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1.
Molecules ; 29(13)2024 Jul 03.
Article in English | MEDLINE | ID: mdl-38999118

ABSTRACT

In this study, a liquid chromatographic method was developed for the fast determination of lincomycin, polymyxin and vancomycin in a preservation solution for transplants. A Kinetex EVO C18 (150 × 4.6 mm, 2.6 µm) column was utilized at 45 °C. Gradient elution was applied using a mixture of mobile phases A and B, both including 30 mM phosphate buffer at pH 2.0 and acetonitrile, at a ratio of 95:5 (v/v) for A and 50:50 (v/v) for B. A flow rate of 1.0 mL/min, an injection volume of 20 µL and UV detection at 210 nm were used. A degradation study treating the three antibiotics with 0.5 M hydrochloric acid, 0.5 M sodium hydroxide and 3% H2O2 indicated that the developed method was selective toward lincomycin, polymyxin, vancomycin and their degradation products. Other ingredients of the preservation solution, like those from the cell culture medium, did not interfere. The method was validated with good sensitivity, linearity, precision and accuracy. Furthermore, lincomycin, polymyxin and vancomycin were found to be stable in this preservation solution for 4 weeks when stored at -20 °C.


Subject(s)
Lincomycin , Polymyxins , Vancomycin , Lincomycin/analysis , Vancomycin/analysis , Polymyxins/analysis , Chromatography, Liquid/methods , Organ Preservation Solutions , Anti-Bacterial Agents/analysis , Reproducibility of Results , Chromatography, High Pressure Liquid/methods
2.
Biophys J ; 121(18): 3486-3498, 2022 09 20.
Article in English | MEDLINE | ID: mdl-35964158

ABSTRACT

Polymyxins are increasingly used as the last-line therapeutic option for the treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, efforts to address the resistance in superbugs are compromised by a poor understanding of the bactericidal modes because high-resolution detection of the cell structure is still lacking. By performing molecular dynamics simulations at a coarse-grained level, here we show that polymyxin B (PmB) disrupts Gram-negative bacterial membranes by altering lipid homeostasis and asymmetry. We found that the binding of PmBs onto the asymmetric outer membrane (OM) loosens the packing of lipopolysaccharides (LPS) and induces unbalanced bending torque between the inner and outer leaflets, which in turn triggers phospholipids to flip from the inner leaflet to the outer leaflet to compensate for the stress deformation. Meanwhile, some LPSs may be detained on the inner membrane (IM). Then, the lipid-scrambled OM undergoes phase separation. Defects are created at the boundaries between LPS-rich domains and phospholipid-rich domains, which consequently facilitate the uptake of PmB across the OM. Finally, PmBs target LPSs detained on the IM and similarly perturb the IM. This lipid Scramble, membrane phase Separation, and peptide Translocation model depicts a novel mechanism by which polymyxins kill bacteria and sheds light on developing a new generation of polymyxins or antibiotic adjuvants with improved killing activities and higher therapeutic indices.


Subject(s)
Lipopolysaccharides , Polymyxins , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Cell Membrane/metabolism , Gram-Negative Bacteria/chemistry , Homeostasis , Lipopolysaccharides/chemistry , Lipopolysaccharides/pharmacology , Phospholipids/chemistry , Polymyxin B/pharmacology , Polymyxins/analysis , Polymyxins/metabolism , Polymyxins/pharmacology
3.
J Antimicrob Chemother ; 74(1): 48-57, 2019 01 01.
Article in English | MEDLINE | ID: mdl-30357331

