CLINICAL SCIENCES
`
`Analysis of Topical Cyclosporine Treatment
`of Patients With Dry Eye Syndrome
`
`Effect on Conjunctival Lymphocytes
`
`Kathleen
`
`Kunert MD Ann
`
`Tisdale MS Michael
`
`Stern PhD Smith Ilene
`
`Gipson PhD
`
`Objective To study the effect of topical cyclosporine
`on lymphocyte activation within the conjunctiva of
`patients with moderate to severe dry eye syndrome
`Sjogren and non-SjOgren
`
`Methods Biopsy specimens were obtained at baseline
`and after months of cyclosporine treatment from eyes
`of 32 patients with moderate to severe dry eye syn
`drome 19 were cyclosporine treated 0.05% cyclospor
`in n13 0.1% cyclosporine n6 and 13 were ve
`hicle treated Within this group there were 12 with SjOgren
`syndrome and 20 with nonSjOgren syndrome Biopsy
`tissue was analyzed using immunohistochemical
`local
`ization of binding of monoclonal antibodies to lympho
`cytic markers CD3 CD4 and CD8 as well as lympho
`cyte activation markers CD la and HLA-DR
`
`tive for CD3 CD4 and CD8 while in vehicle-treated eyes
`results showed increases in these markers although these
`differences were not statistically significant Following
`treatment with 0.05% cyclosporine there was
`signifi
`in the number of cells expressing the lym
`cant decrease
`.05 and HLA-DR
`phocyte activation markers CD la
`.05 indicating less activation of lymphocytes as com
`pared with vehicle
`treatment Within the SjOgren pa
`tient subgroup those treated with 0.05% cyclosporine
`also showed
`significant decrease in the number of cells
`positive for CD11a P.001 as well as CD3 P.03
`reduction in number of activated lympho
`
`indicating
`
`cytes
`
`Conclusion Treatment of dry eye syndrome with topi
`cal cyclosporine significantly reduced the numbers of ac
`tivated lymphocytes within the conjunctiva
`
`Results In cyclosporine-treated eyes biopsy results of
`conjunctivae showed decreases in the number of cells posi
`
`Arch Ophthalmol 2000 181489-1496
`
`tact
`
`ERATOCONJUNCTIVITIS sicca
`
`KCS or dry eye syn
`drome is characterized by
`chronic dryness of the cor
`nea and conjunctiva Pa
`tients with KCS typically show symp
`ranging from
`toms of ocular discomfort
`irritation to severe pain Redness burn
`ing itching foreign body sensation con
`and
`lens intolerance photophobia
`blurred vision can occur.2
`Although KCS can arise from vari
`ous types of diseases common to all
`is the
`involvement of immune-mediated or in
`flammatory-mediated pathways.3 Immu
`nopathologic studies of the lacrimal gland
`in patients with SjOgren syndrome show
`progressive lymphocytic infiltration pri
`marily consisting of CD4 and
`cells.45
`This infiltration is believed to be respon
`sible for the destruction of normal secre
`tory function.6 Lymphocytic infiltration of
`the lacrimal gland has also been de
`scribed in patients with non-SjOgren
`KCS.78 Although the immunopathologic
`
`analysis of the lacrimal gland has re
`ceived considerable attention less work
`changes oc
`has been done on pathological
`curring in the ocular surface The chronic
`the ocular surface in SjOgren
`dryness of
`syndrome has been attributed to deterio
`ration of lacrimal gland function with de
`creased tear production.9 However
`in
`SjOgren syndrome conjunctival epithe
`infiltration pre
`hal and stromal T-cell
`dominantly CD3 and CD4
`lympho
`cytes has also been shown to occur along
`with drying of the ocular surface.9
`role for an immuno
`Supporting
`pathogenesis of KCS are the reports of ac
`tivated lymphocytes as demonstrated by
`expression of lymphocyte activation mark
`ers such as HLA-DR MHC class
`II and
`ICAM-1 intercellular adhesion mol
`in the conjunctiva of patients with
`ecule-i
`SjOgren syndrome.23 To date there is
`information on the effect of modu
`lating these molecules in the conjunctiva
`of patients with SjOgren and non
`SjOgren syndrome
`
`little
`
`From the Schepens Eye
`Institute and
`Research
`
`Department of Ophthalmology
`Harvard Medical School
`Boston Mass Drs Kunert and
`Gipson Ms Tisdale Allergan
`mc Irvine Calif Dr Stern
`and the National Eye Institute
`Bethesda Md Ms Smith
`Dr Stern is an employee of
`
`Allergan Inc
`
`REPRINTED
`
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`1489
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`
`.Assodation
`
`ii rights reserved
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`
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`
`

