UNITED STATES PATENT AND TRADEMARK OFFICE
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`BEFORE THE PATENT TRIAL AND APPEAL BOARD
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`MYLAN PHARMACEUTICALS INC.,
`Petitioner
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`v.
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`ALLERGAN, INC.,
`Patent Owner
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`Case IPR2016-01128
`Patent 8,629,111
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`DECLARATION OF ERIC RUBINSON
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`1
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`ALL 2026
`MYLAN PHARMACEUTICALS V. ALLERGAN
`IPR2016-01128
`
`

`

`I, Eric Rubinson, declare and certify as follows:
`
`1.
`
`I am Executive Director, DDO Business Operations at Allergan, Inc.
`
`(“Allergan”).
`
`2.
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`3.
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`4.
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`I make this declaration based on my personal knowledge.
`
`I have been employed with Allergan for 11 years.
`
`Attached hereto as Exhibit A is an Interoffice Memorandum dated
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`October 29, 1998 titled “Phamacokinetics and Drug Metabolism Report PK—98-
`
`074.”
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`5.
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`6.
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`Exhibit A is a true and accurate copy of the original document.
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`I have personal knowledge of Allergan’s regularly conducted business
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`practices
`
`and activities
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`for
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`creating,
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`recording, maintaining,
`
`and storing
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`information and documents. The regular practice of Allergan when creating a
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`record of an act, event, condition, or
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`information is
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`for an employee or
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`representative of Allergan with knowledge of the respective act, event, condition,
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`or information to make the recordlor to transmit the information to be included in
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`the record at or near the time of the act, event, condition, or information gathering
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`or reasonably soon thereafter.
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`7.
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`The document attached as Exhibit A is a copy of an original document
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`that was created and kept by Allergan in the course of its regularly conducted
`
`business practices and activities.
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`I declare under penalty of perjury under the laws of the United States of
`
`America that the foregoing declaration is true and correct.
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`Executed on: March 17, 2017
`
`Executive Director, DDO Business Operations
`Allergan, Inc.
`Harborside Financial Center, Plaza V
`Jersey City, NJ 07311
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`2
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`

`

`
`
`EXHIBIT A
`EXHIBIT A
`
`3
`
`

`

`Table of Contents
`
`PK-98-074 ................................................................................................................................... .. 3
`
`Title Page ................................................................................................................................... .. 3
`
`SignaturePage 3
`
`Statement Of Compliance .......................................................................................................... .. 4
`
`Summary .................................................................................................................................... .. 5
`
`KeyWords 5
`
`Introduction ............................................................................................................................... .. 6
`
`Materials and Methods .............................................................................................................. .. 6
`
`Chemical and Reagents ......................................................................................................... .. 6
`
`Test Articles
`
`6
`
`Formulation Manufacture ..................................................................................................... .. 7
`
`Formulation Analysis ............................................................................................................ .. 7
`
`Animals ................................................................................................................................. .. 8
`
`Experimental ......................................................................................................................... .. 9
`
`Tissue Bioanalysis ................................................................................................................ .. 9
`
`Data Analysis ....................................................................................................................... ..l0
`
`Data and Report Handling and Storage ................................................................................ ..l0
`
`Protocol Deviations ................................................................................................................... ..l0
`
`Results and Discussion .............................................................................................................. ..ll
`
`Formulations ......................................................................................................................... ..l1
`
`Animals ................................................................................................................................ ..ll
`
`Bioanalysis
`
`Tissue Concentrations and Pharmacokinetic Parameters ..................................................... ..l2
`
`Conclusions ............................................................................................................................. .. 14
`
`References ............................................................................................................................... .. 14
`
`Tables ...................................................................................................................................... .. 16
`
`Table I. Study Design and Formulation and Dosing Data for Ocular and Systemic
`Absorption of Radioactivity in Rabbits .............................................................................. .. 16
`
`Table II. Results of Predose, Interim, and Postdose 31-I-Cyclosporine Formulation
`Analyses
`
`.. l7
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`mn_Rzso2s4e23
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`4
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`

