Erythromycin is a macrolide antibiotic used mainly in the treatment of infections caused by gram-positive organisms. Erythromycin base is rap idly degraded in acidic media necessitating the use of structurally modified erythromycin derivatives or acid resistant dosage forms in order to decrease gastric inactivation of the drug. The majority of pharmacokinetic studies to-date have utilized relatively non-specific microbiological assay procedures which are unable to differentiate between concentrations of active erythromycin base and the inactive pro-drug derivatives. A high-performance liquid chromatographic (HPLC) technique is described for the simultaneous determination of erythromycin base and propionate (inactive pro-drug form) in human serum and urine following the oral administration of erythromycin estolate, an acid stable derivative of erythromycin. The method involves a solid-phase extraction step prior to chromatography on a C18 reversed-phase column with coulometric electrochemical detection. Sample handling and storage techniques are presented which minimize hydrolysis of the inactive ester moiety between sample collection and analysis, thereby more accurately reflecting the in vivo situation than in previously published studies. Results from single dose pharmacokinetic studies indicate that only 10-15% of the total erythromycin concentration in vivo is present as the active base component following oral administration of erythromycin estolate. This percentage increases to approximately 25% during multiple dose administration. Novel urinary excretion data are presented which reveal that approximately 40% and 55% of the total erythromycin excreted in urine is excreted as erythromycin base following single and multiple dosages respectively. Computer fitting of mean serum concentration-time data revealed that an open one compartment model with linear first order absorption and elimination best described the absorption and disposition of erythromycin, although poor computer fits for individual data sets were observed. Some evidence of non-linear elimination is presented utilizing both compartmental and non-compartmental pharmacokinetic techniques. Large intra-and inter-personal variability in erythromycin absorption and disposition was experienced which was evaluated in five subjects who each received one 500 mg erythromycin estolate tablet from the same batch, on three separate occasions. In addition. an HPLC method is described for the analysis of "total erythromycin" concentrations following erythromycin estolate administration which involves hydrolysis of the ester component prior to chromatography. as well as an HPLC method utilizing amperometric electrochemical detection capable of monitoring the stability of erythromycin base in stored biological fluids. These methods were uti I ized in various stability studies involving erythromycin base and propionate as well as for the analysis of erythromycin estolate dosage forms.

The population for this study consisted of 4013 sexually active women seen for family planning. Culture for Chlamydia trachomatis yielded an isolation rate of 6.1%. Women aged 16 to 25 accounted for 81.7% of the C. trachomatis infections, while those younger than 16 or older than 35 accounted for only 2.4% of the infections. Of the 246 patients whose cultures were positive for C. trachomatis, 159 (65%) were asymptomatic. The incidence of C. trachomatis was 11.2% among those with symptoms but only 6.4% among the asymptomatic group. Among 63 patients with Neisseria gonorrhoeae (who were excluded from the study), 26 (41.3%) also were infected by C. trachomatis. There were no microbiologic drug failures with erythromycin or clindamycin. Of 56 patients who enrolled in the clindamycin arm of the protocol, 48 (85.7%) completed therapy and experienced microbiologic and clinical cures. In contrast, erythromycin therapy was completed by only 25 of 57 women (43.9%) enrolled. The number of side effect failures for erythromycin was 22 of 57 (38.6%). This was more than five times the number of side effect failures for clindamycin (4 of 56, or 7.1%).

Im Rahmen der vorliegenden Arbeit wurden analytische Methoden zur Bestimmung des Verunreinigungsprofils von Erythromycin entwickelt, die der bestehenden Ph.Eur.-Methode überlegen sind. Die neue HPLC-Methode ist in der Lage, alle verwandten Verbindungen mit angemessener Präzision nachzuweisen und zu quantifizieren. Mit Hilfe der Massenspektrometrie konnten alle Hauptkomponenten und verwandten Verbindungen der Base, ihrer Ester und Salze eindeutig identifiziert und quantifiziert werden. Zudem konnten zwei neue verwandte Verbindungen von Erythromycin gefunden und als N,N-Didemethylerythromycin A und Anhydroerythromycin F identifiziert werden. / Within this work analytical methods for determination of the impurity profile of erythromycin were developed. These methods show clear improvements compared to the current methods described in the Ph.Eur. The new HPLC method is able to separate and quantify all related substances with an acceptable precision. The mass spectrometry shows the suitability for the direct qualitative and quantitative analysis of the base, ester and salts of erythromycin. Moreover using ion trap two new, unexpected EA related substances could be detected and identified as N,N-didemethylerythromycin A and anhydroerythromycin F.

