Synthesis and application of fluorinated carbohydrates and other bioactive compounds

Die vorliegende Doktorarbeit beschäftigt sich sowohl mit der Synthese und Anwendung von fluorierten Kohlenhydraten als auch mit der Herstellung von Diclofenac Metaboliten für mechanistische Untersuchungen in der Allergieforschung. Das Interesse an fluorierten organischen Verbindungen ist in den letzten Jahren stark gestiegen. Diese Entwicklung wird einerseits auf die veränderten biologischen und physikalisch-chemischen Eigenschaften zurückgeführt, andererseits ermöglicht der hoch empfindliche 19F Kern die Anwendung von speziellen NMR Techniken. Zur Untersuchung von Proteinbindungen mittels 19F-NMR wurde eine neue Strategie entwickelt. 2 19F-markierte Maltose wird als Sonde genutzt, um Protein-Protein Wechselwirkungen zum „maltose binding protein“ (MBP) mittels 19F-NMR zu verfolgen. Für diese Zwecke wurde eine Vielzahl von stereoselektiv fluorierten Kohlenhydraten hergestellt. Weiters wird die Herstellung von 6-Desoxy-6-fluor-glukose und –galaktose Derivaten als potentielle Substrate zur enzymatischen Herstellung von fluormarkierten Disacchariden beschrieben. Diclofenac (VoltarenTM) gehört zu der Gruppe der nichtsteroidalen Antirheumatika und wird aufgrund der antipyretischen, analgetischen und entzündungshemmenden Wirkung zur Behandlung von Rheuma, Arthritis, Spondylitis ankylopoetica sowie bei leichten bis mittleren Schmerzen und Entzündungen eingesetzt. Obwohl Diclofenac schon seit vielen Jahren ein sehr beliebter und häufig angewandter Arzneistoff ist, gibt es immer wieder Meldungen über allergische Reaktionen, einschließlich Leberversagen. Trotz zahlreicher mechanistischer Vermutungen konnte ein IgE-basierender Mechanismus nie bewiesen werden. Um mechanistische Studien in diese Richtung voranzutreiben, wurde ein 5-Hydroxy-Diclofenac Metabolit hergestellt.This PhD Thesis describes the synthesis and application of various fluorinated carbohydrate derivatives and the synthesis of a Diclofenac metabolite to investigate a potential immunologic mechanism underlying Diclofenac-hypersensitivity. The increasing interest in fluorinated organic compounds is due to the fact that these derivatives not only exhibit challenging biological and physico-chemical properties, but also allow the application of high end NMR techniques as a result of the highly sensitive 19F nucleus. The development of a novel reporter system to study protein interactions via 19F-NMR was established. This approach uses 2-19F-labeled maltose as a spy ligand to indirectly probe protein-protein interactions of proteins fused or tagged to maltose binding protein. For that purpose, a variety of stereoselective fluorinated carbohydrate derivatives was synthesized. Synthetic efforts to 6-deoxy-6-fluoro-glucose and –galactose derivatives as potential substrates for enzymatic synthesis of disaccharides are described. Diclofenac (VoltarenTM) is a member of nonsteroidal anti-inflammatory drugs (NSAIDs) and has been used for its antipyretic, analgetic and anti-inflammatory activities in the treatment of rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, and acute muscle pain. Although this drug has proven to be highly effective and secure, various adverse drug reactions have been reported in the past, including hepatotoxicity. There are several postulations of an involvement of IgE, but mechanistic evidence is still not available. To prove drug-specific IgE mediated reactions against the nonsteroidal anti-inflammatory drug Diclofenac, 5-Hydroxy-Diclofenac metabolites were synthesized

Diclofenac Hypersensitivity: Antibody Responses to the Parent Drug and Relevant Metabolites

Background: Hypersensitivity reactions against nonsteroidal antiinflammatory drugs (NSAIDs) like diclofenac (DF) can manifest as Type I-like allergic reactions including systemic anaphylaxis. However, except for isolated case studies experimental evidence for an IgE-mediated pathomechanism of DF hypersensitivity is lacking. In this study we aimed to investigate the possible involvement of drug-and/or metabolite-specific antibodies in selective DF hypersensitivity. Methodology/Principal Findings: DF, an organochemically synthesized linkage variant, and five major Phase I metabolites were covalently coupled to carrier proteins. Drug conjugates were analyzed for coupling degree and capacity to crosslink receptor-bound IgE antibodies from drug-sensitized mice. With these conjugates, the presence of hapten-specific IgE antibodies was investigated in patients' samples by ELISA, mediator release assay, and basophil activation test. Production of sulfidoleukotrienes by drug conjugates was determined in PBMCs from DF-hypersensitive patients. All conjugates were shown to carry more than two haptens per carrier molecule. Immunization of mice with drug conjugates induced drug-specific IgE antibodies capable of triggering mediator release. Therefore, the conjugates are suitable tools for detection of drug-specific antibodies and for determination of their anaphylactic activity. Fifty-nine patients were enrolled and categorized as hypersensitive either selectively to DF or to multiple NSAIDs. In none of the patients' samples evidence for drug/metabolite-specific IgE in serum or bound to allergic effector cells was found. In contrast, a small group of patients (8/59, 14%) displayed drug/metabolite-specific IgG. Conclusions/Significance: We found no evidence for an IgE-mediated effector mechanism based on haptenation of protein carriers in DF-hypersensitive patients. Furthermore, a potential involvement of the most relevant metabolites in DF hypersensitivity reactions could be excluded