ABSTRACT

Background: Current inhaled polymyxin therapy is empirical and often large doses are administered, which can lead to pulmonary adverse effects. There is a dearth of information on the mechanisms of polymyxin-induced lung toxicity and their intracellular localization in lung epithelial cells. Objectives: To investigate the intracellular localization of polymyxins in human lung epithelial A549 cells. Methods: A549 cells were treated with polymyxin B and intracellular organelles (early and late endosomes, endoplasmic reticulum, mitochondria, lysosomes and autophagosomes), ubiquitin protein and polymyxin B were visualized using immunostaining and confocal microscopy. Fluorescence intensities of the organelles and polymyxin B were quantified and correlated for co-localization using ImageJ and Imaris platforms. Results: Polymyxin B co-localized with early endosomes, lysosomes and ubiquitin at 24 h. Significantly increased lysosomal activity and the autophagic protein LC3A were observed after 0.5 and 1.0 mM polymyxin B treatment at 24 h. Polymyxin B also significantly co-localized with mitochondria (Pearson's R = 0.45) and led to the alteration of mitochondrial morphology from filamentous to fragmented form (n = 3, P < 0.001). These results are in line with the polymyxin-induced activation of the mitochondrial apoptotic pathway observed in A549 cells. Conclusions: Accumulation of polymyxins on mitochondria probably caused mitochondrial toxicity, resulting in increased oxidative stress and cell death. The formation of autophagosomes and lysosomes was likely a cellular response to the polymyxin-induced stress and played a defensive role by disassembling dysfunctional organelles and proteins. Our study provides new mechanistic information on polymyxin-induced lung toxicity, which is vital for optimizing inhaled polymyxins in the clinic.


Subject(s)
Alveolar Epithelial Cells/chemistry , Anti-Bacterial Agents/analysis , Organelles/chemistry , Polymyxins/analysis , A549 Cells , Humans , Microscopy, Confocal
4.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1065-1066: 112-118, 2017 Oct 15.
Article in English | MEDLINE | ID: mdl-28961485

ABSTRACT

Polymyxin B has resurfaced as a last-line treatment for multi-drug resistant Gram-negative bacteria. Accurate characterization and quantification of polymyxin B components is necessary to optimize this therapy. We developed and validated a robust, straightforward LC-MS/MS method to quantify polymyxin B1 and B2, the primary polymyxin B components, in various matrices (cation-adjusted Mueller-Hinton broth (CAMHB), human and rat plasma). Of sample preparation approaches investigated, two protein precipitation/extraction methods were developed as part of an analytical strategy based upon reverse-phase LC-MS/MS using electrospray ionization in positive multiple-reaction monitoring mode. Both methods were validated over therapeutically and experimentally relevant concentration ranges (CAMHB: 0.1-8.0µg/mL, rat and human plasma: 0.05-4.0µg/mL). Quality control samples spanning a relevant concentration range were employed to assess intra- and inter-day accuracy (relative error (%RSD)) and precision (coefficient of variation (CV%)). For polymyxin B1 and B2 in CAMHB, inter-day standard deviations were 1.18-4.59% and 0.777-1.23%, respectively, and accuracies were 94.2-99.3% and 94.4-99.1%. For rat plasma, inter-day standard deviations were 1.53%-5.64% and 4.07%-8.26%. Accuracies were 100.6-108.9% and 96.1-108.1%. For human plasma, inter-day standard deviations were 2.77-7.32% and 1.55-7.29%. Accuracies were 89.6-96.4% and 92.9-102.0%. Extraction recoveries for all matrices were >93.5%. Adsorption, storage, and long-term stability were assessed and were acceptable. Accuracy, precision, and cost-efficiency make this an ideal approach for quantifying polymyxin B in in vitro and in vivo samples including those from rat and human subjects.


