ALLERGAN
`DEPARTMENT OF PHARMACOKINETICS AND DRUG METABOLISM
`DEPARTMENTAL RESEARCH REPORT
`
`Concentrations of Cyclosporin A in Cornea and Conjunctiva After a Single Ophthalmic
`Dose to New Zealand White Rabbits: Evaluation of 7 Ophthalmic Emulsion
`Formulations
`
`Report No.: PK-00-163
`
`Study Number:
`Project Name and Number:
`Study Site:
`
`In-Life Start Date:
`In-Life End Date:
`Bioanalytical Start Date:
`Bioanalytical End Date:
`Laboratory Notebooks:
`Study Technician:
`Bioanalytical Principal Investigator:
`Study Director:
`
`PK-00-P002
`Cyclosporin Allergy, 200301201384
`Allergan, Inc.
`2525 Dupont Drive
`Irvine, CA 92612
`February 24, 2000
`February 25,2000
`March 15, 2000
`March 24,2000
`L-2000-7726, L-2000-7626
`V. Baumgarten
`A. Acheampong
`D. Welty
`
`'88
`
`MYLAN - EXHIBIT 1027
`Mylan Pharmaceuticals Inc. et al. v. Allergan, Inc.
`IPR2016-01127, -01128, -01129, -01130, -01131, & -01132
`
`

`

`Written by:
`
`Reviewed by:
`
`R.M. Matsumoto, Ph.D., Principal Scientist
`Pharmacokinetics and Drug Metabolism
`
`A. Suri, Vh.SyyJS&Dior Scientist
`Pharmacokinetics and Drug Metabolism
`
`/elty, Ph.DA Director
`Pharmacokinetics and Drug Metabolism
`
`Approved by:
`
`D. Tang-Liu, Ph.D., Director
`Pharmacokinetics and Drug Metabolism
`
`luO
`
`IS- 30-Ct,
`Date
`
`(o
`Date
`
`Date
`
`i - i r *
`Date
`
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`TABLE OF CONTENTS
`
`1
`
`Summary...............................................................................................................................8
`
`2 Key Words............................................................................................................................ 8
`
`3
`
`Introduction............................................................................................................................9
`
`3.1
`
`Background.............................. ................................................................................ 9
`
`3.2
`
`Study Objectives......................................................................................................10
`
`3.3
`
`Strategy to Meet Study Objectives......................................................................... 10
`
`4 Materials............................................................................................................................11
`
`4.1
`
`Chemicals, Reagents, and Supplies....................................................................... 11
`
`4.1.1 Chemicals and Reagents........................................................................................11
`
`4.1.2 Materials and Equipment....................................................................................... 12
`
`4.2
`
`Test Articles.............................................................................................................12
`
`4.2.1 Test Compound and Source.................................................................................... 12
`
`4.2.2 Preparation, Batch Size, and/or Source of Test Formulation.............................. 12
`
`4.2.3 Analysis of Test Formulations................................................................................ 12
`
`4.2.4 Storage and Disposal of Test Formulations...........................................................13
`
`4.2.5 Handling of Test Compounds and Test Formulations.......................................... 13
`
`5
`
`Animals.............................................................................................................................. 13
`
`5.1
`
`Animal Welfare Statement.......................................................................... ...........13
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`5.2
`
`Animal Species, Sex, Age, and Source................. ..............................................13
`
`5.2.1 Species, Strain, Sex, Age, Size, Source, and Identification................................ 13
`
`5.2.2 Justification...............................................................................................................14
`
`5.3
`
`Animal Husbandry.................................................................................................... 14
`
`5.4 Animal Acclimation.................
`
`
`
`14
`
`5.5 Animal Tennination and Disposal
`
`........................................................................14
`
`6
`
`Study Design and Experimental Procedures.....................................................................14
`
`6.1
`
`Basic Study Design................................................................................................. 14
`
`6.2
`
`Study Size................................................................................................................ 15
`
`6.2.1 Control Animals, Number, and Purpose................................................................ 15
`
`6.2.2 Treated Animals, Number, and Treatment...........................................
