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Chlorine is widely used as a drinking water disinfectant to ensure water security. However, the transformation mechanisms of its degradation of emerging pollutants within the water distribution system (WDS) is insufficiently understood. Thus, the kinetics, degradation byproducts, and toxicity of the chlorination of enoxacin (ENO, a type of emerging pollutant) were explored in a pilot-scale WDS for the first time. It was found that the chlorination rate of ENO was higher in deionized water (DW) than in the pilot-scale WDS, and the degradation followed second-order kinetics in DW. The degradation efficiency was found to be sensitive to pH, and was highest at a pH of 7.4. The chlorination rate of ENO increased with increasing temperature in both DW and WDS. For different pipe materials, the relative performance of ENO chlorination efficiency followed the order of steel pipe > ductile iron pipe > polyethylene (PE) pipe. Seven intermediates were identified during ENO chlorination, and the primary oxidation reaction involved the cleavage of the piperazine group. Finally, it was found that the potential for chlorine toxicity in treated drinking water in the presence of ENO is higher than it is without this pollutant., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Multiple changes in metabolic levels could be useful for understanding physiological toxicity. To explore further risk factors for the convulsions induced by the interaction of nonsteroidal anti-inflammatory and new quinolone antimicrobial drugs, the effect of sparfloxacin, enoxacin, and felbinac on fatty acid metabolism and glucose concentrations in the liver, brain, and blood of rats was investigated. The levels of long-chain acyl-CoAs (C(18:1) and C(20:4)) in the liver and brain were decreased at the onset of convulsions induced by the coadministration of enoxacin with felbinac. Then, glucose concentrations in the liver and blood were decreased, whereas they were increased in a dose-dependant manner in the brain. However, the formation of acyl-CoAs and glucose levels in the liver, brain, and blood was not significantly influenced by enoxacin, felbinac, and sparfloxacin alone, respectively. The disturbance of both fatty acid metabolism and glucose levels might be associated with the increased susceptibility to convulsions, which may contribute to further understanding of the toxic effects associated with these drugs.

The poor bioavailability and therapeutic response exhibited by conventional ophthalmic solutions due to rapid precorneal elimination of the drug may be overcome by the use of a gel system. The present work describes the formulation and evaluation of an ophthalmic delivery system containing an antibacterial agent, enoxacin, based on the concept of ophthalmic sustained gel, in which 2-hydroxypropyl-beta-cyclo-dextrin (HP-beta-CD) was used as a penetration enhancer in combination with hydroxypropylmethylcellulose (Methocel F4M) which acted as a vehicle. The developed formulation was therapeutically efficacious, nonirritant, and provided sustained release of the drug over 8 h period in vitro and 7 h period in vivo. The developed system is a viable alternative to conventional eye drops.

