In Vitro Studies of Antiglaucomatous Prostaglandin
`Analogues: Travoprost with and Without Benzalkonium
`Chloride and Preserved Latanoprost
`
`Christtwbe Baudouinfil’i" Luisa Ridncbt),l‘2‘4 jean-Michel Warnet,
`Fr-angmse Brigm'del‘z’4
`
`1,2,4 and
`
`PURPOSE. With use of the Wong—Kilboume derivative Chang
`conjunctiml cell line, this study compared in vitro the ocular
`toxicity of three topical intraocular pressure COM-lowering
`agents:
`travoprost 0.00495 containing 0.015% ben'ulkonium
`chloride (BAX), traVoprost Z 0.004%, a new fomiulation with-
`out BAK, and latanoprost 00059-6 containing 0.02% BAK.
`Mini-tons. Neutral red, Alamar blue, YOPRO—l, and annexin
`V/7-AAD a$ays were used to evaluate the effects of the [OP—
`lowering agents and BAK on cellular viability, membrane in-
`tegrity, and apoptosis in the conjunctival cell line using mi.-
`tTotitrat'ion fluoromelric analysis and flow cytotuetry. All
`assessments were performed in a masked manner.
`“estrus. Assessment of cell viability and membrane integrity
`revealed a significant effect by latanoprost with BAK or BAK
`alone but no dice! by travoprost Z without BAX or buffer
`alone (P < 0.0001). Litanoprost with BAK, travoprost with
`BAK, and BAK alone were cytotoxic in Chang conjunctival
`cells, whereas no cytotoxicity was observed in cells exposed to
`travoprost' Z Without BAK or in cells treated with buffer (P <
`0.0001). No increase in almptosis or necrosis was observed in
`cells treated with control or travoprost 2 without BAK com-
`pared with BAK, travoprost with RAK, and latanoprost with
`BAK (P < 0.0001).
`
`CONCLUSIONS. Latanoprost with BAK, travoprost with BAK, and
`BAK alone have significant cytotoxic effects on human con-
`junctiva-derived cells and are. associated with apoptosis. 'lhese
`effects likely result from BAK used as a preservative [OP-
`Iowering agents with alternative preservatives instead of BAK
`will most likely have fewer ocular surface adverse etfects than.
`agents containing BAK. (Invest ()pbtbalmol Vis Sci. 2007;48:
`4123- 4128) DOI: l 0.1 lCi7/i0vs.07~0266
`
`Management of glaucoma typically involves pharmacother-
`apy with topical ocular agents, such as the prostaglandin
`analogues latanoprost and Imvoprost.‘ Long'term use of topical
`ocular drugs is often associated with ocular inflamrrmtion,
`
`
`From the 'lX‘paruncnl of Ophthalmology 5. QuinlpVingts Nae
`tional Ophthalmology Hospital. Paris. Frantic; ZINSERM U871. Paris.
`‘75006 France; the 5Dcpartment of Ophthalmology. Ambroisc Paré
`Hospital. APlil’. University of Versailles. Versailles. France: and the
`4Department of'l’oxicology, Faculty of Biological and Pharmacological
`Sciences, University of Paris Descartes, Paris. France.
`Supported by unrestricted grants from INSERM U598 and Alcoa
`Inc. (Fort Worth. Texas).
`Submitted for publication March 4, 2007; revised April 25. 2007;
`acceptcdjunc 18. 2007.
`Disclosure: C. Balldouln. Alcon, Allergan. and Pfizer (C); L. Rian-
`Clio. None: J.-M. War-net. None; F. Brignole, None
`The publication costs of this article were defrayed in part by page
`charge payment. This article must therefore be marked “advertise—
`mem” in accordance with 18 U.S.C. §1754 solely to Indicate this fact.
`Corresponding author. Christophe Baudouin. Department of Oph-
`thalmology 5, Quin‘nc-Vingts National Ophthalmology Hospital, .’.8 Rue
`de (Iliarenton. Paris 75012. France; 1mmIouinzlltquinze-viugtsfr.