Subject(s)
Chromatography, Liquid/methods , Polymyxins/analogs & derivatives , Tandem Mass Spectrometry/methods , Animals , Humans , Limit of Detection , Linear Models , Male , Polymyxins/analysis , Rats , Rats, Sprague-Dawley , Reproducibility of Results
5.
J Pharm Sci ; 105(2): 1006-1010, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26869441

ABSTRACT

The frequency of polymyxin-resistant pathogenic Gram-negative bacteria appearing in the clinic is increasing, and the consequences are largely mediated by modification of lipopolysaccharide (LPS) in the outer membrane. As polymyxins exert their antibacterial effect by binding to LPS, understanding their mode of binding will prove highly valuable for new antibiotic discovery. In this study, we assess the potential of MIPS-9451, a fluorescent polymyxin analogue designed for imaging studies, as a fluorescent reporter molecule, titrating it against 17 different Gram-negative species and/or strains of LPS. MIPS-9451 bound to the various species and/or strains of LPS with a dissociation constant ranging between 0.14 ± 0.01 µM (Escherichia coli) and 0.90 ± 0.42 µM (Porphyromonas gingivalis; mean ± standard error). Furthermore, we assessed the applicability of MIPS-9451 to rank affinities of polymyxin B to different LPS species in a displacement assay which yielded inhibition constants of 6.2 µM ± 33%, 7.2 µM ± 30%, and 0.95 µM ± 13% for Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella enterica, respectively (mean ± coefficient of variation). The results from this study are concordant with those observed with similarly structured polymyxin probes, confirming the potential of MIPS-9451 for quantitation of polymyxin-LPS affinities in discovery programs of novel polymyxin antibiotics.


Subject(s)
Fluorescent Dyes/metabolism , Lipopolysaccharides/metabolism , Polymyxins/metabolism , Anti-Bacterial Agents/analysis , Anti-Bacterial Agents/metabolism , Drug Interactions/physiology , Fluorescent Dyes/analysis , Humans , Klebsiella pneumoniae/chemistry , Klebsiella pneumoniae/metabolism , Lipopolysaccharides/analysis , Polymyxins/analysis , Protein Binding/physiology , Pseudomonas aeruginosa/chemistry , Pseudomonas aeruginosa/metabolism
6.
Anal Chem ; 87(3): 1590-5, 2015 Feb 03.
Article in English | MEDLINE | ID: mdl-25553489

ABSTRACT

Polymyxin is the last-line therapy against Gram-negative 'superbugs'; however, dose-limiting nephrotoxicity can occur in up to 60% of patients after intravenous administration. Understanding the accumulation and concentration of polymyxin within renal tubular cells is essential for the development of novel strategies to ameliorate its nephrotoxicity and to develop safer, new polymyxins. We designed and synthesized a novel dual-modality iodine-labeled fluorescent probe for quantitative mapping of polymyxin in kidney proximal tubular cells. Measured by synchrotron X-ray fluorescence microscopy, polymyxin concentrations in single rat (NRK-52E) and human (HK-2) kidney tubular cells were approximately 1930- to 4760-fold higher than extracellular concentrations. Our study is the first to quantitatively measure the significant uptake of polymyxin in renal tubular cells and provides crucial information for the understanding of polymyxin-induced nephrotoxicity. Importantly, our approach represents a significant methodological advancement in determination of drug uptake for single-cell pharmacology.


Subject(s)
Anti-Bacterial Agents/metabolism , Chemistry, Pharmaceutical , Kidney Tubules/metabolism , Microscopy, Fluorescence/methods , Polymyxins/metabolism , Single-Cell Analysis/methods , Synchrotrons , Animals , Anti-Bacterial Agents/analysis , Cells, Cultured , Fluorescent Dyes , Humans , Iodine Radioisotopes , Kidney Tubules/cytology , Models, Molecular , Oxidative Stress , Polymyxins/analysis , Rats , X-Rays
7.
J Antimicrob Chemother ; 70(3): 827-9, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25377569

