Search

Carrimycin (CA), sanctioned by China's National Medical Products Administration (NMPA) in 2019 for treating acute bronchitis and sinusitis, has recently been observed to exhibit multifaceted biological activities, encompassing anti-inflammatory, antiviral, and anti-tumor properties. Despite these applications, its efficacy in sepsis treatment remains unexplored. This study introduces a novel function of CA, demonstrating its capacity to mitigate sepsis induced by lipopolysaccharide (LPS) and cecal ligation and puncture (CLP) in mice models. Our research employed in vitro assays, real-time quantitative polymerase chain reaction (RT-qPCR), and RNA-seq analysis to establish that CA significantly reduces the levels of pro-inflammatory cytokines, namely tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1β), and interleukin 6 (IL-6), in response to LPS stimulation. Additionally, Western blotting and immunofluorescence assays revealed that CA impedes Nuclear Factor Kappa B (NF-κB) activation in LPS-stimulated RAW264.7 cells. Complementing these findings, in vivo experiments demonstrated that CA effectively alleviates LPS- and CLP-triggered organ inflammation in C57BL/6 mice. Further insights were gained through 16S sequencing, highlighting CA's pivotal role in enhancing gut microbiota diversity and modulating metabolic pathways, particularly by augmenting the production of short-chain fatty acids in mice subjected to CLP. Notably, a comparative analysis revealed that CA's anti-inflammatory efficacy surpasses that of equivalent doses of aspirin (ASP) and TIENAM. Collectively, these findings suggest that CA exhibits significant therapeutic potential in sepsis treatment. This discovery provides a foundational theoretical basis for the clinical application of CA in sepsis management., (Copyright © 2024 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Bitespiramycin, has been shown to have a therapeutic effect against respiratory tract inflammation, including a potential effect against COVID-19. A current clinical trial in China showed that bitespiramycin was an effective treatment for severe pneumonia and intracranial infection. However, there is lack of an analytical method to elucidate the distribution of bitespiramycin. In this study, a highly sensitive, rapid and reliable UPLC-MS/MS method was developed to comprehensively characterize the bitespiramycin distribution in various bio-samples, which is significantly improved upon the published work. A rapid sample preparation method was developed by using n -butanol as the solvent to extract bitespiramycin from different bio-samples. The extract was then directly analyzed by UPLC-MS/MS coupled with an alkaline-resistant column after centrifugation which avoids the time-consuming concentration process under nitrogen and redissolution. The method was employed to accurately quantify bitespiramycin and its metabolites in rat plasma, tissues, and human cerebrospinal fluid. Notably, the presence of bitespiramycin and its metabolites was identified for the first time in various rat organs including brain, testis, bladder and prostate as well as in human cerebrospinal fluid. This newly developed approach shows great promise for drug distribution assays including other antibiotics and can help elucidate the ADME of bitespiramycin.

A liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of residues of spiramycin, a macrolide antibiotic, and its active metabolite neospiramycin in cow's milk as well as in minor species 'milk, goat and ewe. Spiramycin-d3 was used as internal standard for quantification of both analytes. This analytical method was validated using a global accuracy profile as a graphical decision tool built according to the trueness and the precision of the method. A unique and optimal linear model with logarithm transformation (with a determination coefficient of 0.9991) allowed the measurement of both analytes in the milks of the three animal species, in a wide range from 0.2 to 10 times the Maximal Residue Limit (MRL) (40-2000 µg.kg -1 ). The limits of detection and quantification were 13 µg.kg -1 and 40 µg.kg -1 , respectively. The accuracy profile was established to get 80% of future measurements in routine assays that will fall within the acceptance limits. Trueness of the method, expressed as relative bias, was comprised between -1.6% and 5.7% over the whole range of concentrations. The mean relative standard deviation for repeatability and intermediate precision were comprised between 1.1% and 2.7%; 2.5 and 4.2%, respectively, in all levels of concentration for the three milks. Moreover, a two-order polynomial function was used to model the relative expanded uncertainty with a determination coefficient of 0.834. This function aimed to determine the uncertainty of the future quantifications within the validated dosing range. Overall, the global accuracy profile highlighted the reliability of the method for the routine assays of spiramycin and neospiramycin even in milk from minor species (goat, ewe) by using the most accessible milk (often from cow), while guaranteeing a very high proportion of samples within the fixed acceptance limits. The applicability of this method was tested during a depletion study of spiramycin and neospiramycin in the milk of cow, goat and ewe. The developed analytical method will be useful to assess the distribution profile of the antibiotic and its metabolite in milk of minor species where few studies are available., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Bitespiramycin (biotechnological spiramycin, Bsm) is a new 16-membered macrolide antibiotic produced by Streptomyces spiramyceticus WSJ-1 integrated exogenous genes. The gene cluster for Bsm biosynthesis consists of two parts: spiramycin biosynthetic gene cluster (92 kb) and two exogenous genes including 4"-O-isovaleryltransferase gene (ist) and a positive regulatory gene (acyB2) from S. thermotolerans. Four putative regulatory genes, bsm2, bsm23, bsm27 and bsm42, were identified by sequence analysis in the spiramycin gene cluster. The inactivation of bsm23 or bsm42 in S. spiramyceticus eliminated spiramycin production, while the deletion of bsm2 and bsm27 did not abolish spiramycin biosynthesis. The acyB2 gene, homologous with bsm42 gene, cannot recover the spiramycin production in Δbsm42 mutant. The high expression of bsm42 significantly increased the spiramycin production, but overexpression of bsm23 inhibited its production in Δbsm23 and wild-type strain. Bsm23 was shown to be involved in the regulation of the expression of bsm42 and acyB2 by electrophoretic mobility shift assays. The bsm42 gene was also positive regulator for ist expression inferred from the improved yield of 4"-isovalerylspiramycins in the S. lividans TK24 biotransformation test, but adding bsm23 decreased the production of 4''-isovalerylspiramycins. These results demonstrated Bsm42 was a pathway-specific activator for spiramycin or Bsm biosynthesis, but overexpression of Bsm23 alone was adverse to produce these antibiotics although Bsm23 was essential for positive regulation of spiramycin production., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Bitespiramycin is composed of nine main acylated spiramycin components with isovaleryspiramycin as the major component. However, even with excellent therapeutic effects, its application and industrialization are restricted due to its low titer. In this study, the exogenous addition of A-Factor analogue 1,4-butyrolactone (1,4-BL) stimulated an improvement in bitespiramycin biological titer by 29% with a tiny influence on concentration of major component. Moreover, the mechanism of 1,4-BL stimulating effect was preliminarily explored by the analyses of three key enzyme activities, intracellular metabolite profiling and metabolic flux distribution. All results coordinately revealed that the extensive accumulation of methylmalonyl-CoA and acetyl-CoA was the direct reason for the enhanced bitespiramycin biosynthesis. This study would provide theoretical and technical basis for the application of 1,4-BL addition strategy to industrial bitespiramycin production.

