2008

Paper 4237 419

A B S T R A C T

ORIGINAL ARTICLE

Intrinsic cytotoxic effects of fluoroquinolones on human corneal keratocytes and endothelial cellsPadma bezwada, Leslie A. Clark and susan schneiderSanten Incorporated, Napa, CA, USA

Address for correspondence: Padma Bezwada, PhD, Santen Inc., 555 Gateway Drive, Napa, CA 94558, USA. Tel.: +1 707 256 2417; Fax: +1 707 254 1760; pbezwada@santeninc.com

Key words: Cornea – Cytotoxicity – Fluoroquinolones – Keratocytes

Introduction

Topical fluoroquinolones are the antibiotics most commonly used by eye care professionals. Fluoroquin-olones have been associated with rare cases of tendin opathy when used systemically1,2 and with corneal perforation when used topically in patients with bacterial keratitis3. The biologic mechanism is unknown although in vivo and in vitro studies in corneal cells suggest that fluoroquinolones may significantly increase matrix-degrading proteolytic activity, inhibit cell metabolism, and cause degenerative and ultrastructural cell changes secondary to alterations

in regulatory pathways of several cytokines4. Damage to corneal epithelial cells and keratocytes can lead to hazing and lower visual acuity. Because topical fluoroquinolones are applied in high con centrations when corneal integrity and repair processes may already be compromised by infection or trauma, we evaluated the intrinsic cytotoxicity of the fluoroquin-olones available as ophthalmic solutions using human keratocyte and endothelial cell cultures. Cells were exposed to drug concentrations to which corneal tissues might be exposed as topical fluoroquinolones diffuse from the corneal epithelium to the aqueous humor.

Objective: To determine the intrinsic cytotoxicity of five fluoroquinolones (ciprofloxacin, gatifloxacin, levofloxacin, moxifloxacin, ofloxacin) on human corneal keratocytes (HCK) and human corneal endothelial cells (HCE).

Research design and methods: Cultures of replicating HCK and HCE were exposed to ciprofloxacin, gatifloxacin, levofloxacin, moxifloxacin, or ofloxacin concentrations of 1 mg/mL, 100 μg/mL, 10 μg/mL, 1 μg/mL, 100 ng/mL, or 10 ng/mL for 15, 30, 60, or 240 min. Each of the 24 fluoroquinolone concentration–time exposures was tested against its own serum-free minimal essential medium (MEM) control. Cell number was quantified with a fluorescence bioassay.

Main outcome measure: Cytotoxicity was defined as a significant ( p < 0.05) difference in cell number measured as mean calcein

fluorescence product versus control for each fluoroquinolone concentration–time exposure.

Results: Fluoroquinolone-induced cytotoxicity was concentration- and time-dependent in HCK and HCE cultures. The number of cytotoxic concentration–time exposures was highest with ciprofloxacin (23 of 24 exposures in HCK and 24 of 24 exposures in HCE) and lowest with levofloxacin (10 of 24 exposures in both HCK and HCE).

Conclusions: In vitro cell cultures are useful for evaluating cell response to potentially toxic insults, although cell cultures may lack tissue components that may prevent or ameliorate damage in vivo. In this assay, fluoroquinolones displayed the potential to be cytotoxic to human corneal keratocytes and endothelial cells, depending on drug concentration and duration of exposure. The potential for cytotoxicity may differ among fluoroquinolones.

CURREnt MEdICAL REsEARCh And OPInIOn®

VOL. 24, NO. 2, 2008, 419–424

© 2008 LIbRAPhARM LIMItEd

0300-7995

doi:10.1185/030079908X261005

All rights reserved: reproduction in whole or part not permitted

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420 Fluoroquinolone cytotoxicity in human cornea © 2008 LIbRAPhARM Ltd – Curr Med Res 2008; 24(2)

Methodshuman corneal keratocyte and endothelial cell cultures

Primary and subsequent subcultures of human corneal keratocytes (HCK) and human corneal endothelial (HCE) cells were established under in vitro conditions that maintained these cells in a proliferative state, actively undergoing mitosis5. Third-passage cells were maintained in a humidified incubator at 35.5 °C in 95% air : 5% CO

2 throughout the study.

