JOURNAL OF OCULAR PHARMACOLOGY AND THERAPEUTICS
`Volume Ill. Number 5. 2002
`€:> Mary Ann Liebert. Inc.
`
`Blood Concentrations of Cyclosporin A During
`Long-Term Treatment With Cyclosporin A
`Ophthalmic Emulsions in Patients With Moderate
`to Severe Dry Eye Disease
`
`DAVID S. SMALL, J.z ANDREW ACHEAMPONG, 1 BRENDA REIS, 1.3
`KATHERINE STERN, 1 WILLIAM STEWART,4 GREGG BERDY,5
`RANDY EPSTEIN,6 ROBERT FOERSTER/ LANCE FORSTOT,8
`DIANE D-S TANG-LIU 1
`
`1 Allergtm, Inc. Irvine, CA
`2Prese111 affiliation: Eli Lilly and Company, Indianapolis, IN
`3 Presem affiliation: Wyeth Ayers/ Corp., Huntingdon Valley, PA
`4 Plwrmaceutical Research Corp., Charleston. SC
`5Cre"e Coeur, MO
`6Rush Presbyterian Sr. Luke's Medical Center, Chicago. IL
`7Colorado Springs, CO
`Bcomeal Consul tams of Colorado, Littleton. CO
`
`ABSTRACT
`
`To quantify blood cyclo porin A (CsA) concentrations during treatment with CsA top(cid:173)
`ical ophthalmic emulsions. blood was collected from 128 patients enrolled in a Phase 3,
`multicenter, double-masked, randomized, parallel-group study of CsA eyedrops for treat(cid:173)
`ment of moderate to severe dry eye disease. Patients received 0.05% CsA, 0.1 % CsA. or
`vehicle b.i.d. for 6 months; vehicle-treated patients then crossed over to 0.1 % CsA b.i.d. for
`6 months. CsA concentrations were measured using a validated LC/MS-MS assay (quanti(cid:173)
`tation limit = 0.1 ng/mL). No patient receiving 0.05% CsA h·ad any quantifiable CsA in the
`blood (n = 96 samples). All but 7 of 128 (5.5%) trough blood samples from the 0. 1% CsA
`group were below the quantitation limit for CsA; none exceeded 0.3 ng/mL. CsA was also
`below the limit of quantitation in 205 of 208 (98.6%) of serial po tdose blood samples col(cid:173)
`lected from 26 patients during I dosing interval between months 9 and 12. The highest Cmax
`mea ured, 0.105 ng/mL at 3 hours postdose, occurred in a 0.1% CsA-treated patient. The e
`results indicate that long-term use of topical CsA ophthalmic emulsions at doses that are
`clinically efficacious for treating dry eye will not cause any system-wide effects.
`
`INTRODUCTION
`
`Keratoconjunctivitis sicca (KCS). or chronic dJy eye disease, is a frustrating condition chnrac(cid:173)
`lcrized by sensations of ocular grittiness, burning, photophobia. and blurred vision. Despite affecting
`
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`APOTEX 1018, pg. 1
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`miluons worldwide and substantially altering productivity and quality of life (I, 2), therapies for KCS
`have been palliative at best. Recent advances in understanding the pathophysiology of disease allow
`the development of effective therapies for the first time.
`The ocular surface, lacrimal glands, and interconnecting nerves form a homeostatic functional
`unit that maintains normal tear production. Chronic dry eye disease results from aT cell-mediated in(cid:173)
`t1ammatory process leading to a disruption of this nerve traffic. Multiple factors, including an age(cid:173)
`related drop in systemic androg·en levels and chronic ocular surface irritation, create an environment
`in which activated T cells are recruited to the ocu lar surface ·and lacrimal glands in a vicious cycle
`of inflammation that ultimately results in destruction of the lacrimal glands (3). Cyclosporin A (CsA)
`topical ophthalmic emulsion targets the inflammatory basis of disease by acting as a local im(cid:173)
`munomodulator and anti-inflammatory agent. By inhibiting activation of infiltrating T cells on the
`ocular surface and lacrimal glands, production of inflammatory cytokines is prevented (4). CsA treat(cid:173)
`ment has been shown to decrease several molecular markers of immune-based inflammation in con(cid:173)
`junctival biopsies of dry eye patients (5-7).