Objective and Background: Our previous studies suggest that little adverse effect on the growth of the periodontal ligament would be expected, if tetracycline, minocycline, ofloxacin, roxithromycin, clarithromycin, and azithromycin were topically administered to the periodontal pocket at their MIC90 doses required to inhibit the growth of 90% of periodontopathic bacteria, including Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans. In the present study, we investigated the cytocidal effects of eight antibacterial agents on the human gingival epithelial cell line NDUSD-1. We also used NDUSD-1 cells to examine the effects of these agents on the mRNA and protein expressions of genes associated with the proliferation, differentiation, or cellular adhesion important to the epithelial regeneration of the periodontal attachment., Methods: The cytocidal effect of the test agents was measured as a decrease in cell survival. To obtain a quantitative measure of the cytocidal effect, the LD50, i.e. the concentration which results in a 50% decrease in cell survival relative to the controls, was extrapolated from the concentration-response curves. The effects of the agents on the mRNA and protein expressions in NDUSD-1 cells were studied by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses, respectively., Results: The cytocidal effect increased in a concentration-dependent manner as the concentration of each of the eight test agents increased. The order of the agents according to their cytocidal effects (LD50) was minocycline > tetracycline > enoxacin > clarithromycin > roxythromycin approximately ofloxacin > azithromycin > erythromycin. The cytocidal effects of minocycline, tetracycline, enoxacin, clarithromycin, roxythromycin, ofloxacin, and azithromycin ranged from 1.2 to 23.2 times greater than that of erythromycin. The maximum non-cytocidal concentrations (MNCCs) of these agents for NDUSD-1 cells were: 0.3 microm for minocycline, 1 microm for tetracycline, 3 microm for ofloxacin and erythromycin, 10 microm for enoxacin, clarithromycin, and azithromycin, and 100 microm for roxythromycin. The MNCCs of ofloxacin, azithromycin, clarithromycin, and roxythromycin were greater than their MIC90 concentrations for periodontopathic bacteria described above. The effects on the mRNA and protein expressions of epithelial-cell- or cell-adhesion-related genes were examined in NDUSD-1 cells exposed to clarithromycin, roxythromycin, ofloxacin, and azithromycin at their MNCCs. None of the agents affected the mRNA expressions of five genes: keratinocyte growth factor receptor, keratin 18, integrin beta1, integrin beta4, and laminin 5gamma2. Clarithromycin and ofloxacin slightly decreased the protein expression of integrin beta4. Roxythromycin markedly decreased the protein expressions of integrin beta4 and laminin 5gamma2. Azithromycin had little inhibitory effects on the protein expressions of any of the five genes., Conclusions: These results suggest that little, if any, adverse effects on growth, differentiation, and adhesion of basal epithelial cells would be expected with topical administration of clarithromycin, ofloxacin or azithromycin to the periodontal pocket at a dose equivalent to the MIC90. It is important to note, however, that the extrapolation of these findings to in vivo conditions has yet to be undertaken.

Convulsant activity of pazufloxacin mesilate (PZFX mesilate), a new quinolone antibacterial agent for intravenous use, in combination with nonsteroidal anti-inflammatory drug (NSAID) was investigated in mice after intravenous or intracerebroventricular administration. Following results were obtained. 1. In combination with 4-biphenylacetic acid (BPAA) at an oral dose of 100 mg/kg, PZFX mesilate did not induce any convulsions at intravenous doses up to 200 mg/kg. Reference quinolones induced convulsions at the following intravenous doses: Enoxacin (ENX), 3.13 mg/kg or more; norfloxacin (NFLX) and lomefloxacin (LFLX), 6.25 mg/kg or more; ciprofloxacin (CPFX), 50 mg/kg or more; sparfloxacin (SPFX) and temafloxacin (TMFX), 100 mg/kg or more; fleroxacin (FLRX), 200 mg/kg. 2. PZFX mesilate at an intravenous dose of 50 mg/kg did not induce convulsions in mice after oral administration of any of 14 kinds of NSAIDs. It induced convulsions at 200 mg/kg in combination with aspirin at an oral dose of 600 mg/kg, while it did not with the other 13 kinds of NSAIDs. 3. Convulsion-inducing dose of PZFX mesilate after intracerebroventricular administration was 100 mg/body, which was higher than those of reference quinolones (NFLX, CPFX, ENX, LFLX, TMFX, levofloxacin, ofloxacin, FLRX and SPFX) and beta-lactam antibiotics (penicillin G, cefazoline, imipenem/cilastatin and panipenem/betamipron). In addition, concurrent dosing of BPAA (1 microgram/body) did not reduce the convulsion-inducing dose of PZFX mesilate. These results suggest that PZFX mesilate has remarkably weak convulsant activity.

