Analysis of Topical Cyclosporine Treatment
`of Patients With Dry Eye Syndrome
`
`Effect on Conjunctival Lymphocytes
`
`Kathleen S. Kunert, MD; Ann 5. Tisclale, MS; Michael E. Stern, PhD;]. A. Smith; Ilene K. Gipson, PhD
`
`Obie-cIiv_e: 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 6 months of cyclosporine treatment from eyes
`of 32 patients with moderate to severe dry eye syn-
`drome; 19 were cyclosporine treated (0.0S°/o cyclospor-
`ine, n=l3; 0.1% cyclosporine, n=6) and 13 were ve-
`hicle treated. Within this group there were 12 with Sjogren
`syndrome and 20 with non—Sjc")gren 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 CDl1a and HI_A~DR.
`
`tive for CD3, CD4, and CD8, While in vehicle-treated eyes,
`results showed increases in these markeis, although these
`differences were not statistically significant. Following
`treatment with 0.05% cyclosporine, there was a signifi-
`cant decrease in the number of cells expressing the lym-
`phocyte activation markers CD1 la (P< .05) and I-ILA-DR
`(P< 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 a significant decrease in the number of cells
`positive for CDI1a (P<.OOl) as well. as CD3 (P<.O3),
`indicating a reduction in number of adtivated lympho-
`cytes.
`I
`-
`
`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. 2000,11 1.8:1489-1496
`
`
`ERATOCONJUN CTIVITIS sicca
`
`(KCS), or dry eye syn-
`drome, is characterized by
`chroni.c dryness of the cor-
`nea and conjunctiva.‘ Pa-
`tients with KCS typically show symp-
`toms of ocular discomfort ranging from
`irritation to severe pain. Redness, burn-
`ing, itching, foreign body sensation, con-
`tact lens intolerance, photophobia, and
`blurred vision can occur?
`
`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+ T and B cells.”
`This infiltration is believed to be respon-
`sible for the destruction of normal secre-
`tory function.“ Lymphocytic infiltration of
`the lacrimal gland has also been de-
`scribed in patients with non-Sjogren
`KCS.” Although the immunopathologic
`
`From the Schepens Eye
`Research Institute and
`
`Department of Ophthalmology,
`Harvard Medical School,
`Boston, Mass (Drs Kunert and
`Gipson, Ms Tisdale); Allergan,
`Inc, Iwine, Calif (Dr Stern);
`and the National Eye Institute,
`Bethesda, Md (Ms Smith),
`Dr Stern is an employee of
`Allergcm Inc.
`
`analysis of the lacrimal gland has re-
`ceived considerable attention, less work
`has been done on pathological changes oc-
`curring in the ocular surface. The chronic
`dryness of the ocular strrface in Sjogren
`syndrome has been attributed to deterio-
`ration of lacrimal gland function with de-
`creased tear production.9’1° However, in
`Sjogren syndrome, conjunctival epithe-
`.lial and stromal T-cell infiltration (pre-
`dominantly CD3+ and CD4+ T lympho-
`cytes) has also been shoviln to occur along
`with drying of the ocular surface.‘’'“
`Supporting a role for an immuno-
`pathogenesis of KCS are the reports of ac-’
`tivated lyrnphocytes as demonstrated by
`expression of lymphocyte activation mark-
`ers such as I-{LA-DR (MHC class II) and
`lCAM—1 Cintercellularjadhesion mol-
`ecule-I.) in the conjunctiva of patients with
`Sjogren syndrome.”-13 To date, there is
`little information on theleffect of modu-
`lating these molecules in} the conjunctiva
`of patients with Sjogren and non-—
`Sjogren syndrome.
`‘
`
`.m__.__
`(REPRINTED) ARCH O17}-ITHALMOI./VOL 1.18. NOV 2000
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`EXHIBIT 101 2
`
`

`
`SUBJECTS AND METHODS
`
`SUBJECTS
`
`Conjunctival biopsy specimens from 32 pati.ents were ex-
`amined; 13 patients were treated with 0.05% CSA, 6 with
`0.1% CsA, and 13 with vehicle alone. This subject group
`was randomly chosen from a double-masked, vehicle-
`controlled clinical study designed by Allergan, inc, Irvine,
`Calif, to investigate the efficacy and safety of topical CsA
`in the treatment of moderate to severe KCS.“ The study
`was conducted in compliance with Good Clinical Prac-
`tices, investigational site institutional review board regu-
`lations, sponsor and investigator obligations, informed con-
`sent regulations, and the Declaration of Helsinki. Potential
`patients signed a prescreening informed consent form and
`a second written informed consent form prior to actual en-
`rollment.“ The protocol for this study is described briefly
`here. Adult patients of either sex were eligible for partici-
`pation if they had a diagnosis of moderate to severe KCS
`at initial examination as defined by the following criteria:
`(1) Schirnier test results (without anesthesia) less than or
`equal to 5 mm/5 min in at least 1 eye (if Schirmer test re-
`sults without anesthesi.a equaled O mm/5 min, then Schinner
`test results with nasal stimulation had to be >3 mm/5 min
`in the same eye); (2) sum of corneal and inteipalpebral con-
`junctival staining greater than or equal to +5 in the same
`eye where corneal staining was greater than or equal to +2;
`(3) a baseline Ocular Surface Disease index“ score of 0.1
`with no more than 3 responses of “not applicable”; and (4)
`a score greater than or equal to 3 on the Subjective Facial
`Expression Scale.“ Signs and symptoms must have been
`present despite conventional management.