ABSTRACT

OBJECTIVES: Dose-limiting nephrotoxicity remains the Achilles' heel of polymyxin B and polymyxin E (also known as colistin), which are important last-line antibiotics used against infections caused by MDR Gram-negative 'superbugs'. An understanding of the mechanisms of nephrotoxicity, including renal tissue distribution, is crucial for the development of safer polymyxin lipopeptide antibiotics. This is the first study to visualize the kidney distribution of polymyxin B using a mouse nephrotoxicity model and in situ immunostaining of kidney sections. METHODS: Polymyxin B nephrotoxicity in mice was induced over the course of 3 days (accumulated intravenous dose 175 mg/kg) and kidneys were harvested and frozen sectioned. The sections were fixed in cold acetone, dried and treated with 1% hydrogen peroxide. Endogenous mouse immunoglobulins were blocked and the tissue sections were treated with anti-polymyxin B mouse IgM antibody. The sections were incubated with a biotinylated anti-mouse secondary antibody conjugate followed by an Alexa Fluor 647-streptavidin conjugate. Polymyxin B distribution in the kidney sections was then visualized using a fluorescence scanning microscope. Kidney sections were also subjected to haematoxylin and eosin staining to assess pathological damage from the polymyxin-induced nephrotoxicity. RESULTS: Immunostaining of kidney sections from a mouse with polymyxin B-induced nephrotoxicity revealed that polymyxin B distributed predominantly within the renal cortex. More specifically, polymyxin B accumulated within the proximal tubular cells. CONCLUSIONS: The observed accumulation of polymyxin B within proximal tubular cells is consistent with the extensive renal reabsorption of polymyxins and the likely cause of the associated nephrotoxicity.


Subject(s)
Anti-Bacterial Agents/adverse effects , Kidney/chemistry , Kidney/drug effects , Microscopy, Fluorescence , Polymyxins/adverse effects , Renal Insufficiency/chemically induced , Animals , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/analysis , Disease Models, Animal , Female , Kidney/pathology , Mice , Polymyxins/administration & dosage , Polymyxins/analysis , Renal Insufficiency/pathology
8.
Anal Bioanal Chem ; 377(5): 909-21, 2003 Nov.
Article in English | MEDLINE | ID: mdl-12955279

ABSTRACT

The application of liquid chromatography-ion trap mass spectrometry for the characterization of linear and cyclic polypeptide antibiotics was investigated. The aim was on-line identification of impurities in those antibiotic complexes without recourse to time-consuming isolation and purification procedures. Hyphenated techniques, such as liquid chromatography coupled to mass spectrometry, are ideally suited for this purpose. Characterization was performed with an ion trap mass spectrometer offering MS(n) capability; this enables more structural information to be obtained. Liquid chromatography in combination with ion trap mass spectrometry was successfully applied for the characterization of impurities in gramicidin, polymyxin B, polymyxin E, and bacitracin and the study of the degradation products of polymyxins B and E.


Subject(s)
Anti-Bacterial Agents/analysis , Chromatography, Liquid/methods , Mass Spectrometry/methods , Amino Acid Sequence , Anti-Bacterial Agents/chemistry , Bacitracin/analysis , Bacitracin/chemistry , Colistin/analysis , Colistin/chemistry , Gramicidin/analysis , Gramicidin/chemistry , Polymyxins/analysis , Polymyxins/chemistry , Time Factors
9.
J Pharm Biomed Anal ; 15(4): 505-11, 1997 Jan.
Article in English | MEDLINE | ID: mdl-8953494

ABSTRACT

The development of a liquid chromatographic essay system for the stability study of a formulation containing polymyxin, gramicidin and neomycin is described. For the determination of each group of antibiotics, poly(styrenedivinylbenzene) is used as the stationary phase. The mobile phase for the determination of polymyxin consists of an aqueous solution containing 7 g l-1 of sodium sulfate, 50 ml l-1 of 1 M phosphoric acid and 160 ml l-1 of acetonitrile. UV detection is performed at 215 nm. An aqueous solution containing 70 g l-1 of sodium sulfate, 1.4 g l-1 of sodium octanesulfonate and 50 ml l-1 of 0.2 M phosphate buffer pH 3.0 is used as the mobile phase for the determination of neomycin. Since neomycin has no UV-absorbing chromophore, pulsed electrochemical detection is chosen to determine neomycin. For each method, the influence of the different chromatographic parameters on the separation, the selectivity towards the other active compounds and the excipients, the repeatability and the linearity were investigated. The stability of the formulation was examined at 0, 6 and 12 months.


