`
`ISSN: 0731-3829 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/icot19
`
`Evaluation of the Toxicity of Benzalkonium
`Chloride on the Ocular Surface
`
`CAROLINE DEBBASCH, PATRICE RAT, JEAN-MICHEL WARNET, MAGDA DE
`SAINT JEAN, CHRISTOPHE BAUDOUIN & Pisella Pierre-Jean
`
`To cite this article: CAROLINE DEBBASCH, PATRICE RAT, JEAN-MICHEL WARNET, MAGDA DE
`SAINT JEAN, CHRISTOPHE BAUDOUIN & Pisella Pierre-Jean (2000) Evaluation of the Toxicity of
`Benzalkonium Chloride on the Ocular Surface, Journal of Toxicology: Cutaneous and Ocular
`Toxicology, 19:2-3, 105-115, DOI: 10.3109/15569520009051506
`
`To link to this article: http://dx.doi.org/10.3109/15569520009051506
`
`Published online: 27 Sep 2008.
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`Download by: [Emily Florio]
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`Date: 14 September 2015, At: 06:37
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`SENJU EXHIBIT 2064
`LUPIN v. SENJU
`IPR2015-01099
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`Page 1 of 12
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`J. Toxico1.-Cut. & Ocular Toxicol., 19(2&3), 105-115 (2000)
`
`EVALUATION OF THE TOXICITY OF
`BENZALKONIUM CHLORIDE ON THE
`OCULAR SURFACE
`CAROLINE DEBBASCH, Pharm.D. *
`PATRICE RAT, Pharm.D., Ph.D.
`JEAN-MICHEL WARNET, M. D.
`Departments of Cellular Pharmacotoxicology and Ophthalmology
`XV-XX Hospital
`Faculty of Pharmacy
`University of Paris, V
`Paris, France
`MAGDA DE SAINT JEAN, M. D.
`CHRISTOPHE BA UDOUIN, M. D., Ph. D.
`PIERRE-JEAN PISELLA, M.D.
`Department of Ophthalmology
`Ambroise Park Hospital, AP-HP
`University of Paris V Boulogne, France
`
`Abstract
`
`Benzalkonium chloride (BAC), the most widely used preservative
`in ophthalmic solutions, acts on the ocular surface through toxic or im-
`munoallergic reactions. Due to its surfactant properties, this quaternary
`ammonium strongly decreases lachrymal fluid stability. It also causes
`toxic effects to epithelial cells and increases corneal permeability. In
`vivo, strong histopathological changes are observed after topical treat-
`ments with preservatives, including infiltration by inflammatory cells,
`similar to those induced in humans by long-term topical treatments. We
`designed a series of in vitro experiments confirming the toxicity of BAC,
`
`* Address reprint requests to: Caroline Debbasch, Pharm.D., Department of Cellular Pharmacotoxicology,
`XV-XX Hospital, 28, Rue de Charenton, 75012 Paris, France.
`
`Copyright 0 2000 by Marcel Dekker, Inc.
`
`www .dekker.com
`
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`106
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`DEBBASCH ET AL.
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`even at very low concentrations. In vitro, BAC induces cell necrosis at
`the concentrations found in most eye drops after a few minutes, and
`apoptosis when applied at lower concentrations. This apoptotic phenom-
`enon is confirmed by 4’,6-diamidimo-2-phenylindole, dilactate (DAPI)
`coloration and with the use of flow cytometry. A decrease in cell size
`is observed with BAC at 0.001% and is confirmed by morphological
`assay. An overexpression of Apo 2.7 associated with an increase of sub
`G1 phase cells is also shown. BAC induces irreversible cytotoxic dam-
`ages with some characteristics of apoptosis in a concentration-dependent
`manner.
`
`Introduction
`
`Long-term use of antiglaucoma drugs has been associated with toxic as well
`as inflammatory changes of the ocular surface. However, little is known concerning
`the accurate mechanisms of such toxic side effects in long-term and/or multitreated
`glaucoma patients. There is, however, growing evidence that long-term use of topical
`drugs may produce damage to conjunctival and corneal epithelial cells. Histopatho-
`logical studies have confirmed that topically applied drugs may exert toxic effects
`to the corneoconjunctival surface and induce chronic infraclinical inflammation, as
`shown by the presence of immunological changes and inflammatory infiltrates in
`multitreated eyes.