The 14- and 16-membered macrolide antibiotics are an important structural class. Ubiquitously produced by a number of bacterial strains, namely actinomycetes, purification and structure elucidation of the wide array of analogs is challenging, both for discovery efforts and methodologies to monitor for byproducts, metabolites, and contaminants. Collision-induced dissociation mass spectrometry offers an attractive solution, enabling characterization of mixtures, and providing a wealth of structural information. However, interpretation of these spectra can be difficult. We present a study of 14- and 16-membered macrolide antibiotics, including MS n analysis for unprecedented depth of coverage, and complimentary analysis with D 2 O and H 2 18 O labeling to elucidate fragmentation mechanisms. These analyses contrast the behaviors of varying classes of macrolides and highlight how analogues can be identified in relation to similar structures, which will provide utility for future studies of novel macrolides, as well as impurities, metabolites, and degradation products of pharmaceuticals. Graphical Abstract.

Background: Bitespiramycin (BT) is produced by recombinant spiramycin (SP) producing strain Streptomyces spiramyceticus harboring a heterologous 4″-O-isovaleryltransferase gene (ist). Exogenous L-Leucine (L-Leu) could improve the production of BT. The orf2 gene found from the genomic sequence of S. spiramyceticus encodes a leucine-responsive regulatory protein (Lrp) family regulator named as SSP_Lrp. The functions of SSP_Lrp and L-Leu involved in the biosynthesis of spiramycin (SP) and BT were investigated in S. spiramyceticus., Results: SSP_Lrp was a global regulator directly affecting the expression of three positive regulatory genes, bsm23, bsm42 and acyB2, in SP or BT biosynthesis. Inactivation of SSP_Lrp gene in S. spiramyceticus 1941 caused minor increase of SP production. However, SP production of the ΔSSP_Lrp-SP strain containing an SSP_Lrp deficient of putative L-Leu binding domain was higher than that of S. spiramyceticus 1941 (476.2 ± 3.1 μg/L versus 313.3 ± 25.2 μg/L, respectively), especially SP III increased remarkably. The yield of BT in ΔSSP_Lrp-BT strain was more than twice than that in 1941-BT. The fact that intracellular concentrations of branched-chain amino acids (BCAAs) decreased markedly in the ΔSSP_Lrp-SP demonstrated increasing catabolism of BCAAs provided more precursors for SP biosynthesis. Comparative analysis of transcriptome profiles of the ΔSSP_Lrp-SP and S. spiramyceticus 1941 found 12 genes with obvious differences in expression, including 6 up-regulated genes and 6 down-regulated genes. The up-regulated genes are related to PKS gene for SP biosynthesis, isoprenoid biosynthesis, a Sigma24 family factor, the metabolism of aspartic acid, pyruvate and acyl-CoA; and the down-regulated genes are associated with ribosomal proteins, an AcrR family regulator, and biosynthesis of terpenoid, glutamate and glutamine., Conclusion: SSP_Lrp in S. spiramyceticus was a negative regulator involved in the SP and BT biosynthesis. The deletion of SSP_Lrp putative L-Leu binding domain was advantageous for production of BT and SP, especially their III components.

Rationale: Toxoplasma gondii is distributed worldwide, infecting a large population. It can cause focal necrotic retinitis or retinochoroiditis in the human eyes and is one of the most common causes of posterior uveitis., Patient Concerns: A 68-year-old patient with normal immunity was complained about blurred vision and black shadow in the right eye for 1 week., Diagnoses: Combined Yellow-and-white bulged lesions in the fundus of the right eye with the Goldmann-Witmer coefficient = 2 and based on the serological indicators, we considered the diagnosis of T. gondii infection-induced retinochondritis., Interventions: Acetylspiramycin 0.4 QID × 3 weeks, concussive 20 days treatment after 3 days, for a total of 3 months, prednisone 20 mg/day with a weekly reduction of 5 mg for 1 month., Outcomes: After oral acetylspiramycin, topical and systemic corticosteroids for 3 months, the retinal lesions were scarred, and inflammation of the anterior chamber and vitreum disappeared. After a 9-month follow-up, the visual acuity was 0.6, and no active lesions were observed in the fundus., Lessons: The immunocompetent elderly who are in contact with domestic cats may have an opportunistic infection with toxoplasmosis leading to primary retinochoroiditis. Prompt diagnosis and effective treatment can get a good clinical prognosis.

The antimicrobial activity of bitespiramycin (BT) against Chlamydia trachomatis (Ct), Chlamydia pneumoniae (Cp), Ureaplasma urealyticum (Uu), and Mycoplasma pneumoniae (Mp), was compared with those of azithromycin (AZM) and acetylspiramycin (AT-SP) in vitro. Furthermore, the anti-Mp activities of BT and AZM were evaluated in a hamster model. The activities of BT in vitro were similar to those of AZM but were more effective than those of AT-SP. BT effectively inhibited Mp infection at a dose of 200mg/kg in a hamster model., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Carbomycins are 16-membered macrolide antibiotics produced by Streptomyces thermotolerans ATCC 11416 T . To characterize gene cluster responsible for carbomycin biosynthesis, the draft genome sequences for strain ATCC 11416 T were obtained, from which the partial carbomycin biosynthetic gene cluster was identified. This gene cluster was approximately 40 kb in length, and encoding 30 ORFs. Two putative transcriptional regulatory genes, acyB2 and cbmR, were inactivated by insertion of the apramycin resistance gene, and the resulting mutants were unable to produce carbomycin, thus confirming the involvement of two regulatory genes in carbomycin biosynthesis. Overexpression of acyB2 greatly improved the yield of carbomycin; however, overexpression of cbmR blocked carbomycin production. The qPCR analysis of the carbomycin biosynthetic genes in various mutants indicated that most genes were highly expressed in acyB2-overexpressing strains, but few expressed in cbmR-overexpressing strains. Furthermore, acyB2 co-expression with 4″-isovaleryltransferase gene (ist), resulted in efficient biotransformation of spiramycin into bitespiramycin in S. lividans TK24, whereas ist gene regulated by acyB2 and cbmR would cause the lower efficiency of spiramycin biotransformation. These results indicated that AcyB2 was a pathway-specific positive regulator of carbomycin biosynthesis. However, CbmR played a dual role in the carbomycin biosynthesis by acting as a positive regulator, and as a repressor at cbmR high expression levels.

