 mSv/MBq, and the effective dose was reduced to 0.015 ± 0.0010 mSv/MBq., Conclusion: On the basis of the distribution and biokinetic data, the determined radiation dose for (18)F-FSPG was calculated to be 9.5 ± 1.0 mSv at a patient dose of 300 MBq, which is of similar magnitude to that of (18)F-FDG (5.7 mSv). The effective dose can be reduced to 4.5 ± 0.30 mSv (at 300 MBq), with a bladder-voiding interval of 0.75 h.

Published
2013
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28. Exploratory clinical trial of (4S)-4-(3-[18F]fluoropropyl)-L-glutamate for imaging xC- transporter using positron emission tomography in patients with non-small cell lung or breast cancer.

Author

Baek S, Choi CM, Ahn SH, Lee JW, Gong G, Ryu JS, Oh SJ, Bacher-Stier C, Fels L, Koglin N, Hultsch C, Schatz CA, Dinkelborg LM, Mittra ES, Gambhir SS, and Moon DH

Subjects
Adult, Aged, Amino Acid Transport System y+ chemistry, Amino Acid Transport System y+ metabolism, Animals, Breast Neoplasms pathology, Carcinoma, Non-Small-Cell Lung pathology, Female, Humans, Mice, Middle Aged, Breast Neoplasms diagnosis, Carcinoma, Non-Small-Cell Lung diagnosis, Contrast Media administration & dosage, Contrast Media adverse effects, Contrast Media pharmacokinetics, Glutamates administration & dosage, Glutamates adverse effects, Glutamates pharmacokinetics, Positron-Emission Tomography
Abstract

Purpose: (4S)-4-(3-[(18)F]fluoropropyl)-l-glutamate (BAY 94-9392, alias [(18)F]FSPG) is a new tracer to image x(C)(-) transporter activity with positron emission tomography (PET). We aimed to explore the tumor detection rate of [(18)F]FSPG in patients relative to 2-[(18)F]fluoro-2-deoxyglucose ([(18)F]FDG). The correlation of [(18)F]FSPG uptake with immunohistochemical expression of x(C)(-) transporter and CD44, which stabilizes the xCT subunit of system x(C)(-), was also analyzed., Experimental Design: Patients with non-small cell lung cancer (NSCLC, n = 10) or breast cancer (n = 5) who had a positive [(18)F]FDG uptake were included in this exploratory study. PET images were acquired following injection of approximately 300 MBq [(18)F]FSPG. Immunohistochemistry was done using xCT- and CD44-specific antibody., Results: [(18)F]FSPG PET showed high uptake in the kidney and pancreas with rapid blood clearance. [(18)F]FSPG identified all 10 NSCLC and three of the five breast cancer lesions that were confirmed by pathology. [(18)F]FSPG detected 59 of 67 (88%) [(18)F]FDG lesions in NSCLC, and 30 of 73 (41%) in breast cancer. Seven lesions were additionally detected only on [(18)F]FSPG in NSCLC. The tumor-to-blood pool standardized uptake value (SUV) ratio was not significantly different from that of [(18)F]FDG in NSCLC; however, in breast cancer, it was significantly lower (P < 0.05). The maximum SUV of [(18)F]FSPG correlated significantly with the intensity of immunohistochemical staining of x(C)(-) transporter and CD44 (P < 0.01)., Conclusions: [(18)F]FSPG seems to be a promising tracer with a relatively high cancer detection rate in patients with NSCLC. [(18)F]FSPG PET may assess x(C)(-) transporter activity in patients with cancer.

Published
2012
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29. Direct labelling of peptides with 2-[18F]fluoro-2-deoxy-d-glucose ([18F]FDG).

Author

Wuest F, Hultsch C, Berndt M, and Bergmann R

Subjects
Amino Acid Sequence, Molecular Sequence Data, Oximes chemistry, Fluorodeoxyglucose F18 chemistry, Peptides analysis
Abstract

The study describes the use of [(18)F]FDG as (18)F building block for the direct labelling of various aminooxy-functionalised peptides via chemoselective oxime formation.

Published
2009
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30. (18)F-Fluoroglucosylation of peptides, exemplified on cyclo(RGDfK).