`The origin of topical inflammation has not yet been clearly determined but
`benzalkonium chloride (BAC), which is used as a preservative in almost all antiglau-
`coma preparations, has shown strong evidence of toxicity to the ocular surface. It
`may act on ocular surface through toxic’ or immunoallergic reactions.* The toxicity
`can be direct on epithelial and goblet cells or indirect on the lachrymal film, resulting
`from its tensioactive properties. Furthermore, repeated doses of preserved eyedrops
`can lead to a cumulative effect and preservatives transferred with ophthalmic devices
`will have prolonged contact with the epithelium. An iatrogenic disorder may result
`that could lead to chronic irritation and subsconjuctival fibrosis, which could worsen
`keratoconjunctivis sicca or increase the risk of failure of trabeculectomy in patients
`with g l a u c ~ m a . ~ . ~
`The effects of ophthalmic preservatives on corneoconjunctival cells have been
`
`investigated by scanning electron micro~copy,~*~ tandem scanning confocal micros-
`copy:
`in vitro studies of cytotoxicity of preservatives to cultured corneal epithelial
`cells,’ quantitative evaluation of corneal epithelial permeability, and histological or
`cytological analyses after exposure to eyedrops of varying composition in humans’-
`’* or animals. In vivo studies have been conducted after topical treatment with preser-
`vatives and in vitro experiments on human cultured conjunctival cells have also been
`done. Rare clinical studies have been conducted to compare preservative-free and
`preserved eyedrops. Conjunctival biopsies taken at the time of glaucoma surgery
`
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`OCULAR SURFACE BAC TOXICITY
`
`107
`
`have demonstrated a significant increase in immune cells and fibroblasts, possibly
`related to cumulative treatment with antiglaucoma drugs.’-’ ’ Impression cytological
`specimens from long-term-treated patients have shown abnormal induction of in-
`flammatory markers by epithelial cells in about 50% of eyes.” These inflammatory
`and fibrotic anomalies may result in pseudopemphigoid or significant reduction of
`success rate following glaucoma surgery.13
`To understand the relative role of preservatives such as benzalkonium chloride
`in the toxicity of long-term use of antiglaucomatous drugs, we performed a series
`of in vivo and in vitro experiments.
`Different in vivo studies have been conducted. The preservatives most often
`currently used in topical ophthalmic preparations were tested in rats (cetrimide, ben-
`zalkonium chloride, benzododecinium bromide, thiomersal, methylparahydroxyben-
`zoate). Topical drops were administered in both eyes, three times daily for 1 month.
`Histopathological evaluations and indirect immunochemistry studies were per-
`formed. This study demonstrated strong toxic effects of preserved solutions. Preser-
`vatives induced corneal epithelial damages and limbal and conjunctival infiltration
`by immunocompetent cells.14 Comparison of the different preservatives showed no
`significant difference in the intensity of pathological effects between the five preser-
`vatives tested.
`To address the eventual toxicity of beta blockers with or without preservative,
`another study was conducted in rats using immunochemistry. Benzalkonium chloride
`as well as unpreserved and preserved timolol were administered four times daily for
`
`Figure 1. Evaluation of cellular viability after 15 min treatment with BAC. Results
`are expressed in variation from the control values: ** p < 0.01 as compared to control;
`*** p < 0.0001 as compared to control.
`
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`
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`108
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`DEBBASCH ET AL.
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`mean Fs 355
`
`MI
`
`FSC-Height
`
`manFS 310
`
`MI
`
`a
`0
`
`b
`
`1023
`
`FSC-Height
`
`Cell size evaluation by flow cytometry after treatment
`Figure 2.
`BAC 0.001%. c. BAC 0.01%. d. BAC 0.1%.
`
`with BAC. a. Control. b.
`
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`OCULAR SURFACE BAC TOXICITY
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`109
`
`Figure 2. Continued
`
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`Page 6 of 12
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`
`
`Control
`
`a
`
`FL2-Height
`
`BAG 0.0001%
`
`b
`
`FL2-Height
`
`Figure 3.
`Apo 2.7 evaluation by flow cytometry after treatment with BAC. a. Control. b.
`BAC 0.001%. c. BAC 0.01%. d. BAC 0.1%.