`
`
`
`15
`
`6.3
`
`Dosing Regimen......................................................................................................15
`
`6.3.1 Route of Dosing and Justification.......................................................................... 15
`
`6.3.2 Dose Selection and Justification............................................................................ 16
`
`6.3.3 Dosing Frequency and Justification....................................................................... 16
`
`6.4
`
`Animal Assignment............................................................................
`
`
`
`16
`
`6.5
`
`Dosing....................................................................................................................... 17
`
`6.6
`
`Animal Preparation Prior to Sampling...................................................................17
`
`6.7
`
`Sample Collection and Processing.......................................................................... 17
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`6.8
`
`Sample Transfer and/or Shipment.............................................................................17
`
`7
`
`Bioanalysis................................................... ......................................................................18
`
`7.1
`
`Preparation of Ocular Tissues for LC-MS/MS.................................................. ,...18
`
`7.2 LC-MS/MS Bioanalysis of Ocular Tissue Extracts................................................ 18
`
`8
`
`Data Treatment....................................
`
`
`
`19
`
`8.1 Data Acquisition........................................................................................................19
`
`8.2 Data Calculation and Outlier Analysis.....................................................................19
`
`8.3
`
`Pharmacokinetic Analysis......................................................................................... 19
`
`8.4
`
`Statistical Analysis and Number Rounding.............................................................20
`
`8.5
`
`Storage........................................................................................................................20
`
`9
`
`Results..............................................................................................................
`
`
`
`21
`
`9.1
`
`Effect of CsA Concentration on Ocular Tissue Distribution................................. 21
`
`9.2 Effect of Cyclosporin/Castor Oil Ratio..................................................
`
`
`
`21
`
`9.3 Effect of Globule Size................................................................................................21
`
`10 Conclusions...................
`
`
`
`— 22
`
`11 References............................................................................................................................. 22
`
`Table I
`
`Study design for assessment of ocular pharmacokinetics of cyclosporin in
`NZW rabbits after a single ocular dose of cyclosporin....................
`
`24
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`Table II
`
`Table m
`
`Table IV
`
`Table V
`
`Cyclosporin A concentrations in the corneas of NZW rabbits given a single
`28.5 pL eyedrop of 0.01% to 0.4% cyclosporin ophthalmic emulsion.......... 25
`
`Cyclosporin A concentrations in the conjunctivae of NZW rabbits given a
`single 28.5 pL eyedrop of 0.01% to 0.4% cyclosporin ophthalmic emulsion.26
`
`Pharmacokinetic parameters of cyclosporin A in the corneas of NZW rabbits
`given a single 28.5 pL eyedrop of 0.01% to 0.4% cyclosporin ophthalmic
`emulsion............................................................................................................... 27
`
`Pharmacokinetic parameters of cyclosporin A in the conjunctivae of NZW
`rabbits given a single 28.5 pL eyedrop of 0.01% to 0.4% cyclosporin
`ophthalmic emulsion........................................................................................... 28
`
`Figure 1 Cyclosporin A concentrations in the cornea and conjunctiva of NZW rabbits given
`a single 28.5 pL eyedrop of CsA ophthalmic emulsion at 0.01% to 0.4%
`concentrations.................................
`
`
`29
`
`Figure 2 Cyclosporin A concentrations in the cornea and conjunctiva of NZW rabbits given
`a single 28.5 pL eyedrop of 0.05% CsA ophthalmic emulsion at two different
`‘^’"'“ ' ^ “/castoroi.raUos..................................................................................................30
`
`Figure 3 Cyclosporin A concentrations in the cornea and conjunctiva of NZW rabbits given
`a single 28.5 pL eyedrop of 0.4% CsA ophthalmic emulsion at two globule sizes
`.....................................................................................................................................31
`
`Figure 4 Ocular tissue CsA distribution after ophthalmic application of 0.01% to 0.4% CsA
`emulsions in rabbits................................................................................................... 32
`
`Figure 5 Effect of CsA concentration in castor oil phase on ocular tissue distribution after
`0.05% CsA ophthalmic emulsions to rabbits...........................................................33
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`Figure 6 Effect of globular size on ocular tissue distribution of 0.4% CsA ophthalmic
`emulsions in rabbits ................................................................................................