Tissue culture plates (96 well) were seeded with 1 × 103 HCK or HCE cells in a final volume of 200 μL of chondroitin sulfate medium (CSM, Insight Biomed, Inc., Minneapolis, MN, USA), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA). After 4 days of incubation, the medium was removed and the cells were rinsed once with serum-free minimal essential medium (MEM, Sigma-Aldrich, St. Louis, MO, USA).

The cells were then incubated with the test fluoro-quinolone solution or serum-free MEM. Test fluoro-quinolone solutions (levofloxacin and ofloxacin, Daiichi Pharmaceutical Ltd, Japan; ciprofloxacin, moxifloxacin, gatifloxacin, LKT Laboratories, Inc., St. Paul, MN, USA) were prepared in MEM and pH was adjusted to 6.8–7.2. Each fluoroquinolone solution was applied at six concentrations ranging from 1 mg/mL to 10 ng/mL and incubated for 15, 30, 60, or 240 min (six concentrations at four time exposures). Control MEM was incubated with each fluoroquinolone solution at every time exposure (i.e., a separate control MEM for each fluoro-quinolone concentration–time exposure). After incuba-tion for the specified exposure time, wells were rinsed twice with 200 μL of serum-free MEM and incubated with 200 μL of fresh CSM medium, supplemented with 10% fetal bovine serum, for the remainder of 72 h. Each fluoroquinolone concentration and corresponding control was tested in six replicate wells at each time exposure.

Cell number measurement

After 72 h, each well was rinsed twice with 200 μL of commercial balanced salt solution (BSS, Cytosol Laboratories, Inc., Braintree, MA, USA). Cells were then incubated with 100 μL/well of 2 μM Calcein-AM solution (Molecular Probes, Inc., Eugene, OR, USA) and immediately read on a Millipore CytoFluor 2300 Fluorescence Measurement System (Applied Biosystems, Foster City, CA, USA). Non-fluorescent calcein-AM that permeates a cell is enzymatically hydrolyzed to the fluorescent calcein, which produces a uniform, intense green (530 nm) fluorescence that can be read at 485/20 nm excitation and 530/25 nm emission wave length6,7. The fluorescent product is directly proportional to cell number.

statistical analysis

At each time point (15, 30, 60, and 240 min), the mean fluorescent values from the six replicate wells were calculated, and the percent difference in mean total fluorescent units between each fluoroquinolone concen tration and its MEM control was determined. A Wilcoxon signed-rank test was used to evaluate the statistical significance of this difference. Cytotoxicity of drug exposure was defined as a statistically significant ( p < 0.05) difference in cell count (fluorescent product) versus the corresponding MEM control for each fluoro-quinolone concentration–time exposure.

Resultshuman corneal keratocyte cultures

The percent difference between mean total fluorescence of each fluoroquinolone test solution and its corres ponding control at each time point in HCK is shown in Table 1. The only concentration at which all fluoro quinolones were consistently cytotoxic at all time points was 1 mg/mL. With the exception of one time point, ciprofloxacin was cytotoxic to keratocytes at all concen trations, regardless of exposure time. For the other fluoroquinolones, 100 ng/mL and 10 ng/mL were not cytotoxic except with prolonged (60 and 240 min) exposure in the case of ofloxacin. Although ≥ 1 μg/mL concentrations of moxifloxacin, gatifloxacin, and levo floxacin were cytotoxic to keratocytes, the effects were time-dependent. Moxifloxacin was cytotoxic within 15 min whereas cytotoxicity was not observed until 60 min of exposure to levofloxacin.