`Phase 2 and Phase 3 clinical trials have established both the safety and efficacy of topical CsA
`at concentrations ranging from 0.05% to 0.4% (8, 9). CsA is currently approved for systemic treat(cid:173)
`ment of immune-related disorde rs at much higher doses than those given for KCS. Oral administra(cid:173)
`tion for treatment of psoriasis or rheumatoid arthritis produces blood CsA concentrations in the range
`of 75 ng/mL (Cm;n) to 655 ng/mL (Cmax) ( I 0). Because it is a potent immunosuppressant when used
`at these high doses, it is important to assess syste mic exposure to CsA upon administration of the
`very low doses needed for treatment of KCS.
`Topical ophthalmic treatment with CsA requires 2600-fold lower dosage than does systemic us(cid:173)
`age (10). A preliminary study of topical ophthalmic CsA conducted during Phase 2 development found
`that topical instillation produced negligible blood CsA concentrations, although sampling was sparse
`(8). The present study examines blood CsA concentrations of KCS patients participating in a much
`larger Phase 3 study of CsA topical ophthalmic emulsions. We show conclusively that CsA is prac(cid:173)
`tically undetectable in the blood of patients treated with these ophthalmic preparations.
`
`PATIENTS AND METHODS
`
`Clinical Trial Overview
`
`Therapeutic blood monitoring was done at selected sites on a subset of patients enrolled in a
`Phase 3, multicenter, double-masked, randomized, parallel group study of cyclosporin A topical oph(cid:173)
`thalmic emulsions for the treatment of dry eye disease (9). Eligible study participants had moderate
`to severe dry eye disease as defined by I) Schirmer test of 2:: 5 mm/5 min (without anesthesia) in at
`least one eye; 2) sum of corneal and interpalpebral conjunctival staining of 2! + 5 in the same eye in
`which corneal staining was 2! +2; 3) indication of moderate to severe dry eye disease by the Ocular
`Surface Disease Index ( 11) and Subjective Facial Expression Scale. Patients were excluded if they
`had end-stage lacrimal disease, puncta! plugs, were nsing medications that could interfere with the
`study results, wore contact lenses during the study, or were pregnant or lactating. The study was con(cid:173)
`ducted from July 1997 to January 1999 and was in compliance with Good Clinical Practices, inves(cid:173)
`tigational site Institutional Review Board Regulations, Sponsor and Investigator Obligations, Informed
`Consent Regulations, and the Declaration of Helsinki.
`After a 2-week run-in period with Refresh® artificial tears (Allergan, Inc., Irvine, CA), patients
`were randomly assigned 0.05% CsA topical ophthalmic emulsion eyedrop b.i.d., 0.1% CsA b.i.d., or
`vehicle for 6 months. The vehicle consisted of a sterile, non-preserved castor oil-in-water emulsion
`that was identical to the 0.1% CsA preparation except for the presence of medication. Patients in(cid:173)
`stilled one drop in each eye every morning and evening, no later than 8:00p.m. the night before clin(cid:173)
`ical visits. They could use Refresh® as needed through month 4, but no more than 8 times dai ly af-
`
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`5
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`ter that. At month 6. patients receiving vehicle were reassigned to 0.1% CsA b.i.d. for a further 6
`months. Those initially placed in the 0.05% and 0.1% CsA treatment groups continued their assigned
`treatments. Table I lists sample number collected at each clinjcal visit.
`Total daily doses of CsA were 0.057 mg/day (0.05% CsA treatment group) and 0.114 mg/day
`(0. 1% CsA group).
`
`Blood Sampl i n~
`
`Trough blood samples were collected immediately before the morning dose at day 0 and months
`I and 6. A final sample was collected at a single visit that occurred between months 9 and 12 (caUed
`the "month ~12 visit"). In order to quantify peak CsA concentrations. 26 patients also provided se(cid:173)
`rial blood samples at l , 2, 3. 4, 6, 8. I 0, and 12 hours after the morning dose during the month 9 -
`12 visit. All blood samples were frozen until shipment to Allergan, [nc. (Irvine, CA) for analysis.
`
`Blood Bioanalysis
`
`A validated HPLC/tandem mass spectroscopy assay was used to quantify blood concentrations
`of cyclosporin A. Briefly, 1 mL of blood wns acidified with 0.2 mL of 0.1 N HCI, then extracted with
`5 mL methyl-t-butyl ether. The organic phase was neutralized by addition of 2 mL 0.1 N NaOH, evap(cid:173)
`orated, recon tituted in a water/acetonitrile-based mobile phase, and injected onto a 2.1 X 50 mm. 3
`J.Lm pore size C-8 reverse phase HPLC column (Keystone Scientific. Bellefonte, PA). Compounds
`were gradient-eluted at 0.2 mL/min and detected using an API lll triple quadrupole mass spectrom(cid:173)
`eter with a turbo-ionspray source (PE-Sciex, Concord, Ontario, Canada). Molecular reaction moni(cid:173)
`toring enhanced the sensitivity and selectivity of this assay. Protonated molecules for the analyte and
`internal standard were collisionally dissociated and product ions at m/z 425 were monitored for the
`analyte and internal standard. Under these conditions, cyclosporin A and the internal standard cy(cid:173)
`closporin G eluted with retention times of - 3.8 minutes. The lower limit of quantitation was 0. 1
`ng/mL. at which concentration the coefficient of variation and deviation from nominal concentration
`was < 15%.