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`1 SUMMARY
`
`Purpose. Assess the influence of 3 formulation parameters on concentrations of cyclosporin
`A (CsA) in rabbit cornea and conjunctiva, target tissues for the topical treatment of dry eye.
`Methods. Seven castor oil-in-water emulsions were made to contain (w/w) 0.125% to 5%
`castor oil (CO), 0.1% to 3% polysorbate 80 (PS80), 0.01% to 0.4% CsA, 0.05% Carbomer
`1342 (as secondary emulsifier), glycerin (for tonicity), and NaOH (for pH adjustment).
`Formulations were designed to evaluate; 1) Relationship of tissue concentration and instilled
`dose at constant CsA/CO ratio and PS80 concentration, 2) Effect of CsA/CO ratio at constant
`CsA dose, and 3) Effect of globule size. Female albino rabbits received one 28.5 pL dose of
`CsA emulsion to each eye. Animals were euthanized at 0.5,1.5, 3,6, and 12 hr postdose,
`immediately after which cornea and conjunctiva were collected from each eye. CsA was
`extracted with methanol and then quantified by LC-MS/MS. Results. Comeal
`concentration-time profiles were steady between 0.5 and 12 hr. At a constant CsA/CO ratio
`of 8% and a CsA emulsion concentration of 0.05%, tissue concentrations (mean + SD)
`increased to an asymptotic concentration of 718 + 262 ng/g in cornea and 765 + 182 ng/g in
`conjunctiva. Decreasing the CsA/CO ratio to 1%, but keeping total CsA dose unchanged,
`decreased comeal and conjunctival maximal concentrations by 62-72%. Reducing PS80
`concentration from 3% to 1% increased mean globule diameter from 1.5 pm to 3.8 pm, but
`had no effect on ocular tissue concentrations. Conclusions. Increasing the CsA/CO ratio in
`CsA emulsions increased comeal and conjunctival bioavailability. Ocular tissue
`concentrations increased with increasing dose and were independent of globule size.
`
`2 KEYWORDS
`
`Cyclosporin A, AGN 192371, ocular tissue distribution, ophthalmic, formulation screen,
`
`rabbit
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`3 INTRODUCTION
`
`.
`
`3.1 BACKGROUND
`
`Cyclosporin A (CsA, AGN 192371) is an inununosuppressive drug widely used in organ
`transplantation, and is being developed for the treatment of moderate to severe
`keratoconjunctivitis sicca with or without Sjdgren’s Syndrome.
`
`Cyclosporin USP is manufactured by Novartis Pharma AG, Basel, Switzerland. It has poor
`water solubility, but is more soluble in a lipophilic medium such as castor oil. The 0.05% or
`0.1% cyclosporin emulsion formulations (RESTASIS™, 9054X and 8735X, respectively)
`developed for the treatment of dry eye are sterile, droppable oil-in-water emulsions
`containing 0.05% or 0.1% cyclosporin USP. Inactive ingredients and the function of each are
`listed below. The formulation is preservative-free and has a target pH of 7.4.
`
`Ingredient
`
`Cyclosporin
`Castor Oil
`Glycerin
`Polysorbate 80
`Carbomer 1342
`1-N Sodium Hydroxide
`Purified Water USP
`
`Concentration (% w/w)
`0.05 or 0.1
`1.25
`2.2
`1.0
`0.05
`0.397
`95.0
`
`Function
`Active Ingredient
`Lipophilic Vehicle
`Tonicity Agent
`Primary Emulsifier
`Secondary Emulsifier
`pH adjustment
`Hydrophilic Vehicle
`
`Studies in rabbits have shown that cyclosporin concentrations in surface ocular tissues
`increase with increasing formulation concentration (Allergan reports PK-96-011 and PK-98-
`074). These studies compared formulations in which oil concentration was kept constant as
`cyclosporin concentration increased, thereby making the
`concentration ratio a
`formulation variable. The upper limit to cyclosporin concentrations in these emulsion
`formulations was approximately 0.4% based on the castor oil concentration used in these
`formulations and on cyclosporin’s solubility in castor oil.