Spiramycin is a multicomponent antibiotic, and different components have different antibacterial activities. In Streptomyces spiramyceticus 16-10-2, spiramycin II and spiramycin III (SPMII and SPMIII) are the main components, while spiramycin I (SPMI) needs to be controlled below 12%. Based on this, the influences of Al 3+ on total spiramycin titer and components were investigated in this work. Those experiments were mainly performed in 15 L fermentor and Al 3+ made a great improvement in spiramycin titer. The optimal adding concentration and adding time of Al 3+ were 0.32 g/L at 12 hr. Under this condition, spiramycin titer was increased by 19.51% compared with the control. Moreover, the percentage of SPMII and SPMIII was increased by 7.14%. At the same time, the time of mycelia autolysis was lengthened. In addition, the specific activities of acetyl-CoA synthetase, acetate kinase, acetylphosphotransferase, and acylating enzyme were much higher than those of control. The content of acetic acid and succinic acid was beyond 3 and 4.5 times than that of control, respectively.

Huisgen cycloaddition allowed obtaining of novel triazole-bridged antibiotics (6-16) with the reconstructed C(5) arm of spiramycin. (1)H-(1)H NOESY couplings indicated the structure of novel derivatives in solution and demonstrated that the rebuilt C(5) arm is slightly differently oriented relative to the aglycone part if compared to that of spiramycin (1). Combined analysis of biological data together with experimentally determined lipophilicity (clogP) and solubility show the importance of the chemical nature of the newly introduced triazole C(5) arm in the presence of attractive antibacterial and anticancer potency. The most cytotoxic active triazole conjugates having a hydrophobic and bulky C(5) arm showed higher selectivity toward cancer cell lines (HeLa, KB, MCF-7, Hep-G2, and U87) relative to HDF normal cells than that of the parent spiramycin. Our studies have demonstrated that the aldehyde group is not crucial for the presence of interesting antibacterial [MIC(S. pneumoniae) ∼ 1.2 μM] and anticancer [IC50(HepG2) ∼ 6 μM] properties of 16-membered lactone macrolides based on spiramycin's aglycone.

Cryptosporidiosis affects humans of all ages, particularly malnourished children and those with compromised immune systems such as HIV/AIDS. This study investigated the therapeutic effects of acetylspiramycin and garlicin on Cryptosporidium infection in institutionalized male drug users receiving rehabilitative treatment. Examination of stool specimens from 903 drug users via modified acid-fast bacilli staining resulted in 172 positive cases. Among them 151 subjects consented to participate in a randomized trial of acetylspiramycin and garlicin in four groups: acetylspiramycin plus garlicin, acetylspiramycin only, garlicin only, and placebo control. The cryptosporidiosis rate was higher in younger subjects with longer drug use history than subjects who are older with shorter history of drug use. After two segments of treatments, 76.2% of the cases achieved negative test results, with the four groups achieving the rates of 92.1%, 76.7%, 72.2%, and 61.8%, respectively (χ(2) = 9.517, P = 0.023). These results indicate clinical potential of garlicin in conjunction with acetylspiramycin in treating cryptosporidiosis., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

4"-O-isovaleryltransferase gene (ist) was regulated by positive regulatory genes of midecamycin 4"-O-propionyltransferase gene (mpt) in Streptomyces lividans TK24. A BamH I ~8.0 kb fragment from Streptomyces mycarofaciens 1748 was proved that it contained mpt gene and linked with two positive regulatory genes, orf27 and orf28. Orf of mpt was replaced by orf of ist and linked with two regulatory genes or orf27 single, and individually cloned into the vectors pKC1139 or pWHM3 (high copy number), and then transformed into S. lividans TK24. The levels of mpt and ist expression were evaluated by the bio-tramsformation efficacy of spiramycin into 4"-O-acylspiramycins in these transformants. The results showed that 4"-O-isovalerylspiramycins could be detected only in the transformants containing the plasmids constructed with pWHM3. The efficacy of bio-transformation of the transformants containing two regulatory genes was higher than that of orf27 single. So, the positive regulatory genes system of mpt gene could enhance ist gene expression.

The production of secondary metabolites with antibiotic properties is a common characteristic to Bacillus spp. These metabolites not only have diverse chemical structures but also have a wide range of bioactivities with medicinal and agricultural interests such as antibiotic. Bacillus sp. fmbJ has been found to produce lipopeptides fengycin and surfactin in accordance with our previous report. In this study, another antimicrobial substance was separated and purified from the culture supernatant of strain fmbJ using the silica gel column chromatography and preparative reversed-phase high-performance liquid chromatography. By means of electrospray ionization mass spectroscopy, infrared spectroscopy, and nuclear magnetic resonance, the antagonistic compound was determined to be 4″-isovaleryl-spiramycin III with the molecular weight of 982 Da. This report is the first to introduce the finding of spiramycin produced from Bacillus sp. The study provides a novel source for the production of spiramycin in pharmaceutical industries.