Phototoxicity of pazufloxacin mesilate (PZFX mesilate), a novel parenteral quinolone antimicrobial agent, were evaluated in vitro and in vivo studies. In vitro, phototoxicity for cultured cells of PZFX, which is active principle of PZFX mesilate, was studied, and stability for long-wavelength ultraviolet (UVA) was examined. In vivo, phototoxicity tests in guinea pigs and rats, and photoallergenicity tests in guinea pigs were conducted. In the phototoxicity test on cultured cells, CHL/IU cells were irradiated UVA of 300-3000 mJ/cm2 in the presence of PZFX, ofloxacin (OFLX), lomefloxacin (LFLX) or sparfloxacin (SPFX) at 10 micrograms/mL. Phototoxic potencies for cultured cells of the quinolones tested were SPFX > LFLX > OFLX > PZFX. In addition, changes in ultraviolet absorption spectrum and residual rate of PZFX, OFLX, LFLX and SPFX were examined after UVA irradiation of 300-3000 mJ/cm2 to each solution. PZFX was stable for UVA compared with OFLX and LFLX. In the phototoxicity test of guinea pigs, each quinolone was administered intraperitoneally daily for 7 days, and UVA of about 11 J/cm2 was irradiated at 30 minutes after the last administration. Dose levels of each quinolone were 65 and 130 mg/kg of PZFX mesilate (dose levels converted to PZFX: 50 and 100 mg/kg), 50 and 100 mg/kg of nalidixic acid (NA), 100 mg/kg of OFLX, enoxacin (ENX), ciprofloxacin (CPFX), LFLX and SPFX. Grade of skin reaction (erythema) at 24 hours after UVA irradiation decreased in the order: SPFX > CPFX > NA > ENX = OFLX > LFLX > PZFX mesilate. Thus, PZFX mesilate was found to have the weakest phototoxicity. In the maximum plasma concentration of quinolones from 0.5 to 2.5 hours after administration, corresponding to the time of UVA irradiation, the concentration of the group administered PZFX mesilate was about 4.1 times higher than that of CPFX group, and about 1.3 times higher than that of SPFX group. The area under the blood concentration-time curve (AUC0.5-2.5) of the group administered PZFX mesilate was the same as that of SPFX group, and about 3.2 times larger than that of CPFX group. These data showed that phototoxicity of PZFX mesilate was also weaker than that of CPFX or SPFX in consideration of AUC0.5-2.5. In the phototoxicity test of rats injected intravenously, no phototoxicity was observed at 130 mg/kg of PZFX mesilate. In the photoallergenicity test of guinea pigs, no photoallergenicity was observed by PZFX mesilate. As mentioned above, from in vitro studies PZFX was found to be stable for UVA irradiation compared with OFLX and LFLX, and phototoxicity for cultured cells of PZFX was weaker than that of SPFX, LFLX or OFLX. In addition, from in vivo studies phototoxicity of PZFX mesilate was found to be weaker than that of NA, OFLX, ENX, CPFX, LFLX or SPFX, and no photoallergenicity was observed. Therefore, photosensitive potency of PZFX mesilate might be less than that of other quinolones.

The toxicity of Enoxacin (ENX), a fluoroquinolone antibacterial agent, on the testis and epididymis was studied in rats. ENX was administered to 5 male rats orally once daily for 2 weeks at the dose level of 3000 mg/kg/day. ENX-treated rats showed a marked decrease in body weight, and two of them died on Day 10. At the end of the dosing period, absolute weights of the epididymis were decreased; in contrast, relative weights of testis were increased in the ENX-treated group. On histopathological examination, testis of ENX-treated rats exhibited the following regressive changes: degeneration of spermatids and spermatocytes, retention of Step 19 spermatids, chromatin margination in nuclei of spermatids, multinucleated giant cell formation, and/or vacuolar degeneration of Sertoli cells. Additionally, desquamated cell debris was observed in the epididymis. Degenerative spermatids and spermatocytes were strongly positive by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). From these results, it is concluded that a 2-week treatment is sufficient to detect toxic effects of ENX on reproductive organs in male rats, and that testicular toxicity induced by ENX is associated with germ cell apoptosis.

1. Contribution of nitric oxide to the convulsive seizures induced by fluoroquinolones (FQs) coadministered with 4-biphenyl acetic acid (BPAA), the active metabolite of fenbufen, was assessed in mice. 2. Enoxacin + 4-biphenyl acetic acid caused clonic seizures in all treated mice, followed by tonic seizures and death. These events were associated with a significant increase in intracerebellar cyclic GMP. 3. Pretreatment with the nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine methylester (L-NAME), but not with D-NAME, significantly reduced the incidence of convulsions and lethality, as well as the increase in cyclic GMP. 4. Pretreatment with N-methyl-D-aspartic acid (NMDA)-receptor antagonist, MK-801, inhibited only the transition of clonic seizure to tonic seizure without affecting the incidence of clonic seizure and lethality. 5. These findings suggest that FQs + BPAA exert convulsions by activating NOS partly through the mediation of the NMDA receptor in the brain cells.

