LABORATORY SCIENCE
Comparison of toxicities of moxifloxacin, cefuroxime, and levofloxacin to corneal endothelial cells in vitro Tomoko Haruki, MD, Dai Miyazaki, MD, Kazuki Matsuura, MD, Yuki Terasaka, MD, Yumiko Noguchi, BSc, Yoshitsugu Inoue, MD, Satoru Yamagami, MD
PURPOSE: To evaluate and compare the toxic effects of moxifloxacin, cefuroxime, and levofloxacin on human corneal endothelial cells in vitro and determine the safe intracameral concentrations for them. SETTING: Tottori University, Tottori, Japan. DESIGN: Experimental study. METHODS: Human corneal endothelial cells in culture were exposed to moxifloxacin, cefuroxime, and levofloxacin at concentrations up to 2000 mg/mL. Evaluation of membrane damage was determined by ethidium homodimer-1 uptake and cell viability, by intrinsic esterase activity. The inhibitory effects of the 3 antibiotics on the constitutive secretion of interleukin-6 (IL-6) by human corneal endothelial cells were determined by enzyme-linked immunosorbent assay. RESULTS: The acute effects (6 hour) of the 3 antibiotics on membrane damage and cell death were dose-dependent for moxifloxacin and levofloxacin (R500 mg/mL). For cefuroxime, membrane damage was not observed at 6 hours and only slight damage was detected at 24 hours at concentrations higher than 500 mg/mL. The half maximum inhibitory concentrations on cell viability of moxifloxacin, levofloxacin, and cefuroxime were 487 mg/mL, 578 mg/mL, and 1600 mg/mL, respectively. The inhibitory effects of the 3 antibiotics on the constitutive secretion of IL-6 were observed at 15.6 mg/mL or higher, indicating the antibiotics can impair the secretion of the protective cytokine even at low concentrations. CONCLUSIONS: Moxifloxacin at more than 500 mg/mL caused damage to the cell membranes of corneal endothelial cells; even higher concentrations decreased cell viability. Considering the lower minimum inhibitory concentration for inhibiting 90% growth by moxifloxacin, intracameral moxifloxacin at 500 mg/mL or less is recommended for prophylactic use. Financial Disclosure: Dr. Inoue is a medical advisor to Alcon Japan Ltd. No author has a financial or proprietary interest in any material or method mentioned. J Cataract Refract Surg 2014; 40:1872–1878 Q 2014 ASCRS and ESCRS
Endophthalmitis after cataract surgery is a visually devastating complication, with an incidence ranging from 0.014% to 1.238%.1–5 However, the incidence varies greatly in different ethnic populations and with different surgical techniques. Intracameral injection of antibiotics has been used prophylactically worldwide to prevent endophthalmitis, and earlier studies indicate that intracameral antibiotics can reduce the incidence of endophthalmitis by 6- to 22-fold.6,7 1872
Q 2014 ASCRS and ESCRS Published by Elsevier Inc.
The effectiveness of intracameral antibiotics appears independent of the region of the world or the rate of endophthalmitis. For example, its preventive effect is recognizable even in facilities with lower rates of infectious endophthalmitis.7 Intracameral cefuroxime has been favored in Europe as an adjunctive perioperative prophylaxis based on the results of pilot studies by Montan et al.8,9 In addition, intracameral cefuroxime was recommended by the European Society of Cataract and Refractive http://dx.doi.org/10.1016/j.jcrs.2014.08.027 0886-3350
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Surgery (ESCRS)10 to prevent endophthalmitis and to treat it after cataract surgery. Currently, cefuroxime is widely used as a prophylactic intracameral antibiotic in Europe based on the ESCRS study.11 However in the United States and other countries, ophthalmic cefuroxime is not readily available or not convenient for routine use. In addition, the efficacy of cefuroxime is time-dependent and requires prolonged exposure; however, the intracameral concentration of cefuroxime has been shown to decrease below the minimum inhibitory concentration (MIC) after 6 to 8 hours.9 Moxifloxacin was introduced as an intracameral antibiotic in about 2007.12–14 Although evidence of its efficacy was not determined in randomized trials, intracameral moxifloxacin has several advantages. Moxifloxacin is a fourth-generation fluoroquinolone and has broad-spectrum coverage. It is readily available in a preservative-free sterile solution as Vigamox. In addition, its bactericidal effect is concentrationdependent and concentrations higher than the MIC kill bacteria within 1 to 2 hours.13 Moxifloxacin inhibits bacterial DNA gyrase and topoisomerase IV, and its multipoint inhibitory effect makes moxifloxacin less likely than levofloxacin, an older generation fluoroquinolone, to cause an emergence of resistant bacteria. Although intracameral moxifloxacin and cefuroxime are being used for perioperative prophylaxis, the information on which drug is better or what the optimum treatment regimen should be is limited.15–18 For example, the currently used concentrations were empirically determined and appear not to be completely tested for their purpose or safety. In addition, there has been some concern about their toxicity to corneal endothelial cells when used intracamerally. There are anecdotal reports by members of the American Society of Cataract and Refractive Surgery (ASCRS) of corneal endothelial injury after the intracameral use of antibiotics.19 Thus, the purpose of this
Submitted: April 3, 2014. Final revision submitted: August 2, 2014. Accepted: August 4, 2014. From the Division of Ophthalmology and Visual Science (Haruki, Miyazaki, Matsuura, Terasaka, Noguchi, Inoue), Faculty of Medicine, Tottori University, Yonago Tottori, and the Corneal Transplantation Section (Yamagami), University of Tokyo Graduate School of Medicine, Tokyo, Japan. Corresponding author: Dai Miyazaki, MD, Division of Ophthalmology and Visual Science, Faculty of Medicine, Tottori University, 36-1 Nishi-cho, Yonago Tottori 683-8504, Japan. E-mail: dm@ grape.med.tottori-u.ac.jp.
