Thesis (M.S.)--University of Michigan, 1970.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2001. / Härtill 4 uppsatser.

La recherche de nouveaux métabolites d'intérêt médical est toujours d'actualité, surtout si l'on considère que d'anciennes, mais aussi des nouvelles infections bactériennes ou virales apparaissent régulièrement. Les actinomycètes, et plus particulièrement les Streptomyces, sont les principaux producteurs de molécules anti-microbiennes. En effet, ils produisent presque 60-70% des produits naturels d'origine microbienne. La plupart de ces métabolites appartient à la classe des polycétides, qui sont synthétisés par des complexes multienzymatiques, les polycétide-synthétases (PKS). Les PKS utilisent des acides gras simples pour assembler des structures polycétidiques très diversifiées. Une approche très prometteuse pour identifier des nouvelles voies de biosynthèse de métabolites secondaires est basée sur une approche génomique ou « genome mining ». Le séquençage du chromosome linéaire de Streptomyces ambofaciens ATCC23877 a, entre autre, révélé sur le bras chromosomique droit, un cluster cryptique de gènes de PKS de type I de grande taille. Ce cluster contient 25 gènes, dont 9 gènes de biosynthèse, pour un total de 25 modules, tous fonctionnels. Les analyses in silico des gènes de PKS ont permis de prédire que le cluster serait responsable de la synthèse d'une molécule appartenant à la famille des macrolides. Dans des conditions standard de laboratoire, le cluster était silencieux. Afin d'activer l'expression du cluster, un gène de régulation, samR0484, présent dans le cluster et codant un régulateur de la famille LAL (Large ATP binding protein of the LuxR family), a été surexprimé dans la souche sauvage. Les analyses transcriptionelles ont montré que cela se traduisait par l'induction de l'expression des gènes de biosynthèse. Par conséquence, une stratégie de « comparative metabolic profiling » a été menée entre la souche sauvage et la souche mutante afin d'identifier le nouveau métabolite. Quatre formes différentes d'un macrolide avec un cycle lactonique de 50 carbones, ont été isolées et caractérisées. Ces composants, nommés sambomycine A, B, C et D, ont montré une activité antibactérienne et une activité antiproliférative intéressante. La détermination de la structure de la sambomycine a révélé des caractéristiques uniques et intéressantes, concernant la réaction de cyclisation et la synthèse d'un précurseur atypique. Ces mécanismes de biosynthèse ont fait l'objet d'une étude plus approfondie. Nous nous sommes également intéressés à la régulation de ce cluster. Le régulateur LAL agit comme un activateur transcriptionnel essentiel. Des analyses préliminaires indiquent que ce régulateur se fixe aux régions promotrices de certains gènes, notamment celles des gènes de biosynthèse ainsi que celles d'autres gènes de post-modification, activant ainsi leur transcription. / The constant and urgent need of novel bioactive compounds is the result of the emergence in the last decades of new and old infectious diseases, a sore for humankind. Actinomycetes and especially the genus Streptomyces are the principal producers of microbial drugs producing nearly 60-70% of the natural products. The majority of secondary metabolites belong to the class of polyketides that are synthesised by multienzymatic complexes named polyketides synthases (PKS). PKSs condensate simple small carboxylic acids to generate a wide range of complex polyketide structures. In the search for new drugs, the genome mining approach proved to be a powerful tool in the identification of cryptic secondary metabolite pathways. The sequencing and the analysis of Streptomyces ambofaciens ATCC23877 genome has revealed a large type I PKS cluster, on the right arm of the chromosome. The cluster contains 9 PKS, composed of 25 functional modules. In silico analysis of the PKS genes and of the tailoring genes enabled to predict the structure of the expected metabolite, a macrolide. In the laboratory standard conditions, the cluster showed to be silent. Therefore, to promote the expression of the cluster, the regulatory gene samR0484, encoding a LAL regulator (Large ATP binding protein of the LuxR family) was overexpressed in the wt strain. Transcriptional analyses showed that the PKS genes were expressed. Subsequently, by comparative metabolic profiling between the mutant strain and the wt, we were able to detect the novel metabolite produced by S. ambofaciens. Structural elucidation revealed four form of a 50-membered macrolide, named sambomycin. The compounds endow antibacterial and antitumoral activities. The structure unveiled unique and interesting characteristics of sambomycin, i.e. the cyclization reaction and the presence of an atypical extender unit. The mechanisms of biosynthesis have been analysed more in details in this work. We also investigated in the regulation of the cluster. The LAL regulator was shown to be an essential transcriptional activator, binding to the promoter regions of the PKS genes and probably to other genes in the cluster.