`
`Investigative Ophthalmology & Visual Science. September 200?. Vol. «ta. No. 9
`Copyright 4'3: Association for le‘ch in Vision and Ophthalmology
`
`allergy, dry eye syndrome, and failure of filtration surgery?8
`Several lines of evidence indicate that these agents are associ-
`ated with decreased tear turnover and tear film break-up time,
`infiltration of the conjunctiva by inflammatory cells and fibro-
`blasts, and overexpresskm of inflammatory markers such as
`HlA-DR, intercellular adhesion molecule (lCAM-l), FAS anti-
`gen, or the apoptotic marker Apo 2.7.94“
`The. preservative hermlkonium chloride (BAK), the most com-
`monly used ocular prtservative,
`is largely responsible for the
`ocular toxicities and inflammation associated Willi the chronic
`
`use of many ophthalmic. solutions.9"5'l7 BAK is retained in tissues
`and can be found 168 hours after a single 504d drop of 0.01%
`BAK in rabbits. '3 The half—life of BAK from corneal epithelium and
`conjunctival tissues is 20 hours."3 Prolonged exposure to BAK
`causes indirect and direct toxic ellecls to the ocular surface,
`including infiltration by inflammatory cells, reduced cellular via-
`bility, and compromised epithelial barrier function through in-
`creased corneal epithelial cell [)Cl'l'llt’TabilltY.u‘m'W-z' The toxic.
`effects of BAK are dose dependent, with progressive, slow cell
`growth arrest occurring at concentrations as low as 0.0001%,
`more rapid apoptosis at 0.01%, and immediate epithelial cell
`necrosis at higher concentrations of 00596.23
`The development of ocular preservatives that provide a
`nontoxic alternative to BAK will likely become useful in the
`rmnagement of chronic diseases arch as glaucoma. Using a
`human conitulctiva— derived cell line, we specifically investi-
`gated in vitro the first commercially available BAK-free prosta-
`glandin solution, travopmst Z 0.004%, preserved without BAK,
`and compared the ocular toxicity of this topical solution Willi
`that of standard travoprost 0.004% containing 0.015% BAK,
`latanoprost 0.005% containing 0.02% BAK, and BAK alone. We
`evaluated the effects of commercial formulations of these 10P-
`lowering agents and BAK on cellular viability, membrane in-
`tegrity, and apoptosis through microtitration Iluorometric as
`says and flow cytornetry.
`Several types of assays were chosen to explore various
`mechanisms of cell damage caused by the preservative BAK on
`cortjuttctivaderived cells. The neutral red cytotoxicity assay is
`a cell survival/viability assay that measures the integrity of the
`cell membrane. 17 Alamar blue assly measures cell proliferation
`and cytotoxicity.” YOPRO-l, a cell-impermeable nuclear dye
`that stains only cells that have lost membrane integrity, serves
`as an indication of apoptosis.“ The double-slanting annexin
`V/7-AAD assay discriminates cells that are undergoing early
`apoptosis, late apoptosis, and necrosis. These assays were used
`to provide a broader understanding of the cytotoxic. effects of
`preservatives on the conjunctiva.
`
`MATERIAIS AND ME'rHons
`
`Materials
`
`The Wong-Kilhourne derivative of Chang conitu'lcl‘iva, clone l—Sr—i.
`was obtained from American Type Culture Collection ((11—2012; Ma-
`nuSsas, VA). Kanamycin and phosphatebuffcrerI saline (PBS) without
`
`4123
`
`Argentum Pharm. LLC v. Alcon Research, Ltd.
`Case IPR2017-01053
`
`ALCON 2134
`
`

`

`4124 Baudouln et al.
`
`calcium and magnesium (PBS) were purchased from Eurobio. amoxiv
`cillin from GlaxoSmithKline (London, UK). EDTA from Signia~Aldricl1
`(Saint Quentin Fullavicr. France). Lnlll fetal calf serum (FCS)
`l'tuni
`Dominique Dutscher (Brumath. France). Dulbccco minimum essential
`medium (DMEM 1X) with L-glutztmine substitute (GlutttMAX-I). Alamar
`blue. and YOPRO‘I were obtained from lnvitrogen (Cergy—l’ontoise.
`France); annexin \-’/7-aminoactlnomycin D (7-AAD) was obtained from
`Immunotech (Ltuniny, France): and neutral red was obtained from
`Fluka (Buclis, Swityerland). The cytotoxic effects of tmvoprosi Z
`0.004% preserved without BAK and travoprost 0.004% containing
`0.015% BAK ('l'ravalan 7.. and ’l‘ravatan, respectively; Alcon. Fort Worth.
`TX). and Iatanoprost 0.005% containing 0.02% BAK (Xalatan; Pfizer.