Objective: To explore an effective therapy for pregnant Toxoplasma gondii infection by using acetyl spiramycin combined with azithromycin., Methods: ELISA and PCR were used to diagnose and evaluate the therapy efficiency to toxoplasmosis in pregnant women., Results: The serological test showed that the positive rates of specific antibodies IgM and IgG to Toxoplasma gondii in 285 pregnant women were 1.05% (3/285) and 5.97% (17/285), respectively. All the 3 cases of serum IgM positive pregnant women received the amniotic fluid PCR tests for Toxoplasma gondii DNA and 2 were positive, and they received spiramycin combined with azithromycin. After the therapy, their serum IgM antibody specific to Toxoplasma gondii and positive amniotic fluid PCR test for Toxoplasma gondii DNA turned to be negative., Conclusion: Acetyl spiramycin in combination with azithromycin is effective in the treatment of pregnant toxoplasmosis.

Macrolide antibiotics are broad-spectrum, with activity against a range of Gram-positive, Gram-negative organisms and some anaerobes. The components of macrolide antibiotics are generally complicated. Therefore, it is very important to establish impurity profiles of these antibiotics to ensure their safety and process control. Compared with classical methods, the liquid chromatography-mass spectrometry method is particularly advantageous to characterize minor components at trace levels in terms of sensitivity, efficiency and selectivity, thus more and more widely used in establishments of impurity profiles. In this study, the general approaches to characterize minor components in complex pharmaceutical matrix, fragmentation pathways of 14- and 16-membered macrolide antibiotics and the establishment of the impurity profile of acetylspiramycin were given to provide valuable enlightenments to establish the impurity profiles of pharmaceutical products.

Reversed-phase liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was used to characterize the components of bitespiramycin, a group of 4"-acylated spiramycins produced by bioengineered strains. In total 38 components were characterized in commercial samples, including 12 impurities that had never been reported before and 12 other that were partially characterized. The structures of these unknown compounds were deduced by comparison of their fragmentation patterns with those of known major components. Their ultraviolet spectra were used to confirm the presence of an α-, β-, γ-, δ-unsaturated butadiene in the macrocyclic lactone. Compared with the classical method, LC/ESI-MS/MS is particularly advantageous in terms of sensitivity and efficiency to characterize minor components at trace levels in multi-component antibiotics., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Context: Neuropathic pain in patients with cancer can be difficult to treat effectively., Objectives: The purpose of the study was to determine safety and efficacy of KRN5500, a novel, spicamycin-derived, nonopioid analgesic agent, in patients with advanced cancer and neuropathic pain of any etiology., Methods: The study was a Phase 2a, multicenter, double-blind, placebo-controlled, dose escalation clinical trial. Patients with refractory neuropathic pain and advanced cancer were randomly assigned 2:1 to receive a maximum of eight single escalating doses of KRN5500 or placebo, ranging from 0.6 to 2.2 mg/m(2). The primary objective was safety and tolerability. The secondary objective was efficacy, measured by change in average pain intensity on a 0-10 numeric rating scale administered one week after the patient's final dose., Results: Nineteen patients received treatment (KRN5500 n=12; placebo n=7). The most frequently reported adverse events were gastrointestinal symptoms, which were more frequent and severe with KRN5500 than placebo; two (17%) KRN5500 patients discontinued the study because of nausea and vomiting. At study endpoint, KRN5500 exhibited a significant median decrease in pain intensity from baseline of 24% compared with 0% for placebo (P=0.03). The median for largest weekly reduction in target pain intensity was 29.5% for KRN5500 and 0% for placebo patients (P=0.02)., Conclusion: This proof-of-concept study for KRN5500 in patients with advanced cancer and any type of neuropathic pain found gastrointestinal adverse events to be the predominant safety concern. The results also provided the first indication of clinical and statistical efficacy in reducing pain intensity., (Copyright © 2012 U.S. Cancer Pain Relief Committee. Published by Elsevier Inc. All rights reserved.)

Bitespiramycin (BT), a multi-component antibiotic consisted mainly of 4"-isovalerylspiramycin I, II and III, is produced by Streptomyces spiramyceticus WSJ-1, a recombinant spiramycin-production strain that harbored the 4"-O-acyltransferase gene (ist) from Streptomyces mycarofaciens 1748, which could isovalerylate the 4"-OH of spiramycin. To eliminate the production of components 4"-isovalerylspiramycin II and III, therefore reducing the component complexity of BT, inactivation of the sspA gene, which encodes the 3-O-acyltransferase responsible for the acylation of spiramycin I to spiramycin II and III, was performed in Streptomyces spiramyceticus WSJ-1, by in-frame partial deletion. The resulting strain, Streptomyces spiramyceticus WSJ-2, is a 4"-isovalerylspiramycin-I-producing strain as expected.

Investigation of acetylspiramycin (ASPM) and its related substances was carried out using a reversed-phase liquid chromatography/tandem mass spectrometry method. The identification of impurities in the ASPM complex was performed with a quadrupole ion trap mass spectrometer, with an electrospray ionization (ESI) source in the positive ion mode which provides MS(n) capability. A total of 83 compounds were characterized in commercial samples, among which 31 impurities that had never been reported and 31 partially characterized impurities were deduced using the collision-induced dissociation (CID) spectra of major ASPM components as templates. Most of the major impurities arise from the starting materials and the synthesis process. This work provides very useful information for quality control of ASPM and evaluation of its synthesis process., (Copyright © 2010 Elsevier B.V. All rights reserved.)

LC-hyphenated techniques were applied to the on-line identification of isovalerylspiramycin I (isp I), a spiramycin-like macrolide in the crude extract of fermentation broth from a genetically engineered strain of S. spiramyceticus F21. In the structural characterization of the large molecular secondary metabolite of isp I, LC-DAD-UV-ESI-MS(n) analysis played a crucial role, and stop-flow LC-(1)H NMR measurement, with bitespiramycin used as reference, was a valuable complement approach. This rational approach proved to be an efficient means for the rapid and accurate structural determination of known microbial secondary metabolites, by which targeted isolation of component(s) of interest can be subsequently performed for further biological and pharmacological studies in drug development., (Copyright 2009 Elsevier B.V. All rights reserved.)