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study was to evaluate the in vitro toxicity of moxifloxacin, cefuroxime, and levofloxacin by examining corneal endothelial cells for cell damage, viability, and cytokine secretion in vitro. MATERIALS AND METHODS Cells A human corneal endothelial cell line was established by transduction with human telomerase reverse transcriptase (hTERT) and the large T gene using retroviral vectors, BABE-hygro-hTERT for hTERT and MFG-tsT-IRES-neo for simian virus 40 large T antigen.20 The human corneal endothelial cells were propagated to confluence on 96-well plates in Dulbecco modified Eagle's medium (Gibco, Life Technologies Corp.) supplemented with 15% fetal bovine serum.
Assay of Cell Viability and Membrane Damage To determine whether moxifloxacin, levofloxacin, and cefuroxime were toxic to human corneal endothelial cells, the study examined whether the cell membrane was damaged and also whether the cells were alive. Cell viability after exposure to the antibiotics was evaluated by measuring the esterase activity of the cells in serum-depleted medium. The human corneal endothelial cells (1 104 cells/well) were grown in 96-well plates for 24 hours and treated with serially diluted cefuroxime, levofloxacin, or moxifloxacin (Vigamox). After the selected times, membrane damage and cell death were measured by the uptake of ethidium homodimer-1 (EthD-1, Molecular Probes), which enters cells with damaged membrane and produce a red fluorescence. The study assessed how exposure to the 3 antibiotics affected ethidium homodimer-1 (EthD-1, Molecular Probes) uptake by the human corneal endothelial cells. The EthD-1 uptake was measured with a fluorescent microplate reader (Tecan Group AG) with excitation by 495 nm and emission at 635 nm. Dead cells were used as a positive control and were prepared by exposing human corneal endothelial cells to saponin 0.1% (Sigma-Aldrich Co.) for 10 minutes. To evaluate the toxicity of the antibiotics, the study evaluated the cell viability by measuring the intracellular esterase activity using calcein-acetoxymethyl ester (calcein-AM, Molecular Probes) as an esterase substrate. Human corneal endothelial cells were exposed to nonfluorescent calceinAM, which is converted to fluorescent calcein by the esterase activity of living cells. The optical density of the fluorescence due to the esterase activity was measured by a fluorescent microplate reader with excitation at 495 nm and emission at 530 nm. The percentages of living cells and degree of membrane damage were calculated as follows: % Z (antibiotics-treated cell emission untreated cell emission)/(positive control cell emission untreated cell emission) 100.
Enzyme-Linked Immunosorbent Assay After microbial infections, IL-6 is abundantly produced by corneal endothelial cells and the cells also induce numerous antimicrobial proteins and cytokines.20,21 Because IL-6 is constitutively produced by corneal endothelial cells,21 the study assessed whether exposure to the 3 antibiotics affected the constitutive IL-6 synthesis. The supernatants of the human corneal endothelial cells were collected 72 hours after
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antibiotic exposure, and the level of interleukin-6 (IL-6) was measured with an enzyme-linked immunosorbent assay (ELISA) kit (Peprotech). The supernatants were diluted 20fold with the diluent of the kit according to the manufacturer's instructions. The supernatant was incubated on the captured antibody-coated plates overnight at 4 C and processed for ELISA measurements.
Statistical Analysis Data are presented as the means G standard error of the means. Statistical analyses were performed using t tests or analysis of variance as appropriate.
was observed with 500 mg/mL of moxifloxacin and with 1000 mg/mL of levofloxacin. In contrast, cefuroxime did not lead to a significant increase in the number of dead cells or membrane damage up to 4000 mg/mL. Thus, moxifloxacin and levofloxacin were more toxic and increased the percentage of dead or membrane damaged human corneal endothelial cells when the concentration was over 500 mg/mL.
RESULTS
Evaluation of Antibiotic Toxicity of Moxifloxacin, Levofloxacin, and Cefuroxime by Ethidium Homodimer-1 Uptake and by Cellular Intrinsic Esterase Activity at 24 Hours
Comparisons of Antibiotic Toxicity to Human Corneal Endothelial Cells in Vitro Measured by Ethidium Homodimer-1 Uptake After 6 Hours At 6 hours, no gross abnormalities were observed by phase contrast microscopy. However, the exposure to moxifloxacin and levofloxacin led to cell membrane damage and the degree of damage was dosedependent at 6 hours (Figure 1, A). A significant increase in the number of membrane-damaged cells
Similar to the outcome after 6-hour exposure, a significant increase in the uptake of EthD-1 was detected with 500 mg/mL of moxifloxacin and levofloxacin (Figure 1, B). Thus, the 24-hour exposure to moxifloxacin and levofloxacin did not intensify the toxic effects at 6 hours. For cefuroxime, a significant increase in D1 uptake was observed with 500 mg/mL or more. A significant decrease of esterase activity indicating a decrease in cell viability was observed with
Figure 1. Comparative effects of moxifloxacin (MFLX), levofloxacin (LVFX), and cefuroxime (CXM) on membrane integrity of human corneal endothelial cells in vitro (n Z 6). Human corneal endothelial cells were exposed to the 3 antibiotics individually at the indicated concentrations and assessed for cell membrane damage and/or viability at 6 hours (A) and 24 hours (B). The uptake of EthD-1 was used (* Z P!.05; ** Z P!.01; *** Z P!.005; **** Z P!.001). J CATARACT REFRACT SURG - VOL 40, NOVEMBER 2014
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1000 mg/mL or more of moxifloxacin and levofloxacin (P!.05) (Figure 2). For cefuroxime, a significant decrease in esterase activity was observed at concentrations of 2000 mg/mL or more (P!.05). After exposure to 1000 mg/mL cefuroxime, the human corneal endothelial cells retained significantly more esterase activity than after moxifloxacin and levofloxacin (Figure 2). Regarding the concentrations of antibiotics that inhibited cell growth by 50% (IC50), after 24-hour exposure, the IC50 of moxifloxacin, levofloxacin, and cefuroxime was 487 mg/mL, 578 mg/mL, and 1600 mg/mL, respectively, indicating that cefuroxime was less toxic when used at the same concentration. Within the range of 31.25 to 125 mg/mL, an increase in esterase activity was observed after antibiotic exposure, presumably due to the exposure-induced cell proliferation.