Subject(s)
Anti-Bacterial Agents/analysis , Chromatography, Liquid/methods , Gramicidin/analysis , Neomycin/analysis , Polymyxins/analysis , Drug Combinations , Drug Stability
10.
Antibiot Khimioter ; 34(1): 20-4, 1989 Jan.
Article in Russian | MEDLINE | ID: mdl-2543343

ABSTRACT

The effect of proteinases of plant and microbial origin on polymyxin M was studied. It was shown that this antibiotic was absolutely stable to the effect of papain and ficin. On hydrolysis with subtilisin there formed polymyxin decyclized analogs not described earlier. Their isolation, purification and biological activity are described. The structure of these compounds was assessed by one- and two-dimensional 1H NMR spectroscopy. The role of various functional groups, their space orientation and impact on antimicrobial activity of the compounds are discussed.


Subject(s)
Polymyxins/analysis , Ficain/pharmacology , Papain/pharmacology , Peptide Hydrolases/pharmacology , Structure-Activity Relationship
12.
Mikrobiologiia ; 57(3): 410-4, 1988.
Article in Russian | MEDLINE | ID: mdl-2846989

ABSTRACT

The synthesis of the antibiotic polymyxin M was studied under the conditions of batch and continuous cultivation of Bacillus polymyxa var. Ross whose growth was limited with glucose, phosphate and ammonium nitrogen. Polymyxin M was synthesized when the culture growth decelerated as a result of its limitation with the above compounds. Different amounts of the antibiotic were synthesized depending on the type of a limiting factor. The highest productiveness was found in the case of glucose limitation. The optimal conditions for polymyxin M synthesis were established under the conditions of one-step continuous cultivation.


Subject(s)
Bacillus/growth & development , Polymyxins/biosynthesis , Bacillus/metabolism , Culture Media/metabolism , Hydrogen-Ion Concentration , Polymyxins/analysis
13.
Antibiot Khimioter ; 33(4): 262-6, 1988 Apr.
Article in Russian | MEDLINE | ID: mdl-2839125

ABSTRACT

LPCC and HPLC revealed that polymyxin M was a mixture of five components of the polymyxin nature: PM1, PM2, PMx, PMy and PMz. The individual compounds PM1, PM2 and PMz were isolated. Their physicochemical properties and data on antimicrobial activity are presented.


Subject(s)
Polymyxins/isolation & purification , Bacteria/drug effects , Chromatography, High Pressure Liquid , Polymyxins/analysis , Polymyxins/pharmacology
14.
Antibiot Med Biotekhnol ; 32(10): 738-43, 1987 Oct.
Article in Russian | MEDLINE | ID: mdl-2827564

ABSTRACT

Spatial structure of polypeptide antibiotic polymyxin M in water was studied by one-and two-dimensional (COSY, COSY-45, RELAY) H NMR spectroscopy. Analysis of the signal spectral parameters revealed two intramolecular hydrogen bonds in the cyclic part of the molecule which was analogous to the structure of polymyxin B. However, configuration of both the beta-turns in the polymyxin M structure differed from that of the detected earlier beta-turns in the structure of polymyxin B.


