Protective effect of ascorbic acid on netilmicin-induced lipid profile and peroxidation parameters in rabbit blood plasma.

A drug may cause alteration in blood-lipid profile and induce lipid peroxidation phenomena on administration in the body. Antioxidant may play beneficial role to control the negative alteration in lipid profile and lipid peroxidation. In view of this context, the present in vivo study was carried out to evaluate the role of ascorbic acid as antioxidant on netilmicin-induced alteration of blood lipid profile and peroxidation parameters. Rabbits were used as experimental animals and blood was collected to estimate blood-lipid profiles, such as total cholesterol (TCh), high density lipoprotein cholesterol (HDL-Ch), low density lipoprotein cholesterol (LDL-Ch), very low density lipoprotein cholesterol (VLDL-Ch), triglycerides (Tg), phospholipids (PL), and total lipids (TL), as well as peroxidation parameters, such as malondialdehyde (MDA), 4-hydroxy-2-nonenal (HNE), reduced glutathione (GSH) and nitric oxide (NO). The results revealed that netilmicin caused significant enhancement of MDA, HNE, TCh, LDL-Ch, VLDL-Ch, Tg levels and reduction in GSH, NO, HDL-Ch, PL, TL levels. On co-administration, ascorbic acid was found to be effective in reducing netilmicin-induced negative alterations of the above parameters.

Comparison of the Anti-Inflammatory and Cytotoxic Potential of Different Corticosteroid Eye Drop Preparations.

PURPOSE: To compare the immunosuppressive and cytotoxic effects of three anti-inflammatory eye drops formulations containing betamethasone plus chloramphenicol (B+C), dexamethasone plus netilmicin (D+N) or dexamethasone plus tobramycin (D+T).Methods: The eye drops formulations have been tested at different dilutions on cytokine synthesis by mouse or human cultured macrophages, as well as proliferation and viability of cultured human corneal cells (HCE).Results: B+C reduced IL6 and TNFα production by cultured mouse or human macrophages more potently than D+N and D+T, with the tree formulations having the same impact on IL-10 expression. We also found that the eye drops preparations reduced proliferation of HCE cells, with D+T showing the higher anti-proliferative potency and B+C showing the lower cytotoxic potential.Conclusion: Our study points out that it may be erroneous to consider routinely-used anti-inflammatory eye drops preparations with analogous formulations as readily interchangeable and of similar potency and tolerability.

In vitro effects of fluoroquinolone and aminoglycoside antibiotics on human keratocytes.

PURPOSE: The purpose of this study was to assess the cytotoxic effects of the fluoquinolone ofloxacin and the aminoglycoside netilmicin on stromal human keratocytes in vitro. METHODS: Cultured human keratocytes were exposed to various concentrations of ofloxacin or netilmicin (0.16-5.0 mg/mL). Both cell proliferation (MTT assay) and cell morphology (phase-contrast microscopy) were evaluated after 1, 4, 12, and 24 hours of incubation. Measurement of annexin V binding performed in association with the dye exclusion test using propidium iodide (PI) was also performed by FACS analysis after 4 hours of exposure. RESULTS: Both antimicrobials induced dose- and time-dependent morphologic changes in keratocytes, yet the effects of netilmicin were minimal. After 24 hours of exposure, both drugs induced a dose-dependent inhibition of cell proliferation; however, ofloxacin demonstrated significantly more toxic effects than netilmicin (t test for ED50 values, P < 0.0001). Statistical differences between 2 antibiotics start at concentrations above 1.25 mg/mL (ANOVA with post-hoc test, P < 0.01). Expression of the apoptotic marker annexin V was unaffected by antibiotic exposure, whereas the uptake of the necrotic marker PI was increased by ofloxacin (5 mg/mL) but not by netilmicin (ofloxacin versus netilmicin, ANOVA, P < 0.05). CONCLUSIONS: Relative effects of aminoglycosides and fluoroquinolones on stromal keratocytes appear to be different: netilmicin was shown to be significantly less toxic than ofloxacin. This finding is particularly relevant in deciding the optimal antibiotic to be applied in clinical situations in which the epithelium is absent or compromised, as after photorefractive keratectomy, alkali burns, or ulcerative keratitis.

In vivo toxicity of netilmicin and ofloxacin on intact and mechanically damaged eyes of rabbit.