`

`SUBJECTS AND METHODS
`
`SUBJECTS
`
`at
`
`equal
`
`from 32 patients were ex
`Conjunctival biopsy specimens
`amined 13 patients were treated with 0.05% CsA
`with
`0.1% CsA and 13 with vehicle alone This subject group
`was randomly chosen from double-masked
`vehicle-
`controlled clinical study designed by Allergan mc Irvine
`the efficacy and safety of topical CsA
`Calif to investigate
`in the treatment of moderate to severe
`The study
`in compliance with Good Clinical Prac
`was conducted
`tices investigational site institutional review board regu
`lations sponsor and investigator obligations informed con
`sent regulations and the Declaration of Helsinki Potential
`form and
`prescreening informed consent
`patients signed
`form prior to actual en
`second written informed consent
`rollment.2 The protocol
`for this study is described briefly
`here Adult patients of either sex were eligible for partici
`diagnosis of moderate to severe KCS
`they had
`pation if
`initial examination as defined by the following criteria
`Schirmer test results without anesthesia less than or
`to mmJS mm
`eye if Schirmer test re
`in at
`least
`sults without anesthesia equaled mmJS mm then Schirmer
`mmJS mm
`test results with nasal stimulation had to be
`in the same eye
`sum of corneal and interpalpebral con
`than or equal
`in the same
`to
`junctival staining greater
`eye where corneal staining was greater
`than or equal to
`baseline Ocular Surface Disease Index22 score of 0.1
`responses of not applicable and
`with no more than
`than or equal
`on the Subjective Facial
`to
`score greater
`Expression Scale.2 Signs and symptoms must have been
`present despite conventional management
`they had par
`Patients were excluded from the study if
`trial with CsA ophthalmic
`ticipated in an earlier clinical
`emulsion or had used systemic or topical ophthalmic CsA
`within 90 days prior to the study Other exclusion criteria
`were the presence or history of any systemic or ocular dis
`order or condition including ocular surgery trauma and
`disease current or recent use of topical ophthalmic or sys
`temic medications that could affect
`dry eye condition
`known hypersensitivity to any component of the drug or
`such as stains or anesthetics
`procedural medications
`
`lens wear during the study recent within
`required contact
`use of temporary punctal plugs dur
`month or anticipated
`ing the study permanent occlusion of lacrimal puncta within
`lac
`months of the study or if
`the patients were pregnant
`pregnancy Patients were also ex
`tating or planning
`cluded if
`they appeared to have end-stage lacrimal gland
`mmJS
`disease Schirmer reading with nasal stimulation
`mm or if
`their KCS was secondary
`to the destruction of
`conjunctival goblet cells or scarring
`diagnosis of Sj Ogren syndrome was used
`retrospective
`to ensure
`with modified criteria reported by Vitali et a123
`consistent definition of SjOgren syndrome was as
`that
`signed to the patients enrolled Diagnosis included pres
`ence of at least one of the following autoantibodies in sera
`antinuclear antibody ANA rheumatoid factor RF and
`SjOgren syndrome autoantibodies class SS-A Ro and class
`SS-B La In addition oral and ocular symptoms were used
`to classify patients with SjOgren syndrome
`drop of 0.05% or 0.1% CsA oph