`Patients were excluded from the study if they had par-
`ticipated in an earlier clinical trial with CSA ophthalmic
`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 medit:ations that could affect a dry eye condition;
`known hypersensitivity to any component of the drug or
`procedural medications such as stains or anesthetics;
`
`
`required Contact lens wear during the study; recent (within
`1 month) or anticipated. use of temporary punctal plugs dur-
`ing the study; permanent occlusion of lacrimal puncta within
`3 months of the study; or if the patients were pregnant, lac-
`tating, or planning a pregnancy. Patients were also ex-
`cluded if they appeared to have end-stage lacrimal gland
`disease (Schirmer reading with nasal stimulation <3 mm/5
`min) or if their KCS was secondary to the destruction of
`conjunctival goblet cells or scarring.
`A retrospective diagnosis of Sjogren syndrome was used
`with modified criteria reported by Vitali. et al” to ensure
`that a consistent definition of Sjogren syndrome was as-
`signed to the patients enrolled. Diagnosis included. pres-
`ence ofat least one of the following autoantibodies in sera:
`antinuclear antibody (ANA), rheumatoid factor (RF), and
`Sjogren syndrome autoantibodies class SS-A (R0) and class
`SS-B (La). In addition, oral and ocular symptoms were used
`to classify patients with Sjogren syndrome.
`Patients instilled 1. drop of 0.05% or 0.1% CSA oph-
`thalmic emulsions or vehicle of CSA ophthalmic emulsion
`twice daily in each eye for 6 months; once on waking in
`the morning and once at bedtime. Patients were allowed
`to use assigned artificial tears (REFRESH Lubricant Eye
`Drops; Allergan Inc) as needed up to month 4.
`Full-thickness conjunctival biopsy specimens of a stan-
`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 (wi.thout 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
`visit, the conjunctival biopsy specimen was obtained from
`the inferonasal quadrant close to midline. At the 6-month
`visit, the sample was removed from the same eye but from
`the inferotemporal quadrant, also close to midline.
`
`TISSUE PROCESSING FOR
`IMMUNOHISTOCHEMICAL ANALYSIS
`
`After removal, the baseline biopsy specimens were imme-
`diately frozen in OCT embedding compound (Tissue-Tek;
`Miles Laboratories, Elkhart, 1nd) in a cryomold (Miles
`Laboratories) and stored at —80°C until patient-matched
`
`Currently, administration of artificial tears is the most
`common therapy available for lubricating a dry ocular sur-
`face. This palliative treatment gives only temporary and in-
`complete symptomatic relief and does n ot 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 a first attempt to treat
`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.“ Topical
`CsA has been used as treatment of ocular con.diti.on.s such
`
`as Vernal keratoconjunctivitis,'5 corneal transplan.ts,“‘ cor-
`neal ulcers,” and herpetic stromal keratitis.‘8 The effect of
`this drug on inflammatoiy diseases is due to its ability to
`
`inhibit I-cell—mediated inflammation by preventing the ac-
`tivation of T cells (by antigen-presenting cells or
`cytol<ines).l9‘2° Activated T 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 T cells and the production of even more
`inflammatory substances.
`Clinical trials with this drug have shown improve-
`ment in various objective measures of KCS such as cor-
`neal staining and Schirmer test. values.“ To attempt to
`find tissue correlates in these patients, conjunctival bi-
`opsy specimens from patients with Sjogreii and non-
`Sjogren KCS treated with CSA or Vehicle were evaluated
`irnrnunohistochernically for the presence of activated T
`cells (CD3+ [Pan—T cell], CD4+ [T helper cell], and CD8+
`[cytotoxic T cell]) and l.ymphocyte-activation markers
`
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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. Secti.oning of tissue blocks and
`immunohistochernical experiments were performed as pairs
`of biopsies, pretreatment and posttreatment, to minimize
`differences due to experimental conditions.
