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.

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.

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.

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.

Apoptosis in renal proximal tubules of rats treated with low doses of aminoglycosides.

Kidney cortex apoptosis was studied with female Wistar rats treated for 10 days with gentamicin and netilmicin at daily doses of 10 or 20 mg/kg of body weight and amikacin or isepamicin at daily doses of 40 mg/kg. Apoptosis was detected and quantitated using cytological (methyl green-pyronine) and immunohistochemical (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining, in parallel with a measurement of drug-induced phospholipidosis (cortical phospholipids and phospholipiduria), cortical proliferative response ((3)H incorporation in DNA and histoautoradiography after in vivo pulse-labeling with [(3)H]thymidine), and kidney dysfunction (blood urea nitrogen and creatinine). Gentamicin induced in proximal tubules a marked apoptotic reaction which (i) was detectable after 4 days of treatment but was most conspicuous after 10 days, (ii) was dose dependent, (iii) occurred in the absence of necrosis, and (iv) was nonlinearly correlated with the proliferative response (tubular and peritubular cells). Comparative studies revealed a parallelism among the extents of phospholipidosis, apoptosis, and proliferative response for three aminoglycosides (gentamicin >> amikacin congruent with isepamicin). By contrast, netilmicin induced a marked phospholipidosis but a moderate apoptosis and proliferative response. We conclude that rats treated with gentamicin develop an apoptotic process as part of the various cortical alterations induced by this antibiotic at low doses. Netilmicin, and still more amikacin and isepamicin, appears safer in this respect. Whereas a relation between aminoglycoside-induced tubular apoptosis and cortical proliferative response seems to be established, no simple correlation with phospholipidosis can be drawn.

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.

Activation of aminoglycoside antibiotics to cytotoxins.

We have previously postulated an enzymatic transformation of gentamicin (to a metabolite or an 'activated' molecule) as part of its ototoxic action. Here we test with eight aminoglycosides whether the proposed mechanism applies to these antibiotics as a group. Drugs were activated by incubation with a subcellular fraction from liver, and cytotoxicity was tested in a bioassay using isolated outer hair cells from guinea pig. None of the aminoglycosides compromised the viability of the cells when assayed directly, i.e. without a preceding activation. In contrast, all clinically ototoxic aminoglycosides tested were significantly cytotoxic following the incubation. Neamine, considered to be non-ototoxic, did not yield a cytotoxin. A subcellular fraction from cochlear lateral wall tissues also converted gentamicin to a cytotoxin. The results support the hypothesis that activation of aminoglycosides precedes their toxic actions and demonstrate that the capability for activation is not confined to liver.

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.

Effects of diltiazem on netilmicin-induced nephrotoxicity in rabbits.

Aminoglycoside nephrotoxicity remains a common clinical problem and is the major cause of acute toxic renal failure in hospitalized patients. In recent studies, calcium channel blockers gave controversial results in the prevention of acute ischemic or toxic renal failure. The aims of the study were (i) to describe a rabbit model of mild renal failure (50% reduction in glomerular filtration rate with a mean value of 1.78 +/- 0.46 ml/kg/min) induced by netilmicin given intramuscularly at 20 mg/kg of body weight every 8 h for 5 days, (ii) to investigate the protective effect of diltiazem given at a therapeutic dose (1 mg/kg given intramuscularly every 8 h for 5 days), and (iii) to investigate the mechanisms of this protection through evaluation of function tests, optic histology, and glomerular morphometry. Animals treated with netilmicin and diltiazem exhibited an unchanged glomerular filtration rate compared with controls (3.39 +/- 0.58 versus 3.68 +/- 0.78 ml/kg/min, respectively). This protective effect was not associated with any change in systemic or renal hemodynamics (i.e., no change in renal plasma flow) or changes in the pharmacokinetics of netilmicin, as assessed by fractional excretion and cortical uptake. Netilmicin-induced tubular toxicity was unchanged by diltiazem. Our results suggest that (i) netilmicin exhibits a toxic effect at both the glomerular and the tubular levels, (ii) diltiazem, a calcium channel blocker, when given at low therapeutic doses, is able to prevent the aminoglycoside-induced renal failure through a potential glomerular mechanism. The precise mechanisms of the protection remain to be elucidated. These results deserve clinical evaluation in high-risk patients.

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.

[Study of the ototoxicity of amikacin and netilmicin using provoked acoustic oto-emissions and high-frequency audiometry]. / Etude de l'ototoxicité de l'amikacine et de la nétilmicine au moyen des oto-émissions acoustiques provoquées et de l'audiométrie hautes fréquences.

