Analysis of histamine and sisomicin in gentamicin: Search for the causative agents of adverse effects.

In 1998, the aminoglycoside antibiotic gentamicin sulfate caused several cases of deaths in the United States, after the switch from twice- to once-daily application. Endotoxins were discussed as the cause for the adverse effects and sisomicin was identified as the lead impurity; batches containing sisomicin were contaminated with more impurities and were responsible for the fatalities. In 2016, anaphylactic reactions in horses, and later in humans with one fatality, were observed after application of gentamicin sulfate contaminated with histamine. To determine whether histamine was responsible for the 1990s death cases as well, histamine was quantified by means of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in 30 samples of gentamicin sulfate analyzed in previous studies. Furthermore, a relative quantification of sisomicin was performed to check for a correlation between histamine and the lead impurity. A maximum amount of 11.52 ppm histamine was detected, which is below the limit for anaphylactic reactions of 16 ppm, and no correlation of the two impurities was observed. However, the European Medicines Agency recommends a stricter limit with regard to the maximum single dose of gentamicin sulfate to reach a greater gap between the maximum histamine exposition of 4.3 µg and the quantity known to cause hypotension of 7 µg. The low amounts of histamine and the fact that there is no connection with the contamination with sisomicin showed that histamine was not the cause for the death cases in the United States in 1998, and endotoxins remain the most probable explanation.

Hyperosmotic sisomicin infusion: a mouse model for hearing loss.

Losing either type of cochlear sensory hair cells leads to hearing impairment. Inner hair cells act as primary mechanoelectrical transducers, while outer hair cells enhance sound-induced vibrations within the organ of Corti. Established inner ear damage models, such as systemic administration of ototoxic aminoglycosides, yield inconsistent and variable hair cell death in mice. Overcoming this limitation, we developed a method involving surgical delivery of a hyperosmotic sisomicin solution into the posterior semicircular canal of adult mice. This procedure induced rapid and synchronous apoptotic demise of outer hair cells within 14 h, leading to irreversible hearing loss. The combination of sisomicin and hyperosmotic stress caused consistent and synergistic ototoxic damage. Inner hair cells remained until three days post-treatment, after which deterioration in structure and number was observed, culminating in a complete hair cell loss by day seven. This robust animal model provides a valuable tool for otoregenerative research, facilitating single-cell and omics-based studies toward exploring preclinical therapeutic strategies.

Hyperosmotic Sisomicin Infusion: A Mouse Model for Hearing Loss.

Hearing impairment arises from the loss of either type of cochlear sensory hair cells. Inner hair cells act as primary sound transducers, while outer hair cells enhance sound-induced vibrations within the organ of Corti. Established models, such as systemic administration of ototoxic aminoglycosides, yield inconsistent and variable hair cell death in mice. Overcoming this limitation, we developed a method involving surgical delivery of a hyperosmotic sisomicin solution into the posterior semicircular canal of adult mice. This procedure induced rapid and synchronous apoptotic demise of outer hair cells within 14 hours, leading to irreversible hearing loss. The combination of sisomicin and hyperosmotic stress caused consistent and synergistic ototoxic damage. Inner hair cells remained intact until three days post-treatment, after which deterioration in structure and number was observed, culminating in cell loss by day seven. This robust animal model provides a valuable tool for otoregenerative research, facilitating single-cell and omics-based studies toward exploring preclinical therapeutic strategies.

Enhanced Production of Sisomicin in Micromonospora inyoensis by Protoplast Mutagenesis and Fermentation Optimization.

Sisomicin is a broad-spectrum aminoglycoside antibiotic and is the precursor of netilmicin and plazomicin. However, the fermentation level of sisomicin is still low compared with other antibiotics, which restricts the application of sisomicin and its derivatives. In this study, to improve sisomicin production, breeding of high-yielding sisomicin strains was conducted with chemical mutagenesis using Micromonospora inyoensis OG-1 (titer, 1042 U·mL-1) as the starting strain. Protoplast preparation was conducted under optimal conditions, and protoplast mutagenesis was performed with a suitable concentration of diethyl sulfate. Subsequently, a high-yielding and genetically stable strain (H6-32) was obtained by screening, with a sisomicin titer of 1486 U·mL-1 (an increase of 42.6%). Finally, carbon and nitrogen sources were optimized to further improve sisomicin production, and a sisomicin titer of 1780 U·mL-1 was ultimately obtained by controlling the dissolved oxygen level at 30% in a 5-L fermenter, which to the best of our knowledge is the highest reported titer ever achieved by fermentation. Comparative genome analysis showed that a total of 13 genes in the genome of the mutant strain H6-32 were mutated compared to the original strain. This study not only provides a reference for further breeding of high-yielding strains and fermentation optimization, but also enhances our understanding of sisomicin production.

Manufacture of pH- and HAase-responsive hydrogels with on-demand and continuous antibacterial activity for full-thickness wound healing.