Access to abstract restricted until May 2014 / Access to thesis restricted until May 2014 / Department of Biology

Capillary Electrophoresis (CE) is a high resolution analytical technique that may be employed in the separation and quantification of a wide range of analytes. The enormous efficiency obtained in CE are well suited for complex mixtures in which resolution of a large number of peaks in a short time is desirable. Therefore, CE has a promising future in pharmaC-eutical analysis. The separation mechanism of CE is based on the differential electrophoretic mobility of the solutes inside a buffer filled capillary upon the application of a voltage. Capillary electrophoresis is especially suitable for ionic species. The full potential of this technique can only be realised through the manipulation of numerous experimental parameters. In the present study, a CE method has been developed for the analysis of the macrolide antibiotics: erythromycin, oleandomycin, troleandomycin and josamycin. The selection of initial analysis conditions and optimisation of selectivity are reviewed. A systematic approach to method development was used to maximise analyte differential electrophoretic mobilities, by adjusting the pH. Thereafter, the influences of electrolyte molarity and electrolyte additives were investigated. In addition, some instrumental parameters, such as capillary length emf diameter, applied voltage and injection conditions were varied. The effect of the sample solvent and oncapillary concentration techniques such as FASI, were investigated. Also, the influence of injecting a water plug on the quantity of sample injected was demonstrated. Full resolution was achieved with the addition of methanol to the electrolyte. The applicability of CE for the assay of erythromycin and its related substances was investigated. Two methods were developed and successfully validated using CE: one for the quantitative determination of erythromycin alone and another for erythromycin related substances in the presence of large quantities of erythromycin A. Several related substances and impurities that result from the fermentation process used to produce erythromycin as well as degradation products are known to be present in commercial sa~ples. These impurities include erythromycin B, C, D, E, F, erythromycin enol ether, anhydroerythromycin and N-demethylerythromycin. Currently both the USP and BP official assays for the analysis of erythromycin involve the use of microbiological assays. These methods are limited as they are unable to differentiate between erythromycin and its related substances and degradation products. Furthermore, the microbiological assays are time-consuming and tedious to perform. 11 The CE methods developed for the analysis of erythromycin and for its related substances were fully validated in terms of precision, linearity, accuracy, sensitivity and stability. In addition, erythromycin was subjected to six stress modes and the stressed samples were analysed. An intemal standard was employed to provide acceptable precision for the migration time « 1.80 % RSD) and peak area « 4.44 % RSD). Optimum sensitivity was obtained using low UV wavelengths, with LOO values of less than 10 % for the related substances. The developed method was accurate for erythromycin C, anhydroerythromycin and N-demethylerythromycin, even in the presence of large concentrations of the parent. The method for~ erythromycin related substances was applied to the determination of impurities in three commercial erythromycin bases. The CE methods developed were rapid, precise, specific and stability-indicating and may be used to provide additional information to augment that attained by HPLC for purity assessment and in stability studies of erythromycin. Capillary electrophoresis is a simple, cost-effective technique that is capable of generating high quality data. This technique will become firmly established within pharmaceutical analysis for main peak and related impurity determination assays as familiarity becomes more widespread across the pharmaceutical industry and improvements in instrumentation are performed.

