| Bitespiramycin (Shengjimycin) was developed by the Institute of Medical Biotechnology, Chinese Academy of Medical Science. It is a group of 4"-acylated spiramycins with 4"-isovalerylspiramycins as the major components, produced by recombinant Streptomyces spiramyceticus F21. The contents of isovalerylspiramycin I, II, III in bitespiramycin are 7.4%, 22.5% and 37.7%, respectively. Minor components in bitespiramycin include about 6 derivatives of spiramycin such as (iso) butanoylspiramycin, propionylspiramycin and acetylspiramycin. Phase II clinical trial of bitespiramycin will be performed soon.Although spiramycin has been used in clinical therapy for more than 40 years, the research about its metabolism in vivo was limited. This is due in part to difficulties that have been encountered in establishing a sensitive and specific assay for spiramycin and its metabolites. Bitespiramycin is a complex mixture of more than 10 kinds of spiramycin derivatives. The research about its metabolism and pharmacokinetics in vivo was a challenge for the common analytical methods. The aim of this work is to investigate the metabolism and pharmacokinetics of such a complex multicomponents drug in vivo using the advanced LC/MSn and LC/MS/MS method.1. Identification of the metabolites of bitespramycin in rats by LC/MSnMetabolites in the urine, bile and feces of 4 rats following a single oral dose of 40 mg bitespiramycin were investigated. A total of 49 metabolites were found in bile, urine and feces by HPLC with ion trap mass spectrometric detection. Using the multi-stage MS (MSn) analysis of bitespiramycin and its metabolites, the characteristic fragment ions were obtained.The metabolites of isovalerylspiramycin III (MO) were identified as deforosamine derivative of MO (Ml), deisovalery derivative of Ml (M2), reduction derivative of MO (M3), lactone hydrolyzed derivative of MO (M4), cysteine conjugate of MO (M5), deisovalery derivative of MO (M6), reduction derivative of M6 (M7), cysteine conjugate of M6 (M8), demycarose derivative of MO and M6 (M9), cysteine conjugate of M9 (M10), lactone hydrolyzed derivative of M6 (M11). The other components in bitespiramycin have similar metabolic pathways with isovalerylspiramycin III.The facile procedure led to identification of all the 10 known components of bitespiramycin, in addition to the characterization of at least 49 metabolites including spiramycin I, II, III, platenomycin A1, josamycin, leucomycin A1 and midecamycin A1 which have been used in clinical therapy for decades. Structures of 16 major metabolites (M1a-c, M1f, M3a, M5a, M6a-c, M7a-c, M8a-c, and M9c) were established by chromatographic and mass spectrometric analyses andcomparison with synthesized reference substances. The aldehyde reduction and hydrolysis of the forosamine represent two novel biotransformation pathways for spiramycin derivatives in vivo.2. The development of the quantitation methodsA sensitive and specific LC/MSn method was developed for the simultaneous determination of major components (isovalerylspiramycin I, II, III) or their major metabolites (spiramycin I, II, III) in biological samples of rats.A more sensitive and fast LC/MS/MS quantitative method was developed to simultaneously determine six components (isovalerylspiramycin I, II, III and spiramycin I, II, III) in human plasma and urine.3. Kinetics of acid-catalyzed hydrolysisThe developed LC/MSn method was used to clarify the degradation pathways and validated for monitoring degradation process of spiramycin derivatives at 37C in synthetic gastric fluid to simulate human gastric environment. Spiramycin III, acetylspiramycin III, propionylspiramycin III, (iso)butanoylspiramycin III and isovalerylspiramycin I, II, III were also the major components of bitespiramycin, which was developed as a novel antibiotic. These components were found susceptible to degradation from exposure to acidic condition (pH 1.3). Furthermore, the degradation rate constants (Ke) and half-life (t1/2) of spiramycin derivatives were calculated...
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