PURPOSE: To evaluate the in vivo toxicity of netilmicin and ofloxacin using both normal and mechanically damaged eyes of rabbit. METHODS: Male albino New Zealand rabbits were given either 0.3% netilmicin, 0.3% ofloxacin, or 0.9% sodium chloride solution by topical instillation (50 microL) into the conjunctival sac 6 times daily for 5 days. In some animals a 6-mm-diameter epithelial wound was mechanically made to the center of the cornea. Ocular toxicity on normal eyes was evaluated by impression cytology of the conjunctiva, histology of the entire globes, and scanning electron microscopy (SEM) of the cornea. Analysis of toxicity and reepithelialization on wounded corneas was evaluated by SEM with observations being made 48 and 72 hours after induction of the wound. RESULTS: Cytologic, histopathologic, and SEM analyses of normal healthy eyes following netilmicin treatment revealed no signs of toxicity, whereas those treated with ofloxacin revealed alterations in the cornea (stromal swelling) and conjunctiva (infiltration of polymorphonuclear cells) with reduced goblet cell numbers. Wounded corneas treated with netilmicin exhibited normal morphology and reepithelialization, whereas the administration of ofloxacin resulted in disordered cellular organisation and slower rates of epithelial recovery. CONCLUSIONS: Netilmicin, an antibacterial aminoglycoside, is well tolerated even in an experimental wound-healing model where the integrity of the ocular surface is compromised, whereas ofloxacin, a fluoroquinolone, appears to provoke an inflammatory response in the normal eye and a clear alteration of reepithelialization in the wounded eye. These findings suggest that netilmicin may offer a superior toxicological profile in both normal eyes and clinical situations where the integrity of the ocular epithelium is suspect.

Risk factors and comparisons of clinical nephrotoxicity of aminoglycosides.

Controversy continues regarding risk factors for nephrotoxicity and the possible differences in nephrotoxicity rates associated with the aminoglycosides commonly used in clinical practice. Review of published data, including 27 comparative clinical studies, indicates the following: sensitive markers of tubular toxicity do not correlate directly with clinically significant diminution in glomerular filtration rate; of all the proposed risk factors, few are consistently found; despite numerous studies, some with potential drawbacks, no current aminoglycoside has been shown to be consistently and conclusively less nephrotoxic than another; and, as per a previous recommendation, the choice of an aminoglycoside should not be made on the basis of nephrologic criteria.

Choice of drug and dosage regimen. Two important risk factors for aminoglycoside nephrotoxicity.

Since the clinical use of aminoglycosides may be limited by the development of nephrotoxicity, it is important to be aware of those risk factors associated with a greater incidence of renal damage. Some of these factors are related to the drug and its administration and others are related to the patient's clinical condition. In the human kidney, the toxicity mechanism is very likely the same for all aminoglycosides, although the risk of nephrotoxicity increases for a given aminoglycoside as cortical concentrations increase. Kinetic studies in the rat demonstrated a nonlinear increase in renal cortical uptake of gentamicin and netilmicin, a linear relationship for tobramycin uptake, and a mixed kinetic pattern for amikacin, that is, saturation kinetics at low serum concentrations and a linear pattern at high serum levels. At comparable steady-state low serum levels, amikacin and tobramycin showed lower cortical concentrations than gentamicin or netilmicin, demonstrating a lower affinity for the uptake of amikacin and tobramycin in the rat. Since drug uptake kinetics determine the extent of cortical concentrations achieved, dosing strategies may affect cortical accumulation of aminoglycosides. Our kinetic data show that continuous infusions of low doses of gentamicin and amikacin resulted in higher cortical levels, but the differences between regimens were more remarkable for gentamicin than for amikacin. For tobramycin, however, cortical concentrations were similar regardless of the dosing strategy used. In addition, our data show that dosage regimens also determine cortical accumulation in humans. A second major determinant of nephrotoxicity is intrinsic toxicity. At therapeutic doses, gentamicin, tobramycin, netilmicin, and amikacin induce an early lysosomal phospholipidosis in the human kidney cortex comparable to that observed in animals treated with low doses of these drugs. However, animal and human studies have shown that amikacin induces significantly less lysosomal overloading than the other aminoglycosides with no loss of phospholipase A1 activity. Based on the examination of cortical drug levels and the detection of early biochemical and morphologic alterations induced by aminoglycosides, the data suggest that amikacin has less pronounced nephrotoxic effects than gentamicin, netilmicin, or tobramycin, when used in strictly comparable clinical conditions.

Synthesis and antimicrobial and toxicological studies of amino acid and peptide derivatives of kanamycin A and netilmicin.