`
`IMMUNOHISTOCHEMICAL ANALYSIS
`
`lmrriunohistochemical staining for lymphocytic markets as
`well as lymphocyl:e activation markers was conducted us-
`ing monoclonal antibodies to CD3 (PharMingen, San Diego,
`Calif), CD4 (Becton-Dickinson, Sanjose, Calif), CD8 (Bec-
`ton-Dickinson, Sanjose), CD1Ia (PharMingen. San Diego),
`and I-ILA-DR (PharMingen). Cryostat sections were fixed
`in cold acetone (-20°C) for 3 minutes and air dried at room
`temperature for 30 to 45 minutes. They were then rinsed
`in 3 changes of phosphate-buffered saline (PBS) and incu-
`bated in PBS with 1% bovine serum albumin (BSA) (Sigma
`Chemical Co, St Louis, M0) for 10 minutes. Sections were
`incubated for 1 hour at room temperature in primary an-
`tibodies at concentrations derived empirically: CD3, 1.0
`ug/mI.; CD4, 5.0 pg/mL; CD8, 2.5 pg/rnI_; CD11a, 10.0
`pg/mL; and HLA-DR, 1.0 pg/rnI-. Sections were rinsed in
`PBS alone, followed by 10 minutes in PBS with 1% BSA be-
`fore incubation for 1 hour at room t.emperature in the sec-
`ondary antibody, fluorescein isothiocyanate-conj ugated Af-
`finipure Donkey Anti-Mouse lgG (Jackson Immunoresearch,
`West Grove, Pa) at a dilution of 1/50. Sections were then
`rinsed in PBS, mounted in Vectashield (Vector Labs, Bur-
`lingame, Calif), cover-slipped, and viewed under a micro-
`scope (Eclipse E800; Nil<on,Melvil1e, NY) interfaced with
`a digital camera (Spot Digital Camera; Diagnostic lnstru-
`ments Inc, Micro Video Instruments, Avon, Mass). Sec-
`ondary antibody controls omitting the primary antibody
`for all biopsy specimens for each immunohistochemical
`analysis were run.
`Three separate images were acquired for each anti-
`body and biopsy specimen under a ><2O objective using 21
`Spot acquisition program (Diagnostic Instruments Inc). The
`first field selected for imaging was the field with the high-
`est number of positive cells, followed by images to the left
`
`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 Inc, Sanjose, Calif). The total data
`area, measured in pixels, was acquired through the “Im-
`age: Histograin” 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 millimeter squared
`of real tissue area, based on 28.346 pixels per centimeter
`in Photoshop and the fact that 1 mm equals 67.8 cm equals
`1922 pixels at X20 magnification on the Nikon micro-
`scope. Data were recorded as cells per millimeter squared
`for all markers, and sl.atist.ical analysis was based on these
`measurements.
`
`STATISTICAL METHODS
`
`Baseline characteristics were tabulated and summarized by
`treatment groups. Overall differences among treatment
`groups were tested using a 2-way analysis of variance
`(ANOVA) for continuous variables and the Fish er exact test
`for categorical variables.
`Percent changes in the number of cells expressing
`lyinphocytic and/or lymphocyte activation markers were
`summarized using descriptive statistics (ie, sample size.
`mean, SD, minimuin, maximum, and median). A 1-way
`ANOVA with main effect for treatment was used to test
`for differences in percent change from baseline and
`ratios among treatment groups by visit. If the test. for
`among-group differences in main effect was significant,
`then all 3 pairwise comparisons were made. Within-
`group changes from baseline were analyzed by the
`paired t test method.
`The same analysis was performed on Sjogren and
`non-Sjogren subpopulations, excluding the 0.1% CsA
`. treatment group in which there was only 1 patient in the
`Sjogren subset.
`
`(CD1 1a and HLA-DR) to further understand the under-
`lying mechanism of CsA treatment.
`
`——J————
`PATIENT POPULATION
`
`The mean:SD age of our subjects was 59.0: 13.5 years
`(range, 28.8-84.2 years), including 27 women and 5 men.
`Within this group, there were 12 Sjogren and 20 non-
`Sjogren patients.
`
`LYMPHOCYTIC MARKERS
`
`In general, there was a decrease from baseline in the num-
`ber of cells positive for CD3, CD4, and CD8 following
`
`treatment with either concentration of CsA. The only ex-
`ception was that there was a mean increase from base-
`li.ne in the CD4-positive T helper cell population follow-
`ing 0.05% CsA treatment. In comparison, all cells positive
`for the lymphocytic markers increased from baseline fol-
`lowing vehicle treatment.