`New York. NY) were compared With those of the culture medium,
`tsing PBS as a negative control. and with those of BAK (Sigma-Aldrich)
`dissolved in PBS to obtain the concentration of 0.02% as a positive
`control of cytotoxicity.
`
`Methods
`
`Coniunetival Cell Culture. Chang conjunctivaderived cells
`were cultured under standard conditions (moist atmosphere of 5% (:02
`at 37°C) in 75cm" bottles in DMEM with l.-glutamine substitute
`(GlutaMAX—I: Invitrogen) containing 10% FCS. 4.5 g/L glucose. 1%
`kanamycin. and 50 mg/mL amoxicillin. For each assay. culture dishes
`were seeded with 10S cells/ml. and were treated when they reached
`80% confluence. For tests using microtitration fluoromctric assays.
`cells were cultured in 9Gwell microplatcs. Cells used for [low cytom-
`etry assays were cultured in six-well plates. At the end of the incuba-
`tion in the presence of the drugs tested. they were collected after 5
`minute-incubation in 2 mL of 1 mM EDTA. These cells had been used
`
`previously to assess the toxicity of various prostaglandin analogues and
`served as a basis for toxicological uses with similar assessment of cell
`viability and apoptosis."'”
`Assessment of Cell Viability and Membrane Integrity
`with Neutral Red. Neutral red is a fluorescent molecule that enters
`the lysosomcs of living cells. Thus. coloration depends on cell viabil-
`ity?‘l7 Cells were incubated for 50 minutes at 57‘C with 50 [LL
`undiluted test solution and then were washed with .200 ML PBS. A
`200M. volume of medium containing 50 ug/mL neutral red was added
`to each Welland was incubated for 3 hours at 371‘. with an atmosphere
`containing 5% CO}. After another washing. 20t)-;.LL of ethanol—acetic
`acid was added, and the coloration was homogenized by agitation for
`15 minutes. The fluorescence of neutral red taken tip by viable cells
`was measured Wllll a satire microplate reader ('I'ecan Instruments.
`'l‘mppcs, France). with excitation at 555 nm and emission at 600 nm.
`Assessment of Cytotoxicity with Alamar Blue. Alamfl!‘
`blue is made of resazurin. which is blue and nonfluoreseent and can be
`reduced in the cells through enzyme activity to resorufin. which is pink
`and highly fluorescent.“ Fifty microliters of the undiluted test solu-
`tions was added to each well of a microtiter plate containing conjunc-
`tival cells and was incubated for :50 minutes at 37"C. The cells were
`then rinsed with 200 uL PBS. Two hundred microliters ofresazurin at
`a concentration of 50 ug/ml. was added to the cells and incubated for
`6 hours at 37"C in an atmosphere containing 5% C02. The conversion
`of resazurin to rcsorulin by the cells was measured by fluorescence
`with an excitation wavelength at 53.5 no: and an emission war '5»
`at 600 nm.
`
`Assessment of Apoptosis with YOPRO-l. YOPRCH is a
`cell-impermeable nuclear dye that stains cells when they have lost their
`membrane integrity:
`it
`is a useful probe to assess apoptosis.“ The
`opening of specific membrane pores that appear during apoptosis
`induces cationic movements. and reactive oxygen species (ROS) deliv-
`ery from mitochondria. The fluorescence of YUPRO-l is related to the
`generation of ROS when apoptosis occurs. Fifty microlitcrs ot‘ undi-
`luted test solution was added to each well of a microtiter plate con-
`taining conjunctiva] cells. incubated for 50 minutes at 57C, and then
`rinsed with 200 to]. PBS. YOPRO l. at a concentration of 2 FM in PBS.
`was added to the conjunctival cells. and the degree of apoptosis was
`
`IOVS, September 2007, Vol. 48, No. 9
`
`measured by fluorescence with excitation at 491 nm and emission at
`509 nm. Results of the YOPROI assay were expressed as a ratio to the
`resulls ul' llit' neutral red assay to correlate uptiplusis with cellular
`viability.
`Assessment of Apoptosis and Necrosis with Annexin
`V/7-AAI). Annexin V binds to the membrane phosphatidyI-sciine.
`which becomes exposed from the inner part of the cell membrane to
`the outer part during apoptosls and is an indicator of the first step of
`cell membrane alteration that occurs during the early phase of apopto-
`sis. By reacting like propidium iodide, 1AM) binds to DNA when the
`cell membrane is disrupted in the late phase of apoptosis and in
`necrosis. Conjunctiva—dcrived cells. cultured in six-well plates, were
`incubated for 30 minutes with SO—uL of undiluted test solution and
`were collected after incubation for 5 minutes in 2 mL of 1 mM EDTA.
