Structural and phylogenetic analyses of resistance to next-generation aminoglycosides conferred by AAC(2') enzymes.

Plazomicin is currently the only next-generation aminoglycoside approved for clinical use that has the potential of evading the effects of widespread enzymatic resistance factors. However, plazomicin is still susceptible to the action of the resistance enzyme AAC(2')-Ia from Providencia stuartii. As the clinical use of plazomicin begins to increase, the spread of resistance factors will undoubtedly accelerate, rendering this aminoglycoside increasingly obsolete. Understanding resistance to plazomicin is an important step to ensure this aminoglycoside remains a viable treatment option for the foreseeable future. Here, we present three crystal structures of AAC(2')-Ia from P. stuartii, two in complex with acetylated aminoglycosides tobramycin and netilmicin, and one in complex with a non-substrate aminoglycoside, amikacin. Together, with our previously reported AAC(2')-Ia-acetylated plazomicin complex, these structures outline AAC(2')-Ia's specificity for a wide range of aminoglycosides. Additionally, our survey of AAC(2')-I homologues highlights the conservation of residues predicted to be involved in aminoglycoside binding, and identifies the presence of plasmid-encoded enzymes in environmental strains that confer resistance to the latest next-generation aminoglycoside. These results forecast the likely spread of plazomicin resistance and highlight the urgency for advancements in next-generation aminoglycoside design.

Micellar Electrokinetic Chromatography of Aminoglycosides.

The components of the aminoglycosides, e.g., gentamicin, sisomicin, netilmicin, kanamycin, amikacin, and tobramycin, and related impurities of these antibiotics can be separated by means of micellar electrokinetic chromatography (MEKC). Derivatization with o-phthaldialdehyde and thioglycolic acid is found to be appropriate for these antibiotics. The background electrolyte was composed of sodium tetraborate (100 mM), sodium deoxycholate (20 mM), and ß-cyclodextrin (15 mM) having a pH value of 10.0. This method is valid for evaluation of gentamicin, kanamycin, and tobramycin. It has to be adopted for amikacin, paromomycin, neomycin, and netilmicin.

Polymer assisted entrapment of netilmicin in PLGA nanoparticles for sustained antibacterial activity.

This study was aimed to develop poly(dl-lactide-co-glycolide) (PLGA) nanoparticle of highly water soluble antibiotic drug, netilmicin sulfate (NS) with improved entrapment efficiency (EE) and antibacterial activity. Dextran sulfate was introduced as helper polymer to form electrostatic complex with NS. Nanoparticles were prepared by double emulsification method and optimized using 2(5-1) fractional factorial design. EE was mainly influenced by dextran sulfate: NS charge ratio and PLGA concentration, whereas particle size (PS) was affected by all factors examined. The optimized NS-loaded-NPs had EE and PS of 93.23 ± 2.7% and 140.83 ± 2.4 nm respectively. NS-loaded-NPs effectively inhibited bacterial growth compared to free NS. Sustained release protected its inactivation and reduced the decline in its killing activity over time even in presence of bronchial cells. A MIC value of 18 µg/mL was observed for NPs on P. aeruginosa. Therefore, NPs with sustained bactericidal efficiency against P. aeruginosa may provide therapeutic benefit in chronic pulmonary infection, like cystic fibrosis.

Streptomycin interference in Jaffe reaction - possible false positive creatinine estimation in excessive dose exposure.

OBJECTIVES: To study the potential of commonly used aminoglycoside antibiotics to form non-creatinine chromogen with alkaline picrate reagent. DESIGN AND METHODS: We studied the non-creatinine chromogen formation of various concentrations of streptomycin, amikacin, kanamycin, netilmicin, gentamicin and tobramycin added to known creatinine concentrations by the Jaffe reaction based creatinine estimation. RESULTS: Only streptomycin above therapeutic concentrations of 10mg/mL interfered in the Jaffe reaction and acted as non-creatinine chromogen. CONCLUSIONS: Therapeutic doses of the aminoglycosides do not form non-creatinine chromogens.

Cosubstrate tolerance of the aminoglycoside resistance enzyme Eis from Mycobacterium tuberculosis.