`Figure ‘I shows the percent change from baseline
`for cells expressing the lymphocytic markers (CD3, CD4,
`and CD8) after 6 months of treatment for the overall pa-
`tient population. Note that there was a reduction from
`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
`in the vehicle group, with a smaller increase in the 0.05%
`CsA group and a slight decrease in the 0.1% CsA group.
`
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`
`100-
`
`A3AO)DU(36G@..
`
`Mean%Change
`
`
`
`
`
`
`
`CD3
`
`CD4
`
`CD8
`
`CD11a
`
`HLA-DR
`
`Figure 1. Percent change for cells positive for the lymphocytic markers CD3,
`CD4, and CD8 in the overall patient population. Values presented are mean
`percent change:«SE from baseline at month 6. CsA indicates oyc/osporine.
`
`Figure 3. Percent change for cells positive for the lymphocyte activation
`markers CD7 7a and HLA~DR in the overall patient population. Values
`presented are mean percent change:SE from baseline at month 6, The P
`values are relative to pairwise comparisons ( P<.05) and within-group
`differences (P<.03). Cs/1 indicates cyclosporine.
`
`
`
`
`Mean%Change
`
`Siogren Syndrome
`
`CD3
`
`
`“ Non—.~Si6gren Syndrome
`
`Figure 2. Percent change for GD3—positive cells from the Sjiigren syndrome
`and non—Sjo'gren syndrome suhpopulations. Values presented are mean
`percent changerSE from baseline at month 6. The P value is relative to
`pairwise comparisons from 1-way analysis of variance. 0sA indicates
`cyciosporine.
`
`Siiigren Syndrome
`
`CD113
`
`Non—Sifigren Syndrome
`
`Figure 4. Percent change for 0011a-positive cells from the Sjcigren
`syndrome and non—S/figren syndrome subsets. Values presented are mean
`percent change:rSE from baseline at month 6. The P value is relative to
`pairwise comparisons from l-way analysis of variance. Us/l indicates
`cyclosporlne.
`
`lowing vehicle treatment for the overall patient popula-
`tion.
`
`The CD8-positive cells exhibited the same pattern. as CD3-
`positive cells but with less of a decrease from baseline
`following CsA and less of an increase from baseline fol-
`lowing vehicle treatment. However, the change from base-
`line in the number of T lymphocytes (CD3+, CD4+, and
`CD8+) did not reach statistical significance, either among
`or Within treatment groups (Figure 1).
`Within the Sjogren subgroup, 0.5% CsA treatment
`resulted in significantly greater (P< .03) decreases in CD3-
`positive cells than did vehicle. The CD3-positive cells de-
`creased from baseline in all treatm.ent groups among the
`non—S_jogren subgroup. However, this decrease was not
`statistically significant in either group (Figure 2).
`
`LYMPHOCYTEACTIVATION MARKERS
`
`In general, there was a decrease from baseline in the num-
`ber of cells positive for lymphocyte activation markers
`CDl1a and HLA-DR following CsA treatment com-
`pared with an increase from baseline i.n these cells fol-
`
`Statistical analysis revealed a significant among-'
`group difference in change from baseline for cells ex-
`pressing CD11a (P=.O4) and I-ILA-DR (P=.O2) 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
`markers CDl1a (P = .05) and HLA-DR (P = .0 16)
`(Figure 3). Furthermore, a cornparison within indi-
`vidual treatment groups, comparing pretreatment to post-
`treatment results, revealed a statistically significant de-
`crease from baseline for I-ILA-DR in the 0.05% CsA group
`(P: .03) (Figure 3).
`\Vithin the Sj ogren subgroup treated with 0.5% CsA,
`there were significantly greater (P< .001), decreases in cells
`positive for CDl1a than i.n Vehicle. There was a de-
`crease from baseline in both treatment groups (CSA and
`vehicle) among the non-Sjogren subgroup (Figure 4).
`This decrease did not reach statistical significance.
`
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`

`
`

`
`Accepted for publication March 27, 2000.
`The authors wish to thank specifically Brenda L. Reis,
`PhD (Allergan, Inc), for her help and input in organizing
`this project and Bruce R. Ksancler, PhD (Schepens Eye Re-
`search Institute), for his critical review of the manuscript.
`This project was supported by Allergan Inc, Irvine, Calif
`Reprints: Ilene K. Gipson, PhD, Schepens Eye Re-
`search Institute, Boston, MA 02114 (e-mail: gipson
`@vision.eri.harvarctedu).
`
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