`Cells were suspended in binding buffer at a concentration of 100,000
`cells/mL and SGML of cell suspension were combined Willi 5 [LL
`annexin V as a marker of early apoptosis and with l0 uI. 7-AAD as a
`marker of necrosis. and were incubated for 15 minutes on ice. After the.
`incubation period. 500 51,1. binding buffer was added. and ill: satilplcs
`were analyzed by flow cytometry (Beekman Coulter XL-MCL. Miami.
`Fl.) with discrimination of annexin V and 7-AAD on a biparametric
`histogram giving four cell populalions, cells negative to both market's
`(normal viable cells). cells positive only to annexin V (early apoptotic).
`cells positive to both annexin V and 1MB date apoptotic), and cells
`umahuv— nnlv Or\
`'.'.A In lmnml$n\
`rvuluv\ um: u; .- sun.“ \J—n—yluunl.
`
`Statistical Analysis
`All analyses were performed in a masked manner toward the drug
`tested and were repeated in independent assays. Results were ex-
`pressed as percentages of the untreated cell control and were. the
`man".- at ‘0 w-nll» laiv- W-A'Iu :u Ok—hh 2:“).
`In-
`unalla m m m m \BIA «cm in mm. mm
`a nu
`
`each concentration were analyzed with a one-way ANOVA test fol-
`lowed by Bonferroni
`test using statistics software (Statview IV for
`Windows; Abacus, Berkeley, CA); the level of significance was fixed at
`0.05.
`
`RESULTS
`
`Assessment of Cell Viability and Membrane
`Integrity with Neutral Red
`There was no decrease in the viability of cells exposed to PBS
`or travoprost 7. 0.004% preserved without BAK to incorporate
`neutral red compared with cells exposed to culture medium
`(Fig. 1). By comparison, latanoprost 0.005% containing 0.02%
`BAK, travoprost 0.004% with 0.015% BAK, and 0.02% BAK
`alone had signifiumt effects on cell viability, as assessed by
`neutral red uptake.
`
`Assessment of Cytotoxicity with Alainar Blue
`
`Travoprost 7. preserved without BAK had no cytotoxic effects
`on Chang conjunctiva] cells compared with cells treated with
`PBS (Fig. 2). There was a significant cytotoxic effect of latano-
`prost with BAK and 0.02% BAK alone. 'l’ravoprost with 0.015%
`BAK evoked an intermediate response that was significantly
`different from that in the latanoprost group. The reduction of
`resazurin to resorufin in the BAK- or latanoprost with 0.02%
`BAK-trcatcd cells was less than 10%.
`
`Assessment of Apoptosis by YOPRO-l
`
`'l‘here was no increase in apoptosis, measured by fluorescence
`of YOPRO-l, in cells treated with travoprost 2 without BAK
`compared with control medium.
`In contrast, apoptosis in-
`creased in cells treated with latanoprost will] BAK, travoprost
`with BAK, and BAK alone (P < 0.0001 compared with PBS and
`tmvoprost Z without BAK). The degree of apoptosis for each
`treatment was adjusted for cell viability by the ratio of
`
`

`

`IOVS, September 2007, Vol. :18, No. 9
`
`Antiglaucomatous Prostaglandin Analogues
`
`4125
`
`
`
`%ofMediumControl
`
`FIGURE 1. Cellular viability and mem
`brane integrity were preserved in cells
`exposed to PBS and travoprost Z with-
`out BAK compared with medium. as
`assessed by the neutral red assay. la-
`tanoprost and travoprost containing
`BAK and BAK alone showrrd signifir
`cantlv decreased cellular viability. 'P <
`0.0001 compared with medium (re-
`ferred to as 100%). PBS. and tmvo-
`pros! Z.
`
`140
`
`120
`
`100
`
`I
`
`
`
`IDPBS
`
`I
`
`:1 Travoprost 2
`
`lil Travoprost
`
`I Lata noprost
`
`a 9.02% BAK
`
`YOPRO—l and neutral red (Fig. 3). ’lhe ratio was not signifi-
`cantly different in cells treated with travoprost Z and culture
`medium, but it was increased approximately 38-fold in cells
`exposed to either latanoprost or BAK (P < 0.001 compared
`with medium, PBS, and travoprost Z). Travoprost with 0.015%
`BAK showed an intein‘iediate response that was significzu‘itiy
`different from tlte response in the latanoprost with 0.02% BAK
`and BAK groups.