4''-O-isovalerylspiramycins are the major components of bitespiramycin complex consisting of a group of 4''-O-acylated spiramycins. The availability of isovaleryl group, usually in vivo derived from leucine, one of the branched-chain amino acids, affects the content of isovaleryispiramycin significantly. In this study, the effect of glucose on the activity of branched-chain alpha-keto acid dehydrogenase (BCKDH), which catalyzed the rate-limiting as well as the first irreversible reaction oxidative decarboxylation for branched-chain amino acids degradation, and isovaleryispiramycin biosynthesis was investigated. In the initial glucose concentration experiment, when the residual glucose concentration in the medium declined to 2-4 g/L, the BCKDH activity rose rapidly, and glucose deprivation and the summit of BCKDH activity appeared nearly at the same time. After a delay of about 6 h, the maximal isovalerylspiramycin content was observed. However, the shortage of glucose at the later production phase resulted in the marked decrease in BCKDH activity and isovaleryispiramycin content. In the fermentation in a 50 L fermentor, glucose feeding at the late production phase helped to maintain the residual glucose concentration between 0 and 1 g/L, leading to the high level of BCKDH activity and thus isovalerylspiramycin content. These suggested that glucose concentration could be used as a key parameter to regulate BCKDH activity and isovaleryispiramycin biosynthesis in the bitespiramycin production.

The paper is to report the establishment of an HPLC method for determination of the components of bitespiramycin and its products, and to evaluate the components profile of bitespiramycin and its related products. Liquid chromatography combined with mass spectrometry (LC-MS) was used to identify the nine major components of the reference substance of bitespiramycins. The nine components of bitespiramycins and its products have been quantified by HPLC with gradients elution methods. The content of the component of 4"-O-isovalerylspiramycin III was not less than 35%, the content of the components of 4"-O-isovalerylspiramycin (I + II + III) was not less than 60%, and the contents of the nine components of bitespiramycin were not less than 80%, separately. In addition to the components mentioned above, there also exists some other impurities, however, with lower contents. This gradient elution HPLC method reported by this paper is considered to be suitable for the quality control of bitespiramycin.

Bitespiramycin, a group of 4''-O-acylated spiramycins with 4''-O-isovalerylspiramycins as the major components, is produced by recombinant spiramycin-producing strain Streptomyces spiramyceticus harboring a 4''-O-acyltransferase gene from a carbomycin-producing strain S. mycarofaciens 1748. The effects of leucine feeding on the bitespiramycin fermentation, especially the synthesis of isovalerylspiramycin components, were investigated. The experiment was initially performed in flask culture under the condition of feeding 15.4 mmol/l of leucine at 72 h fermentation, and the culture without leucine feeding was used as control. When 15.4 mmol/l leucine was fed at 72 h, 51.3 +/- 0.33% total isovalerylspiramycins was recorded compared to 40.9 +/- 0.26% under the control condition after 96 h of fermentation. The improvement of total isovalerylspiramycin content was further achieved in 15 l fermentation when 15.4 mmol/l of leucine was supplemented from 65 to 72 h. These results indicated that isovaleryl group derived from leucine catabolism could act as the precursor of the 4'' side chain of bitespiramycin, which profoundly enhanced the synthesis of isovalerylspiramycins in the bitespiramycin complex.

A simple confirmatory method for the determination of spiramycin and its metabolite neospiramycin in raw milk using LC ESI MS/MS is presented. Macrolide residues in raw milk were extracted by ACN, and sample extracts were further cleaned up and concentrated using SPE cartridges. Both spiramycin and neospiramycin were protonated in electrospray positive ion mode to form singly and/or doubly charged pseudomolecular ions. Data acquisition was achieved using multiple reaction monitoring, i.e., two transitions, for quantification and confirmation. Matrix-matched standard calibration curves were utilized to achieve the best accuracy for the method. The method performance was evaluated according to both a conventional validation procedure and a designed experimental result. The measurement uncertainty arising from accuracy and precision was estimated. The method accuracy, expressed as a percentage of an overall recovery, was from 82.1 to 108.8%, and its intermediate precision was less than 20%. LC/ESI-MS/MS method LODs (S/N greater, not dbl equals 3:1) of spiramycin and neospiramycin were less than 1.0 microg/kg.

Objective: To explore the effect of usnic acid on Toxoplasma gondii tachyzoites in vitro., Methods: There are four groups named as (+)-usnic acid group, acetylspiramycin group, DMSO group and normal saline group. Groups of (+)-usnic acid and acetylspiramycin were further divided into 4 subgroups with final concentration of 5, 10, 25, 50 microg/ml respectively. Normal saline group and DMSO group were respectively given equal volume normal saline and 1% DMSO. Each group have 15 parallel tubes with 1 ml (1 x 10(6)/ml) T. gondii tachyzoites aqueous suspension. At 1 h, 2h and 4 h after drug treatment, tachyzoites were counted by light microscope with 0.4% Trypan blue staining. Tachyzoites in aqueous suspension was collected, and washed 3 times by PBS solution. Normal mice were inoculated intraperitoneally and observed for three generations. The cultivated rat cardiofibroblasts were then infected in vitro with T. gondii tachyzoites. At the same time, rat cardiomyocytes invasion by T. gondii tachyzoites was investigated., Results: At 4 h treated by 10, 25 and 50 microg/ml (+)-usnic acid, 100% T. gondii tachyzoites were stained. Some tachyzoites were swelling, blunt or round in the two ends; and granules appeared in the cytoplasm, the nuclei were deep stained. The changes of tachyzoites in acetylspiramycin group were similar to (+)-usnic acid group, 100% T. gondii tachyzoites were stained in 50 microg/ml acetylspiramycin subgroup. In inoculation tests, mice died at 8th to 9th days in 5 microg/ml (+)-usnic acid subgroup and numerous tachyzoites were detected in ascites. However, most mice survived to be killed in the other (+)-usnic acid subgroups and the tachyzoites were not found in ascites. All mice in acetylespirmycin groups died at 6th to 8th days after inoculation and many tachyzoites or pseudocysts were observed in mice ascites. In infecting cell tests, the cultivated rat cardiofibroblasts were infected in vitro by the tachyzoites after treated with 5 microg/ml (+)-usnic acid for 4 h, and pseudocysts were formed in infected cells. It was negative in the other subgroups of (+)-usnic acid. But the cultivated rat cardiofibroblasts were infected to varying degree in acetylspiramycin groups, normal saline group and DMSO group., Conclusion: (+)-Usnic acid has a remarkable effect on T. gondii tachyzoites.