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dose-dependent manner and reached a plateau at approximately 1000 mg/mL. The 3 antibiotics also led to a decrease in the secretion of IL-10, another inflammatory cytokine (data not shown). Cefuroxime exposure led to less impairment of IL-6 secretion than moxifloxacin and levofloxacin at concentrations of 15.6 mg/mL or higher (Figure 3). DISCUSSION
Antibiotic Effect on Constitutive Secretion of Cytokine Interleukin-6 All 3 antibiotics significantly impaired IL-6 secretion at concentrations as low as 15.6 mg/mL (P!.005) (Figure 3). This impairment was exacerbated in a
At present, the most widely used intracameral antibiotics are moxifloxacin and cefuroxime.2,4,12,14,22,23 However, it has not been definitively determined whether one is better than the other or whether one is more toxic to corneal endothelial cells. Our results showed that the toxic properties of moxifloxacin and cefuroxime on human corneal endothelial cells are different. To our knowledge, there are no clinical data directly comparing cefuroxime and moxifloxacin. Because the clinical efficacy of prophylactic antibiotics highly depends on the microbial spectrum causing the endophthalmitis, we focused on the safety of cefuroxime and moxifloxacin. We believed that the comparative toxicity should be evaluated using the same platform
Figure 2. Comparative toxicity of moxifloxacin (MFLX), levofloxacin (LVFX), and cefuroxime (CXM) on viability of human corneal endothelial cells (n Z 6). The human corneal endothelial cells were exposed to the antibiotics treated with moxifloxacin, levofloxacin, or cefuroxime at the indicated concentrations and assessed for cell viability at 24 hours by measuring the intrinsic esterase activity of the cells (* Z P!.05; ** Z P!.005; *** Z P!.001).
Figure 3. Comparative effects of moxifloxacin (MFLX), levofloxacin (LVFX), and cefuroxime (CXM) at the indicated concentrations on cytokine secretion by human corneal endothelial cells in vitro (n Z 6). Corneal endothelial cells were exposed to moxifloxacin, levofloxacin, or cefuroxime at the indicated concentration for 72 hours. The supernatant was measured for secreted IL-6 by ELISA (* Z P!.05; ** Z P!.005; *** Z P!.001; IL-6 Z interleukin-6).
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in the same experiments. For example, as we show in Figures 2 and 3, the IC50 values of each antibiotic was markedly different and depended on the method of testing the toxicity. Thus, extrapolation of IC50 values from other studies using different platforms does not ensure that the interpretation of the toxicity comparisons is valid. For cefuroxime, the currently used intracameral dose of 1000 mg/0.1 mL is reduced to 2614 mg/mL G 209 (SD) within 30 seconds by dilution in the aqueous humour.9 For moxifloxacin, an intracameral dose of 100 to 1700 mg/mL is used.12,14,22–24 Thus, in this study, the concentrations of toxicity reported in previous studies were tested. Our in vitro results suggest that both antibiotics can be toxic when high concentrations are maintained for 6 to 24 hours. However, no clear evidence of adverse effects has been reported when their effects were evaluated by slitlamp and specular microscopy.10 Although the 2007 ASCRS member survey19 showed that corneal endothelial injury can arise from intracameral antibiotics, it was not stated which antibiotic was used and whether the toxicity was caused by the preparation process for intracameral use. Thus, our results provide information on the toxic effects of the 3 antibiotics tested. Together with the known MIC for 90% growth (MIC90) of each antibiotic, our findings should help surgeons optimize their administration protocol to balance safety and effectiveness. We found that the toxicity of moxifloxacin on the human corneal endothelial cells occurred much earlier and at lower concentrations than that after cefuroxime. For cell viability, the IC50 of moxifloxacin was 487 mg/mL and that of cefuroxime was 1600 mg/mL. The IC50 of moxifloxacin for 24-hour exposures is in good agreement with the reported value of 500 mg/mL for corneal endothelial cells.18 When other ocular cells were examined for toxicity or viability for exposures of 24 hours, the IC50 of moxifloxacin was 350 mg/mL for trabecular meshwork cells and retinal pigment epithelial cells.15,18 When membrane damage was measured by EthD-1 uptake, damage was observed at 6 hours for moxifloxacin. This was unexpectedly early based on findings in earlier studies.15,18 The acute membrane damage effect was not detectable with less than 500 mg/mL of moxifloxacin and levofloxacin; however, the damage was greatly increased when moxifloxacin and levofloxacin concentrations higher than 500 mg/mL were used. Thus, the toxicity of high doses of moxifloxacin and levofloxacin can occur at 6 hours or perhaps earlier and does not need a long exposure of 24 hours. The toxicity of moxifloxacin on corneal endothelial cell indicates that high doses of moxifloxacin have to be used judiciously. For example, it is known that
the intracameral concentration of antibiotics can be prolonged by inflammation or the condition of the patient.25,26 In addition, corneal endothelial cells with preexisting subclinical dysfunction, such as cornea guttata, pseudoexfoliation, or Fuchs dystrophy, would be more sensitive to the antibiotics. For cefuroxime, toxicity on the membrane integrity was not observed at 6 hours but rather required 24 hours and with 500 mg/mL or more. For intracamerally administered cefuroxime based on the ESCRS recommendation, the concentration is reduced to 1027 G 43 mg/mL in the aqueous humor at 1 hour after injection.9 In general, the half-life of intracameral antibiotics is approximately 1 hour.27 Thus, the clinically used cefuroxime concentration is assumed to be at levels without a toxic effect on cell viability. Considering the effectiveness of cefuroxime, the MIC90 of cefuroxime is less than 10 mg/mL for susceptible bacteria and a concentration exceeding MIC90 should be maintained for approximately 7 hours. This would validate the safety and efficacy of an intracameral