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The kinetics of delayed release neomycin-xanthan complex, and its antimicrobial activity, were studied. Delayed release neomycin (53.13% free neomycin) had zero-order release kinetics, when artificial tear solution was used as eluent. Microbiological tests revealed an activity of 380 units/mg, 10% higher than neomycin in its free form.
Porcine PDGF was found to increase [3H]inositol trisphosphate, [3H]thymidine incorporation and 32P-labelling of polyphosphoinositides in C3H/10T1/2 Cl 8 fibroblasts. These responses to PDGF stimulation were all inhibited by 5 mM neomycin, a polycationic aminoglycoside formerly known to inhibit polyphosphoinositide turnover. PDGF also markedly increased the cellular uptake of inorganic [32P]Pi. This response of PDGF was not inhibited by neomycin (5 mM). Thus, neomycin inhibited PDGF-induced IP3 formation, 32P-labelling of polyphosphoinositides and DNA synthesis, but not cellular uptake of inorganic phosphate. These effects of neomycin suggest a bifurcation of the initial part of the PDGF-induced signal transduction, separating at the receptor level or before phospholipase C activation.
1. The influence of pertussis toxin has been studied on the effects of neomycin on CA1 field potentials in rat hippocampal slices in order to determine a role played by G protein in the modulation of synaptic transmission by the drug. 2. Neomycin (500 microM), within 30 min significantly (P < 0.01) decreased the magnitude of the somatic CA1 excitatory postsynaptic potentials (EPSP) and population spike (PS) in control hippocampal slices. 3. Neomycin (500 microM), within 30 min failed to significantly affect the magnitude of the somatic CA1 EPSP and PS in slices obtained from animals treated intracerebroventricularly (ICV) with 1-2 micrograms of pertussis toxin 3 days before. 4. The results demonstrated that pertussis toxin prevents some electrophysiological effects of neomycin, suggesting a role of G protein in the modulation of the aminoglycoside antibiotic on central synaptic transmission.
We quantitated the activity of ornithine decarboxylase (ODC) in homogenates and subcellular fractions of inner ear tissues from the rat and guinea pig and demonstrate inhibition of cochlear ODC by the aminoglycoside neomycin. Subcellular fractionation showed the enzyme associated with the post-mitochondrial supernatant fraction in each of the tissues: Specific activities of ODC, defined as alpha-difluoromethylornithine (DFMO)-sensitive decarboxylation of ornithine, in the supernatant fractions of combined inner ear tissues were: guinea pig = 44 +/- 4 pmoles CO2 produced/hour/mg protein, and rat = 133 +/- 30. In the guinea pig, supernatant fractions of the lateral wall tissues (stria vascularis and spiral ligament) had specific activities of 62 +/- 25, those of the organ of Corti (plus VIIIth nerve) 64 +/- 41. The ototoxic aminoglycoside neomycin produced a dose-dependent inhibition of ODC with half-maximal inhibition observed at 50 microM drug and almost complete inhibition at 100 microM. This is the first report of the presence of ODC in the inner ear and its inhibition by neomycin. Since both the ODC-inhibitors, DFMO and neomycin, can cause hearing loss in patients and experimental animals it is suggested that inhibition of ODC may be an important factor in the ototoxicity of these drugs.
The effects that the aminoglycoside-aminocyclitol antibiotics amikacin, dihydrostreptomycin, gentamicin, neomycin, and spectinomycin, the neomycin fragment neamine, and the polybasic compounds spermine and poly-L-lysine, have on outer hair cells in cochlear cultures prepared from the early post-natal mouse have been assessed using both scanning and transmission electron microscopy (SEM and TEM). The antibiotics were used at concentrations ranging from 0.25-1.0 mM, spermine from 10 microM to 3.0 mM, and poly-L-lysine from 0.05-2 microM. Qualitative assessment of apical surface damage allows the antibiotics to be ranked in the following order: neomycin > gentamicin > dihydrostreptomycin > amikacin > neamine > spectinomycin. At a concentration of 1 mM spectinomycin is essentially non-toxic and the effects of neamine are marginal. Poly-L-lysine and spermine also cause surface damage, with poly-L-lysine being substantially more toxic than any of the antibiotics, and spermine ranking, on the basis of SEM observations, between dihydrostreptomycin and amikacin. TEM indicates that although all toxic compounds cause damage to the apical surface of the hair cell, only neomycin, poly-L-lysine and spermine induce the formation of whorls of tightly packed membrane resembling myelin within the apical surface lesions to any great extent. Apical-surface changes induced by dihydrostreptomycin and amikacin are simply large distensions of the cell filled with cytoplasmic organelles of normal appearance. Although the effects of the aminoglycoside antibiotics are largely limited to the apical surface of the cell, poly-L-lysine induces complete necrosis of the cell, and spermine causes a dramatic increase in cytoplasmic electron density and condensation of the nuclear chromatin.