An efficient multiresidue method for the simultaneous determination of metronidazole (MET) and spiramycin (SPY) in tilapia fish muscle, based on high performance liquid chromatography with UV detection (HPLC-UV), has been developed. The drugs were extracted with 0.2% orthophosphoric acid-methanol (6:4), and the extracts were cleaned up on a solid phase extraction cartridge, C18 Sep-Pak light column. The LC separation was performed on a RP stainless-steel C-18 analytical column (150 mm x 4.6 mm, 5 microm) with a gradient elution system of 0.05 M phosphate buffer adjusted to pH 2.4-acetonitrile as the mobile phase at the flow rate of 1.0 ml min(-1). A wavelength programming was applied for the UV detection of the analytes. The method not only enabled the determination of the parent drugs, MET and SPY, but also permitted the determination of their metabolites, hydroxymetronidazole (HMET) and neospiramycin (NSPY). The calibration graphs for each drug were rectilinear in the range of 0.005-1.000 microg g(-1) for MET and HMET and 0.025-1.000 microg g(-1) for SPY and NSPY. With this method, the cited drugs with their metabolites were determined in fortified fish muscle tissues at levels of 0.025, 0.1 and 1.0 microg g(-1) with good accuracy and precision. LOD and LOQ obtained for each drug were as follows: 0.002 and 0.005 microg g(-1) for MET and HMET and 0.005 and 0.025 microg g(-1) for SPY and NSPY. Utilization of the method to successfully analyze tilapia fish muscle samples incurred with MET and SPY was described.

Kernel independent component analysis (KICA), a kind of independent component analysis (ICA) algorithms based on kernel, was preliminarily investigated for blind source separation (BSS) of source spectra profiles from troches. The robustness of different ICA algorithms (KICA, FastICA and Infomax) was first checked by using them in the retrieval of source infrared (IR), ultraviolet (UV) and mass spectra (MS) from synthetic mixtures. It was found that KICA is the most robust method for retrieval of source spectra profiles. KICA algorithm is subsequently adopted in the analysis of diffuse reflection IR of acetylspiramycin (ASPM) troches. It is observed that KICA is able to isolate the theoretically predicted spectral features corresponding to the ASPM active components, excipients and other minor components as different independent (spectral) component. A troche can be authenticated and semi-quantified using the estimated ICs. KICA is an useful method for estimation of source spectral features of molecules with different geometry and stoichiometry, while features belonging to very similar molecules remain grouped.

18-amino-4''-O-benzoyl-4'''-N-demethyl-18-deoxospiramycins were designed and synthesized. Synthetic strategy involved selective demethylation of the dimethylamino group in forosamine, benzoylation of the hydroxyl group at the C4'' position and reductive N-amination of the formyl group. Antibacterial characteristics of spiramycin derivatives were tested. The derivatives exhibited promising activity against drug-resistant bacterial strains.

Aim: To propose a novel polarographic method for the determination of acetylspiramycin (ASPM) is proposed., Methods: In 0.1 mol x L(-1) NH4Cl-NH3 x H2O (pH 8.9) buffer containing dissolved oxygen, ASPM yielded a sensitive parallel catalytic hydrogen wave with the peak potential of -1.63 V (vs SCE) by single sweep polarography., Results: The 2nd order derivative peak currents (i(p)") of the parallel catalytic hydrogen waves of ASPM showed a linear relationship with its concentrations in the range from 1.74 x 10(-3) microg x mL(-1) to 3.84 microg x mL(-1) (r = 0.9979, n=13). Its detection limit was 5.80 x l0(-4) microg x mL(-1) (3sigma) and RSD (n=13) was 1.24% at the concentration level of 0.871 microg x mL(-1)., Conclusion: The proposed method could be applied to the determination of ASPM in ASPM tablets.

Bitespiramycin is a macrolide antibiotic consisting of a mixture of some nine spiramycin ester derivatives. It has a similar spectrum of antibiotic activity to that of spiramycin but has superior pharmacokinetic properties. In this study, a rapid and facile LC/ESI-MSn method was applied to study the metabolism of bitespiramycin in rat following a single oral dose (80 mg kg-1). Concentrations of parent drug constituents and metabolites were determined in plasma, urine, feces and bile. Concentrations of parent drug constituents and metabolites in plasma were very low. In urine, feces and bile, parent drug constituents and 38 metabolites were identified on the basis of their chromatographic and mass spectrometric properties. The identity of 17 metabolites was confirmed by comparison with reference substances. The principal metabolites were the corresponding spiramycins formed by hydrolysis of the 4''-(3-methylbutanoate) groups. Other important metabolic pathways were: hydrolytic loss of the forosamine and mycarose sugars; aldehyde reduction; cysteine conjugation of the aldehyde group; and hydrolysis of the lactone ring. Products formed by lactone ring opening were found only in urine, and those formed by aldehyde reduction were found only in feces. Aldehyde reduction and hydrolytic loss of forosamine represent novel biotransformation pathways for spiramycin derivatives.

Objective: To analyze the mechanisms of macrolide resistance in Streptococcus pneumoniae from children in Beijing., Methods: The MICs of penicillin and erythromycin were determined by the E-test methods for 200 Streptococcus pneumoniae isolates collected from 2002 to 2003 at Beijing Children's Hospital. MICs of azithrhomycin, clarithromycin, acetylspiramycin and clindamycin for 147 erythromycin-resistant isolates were detected by the agar dilution methods. For phenotyping, macrolide resistance induction tests were used in erythromycin-resistant isolates. PCR was used to determine the presence of the erythromycin-resistant genes., Results: Of 200 Streptococcus pneumoniae isolates, 89.5% were resistant to erythromycin. In 147 erythromycin-resistant isolates, resistance rates were as follows: azithromycin, 100%; clarithromycin, 100%; acetylspiramycin, 95.2%; and clindamycin, 95.9%. The most common macrolide resistance phenotype was the cMIS phenotype (95.9%), 1.4% had the iMLS phenotype and 2.7% the M phenotype. Erythromycin-resistant isolates were characterized for the underlying resistance genotype, with 79.6% having the ermB genotypes, 17.7% having both ermB and mefA, 2.7% having the mefA, and none having neither ermB nor mefA genotypes., Conclusions: The rates of carriage of macrolide-resistant Streptococcus pneumoniae by children were high in Beijing during 2002 - 2003. cMLS was the most prevalent phenotype among erythromycin-resistant Streptococcus pneumoniae isolates, and ribosomal modification (ermB gene coded) was the main resistance mechanism against macrolides in Beijing region.

