ALCON 2016
`Apotex Corp. v. Alcon Research, Ltd.
`Case IPR2013-00428
`
`

`

`4124 Baudouin et a1.
`
`calcium and magnesium (PBS) were purchased from Eurohio, amoxi»
`cillin from GlaxoSmithKline (London, UK), EDTA from SigmaAldrich
`(Saint Quentin Fallavier, France), and fetal calf serum (FCS) from
`Dominique Dutscher (Brumath, France). Dulbecco minimum essential
`medium (DMEM 1 X) with L—glutamine substitute (GlutaMAX—l), Alamar
`blue, and YOPRO-l were obtained from Invitrogen (CergyLPontoise,
`France); annexin V’fi-aminoactinomycin D (7HAAD) was obtained from
`lmmunotech (Luminy, France); and neunal red was obtained from
`Fluka (Buchs, Switzerland). The cytotoxic effects of travopros‘t Z
`0.004% preserved without BAK and travoprost 0.004% containing
`0.015% BAK (Travatan Z and ’lf'ravatan, respectively; Alcon, Fort Worth,
`TX), and latanoprost 0.005% containing 0.02% BAK (Xalatan; Pfizer,
`New York, NY) were compared with those of the culture medium,
`using PBS as a negative control, and with those of BAK (SigmaAldrich)
`dissolved in PBS to obtain the concentration of 0.02% as a positive.
`control of cytotoxicity.
`
`Methods
`
`Conjunctival Cell Culture. Chang conjunctivaaderived cells
`were cultured under standard conditions (moist atmosphere of 5% CO2
`at 57°C) in 75111112 bottles in DMEM with Lglutamine substitute
`(GlutaMAXl; Invitrogen) containing 10% FCS,
`(1.5 g/L glucose, 1%
`kanamycin, and 50 mg/ml. amoxicillin. For each assay, culture dishes
`were seeded with 105 cells/mL and were treated when they reached
`80% confluence. For tests using microti [ration iluorometric assays,
`cells were cultured in 96—well microplates. Cells used for flow cytonr
`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 apoptosisxg’l7
`Assessment of Cell Viability and Membrane Integrity
`With Neutral Red. Neutral red is a fluorescent molecule that enters
`the lysosomes of living cells. Thus, coloration depends on cell viabil—
`ity.9’l7 Cells were incubated for 30 minutes at 37C with 50 ML
`undiluted test solution and then were washed with 200 [iL PBS. A
`2004M. volume of medium containing :70 ug/m L neutral red was added
`to each well and was incubated for 5 hours at 37°C with an atmosphere
`containing 5% C02. After another washing, 200-,uL of ethanol—acetic
`acid was added, and the coloration was homogenized by agitation for
`15 minutes. The fluorescence of neutral red taken up by viable cells
`was measured with a Safire microplate reader (’l'ecan instruments,
`’l’rappes, France), with excitation at 555 nm and emission at 600 nm.
`Assessment of Cytotoxicity With Alamar Blue. Alamar
`blue is made of resazurin, which is blue and nonfluorescent and can be
`reduced in the cells through enzyme activity to resorufln, 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—
`tiva] cells and was incubated for 30 minutes at 57°C. The cells were
`then rinsed with 200 uL PBS. Two hundred microliters of resazurin at
`a concentration of :30 pig/ml. was added to the cells and incubated for
`6 hours at 57°C in an atmosphere containing 5% C02. The conversion
`of resazurin to resorufln by the cells was measured by fluorescence
`with an excitation wavelength at 535 nm and an emission wavelength