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`A separate study also investigated the effect of oil globule size on cyclosporin's ocular
`bioavailability in emulsions with oil globules of small (majority <10 |xm), intermediate
`(majority ~50-100 pm), and large (majority >200 pm) size (Allergan report PK-95-074).
`Results in rabbits showed that the emulsions containing larger globules produced higher
`cyclosporin concentrations in ocular surface and intraocular tissues than did the small
`globule emulsion. However, the intermediate and large globule emulsions were physically
`unstable, making them unacceptable for further development.
`
`This study assessed the influence of 3 formulation parameters: Cyclosporin concentration,
`cyciosporin^^^ oil concentration ratio, and globule size, which is controlled by the concentration
`of polysorbate 80. Polysorbate 80 concentrations was chosen by the requirement to either
`maintain or alter emulsion globule size distribution. The
`*°/caswr oil ratio was kept
`comparable between formulations in order to minimize the effect of this variable.
`
`3.2 STUDY OBJECTIVES
`
`This study was designed to meet the following objectives:
`
`• Evaluate the dose-relationship of conjunctival and comeal cyclosporin concentrations in
`5 emulsion formulations varying in cyclosporin concentration from 0.01% to 0.4%, but
`comparable in '='‘='“ *^”/cas.oroii and
`®°/castoroii ratios.
`
`• Evaluate the effect of the ‘^^‘^’“ ’^"/castoroii ratio on ocular tissue distribution.
`
`• Evaluate the effect of globule size on ocular tissue distribution.
`
`3.3 STRATEGY TO MEET STUDY OBJECTIVES
`
`The following strategy was used to meet the stated study objectives:
`
`• Cyclosporin concentration in 5 formulations ranged from 0.01% to 0.4%, castor oil
`concentration ranged from 0.125% to 5% such that the cyclosporin concentration in oil
`was 8%, and polysorbate 80 concentration ranged from 0.1% to 3% as required to
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`maintain consistent globule size distribution between formulations (Formulations A-E,
`see Section 4.2.2). The
`concentration ratio in the 0.4% cyclosporin
`emulsion was slightly lower than that in the other formulations in this series, but
`polysorbate 80 concentration was limited to 3% or less in all formulations in order to
`keep the emulsion nonirritating to rabbit eyes, thus avoiding excessive tearing and
`blinking that could confound pharmacokinetic analysis.
`
`The effect of the ‘^''“ ’^ “/castoroii ratio was evaluated in 2 emulsion formulations
`containing 0.05% cyclosporin, 0.625% or 5% castor oil, and 0.5% or 3% polysorbate 80
`(Formulations B and F, see Section 4.2.2). The predominant difference between these
`formulations was the cyclosporin concentration in oil, which was 1% or 8%. The
`poiysoibate
`j-atio was 0.8 or 0.6, respectively, but cannot be kept constant because to
`do so would require a polysorbate 80 concentration of 4%. This concentration would
`likely be irritating to rabbit eyes and would likely produce excessive tearing and blinking
`that could confound pharmacokinetic analyses.
`
`The effect of globule size was evaluated in 2 emulsion formulations containing 0.4%
`cyclosporin, 5% castor oil, and a polysorbate 80 concentration of 1% or 3%
`(Formulations E and G, see Section 4.2.2). Globules in these formulations were expected
`to have mean particle sizes of approximately 1.8 pm (90% ^ . 8 pm) or 0.54 pm (90%
`<1.5 pm). These formulations were physically stable during the course of the study.
`
`4 MATERIALS
`
`4.1 CHEMICALS, REAGENTS, AND SUPPLIES
`
`4.1.1 CHEMICALS AND REAGENTS
`
`Chemicals, solvents, and reagents were HPLC-grade or better unless otherwise noted.