Bitespiramycin is a novel antibiotic containing a number of 4''-acylated spiramycin derivatives (isovalerylspiramycins I-III, butanoylspiramycin III, propanoylspiramycin III and acetylspiramycin III) as major components. These spiramycin derivatives are susceptible to degradation in acid solution. Liquid chromatography-ion trap mass spectrometry (LC/MS(n)) was used to study the degradation of these spiramycin derivatives in simulated gastric fluid at 37 degrees C. All derivatives degraded by first-order reactions for which rate constants (k) and half-lives (t(1/2)) were calculated. Acyl groups at position 3 had less effect on acid-stability of spiramycin derivatives than acyl groups at position 4''. The introduction of 4''-acyl groups enhanced the acid-stability of spiramycin derivatives and altered the degradation pathway in simulated gastric fluid such that loss of forosamine rather than loss of mycarose becomes the major degradation pathway.

Aim: To investigate the tissue distribution of bitespiramycin (BSPM) and spiramycin (SPM) in rats., Methods: Liquid chromatographic-mass spectrometric assay was applied for the determination of three major components (isovalerylspiramycins, ISV-SPMs) of BSPM and their major active metabolites (SPMs) in rat tissues and plasma after an oral dose of bitespiramycin, as well as SPMs., Results: High levels of drug concentrations were observed in most tissues, especially in the liver, stomach, intestine, spleen, lung, womb, and pancreas. BSPM persisted long time in many rat tissues such that the drug concentration in spleen was 69.4 nmol/g at 60 h post-dose and it was still above the minimum inhibitory concentration of many susceptible pathogens. At 2.5 h post-dose, the total concentrations of ISV-SPMs and SPMs achieved in tissues were from 6 to 215 times higher than the corresponding concentrations in plasma. At 2.5 h post-dose, the mean C(t)/C(p) of BSPM appeared to be 2- or 3-fold those of SPM in most tissues. The tissue to plasma concentration ratios following oral dose of BSPM were higher than those of SPM in most tissues. The drug was not detected in brain and testis after a single dose of BSPM and SPM., Conclusion: Both BSPM and SPM penetrate into rat tissues well and BSPM has higher tissue affinity than SPM.

Aim: To establish a new and simple flow injection method for the rapid determination of acetylspiramycin (ASPM)., Methods: ASPM was determined by chemiluminescence (CL) method combined with flow injection (FI) technology, which was based on the inhibitive effect of ASPM on the chemiluminescence reaction of the luminol-K3Fe (CN)6 system., Results: The decrease of chemiluminescence intensity was proportional to the logarithm of ASPM concentration (0.1-100) microgram.L-1, the detection limit was 40 ng.L-1 (3 sigma). The whole process, including sampling and washing, could be completed in 0.5 min with a RSD less than 3.0% (n = 5)., Conclusion: The FI-CL method is of both high sensitivity and good selectivity giving a throughput of 120 h-1. The proposed method was applied successfully to the determination of ASPM in pharmaceutical preparations and human urine without any pre-treatment. It was found that the ASPM concentration reached its maximum after being orally administrated for two hours.

Aim: To study the activities of in vitro resistance to the tachyzoite of Toxoplasma gondii by murine lymphocytes., Methods: The rat's splenocyte culture method was used to observe the effects of lymphocytes themselves and lymphocytes together with Mandelic( MA) on the invasion and proliferation of T. gondii in lymphocytes. At the same time acetylspiramycin was used as positive control., Results: As compared with other somatic cells, the lymphocytes invaded by T. gondii still inhibited and killed the toxoplasma organisms in the presence of immunity, the effect safety dose of MA on inhibition of the invasion of T. gondii was not notable while the inhibition of the proliferation of T. gondii in lymphocytes was remarkable., Conclusion: Cell-mediated immunity(CMI) was an important factor that host resists the T. gondii infection. So, we should pay attention to improving the organism's CMI and take proper medicine so as to enhance the effect of lymphocyte's resistance to T. gondii.

Toxoplasma infection during pregnancy is widely treated with oral spiramycin to reduce the risk of congenital toxoplasmosis in the infant. Failures of therapy have been observed, however. In this study, a sensitive high-performance liquid chromatography technique was used to measure concentrations of spiramycin and neospiramycin, one of the major metabolites of spiramycin, in maternal serum and amniotic fluid. Samples were obtained from 18 women who underwent amniocentesis for polymerase chain reaction (PCR) diagnosis of fetal infection 5-109 days following the prescription of spiramycin therapy (3 g/day). Concentrations of spiramycin and neospiramycin in both serum and amniotic fluid were highly variable, ranging from nondetectable values to 1 microg/ml. None of the concentrations measured were within the range reported to inhibit growth of the parasite in vitro. Consistent with previous reports, part of the observed variability in maternal and fetal drug concentrations could be explained by individual differences in several pharmacokinetic parameters: intestinal absorption, tissue distribution, cellular uptake, metabolism, transfer across the placenta, drug accumulation in fetal tissue, and maternal and fetal drug elimination. The heterogeneity of the data could also be related to differences in patient compliance with the medication prescribed. By addressing factors that could impair adequate treatment of toxoplasmosis during pregnancy, the data presented call for a larger-scale controlled study to determine individual and diurnal variations in maternal drug levels, patient compliance, and outcomes of the offspring. The activity of neospiramycin against Toxoplasma gondii should be assessed.