We previously demonstrated that aminoglycoside acetyltransferases (AACs) display expanded cosubstrate promiscuity. The enhanced intracellular survival (Eis) protein of Mycobacterium tuberculosis is responsible for the resistance of this pathogen to kanamycin A in a large fraction of clinical isolates. Recently, we discovered that Eis is a unique AAC capable of acetylating multiple amine groups on a large pool of aminoglycoside (AG) antibiotics, an unprecedented property among AAC enzymes. Here, we report a detailed study of the acyl-coenzyme A (CoA) cosubstrate profile of Eis. We show that, in contrast to other AACs, Eis efficiently uses only 3 out of 15 tested acyl-CoA derivatives to modify a variety of AGs. We establish that for almost all acyl-CoAs, the number of sites acylated by Eis is smaller than the number of sites acetylated. We demonstrate that the order of n-propionylation of the AG neamine by Eis is the same as the order of its acetylation. We also show that the 6' position is the first to be n-propionylated on amikacin and netilmicin. By sequential acylation reactions, we show that AGs can be acetylated after the maximum possible n-propionylation of their scaffolds by Eis. The information reported herein will advance our understanding of the multiacetylation mechanism of inactivation of AGs by Eis, which is responsible for M. tuberculosis resistance to some AGs.

Impact of metal ions on netilmicin-melanin interaction.

Netilmicin, which is mainly used as the sulfate, is a semisynthetic, water soluble aminoglycoside antibiotic obtained by chemical modification of sisomicin. It is active against both Gram-positive and Gram-negative bacteria, including strains which are resistant to other aminoglycosides. Netilmicin form complexes with melanin. The aim of the presented work was to examine the effect of Cu2+, Zn2+, Ca2+ and Mg2+ on netilmicin binding to synthetic DOPA-melanin. It has been demonstrated that metal ions decrease the amount of antibiotic bound to melanin as compared with netilmicin-melanin complexes obtained in the absence of metals. It has been also shown that only one class of binding sites participates in netilmicin-[melanin-metal ion] complexes formation with the association constant K approximately 10(3) M(-1). The obtained results demonstrate that Cu2+, Zn2+, Ca2+ and Mg2+ ions modify the interaction between netilmicin and melanin biopolymer. The blocking of some active centers in melanin molecules by metal ions, which potentially exist in living systems, may influence the clinical therapeutic efficiency as well as the undesirable side effects of netilmicin.

Development and validation of a RP-HPLC method for the estimation of netilmicin sulfate and its related substances using charged aerosol detection.

Netilmicin is a semi-synthetic aminoglycoside antibiotic used against a broad spectrum of Gram-negative bacteria. A reversed-phase high-performance liquid chromatographic method has been developed to determine the composition of netilmicin sulfate and to estimate its related substances without pre- or post-column derivatization. A UV detector cannot be used to detect low levels of known and unknown related substances of netilmicin, as it has only a weak UV chromophore. A charged aerosol detector was used instead to obtain the high sensitivity that was necessary for the intended purpose of the method. This method can separate all related substances of netilmicin. A (10 cm x 4.6 mm) pentafluorophenyl high-performance liquid chromatographic column from Restek was used with a mobile phase consisting of (A) pentafluoropropionic acid-water-acetonitrile (0.1:96:4, v/v/v) and (B) trifluoroacetic acid-water-acetonitrile (1:96:4, v/v/v).

In vitro adsorption of gentamicin and netilmicin by polyacrylonitrile and polyamide hemofiltration filters.

Adsorption of gentamicin and netilmicin by new polyacrylonitrile and polyamide hemofiltration filters was studied over 4 h, using a single-compartment in vitro continuous venovenous hemofiltration model. After the first dose (16.6 mg of gentamicin, 19.3 mg of netilmicin), 14.9 mg of gentamicin and 19.2 mg of netilmicin were adsorbed to polyacrylonitrile filters in vitro. Adsorption by polyacrylonitrile filters was rapid and irreversible and could be increased by repeated dosing. Adsorption by polyamide filters was substantially less.

Polyhydroxyethylaspartamide-based micelles for ocular drug delivery.