`at 600 nm.
`
`is a
`Assessment of Apoptosis with YOPRO-l. YOPRO-t
`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—
`cry from mitochondria The fluorescence of ‘YOPROAl is related to the
`generation of ROS when apoptosis occurs. Fifty microliters of undr
`luted test solution was added to each well of a microtiter plate con-
`taining conjunctival cells, incubated for 30 minutes at 37°C, and then
`rinsed with 200 [LL PBS. YOPROI, at a concentration of 2 MM in PBS,
`was added to the conjunctiva] cells, and the degree of apoptosis was
`
`IOVS, September 2007, Vol. 48, No. 9
`
`measured by fluorescence with excitation at 1491 nm and emission at
`509 nm. Results of the YOPRO-l assay were expressed as a ratio to the
`results of the neutral red assay to correlate apoptosis with cellular
`viability
`Assessment of Apoptosis and Necrosis with. Annexin
`V/7-AAD. Annexin V binds to the membrane phosphatidyl—seiine,
`which becomes exposed from the inner part of the cell membrane to
`the outer part during apoptosis 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, 7AAAI) binds to DNA when the
`cell membrane is disrupted in the late phase of apoptosis and in
`necrosis. Conjunctiva—derived cells, cultured in six-well plates, were
`incubated for '50 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 fro-ML of cell suspension were combined with 5 ML
`annexin V as a marker of early apoptosis and with 10 ,uL 71AM) as a
`marker of necrosis, and were incubated for 1.5 minutes on ice. After the
`incubation period, 500 [LL binding buffer was added, and the samples
`were analyzed by flow cytometry (Beckman Coulter XL—MCL, Miami,
`FL) with discrimination of annexin V and 7—AAD on a biparametric
`histogram giving four cell populations, cells negative to both markers
`(normal viable cells), cells positive only to annexin V (early apoptotic),
`cells positive to both annexin V and 7‘AAI) (late apoptotic), and cells
`positive only to 7—AAD (necrotic).
`
`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
`means of 18 wells (six wells in three different assays). Mean values for
`each concentration were analyzed with a one—way ANOVA test fol-
`lowed by Bonferroni test using statistics software (Statview IV for
`W’indows; 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 Z 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 significant effects on cell viability, as assessed by
`neutral red uptake.
`
`Assessment of Cytotoxicity with Alamar Blue
`
`Travoprost Z preserved without BAK had no cytotoxic effects
`on Chang conjunctival 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. Travoprost with 0.01 5%
`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—treated cells was less than 10%.
`
`Assessment of Apoptosis by YOPROJ
`
`There was no increase in apoptosis, measured by fluorescence
`of YOPRO—l, in cells treated with. travoprost Z Without BAK
`compared with control medium.
`In contrast, apoptosis in-
`creased in cells treated with latanoprost with BAK, travoprost
`with BAK, and BAK alone (P < 0.0001 compared with PBS and
`travoprost Z without BAK). The degree of apoptosis for each
`treatment was adjusted for cell viability by the ratio of
`
`