To compare the ototoxicity of amikacin and netilmicin, tone audiometry, high-frequency audiometry, early auditory evoked potentials, and evoked otoacoustic emission testing were used to evaluate 30 patients (15 under amikacin and 15 under netilmicin). Ototoxicity was not significantly different in the two groups.

Pharmacokinetic and toxicological evaluation of a once-daily regimen versus conventional schedules of netilmicin and amikacin.

The safety and pharmacokinetics of netilmicin (6.6 mg/kg) and amikacin (14.5 mg/kg) once daily (od) have been compared to their corresponding conventional schedules thrice daily (tid), and twice daily (bd), in patients (20 per group) suffering from pelvic inflammatory disease. Sensitive criteria of early renal and auditory alterations, namely urinary excretion of phospholipids and audiometry over a wide frequency range (0.25-18 kHz), respectively, were used. The first criterion (phospholipiduria) was validated by an animal study which demonstrated that rats receiving poly-L-aspartic acid, which protects against gentamicin-induced nephrotoxicity, are also protected against renal phospholipidosis and phospholipiduria caused by this antibiotic. On that basis, netilmicin od was better tolerated than netilmicin tid. Amikacin caused less phospholipiduria than netilmicin, and, given od, resulted in little increase over baseline (95% CI, 95-147% increase). Reduction in threshold by greater than or equal to 15 dB for frequencies between 10-18 kHz occurred in nine of 19 patients receiving netilmicin tid compared with three or four of 19 or 20 patients treated with netilmicin od or amikacin (od or bd). However, changes at lower frequencies (0.25-8 kHz) were infrequent with all regimens (from 0/19 to 2/20). In conclusion, these very sensitive tests of nephro- and oto-toxicity suggest that od dosing of amikacin or netilmicin is, if anything, safer than bd or tid dosing.

The impact of different dosing regimens of the aminoglycosides netilmicin and amikacin on vestibulotoxicity in the guinea pig.

The purpose of this study was to assess whether vestibulotoxicity caused by aminoglycoside antibiotics is influenced by the treatment schedule used: i.e., a single high dose given once daily (o.d.) vs multiple divided doses (tres in die, t.i.d.). Two groups of guinea pigs (5 animals/group) were injected intramuscularly for 21 days with either netilmicin or amikacin 150 mg/kg o.d., whereas other groups received each drug at 50 mg/kg t.i.d. at 8-h intervals. Amikacin was also given at 225 mg/kg o.d. and 75 mg/kg t.i.d. Vestibular functions were assessed by measuring vestibulo-ocular reflexes. Acoustic function was also evaluated by measuring Preyer's pinna reflex. Sensory epithelia of the inner ears were evaluated histologically under a scanning electron microscope. Netilmicin failed to affect either the vestibular or the acoustic apparatus in the animal groups receiving the two dosing regimens. Amikacin in a dose of 150 mg/kg per day induced an acoustic deficit which was more severe in the t.i.d. group. The higher dose of amikacin provoked significant lesions of acoustic and vestibular function, irrespective of the dosing regimen used. These data suggest that the o.d. dosing regimen of the aminoglycoside antibiotics might provide effective treatment for infectious diseases without enhancing the risk for vestibular and acoustic side effects.

Does administration of an aminoglycoside in a single daily dose affect its efficacy and toxicity?

Treatment efficacy, oto- and nephrotoxicity, and aminoglycoside pharmacokinetics were evaluated in a prospective, comparative, randomized clinical study of aminoglycosides given once a day or three times a day for severe infections. Sixty patients were treated with netilmicin or gentamicin 4.5 mg/kg bodyweight/day, either once a day or divided into three doses a day. The patients were allocated randomly to the different groups. The clinical effect was difficult to compare in the different groups, because of the small numbers of patients. Therapeutic failures were seen in seven patients (three after one and four after three doses per day). Two patients, one with Staphylococcus aureus endocarditis and one with streptococcal endocarditis, on netilmicin once daily and conventional high-dose therapy with a penicillin had positive blood cultures after five and seven days of treatment, respectively. Vestibular function and hearing acuity were examined by serial audiograms and electronystagmograms. In spite of extensive diagnostic evaluation, only two cases of ototoxicity were detected. One patient treated with gentamicin three times a day developed vertigo and a severe abnormality of her electronystagmogram. One young patient treated with gentamicin once daily had a slight bilateral reduction of hearing. Nephrotoxicity was mild and did not differ in the four treatment groups. This was the first investigation of a once-daily dosing regimen conducted in seriously ill patients with systemic infections. We could not demonstrate any evidence that aminoglycoside treatment once daily has greater oto- or nephrotoxicity than the traditional three times daily regimen.