Diluting ferric carboxymaltose in sodium chloride infusion solution (0.9% w/v) in polypropylene bottles and bags: effects on chemical stability

OBJECTIVES: This study was designed to assess the physicochemical stability of colloidal ferric carboxymaltose solution (Ferinject) when diluted and stored in polypropylene (PP) bottles and bags for infusion. METHODS: Two batches of ferric carboxymaltose solution (Ferinject) were diluted (500 mg, 200 mg and 100 mg iron in 100 mL saline) in PP bottles or bags under aseptic conditions. The diluted solutions were stored at 30°C and 75%±5% relative humidity (rH) for 72 h, and samples were withdrawn aseptically at preparation and after 24 h, 48 h and 72 h. Multiple parameters were used to test stability-related measures (pH, total iron and iron (II) content, molecular weight range determination, microbial contamination and particles count ≥10 μm). RESULTS: Overall, Ferinject diluted in 0.9% (w/v) NaCl solution and stored in PP bottles and bags was stable within the specifications for the complex and the acceptability limits set for all assays. In both containers, total iron content remained stable, within 10% of the theoretical iron content, and levels of iron (II) remained far below the threshold of acceptability. All preparations were free from sediments, particle numbers were acceptable and there was no microbial contamination. The molecular weight distribution and polydispersity index were also acceptable. CONCLUSIONS: Under the tested experimental conditions, colloidal ferric carboxymaltose solution (Ferinject) diluted in saline in PP infusion bottles or bags demonstrated physical and chemical stability for up to 72 h at 30°C and 75% rH. Because of the lack of additional clinical data, when using ferric carboxymaltose, physicians/pharmacists should refer to the dilution and storing recommendations given in the product's summary of product characteristics

Diluting ferric carboxymaltose in sodium chloride infusion solution (0.9% w/v) in polypropylene bottles and bags: effects on chemical stability

This study was designed to assess the physicochemical stability of colloidal ferric carboxymaltose solution (Ferinject) when diluted and stored in polypropylene (PP) bottles and bags for infusion.; Two batches of ferric carboxymaltose solution (Ferinject) were diluted (500 mg, 200 mg and 100 mg iron in 100 mL saline) in PP bottles or bags under aseptic conditions. The diluted solutions were stored at 30°C and 75%±5% relative humidity (rH) for 72 h, and samples were withdrawn aseptically at preparation and after 24 h, 48 h and 72 h. Multiple parameters were used to test stability-related measures (pH, total iron and iron (II) content, molecular weight range determination, microbial contamination and particles count ≥10 μm).; Overall, Ferinject diluted in 0.9% (w/v) NaCl solution and stored in PP bottles and bags was stable within the specifications for the complex and the acceptability limits set for all assays. In both containers, total iron content remained stable, within 10% of the theoretical iron content, and levels of iron (II) remained far below the threshold of acceptability. All preparations were free from sediments, particle numbers were acceptable and there was no microbial contamination. The molecular weight distribution and polydispersity index were also acceptable.; Under the tested experimental conditions, colloidal ferric carboxymaltose solution (Ferinject) diluted in saline in PP infusion bottles or bags demonstrated physical and chemical stability for up to 72 h at 30°C and 75% rH. Because of the lack of additional clinical data, when using ferric carboxymaltose, physicians/pharmacists should refer to the dilution and storing recommendations given in the product's summary of product characteristics

Synthesis of fluorinated maltose derivatives for monitoring protein interaction by 19F NMR

A novel reporter system, which is applicable to the 19F NMR investigation of protein interactions, is presented. This approach uses 2-F-labeled maltose as a spy ligand to indirectly probe protein–ligand or protein–protein interactions of proteins fused or tagged to the maltose-binding protein (MBP). The key feature is the simultaneous NMR observation of both 19F NMR signals of gluco/manno-type-2-F-maltose-isomers; one isomer (α-gluco-type) binds to MBP and senses the protein interaction, and the nonbinding isomers (β-gluco- and/or α/β-manno-type) are utilized as internal references. Moreover, this reporter system was used for relative affinity studies of fluorinated and nonfluorinated carbohydrates to the maltose-binding protein, which were found to be in perfect agreement with published X-ray data. The results of the NMR competition experiments together with the established correlation between 19F chemical shift data and molecular interaction patterns, suggest valuable applications for studies of protein–ligand interaction interfaces