In this paper three copolymers of polyhydroxyethylaspartamide (PHEA), bearing in the side chains polyethylene glycol (PEG) and/or hexadecylamine (C(16)) (PHEA-PEG, PHEA-PEG-C(16) and PHEA-C(16) respectively) have been studied as potential colloidal drug carriers for ocular drug delivery. The physical characterization of all three PHEA derivatives, using the Langmuir trough (LT) and micellar affinity capillary electrophoresis (MACE) techniques allowed to assume that whereas alone PHEA backbone is an inert polymer with respect to the interactions with lipid membranes and drug complexation, when PHEA chains are grafted with long alkyl chains like C(16) or in combination C(16) chains and hydrophilic chains like PEG, copolymers with lipid membrane interaction ability and drug complexation capability are obtained. In vitro permeability studies performed on primary cultured rabbit conjunctival and corneal epithelia cells, using PHEA-C(16) and PHEA-PEG-C(16) as micelle carriers for netilmicin sulphate, dexamethasone alcohol and dexamethasone phosphate, demonstrated that in all cases drug loaded PHEA-C(16) and PHEA-PEG-C(16) micelles provide a drug permeation across ocular epithelia greater than simple drug solutions or suspensions. In particular PHEA-PEG-C(16) acts as the best permeability enhancer in our experimental model. In vivo bioavailability studies conducted with PHEA-PEG-C(16) micelles loaded with dexamethasone alcohol, confirmed that this system also provides a drug bioavailability greater in comparison with that obtained with water suspension of the same drug after ocular administration to rabbits.

Characterization of impurities in sisomicin and netilmicin by liquid chromatography/mass spectrometry.

The characterization of unknown impurities present in netilmicin and sisomicin by liquid chromatography (LC) coupled with mass spectrometry (MS) is described. The volatile ion-pairing agent trifluoroacetic acid (TFA) was used for the retention of the main compounds and their impurities on a reversed-phase (RP) C18 column, because they are highly hydrophilic and basic compounds. The method showed good separation between netilmicin and its four potential related substances prescribed in the European Pharmacopoeia, which were identified by comparison of their retention times with those of the reference substances. Furthermore, in total 16 unknown impurities in a netilmicin sample and six in a sisomicin sample with unknown identity were detected. The structures of the unknown compounds were deduced based on comparison of fragmentation patterns with those of the reference substances investigated in LC/MSn experiments by the use of electrospray ion trap mass spectrometry.

Adsorptive stripping voltammetric determination of netilmicin in the presence of formaldehyde.

A linear sweep adsorptive stripping voltammetric method for the determination of netilmicin in the presence of formaldehyde has been proposed for the first time. In the presence of 3.0 x 10(-3) g ml(-1) formaldehyde, netilmicin exhibits a sensitive cathodic peak at -1.30 V (vs. the saturated calomel electrode, SCE) in a medium of Britton-Robinson buffer (pH 8.7) with a scan rate of 100 mV s(-1) after a preconcentration period of 120 s at -1.10 V (vs. SCE). The peak current showed a linear dependence on the netilmicin concentration over the range 4.2 x 10(-9)-1.0 x 10(-7) g ml(-1). The achieved limits of detection and quantitation were 1.0 x 10(-10) and 3.3 x 10(-10) g ml(-1) netilmicin, respectively. It was deduced from the experiments that the amine-aldehyde condensation product formed between netilmicin and formaldehyde is mainly responsible for the appearance of the peak. The electrochemical behavior of netilmicin in the presence of formaldehyde has been studied. The method was applied to the direct determination of netilmicin in injectable formulations and spiked human urine and serum samples.

Capillary electrophoresis assay of netilmicin sulphate.

An effective method based on capillary electrophoresis (CE) for the determination of netilmicin sulphate in commercial ophthalmic formulations was developed and validated. The use of a polymer-coated capillary and a non-absorbing running buffer permitted the elution of netilmicin in cationic mode at pH 3.0 and with direct UV detection at lambda=195 nm. Since pre-treatment of the samples is not required, this procedure may be straightforwardly applied to the other aminoglycosides provided that their extinction coefficient is not too low.

Shelf lives of aseptically prepared medicines--stability of netilmicin injection in polypropylene syringes.

There is no published information on the stability of netilmicin solutions in prefilled syringes. The purpose of this study was to evaluate the stability of netilmicin in polypropylene syringes and to determine the optimum validated shelf life so that they may be prepared in bulk in appropriately licensed facilities. The syringes containing netilmicin 10 or 100mg/ml were stored at 7 degrees C, room temperature in the light (RTL) and 25 degrees C/60% relative humidity for up to 300 days. Netilmicin concentration was determined by reversed phase high performance liquid chromatography (RP-HPLC) of the isoindole derivative formed with o-phthalaldehyde (OPA). The shelf lives were calculated using the maximum rate method applied to the netilmicin analytical data. At 7 degrees C 10 and 100mg/ml solutions were stable for 90 days falling to 30 days at 25 degrees C and 60% RH. At RTL the 10mg/ml solution was stable for 9 days.