human corneal endothelial cell cultures

The percent difference between mean total fluorescence of each fluoroquinolone test solution and its corres-ponding control at each time point in HCE is shown in Table 2. The findings were similar to those with HCK cells in that 1 mg/mL of all fluoroquinolones was cytotoxic regardless of exposure duration. Ciprofloxacin was cytotoxic at all concentrations and all time points. Gatifloxacin, levofloxacin, and ofloxacin were not cytotoxic at 100 ng/mL or 10 ng/mL concentrations, except with prolonged (240 min) exposure to ofloxacin. The effects of moxifloxacin at these lower concentrations were inconsistent; 10 ng/mL did not appear to be cytotoxic to endothelial cells except at 240 min exposure. As with keratocytes, ≥ 1 μg/mL concentrations of moxifloxacin, gatifloxacin, and levofloxacin could be cytotoxic; the effects were time-dependent.

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© 2008 LIbRAPhARM Ltd – Curr Med Res 2008; 24(2) Fluoroquinolone cytotoxicity in human cornea Bezwada et al. 421

Based on the proportion of concentration–time exposures in which fluoroquinolones were cytotoxic (Table 3), ciprofloxacin was the most cytotoxic to HCK (96%), followed by moxifloxacin (67%), ofloxacin (62.5%), gatifloxacin (58%), and levofloxacin (42%); ciprofloxacin was also the most cytotoxic to HCE (100%), followed by moxifloxacin (79%), gatifloxacin (67%), ofloxacin (67%), and levofloxacin (42%).

Discussion

The results of this in vitro study show that fluoro-quinolones, in the absence of preservatives, have both concentration- and time-dependent cytotoxic effects on corneal keratocytes and endothelial cells. The propensity for cytotoxicity appears to vary according to the fluoro quinolone, with ciprofloxacin

Table 1. Human corneal keratocytes: concentration- and time-dependence of fluoroquinolone-induced cytotoxicity measured as percent difference in mean calcein fluorescence product, which is directly proportional to cell number

Exposure time

15 min 30 min 1 h 4 h

Ciprofloxacin

1 mg/mL –20.8 –23.9 –35.8 –72.2

100 μg/mL –14.4 –20.6 –12.3 –17.2

10 μg/mL –8.2 –13.5 –10.6 –14.8

1 μg/mL –10.0 –10.9 –12.8 –14.2

100 ng/mL –7.1 –4.8 –4.7 –7.6

10 ng/mL –5.1 –5.2 –8.9 –13.4

Gatifloxacin

1 mg/mL –21.8 –27.6 –26.4 –24.2

100 μg/mL –13.0 –17.9 –16.2 –15.9

10 μg/mL –3.4 –7.0 –6.7 –7.2

1 μg/mL –2.7 –2.6 –6.2 –9.9

100 ng/mL 1.9 0.8 < –0.1 2.9

10 ng/mL 1.9 0.9 –0.9 0.8

Levofloxacin

1 mg/mL –16.0 –18.2 –33.4 –24.6

100 μg/mL 1.4 –4.5 –18.2 –13.6

10 μg/mL 0.2 –1.6 –7.1 –3.4

1 μg/mL 1.2 –0.7 –9.7 –5.7

100 ng/mL 0.9 2.1 –1.7 1.8

10 ng/mL –1.9 –0.5 –2.5 –2.2

Moxifloxacin

1 mg/mL –35.7 –31.7 –31.2 –27.4

100 μg/mL –18.0 –20.4 –18.6 –17.8

10 μg/mL –5.8 –7.0 –8.1 –6.7

1 μg/mL –6.6 –7.8 –8.9 –6.7

100 ng/mL 1.9 0.4 –2.9 –2.2

10 ng/mL –0.5 0.4 –2.0 –0.9

Ofloxacin

1 mg/mL –17.2 –27.5 –27.9 –21.4

100 μg/mL –7.2 –8.5 –17.8 –15.9

10 μg/mL 1.0 –5.5 –10.3 –9.0

1 μg/mL –2.4 –1.6 –14.5 –10.0

100 ng/mL 2.2 –1.5 –6.3 –5.2

10 ng/mL –1.9 –5.6 –10.7 –8.8

Shaded values represent statistically significant ( p < 0.05) difference versus control for fluoroquinolone concentration–time exposure

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422 Fluoroquinolone cytotoxicity in human cornea © 2008 LIbRAPhARM Ltd – Curr Med Res 2008; 24(2)

having the greatest potential for cytotoxicity. Since fluoroquinolone solutions were adjusted to neutral pH, ciprofloxacin toxicity was not due to its lower pH when in solution or to formation of a precipitate.