Amino acid and peptide derivatives of aminoglycosides have been obtained by substitution of the 1-N or 6'-N amino functions of kanamycin A and netilmicin via the temporary complexation of vicinal and nonvicinal amino and hydroxy functions by copper ion [1-N kanamycin A derivatives: L-Ala (6a), D-Ala (6b), Gly (6c), L-Asp (6d), L-Ala-L-Ala (6e). 6'-N kanamycin A derivatives: L-Ala (3a), D-Ala (3b), Gly (3c), L-Ala-L-Ala (3e), L-Leu (3f). 6'-N netilmicin derivatives: L-Ala (9a), D-Ala (9b), Gly (9c), L-Asp (9d), L-Ala-L-Ala (9e)]. Characterization was made by FAB-MS, IR, 1H-NMR, and 13C-NMR. All derivatives were essentially inactive. The nephrotoxic potential of the derivatives obtained in sufficient quantities (3b,e and 9a-e) was assessed by measuring their inhibitory potential toward the activity of lysosomal phospholipase A1 acting on phosphatidylcholine embedded in negatively-charged membranes. One compound, 6'-N-L-Ala-netilmicin (9a), showed a 2-fold decrease of inhibitory potency compared to its parent drug. A conformational analysis revealed that it adopts two equally probable conformations and orientations when interacting with phosphatidylinositol. The first in which the drug lies parallel to the hydrophobic-hydrophilic interface, is similar to that of netilmicin. The second, in which the drug inserts itself in the bilayer across the hydrophilic/hydrophobic interface, is similar to that described for streptomycin, an almost non-nephrotoxic aminoglycoside.

Experimental drusen formation induced by intravitreal aminoglycoside injection.

A single intravitreal injection of aminoglycoside antibiotics in the rabbit induces changes in the retinal pigment epithelial layer that consist of disseminated yellow-white dots that are apparent six to ten months after injection. With fluorescein angiography, these dots have the characteristics of window defects or drusen. Histologic examination disclosed subepithelial amorphous material that stained positively with both periodic acid-Schiff and oil red O. Ultrastructural examination disclosed lipidic inclusions in the retinal pigment epithelial cells, basally directed cellular evagination, and basal accumulation of granular material, findings consistent with the reported morphologic features of hard drusen. These findings suggest that aminoglycoside-induced lesions may represent a model for retinal pigment epithelial degeneration and drusen formation.

Comparative toxicity of intravitreal aminoglycoside antibiotics.

We compared the toxicity of the aminoglycoside antibiotics (tobramycin, amikacin, netilmicin, and kanamycin) by ophthalmoscopy, light and electron microscopy, and electro-retinography after intravitreal injection in rabbits in doses ranging from 100 to 3,000 micrograms. The earliest manifestations of toxicity were confined to the outer retina with each drug, with lamellar lysosomal inclusions in the retinal pigment epithelium as the earliest finding. However, the aminoglycosides displayed marked differences in the threshold dose required to produce toxic reactions, permitting the following ordering of toxicity: (most toxic) gentamicin greater than netilmicin = tobramycin greater than amikacin = kanamycin (least toxic).

Course of gentamicin nephrotoxicity.

Metabolic balance and morphologic studies were performed on rats receiving gentamicin 100 mg/kg/day for a period of 8--10 days and during the recovery period. Daily urine flow rate increased with the administration of gentamiccin and remained elevated up to 20 days following the discontinuation of gentamicin, although BUN and plasma creatinine were virtually normal 10 days after the discontinuation of gentamicin. During the development of renal failure means daily electrolyte excretion remained normal. During the recovery period, however, sodium and potassium excretion exceeded control values while chloride and net acid excretion remained normal. Proteinuria developed during the administration of gentamicin and returned to normal 6--10 days after the discontinuation of gentamicin. Ten days of netilmicin administration (150 mg/kg/day) resulted in only mild tubular degeneration and no azotemia.

In vitro toxicity of netilmicin and ofloxacin on corneal epithelial cells.

PURPOSE: To evaluate the in vitro cytotoxic effect of the aminoglycoside antibiotic netilmicin on rabbit corneal epithelial cells (SIRC) compared with ofloxacin, a commonly used fluoroquinolone ocular antibiotic. METHODS: SIRC cell cultures were incubated for 8 to 72 h in the presence and absence of netilmicin (1.5, 3, and 6 mg/mL) and equal concentrations of ofloxacin. Cell viability in treated and untreated SIRC cells was measured by both neutral red and MTT colorimetric assays at 8, 24, and 72 h, whereas changes in cell morphology were examined at 8, 24, 48, and 72 hours by the use of phase-contrast microscopy. RESULTS: Netilmicin, at all tested concentrations, failed to alter SIRC cell viability or morphology. In contrast, all concentrations of ofloxacin caused statistically significant dose- and time-dependent reductions in cell viability even after 8 h. After 72 h there was complete loss of cell viability. Morphologic examination of SIRC cells after 8 h of incubation with ofloxacin revealed that the fluoroquinolone antibiotic, at all concentrations, produced large numbers of dead cells, compromised intercellular contacts, and altered general morphology. After 48 h the cell monolayer was observed to be completely destroyed. CONCLUSION: Netilmicin, at the concentrations used, is an antibiotic devoid of obvious cellular toxicity and may also be considered as a suitable first-choice drug in the treatment of those pathologies that compromise the integrity of the ocular surface.

