Note:

Note:

The investigations detailed herein have been conducted to address various aspects of the biopharmaceutics and pharmacokinetics of josamycin which to-date, have received little or no attention in the literature. Areas of investigation have included the selective determination of josamycin in serum and urine samples, the stability of josamycin in stored biological samples, intrinsic dissolution rates, solubility, acid and alkali stability and bioavailability and pharmacokinetics after dosing with a solution, powder and tablets. High performance liquid chromatography (HPLC) was used as the main analytical tool throughout these studies and proved to be highly versatile for the determination of josamycin in a number of different media. HPLC analysis afforded simple yet accurate determination of josamycin in samples from dissolution, solubility, tablet content and stability studies. Furthermore, the specificity afforded by HPLC was particularly useful for the separation of josamycin from degradation products formed in acid and alkali media. Since metabolites of josamycin are microbiologically active, microbiological assays do not determine the concentration solely of josamycin. An analytical method capable of the selective determination of josamycin in serum and urine samples is therefore required for the procurement of reliable bioavailability and pharmacokinetic data. HPLC affords this selectivity and a method for the selective determination of josamycin in serum and urine was successfully developed. The assay was simple yet precise, accurate and sensitive. Furthermore, it was well suited to the determination of josamycin in a large number of biological samples. Its success was largely due to the use of a solid phase extraction step using C₁₈ extraction columns, with a highly specific wash sequence followed by a phase separation step after elution from the extraction column. Chromatography was performed on a C₁₈ reversed-phase analytical column with UV detection of josamycin and internal standard at 231 nm and at 204 nm respectively using a programmable multi-wavelength detector. Only slight modification of the assay described should enable the selective determination of the metabolites of josamycin. This assay, therefore, lays the groundwork for future investigations into the pharmacokinetics of these metabolites. The re-usability of extraction columns was assessed in an attempt to reduce the cost of sample analysis. It was found that extraction columns could be used twice for the extraction of serum samples and up to four times for the extraction of urine samples. The difference between the re-usability of extraction columns for serum and urine samples was ascribed to various differences in the composition of the sample matrix. The stability of josamycin in stored serum and urine samples was also assessed.

A study has been conducted aimed at the generation and characterisation of mutations in the Escherichia coli gyrA gene, resistant to the quinolone group of antibacterial agents. Preliminary studies on quinolone-resistant mutants of strains that over-express the DNA gyrA gene, revealed the over-production of a 60 KDa protein which was partially purified. This 60 KDa protein was found to be similar, but not identical to the E. coli heat shock protein GroEL. The gyrA gene has been recloned in the 8.0 kb plasmid pPH3, which contains the gene under the stringent control of the hybrid tac promoter. The E. coli strain JMtacA containing pPH3 exhibits no expression of the gyrA gene in the absence of the inducer (IPTG), but over-produces the protein at greater than 20% of the total soluble cell protein after induction. The optimisation and purification of the GyrA subunit from JMtacA is also described. A fragment was subjected to site-directed mutagenesis which contained the TCG codon for serine-83, which was mutated to alanine (GCG). The mutant showed a 15x increase in MIC50 compared to wild-type. The mutated GyrA subunit was over-produced, complexed with wild-type GyrB subunit and used in various assays for reactions performed by DNA gyrase. The ID50 was determined for supercoiling, decatenation, relaxation of negatively supercoiled DNA, and cleavage of supercoiled DNA. The cleavage reaction mediated by Ca++ was also investigated. The technique of gap-misrepair mutagenesis, geared to the generation of single, random point mutations on a plasmid was also used on the plasmid pPH3, to generate a quinolone-resistant mutant of the gyrA gene. The mutant isolated (GMIOO) was also over-produced and compared to wild-type in various assays. The mutation was determined by DNA sequencing to be glutamine-106 to arginine.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.