Subject(s)
Polymyxins/analysis , Hydrogen , Magnetic Resonance Spectroscopy , Molecular Conformation , Solutions , Structure-Activity Relationship , Water
15.
Antibiot Med Biotekhnol ; 32(1): 20-4, 1987 Jan.
Article in Russian | MEDLINE | ID: mdl-3032084

ABSTRACT

Polymyxin B and its shortened analog were studied comparatively by 1H-NMR spectroscopy. Analysis of the signal chemical shifts, constants of spin-spin interaction of 3J HN-C alpha H and temperature coefficients of the NH signal chemical shifts revealed absolute structural identity of both molecules cyclic parts. This proved that there was no conformative interaction between the cyclic and linear parts of the polymyxin B molecule. Comparison of the results with the data on the biological activity showed that the hydrophobic N-end moiety of the polymyxin B molecule played a specific role in its antibacterial effect and toxicity.


Subject(s)
Polymyxin B/pharmacology , Polymyxins/pharmacology , Magnetic Resonance Spectroscopy , Polymyxin B/analogs & derivatives , Polymyxin B/analysis , Polymyxins/analysis , Protons , Structure-Activity Relationship
17.
Eur J Biochem ; 142(1): 113-9, 1984 Jul 02.
Article in English | MEDLINE | ID: mdl-6086338

ABSTRACT

Interactions between the antibiotic polymyxin B and monolayers of dipalmitoylglycerophosphoglycerol have been reinvestigated through a study of the structure and dynamics of the complexes by means of an interface fluorimeter of our fabrication. A fluorescence technique has been developed where the use of linearly polarized incident beams gives the simultaneous determination of the orientation and the lateral diffusion rate of a fluorescent probe inserted in the film. The present investigation was carried out with 12-(9-anthroyloxy)-stearic acid, a fluorescent compound which forms non-fluorescent photodimers upon illumination. Orientation of the probe was studied by computing the ratio of the two dimerization constants KD and the ratio of the fluorescence intensities obtained with crossed linearly polarized incident lights. The lateral diffusion rate of the probe was obtained by measuring fluorescence recovery after photobleaching (photodimerization) of the probe. Control experiments, carried out with dimyristoylglycerophosphocholine, a lipid which does not interact with polymyxin B, show that the antibiotic does not significantly modify the behaviour of the probe. Both in terms of orientation and dynamics, with respect to dipalmitoylglycerophosphoglycerol, when the antibiotic is present in the subphase (1 microM, saturating conditions), data indicate that the lipid remains in a liquid-expanded state. This is true even at a high surface pressure (pi approximately equal to 37 mN X m-1), above the apparent 'transition' which can be observed at 30-35 mN X m-1 on its compression isotherm. Computation of the contribution of polymyxin B to the film expansion to the conclusion that this 'transition' would be a structural transition between two models of interaction: one, below the 'transition', where the polypeptide ring penetrates between the film-forming lipid molecules and another one, above the 'transition', were the antibiotic is adsorbed at the lipid-water interface with only its hydrocarbon chain penetrating the film.


Subject(s)
Lipid Bilayers/analysis , Phosphatidylglycerols/analysis , Polymyxin B/analysis , Polymyxins/analysis , Dimyristoylphosphatidylcholine/analysis , Fluorescence , Macromolecular Substances , Photochemistry , Pulmonary Surfactants/analysis , Stearic Acids/analysis
18.
J Chromatogr ; 218: 653-61, 1981 Nov 20.
Article in English | MEDLINE | ID: mdl-6274887

ABSTRACT

Two polymyxin antibiotics, polymyxins B and E (colistin), have been separated analytically into ten to thirteen components on a commercial reversed-phase material by isocratic elution with a mixture of acetonitrile, phosphate/formate and acetate buffer containing sodium sulphate and triethylamine. The analytical system was transferred to a preparative system, using a C18-bonded stationary phase, without extensive impairing of the selectivity. The major components of each product were isolated and characterized by high-performance liquid chromatography, amino acid analysis and identification of the fatty acid. Three components were isolated and characterized for the first time. The fatty acid was also identified in some of the minor components.


Subject(s)
Colistin/analysis , Polymyxin B/analysis , Polymyxins/analysis , Polymyxins/isolation & purification , Chromatography, High Pressure Liquid/methods , Technology, Pharmaceutical
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