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`4.1.2 MATERIALS AND EQUIPMENT
`
`EUTHA-6® was purchased from Western Medical Supply (Vista, CA). LENS PLUS® Sterile
`Saline was supplied by Allergan. Merocel® Sponge Points (catalog #400115) was supplied
`by Merocel Corporation (Mystic, CT).
`
`4.2 TEST ARTICLES
`
`4.2.1 TEST COMPOUND AND SOURCE
`
`Cyclosporin was supplied by Pharmaceutical Sciences Operation (PSO). At least 97.5% of
`this cyclosporin consisted of cyclosporin A.
`
`4.2.2 PREPARATION, BATCH SIZE, AND/OR SOURCE OF TEST FORMULATION
`
`The following formulations were supplied by the Product Development Department and used
`as supplied:
`
`Formulation
`
`A
`B
`C
`D
`E
`F
`G
`
`Cyclosporin
`concentration
`in emulsion
`0.01%
`0.05%
`.0.1%
`,0.2%
`0.4%
`0.05%
`0.4%
`
`Castor oil
`concentration
`in emulsion
`0.125%
`0.625%
`1.25%
`2.5%
`5%
`5%
`5%
`
`Cyclosporin
`concentration
`in oil phase
`8%
`8%
`8%
`8%
`8%
`1%
`8%
`
`Polysorbate 80
`concentration
`
`Polysorbate 80/
`oil
`
`0.1%
`0.5%
`1%
`2%
`3%
`3%
`1%
`
`0.8
`0.8
`0.8
`0.8
`0.6
`0.6
`0.2
`
`4.2.3 ANALYSIS OF TEST FORMULATIONS
`
`4.2.3.1 Cyclosporin Concentration
`
`Formulations were diluted with mobile phase, after which the CsA concentration in each
`dilution was quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS)
`during the same runs containing ocular tissue samples.
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`4.2.3.2 Globule Size
`
`The globule size distribution in each formulation was characterized by the Product
`Development Department using a Coulter LS230 laser light scattering instrument. The
`results of the measurements were supplied to the study director for use in pharmacokinetic
`interpretation of tissue concentration-time data.
`
`4.2.4 STORAGE AND DISPOSAL OF TEST FORMULATIONS
`
`Test formulations were used within 2 weeks of receipt from Product Development, and were
`refrigerated when not in use. Formulations were deemed expired and discarded at the end of
`the study.
`
`4.2.5 HANDLING OF TEST COMPOUNDS AND TEST FORMULATIONS
`
`Consistent with available safety information for cyclosporin.
`
`5 ANIMALS
`
`5.1 ANIMAL WELFARE STATEMENT
`
`This study complied with all requirements of the United States Department of Agriculture
`(USDA), and all regulations issued by the USDA implementing the Animal Welfare Act,
`9 CFR, Parts 1, 2, and 3. The animal procedures used had been approved by Allergan's
`Animal Care and Use Committee (AACUC), and were described in approved AACUC
`Protocol #76 located in Allergy's Laboratory Animal Sciences Department.
`
`5.2 ANIMAL SPECIES, SEX, AGE, AND SOURCE
`
`5.2.1 SPECIES, STRAIN, SEX, SIZE, SOURCE, AND EDENTMCATION
`
`Female New Zealand White (NZW) rabbits were purchased from an approved vendor.
`Rabbits weighed approximately 1.8 kg to 2.2 kg at arrival. Animals were identified by ear
`
`tag.
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`5.2.2 JUSTmCATION
`
`Female New Zealand White rabbits were used because of ease of housing, ease of handling,
`size, relatively low cost, availability of uniform strain, and availability of information
`concerning the ocular disposition of cyclosporin in NZW rabbits.
`
`5.3 ANIMAL HUSBANDRY
`
`All animals were housed in an environmentally-controlled facility with a time-controlled
`fluorescent lighting system providing a daily 12-hour light/12-hour dark period. Room
`temperature was maintained between 18°C and 26°C, and relative humidity between 30%
`and 70%. Analysis of animal feed was conducted by the manufacturer.