A new micellar electrokinetic capillary chromatography method for separation of the components of the aminoglycoside antibiotics.

Aminoglycoside antibiotics are always a mixture of structurally related amino sugars, which do not have a chromophore or fluorophore. The aim of the study was to find one method for evaluation of the components and impurities of the antibiotics. Derivatization with o-phthaldialdehyde and thioglycolic acid is found to be appropriate for all antibiotics. The components of gentamicin (GM), sisomicin, netilmicin, kanamycin, amikacin, and tobramycin were tried to separate by means of micellar electrokinetic chromatography. The background electrolyte was composed of sodium tetraborate (100 mM, pH 10.0), sodium deoxycholate (20 mM), and beta-cyclodextrin (15 mM). This method is valid for evaluation of GM, kanamycin, and tobramycin. It has to be improved for amikacin and netilmicin. In addition, 46 bulk samples of GM of different manufacturer or pharmaceutical companies were investigated. Many samples were found to contain many minor products and different amounts. Beside different patterns of the main compounds GM C1, GM C1a, GM C2a, and GM C2, many lots were found consisting of a substantial number of minor products. The appearance of a high number of minor products is always associated with the existence of sisomicin, which is not found in "pure" samples. However, almost all samples met the requirements of the European Pharmacopoeia (EP) and United States Pharmacopoeia (USP).

Study of aminoglycoside-nucleic acid interactions by an HPLC method.

The interactions of a number of aminoglycoside antibiotics with tRNA and DNA were studied by an HPLC method. based on tRNA and DNA peak size exclusion. Among the compounds studied (deoxystreptamine, neamine, neomycin B, kanamycin A, gentamicin A, netilmicin, streptomycin, and the synthetic neamine analogue BKN3), neomycin B and the synthetic analogue of neamine were proved to be the most potent binders.

Stability and compatibility of an aerosol mixture including N-acetylcysteine, netilmicin and betamethasone.

The physicochemical stability and the compatibility between N-acetylcysteine (1 g/5 ml), betamethasone (4 mg/1 ml) and netilmicin (100 mg/1 ml) were studied at room temperature (25+/-2 degrees C) over 1 h. During this study, drug concentrations were measured using three separate HPLC methods with UV detection at t=0, 5, 10, 20, 30, and 60 min. The pH of the mixture was determined. Degradation products of the drugs were assayed using HPLC. This study demonstrates the stability and compatibility of the mixture over 1 h at room temperature. The pinkish non-remnant coloration observed when pouring N-acetylcysteine into a recipient has no effect on the stability of the drug.

[Osmolarity of solutions used in nebulization]. / Osmolarité des solutions utilisées en nébulisation.

Inhaled medications are widely used in patients suffering from bronchial diseases. Beside their pharmacological properties, nebulised solutions have physico-chemical characteristics that can alter bronchial reactivity. Non-isotonic solutions can induce a bronchial hyperresponsiveness and/or a severe bronchonconstriction. Nevertheless, multiple drugs are used for nebulisation despite their unknown osmolarity. The aim of this study was to measure the tonicity of drug solutions commonly used for nebulisation in patients suffering from bronchial disease. Drug solutions were prepared either according to manufacturer recommendations or by diluting the stock in 5 ml of NaCl (0.9%) or H2CO3 (0.14%). Although bronchodilatator solutions (i.e. salbutamol, terbulatine, ipratropium bromide) were nearly isotonic, some drugs prepared for nebulisation had either a very high (e.g. mesna, netilmicine) or a very low (e.g. gomenol, sodium cromoglycate) tonicity. These values may be responsible for bronchoconstriction. Some hypertonic solutions, prepared with drugs such as acetylcytein or netilmycin, are not commercialised for nebulisation but are commonly used for aerosol therapy. In addition, solutions initially isotonic could become significantly hypertonic towards the end of nebulisation. Taken together, these results suggest that non-isotonic solutions should be used with caution specially in patients with bronchial hyperresponsiveness, even when aerosol therapy is prescribed for upper airways.