Author

Hultsch C, Schottelius M, Auernheimer J, Alke A, and Wester HJ

Subjects
Animals, Fluorodeoxyglucose F18 pharmacokinetics, Fluorodeoxyglucose F18 pharmacology, Glycosylation, Melanoma, Experimental diagnostic imaging, Mice, Mice, Nude, Neoplasm Transplantation, Peptides, Cyclic pharmacokinetics, Peptides, Cyclic pharmacology, Positron-Emission Tomography, Radiopharmaceuticals pharmacokinetics, Radiopharmaceuticals pharmacology, Tissue Distribution, Fluorodeoxyglucose F18 chemistry, Peptides, Cyclic chemistry, Radiopharmaceuticals chemistry
Abstract

Purpose: Oxime formation between an aminooxy-functionalized peptide and an (18)F-labelled aldehyde has recently been introduced as a powerful method for the rapid one-step chemoselective synthesis of radiofluorinated peptides., Materials and Methods: Here, the potential of using routinely produced and thus readily available [(18)F]fluorodeoxyglucose ([(18)F]FDG) as the aldehydic prosthetic group was investigated using an aminooxyacetyl-conjugated cyclic RGD peptide (cyclo(RGDfK(Aoa-(Boc)) as a model peptide., Results: The use of [(18)F]FDG from routine production ([(18)F]FDGTUM) containing an excess of D: -glucose did not allow the radiosynthesis of [(18)F]FDG-RGD in activities >37 MBq in reasonable yield, rendering the direct use of clinical grade [(18)F]FDG for the routine clinical synthesis of (18)F-labelled peptides impossible. Using no-carrier-added (n.c.a.) [(18)F]FDG obtained via HPLC separation of [(18)F]FDGTUM from excess glucose, however, afforded [(18)F]FDG-RGD in yields of 56-93% (decay corrected) and activities up to 37 MBq. Suitable reaction conditions were 20 min at 120 degrees C and pH 2.5, and a peptide concentration of 5 mM. In a preliminary in vivo biodistribution study in M21 melanoma-bearing nude mice, [(18)F]FDG-RGD showed increased tumour accumulation compared to the "gold standard" [(18)F]galacto-RGD (2.18 vs 1.49 %iD/g, respectively, at 120 min after injection), but also slightly increased uptake in non-target organs, leading to comparable tumour/organ ratios for both compounds., Conclusion: These data demonstrate that chemoselective (18)F-labelling of aminooxy-functionalized peptides using n.c.a. [(18)F]FDG represents a radiofluorination/glycosylation strategy that allows preparation of (18)F-labelled peptides in high yield with suitable pharmacokinetics. As soon as the necessary n.c.a. preparation of [(18)F]FDG prior to reaction with the Aoa-peptide can be implemented in a fully automated [(18)F]FDG-synthesis, [(18)F]fluoroglucosylation of peptides may represent a promising alternative to currently used chemoselective one-step (18)F-labelling protocols.

Published
2009
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31. Fast and repetitive in-capillary production of [18F]FDG.

Author

Wester HJ, Schoultz BW, Hultsch C, and Henriksen G

Subjects
Automation, Chemistry, Pharmaceutical methods, Equipment Design, Hydrolysis, Isotope Labeling methods, Positron-Emission Tomography methods, Radiochemistry methods, Solvents chemistry, Technology, Pharmaceutical methods, Temperature, Fluorodeoxyglucose F18 chemical synthesis, Fluorodeoxyglucose F18 pharmacology, Positron-Emission Tomography instrumentation, Radiochemistry instrumentation, Radiopharmaceuticals chemical synthesis, Radiopharmaceuticals pharmacology
Abstract

Purpose: The increasing demand for radiopharmaceuticals to be provided reproducibly and flexibly with high frequency for clinical application and animal imaging would be better met by improved or even new strategies for automated tracer production. Radiosynthesis in microfluidic systems, i.e. narrow tubing with a diameter of approximately 50-500 microm, holds promise for providing the means for repetitive multidose and multitracer production. In this study, the performance of a conceptually simple microfluidic device integrated into a fully automated synthesis procedure for in-capillary radiosynthesis (ICR) of clinical grade [(18)F]FDG was evaluated., Materials and Methods: The instrumental set-up consisted of pumps for reagent and solvent delivery into small mixing chambers, micro-fluidic capillaries, in-process radioactivity monitoring, solid-phase extraction and on-column deprotection of the (18)F-labelled intermediate followed by on-line formulation of [(18)F]FDG., Results: In-capillary(18)F-fluorination of 2.1 micromol 1,3,4,6-tetra-O-acetyl-2-O-trifluoromethanesulphonyl-beta-D-mannopyranose (TATM; precursor for [(18)F]FDG) in acetonitrile (MeCN) at a flow rate of 0.3 ml/min within 40 s and subsequent on-line hydrolysis of the intermediate by treatment with 0.3 M NaOH for 1 min at 40 degrees C resulted in a radiochemical yield of 88 +/- 4% within <7 min. Reproducibility, robustness and suitability as a fast and efficient radiopharmaceutical research tool for (18)F-fluorination was demonstrated by eight independent, sequentially performed ICRs which provided identical tracer quality (radiochemical purity >97%, MeCN <5 microg/ml) and similar absolute yields (approximately 1.4 GBq)., Conclusion: The described ICR process is a simple and efficient alternative to classic radiotracer production systems and provides a comparatively cheap instrumental methodology for the repetitive production of [(18)F]FDG with remarkably high efficiency and high yield under fully automated conditions. Although the results concerning the levels of activity need to be confirmed after installation of the equipment in a suitable GMP hot-cell environment, we expect the instrumental design to allow up-scaling without major difficulties or fundamental restrictions. Furthermore, we are convinced that similar or nearly identical procedures, and thus instrumentation, will allow ICR of other (18)F-labelled radiopharmaceuticals.