`
`Assessment of Apoptosis and Necrosis with
`Annexin V/7-AAD
`
`In [low cytometry, the double-staining amtcxin V/7-AAD dis-
`criminates on a biparametric histogram cells that are undergo-
`ing early apoptosis (armexin positive, 74ml) negative),
`late.
`apoptosis (annexin positive, 7AM) positive), and necrosis (a11-
`nexin negative, 7-AA1) positive). No significant apoptosis or
`necrosis occurred in cells treated with PBS or with travoprost
`7. preserved without BAR (Fig. Ii). Cells treated with iatano-
`prost with O 02% BAK, tmvoprost with 0.015°2. BAK or 0.02%
`BAK alone had sigmftcant late apoptosis and necrosis com-
`pared with cells treated with medium or travoprost Z (P <
`0.0001). Significant early apoptosis occurred in cells treated
`with latanoprost with 0. 02% BAK that was not observed with
`[\ihnr frnnimml The Ininl tnm~ at?“w Aaron-Ska H1514,-
`uunun uyuuunlus.
`tux xuuu lumL \thxt usoLuuus use uaunage
`to conjunctival cells caused by apoptosis and necrosis. latano-
`prost with 0.02% BAK, travoprost with 0.015% BAK, and 0.02%
`BAK alone had similar total toxic effects on conjunctiva-de-
`rived cells (Pig. 5). There were no significant differences be.-
`twccn cells treated with PBS representing spontaneous apo-
`pmsis in the cell line and thOprost Z
`
`DISCUSSION
`
`Glaucoma, a chronic disease characterized by increased in‘
`tmocular pressure that commonly leads to blindnessfs is often
`treated with topical prostaglandins or [Ii-blockers. Unfortu‘
`nately, the chronic use of most IOP-lowen'ng medications is
`associated with some toxicity, such as allergic reactions, ocular
`pseudopemphigoid, allergic contact dermatitis, ptmctate cor—
`neal staining, and failure of filtration surgery.“"’ This toxicity
`has often been associated not with the active component of
`the medication but with the preservative BAK, which damages
`comeai epitheiiai ceiis, even at concentrations as low as
`0.005%." Samples ct alt demonstrated that BAK caused a
`significant inhibition of the growth of trztht-a‘tilar meshwork
`cells at extremely low concentrations. Sherwood ct al.3 re-
`ported in eyes chronically exposed to preservatives an increase
`in macrophages, lymphocytes, and fibroblasts in the conjunc-
`ii‘v'it and Tenor: capsule and a decrease in the tun-libel“ of
`conjunctival goblet cells. Although there are subtle differences
`with primary cultures of human conjunctiva] cells. the Wong-
`Kilboume derivative of the Chang conjunctiva-derived cell line
`has been used largely to determine the effects of toxic
`preservatives and IOP-lowering agents and is a well-recognized
`v )3
`Uri-vcan can».
`"u..- u
`..
`nun/l5]. 1
`‘l l};- nmspno ell-AV "Sud this r!\l\1I Int‘h\1nl veil line 01‘
`assess the direct (vtotoxicity of travoprost Z, the first commer-
`tially available BAK-free prostaglandin analogue (which (Oll-
`tains 0.004% travoprost and a preservative system [Soniaz
`Alcott Laboratories, l‘ort Worth, ’I'XJ), travoprost 0004% with
`0.015% [MK and lamnoprost 0. 0059’u with 002% BAK and to
`assess the (levelnpntent of npnptnsis after exposure to they;
`
`U Tra voptost Z
`
`l DPBS
`a Travoprost
`I BAK 0.02%
`I ILatanoprost
`
`l
`
`l
`
` l
`
`100
`
`90
`
`80
`
`z,
`
`
`
`~
`
`‘
`I
`
`
`
`FIGURE 2. Cytotoxicity assessment g 70 ‘1
`using the Alamar blue assay. latano~ U 50
`prost with BAK and BAK alone were
`cytotoxic in Chang conjunctivacle— g
`50
`rived cells and. to a lesser extent, in
`'6
`BAR-containing travoprost. whereas 2 40
`no cytotoxicity was observed in cells E
`exposed to travoprost Z without
`0
`BAK or cells treated with PBS.