injection of 1 mg/0.1 mL of cefuroxime indicated in the ESCRS guideline.10 However in the ESCRS study, Pseudomonas species, methicillin-resistant Staphylococcus aureus (MRSA), and Enterococcus species were not identified as the causative bacteria for the endophthalmitis. The cephalosporin-resistant species are more prevalent in different countries.28–30 In addition, a nationwide use of intracameral cefuroxime may cause an emergence of cefuroxime-resistant strains.2 This is a concern about the continuous use of cefuroxime as a standard prophylactic treatment. Moxifloxacin is a fourth-generation quinolone and has a wider spectrum of activity and much lower MIC than cefuroxime. In addition, moxifloxacin is conveniently available as a preservative-free solution. Thus, moxifloxacin has the potential of becoming a standard prophylactic drug. However, different from cefuroxime, the recommended dose of moxifloxacin has not been definitively determined. When using moxifloxacin, we believe that standardization of the protocol should be performed to avoid acute toxic effects by potential mishandling of the solution. Moxifloxacin should be used as a preservativefree solution, such as Vigamox 0.5% solution. The concentration of this solution is 5000 mg/mL. In general, the anterior chamber volume is approximately 0.25 to 0.3 mL. To achieve an initial anterior chamber concentration of 100 to 500 mg/mL, Vigamox 0.5% has to be diluted 3 to 10 times and 0.1 mL injected. Clinically, moxifloxacin doses between 100 mg/mL and 1700 mg/mL of estimated anterior chamber concentration have been used for intracameral injections with no apparent reported side effects.12,14,22,24 We recently reported that endothelial dysfunction was
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not observed for 19 000 cases treated with prophylactic intracameral injection of moxifloxacin at ranges from 100 mg/mL to 500 mg/mL.23 Thus, moxifloxacin appears to have been cleared from the anterior chamber after an intracameral injection and without causing permanent damage. In addition, intracameral injections of 0.1 mL of Vigamox 0.5% have not caused long-term toxicity to the corneal endothelium 1 month postoperatively.12 Thus, our cell culture results may indicate a narrower therapeutic safe dose than seen in real-life clinical situations. The MIC90 of moxifloxacin is less than 1 mg/mL for most susceptible bacteria that might cause endophthalmitis. For MRSA, Enterococcus species, or Pseudomonas species, moxifloxacin MIC90 should be within 50 mg/mL. Because the bactericidal properties of the quinolones are concentration-dependent, their action with concentrations more than MIC90 will require only 1 to 2 hours. Considering the half-life of intracameral moxifloxacin is approximately 1 hour, 500 mg/mL of moxifloxacin would achieve a concentration 4 times higher than the MIC90 of the 50 mg/mL for the first 1 to 2 hours. This indicates that this dose would be effective for the refractory strains without noticeable endothelial damage. Thus, we propose a dose of 500 mg/mL for moxifloxacin, which should be safe for general prophylactic use. We also evaluated levofloxacin as a third-generation fluoroquinolone. Levofloxacin had very similar toxic effects on the membrane and viability as moxifloxacin. However, levofloxacin had an MIC90 almost 4-fold higher than that of moxifloxacin. These findings favor the use of moxifloxacin. Interleukin-6 is a canonical mediator to combat infections, and it is a cytokine that is induced in corneal endothelial cells after microbial infection.20,21 Interleukin-6 is abundantly and constitutively secreted by the corneal endothelial cells. When alterations of the cytokine secretion by antibiotics were evaluated, we observed a significant impairment in the constitutive secretion of IL-6 at a surprisingly low concentration of 15.6 mg/mL for all 3 antibiotics. However, no loss of endothelial function by intracameral antibiotics has been reported12,14,22,24; this impairment appears reversible and does not lead to a permanent reduction in cell viability. However, we must be aware that the protective arm mediated by IL-6 can be impaired by intracameral antibiotics. This disarmament might cause adverse effects when intracameral antibiotics are used, especially for resistant strains. Clinically, fluoroquinolone-resistant strains are emerging.29 For fluoroquinolone-resistant S aureus and S epidermidis, moxifloxacin does not have an appreciable bactericidal effect even when 500 mg/mL was used for 3 hours (data not shownA). This suggests
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the necessity and importance of well-organized perioperative prophylactic measures and not solely relying on intracameral antibiotic injections. There are several limitations of this study. The current analysis was based on in vitro study of a corneal endothelial cell line; thus, the data may underestimate or overestimate the adverse effects of intracameral antibiotic use. To determine the optimum dose of intracameral moxifloxacin or other antibiotics more accurately, well-organized clinical trial-based studies will be required. In conclusion, moxifloxacin and cefuroxime have different toxic properties for corneal endothelial cells. Although moxifloxacin is a most promising alternative to cefuroxime, the treatment protocol of its concentration and indications have to be determined. WHAT WAS KNOWN Intracameral moxifloxacin and cefuroxime have been used as adjunctive perioperative prophylaxis. However, the currently used concentrations were empirically determined and appear not to be completely tested for their purpose or safety. In addition, no information is available to directly compare their potential toxicities. WHAT THIS PAPER ADDS Although moxifloxacin is a promising alternative to cefuroxime, moxifloxacin at more than 500 mg/mL causes acute damage (6 hours) to the membranes of corneal endothelial cells, and even higher concentrations decrease cell viability.
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OTHER CITED MATERIAL A. Data available (in Japanese) from Alcon Japan Ltd.