Published
2009
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32. Radiolabeling of multimeric neurotensin(8-13) analogs with the short-lived positron emitter fluorine-18.

Author

Hultsch C, Berndt M, Bergmann R, and Wuest F

Subjects
Benzaldehydes chemistry, Benzoates chemistry, Dimerization, Isotope Labeling, Maleimides chemistry, Succinimides chemistry, Electrons, Fluorine Radioisotopes chemistry, Neurotensin chemistry, Peptide Fragments chemistry
Abstract

Three methods for (18)F-labeling of dimeric and tetrameric neurotensin(8-13) derivatives were evaluated with respect to the labeling yield and the required peptide amounts. Labeling using N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB) gave low radiochemical yield for the dimeric peptides. Coupling of the tetramer with [(18)F]SFB was not successful. High yields were obtained for labeling of the aminooxy-functionalized neurotensin(8-13) dimer using 4-[(18)F]fluorobenzaldehyde ([(18)F]FBA) whilst coupling of the corresponding tetramer gave only low yields. Labeling of sulfydryl-functionalized neurotensin(8-13) derivatives using the maleinimide 4-[(18)F]fluorobenzaldehyde-O-[6-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-hexyl]-oxime ([(18)F]FBAM) resulted in high radiochemical yields for both, the dimer and the tetramer. Therefore, [(18)F]FBAM seems to be the most suitable (18)F-labeling agent for multivalent neurotensin(8-13) derivatives.

Published
2007
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33. Biodistribution and catabolism of 18F-labeled N-epsilon-fructoselysine as a model of Amadori products.

Author

Hultsch C, Hellwig M, Pawelke B, Bergmann R, Rode K, Pietzsch J, Krause R, and Henle T

Subjects
Animals, Fluorine Radioisotopes pharmacokinetics, Lysine pharmacokinetics, Male, Metabolic Clearance Rate, Metabolism, Organ Specificity, Positron-Emission Tomography, Radiopharmaceuticals pharmacokinetics, Rats, Rats, Wistar, Tissue Distribution, Lysine analogs & derivatives, Maillard Reaction
Abstract

Amadori products are formed in the early stage of the so-called Maillard reaction between reducing sugars and amino acids or proteins. Such nonenzymatic glycosylation may occur during the heating or storage of foods, but also under physiological conditions. N-epsilon-fructoselysine is formed via this reaction between the epsilon-amino group of peptide-bound lysine and glucose. Despite the fact that, in certain heated foods, up to 50% of lysyl moieties may be modified to such lysine derivatives, up to now, very little is known about the metabolic fate of alimentary administered Amadori compounds. In the present study, N-succinimidyl-4-[18F]fluorobenzoate was used to modify N-epsilon-fructoselysine at the alpha-amino group of the lysyl moiety. The in vitro stability of the resulting 4-[18F]fluorobenzoylated derivative was tested in different tissue homogenates. Furthermore, the 4-[18F]fluorobenzoylated N-epsilon-fructoselysine was used in positron emission tomography studies, as well as in studies concerning biodistribution and catabolism. The results show that the 4-[18F]fluorobenzoylated N-epsilon-fructoselysine is phosphorylated in vitro, as well as in vivo. This phosphorylation is caused by fructosamine 3-kinases and occurs in vivo, particularly in the kidneys. Despite the action of these enzymes, it was shown that a large part of the intravenously applied radiolabeled N-epsilon-fructoselysine was excreted nearly unchanged in the urine. Therefore, it was concluded that the predominant part of peptide-bound lysine that was fructosylated during food processing is not available for nutrition.