This thesis investigated the physical properties of the macrolide antibiotics: Erythromycin dihydrate (EM-DH), Roxithromycin monohydrate (RM-MH) and Azithromycin dihydrate (AZM-DH). The abovementioned hydrate compounds were investigated in terms of the hydrate-anhydrate crystal structure stability, dehydration and observed polymorphism under controlled temperature heating programs. Identified hydrate and anhydrate polymorphs were subjected to physical stability testing during controlled storage. EM-DH was characterized by thermal analysis (DSC, TGA), X-ray diffraction, FTIR and microscopy. Dehydration of EM-DH at temperatures of 100, 157 and 200°C (followed by supercooling to 25°C) produced the form (I) anhydrate (Tm =142.9°C), form (II) anhydrate (Tm = 184.7°C ) and amorph (II) (Tg = 118°C) respectively. The attempts to produce amorph (I) from melting (in vicinity of form (I) melt over temperature range 133°C to 144°C) and supercooling was unsuccessful due to the high crystallization tendency of the form (I) melt. Brief humidity exposure and controlled temperature (40°C)/ humidity storage for 4 days (0-96% RH) revealed hygroscopic behaviour for the anhydrate crystal (forms (I) and (II)) and amorph (II) forms. Form (II) converted to a nonstoichiometric hydrate where extent of water vapour absorption increased with increased storage humidity (2.1% absorbed moisture from recorded TGA at 96% RH). Amorph (II) exhibited similar trends but with greater water absorption of 4.7% (recorded with TGA) at 96% RH. The pulverization and sieving process of amorph (II) (at normal environmental conditions) was accompanied by some water vapour absorption (1.1%). A slightly lower absorbed moisture content of 3.3% (from TGA) after controlled 4 days storage at 40°C/ 96% RH was recorded. This suggested some physical instability (crystallization tendency) of amorph (II) after pulverization. The thermally induced dehydration of RM-MH by DSC-TG was evaluated structurally (SCXRD), morphologically (microscopy) and by kinetic analysis. Various kinetic analysis approaches were employed (advanced, approximation based integral and differential kinetic analysis methods) in order to obtain reliable dehydration kinetic parameters. The crystal structure was little affected by dehydration as most H-bonds were intramolecular and not integral to the crystal structure stability. Kinetic parameters from thermally stimulated dehydration indicated a multidimensional diffusion based mechanism, due to the escape of water from interlinked voids in crystal. The hygroscopicity of the forms RM-MH, Roxithromycin-anhydrate and amorph glass (Tg = 81.4°C) were investigated. Roxithromycinanhydrate (crystalline) converted readily to RM-MH which were found to be compositionally stable over the humidity range 43-96%RH. Amorphous glass exhibited increased water vapour absorption with increasing storage humidity (40°C/ 0-96% RH). TG analysis suggested a moisture content of 3.5% at 96% RH after 4 storage days. DSC and powder XRD analysis of stored pulverised amorphous glass indicated some physical instability due to water induced crystallization. Commercial AZM-DH and its modifications were characterized by thermal analysis (DSC, TGA), SC-XRD and microscopy. Thermally stimulated dehydration of AZM-DH occurred in a two-step process over different temperature ranges. This was attributed to different bonding environments for coordinated waters which were also verified from the crystal structure. Dehydration activation energies for thermally stimulated dehydration were however similar for both loss steps. This was attributed to similarities in the mode of H- bonding. Different forms of AZM were prepared by programmed temperature heating and cooling of AZM-DH. The prepared forms included amorphous glass (melt supercooling), amorphous powder (prepared below crystalline melting temperature), crystalline anhydrate and crystalline partial dehydrate. Humidity exposure indicated hygroscopic behaviour for the amorphous, crystalline anhydrate and crystalline partial dehydrate modifications. Both the crystalline anhydrate and partial dehydrate modifications converted to the stoichiometric dihydrate form (AZM-DH) at normal environmental conditions at ambient temperature. Both the amorph glass and amorph powder exhibited increased moisture absorption with increased humidity exposure. TG analysis of the pulverised amorph glass indicated a moisture content of 5.1% at 96% RH after 4 storage days. The absence of crystalline melt in DSC and presence of Tg (106.9°C) indicated the sample remained amorphous after pulverisation and storage for 4 days at 40°C/ 96% RH.

