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`
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`
`(cid:45)(cid:46)(cid:10)(cid:11)(cid:7)(cid:10)(cid:21)(cid:15)(cid:6)(cid:9)(cid:3)(cid:6)(cid:12)(cid:3)(cid:21)(cid:39)(cid:22)(cid:3)(cid:13)(cid:6)(cid:14)(cid:15)(cid:16)(cid:15)(cid:21)(cid:18)(cid:3)(cid:6)(cid:12)(cid:3)(cid:47)(cid:22)(cid:9)(cid:48)(cid:10)(cid:11)(cid:49)(cid:6)(cid:9)(cid:15)(cid:7)(cid:40)
`(cid:20)(cid:39)(cid:11)(cid:6)(cid:8)(cid:15)(cid:24)(cid:22)(cid:3)(cid:6)(cid:9)(cid:3)(cid:21)(cid:39)(cid:22)(cid:3)(cid:25)(cid:16)(cid:7)(cid:11)(cid:10)(cid:8)(cid:3)(cid:27)(cid:7)(cid:8)(cid:12)(cid:10)(cid:16)(cid:22)
`
`(cid:20)(cid:50)(cid:51)(cid:25)(cid:52)(cid:26)(cid:28)(cid:45)(cid:3)(cid:53)(cid:45)(cid:47)(cid:47)(cid:50)(cid:27)(cid:20)(cid:54)(cid:55)(cid:3)(cid:37)(cid:50)(cid:13)(cid:51)(cid:26)(cid:20)(cid:45)(cid:3)(cid:51)(cid:50)(cid:13)(cid:55)(cid:3)(cid:5)(cid:45)(cid:50)(cid:28)(cid:4)(cid:56)(cid:26)(cid:20)(cid:54)(cid:45)(cid:52)(cid:3)(cid:57)(cid:50)(cid:51)(cid:28)(cid:45)(cid:13)(cid:55)(cid:3)(cid:56)(cid:50)(cid:58)(cid:53)(cid:50)(cid:3)(cid:53)(cid:45)
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`
`(cid:13)(cid:6)(cid:3)(cid:16)(cid:15)(cid:21)(cid:22)(cid:3)(cid:21)(cid:39)(cid:15)(cid:23)(cid:3)(cid:10)(cid:8)(cid:21)(cid:15)(cid:16)(cid:11)(cid:22)(cid:19)(cid:3)(cid:5)(cid:6)(cid:7)(cid:8)(cid:9)(cid:10)(cid:11)(cid:12)(cid:3)(cid:13)(cid:12)(cid:14)(cid:14)(cid:6)(cid:15)(cid:5)(cid:16)(cid:17)(cid:3)(cid:18)(cid:6)(cid:19)(cid:7)(cid:10)(cid:5)(cid:12)(cid:3)(cid:7)(cid:6)(cid:19)(cid:17)(cid:3)(cid:20)(cid:12)(cid:6)(cid:11)(cid:4)(cid:21)(cid:10)(cid:5)(cid:16)(cid:12)(cid:9)(cid:3)(cid:22)(cid:6)(cid:7)(cid:11)(cid:12)(cid:19)(cid:17)(cid:3)(cid:21)(cid:6)(cid:23)(cid:13)(cid:6)(cid:3)(cid:13)(cid:12)