`
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`OCULAR SURFACE BAC TOXICITY
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`111
`
`BAC 0.001%
`
`
`
`69% mean 42 69% mean 42
`
`
`
`f > f
`
`>
`
`LLI LLI
`
`
`
`0 0
`
`C
`
`
`
`4 4
`
`FL2-HeigM
`
`EAC 0.01%
`
`89% mean 49
`
`MI
`
`d
`
`Figure 3. Continued
`
`FL2-Height
`
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`Page 8 of 12
`
`
`
`#
`N
`7
`
`Control
`
`;
`Iu > w
`
`a
`
`b
`
`FL2-Height
`
`FL2-Height
`
`Figure 4.
`Cellular DNA quantity evaluation after treatment with BAC. a. Control. b. BAC
`0.001%. c. BAC 0.01%. d. BAC 0.1%.
`
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`OCULAR SURFACE BAC TOXICITY
`EAC 0 .0O 1 %
`
`“1
`
`113
`
`C
`
`FLT2-Height
`
`EAC 0.01%
`
`FL2-Height
`
`d
`
`Figure 4. Continued
`
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`Page 10 of 12
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`
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`114
`
`DEBBASCH ET AL.
`
`1 month. Ocular lesions were found with inflammatory infiltrates in the conjunctiva
`induced by both preserved timolol and benzalkonium chloride, unpreserved timolol
`did not result in significant infiltration compared to contr01s.'~ The results of studies
`taken together confirmed that most of the preservatives used in ophthalmic eyedrops
`may induce significant histopathological and inflammatory changes in the ocular
`surface, and that they play a major role in the toxicity of chronically administered
`eyedrops.
`The toxic effects induced by preservatives in vivo were confirmed by a series of
`in vitro experiments on a continuous human conjunctival cell line (Wong Kilbourne-
`derived human conjunctiva). The tests were performed using immunocytological and
`toxicological techniques and flow cytometry.16 Cell viability was evaluated using
`cold light cytofluorimetry. To confirm the apoptotic phenomenon induced by benzal-
`konium chloride, we used a DAPI coloration. Apo 2.7, a mitochondria1 marker that
`appears early in apoptotic mechanisms, cellular DNA quantity, and cell size also
`were analyzed by flow cytometry. Cells were treated with different concentrations
`of benzalkonium chloride (from 0.000 1 % to 0.1 %) over 15 mins.
`Using a neutral red assay (Fig. l), we observed a decrease in cell viability
`after a benzalkonium chloride treatment at a concentration of 0.001% (p < 0.01).
`Benzalkonium chloride 0.01% induced cell necrosis after a few minutes. At lower
`concentrations, no alteration in cell viability was observed but a DAPI coloration
`clearly showed apoptotic nuclei.
`This apoptotic phenomenon was confirmed using flow cytometry. A 30% de-
`crease in cell size was observed when we compared the control and the cells treated
`with BAC 0.01 % for 15 min (Fig. 2). This result was also found using a morphologi-
`cal assay with cytoplasmic retraction, after treatment with benzalkonium chloride
`0.01 %, the concentration used in most eyedrops. An overexpression of Apo 2.7 was
`also shown. This expression was concentration-dependent (Fig. 3). Immunostaining
`confirmed these results. Furthermore, we analyzed cellular DNA quantity to detect
`apoptotic cells. These apoptotic cells had a lower DNA quantity than control cells.
`After treatment with BAC, there was a concentration-dependent increase of sub-GI-
`phase cells, which confirmed the apoptotic mechanism induced by BAC (Fig. 4).
`These studies have proven that most of the preservatives used in eye drops
`may induce strong inflammatory, histopathological, and toxicological changes in the
`ocular surface.
`In animal models, BAC causes epithelial toxicity and inflammatory infiltration
`of ocular surface structures. In vitro, BAC induces growth arrest and cell death in
`a dose-dependent manner at a concentration as low as 0.0001 %. Cells die by necrosis
`after BAC treatment at high concentrations and by apoptosis if lower concentrations
`of BAC are applied. BAC could therefore be involved in ocular surface toxicological
`and/or inflammatory disorders observed in patients undergoing long-term topical
`treatments with preservative-containing drugs.