Although the antimicrobial potency of levofloxacin, the S-isomer of ofloxacin, is twice that of ofloxacin, levofloxacin displayed less potential to be cytotoxic in our assays. Studies suggest a correlation between inhibition of mammalian topoisomerase II and fluoro-

quinolone cytotoxicity8. None of the fluoroquinolones in clinical use, including levofloxacin and ofloxacin, are potent inhibitors of mammalian topoisomerase II, although differences in their inhibitory effect have been observed. In a study evaluating ofloxacin and its stereo-specific isomers9, the relative potency of fluoroquinolone inhibitory effects on mammalian topoisomerase II were R-isomer > ofloxacin > S-isomer (i.e., levofloxacin). In a study comparing ciprofloxacin, ofloxacin, and

Table 2. Human corneal endothelial cells: concentration- and time-dependence of fluoroquinolone cytotoxicity measured as percent difference in mean calcein fluorescence product, which is directly proportional to cell number

Exposure time

15 min 30 min 1 h 4 h

Ciprofloxacin

1 mg/mL –14.6 –20.1 –24.6 –50.3

100 μg/mL –16.7 –19.3 –20.8 –24.7

10 μg/mL –13.2 –14.6 –16.6 –18.5

1 μg/mL –7.3 –9.7 –11.5 –9.1

100 ng/mL –10.6 –14.4 –14.5 –13.0

10 ng/mL –7.0 –10.9 –13.3 –10.8

Gatifloxacin

1 mg/mL –15.8 –18.9 –35.0 –46.1

100 μg/mL –12.4 –14.4 –23.8 –22.5

10 μg/mL –9.0 –8.4 –11.9 –11.9

1 μg/mL –7.3 –7.3 –12.8 –10.4

100 ng/mL –0.6 –0.7 –1.4 –3.6

10 ng/mL –2.7 –0.9 –2.1 –3.7

Levofloxacin

1 mg/mL –3.4 –4.1 –28.2 –29.4

100 μg/mL –0.4 –0.3 –13.6 –13.5

10 μg/mL 3.0 –4.3 –7.4 –4.1

1 μg/mL 0.1 –3.1 –5.5 –4.0

100 ng/mL 1.0 –1.3 0.0 –2.3

10 ng/mL 2.2 –3.0 –0.4 –2.3

Moxifloxacin

1 mg/mL –28.7 –30.9 –32.9 –32.2

100 μg/mL –15.5 –11.5 –20.1 –21.0

10 μg/mL –4.4 –6.0 –11.2 –6.4

1 μg/mL –5.5 –3.1 –9.5 –8.0

100 ng/mL –3.1 –5.2 –1.5 –0.3

10 ng/mL –3.5 –2.0 0.1 –2.2

Ofloxacin

1 mg/mL –20.9 –28.3 –34.4 –27.9

100 μg/mL –16.1 –20.1 –22.2 –14.5

10 μg/mL –4.5 –11.4 –10.5 –11.3

1 μg/mL –3.4 –3.0 –9.2 –12.4

100 ng/mL 1.4 –0.4 –2.0 –4.7

10 ng/mL 2.2 0.3 –0.2 –0.7

Shaded values represent statistically significant ( p < 0.05) difference versus control for fluoroquinolone concentration–time exposure

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© 2008 LIbRAPhARM Ltd – Curr Med Res 2008; 24(2) Fluoroquinolone cytotoxicity in human cornea Bezwada et al. 423

gati floxacin10, relative inhibitory effects on mammalian topo isomerase II were ciprofloxacin > ofloxacin > gatiflox acin. Our findings parallel these observations, i.e., cipro floxacin was associated with the greatest potential for cytotoxicity, while the potential for cytotoxicity was less with levofloxacin versus ofloxacin or ciprofloxacin.