`(cid:15)(cid:6)(cid:10)(cid:11)(cid:19)(cid:3)(cid:20)(cid:12)(cid:6)(cid:11)(cid:17)(cid:3)(cid:5)(cid:16)(cid:7)(cid:10)(cid:15)(cid:19)(cid:8)(cid:18)(cid:16)(cid:12)(cid:3)(cid:14)(cid:6)(cid:24)(cid:13)(cid:8)(cid:24)(cid:10)(cid:11)(cid:3)(cid:25)(cid:3)(cid:18)(cid:26)(cid:27)(cid:28)(cid:29)(cid:29)(cid:30)(cid:3)(cid:18)(cid:26)(cid:28)(cid:31)(cid:31)(cid:28)(cid:4)(cid:20)(cid:28)(cid:30)(cid:32)(cid:3)(cid:33)(cid:34)(cid:35)(cid:35)(cid:35)(cid:36)(cid:3)(cid:12)(cid:37)(cid:30)(cid:29)(cid:38)(cid:30)(cid:39)(cid:26)(cid:40)(cid:32)(cid:3)(cid:40)(cid:41)(cid:3)(cid:39)(cid:42)(cid:28)(cid:3)(cid:19)(cid:40)(cid:43)(cid:26)(cid:44)(cid:26)(cid:39)(cid:45)(cid:3)(cid:40)(cid:41)
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`(cid:19)(cid:40)(cid:43)(cid:26)(cid:44)(cid:40)(cid:29)(cid:40)(cid:50)(cid:45)(cid:17)(cid:3)(cid:52)(cid:53)(cid:51)(cid:34)(cid:4)(cid:54)(cid:17)(cid:3)(cid:52)(cid:35)(cid:55)(cid:4)(cid:52)(cid:52)(cid:55)(cid:17)(cid:3)(cid:13)(cid:8)(cid:10)(cid:51)(cid:3)(cid:52)(cid:35)(cid:56)(cid:54)(cid:52)(cid:35)(cid:53)(cid:57)(cid:52)(cid:55)(cid:55)(cid:58)(cid:53)(cid:55)(cid:34)(cid:35)(cid:35)(cid:35)(cid:53)(cid:35)(cid:55)(cid:52)(cid:55)(cid:35)(cid:58)
`
`(cid:13)(cid:6)(cid:3)(cid:11)(cid:15)(cid:9)(cid:49)(cid:3)(cid:21)(cid:6)(cid:3)(cid:21)(cid:39)(cid:15)(cid:23)(cid:3)(cid:10)(cid:8)(cid:21)(cid:15)(cid:16)(cid:11)(cid:22)(cid:19)(cid:3)(cid:3)(cid:42)(cid:39)(cid:39)(cid:59)(cid:51)(cid:57)(cid:57)(cid:49)(cid:43)(cid:56)(cid:49)(cid:40)(cid:26)(cid:56)(cid:40)(cid:31)(cid:50)(cid:57)(cid:52)(cid:35)(cid:56)(cid:54)(cid:52)(cid:35)(cid:53)(cid:57)(cid:52)(cid:55)(cid:55)(cid:58)(cid:53)(cid:55)(cid:34)(cid:35)(cid:35)(cid:35)(cid:53)(cid:35)(cid:55)(cid:52)(cid:55)(cid:35)(cid:58)
`
`(cid:18)(cid:38)(cid:60)(cid:29)(cid:26)(cid:27)(cid:42)(cid:28)(cid:49)(cid:3)(cid:40)(cid:32)(cid:29)(cid:26)(cid:32)(cid:28)(cid:51)(cid:3)(cid:34)(cid:61)(cid:3)(cid:15)(cid:28)(cid:59)(cid:3)(cid:34)(cid:35)(cid:35)(cid:62)(cid:56)
`
`(cid:15)(cid:38)(cid:60)(cid:48)(cid:26)(cid:39)(cid:3)(cid:45)(cid:40)(cid:38)(cid:31)(cid:3)(cid:30)(cid:31)(cid:39)(cid:26)(cid:44)(cid:29)(cid:28)(cid:3)(cid:39)(cid:40)(cid:3)(cid:39)(cid:42)(cid:26)(cid:27)(cid:3)(cid:63)(cid:40)(cid:38)(cid:31)(cid:32)(cid:30)(cid:29)(cid:3)
`
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`