Validity of N-acetyl-beta-D-glucosaminidase (NAG) determination in assessing netilmicin nephrotoxicity in preterm babies.

The supposed nephrotoxicity of netilmicin has been assessed in preterm neonates using the urinary excretion of a lysosomal enzyme as marker: N-acetyl-beta-D-glucosaminidase (NAG). 17 male preterm neonates with birth weight appropriate for gestational age were enrolled in a study where 9 received netilmicin therapy since the first day of life and 8 served as control group. We observed a significant increase in urinary NAG/creatinine ratio during the postnatal days in the netilmicin group babies followed by a regular decrease during the days after the end of therapy. If this increase in lysosomal enzymuria such as NAG could reflect netilmicin nephrotoxicity on the proximal tubular cell, many questions remain unanswered about the exact significance of this finding. In particular, its relation with tubular cell dysfunction remains to be established.

High-performance liquid chromatographic determination of netilmicin in guinea-pig and human serum by fluorodinitrobenzene derivatization with spectrophotometric detection.

A high-performance liquid chromatographic procedure for netilmicin determination in guinea-pig and human serum using pre-column derivatization with 1-fluoro-2,4-dinitrobenzene and UV detection is described. Linearity was established over the range 0.5-40 micrograms/ml using only 50 microliters of serum. Accuracy and precision were good, with a mean coefficient of variation less than 5% and a mean relative error less than 4%. This procedure correlates well with an enzyme multiplied immunoassay technique and has a sensitivity similar to those of published fluorescence derivatization methods.

[Nephrotoxicity of gentamicin and netilmicin in the rat. Quantification and qualification of glomeruli]. / Nefrotoxicidad de gentamicina y netilmicina en rata. Cuantificación y cualificación de glomérulos.

The application via i.v. jugular, in rats, of two fluorochromes (rhodamine 6GO and phosphine 3R) with a 10 min interval, allows the direct observation of nephronal corpuscles using an epifluorescent microscopical technique, as well as their eventual quantification and qualification into normal or semifunctional corpuscles. In rats previously treated by i.p. injection of gentamicin or netilmycin a decrease of the active nephronal population as well as an increase of the percentage of semifunctionality has been noticed.

Aminoglycoside and nephrotoxicity.

The effect of three aminoglycosides--gentamicin, netilmicin and amikacin--on renal acid excretion was studied in male rats treated with doses equivalent to those clinically used. The amikacin and netilmicin groups showed no important changes in the values of glomerular filtration rate (GFR), effective renal plasma flow (ERPF) and U/P inulin ratio during normal and acidotic conditions. The gentamicin group, however, showed a clear tendency to decreases in these functional parameters even in normal conditions, a finding that reinforces the concept that gentamicin is more nephrotoxic than other aminoglycosides. During normal conditions net acid excretion (BH) did not change with any of the three tested drugs. However, after an acute acid load BH markedly fell regardless of the antibiotic used. The capacity to elevate the urine-blood pCO2 was preserved after an alcaline overload, suggesting that the distal tubule was not significantly affected by aminoglycoside treatment. These data suggest that the clinical use of aminoglycosides during metabolic acidosis deserves close attention due to the possible deleterious effect that can emerge as the result of an inappropriate retention of acid loads.

In utero aminoglycosides-induced nephrotoxicity in rat neonates.

Pregnant Wistar females were treated with gentamicin (G), netilmicin (N) or amikacin (A) during two periods of pregnancy covering organogenesis and the beginning of nephrogenesis. Deliveries occurred normally. We studied functional effects--influence of sex, litter size, diuresis, creatinine clearance, G-kidney concentration, and kidney morphological alterations--in rat neonates on day 1 of life. After G and N, the creatinine clearance of the neonates was decreased according to the dosage given to the mother. Whatever the aminoglycoside, kidneys presented proximal tubular alterations (close to those observed in adults) at protonic microscopy and, with electron microscopy, some modifications of distal tubules and of mature and immature glomeruli. It is concluded that the developing kidney can be altered after treating pregnant mothers with aminoglycosides. This model of in utero-induced nephrotoxicity is dose-dependent. Mature and/or immature structures could be affected. The toxicity of the investigated antibiotics could be asserted as G greater than or equal to N greater than A.