Numerous studies have examined cellular effects of fluoroquinolones in various in vitro and in vivo test systems using different measures and conditions. How ever, few published studies have evaluated all ophth almic fluoroquinolones simultaneously using identical methodology; studies evaluating topical fluoroquinolones have generally examined their effects on corneal epi thelial cells. In a rabbit study by Kovoor and colleagues, epithelial thickness measured by in vivo confocal microscopy was significantly ( p < 0.05) reduced after 7 days’ repeated exposure to commercial solutions of ciprofloxacin, gatifloxacin, levofloxacin, and ofloxacin but it was not reduced with moxifloxacin or the control (preservative-free artifical tears)11. However, the difference favoring moxifloxacin may have reflected the lack of preservatives in the moxifloxacin formulation versus the preservatives present in varying concentrations in the other fluoroquinolone solutions. As for studies in human ocular tissues, Matsumoto et al.12 compared all currently available ophthalmic fluoroquinolones in human corneal epithelial cell cultures, without the confounding factor of preservatives. All fluoroquinolones were associated with concentration-dependent inhibition of epithelial cell proliferation. After 4–7 days of exposure (0.3–3.0 mmol/L), ciprofloxacin and moxifloxacin were the most cytotoxic and significantly ( p < 0.05) more cytotoxic than gatifloxacin. The rank order of pro liferative inhibition was ciprofloxacin = moxifloxacin > gatifloxacin > levofloxacin > ofloxacin. Effects on corneal epithelial cell migration mirrored effects on proliferation in that all fluoroquinolones showed dose-dependent inhibition (0.2–0.6 mmol/L) with ciprofloxacin being the most toxic, with a rank

order of ciprofloxacin > moxi floxacin > gatifloxacin > levofloxacin > ofloxacin12.

In contrast to the study of Matsumoto et al.12, our study evaluated fluoroquinolone effects on human corneal keratocyte and endothelial cell cultures. The potential for cytotoxicity and inhibition of cell proliferation in these cells is of particular interest, given their role in maintaining corneal transparency and visual acuity. If keratocytes are lost, they may be replaced by aggressive stromal cells involved in scar tissue formation and corneal haze13. Cell replication in the fragile corneal endothelial monolayer may not occur or may be too slow to adequately replace dead or injured cells. Loss of corneal endothelial cells and consequent reduction in endothelial cell density can diminish visual acuity as the stroma becomes edematous14.

This study spanned a range of fluoroquinolone concen-trations, from a concentration (1 mg/mL) at which all fluoroquinolones were cytotoxic, regardless of expos ure time, to ‘no effect’ concentrations (100 and 10 ng/mL), despite prolonged exposure, for several fluoro-quinolones. Although data are limited, concen trations in these assays encompass reported tear, conjunctival, and aqueous humor concentrations in humans. They represent a concentration gradient to which corneal keratocytes and endothelial cells might be exposed as fluoroquinolones diffuse from tear film into the aqueous humor. However, published reports of in vivo fluoroquinolone concen trations in ocular tissues are typically based on single-point samples.

Data relevant to human corneal keratocyte exposure to fluoroquinolones are limited to the few studies that have measured corneal tissue concentrations in patients undergoing penetrating keratoplasty or corneal trans plantation. Mean corneal stroma concentrations follow ing 0.5% levofloxacin, 0.3% ofloxacin, and 0.3% ciprofloxacin were 18.2, 10.8, and 9.9 μg/g, respect-ively15, and 65 μg/g following 1.5% levofloxacin admin-istration. Mean conjunctival fluoroquinolone concen-trations 20 min after dosing with 0.5% moxifloxacin, 0.3% gatif loxacin, 0.3% ciprofloxacin, 0.5% levofloxacin, and 0.3% ofloxacin were 18.0, 2.5, 2.6, 2.3, and 1.3 μg/g, respectively16. Based on our in vitro assays, those tissue concentrations of moxifloxacin and cipro floxacin may have cytotoxic effects on keratocytes with relatively short duration (≤ 15 min) of exposure; gatifloxacin may be toxic with ≥ 30 min exposure whereas levofloxacin and ofloxacin might be toxic if exposure was ≥ 60 min at those concentrations.