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First author: Tomoko Haruki, MD Division of Ophthalmology and Visual Science, Faculty of Medicine, Tottori University, Yonago Tottori, Japan
Comparison of toxicities of moxifloxacin, cefuroxime, and levofloxacin to corneal endothelial cells in vitro Tomoko Haruki, MD, Dai Miyazaki, MD, Kazuki Matsuura, MD, Yuki Terasaka, MD, Yumiko Noguchi, BSc, Yoshitsugu Inoue, MD, Satoru Yamagami, MD
PURPOSE: To evaluate and compare the toxic effects of moxifloxacin, cefuroxime, and levofloxacin on human corneal endothelial cells in vitro and determine the safe intracameral concentrations for them. SETTING: Tottori University, Tottori, Japan. DESIGN: Experimental study. METHODS: Human corneal endothelial cells in culture were exposed to moxifloxacin, cefuroxime, and levofloxacin at concentrations up to 2000 mg/mL. Evaluation of membrane damage was determined by ethidium homodimer-1 uptake and cell viability, by intrinsic esterase activity. The inhibitory effects of the 3 antibiotics on the constitutive secretion of interleukin-6 (IL-6) by human corneal endothelial cells were determined by enzyme-linked immunosorbent assay. RESULTS: The acute effects (6 hour) of the 3 antibiotics on membrane damage and cell death were dose-dependent for moxifloxacin and levofloxacin (R500 mg/mL). For cefuroxime, membrane damage was not observed at 6 hours and only slight damage was detected at 24 hours at concentrations higher than 500 mg/mL. The half maximum inhibitory concentrations on cell viability of moxifloxacin, levofloxacin, and cefuroxime were 487 mg/mL, 578 mg/mL, and 1600 mg/mL, respectively. The inhibitory effects of the 3 antibiotics on the constitutive secretion of IL-6 were observed at 15.6 mg/mL or higher, indicating the antibiotics can impair the secretion of the protective cytokine even at low concentrations. CONCLUSIONS: Moxifloxacin at more than 500 mg/mL caused damage to the cell membranes of corneal endothelial cells; even higher concentrations decreased cell viability. Considering the lower minimum inhibitory concentration for inhibiting 90% growth by moxifloxacin, intracameral moxifloxacin at 500 mg/mL or less is recommended for prophylactic use. Financial Disclosure: Dr. Inoue is a medical advisor to Alcon Japan Ltd. No author has a financial or proprietary interest in any material or method mentioned. J Cataract Refract Surg 2014; 40:1872–1878 Q 2014 ASCRS and ESCRS
Endophthalmitis after cataract surgery is a visually devastating complication, with an incidence ranging from 0.014% to 1.238%.1–5 However, the incidence varies greatly in different ethnic populations and with different surgical techniques. Intracameral injection of antibiotics has been used prophylactically worldwide to prevent endophthalmitis, and earlier studies indicate that intracameral antibiotics can reduce the incidence of endophthalmitis by 6- to 22-fold.6,7 1872
Q 2014 ASCRS and ESCRS Published by Elsevier Inc.
The effectiveness of intracameral antibiotics appears independent of the region of the world or the rate of endophthalmitis. For example, its preventive effect is recognizable even in facilities with lower rates of infectious endophthalmitis.7 Intracameral cefuroxime has been favored in Europe as an adjunctive perioperative prophylaxis based on the results of pilot studies by Montan et al.8,9 In addition, intracameral cefuroxime was recommended by the European Society of Cataract and Refractive http://dx.doi.org/10.1016/j.jcrs.2014.08.027 0886-3350
LABORATORY SCIENCE: ANTIBIOTIC TOXICITY TO CORNEAL ENDOTHELIAL CELLS
Surgery (ESCRS)10 to prevent endophthalmitis and to treat it after cataract surgery. Currently, cefuroxime is widely used as a prophylactic intracameral antibiotic in Europe based on the ESCRS study.11 However in the United States and other countries, ophthalmic cefuroxime is not readily available or not convenient for routine use. In addition, the efficacy of cefuroxime is time-dependent and requires prolonged exposure; however, the intracameral concentration of cefuroxime has been shown to decrease below the minimum inhibitory concentration (MIC) after 6 to 8 hours.9 Moxifloxacin was introduced as an intracameral antibiotic in about 2007.12–14 Although evidence of its efficacy was not determined in randomized trials, intracameral moxifloxacin has several advantages. Moxifloxacin is a fourth-generation fluoroquinolone and has broad-spectrum coverage. It is readily available in a preservative-free sterile solution as Vigamox. In addition, its bactericidal effect is concentrationdependent and concentrations higher than the MIC kill bacteria within 1 to 2 hours.13 Moxifloxacin inhibits bacterial DNA gyrase and topoisomerase IV, and its multipoint inhibitory effect makes moxifloxacin less likely than levofloxacin, an older generation fluoroquinolone, to cause an emergence of resistant bacteria. Although intracameral moxifloxacin and cefuroxime are being used for perioperative prophylaxis, the information on which drug is better or what the optimum treatment regimen should be is limited.15–18 For example, the currently used concentrations were empirically determined and appear not to be completely tested for their purpose or safety. In addition, there has been some concern about their toxicity to corneal endothelial cells when used intracamerally. There are anecdotal reports by members of the American Society of Cataract and Refractive Surgery (ASCRS) of corneal endothelial injury after the intracameral use of antibiotics.19 Thus, the purpose of this
Submitted: April 3, 2014. Final revision submitted: August 2, 2014. Accepted: August 4, 2014. From the Division of Ophthalmology and Visual Science (Haruki, Miyazaki, Matsuura, Terasaka, Noguchi, Inoue), Faculty of Medicine, Tottori University, Yonago Tottori, and the Corneal Transplantation Section (Yamagami), University of Tokyo Graduate School of Medicine, Tokyo, Japan. Corresponding author: Dai Miyazaki, MD, Division of Ophthalmology and Visual Science, Faculty of Medicine, Tottori University, 36-1 Nishi-cho, Yonago Tottori 683-8504, Japan. E-mail: dm@ grape.med.tottori-u.ac.jp.