`For serial post-dose blood samples, the area under the blood concentration-time curve (AUCo.12)
`was calculated using the linear trapezoidal rule ( 12). Since most concentrations were below the limit
`
`Table l. Numbers of Trough Blood Samples Collected for CsA Quantitation
`
`Treatment
`Group
`
`Vehicle
`0.05% CsA
`0.1 % C A
`All treatment
`groups:
`
`Samples Per Treatmem Group Per Visit
`
`Day 0
`( Presflldy)
`45
`43
`43
`131 3
`
`Momh I
`
`Montll6
`
`Momll 9- 12
`
`All Visits
`
`37
`40
`36
`113
`
`30
`30
`34
`94
`
`Ob
`26
`sse
`84
`
`112
`139
`171
`422
`
`•samples were collected from 128 patients. Two patients from the vehicle group and 2 from the 0.1% CsA
`group each gave 2 samples. Baseline was not collected for I patient.
`bPatients assigned to vehicle were reassigned to 0. I % CsA at month 6.
`clncludes patiems who had crossed over from the vehicle group at month 6.
`
`413
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`APOTEX 1018, pg. 3
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`of quamitation. an upper limit to AUC0. 12 was calculated by assuming a concentration equal to the
`limit of quantitation at each ampling time. then expressing the mean AUCo.12 as below this theo(cid:173)
`retical upper limit of 1.2 ng•hr/mL. o statistical calculations were performed on serial postdose or
`trough blood concentrations becau e most data were below the limit of quamitation.
`
`Study Partic ipants
`
`RES ULTS
`
`Blood was collected from a subpopulation of 128 patients out of 405 enrolled in a Phase 3 trial
`of cyclosporin A for the treatment of dry eye disease, yielding a total of 422 trough blood sample~
`over the study period (Table 1 ). Patients ranged from 24 to 88 years of age (n = 128), and 78o/i
`( I 00/ 128) were women (9). Baseline characteristics of the subpopulation were similar to those of the
`larger study population. The rate of completion of the subpopulation (68%, 87/128) was slightly lowe1
`than the study population (77%, 6711877), but the reasons for discontinuation were similar in both
`popu lations and were primarily non-treatment-related.
`
`• I .. ..
`5 .
`~ • ;
`
`Trough Blood CsA Concentrations
`
`Of96 samples from the 0.05% C A treatment group. none contained any quantifiable CsA. Mean
`trough blood CsA concentrations were below the limit of quantitation in both treatment groups at al i
`time points. including month 9-1 2 (Table 2). The only study amples above the limit of quantitatior
`were 7 samples from the 0.1 % C A treatment group, representing only 5.5% (7/128) of the samples
`collected from this group. Quantifiable blood CsA concentrations ranged from 0.104 ng/mL to 0.29~
`ng/mL and came from 7 different patients who had no obvious commonalities with respect to age
`sex, or time point at which CsA was detected (Table 3). No indi vidual patient had quantifiable CsA
`at more than one time point.
`
`Serial Blood CsA Concentrations During One Dosing Interval
`
`Serial post-dose blood samples (n = 208 samples) were collected from 26 patients during the I_
`hours following the morning dose. As was the case with trough blood measurements, no patient i
`the 0.05% CsA treatment group had any quantifiable CsA. Overall, 99% of samples (205/208) did
`not contain quantifiable CsA. T he 3 samples that were above the limit of quantitation came fro m .)
`
`T able 2. Mean T rough Blood CsA Level
`
`Treatment Group
`
`Blood CsA Concentration (nglml)
`
`Vehicle
`0.05% CsA
`0.1 % CsA
`
`Day 0
`( Preswdy)
`< 0. 1
`< 0. 1
`< 0. 1
`
`Momh 1
`
`Momh 6
`
`Month 9- 12
`
`< 0. 1
`< 0. 1
`< 0. 1
`
`< 0.1
`< 0. 1
`< 0. 1
`
`NA
`< 0. 1
`< 0. 1"
`
`"Includes samples from patients in the vehicle group who crossed over to the 0. 1% CsA group.