Synthesis of 5-deoxy-5-fluoro- and 5-deoxy-5,5-difluoro-netilmicin.

5-Deoxy-5-fluoro- (9) and 5-deoxy-5,5-difluoro-netilmicin (27) have been prepared from the corresponding 5-epi and 5-oxo derivatives of netilmicin by treatment with DAST. Structures of the fluorinated by-products (10, 11, and 12) obtained in one of the synthesis of 9 were determined. 5-Epi-netilmicin (13) and 5-epi-6'-N-methyl-netilmicin (21) have also been prepared.

Studies of a potential in vitro test for estimation of toxicity of aminoglycoside antibiotics and polyamines.

The electrophoretic mobility of phosphatidyl inositol liposomes at pH 7.4, 25 degrees C, was reduced by aminoglycoside antibiotics, neamine and several polyamines in general accordance with the number of amino-groups on each molecule. There was good agreement between the relative position of the tested compounds on the mobility-concentration graph and available information about their relative mammalian toxicities in vivo. The slope of the graph for netilmicin was distinctively flat; a comparatively flat dose-response curve for netilmicin has been reported also from in vivo studies of nephrotoxicity. Investigation of a homologous series of alpha,omega straight chain diaminoalkanes revealed that hydrophobicity did not contribute significantly to the observed interaction in this system. L-Lysine showed the weakest effect amongst all compounds tested, supporting the view that the overall positive charge on the molecule was the major determinant of the observed effect. Further structure-activity work is required to confirm whether this 'in vitro' test is predictive of the toxicity of aminoglycoside antibiotics in man.

Vestibulo-ocular reflex (VOR) in guinea pigs. Impairment induced by aminoglycoside antibiotics.

The vestibulo-ocular reflexes (VORs) were studied in guinea pigs receiving daily administration of aminoglycoside antibiotics. The vestibular epithelia were also examined by scanning electron microscope technique (SEM). The treatment with aminoglycosides led to varying degrees of VORs according to (i) the type of aminoglycoside drug; (ii) the duration of the treatment and, (iii) the sensitivity of the various vestibular receptors. Gentamicin caused an earlier and severe reduction of the VOR gain. Dibekacin also caused evident damage, but the onset of its action was delayed. Both drugs affected mainly the vertical responses. Tobramycin and netilmicin altered the VORs slightly. Histological examination revealed damage to the sensory epithelia corresponding to the observed VOR impairments.

Aspects on the ototoxic potential of netilmicin.

The organ culture system using the 16th gestational day inner ear explant from fetal mice was used for ototoxicity screening of netilmicin. Organ cultures were incubated in 1, 10, 100 and 1000 micrograms/ml, respectively, of netilmicin base in the nutrient solution. After constant exposure to the drug during 5 days, the inner ears (N = 56) were analyzed morphologically at the light and electron microscopic levels. All specimens were serially sectioned. Exposure to 1 and 10 micrograms/ml of netilmicin base, doses that are reached during in vivo conditions in man, did not cause morphological damage to hair cells as observed in the light microscope at the end of the 5-day period. At the ultrastructural level, minor changes characteristic for aminoglycosides occurred in hair cell mitochondria. Incubation with 100 and 1000 micrograms/ml of netilmicin base caused morphological changes in many types of epithelia and not only in vestibular hair cells. This indicates a general sign of toxicity of netilmicin in these doses. The dose response relationship with regard to inner ear pathology was obvious in the vestibular part of the labyrinth. In the cochlea, however, few cytological changes occurred independent of dose. As compared with gentamicin, exposure in the same concentrations as in the present study and under similar experimental conditions, the less ototoxic effect of netilmicin was obvious. The results of the present study indicate that the less ototoxic potential of netilmicin as compared with other aminoglycosides is probably due to the drug itself and not primarily due to a poor penetration of netilmicin into the inner ear.

Netilmicin: a review of toxicity in laboratory animals.

