Carrimycin ameliorates lipopolysaccharide and cecal ligation and puncture-induced sepsis in mice.

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-1ß), 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.

Study of Bitespiramycin Distribution in Rats and Cerebrospinal Fluid of Patients by a Sensitive LC-MS/MS Method with Rapid Sample Preparation.

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 coupled to tandem mass spectrometry method for the quantification of spiramycin and its active metabolite neospiramycin in milk of major and minor species: Validation using the accuracy profile.

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.

The regulatory genes involved in spiramycin and bitespiramycin biosynthesis.

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.

A preliminary study on the impact of exogenous A-Factor analogue 1,4-butyrolactone on stimulating bitespiramycin biosynthesis.

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.

Structure Elucidation of Macrolide Antibiotics Using MSn Analysis and Deuterium Labelling.

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 MSn analysis for unprecedented depth of coverage, and complimentary analysis with D2O and H218O 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.

Engineering of leucine-responsive regulatory protein improves spiramycin and bitespiramycin biosynthesis.

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.

Early diagnosis and successful treatment of acquired toxoplasmosis infectious retinochoroiditis: A case report.

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.

Antimicrobial activity of bitespiramycin, a new genetically engineered macrolide.

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.

Identification of two regulatory genes involved in carbomycin biosynthesis in Streptomyces thermotolerans.

Carbomycins are 16-membered macrolide antibiotics produced by Streptomyces thermotolerans ATCC 11416T. To characterize gene cluster responsible for carbomycin biosynthesis, the draft genome sequences for strain ATCC 11416T 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.

Influence of Al3+ on the titer of spiramycin and effective components in fermentor.

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 Al3+ on total spiramycin titer and components were investigated in this work. Those experiments were mainly performed in 15 L fermentor and Al3+ made a great improvement in spiramycin titer. The optimal adding concentration and adding time of Al3+ 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.

Synthesis, Antibacterial, and Anticancer Evaluation of Novel Spiramycin-Like Conjugates Containing C(5) Triazole Arm.

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.

Therapeutic effects of acetylspiramycin and garlicin on cryptosporidiosis among drug users.

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.

[Expression of 4"-O-isovaleryltransferase gene from Streptomyces thermotolerans in Streptomyces lividans TK24].

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.

Identification of 4″-isovaleryl-spiramycin III produced by Bacillus sp. fmbJ.

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.

[Impurity profiling of macrolide antibiotics by liquid chromatography-mass spectrometry].

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.

[Clinical experience on treating Toxoplasma gondii infection during pregnancy by using acetyl spiramycin combined with azithromycin].

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.

Identification of the components of bitespiramycin by liquid chromatography-mass spectrometry.

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-, ß-, 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.

A spicamycin derivative (KRN5500) provides neuropathic pain relief in patients with advanced cancer: a placebo-controlled, proof-of-concept trial.

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.