omega-Conotoxin GVIA (omega-CT) has been reported to block calcium currents at the L- and N-type calcium channels. In neuronal membranes omega-CT, and the aminoglycoside antibiotic neomycin, have been shown to inhibit [125I]omega-CT binding, presumably acting at the N-type calcium channel. We demonstrate here that the concentration curve for neomycin sulfate inhibition of [125I]omega-CT binding is shifted to the right by GTP analogues or fluoride, increasing the IC50 for neomycin. [125I]omega-CT binding is unaffected by these agents and in competition studies the potency of omega-CT, Ca2+, or La3+ is not modulated by GTP analogues or fluoride. These results indicate that the inhibition of [125I]omega-CT binding by neomycin may be mediated by a GTP binding protein.
An approach is described to the design of neomycin-dipeptide conjugates as ligands for Rev responsive element (RRE) RNA, which effectively inhibit Rev-RRE interaction. A library of 256 neomycin-dipeptide conjugates was constructed on TentaGel beads using a split-and-pool combinatorial synthesis. Five conjugates were selected after screening the library with fluorescence linked RRE RNA, and they were identified after sequencing by MALDI-TOF mass spectrometer. The heteroconjugates bind to RRE RNA with moderately improved affinities and highly improved specificity, compared to neomycin as determined by means of fluorescence anisotropy and surface plasmon resonance (SPR) experiments. This strategy, synthesis of the neomycin-peptide heteroconjugate library and selection against RNA target, could provide an efficient way to develop inhibitors against pathogenic RNA.
In the frame of the development of a novel HPLC-ELSD (evaporative light scattering detection) method for the determination of the aminoglycoside antibiotic neomycin sulfate, the influence of mobile phase composition and peak broadening on ELSD response was evaluated. ELSD response was enhanced by: (a) increase of mobile phase volatility (solvents examined: water, acetonitrile, methanol and acetone), (b) increase of molecular mass of ion-pairing species [acidic reagents tested: formic, acetic, trifluoroacetic, trichloroacetic and heptafluorobutyric acid (HFBA)], and (c) decrease of peak width and asymmetry obtained by controlling the concentration of the ion-pairing acidic reagent (HFBA). Utilizing a Waters ODS-2 C18 Spherisorb column, evaporation temperature of 45 degrees C and nitrogen pressure of 3.5 bar, the optimized mobile phase was water-acetone (50:50), containing 11.6 mM HFBA, in an isocratic mode at a rate of 1.0 ml/min. Neomycin was eluted at 4.9 min, with asymmetry factor 1.3. Logarithmic calibration curve was obtained from 2 to 50 microg/ml (r > 0.9997). Limit of detection (LOD) was 0.6 microg/ml and R.S.D. = 1.7% (n = 3, 3.3 microg/ml). In raw materials, the simultaneous determination of sulfate (LOD = 3 microg/ml, R.S.D. = 1.7%, r> 0.9998) and of minor impurities was feasible. The developed method was also applied for the determination of neomycin in pharmaceutical formulations (powder, aerosol and cream) without any interference from excipients (recovery from spiked samples ranged from 99 to 102%) and a %R.S.D. of <2.1 (n = 3). The HPLC-ELSD method was also found applicable in the determination of neomycin in animal feeds (LOQ=0.2%) without any interference from the feed matrices.