`Very few clinical studies have been conducted to compare preservative-free
`
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`OCULAR SURFACE BAC TOXICITY
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`11s
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`and preserved eyedrops. Studies have shown strong benefits of unpreserved solutions
`to the ocular surface. However, very few preservative-free ophthalmic solutions are
`now available. It is therefore of striking importance to become aware of preservative
`toxicity in order to develop in the near future many more unpreserved drugs, espe-
`cially for long-term use and/or for patients with pre-existing ocular surface disorders.
`
`References
`
`I .
`
`2.
`
`3.
`
`4.
`
`5.
`
`6.
`
`7.
`
`8.
`
`9.
`
`10.
`
`11.
`
`12.
`
`13.
`
`14.
`
`15.
`
`16.
`
`N.L. Burstein, Corneal cytotoxicity of topically applied drugs, vehicles and preservatives, Sum.
`Ophthalmol. 25: 15-30, 1980.
`M.G. Baines, F. Cai, and H.A. Backman, Ocular hypersensitivity to thimerosal in rabbits, Invest.
`Ophthalmol. Vis. Sci. 32:2259-2265, 1991.
`C. Baudouin, Side effects of antiglaucomatous drugs on the ocular surface, Curr. Opin. Ophthal-
`mol. 7(2):80-86, 1996.
`W.N. Wilson, A.J. Duncan, and J.L. Jay, Effects of benzalkonium chloride on the stability of the
`precorneal tear film in rabbit and man, Br. J. Ophthalmol. 105:670-673, 1988.
`A.R. Gassett, Y. Ishii, H.E. Kaufman, T. Miller, Cytotoxicity of ophthalmic preservatives, Am.
`J. Ophthalmol. 78:98-105, 1974.
`N.L. Burstein, The effects of topical drugs and preservatives on the tears and corneal epithelium
`in dry eye, Trans. Ophthalmol. SOC. UK, 104:402-409, 1985.
`H. Ichijima, W.M. Petroll, J.V. Jester, and H.D. Cavanagh, Confocal microscopic studies of living
`rabbit cornea treated with benzalkonium chloride, Cornea 11:221-225, 1992.
`P.S. Imperia, H.M. Lazarus, R.E. Botti, and J.H. Lass, An in vitro method for measuring ophthal-
`mic preservative cytotoxicity, J. Cut. Ocular Toxicol. 5309-3 17, 1986.
`D.C. Broadway, I. Grierson, C. O’Brien, and R.A. Hitchings, Adverse effects of topical antiglau-
`coma medications. I. The conjunctival cell profile, Arch. Ophthalmol. 112: 1437-1445, 1994.
`D.C. Broadway, I. Grierson, C. O’Brien, and R.A. Hitchings, Adverse effects of topical antiglau-
`coma medications. 11. The outcome of filtration surgery, Arch. Ophthalmol. 112: 1446-1 454,1994.
`M.B. Shenvood, I. Grierson, L. Millar, and R.A. Hitchings, Long-term morphologic effects of
`antiglaucoma drugs on the conjunctiva and Tenon’s capsule in glaucomatous patients, Ophthal-
`mology 96327-335, 1989.
`C. Baudouin, C. Garcher, N. Haouat, A. Bron, and P. Gastaud, Expression of inflammatory mem-
`brane markers by conjunctival cells in chronically treated glaucoma patients, Ophthalmology 101:
`454-460, 1994.
`M.J. Lavin, R.P.L. Wormald, C.S. Migdal, and R.A. Hitchings, The influence of prior therapy
`on the success of trabeculectomy, Arch. Ophthalmol. 108:1543-1550, 1990.
`F. Becquet, M. Goldschild, M.S. Moldovan, M. Ettaiche, P. Gastaud, and C. Baudouin, Histopath-
`ological effects of topical ophthalmic preservatives on rat corneoconjunctival surface, Curr. Eye
`Res. 17(4):419-425, 1998.
`C. Baudouin, P.J. Pisella, K. Fillacier, M. Goldschild, F. Becquet, M. De Saint Jean, and A.
`Bechetoille, Ocular surface inflammatory changes induced by topical antiglaucoma drugs. Human
`and animal studies. Ophthalmology 1998, in press.
`P. Rat, C. Konvin-Zmilowska, J.M. Warnet, and M. Adolphe, New in vitro fluorimetric microtitra-
`tion assays for toxicological screening of drugs, Cell Bid. Toxicol. 10:329-337, 1994.
`
`Downloaded by [Emily Florio] at 06:37 14 September 2015
`
`Page 12 of 12