`Patients instilled
`thalmic emulsions or vehicle of CsA ophthalmic emulsion
`twice daily in each eye for months once on waking in
`the morning and once at bedtime Patients were allowed
`tears REFRESH Lubricant Eye
`to use assigned artificial
`Drops Allergan mc as needed up to month
`biopsy specimens of stan
`Full-thickness conjunctival
`dard size 2-3 mm were removed from the worse eye
`by surgeons following standard procedure The worse eye
`was defined as the eye with the worse Schirmer tear
`test
`value without anesthesia and the worse sum of corneal
`and interpalpebral conjunctival staining If both eyes were
`comparable then the right eye was used At the baseline
`the conjunctival biopsy specimen was obtained from
`visit
`the inferonasal quadrant close to midline At the 6-month
`the sample was removed from the same eye but from
`visit
`the inferotemporal quadrant also close to midline
`
`TISSUE PROCESSING FOR
`IMMUNOHISTOCHEMICAI
`
`ANAIXSIS
`
`the baseline biopsy specimens were imme
`After removal
`diately frozen in OCT embedding compound Tissue-Tek
`Miles Laboratories Elkhart md in
`cryomold Miles
`and stored at 80C until patient-matched
`Laboratories
`
`tears is the most
`Currently administration of artificial
`common therapy available for lubricating dry ocular sur
`face This palliative treatment gives only temporary and in
`complete symptomatic relief and does not address the cause
`of the symptoms which may include immune-mediated
`inflammation of the ocular surface Evidence of inflamma
`tory processes in the pathogenesis of KCS led to the de
`velopment of cyclosporine CsA as
`first attempt
`this condition therapeutically Cyclosporine is an immu
`nosuppressive agent commonly used systemically to treat
`inflammatory diseases such as psoriasis or rheumatoid ar
`thritis or to prevent organ transplant rejection.4 Topical
`CsA has been used as treatment of ocular conditions such
`as vernal keratoconjunctivitis5 corneal transplants6 cor
`neal ulcers7 and herpetic stromal keratitis.8 The effect of
`this drug on inflammatory diseases is due to its ability to
`
`to treat
`
`inhibit T-cellmediated inflammation by preventing the ac
`cells by antigen-presenting cells or
`tivation of
`cytokines.92 Activated
`cells are responsible for the pro
`duction of inflammatory substances such as cytokines
`which lead to further tissue damage and in turn to the ac
`tivation of more
`cells and the production of even more
`inflammatory substances
`trials with this drug have shown improve
`Clinical
`ment in various objective measures of KCS such as cor
`neal staining and Schirmer test values.2 To attempt to
`find tissue correlates in these patients conjunctival bi
`from patients with SjOgren and non
`opsy specimens
`SjOgren KCS treated with CsA or vehicle were evaluated
`for the presence of activated
`immunohistochemically
`cells CD3
`cell CD4 FT helper cell and CD8
`cell and lymphocyte-activation markers
`
`Fcytotoxic
`
`REPRINTED
`
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`1490
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`
`2000 American Medical
`
`.Assodation
`
`ii rights reserved
`
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`
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`
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`
`