`
`Rabbits were individually housed in stainless steel cages and fed approximately 145 g of
`approved rabbit chow daily. Drinking water puriHed by reverse osmosis was offered
`ad libitum. Water was periodically analyzed for any contaminants that may interfere with the
`conduct of this study.
`
`5.4 ANIMALACCLIMATION
`
`Rabbits were quarantined for at least 7 days prior to the start of the study. Only rabbits that
`appear healthy were used.
`
`5.5 ANIMAL TERMINATION AND DISPOSAL
`
`Rabbits were euthanized with an approximately 1 mL injection of EUTHA-6® to a marginal
`ear vein.
`
`6 STUDY DESIGN AND EXPERIMENTAL PROCEDURES
`
`6.1 BASIC STUDY DESIGN
`
`Single dose, open label, (see Table I)
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`6.2 STUDY SIZE
`
`Seventy-four rabbits (Four control, 70 treated).
`
`6.2.1 CONTROL ANIMALS, NUMBER, AND PURPOSE
`
`Four rabbits (8 eyes) remained untreated:
`
`• Two rabbits (4 eyes) served as bioanalytical controls.
`
`• Two rabbits (4 eyes) remained untreated and were sacrificed to collect blank ocular
`tissues that were used to prepare bioanalytical reagents. Comea and conjunctivae were
`collected from these eyes and were added separately by tissue to 2 vials of blank
`methanol and allowed to soak overnight. Methanol from these undosed tissue extracts
`was used during the preparation of calibration and quality control standards.
`Components of tissue extract may enhance or suppress the detector response to CsA
`during LC-MS/MS bioanalysis, and the use of tissue-exposed methanol in the preparation
`of calibration curves minimized the bioanalytical effect of these components.
`
`6.2.2 TREATED ANIMALS, NUMBER, AND TREATMENT
`
`Seventy rabbits (140 eyes) were dosed bilaterally.
`
`6.3 DOSING REGIMEN
`
`6.3.1 ROUTE OF DOSING AND JUSTinCATlON
`
`Test formulations were applied topically to the eye because this is the intended clinical route
`of administration for these formulations. Both eyes were dosed because a previous study
`(Allergan report PK-95-010) has shown that contralateral crossover of cyclosporin was
`negligible, and the use of both eyes minimized the number of animals used.
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`6.3.2 DOSE SELECTION AND JUSTMCATION
`
`Cyclosporin emulsion formulations of 0.05% to 0.4% have been investigated previously
`(Allergan Report PK-94-012, Allergan Report PK-96-011). Ocular tissue concentrations
`after a single dose of these emulsions generally increased with increasing cyclosporin
`concentration in the emulsion. However, the formulations assessed in those studies
`contained a constant concentration of oil, thereby making
`gg
`gj
`cyclosporin concentration a formulation variable.
`
`Based on the known safety and efficacy of 0.05% to 0.4% cyclosporin ophthalmic emulsion
`in humans (Allergan report PK-96-018), the likelihood for nonchronic treatment for future
`indications such as ocular allergy, the limited solubility of cyclosporin in castor oil, and the
`reported lack of efficacy during treatment with 0.01% cyclosporin emsulsion in animals, a
`range of 0.05% to 0.4% cyclosporin is reasonable within which to conduct the comparisons
`intended for this study. The 0.01% formulation was included because it's likely that this dose
`will be included as a "no effect dose" in clinical trials.
`
`The dosing volume of 28.5 pL used in this study was established as the mean drop size for
`RESTASIS™ cyclosporin 0.05% ophthalmic emulsion (Product Development Research
`Memo, C. Ramirez, August 27,1998), and was smaller than the drop sizes used in
`cyclosporin studies conducted before drop volume was measured.
`
`6.3.3 DOSING FREQUENCY AND JUSTMCATION
`
`A single dose was given because a single dose is sufficient to elucidate the ocular
`pharmacokinetic parameters of interest.
`
`6.4 ANIMAL ASSIGNMENT
`
`Animals were distributed randomly between the 7 treatment groups.