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`
`(cid:5)(cid:26)(cid:39)(cid:26)(cid:32)(cid:50)(cid:3)(cid:30)(cid:31)(cid:39)(cid:26)(cid:44)(cid:29)(cid:28)(cid:27)(cid:51)(cid:3)(cid:52)(cid:3)(cid:66)(cid:26)(cid:28)(cid:64)(cid:3)(cid:44)(cid:26)(cid:39)(cid:26)(cid:32)(cid:50)(cid:3)(cid:30)(cid:31)(cid:39)(cid:26)(cid:44)(cid:29)(cid:28)(cid:27)(cid:3)
`
`(cid:67)(cid:38)(cid:29)(cid:29)(cid:3)(cid:19)(cid:28)(cid:31)(cid:48)(cid:27)(cid:3)(cid:25)(cid:3)(cid:5)(cid:40)(cid:32)(cid:49)(cid:26)(cid:39)(cid:26)(cid:40)(cid:32)(cid:27)(cid:3)(cid:40)(cid:41)(cid:3)(cid:30)(cid:44)(cid:44)(cid:28)(cid:27)(cid:27)(cid:3)(cid:30)(cid:32)(cid:49)(cid:3)(cid:38)(cid:27)(cid:28)(cid:3)(cid:44)(cid:30)(cid:32)(cid:3)(cid:60)(cid:28)(cid:3)(cid:41)(cid:40)(cid:38)(cid:32)(cid:49)(cid:3)(cid:30)(cid:39)
`(cid:42)(cid:39)(cid:39)(cid:59)(cid:51)(cid:57)(cid:57)(cid:64)(cid:64)(cid:64)(cid:56)(cid:39)(cid:30)(cid:32)(cid:49)(cid:41)(cid:40)(cid:32)(cid:29)(cid:26)(cid:32)(cid:28)(cid:56)(cid:44)(cid:40)(cid:48)(cid:57)(cid:30)(cid:44)(cid:39)(cid:26)(cid:40)(cid:32)(cid:57)(cid:63)(cid:40)(cid:38)(cid:31)(cid:32)(cid:30)(cid:29)(cid:10)(cid:32)(cid:41)(cid:40)(cid:31)(cid:48)(cid:30)(cid:39)(cid:26)(cid:40)(cid:32)(cid:68)(cid:63)(cid:40)(cid:38)(cid:31)(cid:32)(cid:30)(cid:29)(cid:5)(cid:40)(cid:49)(cid:28)(cid:69)(cid:26)(cid:44)(cid:40)(cid:39)(cid:52)(cid:53)
`
`(cid:53)(cid:6)(cid:43)(cid:9)(cid:11)(cid:6)(cid:10)(cid:24)(cid:3)(cid:61)(cid:18)(cid:19)(cid:3)(cid:70)(cid:12)(cid:48)(cid:26)(cid:29)(cid:45)(cid:3)(cid:67)(cid:29)(cid:40)(cid:31)(cid:26)(cid:40)(cid:71)
`
`(cid:53)(cid:10)(cid:21)(cid:22)(cid:19)(cid:3)(cid:52)(cid:65)(cid:3)(cid:15)(cid:28)(cid:59)(cid:39)(cid:28)(cid:48)(cid:60)(cid:28)(cid:31)(cid:3)(cid:34)(cid:35)(cid:52)(cid:55)(cid:17)(cid:3)(cid:6)(cid:39)(cid:51)(cid:3)(cid:35)(cid:58)(cid:51)(cid:54)(cid:61)
`
`SENJU EXHIBIT 2064
`INNOPHARMA v. SENJU
`IPR2015-00903
`
`Page 1 of 12
`
`

`
`J. Toxicol.-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 BAUDOU/N, M.D., Ph.D.
`PIERRE-JEAN PISELLA, M.D.