`NA = Not applicable.
`
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`APOTEX 1018, pg. 4
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`

`Table 3. Quantiliable Trough CsA Concentrations in Individual Patient:.
`
`\ge. Sex
`
`Treatmem
`Group
`
`Blood CsA Co11cemration (nglmL)
`
`DayO
`(Prestudy)
`BLQ
`BLQ
`BLQ
`BLQ
`BLQ
`BLQ
`BLQ
`
`Momh I
`
`Month 6
`
`Month 9- 12
`
`0.1 34
`0. 110
`0.143
`BLQ
`BLQ
`BLQ
`BLQ
`
`BLQ
`NS
`BLQ
`0.299
`0.144
`0.125
`BLQ
`
`BLQ
`NS
`BLQ
`BLQ
`BLQ
`BLQ
`0.104
`
`56. M
`56. F
`64. F
`86. F
`8 1. F
`52. F
`76. M
`
`0. 1% CsA
`0. 1% CsA
`0.1 % CsA
`0. 1% CsA
`0. 1% CsA
`0.1% CsA
`vehicle ~ 0.1%
`CsA
`
`BLQ = Below limit of quantitation.
`NS = No sample collected.
`
`Jifferent patients in the 0. 1% treatment group. One of these patients had received 0.1 % CsA for 9-
`12 months, while the other 2 had received vehicle for 6 months before being reassigned to the 0. 1%
`CsA treatment group 3 to 6 months prior to the sampling period. CsA was barely detectable in these
`. sample . with Cmax ranging from 0.1 02 nglmL to 0.105 ng/mL. The e values occurred early in the
`12-hour time course, with tmax ranging from I to 3 hours (Table 4). Mean Cmax was not calculable
`because most samples were below the limit of quantitation. Based on these results, the AUCo. 12 was
`ess than 1.2 ng•hr/mL in both treatment groups.
`
`DISCUSSION
`
`Systemic exposure to CsA during treatment with CsA ophthalmic emulsions is barely detectable,
`' ven with an extremely sensitive a say capable of measuring blood CsA concentrations as low as 0.1
`nglmL. A striking result of this study i that no patient receiving 0.05% CsA had quantifiable CsA
`m the blood at any rime during t11e yearlong study. Phase 2 and Pha e 3 clinical trials have demon-
`trated this formulation of CsA to be clinically effective for the treatment of dry eye disease.
`For those very few patients who did have quantifiable blood CsA in the present study, concen(cid:173)
`trations were no more ilian 2-fold higher than the limit of quantitation. No patient had quantifiable
`blood CsA at more than one time point, for either trough or serial postdose sampling, even for pa(cid:173)
`tients treated up to I year, demonstrating that long-term use of topical ophthalmic CsA emulsions
`1lroduces insignificant systemic exposure. This is consistent with animal pharmacokinetic studies that
`have demonsLrated ready penetratjon of topical CsA into ocular surface tissues bul minimal absorp(cid:173)
`tion into the blood ( 13).
`
`Table 4. Quantiliable Serial Postdose Blood CsA Concentrations in Individual Patiems
`
`Age, Sex
`
`59, F
`64, F
`SO, M
`
`Treatmenr Group
`
`Blood Cmax (nglmL)
`
`f 111a.r (hr)
`
`0.1% CsA
`Vehicle ~ 0.1 % CsA
`Vehicle ~ 0.1% CsA
`
`0.104
`0.102
`0.105
`
`2
`I
`3
`
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`

`Trough blood CsA concentTations measured in this study should be assessed within the context
`of concentrations produced by approved treatments whose safety has been demonstrated. The ap(cid:173)
`proved oral treatment producing the lowest systemic exposure is the use of oral NEORAL® Soft
`Gelatin Capsules for treatment of rheumatoid arthritis and psoriasis (10). Table 5 compares blood
`concentrations produced by topical treatment with CsA emulsions to those produced by systemic treat(cid:173)
`ment with NEORAL® Soft Gelatin Capsules (10). On a weight-normalized basis assun"ling a typical
`60 kg patient, the recommended starting dose of NEORAL® is 150 mg/60 kg/day taken as a divided
`dose of 75 mg/60 kg/day. This produces trough blood CsA concentrations (mean :!: S.D.) of 74.9 :!:
`46.7 ng/mL and 96.4 :!: 37.7 nglmL, as reported in 2 studies summarized Ln the approved label for
`NEORAL ® treatment of rheumatoid arthritis and psoriasis ( I 0).