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study was to evaluate the in vitro toxicity of moxifloxacin, cefuroxime, and levofloxacin by examining corneal endothelial cells for cell damage, viability, and cytokine secretion in vitro. MATERIALS AND METHODS Cells A human corneal endothelial cell line was established by transduction with human telomerase reverse transcriptase (hTERT) and the large T gene using retroviral vectors, BABE-hygro-hTERT for hTERT and MFG-tsT-IRES-neo for simian virus 40 large T antigen.20 The human corneal endothelial cells were propagated to confluence on 96-well plates in Dulbecco modified Eagle's medium (Gibco, Life Technologies Corp.) supplemented with 15% fetal bovine serum.
Assay of Cell Viability and Membrane Damage To determine whether moxifloxacin, levofloxacin, and cefuroxime were toxic to human corneal endothelial cells, the study examined whether the cell membrane was damaged and also whether the cells were alive. Cell viability after exposure to the antibiotics was evaluated by measuring the esterase activity of the cells in serum-depleted medium. The human corneal endothelial cells (1 104 cells/well) were grown in 96-well plates for 24 hours and treated with serially diluted cefuroxime, levofloxacin, or moxifloxacin (Vigamox). After the selected times, membrane damage and cell death were measured by the uptake of ethidium homodimer-1 (EthD-1, Molecular Probes), which enters cells with damaged membrane and produce a red fluorescence. The study assessed how exposure to the 3 antibiotics affected ethidium homodimer-1 (EthD-1, Molecular Probes) uptake by the human corneal endothelial cells. The EthD-1 uptake was measured with a fluorescent microplate reader (Tecan Group AG) with excitation by 495 nm and emission at 635 nm. Dead cells were used as a positive control and were prepared by exposing human corneal endothelial cells to saponin 0.1% (Sigma-Aldrich Co.) for 10 minutes. To evaluate the toxicity of the antibiotics, the study evaluated the cell viability by measuring the intracellular esterase activity using calcein-acetoxymethyl ester (calcein-AM, Molecular Probes) as an esterase substrate. Human corneal endothelial cells were exposed to nonfluorescent calceinAM, which is converted to fluorescent calcein by the esterase activity of living cells. The optical density of the fluorescence due to the esterase activity was measured by a fluorescent microplate reader with excitation at 495 nm and emission at 530 nm. The percentages of living cells and degree of membrane damage were calculated as follows: % Z (antibiotics-treated cell emission untreated cell emission)/(positive control cell emission untreated cell emission) 100.
Enzyme-Linked Immunosorbent Assay After microbial infections, IL-6 is abundantly produced by corneal endothelial cells and the cells also induce numerous antimicrobial proteins and cytokines.20,21 Because IL-6 is constitutively produced by corneal endothelial cells,21 the study assessed whether exposure to the 3 antibiotics affected the constitutive IL-6 synthesis. The supernatants of the human corneal endothelial cells were collected 72 hours after
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antibiotic exposure, and the level of interleukin-6 (IL-6) was measured with an enzyme-linked immunosorbent assay (ELISA) kit (Peprotech). The supernatants were diluted 20fold with the diluent of the kit according to the manufacturer's instructions. The supernatant was incubated on the captured antibody-coated plates overnight at 4 C and processed for ELISA measurements.
Statistical Analysis Data are presented as the means G standard error of the means. Statistical analyses were performed using t tests or analysis of variance as appropriate.
was observed with 500 mg/mL of moxifloxacin and with 1000 mg/mL of levofloxacin. In contrast, cefuroxime did not lead to a significant increase in the number of dead cells or membrane damage up to 4000 mg/mL. Thus, moxifloxacin and levofloxacin were more toxic and increased the percentage of dead or membrane damaged human corneal endothelial cells when the concentration was over 500 mg/mL.
RESULTS
Evaluation of Antibiotic Toxicity of Moxifloxacin, Levofloxacin, and Cefuroxime by Ethidium Homodimer-1 Uptake and by Cellular Intrinsic Esterase Activity at 24 Hours
Comparisons of Antibiotic Toxicity to Human Corneal Endothelial Cells in Vitro Measured by Ethidium Homodimer-1 Uptake After 6 Hours At 6 hours, no gross abnormalities were observed by phase contrast microscopy. However, the exposure to moxifloxacin and levofloxacin led to cell membrane damage and the degree of damage was dosedependent at 6 hours (Figure 1, A). A significant increase in the number of membrane-damaged cells
Similar to the outcome after 6-hour exposure, a significant increase in the uptake of EthD-1 was detected with 500 mg/mL of moxifloxacin and levofloxacin (Figure 1, B). Thus, the 24-hour exposure to moxifloxacin and levofloxacin did not intensify the toxic effects at 6 hours. For cefuroxime, a significant increase in D1 uptake was observed with 500 mg/mL or more. A significant decrease of esterase activity indicating a decrease in cell viability was observed with
Figure 1. Comparative effects of moxifloxacin (MFLX), levofloxacin (LVFX), and cefuroxime (CXM) on membrane integrity of human corneal endothelial cells in vitro (n Z 6). Human corneal endothelial cells were exposed to the 3 antibiotics individually at the indicated concentrations and assessed for cell membrane damage and/or viability at 6 hours (A) and 24 hours (B). The uptake of EthD-1 was used (* Z P!.05; ** Z P!.01; *** Z P!.005; **** Z P!.001). J CATARACT REFRACT SURG - VOL 40, NOVEMBER 2014
LABORATORY SCIENCE: ANTIBIOTIC TOXICITY TO CORNEAL ENDOTHELIAL CELLS
1000 mg/mL or more of moxifloxacin and levofloxacin (P!.05) (Figure 2). For cefuroxime, a significant decrease in esterase activity was observed at concentrations of 2000 mg/mL or more (P!.05). After exposure to 1000 mg/mL cefuroxime, the human corneal endothelial cells retained significantly more esterase activity than after moxifloxacin and levofloxacin (Figure 2). Regarding the concentrations of antibiotics that inhibited cell growth by 50% (IC50), after 24-hour exposure, the IC50 of moxifloxacin, levofloxacin, and cefuroxime was 487 mg/mL, 578 mg/mL, and 1600 mg/mL, respectively, indicating that cefuroxime was less toxic when used at the same concentration. Within the range of 31.25 to 125 mg/mL, an increase in esterase activity was observed after antibiotic exposure, presumably due to the exposure-induced cell proliferation.