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`6.5 DOSING
`
`Test formulations were applied by Eppendorf pipette or equivalent. The lower eyelid was
`pulled away from the eye, and 28.5 pL was instilled directly into the lower cul-de-sac. The
`eye was gently hand-held closed for 5 seconds, after which the eye was released and left
`alone until sampling.
`
`6.6 ANIMAL PREPARATION PRIOR TO SAMPLING
`
`Rabbits were euthanized with EUTHA-6® immediately before sampling.
`
`6.7 SAMPLE COLLECTION AND PROCESSING
`
`Samples were collected from both eyes of each animal predose and at 0.5,1.5, 3,6, and
`12 hours postdose. These sampling times were based on cyclosporin's relatively rapid
`attainment of Cmax and flat concentration-time profile in cornea and conjunctiva after a single
`ocular dose of 0.2% emulsion (Allergan report PK-95-010) and after multiple doses of 0.05%
`and 0.1% emulsions to NZW rabbits (Allergan report PK-98-074).
`
`Both eyes were thorougjily rinsed with LENS PLUS® in order to deaf residual surface
`formulation remaining in the precorneal area. Aqueous humor was aspirated from the
`anterior chamber and discarded. A section of the upper and lower conjunctiva was removed
`and pooled into a single screw-cap polypropylene vial, which was then sealed. The entire
`cornea was removed and sealed into a separate single screw-cap polypropylene vial.
`
`Samples were stored at or below -15®C imtil bioanalysis.
`
`6.8 SAMPLE TRANSFER AND/OR SHIPMENT
`
`All study samples remained in the custody of the Department of Pharmacokinetics and Drug
`Metabolism throughout the study.
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`7 BIOANALYSIS
`
`7.1 PREPARATION OF OCULAR TISSUES FOR LC-MS/MS
`
`Comeal and conjunctival samples were extracted using a procedure previously shown to
`quantitatively extract cyclosporin ftom rabbit ocular tissues (Allergan reports PK-99-073 and
`PK-00-013). Individual samples were extracted twice with methanol, with each extraction
`consisting of soaking for at least 1 hour in 2 mL of solvent. The serial extracts from each
`sample were combined and centrifuged, after which 1 mL was transferred to a silanized glass
`tube and 10 pL of the 1,000 ng/mL cyclosporin G (CsG) internal standard was added. This
`solution was evaporated with nitrogen and then reconstituted with 200 pL of 50% mobile
`phase A/50% mobile phase B, after which 20 pL was analyzed by LC-MS/MS.
`
`Calibration curves used to quantify the mass of CsA in these samples were prepared by
`spiking appropriate working solution into methanolic extract of blank tissue.
`
`7.2 LC-MS/MS BIO ANALYSIS OF OCULAR TISSUE EXTRACTS
`
`Quantitation of CsA concentrations in cornea and conjunctiva were based on an electrospray
`LC-MS/MS method validated for the quantitation of CsA in human tears (Allergan report
`PK-99-029). These methods were subsequently shown to be appropriate for the quantitation
`of CsA in rat and dog ocular tissues (Allergan report PK-00-013).
`
`Reconstituted extracts were injected onto an HPLC system consisting of a Keystone
`2 X 50 mm 3 pm Cg column using a mobile phase flow rate of 0.2 mL/minute and fast
`gradient elution to separate the analyte and internal standard from the bulk of the remaining
`matrix components. The mobile phase consisted of 2 mM ammonium acetate and 0.4%
`formic acid in both water and acetonitrile.
`
`The HPLC system was coupled to a PE-Sciex triple quadrupole mass spectrometer with a
`turbo-ionspray source for detection of CsA and the internal standard cyclosporin G (CsG).
`The sensitivity and selectivity of the assay were enhanced by the use of positive ion
`molecular reaction monitoring (MRM), which monitored the specific ion firagmentations of
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`m/z 1,203 (M+HT) to 425 for CsAand m/z 1,217 to 425 for CsG. The retention time of CsA
`and CsG was approximately 3.8 minutes.
`
`The lower limit of CsA quantitation was 1 ng in cornea and conjunctiva.