Studies toward synthesis of subunits of sanglifehrin A, an immunosuppressant featuring a highly substituted [5,5]-spirolactam moiety as well as a 22-membered macrocycle are described. The macrolactone contains a peptidic backbone characterized by an unusual [beta]-substituted (S)-piperazic acid and (S)-m-hydroxyphenylalanine units. These studies resulted in the synthesis of advanced intermediate 358 which contains all of the carbon atoms of the C1-C25 macrolactone of sanglifehrin A, and 251 which bears the C31-C41 carbon skeleton of the [5,5]-spirolactam moiety of sanglifehrin A. A Masamune anti-aldol reaction of aldehyde 294 and ester 285 furnished alcohol 295 in a second generation approach to carboxylic acid 242, while a third generation route toward 242 improved the yield and required fewer synthetic steps. An asymmetric, catalytic phase-transfer method was used to introduce an [alpha]-amino function into 331 in the synthesis of (S)-m-hydroxyphenylalanine derivative 244. Assembly of 244, piperazic acid 113 and L-valine derivative 336 into tripeptide 241 using a racemization-free peptide coupling method is described. The synthesis of C31-C37 aldehyde 253 exploited double asymmetric crotylation to set in place the correct configuration of alternating hydroxyl and methyl groups at C33, 34, 35 and 36. / Graduation date: 2013

The main research of this thesis includes three sections. The purpose of first part is to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of 8 macrolide antibiotics and lincosamides inside fish tissue, including erythromycin (ERM), oleandomycin (OLD), kitasamycin (KIT), tylosin (TYL), josamycin (JOS), spiramycin (SPM), tilmicosin (TIL), and lincomycin (LIN). Homogenized samples are first extracted with acetonitrile, dehydrated with sodium sulphate anhydrous, and then condensed. After the residue was redissolved in methanol and the extracts were partitioned with n-hexane to remove lipids, the sample is filterced and detected by LC/MS-MS using chromatography columns of Agilent HC-C18 (5£gm, 150 mm ¡Ñ4.6 mm). The mobile phase A was 5mM ammonium acetate containing 0.1% formic acid, while the mobile phase B was acetonitrile. The analysis of 8 macrolide antibiotics and lincosamides can be achieved within 10 minutes with electrospray ionization-tandem mass spectrometry in positive mode using multiple reaction monitoring (MRM) for simultaneous detection. The second part is to verify the method by regulation of European Union (EU) resolution scheme (2002/657/EC). In the case where the drug is set as allowed drug, the recovery rate under gradient addition according to MRL is between 93.64% to 106.67%, and the CV is between 0.27% to 7.17%. In the case where the drug is set as prohibited drug, the recovery rate under gradient addition according to MRPL is between 96.35%~104.88%, and the CV is between 6.77%~13.91%. As a result, the decision limit (CC£\) and the Detection capability (CC£]) of the 8 macrolide antibiotics and lincosamides is between 0.24 to 0.40£gg kg-1 and 0.33 to 0.49£gg kg-1. The last section is to evaluate the stability of drugs in fish body under domestic preservation and process methods on fish, including refrigeration at -20¢J and cold storage at 4 ¢J. The test is implemented by adding the drug into fish tissue according to MRL and detecting the antibiotics residue after regulated 40 days. Besides, the effect on activity of drug residue in fish body after boiling at 100 ¢J is compared. The results show that the residual amount of spiramycin, josamycin, tilmicosin, and lincomycin is below 35% while that of erythromycin, oleandomycin, kitasamycin, and tylosin will be below 20%. Therefore, the drugs including erythromycin, josamycin, tylosin, and lincomycin will stay stably in fish tissue if they are stored under -20 ¢J. However, it may affect human health if the fish contains such antibiotic residues is not boiled.

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Specific bacterial commensals demonstrating multidrug resistance (MDR) are opportunistic pathogens for immunocompromised patients, including Corynebacterium species (spp.). Severe infections due to MDR corynebacteria are being increasingly reported where several MDR phenotypes have been described. One such phenotype, the macrolide-lincosamide-streptogramin B phenotype (MLSB), is characterized by high-level resistance to macrolides, lincosamides, and streptogramin B. Resistance is thought to be attributable to acquisition of the ermX gene, a methyltransferase that alters the ribosomal macrolide binding site. Until recently, ermX had been reported in only six Corynebacterium spp. We have observed other corynebacteria can also display high-level resistance to MLSB antimicrobials and are ermX positive. Hypotheses being tested include: 1) high-level macrolide and lincosamide resistance in Corynebacterium spp. is caused by acquiring ermX; 2) distribution of ermX is more widespread than previously published; 3) ermX is associated with transposon Tn5432; 4) multidrug resistance has spread to Canadian C. afermentans and C. aurimucosum strains.