Convulsions induced by the interaction of new quinolone antimicrobial agents (NQs) and nonsteroidal anti-inflammatory drugs (NSAIDs) were previously reported, and blockade of GABA(A) receptor by NQs and its potentiation with NSAIDs were considered as one of its possible mechanisms. However, useful methodology for prediction of convulsive potencies of NQs with or without NSAIDs in vivo based on in vitro screening was not established. Therefore, we applied the Xenopus oocytes translation system of exogenous messenger RNA (mRNA) to examine the mechanism of convulsion induced by interaction of NQs and NSAIDs, and the relationship between convulsive potencies in vivo and inhibitory effect on GABA-induced current response in vitro was investigated. This system also has alternative possibility for the in vivo toxicological studies sacrificing innumerous animals. Glutamic acid, kainic acid, quisqualic acid, NMDA, and serotonin-induced currents were not modified by ENX of NQs and/or FLB of NSAIDs, while glycine- and ACh-induced currents were slightly inhibited. GABA (10 microM)-induced current was inhibited by norfloxacin (NFLX), ciprofloxacin, ENX, and ofloxacin (OFLX) with IC50 of 17, 33, 58, and 280 microM, respectively. IC50 of NQs decreased to 1/3 (OFLX)-1/165 (NFLX) in the presence of 10 microM FLB, while FLB did not modulate the GABA response in the absence of NQs. CSF concentration of ENX at the time of convulsion in clinical situation approximated the IC50 of ENX for the GABA response. The increase of incidence for NQs-induced convulsion by concomitant NSAIDs in vivo could also be explained by the potentiation of inhibitory effects of NQs with FLB in the normal range of CSF concentration of these drugs. We also examined convulsive potency (threshold dose for convulsion) in CNS by intracerebral infusion of NQs to mice with or without FLB pretreatment, and significant correlations between the convulsive potencies and IC50 of NQs for the GABA response were observed. These findings suggested that the blockade of GABA-ersic neurotransmission in CNS is a dominant mechanism of convulsion induced by NQs and that the convulsant-adverse reaction of NQs in vivo may be predicted from the inhibitory effect on the GABA(A) receptor in vitro using the Xenopus oocytes translation system of exogenous mRNA.

We investigated the effect of acute renal failure on the neurotoxicity of enoxacin (ENX) in rats. Experimental acute renal failure was produced by bilateral ureteral ligation. ENX was intravenously infused to ureter ligated (UL) and control rats, and its concentration in plasma, brain and cerebrospinal fluid (CSF) was compared. Plasma concentration of ENX increased rapidly in UL rats as compared with control rats. Brain/plasma concentration ratio (Kp)-time profile of ENX was similar in UL and control rats. Brain concentration of ENX at the occurrence of convulsion did not depend on the infusion rate, suggesting that in the brain tissue it equilibrates rapidly with the site of action for clonic convulsion. Brain concentration of ENX in UL rats at the occurrence of clonic convulsion was lower than that in control rats. A similar tendency was also observed with CSF concentration. In conclusion, the potentiation of neurotoxicity of ENX with acute renal failure may be caused by not only decreased capability for renal elimination of ENX but also increased sensitivity to convulsant activity of ENX in the central nervous system.