`'P <
`32
`0.0001 compared with medium (re-
`ferred to as 100%) and travoprost Z.
`'.‘P < 0.0001 compared with latanrr
`prost with BAK and BAK alone.
`
`30
`20
`
`10
`
`0
`
`

`

`41 26 Baudouin ct al.
`
`IOVS, September 2007, Vol. 4’8, No. 9
`
`
`
`YOPFIO-1/NeutralRedRatio
`
`ANNww0"DM€0!
`
`.A c:
`
`:01
`
`El Tra voprost Z
`
`EPBS
`
`aTravoprog
`
`.0. 020/0 BAK
`ILatanoprost
`
`l
`
`i
`
`I
`
`FIGURF 5.
`
`.-\poptosis measured by
`
`the ratio of YOPRO-I and neutral
`
`red. No increase was obserVed in
`cells treated with PBS or travoprost 7.
`without BAK comparcd with PBS,
`whereas s‘gnificant aoostos‘s was
`'
`‘
`1
`‘
`observed with BAK and latanoprost
`with [MK and.
`lesser extent.
`to :1
`with
`BAK-conlaining
`travoprost
`‘P’0 0001 compared with PBS and
`travoprost Z without BAK. LP «1
`0.0001 compared with latanoprost.
`
`agents. These agents are aii prostagiandin analogues but they
`differ in the concentration and tltc type of preservative used
`'lhe neutral red assay measures the ability of viable cells to
`incorporate dye in their lysosomes, and the Alamar blue assay
`assesses intracellular redttction of resazurin by intact enzymatic
`systems in viable cells Both assa3s assess cell viabilit3 by
`.l. ‘1“... ".1 n.
`..I....eh...
`I .. I...“ oh...
`I
`n ... .11..“
`Wlklklll ILIKLJIIlIUDUAS llllkl‘ WIILII Kllk -' all RISKKl ui ilshlk IIIIL‘JJL‘
`improve the analysis of cytotoxic effects. In both assays,
`la-
`tanoprost with O. 029'm BA,K travoprost with 0. 015% DAR and
`IiAK alone demonstrated deleterious effec ts on the viability of
`the (.hang tonttlntt1v+dcm((1 cells, with tmvoprost 0.015%
`HAK the least toxic of the three, which is consistent with the
`well-demonstrated dose-dependent toxicity of 8.5.1933 Indeed
`previous studies comparing the three commercially available
`prostaglandin analogues found dill‘erences in their toxic pro-
`files corresponding to their co_ncenlmtion in BAK (the least
`concentr.1ted_.tl1e least toxic.)l
`YOPRO-l is onIyttaken up by membrane pores within cells
`undergoing apoptosis. Atmexin V binds to phosphatidyl scrim:
`exposed on cell membranes only during apoptosis. Therefore,
`test results are positive only in cells undergoing apoptosis.
`However, the combined assay annexin V/T—AAI) discriminates
`cells undergoing early apoptosis (annexin positive. 7-AA I) neg;
`ative), late apoptosis (amtexin positive, 7-AAI) positive). and
`necrosis (annexin negative, 1AM) positive). As with the cyto-
`toxicity assays, cells exposed to latattoprost with BAK,
`tra-
`voprost containing BAK, and BAK alone reached a significant
`level of apoptosis compared with media or traioprost 7. with-
`out BAK. These results are consistent with previous studies in
`
`which the Chang ceiis were exposed to iatanoprost with BAK,
`travoprost with BAK, and bimatoprost with BAK.” In these
`studies, the development of cell cytotoxicity and apoptosis was
`clearly related to ISAK.
`As with any other experimental in vitro models, the present
`. .HI 4‘I\r-
`.II I'..... I .1 AS
`.I1
`.01: I. .....
`study did have some. limitations. Evaluations were conducted
`in Uluv \Iuk 'LKLI aux, uut IIIKSK 1K ‘qu‘ aux Lunc‘wlkul 'V‘Vuu Ulu
`
`previous results and with those previously published in other
`cell lines. In vitro studies remove the cells front the influence
`of circulating substances, such as hormones and inflammatory
`mediators, and from other cells present in the tissue, especially
`inflammatory cells. HoWever, the present study is valuable for
`aiding our understanding of the effects of preservatives be-
`cause the experimental stimulus can be well controlled. and
`the resultant cellular response can he quantitated. It would be
`of interest to extend these studies to other clones and to other
`
`cell types, such as corneal epithelial and trahecular meshwork
`cells. Additional studies evaluating the. effects of BAK on in
`IIanunatory mediators such as prostaglandins and leukotrienes
`would be of value and interest.