The data on the toxicity of netilmicin in laboratory animals as well as preliminary data in man are reviewed. Netilmicin is less toxic to the VIIIth nerve than is gentamicin in all species tested. The data suggest that it probably is less ototoxic than tobramycin, although confirmatory studies should be performed. Netilmicin is also nephrotoxic than gentamicin in all species tested. It is less nephrotoxic than tobramycin in the rat and dog. Comparisons in the rat suggest that netilmicin has a flat dose--response curve that resembles the curve produced by streptomycin. In animals, netilmicin produces more neuromuscular blockade than gentamicin; however, neuromuscular blockade with aminoglycosides in man is rare and thus far no episodes have been associated with netilmicin during clinical investigation. Initial clinical studies in man indicate that netilmicin is efficacious and well tolerated. Presently available data suggest that netilmicin offers distinct advantages over older aminoglycosides. Final conclusions must await prospective randomized double-blind trials in man.

Pharmacokinetic parameters of netilmicin and protective effect of piperacillin regarding nephrotoxicity caused by netilmicin.

The pharmacokinetic interaction of Netilmicin and Piperacillin has been studied as well as the potential protective effect that Piperacillin exert on nephrotoxicity caused by Netilmicin, when both antibiotics are administered to rabbits by single and multiple dosage regimens. Netilmicin was administered at a dose of 7 mg/kg and 12 h interval, which allometrically correspond to 5 mg/kg at 24 h interval for men. Piperacillin was administered at a dose of 280 mg/kg at 12 h interval (the total number of doses of both antibiotics was 20). After single and multiple dose regimens plasma level curves of Netilmicin and renal concentration were determined using an HPLC technique. Besides that, an histologic study was carried out by electronic microscopy to determine the renal damage. A significant variation of some pharmacokinetic parameters of Netilmicin such as Vc and t(1/2) was observed when Netilmicin is administered together with Piperacillin; a similar modification in the renal accumulation and renal damage caused by Netilmicin was shown.

[Reproduction study on netilmicin. (1) Teratological study in rats (author's transl)].

Teratological study on netilmicin (NTL), a new aminoglycoside antibiotic, was carried out in Sprague-Dawley rats (Slc : SD). NTL was administered intramuscularly to female rats from day 7 to day 17 of gestation at the dosages of 12.5, 25, 50 and 100 mg/kg. The decrease of food intake at the dosage of 50 mg/kg and more, and the resultant depression of maternal body weight gain at the dosage of 100 mg/kg were observed in dams receiving NTL. The depression of fetal growth, such as body weight and ossification of the sternebrae and caudal vertebrae, were detected in animals treated with 50 and 100 mg/kg of NTL. However, NTL failed to induce the external, visceral and skeletal anomalies in fetuses. Also, NTL did not cause any significant changes in birth rate, suckling rate, weaning rate, body weight, postnatal development, behavior and reproductive performance in F1. These results suggest that NTL has no adverse effect on rat fetuses and F1 generation at the dosage of 25 mg/kg or less.

[Reproduction study on netilmicin. (2) Fertility study in rats (author's transl)].

Fertility study on netilmicin (NTL), a new aminoglycoside antibiotic, was carried out in Sprague-Dawley rats (Slc : SD). NTL was administered intramuscularly to male rats at the daily dose of 12.5, 25, 50 and 100 mg/kg from 6 to 15 weeks of age for 9 weeks before mating and during the mating period, and to 10 weeks old female rats at the daily dose levels from day 14 before mating through day 7 after gestation. The increase of kidney weight at the dose of 12.5 mg/kg and more, the decreases of food intake and body weight were observed in treated male rats. The decreases of food intake and body weight were observed in female rats treated with the dose of 50 and 100 mg/kg. No dose-related changes were observed in mating and fertility ratios of parent animals, numbers of corpora lutea and implantations, fetal mortality, external, visceral and skeletal anomalies, body weight, body length and tail length of fetuses. Therefore, it can be concluded that maximum non-toxic dose level of NTL on rat fertility is 100 mg/kg.

[Reproduction study on netilmicin. (3) Perinatal and postnatal study in rats (author's transl)].

Perinatal and postnatal study on netilmicin (NTL), a new aminoglycoside antibiotic, was carried out in Sprague-Dawley rats (Slc : SD). NTL was administered intramuscularly from day 17 of gestation throughout day 20 after delivery at the daily dose of 12.5, 25, 50 and 100 mg/kg. Water intake of pregnant and nursing dams was increased in the animals treated with 50 mg/kg or more of NTL. The increase of cecum weight was observed in F1 animals in all treated groups at 3 weeks of age. However, birth rate, suckling rate, weanling rate, body weight, postnatal development, behavior and reproductive function remained within normal ranges in all treated groups.