A simple and rapid capillary electrophoresis method, with indirect UV detection, for the simultaneous determination of neomycin sulfate and polymyxin B sulfate in pharmaceutical formulations was developed. Critical parameters such as pH, buffer composition and concentration, voltage and injection time have been studied to evaluate, how they affect responses, such as resolution and migration times. Separation was performed on a fused silica capillary with 50 microm i.d. and 27 cm total length at an applied voltage of 6 kV with a 15 mM phosphate run buffer (pH 5.0) containing 40 mM N-(4-hydroxy-phenyl)acetamide and 50 mM tetradecylammonium bromide (TTAB). The detection wavelength was set at 280 nm. Quantitative analysis was validated by testing the reproducibility of the method, giving a relative standard deviation less than 0.4 and 2.4% for the repeatability of migration time and corrected peak area, respectively. Accuracy was tested by spiking eye-ear formulations with standards and the recoveries of neomycin sulfate and polymyxin B sulfate were found to be between 97.44-103.18% and 96.85-101.68%, respectively. Linearity of neomycin sulfate and polymyxin B sulfate were obtained in the ranges of 17-682 and 24-608 microg/mL, respectively, with r(2) values above 0.999. The established TLC-densitometric method was applied to evaluate the proposed CE method, and comparable results were obtained by using CE with much shorter analysis time and a small quantity of solvents consumed. The developed method is also the first report on the simultaneous determination of neomycin sulfate and polymyxin B sulfate in pharmaceutical preparations by CE.
A chemostat model of the healthy human large bowel ecosystem was used to establish no effect levels for tetracycline, neomycin, and erythromycin. For each compound, the equivalent to four oral doses (0, 1.5, 15, and 150 mg/60 kg person/d) was studied. Concentrations of the test compounds in the chemostat medium were intended to simulate fecal levels that might be expected following consumption of food containing antibiotic residue and were based on published oral doses and fecal levels. We monitored the following parameters: short chain fatty acids, bile acids, sulfate reduction, azoreductase and nitroreductase activities, beta-glucosidase and beta-glucuronidase activities, a range of bacterial counts and, lastly, the susceptibility among sentinel bacteria to each test compound. Neomycin and erythromycin reduced bile acid metabolism. Neomycin elevated propionate levels and caused a marginal diminution in azoreductase activity. Based on our results, the no observed effect level (NOEL) of both tetracycline and erythromycin was 15 mg/60 kg person/d. The NOEL for neomycin was 1.5 mg/60 kg person/d.
The effect of neomycin on the Herpes Simplex Virus (HSV) type 1 and 2 infection of baby hamster kidney cells was studied. Neomycin concentrations of 3 mM or more caused a more than 90% inhibition of HSV 1 proliferation, while it had no effect on HSV 2 proliferation, measured as plaque-forming units. Furthermore, neomycin must be present at the time of infection in order to exert full effect, addition 1 hour postinfection was comparable to untreated cells. This indicates that neomycin may specifically interfere with very early stages of HSV 1 infection.
Neomycin is known for its ototoxicity after many different methods of administration of the drug. Neomycin sometimes is used for bladder irrigation in patients with urinary tract infections when other treatments fail. To our knowledge toxicity after bladder instillation of neomycin has not been reported previously. We report a case of perception deafness in a boy with end stage renal disease who underwent neomycin bladder instillations following augmentation of the bladder with a megaureter.
Fourth instar Chironomus sp. larvae were used. Isolated salivaries (1/2) were incubated in RINGER containing various amounts of neomycin sulphate for various durations. Another half of salivaries, incubated in RINGER for corresponding durations served for controls. for in vivo experiments larvae were exposed to distilled water containing neomycin. Analysis of squashed preparations has revealed that puffing varies with incubation period in in vitro experiments with low concentration of drug. In in vivo studies also puff induction was evident. Higher concentrations of neomycin both in in vivo or in vitro treatments disorganized chromosomes. Present results suggest that neomycin can activate genes in an insect (or in other words in eukaryotes).
The purpose of the present study was to analyze the Ca2+ channel involved in GABA release under resting and K(+)-evoked conditions in vivo. We used microdialysis to investigate the effects of the voltage-dependent calcium channel (VDCC) blockers neomycin, kanamycin, and omega-conotoxin GVIA, and the voltage-dependent Na+ channel blocker tetrodotoxin, in the frontal cortex of awake rats. The GABA content in frontal dialysates was analyzed by high performance liquid chromatography coupled to fluorescence detection. Basal GABA release was kanamycin, omega-conotoxin, and tetrodotoxin resistant, whereas neomycin induced a significant increase from the basal level. The K(+)-evoked release of GABA was kanamycin and omega-conotoxin resistant, but tetrodotoxin sensitive. The effects of neomycin were masked by the action of this drug on basal release. These results suggest that neomycin may affect GABA release in the frontal cortex through a mechanism independent of VDCC. In addition, the K(+)-evoked release of GABA in this cortical area was not mediated by the N-type voltage-dependent calcium channels, but was dependent on neural activity or TTX sensitive.
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.