Published
2006
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34. Synthesis and evaluation of novel multimeric neurotensin(8-13) analogs.

Author

Hultsch C, Pawelke B, Bergmann R, and Wuest F

Subjects
Amino Acid Sequence, HT29 Cells, Humans, Inhibitory Concentration 50, Neurotensin chemistry, Neurotensin pharmacology, Peptide Fragments chemistry, Peptide Fragments pharmacology, Protein Binding, Receptors, Neurotensin chemistry, Receptors, Neurotensin metabolism, Neurotensin chemical synthesis, Neurotensin metabolism, Peptide Fragments chemical synthesis, Peptide Fragments metabolism
Abstract

Neurotensin(8-13) is a hexapeptide with subnanomolar affinity to the neurotensin receptor 1 which is expressed with high incidence in several human tumor entities. Thus, radiolabeled neurotensin(8-13) might be used for tumor targeting. However, its application is limited by insufficient metabolic stability. The present study aims at improving metabolic stability by the synthesis of multimeric neurotensin(8-13) derivatives rather than commonly employed chemical modifications of the peptide itself. Thus, different dimeric and tetrameric peptides carrying C- or N-terminal attached neurotensin(8-13) moieties have been synthesized and their binding affinity toward the neurotensin receptor has been determined. The results demonstrate that branched compounds containing neurotensin(8-13) attached via its C-terminus only show low receptor affinities, whilst derivatives with neurotensin(8-13) attached via the N-terminus show IC50 values in the nanomolar range. Moreover, within the multimeric neurotensin(8-13) derivatives with neurotensin(8-13) attached via the N-terminus an increasing number of branching units lead to higher binding affinities toward the neurotensin receptor.

Published
2006
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35. Fluorescein-labeled stable neurotensin derivatives.

Author

Maes V, Hultsch C, Kohl S, Bergmann R, Hanke T, and Tourwé D

Subjects
Fluorescein-5-isothiocyanate chemistry, HT29 Cells, Humans, Microscopy, Fluorescence methods, Molecular Structure, Neurotensin chemistry, Neurotensin metabolism, Protein Binding, Protein Transport, Receptors, Neurotensin metabolism, Fluorescent Dyes chemistry, Neurotensin analogs & derivatives
Abstract

Neurotensin(8-13) analogs containing a glycine or 5-aminovaleroyl spacer were labeled with fluorescein through formation of an N-terminal thiourea function. The receptor binding was measured in HT-29 cell cultures and showed a substantial decrease in affinity, especially for the metabolically stabilized [MeArg(9), Tle(11)] analog. Using fluorescence microscopy, the internalization of the fluorescent neurotensin analogs into HT-29 cells was observed., (Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd.)

Published
2006
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36. Fluorine-18 radiolabeling of low-density lipoproteins: a potential approach for characterization and differentiation of metabolism of native and oxidized low-density lipoproteins in vivo.

Author

Pietzsch J, Bergmann R, Rode K, Hultsch C, Pawelke B, Wuest F, and van den Hoff J

Subjects
Cell Line, Feasibility Studies, Humans, Liver Neoplasms diagnostic imaging, Liver Neoplasms metabolism, Metabolic Clearance Rate, Positron-Emission Tomography methods, Radiopharmaceuticals pharmacokinetics, Benzoates pharmacokinetics, Carcinoma, Hepatocellular diagnostic imaging, Carcinoma, Hepatocellular metabolism, Lipoproteins, LDL pharmacokinetics, Monocytes diagnostic imaging, Monocytes metabolism, Succinimides pharmacokinetics
Abstract

Oxidative modification of low-density lipoprotein (LDL) is regarded as a crucial event in atherogenesis. Assessing the metabolic fate of oxidized LDL (oxLDL) in vivo with radiotracer techniques is hindered by the lack of suitable sensitive and specific radiolabeling methods. We evaluated an improved methodology based on the radiolabeling of native LDL (nLDL) and oxLDL with the positron emitter fluorine-18 ((18)F) by conjugation with N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). We investigated whether radiolabeling of LDL induces adverse structural modifications. Results suggest that radiolabeling of both nLDL and oxLDL using [(18)F]SFB causes neither additional oxidative structural modifications of LDL lipids and proteins nor alteration of their biological activity and functionality, respectively. Thus, radiolabeling of LDL using [(18)F]SFB could prove to be a promising approach for studying the kinetics of oxLDL in vivo.

Published
2004
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