`

`6-month biopsy specimens were obtained and similarly fro
`zen Six-micrometer sections were taken from each block
`mounted on gelatin-coated slides and processed for im
`munohistochemical analysis Sectioning of tissue blocks and
`immunohistochemical experiments were performed as pairs
`to minimize
`of biopsies pretreatment and posttreatment
`due to experimental conditions
`differences
`
`IMMUNOHISTOCHEMICAI
`
`ANAIXSIS
`
`Immunohistochemical staining for lymphocytic markers as
`well as lymphocyte activation markers was conducted us
`ing monoclonal antibodies to CD3 PharMingen San Diego
`Sanjose Calif CD8 Bec
`Calif CD4 Becton-Dickinson
`ton-Dickinson Sanjose CD1 la PharMingen San Diego
`and HLA-DR PharMingen Cryostat sections were fixed
`in cold acetone 20C for minutes and air dried at room
`temperature for 30 to 45 minutes They were then rinsed
`in changes of phosphate-buffered saline PBS and incu
`bated in PBS with 1% bovine serum albumin BSA Sigma
`Chemical Co St Louis Mo for 10 minutes Sections were
`hour at room temperature in primary an
`incubated for
`tibodies at concentrations derived empirically CD3 1.0
`pg/mL CD4 5.0 pg/mL CD8 2.5 pg/mL CD11a 10.0
`pg/mL and HLA-DR 1.0 pg/mL Sections were rinsed in
`PBS alone followed by 10 minutes in PBS with 1% BSA be
`hour at room temperature in the sec
`fore incubation for
`ondary antibody fluorescein isothiocyanateconjugated Af
`finipure Donkey Anti-Mouse IgG Jackson Immunoresearch
`West Grove Pa at
`dilution of 1/50 Sections were then
`rinsed in PBS mounted in Vectashield Vector Labs Bur
`lingame Calif cover-slipped and viewed under micro
`scope Eclipse E800 Nikon Melville NY interfaced with
`digital camera Spot Digital Camera Diagnostic Instru
`ments mc Micro Video Instruments Avon Mass Sec
`ondary antibody controls omitting the primary antibody
`for each
`immunohistochemical
`for all biopsy specimens
`analysis were run
`Three separate images were acquired for each anti
`body and biopsy specimen under 20 objective using
`Spot acquisition program Diagnostic Instruments mc The
`field selected for imaging was the field with the high
`est number of positive cells followed by images to the left
`
`first
`
`and right of that area In this manner the entire biopsy area
`was usually captured
`
`COUNTING PROCEDURE
`
`Measurement of the entire area of epithelium and stroma
`substantia propria was achieved by tracing the area us
`ing the lasso tool under the Adobe Photoshop computer
`program Adobe Systems mc Sanjose Calif The total data
`in pixels was acquired through the Im
`area measured
`age Histogram command in Photoshop Two indepen
`dent counts were recorded for cells positive for each anti
`body within the traced area Cells per unit area of pixels
`were adjusted to real unit area or cells per millimetersquared
`of real tissue area based on 28.346 pixels per centimeter
`and the fact that mm equals 67.8 cm equals
`in Photoshop
`20 magnification on the Nikon micro
`1922 pixels at
`scope Data were recorded as cells per millimeter squared
`for all markers and statistical analysis was based on these
`measurements
`
`STATISTICAI METHODS
`
`test
`
`Baseline characteristics were tabulated and summarized by
`treatment groups Overall differences among treatment
`2-way analysis of variance
`groups were tested using
`ANOVA for continuous
`variables and the Fisher exact
`for categorical
`variables
`changes in the number of cells expressing
`Percent
`lymphocytic and/or
`lymphocyte activation markers were
`summarized using descriptive statistics ie sample size
`mean SD minimum maximum and median
`1-way
`ANOVA with main effect
`for treatment was used to test
`from baseline and
`for differences in percent
`change
`ratios among treatment groups by visit
`for
`the test
`among-group differences in main effect was significant
`pairwise comparisons were made Within-
`then all
`from baseline were analyzed by the
`group changes
`test method
`paired
`The same analysis was performed on SjOgren and
`non-SjOgren subpopulations excluding the 0.1% CsA
`treatment group in which there was only
`in the
`SjOgren subset
`
`If
`
`patient
`
`CD1 la and HLA-DR to further understand the under-
`lying mechanism of CsA treatment
`
`RESULTS
`
`PATIENT POPULATION
`
`The meanSD age of our subjects was 59.0 13.5 years
`range 28.8-84.2 years including 27 women and men
`Within this group there were 12 SjOgren and 20 non
`SjOgren patients
`
`LYMPHOCYTIC MARKERS
`
`decrease from baseline in the num
`In general there was
`ber of cells positive for CD3 CD4 and CD8 following
`
`of CsA The only ex
`treatment with either concentration
`ception was that there was mean increase from base
`helper cell population follow
`line in the CD4-positive
`ing 0.05% CsA treatment In comparison all cells positive
`for the lymphocytic markers increased from baseline fol
`lowing vehicle treatment
`shows the percent change from baseline
`Figure
`for cells expressing the lymphocytic markers CD3 CD4
`and CD8 after months of treatment for the overall pa
`reduction from
`tient population Note that there was
`baseline in the number of CD3-positive cells in the CsA
`treated groups while there was an increase from base
`line in the vehicle-treated group There was also an in
`crease from baseline in the numbers of CD4-positive cells
`smaller increase in the 0.05%
`in the vehicle group with
`CsA group and
`slight decrease in the 0.1% CsA group
`
`REPRINTED
`
`ARCH OPHTHALMOL/ VOL 118 NOV 2000
`1491
`
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`
`2000 American Medical
`
`.Assodation
`
`ii rights reserved
`
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`
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`
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`
`