Shengjimycin is a group of 4"-acylated spiramycins with 4"-isovalerylspiramycin as the major component, produced by recombinant S. spiramyceticus F21 harboring a 4"-O-acyltransferase gene from S. mycarofaciens 1748. A stable bioengineered strain of Streptomyces spiramyceticus WSJ-1 was constructed by integrating the 4"-O-acyltransferase gene (ist) by homologous recombination into the chromosome of the spiramycin-producing strain S. spiramyceticus F21. In this construction, a Streptomyces/E. coli shuttle plasmid pKC1139 (AmR) was used as the vector with the tsr gene used as selection marker for homologous recombination. The constructed strain, S. spiramyceticus WSJ-1,was genetically stable in production titer and proportion of components of shengjimycin as well as in maintaining the tsr selective marker when grown without selection. Southern hybridization confirmed the integrated status of the ist gene in the host genome. The production and the proportion of major component of 4"-isovalerylspiramycin of S. spiramyceticus WSJ-1 was also improved comparing with the strain harboring an autonomous plasmid -S. spiramyceticus F21/pIJ680(311) as shown by HPLC analysis. Physiological studies indicated that increase of the VDH ( valine dehydrogenase ) and LDH ( leucine dehydrogenase ) activities of WSJ-1 may be involved in this improvement.

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of tilmicosin, tylosin, spiramycin, and its major metabolite neospiramycin was developed that is suitable for porcine, bovine, and poultry muscles. Macrolide residues were extracted from muscle with acetonitrile, fat was removed by liquid-liquid extraction with isooctane, and the extract was then cleaned on Bond Elut C18 cartridges. The HPLC separation was performed on an Inertsil ODS3 C18 column (150 x 4 mm) with 0.05% trifluoroacetic acid-acetonitrile in a gradient mode. Two different chromatographic gradients were used for tilmicosin-tylosin and spiramycin-neospiramycin, and the detection wavelengths were 287 and 232 nm, respectively. The method was validated from 1/2 the maximum residue limit (MRL) to 4 times the MRL with pork muscle samples. Mean recoveries were 60, 63.5, 51, and 42% for tilmicosin, tylosin, spiramycin, and neospiramycin, respectively. The detection limits are 15 micrograms/kg for tilmicosin and tylosin, 30 micrograms/kg for spiramycin, and 25 micrograms/kg for neospiramycin. Linearity, precision, and accuracy of the method were also tested.

An interlaboratory study has been performed to determine the relative potencies of spiramycins (SPMs) I, II and III by diffusion or/and turbidimetric assays with Bacillus subtilis or Staphylococcus aureus as the test organisms. Six laboratories from three countries participated. Experimental procedures were according to the European Pharmacopoeia, 3rd ed. The activity of SPM I is markedly higher than that of SPM II and III. By diffusion, the activities of SPM II and III relative to SPM I were found to be 57 and 72%, respectively. The interlaboratory relative standard deviations (RSD) varied from 3.6 to 16.3%. By turbidimetry, the activities of SPM II and III relative to SPM I were found to be 45 and 52%, respectively. The interlaboratory RSD values varied from 2.6 to 7.7%. The results of the study were analyzed according to the ISO 5725-2 guidelines to determine the repeatability, the between-laboratory and the reproducibility variances of both methods.

A stable bioengineered strain of Biotechmycin (Streptomyces spiramyceticus WSJ-1) was constructed by integrating the 4"-isovaleryltransferase gene (ist) through homologous recombination into the chromosome of spiramycin-producing strain S.spiramyceticus F21. In this construction, a Streptomyces/E.coli shuttle plasmid pKC1139 (AmR) was used as the vector and tsr gene was inserted as the marker for selection of homologous recombination. This constructed strain, S.spiramyceticus WSJ-1, was genetically very stable in production titer and in the production of biotechmycin as well as in carrying tsr selective marker when grown without pressure. The fermentation of S.spiramyceticus WSJ-1 was also improved compared with the original strain harboring unintegrated plasmid. Southern hybridization confirmed the integrated status of the ist gene in the host genome.

The first synthesis of siderophore conjugates of two macrolide antibiotics, spiramycin 1 and neospiramycin 2, which are unable to penetrate the outer membrane of gram-negative bacteria are described. These novel conjugates were prepared by regioselective acylation of a hydroxyl function of 1 and 2 with a dihydroxybenzoic Fe(III) complexing ligand linked via a carboxyl group containing spacer to the macrolide antibiotics. The preliminary biological evaluation of these novel conjugates under standard and iron depleted conditions has shown that their antibacterial activity was comparable to that of spiramycin 1 and neospiramycin 2.

We report a case of dermatomyositis (DM) in a 15-year-old female with toxoplasmosis after ingestion of raw bovine liver. Facial erythema and cervical lymphadenopathy preceded myalgia and muscle weakness of the extremities. The diagnostic criteria of DM was fulfilled because of symmetrical and proximal dominant muscle weakness, elevation of myogenic enzyme (CPK, GOT, LDH, myoglobin, aldorase), myogenic pattern of electromyogram, skeletal muscle biopsy showing interstitial myositis with mild destruction of muscle fiber, and facial erythema. Immunological findings showed IgG anti-toxoplasma antibody to be 1340 IU/ml and IgM to be 7.0 (Cut off index 0.7), suggesting acute toxoplasmosis. Treatment with prednisolone for DM and acetylspiramycin for toxoplasmosis was successful. Toxoplasmosis should be considered as a possibility in patients with myositis.

In an attempt to gain information about the peptidyl transferase center at the peptide level we cross-linked the spiramycin derivative dihydrospiramycin to its functional binding site in the 70 S ribosome of Escherichia coli. In this manner ribosomal proteins S12, S14, L17, L18, L27 and L35 were found specifically affinity-labeled. Proteolytic fragmentation of these proteins, separation by C18 reversed-phase high performance liquid chromatography of the peptide mixtures, and subsequent sequence analysis of labeled peptides revealed peptide regions at positions Ala1-Lys9 and Tyr116-Lys119 of S12, Leu47-Asp53 of protein S14, Ser6-Lys35 of protein L17, Ala57-Lys63 of protein L18, Ala5-Lys18 and Val66-Lys71 of protein L27, and Thr5-Lys11 of protein L35. This approach is a valuable tool to characterize the binding site of spiramycin as well as the peptidyl transferase center at the molecular level.