A kind of "intelligent" antibacterial dressing-A-HA/HA-ADH/SS hydrogel was in situ formed quickly via dynamic covalent bonds cross-linking between aldehyde hyaluronic acid (A-HA), adipic acid dihydrazide graft hyaluronic acid (HA-ADH) and sisomicin sulfate (SS). FT-IR, SEM and rheological results displayed that the hydrogels were successfully prepared. The hydrogels had good optical transmittance, injectability, self-healing ability, cytocompatibility, antioxidant activity and hemostatic performance which were beneficial to observe the wound healing condition and provide a good healing environment for wounds. In addition, the hydrogels showed a pH- and HAase- dependent degradability, which allowed them to release more SS at infected wound and then exert on-demand and sustained antibacterial effect against S. aureus and E. coli. The results of wound healing and histological examination revealed that these hydrogels have a good therapeutic effect in the full-thickness mouse skin defect wound. Thus, the hydrogels are expected to be used as potential wound dressings to improve wound healing.

Determination of equilibrium constant of amino carbamate adduct formation in sisomicin by a high pH based high performance liquid chromatography.

Amino carbamate adduct formation from the amino group of an aminoglycoside and carbon dioxide has been postulated as a mechanism for reducing nephrotoxicity in the aminoglycoside class compounds. In this study, sisomicin was used as a model compound for amino carbamate analysis. A high pH based reversed-phase high performance liquid chromatography (RP-HPLC) method is used to separate the amino carbamate from sisomicin. The carbamate is stable as the breakdown is inhibited at high pH and any reactive carbon dioxide is removed as the carbonate. The amino carbamate was quantified and the molar fraction of amine as the carbamate of sisomicin was obtained from the HPLC peak areas. The equilibrium constant of carbamate formation, Kc, was determined to be 3.3 × 10(-6) and it was used to predict the fraction of carbamate over the pH range in a typical biological systems. Based on these results, the fraction of amino carbamate at physiological pH values is less than 13%, and the postulated mechanism for nephrotoxicity protection is not valid. The same methodology is applicable for other aminoglycosides.

Determination of the content of sisomicin sulfate and sodium chloride injection by RP-HPLC / 中国药科大学学报

@#To establish a RP-HPLC method for the determination of content of sisomicin sulfate and sodium chloride injection. Thermo Aminoglycoside RP 18(4. 6 mm ×150 mm, 3 μm)column was used. The mobile phase consisted of Sodium heptane sulfonate solution(take 6 g of sodium heptane sulfonate, add 0. 1 mol/L potassium dihydrogen phosphate solution and dilute to 1 000 mL, adjust the pH to 1. 5 with phosphoric acid)- acetonitrile(77∶23). The detection wavelength was 205 nm, the flow rate was 1. 0 mL/min. and the column temperature was 35 °C. The separation of sisomicin peaks with related substances and the degradation products was good. The linear range of the peak area with sisomicin was 0. 010 024-1. 002 4 mg/mL(Y=4. 210 2×106 X+9. 107 0×103, r=0. 999 9, n=7), the detection limit was 0. 6 ng, the limit of quantification was 2 ng, and the recovery rate was at 99. 1%-100. 9%(RSD< 1. 0%, n=9). The method is sensitive, exclusive, accurate and suitable for the determination of sisomicin. Compared with the antibiotic microbiological test method, the specificity is better, the confidence interval of the result is narrowed, and the test time is saved.

Comparison of Analytical Methods for Related Substances of Sisomicin Sulfate Injection / 中国药学杂志

OBJECTIVE: To establish an HPLC-PAD method to determine the related substances of sisomicin sulfate injection and compare with the statutory method. METHODS: IonPac AMG C18(4.0 mm×150 mm, 3 μm)chromatographic column was used with acetonitrile-0.1 mol·L-1 trifluoroacetic acid (containing 0.025% of pentafluoropropionic acid, 5 mL of 50% NaOH solution without carbonate, pH of the aqueous solution adjusted to 2.3 with 50% NaOH solution.)as mobile phase at a flow rate of 0.7 mL·min-1. NaOH solution of 0.76 mol·L-1 was added post column at a flow rate of 0.35 mL·min-1. The column temperature was maintaine at 30 ℃. PAD detector was operated with the cell temperature set at 35 ℃. The working electrode was a gold electrode (diameter of 3 mm)and a quadruple-potential waveform was selected as detection waveform. The reference electrode was Ag/AgCl, the detection potential was four potential. The determination result of the related substances of sisomicin sulfate injection was compared with that of the statutory method. RESULTS: The peaks of sisomicin sulfate, gentamicin C1a and netilmicin could be completely separated, and other impurities could also be effectively separated. The blank sample had no interferences. The LOD and LOQ of etimicin were found to be 2 and 6 ng respectively, and the RSD of precision test (n=6) was 0.9%. Paired-samples t-test showed significance levels of P=0.034, P=0.364 and P=0.605 for total amount of impurities (%), the biggest single impurity (%)and content (%)respectively between the statutory method and the method of HPLC-PAD. CONCLUSION: Compared with the statutory method, this HPLC-PAD method shows higher sensitivity, and is accurate and reliable. It can be applied to the determination of related substances in sisomicin sulfate injection.