`In contrast to oral therapy, the cyclosporine dose instilled during topical treatment with 0.05%
`and 0.1 % cyclosporine ophtha lmic emulsions is 0.057 mg/day and 0.1 14 mg/day, respectively (Table
`5). Assuming again a typical 60 kg patient, these doses are 0.00095 mg/kg/day and 0.0190 mglkg/day.
`These are 2,630 and 1,320 times lower, respectively. than the recommended starting dose of
`NEORAL ®_ Mean trough blood concentrations produced by topical ophthalmic use of CsA emulsion~
`are less than 0.1 ng/mL, which is at least 750 times lower than mean trough levels seen with systemic
`therapeutic use. in addition, peak CsA concentrations are over 6500 times lower, and the area under
`the blood concentration-time curve is over 1900-fold lower with topical CsA treatment than with sys(cid:173)
`temic treatment (Table 5). Thus, systemic exposure produced by topical ophthalmic CsA is several
`orders of magnitude lower than that produced by cun-ently approved therapies that are themselves
`considered safe for non-life-threatening conditions.
`Our finding that topically-administered CsA is not quantifiable in the blood of patients using
`0.05% ophthalmic CsA, and is barely detectable in patients using O.l % CsA, is consistent with the
`excellent systemjc safety profile of these formulations. In a 12-week Phase 2 study of 0.05% to 0.40k
`CsA ophthalmic emulsions, there were no occurrences of ocular infections or overgrowths of micro(cid:173)
`bial organisms, nor were there any clinicalJy significant changes in visual acuity or measures of live1
`and renal function (8). In 2 much larger vehicle-controlled Phase 3 studies of 0.05% and 0.1 % CsA
`ophthalmic emulsions, only 2 of 877 patients developed ocular infections and those were in the ve(cid:173)
`hicle group (9).
`The results of the cunent study, considered in concert with blood concentrations measured in
`the Phase 2 study (8) and dming NEORAL® treatment (10), indicate that systemic exposure to cy(cid:173)
`closporine during ophthalmic treatment is barely quantifiable and is orders of magnitude lower tha1
`that produced by approved sys temic treatments. ImpoJtantly, CsA was not quantifiable at all in the
`blood of patients using 0.05% ophthalmk CsA, a formulation that is clinically effective for the treat(cid:173)
`ment of dry eye {8,9). Finally, ophthalmic preparations of CsA have an excellent clinical safety record
`
`i • 2 ... ..
`
`Table 5. Comparison of Dosages and Blood CsA Concentrations For Oral Versus Topical CsA Therapies
`
`Oral CsA
`(NEORAL®)a
`
`Topical CsA Ophthalmic
`Emulsions~:>
`
`NEORAL®JOpluhalmic
`Emulsion Ra1io
`
`Starting dose
`(mg/60 kg/day)
`Blood Cmox (ng/mL)
`Blood Cmax (ng/mL)
`AUCo. 12 (ng • hr/mL)
`
`ISO
`
`655
`75
`2,324
`
`0.057 (0.05% CsA)
`0.11 4 (0.1 % CsA)
`< 0.1
`< 0.1
`< J.2C
`
`2,630 (0.05% CsA)
`I ,320 (0.1 % CsA)
`> 6,550
`> 749
`> 1,940
`
`•Data source: (I 0); use of NEORAL ® Soft Gel Capsules for treatment of psoriasis.
`bData source: This study.
`csased on blood concentrations of 0.1 ng/mJ throughout dosing interval.
`
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`These results indicate that treatment with topical CsA ophthalmic emulsions for local immunomod(cid:173)
`ulation is safe and will not exert any system-wide effects.
`
`ACKNOWLEDGMENTS
`
`The authors thank Dr. Cathleen Josaitis for editorial assistance in preparation of the manuscript.
`Drs. Small, Acheampong, Stem, Re is, unci Tang-Liu are employees of Allergan, lnc. A pre liminary
`report of this research was presented at the annual meeting of the Association for Research in Vision
`and Ophthalmology (ARVO). April 30- May 5, 2000; Ft. Lauderdale, FL.
`
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`
`Received: February 27, 2002
`Accepted for Publication: March 27, 2002
`
`Reprint Requests: Diane D-S Tang-Liu, Ph.D.
`Allergan , lnc.
`2525 Dupont Drive
`Irvine, CA 92715
`E-mail: tang-liu_diaJle@allergan.com
`
`•
`~ .. ..
`...
`
`:II i
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`APOTEX 1018, pg. 8
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`