`

`IOVS, September 2007, Vol. 48, No. 9
`
`Antiglaucomatous Prostaglandin Analogues
`
`4125
`
`140
`
`1 20
`
`
`
`aPas'
`
`
`
`E: Travoprost Z
`
`Travoprost
`
`Lata nopmst
`
`I 0.02% BAK
`
`
`
`§‘
`
`OU
`
`5’ 130
`
`30
`
`E
`FIGURE. 1. Cellular viability and men}
`brane integrity were preserved in cells E
`exposed to PBS and travoprost Z witty B fifl
`out BAK compared with medium, as E
`assessed by the neutral red assay. La~
`u..-
`tanoprost and travoprost containing
`G
`BAK and BAK alone showed signifi-
`2:}?
`canl’ly decreased cellular viability. *P <
`0.0001 compared with medium (re
`ferred to as 100%), PBS, and travo—
`pl‘OSt Z.
`
`48
`
`2{)
`
`0
`
`i
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`YOPROJ and neutral red (Fig. 3). The 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 intermediate response that was significantly
`different from the response in the latanoprost with 0.02% BAK
`and BAK groups.
`
`Assessment of Apoptosis and Necrosis with
`Annexin V/7~AAD
`
`In flow cytometry, the double—staining annexin V/7-AAD dis-
`criminates on a biparametric histogram cells that are undergo-
`ing early apoptosis (annexin positive, 7-AAD negative), late
`apoptosis (annexin positive, 7—AAD positive), and necrosis (an~
`nexin negative, 7—AAD positive). No significant apoptosis or
`necrosis occurred in cells treated with. PBS or with travoprost
`Z preserved without BAK (Fig. 4). Cells treated with latano-
`prost with 0.02% BAK, travoprost with 0.015% BAK, or 0.02%
`BAK alone had significant 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
`other treatments. The total toxic effect describes the damage
`to conjunctival cells caused by apoptosis and necrosis. Latano-
`prost with 0.02% BAK, travoprost with 0.01 5% BAK, and 0.02%
`BAK alone had similar total toxic effects on conjunctiva'de—
`rived cells (Fig: 5). There were no significant differences be»
`tween cells treated with PBS representing spontaneous apo-
`ptosis in the cell line and travoprost Z.
`
`DISCUSSION
`
`Glaucoma, a chronic disease characterized by increased in.
`traocular pressure that commonly leads to blindness}5 is often
`treated with topical prostaglandins or B—blockers. Unfortu—
`nately, the chronic use of most IOP~lowering medications is
`associated with some toxicity, such as allergic reactions, ocular
`pseudopemphigoid, allergic contact dermatitis, punctate cor—
`neal staining, and failure of filtration surgerylz‘i‘ This toxicity
`has often been associated not with the active component of
`the medication but with the preservative BAK, which damages
`corneal epithelial cells, even at concentrations as low as
`0.005%.” Samples et 31.27 demonstrated that BAK caused a
`significant inhibition of the growth of trabecular meshwork
`cells at extremely low concentrations. Sherwood et a1.3 re»
`ported in eyes chronically exposed to preservatives an increase
`in macrophages, lymphocytes, and fibroblasts in the conjunc—
`tiva and Tenon capsule and a decrease in the number of
`conjunctival goblet cells. Although there are subtle differences
`with primary cultures of human conjunctiva] cells, the Wong
`Kilbournc derivative of the Chang conjunctiva—derived cell line
`has been used largely to determine the effects of toxic
`preservatives and lOP—lowering agents and is a well-recognized
`model.”*28 The present study used this conjunctiva] cell line to
`assess the direct cytotoxicitv of travoprost Z, the first commer-
`cially available BAK-free prostaglandin analogue (which con—
`tains 0.004% travoprost and a preservative system [SotZia;
`Alcon Laboratories, Fort Worth, TXD, travoprost 0.004% with
`0.015% BAK, and latanoprost 0.005% with 0.02% BAK and to
`assess the development of apoptosis after exposure to these
`
`
`
`100
`
`96 —
`
`iffl “
`
`a 36 -
`&
`a
`FIGURE 2. Cytotoxicitv assessment
`Q
`.
`’
`‘
`.
`usmg the Alamar blue assay. Latanol Q 63
`prosi with BAK and BAK alone were
`E
`cytotoxic in Chang conjunctiva—do
`:
`56
`rived cells and, to a lesser extent, in
`1%
`ENC-containing travoprost, whereas
`‘1’
`no cytotoxicity was observed in cells §
`exposed to travoprost Z without
`a:
`BAK or cells treated with P135. *P <
`39,
`0.0001 compared with medium (re
`feircd to as 100%) and travoprost Z.
`H7 < 0.0001 compared with latancr
`prost with BAK and BAK alone.
`
`43 N
`
`3a “
`29‘ fl
`
`16 a
`
`a ‘
`
`
`
`If~
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
` C] PBS
`c1 Travoprost z
`
`
`a Travoprost
`
`
`
`
`
`a BAK 6.02%
`
`I Latanaprost
`
`
`
`