`

`140
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`20
`
`40
`
`0.05o CsA
`0.10CsA
`
`Vehicle
`
`0.0500 CsA
`
`0.10CsA
`
`Vehicle
`
`P.05
`
`250
`
`200
`
`150
`
`100
`
`50
`
`-50
`
`CD3
`
`CD4
`
`CD8
`
`CD11a
`
`UI
`
`HLA-DR
`
`Percent change for cells positive for the lymphocytic markers C03
`Figure
`CD4 and CD8 in the overall patient population
`Values presented are mean
`percent changeSE from baseline at month
`CsA indicates cyclosporine
`
`20
`
`10
`
`10
`
`20
`
`-30
`
`-40
`
`-50
`
`-60
`
`70
`
`-80
`
`go
`
`0.05o CsA
`
`Vehicle
`
`Sjögren Syndrome
`
`NonSjögren Syndrome
`
`CD3
`
`Figure
`
`for C03-positive cells from the Sjogren syndrome
`Percent change
`Values presented are mean
`and nonSjOgren
`syndrome subpopulations
`percent changeSE from baseline at month
`The
`paiwise comparisons from 1-way analysis of variance GsA indicates
`
`value is relative to
`
`cyclosporine
`
`The CD 8-positive cells exhibited the same pattern as CD3-
`decrease from baseline
`positive cells but with less of
`following CsA and less of an increase from baseline fol
`lowing vehicle treatment However the change from base
`lymphocytes CD3 CD4 and
`line in the number of
`CD
`did not reach statistical significance either among
`or within treatment groups Figure
`Within the SjOgren subgroup 0.5% CsA treatment
`.03 decreases in CD3-
`resulted in significantly greater
`positive cells than did vehicle The CD3-positive cells de
`creased from baseline in all treatment groups among the
`non-Sjogren subgroup However
`this decrease was not
`statistically significant in either group Figure
`
`LYMPHOCYTE-ACTIVATION MARKERS
`
`decrease from baseline in the num
`In general there was
`ber of cells positive for lymphocyte activation markers
`CD11a and HLA-DR following CsA treatment com
`pared with an increase from baseline in these cells fol
`
`Figure
`
`Percent change
`for cells positive for the lymphocyte activation
`la and Hut-DR in the overall patient population
`Values
`markers CDI
`presented are mean percent changeSE from baseline at month
`The
`.05 and within-group
`differences P.03 CsA indicates cyclosporine
`
`values are relative to paiwise comparisons
`
`40
`
`20
`
`-20
`
`-40
`
`-60
`
`-80
`
`100
`
`0.05o CsA
`
`Vehicle
`
`Sjögren Syndrome
`
`NonSjögren Syndrome
`
`CD11a
`
`for CD1Ia-positive cells from the SjOgren
`Percent change
`Figure
`syndrome and nonSjogren syndrome subsets Values presented are mean
`percent changeSE from baseline at month
`The
`value is relative to
`pairwise comparisons from I-way analysis of variance CsA indicates
`
`cyciosporine
`
`lowing vehicle treatment for the overall patient popula
`tion
`
`Statistical analysis revealed
`significant among-
`in change from baseline for cells ex
`group difference
`pressing CD11a P.04 and HLA-DR P.02 for the
`overall patient population Pairwise comparisons showed
`significant reductions with 0.05% CsA treatment com
`pared with treatment with vehicle in cells positive for both
`.05 and HLA-DR 016
`markers CD11a
`Figure
`Furthermore
`comparison within indi
`vidual treatment groups comparing pretreatment to post-
`statistically significant de
`treatment results revealed
`crease from baseline for HLA-DR in the 0.05% CsA group
`.03 Figure
`Within the SjOgren subgroup treated with 0.5% CsA
`.001 decreases in cells
`there were significantly greater
`de
`positive for CD11a than in vehicle There was
`crease from baseline in both treatment groups CsA and
`vehicle among the non-Si Ogren subgroup Figure
`This decrease did not reach statistical significance
`
`REPRINTED
`
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`