A number of studies have reported fluoroquinolone aqueous humor concentrations in humans. Given our findings of in vitro cytotoxicty at low concentrations, the aqueous humor concentrations achieved with 0.5% moxifloxacin, 0.3% gatifloxacin, and 0.3% ciprofloxacin have the potential to be cytotoxic to corneal endothelial

Table 3. Fluoroquinolone cytotoxicity in human corneal keratocytes and endothelial cells: total cytotoxic

concentration–time points*

Fluoroquinolone Human corneal keratocytes

Human corneal endothelial cells

Ciprofloxacin 23/24 (96%) 24/24 (100%)

Moxifloxacin 16/24 (67%) 19/24 (79%)

Ofloxacin 15/24 (62.5%) 16/24 (67%)

Gatifloxacin 14/24 (58%) 16/24 (67%)

Levofloxacin 10/24 (42%) 10/24 (42%)

*Cytototoxicty defined as statistically significant ( p < 0.05) difference in cell number measured as mean calcium fluorescence product versus control for each fluoroquinolone concentration–time exposure

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cells within 15 min after instillation17. At the aqueous humor concentrations achieved with 1.5% levofloxacin (1 μg/mL), the margin of safety with levofloxacin is at least double that of ciprofloxacin, gatifloxacin, and moxifloxacin.

The above data illustrate the limitations of assessing drug exposure based on a single time point since con centrations change over time in vivo. However, concentration over time can be measured in vivo as area under the curve (AUC) providing a measure of exposure that can be more directly compared to assays such as ours. Although studies have evaluated fluoroquinolone concentrations over time in tears, exposure data from tear studies are more relevant to conjunctival and corneal epithelial cells than to the corneal keratocytes and endothelial cells in our assays. A unique study reported by Robertson et al.17 allowed researchers to calculate the AUC for moxifloxacin and gatifloxacin in aqueous humor in patients undergoing cataract surgery. With two different administration schedules, the 3-h exposure (AUC

0–3) was 3.0 and

4.0 μg∙h/mL following adminis tration of 0.5% moxifloxacin and 1.6 and 1.8 μg∙h/mL following 0.3% gatifloxacin administration. Findings from our study suggest that these exposures could be cytotoxic to human corneal endothelial cells.

Obviously, in vitro systems such as those used in our assays lack critical tissue components present in vivo that may prevent, compensate, repair or otherwise ameliorate the cytotoxic effects of fluoroquinolones on corneal keratocytes and endothelial cells. Because bacterial eradication with fluoroquinolones is a function of both concentration and time, high concentrations of fluoro quinolones in tears and ocular tissues are critical to therapeutic success. Although our data suggest that the benefits of high fluoroquinolone exposure may be associated with an increased risk of cytotoxicity, levo-floxacin may have a lower propensity for cytotoxic effects involving corneal keratocytes and endothelial cells.

Conclusion

Findings from this in vitro bioassay indicate that fluoro quinolones have both concentration- and time-dependent cytotoxic effects on human keratocytes and human corneal endothelial cells. Fluoroquinolones may differ in their cytotoxic potential.

Acknowledgments

Declaration of interest: This study was sponsored by Santen Incorporated, Napa, CA, USA. All authors are employees.

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CrossRef links are available in the online published version of this article:http://www.cmrojournal.com

Paper CMRO-4237_3, 11:24-25.01.08Accepted for publication: 28 November 2007

Published Online: 21 December 2007doi:10.1185/030079908X261005

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