To elucidate the mechanism of phototoxicity induced as a side effect by some of the new quinolone antibiotics, we studied sparfloxacin (SPFX), lomefloxacin, enoxacin, ofloxacin, and ciprofloxacin. We first examined the photosensitized formation of reactive oxygen species such as singlet oxygen (1O2) and superoxide anion (O2-) mediated by the new quinolones. Although a large number of studies have been reported, there is no direct evidence that these drugs generate reactive oxygen species. We employed a near-infrared emission spectrometer to detect 1O2-specific emission (1268 nm), and the nitroblue tetrazolium reduction method to detect O2-. All the quinolones investigated in this study were found to produce 1O2. Four drugs, but not SPFX, produced O2-. We also examined photodynamic DNA strand-breaking activity as a possible mechanism to explain the participation of reactive oxygen species in the phototoxicity of the drugs. All the drugs exhibited photodynamic DNA strand-breaking activity. The inhibitory effect of scavengers of reactive oxygen species indicated that the main active species was 1O2. The DNA strand-breaking activity was correlated not with the 1O2-forming ability, but with the affinity of the drugs for DNA. This result may be due to the short lifetime of 1O2. These data suggested that the phototoxicity of the new quinolones was related to DNA damage caused by reactive oxygen species, especially 1O2.

Susceptibility to enoxacin (80 mg/kg, p.o.)-induced convulsions was examined in mice coadministered with fenbufen (100 mg/kg, p.o.) over 24 hr at 3-hr intervals (light 7:00-19:00 hr). There was a marked circadian variation in the incidence of clonic and tonic convulsions and mortality. The susceptibility to enoxacin was higher around 15:00-18:00 hr and lower around 3:00-9:00 hr; the 50% clonic convulsive dose (CD50) at 9:00 and 15:00 hr was 95.0 and 56.5 mg/kg, respectively, its ratio being 1.64. Under these conditions, brain enoxacin level at 15:00 hr was increased 2.43-fold over that at 9:00 hr 30 min after enoxacin administration. Thus, the change of brain enoxacin may contribute to one of the causes of the above circadian variation.

Epileptogenic activity induced by combined treatment with antiinflammatory drugs and enoxacin was investigated in chronic electrode-implanted rats. Ferubinac ethyl and aspirin DL-lysine showed a spike and wave complex in EEG without showing remarkable behavioral changes when they were injected intraventricularly, although a relatively high dose was needed. Enoxacin, on the other hand, elicited potent epileptogenic activity characterized by uninterrupted high voltage spike and wave complex at doses of 50 and 100 micrograms. At the same time, rats showed hyperactivity, jumping and violent convulsion. Combined treatment with enoxacin (p.o.) and ferubinac ethyl (i.v.) caused potent epileptogenic activity characterized by uninterrupted burst of high voltage spike and wave complex. Behaviorally, animals showed forelimb clonus, head nodding and generalized convulsion. High voltage spike and wave complex was also observed after combined treatment with enoxacin (i. vent.) and ferubinac ethyl (i.v. or i. vent.) in association with hyperactivity and jumping and violent convulsion. Nicardipine remarkably inhibited epileptic seizures induced by combined treatment with enoxacin (p.o.) and ferubinac ethyl (i.v.). It is concluded that simultaneous treatment with enoxacin and ferubinac ethyl produced epileptogenic activity when injected intraventricularly, and nicardipine inhibited convulsions induced by combined use of enoxacin (p.o.) and ferubinac ethyl (i.v.).

Convulsions induced by the combination of enoxacin, a new antimicrobial, and nonsteroidal anti-inflammatory drugs including nimesulide, ketoprofen, pranoprofen and loxoprofen sodium, were investigated in mice. The oral administration of nimesulide alone induced clonic convulsions at more than 300 mg/kg. The oral administration of ketoprofen, pranoprofen or loxoprofen sodium induced no convulsion up to 1000 mg/kg, 500 mg/kg and 600 mg/kg, respectively, and that of enoxacin induced no convulsion at more than 5000 mg/kg. The combination of nimesulide at 200 mg/kg and enoxacin at 400 mg/kg induced no convulsion. In contrast, the combination of enoxacin at 100 mg/kg and either ketoprofen at 125 mg/kg or pranoprofen at 500 mg/kg induced clonic convulsions, while that of enoxacin at 400 mg/kg and loxoprofen sodium at 600 mg/kg induced no convulsion. These results suggest that the combination of nimesulide and enoxacin may possibly induce few or less convulsions in the clinical setting.