No / Background Propionibacterium acnes and P. granulosum are widely regarded as the aetiological agents of inflammatory acne. Their proliferation and metabolism are controlled using lengthy courses of oral and/or topical antibiotics. Despite numerous reports of skin colonization by antibiotic-resistant propionibacteria among acne patients, accurate prevalence data are available only for the U.K. Objectives To determine the prevalence of skin colonization by antibiotic-resistant propionibacteria among acne patients and their contacts from six European centres. Methods Skin swabs were collected from 664 acne patients attending centres in the U.K., Spain, Italy, Greece, Sweden and Hungary. Phenotypes of antibiotic-resistant propionibacteria were determined by measuring the minimum inhibitory concentrations (MIC) of a panel of tetracycline and macrolide, lincosamide and streptogramin B (MLS) antibiotics. Resistance determinants were characterized by polymerase chain reaction (PCR) using primers specific for rRNA genes and erm(X), followed by nucleotide sequencing of the amplified DNA. Results Viable propionibacteria were recovered from 622 patients. A total of 515 representative antibiotic-resistant isolates and 71 susceptible isolates to act as control strains were characterized phenotypically. The prevalence of carriage of isolates resistant to at least one antibiotic was lowest in Hungary (51%) and highest in Spain (94%). Combined resistance to clindamycin and erythromycin was much more common (highest prevalence 91% in Spain) than resistance to the tetracyclines (highest prevalence 26·4% in the U.K.). No isolates resistant to tetracycline were detected in Italy, or in Hungary. Overall, there were strong correlations with prescribing patterns. Prevalence of resistant propionibacteria on the skin of untreated contacts of the patients varied from 41% in Hungary to 86% in Spain. Of the dermatologists, 25 of 39 were colonized with resistant propionibacteria, including all those who specialized in treating acne. None of 27 physicians working in other outpatient departments harboured resistant propionibacteria. Conclusions The widespread use of topical formulations of erythromycin and clindamycin to treat acne has resulted in significant dissemination of cross-resistant strains of propionibacteria. Resistance rates to the orally administered tetracycline group of antibiotics were low, except in Sweden and the U.K. Resistant genotypes originally identified in the U.K. are distributed widely throughout Europe. Antibiotic-resistant propionibacteria should be considered transmissible between acne-prone individuals, and dermatologists should use stricter cross-infection control measures when assessing acne in the clinic. Background Propionibacterium acnes and P. granulosum are widely regarded as the aetiological agents of inflammatory acne. Their proliferation and metabolism are controlled using lengthy courses of oral and/or topical antibiotics. Despite numerous reports of skin colonization by antibiotic-resistant propionibacteria among acne patients, accurate prevalence data are available only for the U.K. Objectives To determine the prevalence of skin colonization by antibiotic-resistant propionibacteria among acne patients and their contacts from six European centres. Methods Skin swabs were collected from 664 acne patients attending centres in the U.K., Spain, Italy, Greece, Sweden and Hungary. Phenotypes of antibiotic-resistant propionibacteria were determined by measuring the minimum inhibitory concentrations (MIC) of a panel of tetracycline and macrolide, lincosamide and streptogramin B (MLS) antibiotics. Resistance determinants were characterized by polymerase chain reaction (PCR) using primers specific for rRNA genes and erm(X), followed by nucleotide sequencing of the amplified DNA. Results Viable propionibacteria were recovered from 622 patients. A total of 515 representative antibiotic-resistant isolates and 71 susceptible isolates to act as control strains were characterized phenotypically. The prevalence of carriage of isolates resistant to at least one antibiotic was lowest in Hungary (51%) and highest in Spain (94%). Combined resistance to clindamycin and erythromycin was much more common (highest prevalence 91% in Spain) than resistance to the tetracyclines (highest prevalence 26·4% in the U.K.). No isolates resistant to tetracycline were detected in Italy, or in Hungary. Overall, there were strong correlations with prescribing patterns. Prevalence of resistant propionibacteria on the skin of untreated contacts of the patients varied from 41% in Hungary to 86% in Spain. Of the dermatologists, 25 of 39 were colonized with resistant propionibacteria, including all those who specialized in treating acne. None of 27 physicians working in other outpatient departments harboured resistant propionibacteria. Conclusions The widespread use of topical formulations of erythromycin and clindamycin to treat acne has resulted in significant dissemination of cross-resistant strains of propionibacteria. Resistance rates to the orally administered tetracycline group of antibiotics were low, except in Sweden and the U.K. Resistant genotypes originally identified in the U.K. are distributed widely throughout Europe. Antibiotic-resistant propionibacteria should be considered transmissible between acne-prone individuals, and dermatologists should use stricter cross-infection control measures when assessing acne in the clinic. Background Propionibacterium acnes and P. granulosum are widely regarded as the aetiological agents of inflammatory acne. Their proliferation and metabolism are controlled using lengthy courses of oral and/or topical antibiotics. Despite numerous reports of skin colonization by antibiotic-resistant propionibacteria among acne patients, accurate prevalence data are available only for the U.K. Objectives To determine the prevalence of skin colonization by antibiotic-resistant propionibacteria among acne patients and their contacts from six European centres. Methods Skin swabs were collected from 664 acne patients attending centres in the U.K., Spain, Italy, Greece, Sweden and Hungary. Phenotypes of antibiotic-resistant propionibacteria were determined by measuring the minimum inhibitory concentrations (MIC) of a panel of tetracycline and macrolide, lincosamide and streptogramin B (MLS) antibiotics. Resistance determinants were characterized by polymerase chain reaction (PCR) using primers specific for rRNA genes and erm(X), followed by nucleotide sequencing of the amplified DNA. Results Viable propionibacteria were recovered from 622 patients. A total of 515 representative antibiotic-resistant isolates and 71 susceptible isolates to act as control strains were characterized phenotypically. The prevalence of carriage of isolates resistant to at least one antibiotic was lowest in Hungary (51%) and highest in Spain (94%). Combined resistance to clindamycin and erythromycin was much more common (highest prevalence 91% in Spain) than resistance to the tetracyclines (highest prevalence 26·4% in the U.K.). No isolates resistant to tetracycline were detected in Italy, or in Hungary. Overall, there were strong correlations with prescribing patterns. Prevalence of resistant propionibacteria on the skin of untreated contacts of the patients varied from 41% in Hungary to 86% in Spain. Of the dermatologists, 25 of 39 were colonized with resistant propionibacteria, including all those who specialized in treating acne. None of 27 physicians working in other outpatient departments harboured resistant propionibacteria. Conclusions The widespread use of topical formulations of erythromycin and clindamycin to treat acne has resulted in significant dissemination of cross-resistant strains of propionibacteria. Resistance rates to the orally administered tetracycline group of antibiotics were low, except in Sweden and the U.K. Resistant genotypes originally identified in the U.K. are distributed widely throughout Europe. Antibiotic-resistant propionibacteria should be considered transmissible between acne-prone individuals, and dermatologists should use stricter cross-infection control measures when assessing acne in the clinic.