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dose-dependent manner and reached a plateau at approximately 1000 mg/mL. The 3 antibiotics also led to a decrease in the secretion of IL-10, another inflammatory cytokine (data not shown). Cefuroxime exposure led to less impairment of IL-6 secretion than moxifloxacin and levofloxacin at concentrations of 15.6 mg/mL or higher (Figure 3). DISCUSSION
Antibiotic Effect on Constitutive Secretion of Cytokine Interleukin-6 All 3 antibiotics significantly impaired IL-6 secretion at concentrations as low as 15.6 mg/mL (P!.005) (Figure 3). This impairment was exacerbated in a
At present, the most widely used intracameral antibiotics are moxifloxacin and cefuroxime.2,4,12,14,22,23 However, it has not been definitively determined whether one is better than the other or whether one is more toxic to corneal endothelial cells. Our results showed that the toxic properties of moxifloxacin and cefuroxime on human corneal endothelial cells are different. To our knowledge, there are no clinical data directly comparing cefuroxime and moxifloxacin. Because the clinical efficacy of prophylactic antibiotics highly depends on the microbial spectrum causing the endophthalmitis, we focused on the safety of cefuroxime and moxifloxacin. We believed that the comparative toxicity should be evaluated using the same platform
Figure 2. Comparative toxicity of moxifloxacin (MFLX), levofloxacin (LVFX), and cefuroxime (CXM) on viability of human corneal endothelial cells (n Z 6). The human corneal endothelial cells were exposed to the antibiotics treated with moxifloxacin, levofloxacin, or cefuroxime at the indicated concentrations and assessed for cell viability at 24 hours by measuring the intrinsic esterase activity of the cells (* Z P!.05; ** Z P!.005; *** Z P!.001).
Figure 3. Comparative effects of moxifloxacin (MFLX), levofloxacin (LVFX), and cefuroxime (CXM) at the indicated concentrations on cytokine secretion by human corneal endothelial cells in vitro (n Z 6). Corneal endothelial cells were exposed to moxifloxacin, levofloxacin, or cefuroxime at the indicated concentration for 72 hours. The supernatant was measured for secreted IL-6 by ELISA (* Z P!.05; ** Z P!.005; *** Z P!.001; IL-6 Z interleukin-6).
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in the same experiments. For example, as we show in Figures 2 and 3, the IC50 values of each antibiotic was markedly different and depended on the method of testing the toxicity. Thus, extrapolation of IC50 values from other studies using different platforms does not ensure that the interpretation of the toxicity comparisons is valid. For cefuroxime, the currently used intracameral dose of 1000 mg/0.1 mL is reduced to 2614 mg/mL G 209 (SD) within 30 seconds by dilution in the aqueous humour.9 For moxifloxacin, an intracameral dose of 100 to 1700 mg/mL is used.12,14,22–24 Thus, in this study, the concentrations of toxicity reported in previous studies were tested. Our in vitro results suggest that both antibiotics can be toxic when high concentrations are maintained for 6 to 24 hours. However, no clear evidence of adverse effects has been reported when their effects were evaluated by slitlamp and specular microscopy.10 Although the 2007 ASCRS member survey19 showed that corneal endothelial injury can arise from intracameral antibiotics, it was not stated which antibiotic was used and whether the toxicity was caused by the preparation process for intracameral use. Thus, our results provide information on the toxic effects of the 3 antibiotics tested. Together with the known MIC for 90% growth (MIC90) of each antibiotic, our findings should help surgeons optimize their administration protocol to balance safety and effectiveness. We found that the toxicity of moxifloxacin on the human corneal endothelial cells occurred much earlier and at lower concentrations than that after cefuroxime. For cell viability, the IC50 of moxifloxacin was 487 mg/mL and that of cefuroxime was 1600 mg/mL. The IC50 of moxifloxacin for 24-hour exposures is in good agreement with the reported value of 500 mg/mL for corneal endothelial cells.18 When other ocular cells were examined for toxicity or viability for exposures of 24 hours, the IC50 of moxifloxacin was 350 mg/mL for trabecular meshwork cells and retinal pigment epithelial cells.15,18 When membrane damage was measured by EthD-1 uptake, damage was observed at 6 hours for moxifloxacin. This was unexpectedly early based on findings in earlier studies.15,18 The acute membrane damage effect was not detectable with less than 500 mg/mL of moxifloxacin and levofloxacin; however, the damage was greatly increased when moxifloxacin and levofloxacin concentrations higher than 500 mg/mL were used. Thus, the toxicity of high doses of moxifloxacin and levofloxacin can occur at 6 hours or perhaps earlier and does not need a long exposure of 24 hours. The toxicity of moxifloxacin on corneal endothelial cell indicates that high doses of moxifloxacin have to be used judiciously. For example, it is known that
the intracameral concentration of antibiotics can be prolonged by inflammation or the condition of the patient.25,26 In addition, corneal endothelial cells with preexisting subclinical dysfunction, such as cornea guttata, pseudoexfoliation, or Fuchs dystrophy, would be more sensitive to the antibiotics. For cefuroxime, toxicity on the membrane integrity was not observed at 6 hours but rather required 24 hours and with 500 mg/mL or more. For intracamerally administered cefuroxime based on the ESCRS recommendation, the concentration is reduced to 1027 G 43 mg/mL in the aqueous humor at 1 hour after injection.9 In general, the half-life of intracameral antibiotics is approximately 1 hour.27 Thus, the clinically used cefuroxime concentration is assumed to be at levels without a toxic effect on cell viability. Considering the effectiveness of cefuroxime, the MIC90 of cefuroxime is less than 10 mg/mL for susceptible bacteria and a concentration exceeding MIC90 should be maintained for approximately 7 hours. This would validate the safety and efficacy of an intracameral injection of 1 mg/0.1 mL of cefuroxime indicated in the ESCRS guideline.10 However in the