`Although the results of the present study cannot be directly
`extrapolated to humans, the toxicity to BAK in this human ceil
`line was demonstrated to occur after exposure to the test
`solutions for only 310 minutes The half-life of BAK in the
`conjunctiva is nearl3 12 hours, suggesting that the damage to
`1011111111 oval cells observed in vitro ma3 also occur in vivo.‘
`Because of its complex structure and heterogeneous cell pop-
`ulations, tissue reaction after repeated contact with a topically
`administered drug differs from that of a cell monolayer ex-
`
`MEN" 7MB
`
`*
`
`*
`
`I” ‘1
`
`1“
`
`u
`
`E g
`
`..
`I.
`
`a
`
`
`
`7M0?
`
`:7MD- with BAK and latanoprost with BAK
`I:II'AAD-I'
`I Ann.
`
`thlkt: 4. No increase in apoptosis
`and necrosis, measured by annexin
`V/T-AAI) assays, was observed in
`cells treated with PBS or with tra-
`voprost Z. without BAK compared
`with cells treated with travoprost
`
`or BAK. Anncxin +/T’-.—\AD—. PBS
`compared with all other groups (1’ a’.
`0,001). Anncxin +/7-1\AI)+. no sig-
`nificant
`(liftcrcnccs.
`Annexin-[T—
`AAD+. PBS and tmvoprost Z com-
`
`pared Wi
`travoprost with BAK
`latanopmst with IKAK and hAK (I) 1:
`0.0001).
`'l'ravoprost with BAK corn-
`pared with latanopt'ost with BAK and
`BAX (1’ < 0.001).
`
`

`

`IOVS, September 2007, Vol.
`
`'18, No. 9
`
`Antiglaueomatous Prostaglandin Analogues
`
`4127
`
`10TH. rotoc EFEC'I'
`
`Porem
`
`ll
`
`
`
`FIGURE 5. No increase in the total toxic effect. measured as either apoptosis or as necrosis using annexin VITAAD assays. was observed in cells
`treated with PBS or with Iravoprost Z without BAK compared with cells treated with ttavoprost Willi BAK. latanoprost with BAK. or BAK alone.
`PBS compared with travoprost with BAK (P = 0.0004). lantanoprost with BAK. and BAK alone (1’ < 0.0001). 'l‘ravoprost 7. compared with
`travoprost with BAR (P = 0.0009). iantanoprost with BAK. and BAK:1ione{P < 0.0001). 'i'ravoprost with BAK. iatanoprost with BAK. and BAK
`alone (P = NS).
`
`posed to chemical compounds. Repeated administration over
`the long term of a weakly toxic drug would most likely cause
`increased renewal of epithelial structures and tissue stimula-
`tion on an itL‘lanmtatory mode rather than destruction. indeed,
`many reports show increased inflammatory responses in the
`conjunctiva of patients treated over the long term as a reaction
`to the chronic use of :uttiglaucoma drugs“? '3 and especially to
`their preservatives; this
`'as not fottnd in patients treated with
`unpreserved beta blockers.0 The clinical profile of prostaglan-
`din :malogucs is usually satisfactory, probably because they are
`administered once a day. We reported mild inflammatory reac-
`tions in conjunctiva! specimens from patients receiving these
`treatments." Nevertheless, ocular surface involvement may be
`pathologically enhanced after years or decades of treatment,
`after multiple treatments, or when patients have associated
`ocular surface diseases, such as dry eye disease or mcibornian
`gland dysfunction. In such populations, the use of nontoxic
`compounds may be of critical importance, especially because
`the ocular surface has a major influence on compliance and
`filtering surgery outcome. In a series of in vitro assays,
`the
`absence of toxicity consistently observed with travoprost 2
`Without BAK, compared with the high level of toxicity noted
`with exposure of conjunctival cells to BAK, suggests that use of
`topical prostaglandin analogues without BAK may reduce the
`topical ophthalmic toxicity reported with chronic use of these
`agents. Future studies of these agents in humans will assist in
`further characterizing the presence or absence of topical oph-
`thalmic toxicity.
`
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