`

`4126 Baudouin et a1.
`
`IOVS, September 2007, Vol. 48, N0. 9
`
`40
`
`NNWWOU‘IOQ‘!
`m5‘.
`
`ad» a
`
`YOPRO-fiNeutralRedRatio
`
`
`
`Tra vaprost
`
`
`
`El 0.02% BAK
`
`l Lara neprost
`
`El Tra voprost 2
`PBS
`
`FIGURE 3. Apoptosis measured by
`the ratio of YOPRO—l and neutral
`red. No increase was observed in
`cells treated with PBS or travoprost Z
`without BAK compared with PBS,
`whereas significant apoptosis was
`observed with BAK and latanoprost
`with BAK and,
`to a lesser extent,
`with
`BAK-containing
`travoprost.
`*P < 0.0001 compared with PBS and
`travoprost Z without BAK.
`1'13 <
`0.0001 compared with latanoprost.
`
`agents. These agents are all prostaglandm analogues, but they
`differ in the concentration and the type of presewative used.
`The neutral red assay measures the ability of viable cells to
`incorporate dye in their lysosomes, and the Alamar blue assay
`assesses intracellular reduction of resazurin by intact enzymatic
`systems in viable cells. Both assays assess cell viability by
`different mechanisms and, when they are used in association,
`improve the analysis of cytotoxic effects. In both assays,
`la»
`tanoprost with 0.02% BAK, travoprost with 0.015% BAK, and
`BAK alone demonstrated deleterious effects on the viability of
`the Chang conjunctiva-derived cells, with travoprost 0.015%
`BAK the least toxic of the three, which is consistent with the
`well~demonstrated dose~dependent toxicity of BAK.22 Indeed
`previous studies comparing the three commercially available
`prostaglandin analogues found differences in their toxic pro-
`files corresponding to their concentration in BAK (the least
`concentrated, the least toxic).17
`YOPRO-l is only taken up by membrane pores within cells
`undergoing apoptosis. Annexin V binds to phosphatidyl serine
`exposed on cell membranes only during apoptosis. Therefore,
`test results are positive only in cells undergoing apoptosis.
`I'lowever, the combined assay annexin V/7-AAD discriminates
`cells undergoing early apoptosis (annexin positive, 7-AAD neg-
`ative), late apoptosis (annexin positive, 7-AAD positive), and
`necrosis (annexin negative, 7—AAD positive). As with the cyto—
`toxicity assays, cells exposed to latanoprost with BAK,
`tra—
`voprost containing BAK, and BAK alone reached a significant
`level of apoptosis compared with media or travoprost Z with-
`out BAK. These results are consistent with previous studies in
`
`1
`
`120
`
`which the Chang cells were exposed to latanoprost with BAK,
`travoprost with BAK, and bimatoprost with BAK,” In these
`studies, the development of cell cytotoxicity and apoptosis was
`clearly related to BAK.
`As with any other experimental in vitro models, the present
`study did have some limitations. Evaluations were conducted
`in only one cell line, but these results are consistent with our
`previous results and with those previously publisher! in other
`cell lines. In vitro studies remove the cells from 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 be quantitated. It would be
`of interest to extend these studies to other clones and to other
`
`cell types, such as corneal epithelial and trabecular meshwork
`cells. Additional studies evaluating the effects of BAK on in
`flammatory 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 cell
`line was demonstrated to occur after exposure to the test
`solutions for only 30 minutes The half—life of BAK in the
`conjunctiva is nearly 12 hours, suggesting that the damage to
`conjunctival cells observed in vitro may also occur in vivo.18
`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-
`
`i
`
`100
`
`ANNEX!" “(MD
`
`20
`
`
`
`
`PBS
`
`Tramprosl Z
`
`Tmopmst
`
`Lahnopmst
`
`0.02% BAK
`
`FIGURE 4. No increase in apoptosis
`and necrosis, measured by annexin
`V/7‘AAD assays, was observed in
`cells treated with PBS or with tra-
`voprost Z Without BAK compared
`with cells treated with travoprosl‘
`with BAK and latanoprost with BAK
`or BAK. Annexin +/7—AAD—, PBS
`compared with all other groups (P <
`0001), Annexin +/7-AAD+, no sig—
`niticant
`differences. Annexin-j’?
`AAD+, PBS and travoprost Z com-
`pared with travoprost with BAK
`latanoprost with BAK and BAK (P <
`0.0001). Travoprost with BAK com—
`pared with latanoprost with BAK and
`BAK (P < 0.001).
`
`

`

`IOVS, September 2007, Vol. 48, No. 9
`
`Antiglaucomatous Prostaglandin Analogues
`
`4127
`
`mTfimeMT
`
`129
`
`908““!
`
`100
`
`21'
`
`FIGURE 5. No increase in the total toxic effect, measured as either apoptosis or as necrosis using annexin V/7AAD assays, was observed in cells
`treated with PBS or with travoprosl Z without BAK compared with cells treated with travoprost with BAK, latanoprost with BAK, or BAK alone.
`PBS compared with trayoprost with BAK (P = 0.0004), lantanoprost with BAK, and BAK alone (P < 0,0001). Travoprost Z compared with
`travoprost with BAK (P = 0.0009,), lantanoprost with BAK, and BAK alone (P < 0.0001). Travoprost with BAK, latanoprost 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 inflantmatory 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 antiglaucoma drugs-5‘7712 and especially to
`their preservatives; this was not found in patients treated with
`unpreserved beta blockers.9 The clinical profile of prostaglan—
`din analogues is usually satisfactory, probably because they are
`administered once a day. We reported mild inflammatory reac—
`tions in conjunctiyal specimens from patients receiving these
`treatments.9 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 meibomian
`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 Z
`without BAK, compared with the high level of toxicity noted
`with expo sure 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.
`
`References
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`. Netland PA, Landry T, Sullivan F,K et al. Travoprost compared with
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`. Baudouin C, Hamard P, Liang H, Creuzot—Garcher C, Bensoussan L,
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