Azithromycin, an azalide and member of the macrolide group, is a broad spectrum antimicrobial, representing one of the bestselling antimicrobials worldwide. It is derived from erythromycin and exhibits improved acidic stability as a result of its structural modifications. The stable solid form of azithromycin is its dihydrate, although it also naturally occurs in its metastable forms, i.e. the monohydrate and anhydrate. Because azithromycin is poorly soluble in water, its absorption from the gastro-intestinal tract is negatively influenced, which ultimately affects its bioavailability following oral administration (37 %). Polymorphic (monohydrates and dihydrates) and anhydrous forms of azithromycin were screened and investigated. One anhydrous form also proved to be amorphous, which shifted the focus of this study from polymorphism to amorphism. An amorphous glassy azithromycin was subsequently prepared and fully characterised to present its solid state profile. The stability of this amorphous glassy form was established at a high temperature and relative humidity over a period of four weeks. Exposure to increased relative humidity (up to 95 %) and increased water content (up to 50 %) also served as stability indicating tests. Its solubility in various aqueous media was determined. A solid dosage form (tablet), containing the azithromycin glass, was prepared, whereafter these tablets were subjected to dissolution studies in different aqueous media. The stability of azithromycin glass in tablet form was determined over a period of three months. The permeability of azithromycin glass across excised pig intestinal tissue was further established at various pH values. This amorphous glassy form of azithromycin (AZM-G) proved to be very stable at high temperature and relative humidity, whilst also remaining stable after prolonged exposure to 95 % of relative humidity, as it only adsorbed moisture onto its surface. Water content (up to 50 %) had no plasticising effect on azithromycin glass. It demonstrated a significantly higher water solubility (339 % improvement) in comparison with the commercially available azithromycin dihydrate and was it also 39 % more soluble in phosphate buffer (pH 6.8) than its dihydrate counterpart. The prepared azithromycin glass tablets showed a promising dissolution profile in water, due to the improved water solubility of this glass form. The transport of azithromycin glass at higher pH values (6.8 and 7.2) across the membrane proved to be significantly higher than that of azithromycin dihydrate, thus also illustrating its pH dependence for its transport across pig intestinal tissue. The improved water solubility of the azithromycin glass, together with its faster dissolution rate, its superior stability and its increased permeability, may ultimately result in a higher azithromycin bioavailability following oral administration. These research outcomes hence give rise to the need for investigating the effect of administering lower dosages of azithromycin and to determine whether the same antimicrobial efficacy would possibly be achieved, due to maintaining the same tissue concentration levels at these lower dosages. / Thesis (PhD (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013

Azithromycin, an azalide and member of the macrolide group, is a broad spectrum antimicrobial, representing one of the bestselling antimicrobials worldwide. It is derived from erythromycin and exhibits improved acidic stability as a result of its structural modifications. The stable solid form of azithromycin is its dihydrate, although it also naturally occurs in its metastable forms, i.e. the monohydrate and anhydrate. Because azithromycin is poorly soluble in water, its absorption from the gastro-intestinal tract is negatively influenced, which ultimately affects its bioavailability following oral administration (37 %). Polymorphic (monohydrates and dihydrates) and anhydrous forms of azithromycin were screened and investigated. One anhydrous form also proved to be amorphous, which shifted the focus of this study from polymorphism to amorphism. An amorphous glassy azithromycin was subsequently prepared and fully characterised to present its solid state profile. The stability of this amorphous glassy form was established at a high temperature and relative humidity over a period of four weeks. Exposure to increased relative humidity (up to 95 %) and increased water content (up to 50 %) also served as stability indicating tests. Its solubility in various aqueous media was determined. A solid dosage form (tablet), containing the azithromycin glass, was prepared, whereafter these tablets were subjected to dissolution studies in different aqueous media. The stability of azithromycin glass in tablet form was determined over a period of three months. The permeability of azithromycin glass across excised pig intestinal tissue was further established at various pH values. This amorphous glassy form of azithromycin (AZM-G) proved to be very stable at high temperature and relative humidity, whilst also remaining stable after prolonged exposure to 95 % of relative humidity, as it only adsorbed moisture onto its surface. Water content (up to 50 %) had no plasticising effect on azithromycin glass. It demonstrated a significantly higher water solubility (339 % improvement) in comparison with the commercially available azithromycin dihydrate and was it also 39 % more soluble in phosphate buffer (pH 6.8) than its dihydrate counterpart. The prepared azithromycin glass tablets showed a promising dissolution profile in water, due to the improved water solubility of this glass form. The transport of azithromycin glass at higher pH values (6.8 and 7.2) across the membrane proved to be significantly higher than that of azithromycin dihydrate, thus also illustrating its pH dependence for its transport across pig intestinal tissue. The improved water solubility of the azithromycin glass, together with its faster dissolution rate, its superior stability and its increased permeability, may ultimately result in a higher azithromycin bioavailability following oral administration. These research outcomes hence give rise to the need for investigating the effect of administering lower dosages of azithromycin and to determine whether the same antimicrobial efficacy would possibly be achieved, due to maintaining the same tissue concentration levels at these lower dosages. / Thesis (PhD (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013