`Figure
`
`Immuno fluorescence micrographs
`demonstrating cells positive for the lymphocyte activation marker C/il Ia in conjunctival biopsy specimens of
`and
`vehicle The number of
`0.05% cyclosporine and
`sicca pretreatm8nt and posttreatnient with
`patients with nonSjOgren
`and/i
`group decreased while the number in the vehicle-treated biopsy sample
`positive cells within epithelium and substantia propria in the cyclosporine-treated
`increased bar25 pm
`
`keratoconjunctivitis
`
`show
`and Figure
`Figure
`set
`representative
`of immunofluorescence micrographs for cells positive for
`the markers CD11a and HLA-DR from the non-SjOgren
`subgroup treated with 0.05% CsA or vehicle Figure
`shows immunofluorescence micrographs for cells posi
`tive for the markers CD3 and CD11a from patients with
`SjOgren KCS treated with 0.05% CsA
`
`COMMENT
`
`In the present study immunohistochemical analysis was
`used to evaluate changes in the presence of cells posi
`tive for lymphocytic and lymphocyte activation mark
`biopsy specimens of patients with mod
`ers in conjunctival
`erate to severe KCS following treatment with 0.05% CsA
`0.1% CsA or vehicle We found that CsA treatment re
`duced the number of activated
`lymphocytes within the
`ocular surface of patients with and without SjOgren syn
`drome After months of treatment with 0.05% CsA sta
`tistically significant decreases were seen in cells positive
`for CD la and HLA-DR compared with those in vehicle
`for the overall patient population Within the SjOgren pa
`tient subgroup treated with 0.05% CsA there were also
`significantly greater decreases than with vehicle in the
`number of cells positive for CD3 and CD11a
`
`These findings provide additional evidence that in
`role in the pathogenesis of KCS and
`flammation plays
`suggests that modulating the underlying immune re
`sponse may prove more efficacious
`in the treatment of
`KCS than the frequent use of artificial
`tears Topical CsA
`has been successfully used for the treatment of canine
`dry eye for many years Studies in the canine KCS model
`have demonstrated that CsA decreases the conjunctival
`and lacrimal gland lymphocytic infiltrates.24-26
`However
`limited number of
`there have been only
`reports on the use of topical CsA in the treatment of dry
`eye syndrome in humans27-29 with only
`attempt to look
`cellular level.30 Power
`at the effect of the treatment at
`et al reported
`reduction in CD4-positive
`significant
`lymphocytes in both the conjunctival epithelium and
`the substantia propria of patients with secondary SjO
`gren syndrome compared with nondry eye controls fol
`lowing treatment with CsA The present study also dem
`onstrated
`significant decrease in CD3-positive cells after
`months of 0.05% CsA treatment in patients with SjO
`gren syndrome
`Furthermore the number of cells positive for CD la
`and HLA-DR which are lymphocyte activation mark
`ers decreased significantly in patient populations treated
`with CsA HLA-DR is
`class II major histocompatibility
`
`REPRINTED
`
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`1493
`
`WWW.ARCHOPHTHALMOL.COM
`
`2000 American Medical
`
`.Assodation
`
`ii rights reserved
`
`Downloaded From http//archopht.jamanetwork.com/
`
`by University of Michigan User on 01/25/2016
`
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`

`

`Figure
`
`keratoconjunctivitis
`
`Immuno fluorescence micrographs
`demonstrating cells positive for HL.4-DR in conjunctival biopsy specimens of patients with nonSjögren
`and
`and
`decrease in the number of positive cells
`0.05% cyclosporine and
`sicca pretreatment and positreatment with
`vehicle
`within epithelium and substantia propria in the 0.05% cyclosporine-treated
`group is apparent compared with an increase in number in the vehicle-treated biopsy
`Example of negative control bra vehicle biopsy in which the primaty antibody was omitted Bar25 pm A-C
`sample Eand
`
`complex antigen that is expressed in inflamed regions and
`ligand for the I-cell receptor CD4
`lym
`serves as
`phocytes are activated through signal from HLA-DR mol
`ecules of antigen-presenting cells.3 Immunopathologic
`studies show evidence of immune activation of the con
`junctival epithelium in SjOgren syndrome Compared with
`significantly greater percentage of con
`control eyes
`junctival epithelial cells from patients with SjOgren syn
`drome express the HLA-DR molecule.232 Hingorani et
`in HLA-DR expression on cells in
`decrease
`al3 report
`the substantia propria of patients with atopic keratocon
`junctivitis following months of treatment with CsA In
`
`confirmation of these findings the data presented here
`demonstrate reduction in the number of cells positive
`for the lymphocyte activation marker HLA-DR after
`months of 0.05% CsA treatment
`CD 1aILFA-
`lymphocyte functionassociated an
`tigen is associated with adhesion of lymphocytes mac
`rophages and granulocytes and is
`ligand of intercel
`lular adhesion molecule-i ICAM-i which supports the
`binding of lymphocytes to antigen-presenting cells.34
`CD ia is up-regulated during activation of human lym
`phocytes and with its ligand ICAM-i plays an impor
`interactions and cell migration of
`tant role in cell-to-cell
`
`REPRINTED
`
`ARCH OPHTHALMOL/ VOL 118 NOV 2000
`1494
`
`WWW.ARCHOPHTHALMOL.COM
`
`2000 American Medical
`
`.Assodation
`
`ii rights reserved
`
`Downloaded From http//archopht.jamanetwork.com/
`
`by University of Michigan User on 01/25/2016
`
`FAMY CARE - EXHIBIT 1012-0006
`
`