`Department of Ophthalmology
`Ambroise Pare 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(cid:173)
`munoallergic reactions. Due to its surfactant properties, this quaternary
`ammonium strongly decreases lachrymal fl uid stability. It also causes
`toxic effects to epithelial cells and increases corneal permeability. In
`vivo, strong histopathological changes are observed after topical treat(cid:173)
`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,
`
`Downloaded by [Emily Florio] at 06:37 14 September 2015
`
`* Address reprint requests to: Caroline Debhasch, Pharm.D., Department of Cellular Pharmacotox.icology,
`XV-XX Hospital, 28, Rue de Charenton, 75012 Paris, France.
`
`Copyright © 2000 by Marcel Dekker, Inc.
`
`www .dekker.com
`
`105
`
`Page 2 of 12
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`

`
`106
`
`DEBBASCH ET AL.
`
`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(cid:173)
`enon is confirmed by 4',6-diamidimo-2-phenylindole, dilactate (DAPI)
`coloration and with the use of flow cytometry. A decrease in cell si:z.e
`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
`G I phase cells is also shown. BAC induces irreversible cytotoxic dam(cid:173)
`ages with some characteristics of apoptosis in a concentration-dependent
`marmer.
`
`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(cid:173)
`logical studies have confirmed that topically applied drugs may exert toxic effects
`to the comeoconjunctiva1 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 a11 antiglau(cid:173)
`coma preparations, has shown strong evidence of toxicity to the ocular surface. It
`may act on ocular surface through toxic 1 or immunoallergic reactions.2 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 glaucoma.3.4
`The effects of ophthalmic preservatives on corneoconjunctival cells have been
`investigated by scanning electron microscopy,5•6 tandem scanning confocal micros(cid:173)
`copy/ in vitro studies of cytotoxicity of preservatives to cultured corneal epithelial
`cells,8 quantitative evaluation of corneal epithelial permeability, and histological or
`cytological analyses after exposure to eyedrops of varying composition in humans 9-
`12 or animals. In vivo studies have been conducted after topical treatment with preser(cid:173)
`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. Conjunctiva] biopsies taken at the time of glaucoma surgery
`
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`
`Page 3 of 12
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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.9- 11 Impression cytological
`specimens from long-term-treated patients have shown abnormal induction of in(cid:173)
`flammatory markers by epithelial cells in about 50% of eyes. 12 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(cid:173)
`zalkonium chloride, benzododecinium bromide, thiomersal, methyJparahydroxyben(cid:173)
`zoate). Topical drops were administered in both eyes, three times daily for 1 month.
`Histopathological evaluations and indirect immunochemistry studies were per(cid:173)
`formed. This study demonstrated strong toxic effects of preserved solutions. Preser(cid:173)
`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(cid:173)
`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
`
`.:s 80
`
`-1120
`c
`0100
`~
`·s eo
`:s
`e u AO
`s::
`3
`i 20
`
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`ii:
`
`0
`
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`
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`
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`
`-
`
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`
`'
`'
`'
`
`O,D01
`
`0,01
`
`***
`
`~ 0,00001
`
`O,OGD1
`Conc:e*illkln (~
`
`Figure I. 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.
`
`Downloaded by [Emily Florio] at 06:37 14 September 2015
`
`Page 4 of 12
`
`

`
`108
`
`DEBBASCH ET AL.
`
`meanFS 355
`
`M1
`
`(I') c: Q)
`> w
`
`a
`
`(/) c Q)
`> w
`
`0
`
`0
`
`b
`
`FSC-Height
`
`1023
`
`m.eanFS 310
`
`M1
`
`FSC-Height
`
`1023
`
`Figure 2. Cell size evaluation by flow cytometry after treatment with BAC. a. Control. b.
`BAC 0.001%. c. BAC 0.01 %. d. BAC 0.1 %.