We use spectroscopic and calorimetric techniques to characterize the binding of the aminoglycoside antibiotics neomycin, paromomycin, and ribostamycin to a RNA oligonucleotide that models the A-site of Escherichia coli 16S rRNA. Our results reveal the following significant features: (i) Aminoglycoside binding enhances the thermal stability of the A-site RNA duplex, with the extent of this thermal enhancement decreasing with increasing pH and/or Na(+) concentration. (ii) The RNA binding enthalpies of the aminoglycosides become more exothermic (favorable) with increasing pH, an observation consistent with binding-linked protonation of one or more drug amino groups. (iii) Isothermal titration calorimetry (ITC) studies conducted as a function of buffer reveal that aminoglycoside binding to the host RNA is linked to the uptake of protons, with the number of linked protons being dependent on pH. Specifically, increasing the pH results in a corresponding increase in the number of linked protons. (iv) ITC studies conducted at 25 and 37 degrees C reveal that aminoglycoside-RNA complexation is associated with a negative heat capacity change (Delta C(p)), the magnitude of which becomes greater with increasing pH. (v) The observed RNA binding affinities of the aminoglycosides decrease with increasing pH and/or Na(+) concentration. In addition, the thermodynamic forces underlying these RNA binding affinities also change as a function of pH. Specifically, with increasing pH, the enthalpic contribution to the observed RNA binding affinity increases, while the corresponding entropic contribution to binding decreases. (vi) The affinities of the aminoglycosides for the host RNA follow the hierarchy neomycin > paromomycin > ribostamycin. The enhanced affinity of neomycin relative to either paromomycin or ribostamycin is primarily, if not entirely, enthalpic in origin. (vii) The salt dependencies of the RNA binding affinities of neomycin and paromomycin are consistent with at least three drug NH(3)(+) groups participating in electrostatic interactions with the host RNA. In the aggregate, our results reveal the impact of specific alterations in aminoglycoside structure on the thermodynamics of binding to an A-site model RNA oligonucleotide. Such systematic comparative studies are critical first steps toward establishing the thermodynamic database required for enhancing our understanding of the molecular forces that dictate and control aminoglycoside recognition of RNA.
Synthesis of a BQQ-neomycin conjugate is reported. The conjugate combines two ligands, one known to intercalate triplexes (BQQ) and another known to bind in the triplex groove (neomycin). The conjugate stabilizes T.A.T, as well as mixed base DNA triplex, better than neomycin, BQQ, or a combination of both. The conjugate selectively stabilizes the triplex (in the presence of physiological salt concentrations), with as little as 4 muM of the ligand leading to a DeltaTm of >60 degrees C. Competition dialysis studies show a clear preference for the drug binding to triplex DNA/RNA over the duplex/single strand structures. Modeling studies suggest a structure of neomycin bound to the larger W-H (Watson-Hoogsteen) groove with BQQ intercalated between the triplex bases.
The kinetics of phosphatidylcholine-specific phospholipase D activated by phosphatidylinositol 4,5-bisphosphate (PIP2) and inhibition by neomycin were studied in an enzyme preparation partially purified from human hepatocarcinoma cell line. It was found that phospholipase D was marginally activated by phosphatidyl-4-phosphate (PIP) and phosphatidylethanolamine (PE). In contrast, it was considerably activated by PIP2 in different concentration of phosphatidylcholine (PC). Sphingomyelin (SM), lysophosphatidylcholine (LPC) and phosphatidylserine (PS) were neither substrates nor inhibitors of the phospholipase D. PIP, induced an allosteric effect on phospholipase D and a negative cooperative effect with respect to phosphatidylcholine as indicated in the Lineweaver-Burk plot. In the absence of PIP2, a straight line was obtained, whereas a downward concave curve was observed in the presence of 25 microM of PIP2. The Hill coefficient and the apparent K(m) of phosphatidylcholine in the presence of 25 microM PIP, were calculated to be 0.631 and 10.79 mM, respectively. PIP2 also increased the maximal velocity (Vmax) of the phospholipase D reaction, suggesting that the affinity of substrate to enzyme was decreased, and the turnover number of the enzyme (kcat) was increased by PIP2. The activation of phospholipase D by PIP2 was dose dependent up to 50 microM of PIP2. The Ka of PIP2 was 15.8 mM. Neomycin, a polycationic glycoside, was shown to be an uncompetitive inhibitor of phospholipase D, and revealed the formation of a neomycin-PIP2 complex. The Ki of neomycin was estimated to be 8.7 mM.