Im Zusammenhang mit der Labmagenverlagerung zählen mangelnde Motilität und Entleerungsstörungen zu den häufigsten Problemen im postoperativen Zeitraum. In der vorliegenden Arbeit sollte geprüft werden, ob eine präoperative Erythromycingabe die Entleerung des Labmagens in den ersten 24 Stunden nach Reposition beeinflusst und so die klinische Rekonvaleszenz beschleunigt wird. Untersucht wurden 60 Milchkühe mit linksseitiger bzw.rechtsseitiger Labmagenverlagerung. Die abomasale Entleerungsrate der mit Erythromycin behandelten Tiere war signifikant höher als die der unbehandelten Tiere, sowohl in der Gruppe der linksseitig verlagerten als auch der rechtsseitig verlagerten Kühe.

The ubiquitous presence and chronic effect of pharmaceuticals is one of the emerging issues in environmental field. As a result of incomplete removal by sewage treatment plants, pharmaceuticals are released into the environment and drinking water sources. On the other hand, conventional drinking water treatment processes such as coagulation, filtration and sedimentation are reported to be ineffective at removing pharmaceuticals. Therefore, the potential presence of pharmaceuticals in finished drinking water poses a threat on public health. Antibiotics, as an important group of pharmaceuticals, are given special concerns because the potential development of bacteria-resistance. Ozonation and advanced oxidation processes are demonstrated to be quite effective at removing pharmaceuticals. The oxidation of pharmaceuticals is caused by ozone itself and hydroxyl radicals that are generated from ozone decomposition. Whether ozone or hydroxyl radicals are the primary oxidant depends on the specific pharmaceutical of interest and the background water matrix. In this research, erythromycin, a macrolide antibiotic, was chosen as the target compound because of its high detection frequency in the environment and its regulation status. The objective of this research was to investigate the removal performance of erythromycin by ozonation from the standpoint of kinetics. The effects of pH, carbonate and phosphate buffers, and initial ozone dose on ozonation of erythromycin were also studied. The second-order rate constant for the reaction between deprotonated erythromycin and ozone was determined to be 4.44x10⁹ M⁻¹·s⁻¹ while protonated erythromycin did not react with ozone. Ozone was determined to be the primary oxidant for erythromycin removal by ozonation. pH was found to have great positive impact on the degradation of erythromycin by ozonation due to the deprotonation of erythromycin at high pH. Carbonate and phosphate buffers were found to have negligible effects on the degradation of erythromycin by ozonation. Initial ozone dose showed a positive impact on the total erythromycin removal rate by ozonation. / text

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2006. / Härtill 5 uppsatser.