We investigated the possible potentiation of the convulsive toxicity of enoxacin (ENX) by the concomitant topical application of a felbinac (FLB) patch in rats. A felbinac patch (Seltouch; 0.5%, 3 cm x 4 cm) was attached on the back of rats where their hair has been removed. ENX was infused from the left jugular vein at 8 h after the application of FLB patch under an unanesthetized and unstrained condition. Blood, CSF and brain samples were collected at the occurrence of convulsion, and ENX concentrations of each part were determined. As a result, no significant potentiation by FLB patch was found in the onset time of convulsion or in the ENX concentration of each part. Moreover, based on the assumption that there are no inter-species differences in ENX concentration in the brain at the occurrence of a convulsion (Cbr), the predicted plasma ENX concentration required to elicit convulsions in humans, which was estimated from the Cbr and Kp value of ENX in the brain of rats, was 20 times higher than the therapeutic plasma level.

The phototoxic effects of quinolone antimicrobial agents on mouse auricular skin and retina were examined histologically. Sparfloxacin at 50 or 100 mg/kg, which alone causes no histologic change, was orally administered to albino Balb/c mice, which were irradiated with ultraviolet A for 4 hr immediately after administration. In the auricle, degeneration of basal epidermal cells was sporadically observed at 2 hr (during the irradiation). Foci of slight edema with degenerated fibroblasts were seen in the dermis at 4 hr. Edema and neutrophil infiltration in the dermis became severe up to 96 hr. Initial changes in the retina were observed at 2 hr. Vacuolation of the photoreceptor segments (particularly the inner segment) was occasionally associated with swelling of retinal pigment epithelial cells. The segments became disorganized with time, and the outer nuclear layer showed reduced cellularity. The segments and layer were partially thinned and lost 96 hr later. Enoxacin at 400 and 800 mg/kg induced similar lesions to those of sparfloxacin. Levofloxacin caused similar lesions in the auricle but no change in the retina. The combination of oral administration of quinolone and ultraviolet A irradiation, which never caused apparent morphological changes alone, was shown to be able to induce phototoxic lesions in albino mice. Therefore, this method is thought to be useful to examine morphological changes caused by quinolone phototoxicity.

The phototoxic potential of (-)-(S)-9-fluoro-2,3-dihydro-3-methyl-10-(4- methyl-1-piperazyl)-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6- carboxylic acid hemihydrate (levofloxacin, DR-3355, CAS 100986-85-4) was evaluated by the mice ear swelling reaction. Results were compared with those for enoxacin (ENX). Mice were orally administered DR-3355 or ENX and immediately exposed to ultraviolet-A for 4 h. Phototoxicity was determined by measuring ear thickness developing 24 h after irradiation. DR-3355 provided no changes in the ear at 200 mg/kg, but caused ear swelling at 800 mg/kg. ENX caused severe phototoxicity in a dose dependent manner from 50 mg/kg. These data suggest that DR-3355 has minor phototoxic potential, but is approximately 4-fold less toxic than ENX in these experimental conditions.

Temafloxacin hydrochloride (HCl) was studied in fenbufen-treated mice and for its effects on the locomotor activity of rats. Mice were observed for convulsions for 90 minutes after the administration of temafloxacin HCl (100 mg/kg orally [p.o.]), ciprofloxacin HCl (100 mg/kg p.o.), enoxacin (100 mg/kg p.o.), or oxolinic acid (100 mg/kg p.o.). The quinolones were coadministered with either saline (0.2 mL p.o.) or fenbufen (100 mg/kg p.o.), a nonsteroidal antiinflammatory compound. No convulsions occurred following the administration of the quinolones with saline. Clonic convulsions occurred in 90% of mice receiving enoxacin and fenbufen and in 20% of mice receiving ciprofloxacin HCl and fenbufen. No convulsions occurred in mice receiving temafloxacin HCl or oxolinic acid with fenbufen. At doses of 30, 100, and 300 mg/kg p.o., temafloxacin HCl and enoxacin had no effect on locomotor activity of rats, but oxolinic acid markedly stimulated locomotor activity. The lack of central nervous system (CNS) stimulation and the absence of interaction with fenbufen suggest that temafloxacin may have less potential for adverse CNS effects than other quinolone compounds.