`

`Figure
`
`C03 and
`Immuno fluorescence micrographs
`and
`and
`CD1 la in conjunctival biopsy specimens of patients
`demonstrating cells positive for
`sicca pretreatment and posttreatment with 0.05% cyclosporine Note the decrease in number of positive cells within the
`with SjOgren keratoconjunctivitis
`epithelium and sutistantia propria in the positreatment biopsy specimens bar25pm
`
`lymphocytes into the surrounding tissue such as the con
`junctival epithelium and substantia propria.3537 Cyclo
`sporine has been shown to regulate immune-based in
`flammation within epithelial tissues by inhibiting ICAM-
`production.38 Our data support these results showing re
`duced immune activation by means of
`decrease
`in the
`number of cells positive for CD la after 6-month course
`of 0.05% topical CsA treatment
`Part of the beneficial effect of CsA might be due to
`the reduction in I-cell activation as illustrated by de
`crease of cells positive for HLA-DR By preventing the
`migration of new lymphocytes into the conjunctiva as
`suggested by the reduction in CD la-positive cells CsA
`may help to reduce the inflammatory process The fact
`that the data show
`reduction in positive cells mainly
`for the lymphocyte activation markers CD11a and
`HLA-DR suggests that CsA is promoting lymphocytes to
`more quiescent status rather than eliminating present
`lymphocytes This might explain why the change from
`lymphocytes CD3 CD4
`baseline in the number of
`and CD8 did not reach statistical significance
`for the
`overall patient population However another contribut
`ing factor may be the small patient number and high vari
`ability within each treatment group
`
`These results provide further evidence that topical
`use of CsA may have
`immunoregulatory effect on
`local
`inflammation in the conjunctiva of patients with dry eye
`syndrome This effect
`is evident
`in the reduction of the
`number of cells positive for lymphocyte activation mark
`cells in the con
`ers In preventing the activation of
`junctiva topical administration of CsA may interrupt an
`ongoing immune reaction Even though the reduction
`in Pan-T cells CD3 and CD11a seems to be larger in
`patients with SjOgren syndrome our data on the lym
`phocyte activation markers especially HLA-DR pro
`vide evidence that CsA treatment is providing benefits
`for both types of dry eye syndrome The larger effect
`in
`patients with SjOgren syndrome may be due to the fact
`there are greater numbers of lymphocytic
`infil
`that
`trates in patients with this disease
`In conclusion this study demonstrates
`activated lymphocytes with topical CsA use in patients with
`moderate to severe KCS Consistent with these findings
`clinical symptoms of KCS also appear to improve with the
`use of CsA in the overall patient population of the multi-
`center study conducted by Allergan Inc.21 This suggests that
`CsA treatment may help to reduce the pathophysiological
`factors contributing to the development of KCS
`
`reduction in
`
`REPRINTED
`
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`1495
`
`WWW.ARCHOPHTHALMOL.COM
`
`2000 American Medical
`
`.Assodation
`
`ii rights reserved
`
`Downloaded From http//archopht.jamanetwork.com/
`
`by University of Michigan User on 01/25/2016
`
`FAMY CARE - EXHIBIT 1012-0007
`
`

`

`Accept edfor publication March 27 2000
`The authors wish to thank specifically Brenda
`PhD Allergan mc for her help and input
`in organizing
`Ksander PhD Schepens Eye Re
`this project and Bruce
`search Institute for his critical
`review of the manuscript
`This project was supported by Allergan mc Irvine Calif
`Gipson PhD Schepens Eye Re
`Reprints Ilene
`search Institute Boston MA 02114 e-mail gipson
`@vision en harvard edu
`
`Reis
`
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