`
`Downloaded by [Emily Florio] at 06:37 14 September 2015
`
`Page 5 of 12
`
`

`
`OCULAR SURFACE BAC TOXICITY
`
`109
`
`mean FS 260
`
`M1
`
`FSC-Height
`
`1023
`
`:mean FS 61
`
`M1
`
`FSC-Height
`
`1023
`
`(I) c Q)
`> w
`
`C)
`
`0
`
`c
`
`(I) c Q)
`> w
`
`C)
`
`0
`
`d
`
`Figure 2. Continued
`
`Downloaded by [Emily Florio] at 06:37 14 September 2015
`
`Page 6 of 12
`
`

`
`Control
`
`2% n1eon 22
`
`M1
`
`1 2
`FL2-Height
`
`BAC 0.0001 Yo
`
`44Yo ll\edn 1~
`
`M1
`
`1 4
`
`(I) c Q)
`> w
`
`0
`
`a
`
`'1:1'
`(.0
`
`en c Q) >
`
`UJ
`
`b
`
`Downloaded by [Emily Florio] at 06:37 14 September 2015
`
`Figure 3. Apo 2.7 evaluation by fJow cytometry after treatment with BAC. a. Control. b.
`BAC 0.001%. c. BAC 0.01%. d. BAC 0 .1%.
`
`Page 7 of 12
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`

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`OCULAR SURF ACE BAC TOXICITY
`
`111
`
`BAC 0 .001%
`
`69% n\~C)n 12.
`
`M1
`
`BACO.Ol %
`
`89% meel" 119
`
`M1
`
`(I) c Cl.)
`> w
`
`c
`
`(/) c
`> w
`
`Q)
`
`d
`
`Figure 3. Continued
`
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`Page 8 of 12
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`

`
`co
`N
`
`Control
`
`.sub- G 1
`
`4%
`
`M1
`
`BAC 0.0001%
`
`sub- G~
`
`18%
`
`M1
`
`(/) c U)
`>
`UJ
`
`a
`
`(/) c Q)
`>
`UJ
`
`Downloaded by [Emily Florio] at 06:37 14 September 2015
`
`0~-.--.--...........tL-.,........,..J~--~
`10 2
`10~
`FL2-Height
`
`b
`
`Figure 4. Cellular DNA quantity evaluation after treatment with BAC. a. Control. b. BAC
`0.001%. c. BAC 0.0 1%. d. BAC 0.1%.
`
`Page 9 of 12
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`OCULAR SURFACE BAC TOXICITY
`
`113
`
`BAC 0.001%
`
`sub- G 1.
`
`32%
`
`G1
`
`/
`
`M1
`
`BAC 0.01%
`
`sub. G1.
`
`G1
`
`c
`
`(I) c Q)
`>
`UJ
`
`d
`
`Figure 4. Continued
`
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`Page 10 of 12
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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 controls. 15 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(cid:173)
`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(cid:173)
`konium chloride, we used a DAPI coloration. Apo 2.7, a mitochondrial 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 0.0001% to 0.1%) over 15 mins.
`Using a neutral red assay (Fig. 1), 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(cid:173)
`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(cid:173)
`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). lmmunostaining
`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-G 1-
`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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`115
`
`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(cid:173)
`cially for long-term use and/or for patients with pre-existing ocular surface disorders.
`
`References
`
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`Ophthalmol. 25:15-30, 1980.
`2. M.G. Baines, F. Cai, and H.A. Backman, Ocular hypersensitivity to thimerosal in rabbits, Invest.
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`mol. 7(2):80-86, 1996.
`4. W.N. Wilson, A.J. Duncan, and J.L. Jay, Effects of benzalkonium chloride on the stability of the
`precomeal tear film in rabbit and man, Br. J. Ophthalmol. 105:670-673, 1988.
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`14. F. Becquet, M. Goldschild, M.S. Moldovan, M. Ettaiche, P. Gastaud, and C. Baudouin, Histopath(cid:173)
`ological effects of topical ophthalmic preservatives on rat corneoconjunctival surface, Curr. Eye
`Res. 17(4):419-425, 1998.
`15. 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.
`16. P. Rat, C. Korwin-Zmilowska, J.M. Wamet, and M. Adolphe, New in vitro flunrimetric microtitra(cid:173)
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