ESCRS study, Pseudomonas species, methicillin-resistant Staphylococcus aureus (MRSA), and Enterococcus species were not identified as the causative bacteria for the endophthalmitis. The cephalosporin-resistant species are more prevalent in different countries.28–30 In addition, a nationwide use of intracameral cefuroxime may cause an emergence of cefuroxime-resistant strains.2 This is a concern about the continuous use of cefuroxime as a standard prophylactic treatment. Moxifloxacin is a fourth-generation quinolone and has a wider spectrum of activity and much lower MIC than cefuroxime. In addition, moxifloxacin is conveniently available as a preservative-free solution. Thus, moxifloxacin has the potential of becoming a standard prophylactic drug. However, different from cefuroxime, the recommended dose of moxifloxacin has not been definitively determined. When using moxifloxacin, we believe that standardization of the protocol should be performed to avoid acute toxic effects by potential mishandling of the solution. Moxifloxacin should be used as a preservativefree solution, such as Vigamox 0.5% solution. The concentration of this solution is 5000 mg/mL. In general, the anterior chamber volume is approximately 0.25 to 0.3 mL. To achieve an initial anterior chamber concentration of 100 to 500 mg/mL, Vigamox 0.5% has to be diluted 3 to 10 times and 0.1 mL injected. Clinically, moxifloxacin doses between 100 mg/mL and 1700 mg/mL of estimated anterior chamber concentration have been used for intracameral injections with no apparent reported side effects.12,14,22,24 We recently reported that endothelial dysfunction was
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not observed for 19 000 cases treated with prophylactic intracameral injection of moxifloxacin at ranges from 100 mg/mL to 500 mg/mL.23 Thus, moxifloxacin appears to have been cleared from the anterior chamber after an intracameral injection and without causing permanent damage. In addition, intracameral injections of 0.1 mL of Vigamox 0.5% have not caused long-term toxicity to the corneal endothelium 1 month postoperatively.12 Thus, our cell culture results may indicate a narrower therapeutic safe dose than seen in real-life clinical situations. The MIC90 of moxifloxacin is less than 1 mg/mL for most susceptible bacteria that might cause endophthalmitis. For MRSA, Enterococcus species, or Pseudomonas species, moxifloxacin MIC90 should be within 50 mg/mL. Because the bactericidal properties of the quinolones are concentration-dependent, their action with concentrations more than MIC90 will require only 1 to 2 hours. Considering the half-life of intracameral moxifloxacin is approximately 1 hour, 500 mg/mL of moxifloxacin would achieve a concentration 4 times higher than the MIC90 of the 50 mg/mL for the first 1 to 2 hours. This indicates that this dose would be effective for the refractory strains without noticeable endothelial damage. Thus, we propose a dose of 500 mg/mL for moxifloxacin, which should be safe for general prophylactic use. We also evaluated levofloxacin as a third-generation fluoroquinolone. Levofloxacin had very similar toxic effects on the membrane and viability as moxifloxacin. However, levofloxacin had an MIC90 almost 4-fold higher than that of moxifloxacin. These findings favor the use of moxifloxacin. Interleukin-6 is a canonical mediator to combat infections, and it is a cytokine that is induced in corneal endothelial cells after microbial infection.20,21 Interleukin-6 is abundantly and constitutively secreted by the corneal endothelial cells. When alterations of the cytokine secretion by antibiotics were evaluated, we observed a significant impairment in the constitutive secretion of IL-6 at a surprisingly low concentration of 15.6 mg/mL for all 3 antibiotics. However, no loss of endothelial function by intracameral antibiotics has been reported12,14,22,24; this impairment appears reversible and does not lead to a permanent reduction in cell viability. However, we must be aware that the protective arm mediated by IL-6 can be impaired by intracameral antibiotics. This disarmament might cause adverse effects when intracameral antibiotics are used, especially for resistant strains. Clinically, fluoroquinolone-resistant strains are emerging.29 For fluoroquinolone-resistant S aureus and S epidermidis, moxifloxacin does not have an appreciable bactericidal effect even when 500 mg/mL was used for 3 hours (data not shownA). This suggests
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the necessity and importance of well-organized perioperative prophylactic measures and not solely relying on intracameral antibiotic injections. There are several limitations of this study. The current analysis was based on in vitro study of a corneal endothelial cell line; thus, the data may underestimate or overestimate the adverse effects of intracameral antibiotic use. To determine the optimum dose of intracameral moxifloxacin or other antibiotics more accurately, well-organized clinical trial-based studies will be required. In conclusion, moxifloxacin and cefuroxime have different toxic properties for corneal endothelial cells. Although moxifloxacin is a most promising alternative to cefuroxime, the treatment protocol of its concentration and indications have to be determined. WHAT WAS KNOWN Intracameral moxifloxacin and cefuroxime have been used as adjunctive perioperative prophylaxis. However, the currently used concentrations were empirically determined and appear not to be completely tested for their purpose or safety. In addition, no information is available to directly compare their potential toxicities. WHAT THIS PAPER ADDS Although moxifloxacin is a promising alternative to cefuroxime, moxifloxacin at more than 500 mg/mL causes acute damage (6 hours) to the membranes of corneal endothelial cells, and even higher concentrations decrease cell viability.
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OTHER CITED MATERIAL A. Data available (in Japanese) from Alcon Japan Ltd.
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First author: Tomoko Haruki, MD Division of Ophthalmology and Visual Science, Faculty of Medicine, Tottori University, Yonago Tottori, Japan