`
`8 DATA TREATMENT
`
`8.1 DATA ACQUISITION
`
`Data were captured using PE-Sciex RAD (Routine Data Acquisition and Display) version 2.6
`and PE-Sciex Sample Control version 1.4. PE-Sciex MacQuan version 1.4 no-FPU or
`version 1.6 was used to integrate the peak areas of CsA and CsG and to construct the
`calibration curves. MacQuan software determined sample concentrations automatically
`using ^^/csc peak area ratios (PAR) for test samples and the linear regression equation
`generated for the calibration curve.
`
`8.2 DATA CALCULATION AND OUTLIER ANALYSIS
`
`Calibration curves of PAR vs CsA mass were used to determine analyte concentrations in test
`formulations. Tissue concentrations were used unadjusted and were also dose-normalized by
`dividing the concentration by the CsA mass instilled in 1 eyedrop.
`
`All data were used in calculations unless omitted for reasons justified in the raw data.
`Calculations were performed using Microsoft® Excel® version 7.0a (Redmond, WA), and
`results were plotted using Kaleidagraph® version 3.0.5 (Synergy Software, Reading, PA).
`
`8.3 PHARMACOKINETIC ANALYSIS
`
`Based on CsA's long terminal half-life in rabbit cornea and conjunctiva (Allergan report
`PK 95-010, Allergan report PK-98-074), pharmacokinetic parameters calculated through
`infinity were not accurate in these tissues and therefore not calculated. The following
`pharmacoldnetic parameters were calculated:
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`• The area under the cornea and conjunctiva concentration-time curve through the last
`quantifiable time point (AUCo-uast)- This parameter was calculated using the validated
`Microsoft® Excel® macro AUCRANDOM (Tang-Liu and Burk, 1988).
`
`• Maximum mean concentration (Cmax)-
`
`• Time of maximum mean concentration ( T m a x ) -
`
`CsA dose-tissue concentration relationship of formulations ranging from 0.01% to 0.4% was
`assessed by linear regression analysis. Comeal and conjunctival Cmax
`AUCo-uast hrom
`formulations A through E were compared with their respective CsA formulation
`concentration. The effect of '’''*“ ’^ “/castor oil ratio on ocular tissue concentration was assessed
`by comparing comeal and conjunctival Cmax and AUCo-uast from formulations B and F. The
`effect of oil globule size on ocular tissue concentration was assessed by comparing comeal
`and conjunctival Cmax and AUCo-uast from formulations E and G.
`
`8.4 STATISTICAL ANALYSIS AND NUMBER ROUNDING
`
`Mean, standard deviation (SD) and/or standard error of the mean (SEM) were calculated
`using standard methods (Zar, 1974). Linear regression analysis of tissue concentration data
`and unpaired t-test comparison between formulations were performed by StatView version
`5.0.1.0 (SAS Institute, Cary, NC).
`
`Mean comeal and conjunctival concentrations were reported to 3 significant figures.
`Standard deviation and/or SEM were reported to the same decimal place as their respective
`mean values.
`
`8.5 STORAGE
`
`Study-related data were compiled in Allergan laboratory logbooks and stored in Allergan’s
`R&D Records Management upon study completion.
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`9 RESULTS
`
`CsA concentration-time profile in comeal and conjimctival tissues after ophthalmic
`administration of 7 different formulations are given in Tables II-III and shown in Figures 1-3.
`Comeal concentration-time profiles were steady between 0.5 and 12 hr. Conjunctival
`concentrations declined over time in a multi-phasic fashion. The pharmacokinetic
`parameters of CsA in comea and conjunctival tissue are listed in Tables IV-V.
`
`9.1 EFFECT OF CSA CONCENTRATION ON OCULAR TISSUE DISTRIBUTION
`
`Cyclosporin A concentrations in comea and conjunctival tissue increase with increasing CsA
`formulation strengths of 0.01 to 0.4% (see Figure 4). Mean comea Cmax went fi-om 409 to
`1530 ng/g and maximal conjunctival concentrations increased from 463 to 1730 ng/g. When
`regressing comeal or conjunctival Cmax or AUC values with formulation concentratio