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`Table III. Tissue Sample Masses from Dosed Rabbits and Limits of Quantitation (LPQ)
`of 3H-Radioactivity in Sampled Tissues ............................................................................ .. 18
`
`Table IV. Ocular Tissue Concentrations (n=4) in Albino Rabbits after 9 1/2 Days of
`Twice-Daily Ophthalmic Instillation of ............................................................................. .. 19
`
`Table V. Ocular Tissue Concentrations (n=4) in Albino Rabbits after 9 1/2 Days of
`Twice-Daily Ophthalmic Instillation of.............................................................................. .. 20
`
`Table VI. Ocular Phmmacokinetic Parameters of 3H-Radioactivity in Albino Rabbits
`after 9 1/2 Days of Twice-Daily ......................................................................................... .. 21
`
`Table VII. Ocular Pharmacokinetic Parameters of 3H-Radioactivity in Albino Rabbits
`after 9 1/2 Days of Twice-Daily ......................................................................................... .. 22
`
`Table VIII. Ocular Pharmacokinetic Ratios in Specific Tissues after b.i.d. Instillation of
`0.05% and 0.1% CsA Emulsions ........................................................................................ .. 23
`
`Table IX. Ocular Pharmacokinetic Parameters of Total Radioactivity in Albino Rabbits
`after Instillation of a Single Dose ...................................................................................... .. 24
`
`Figures ..................................................................................................................................... .. 25
`
`Figure 1. Concentrations in Albino Rabbit Tears (top) and Lacrimal Gland (bottom) after
`9 1/2 Days of Twice Daily.................................................................................................. .. 25
`
`Figure 2. Concentrations in Albino Rabbit Upper (top) and Lower (bottom) Conjunctiva
`after 9 1/2 Days of Twice Daily ......................................................................................... .. 26
`
`Figure 3. Concentrations in Albino Rabbit Cornea (top) and Sclera (bottom) after 9 1/2
`Days of Twice Daily Ophthalmic...
`
`.. 27
`
`Figure 4. Concentrations in Albino Rabbit Aqueous Humor (top) and Iris-Ciliary Body
`(bottom) after 9 1/2 Days of Twice...
`
`.. 28
`
`Figure 5. Concentrations in Albino Rabbit Lens (top) and Vitreous Humor (bottom) after
`9 1/2 Days of Twice Daily .................................................................................................. .. 29
`
`Figure 6. Concentrations in Albino Rabbit Choroid-Retina after 9 1/2 Days of Twice
`Daily Ophthalmic Instillation of 0.05 ................................................................................. .. 30
`
`Inspection Statement ............................................................................................................... .. 31
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`5
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`6
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`PK-98-074
`D. Small
`
`Page 2 of 28
`
`STATEMENT OF COMPLIANCE
`
`This study was conducted in accordance with Food and Drug Administration Good
`Laboratory Practice Regulations (21 CFR, Part 58). This report accurately reflects all raw
`data obtained for analysis. There were no significant deviations from Good Laboratory
`Practice Regulations that could have affected the quality or integrity of the study.
`
`Study Director:
`
`
`
`Dave mall, Ph.D., Senior Scientist
`
` /o-ova ~02:
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`PK-98-074
`D. Small
`Page 3 of 28
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`SUMMARY
`
`Cyclosporin A (CsA, AGN 192371) is being developed for the treatment of Sj6gren’s
`and non-Sj6gren's dry eye. We investigated the absorption and disposition of CsA in blood and
`various ocular tissues of albino rabbits after repeated ophthalmic 3H-CsA administration. Forty-
`two rabbits were dosed bilaterally with 50 ul of 3H-CsA 0.05% (N=20) or 0.1% (N=20) 3H-CsA
`emulsions twice daily for 9V2 days or remained undosed (N=2) as analytical controls. Iustilled
`doses were 0.0444 and 0.100 mg/kg/day for 0.05 and 0. 1 %—treated animals, respectively.
`Systemic blood was sampled and the tears, lacrimal gland, upper and lower bulbar conjunctiva,
`sclera, cornea, aqueous humor, iris-ciliary body, lens, vitreous humor, choroid-retina, and optic
`nerve head from each eye of two rabbits were sampled immediately before and 0.33, 1, 3, 6, 12,
`24, 48, 96, and 144 hours after the last dose, after which they were analyzed by liquid
`scintillation analysis with or without tissue combustion. A previous study has shown that CsA is
`not metabolized in albino rabbit ocular tissues, and therefore radioactivity represents intact CsA.
`Results in selected tissues after the last dose are shown below:
`
`Tissue
`
`Cmax
`n-e
`
`AUCN1
`n--hrl)
`
`tm
`)
`
`Cmax
`n-
`
`AUCN2
`)(n-0hr/)
`
`t In
`r)
`
`0.05% CsA
`
`0.1% CsA
`
`Lacrimal gland
`11.9
`66.0
`ND
`15.4
`140
`ND
`Lower conjunctiva
`713
`5,030
`ND
`1,920
`15,600
`31.8
`Cornea
`1,550
`12,300
`50.9
`4,810
`49,300
`52.0
`Sclera
`84.5
`848
`39.2
`262
`2,710
`40.5
`Lens
`18.4
`186
`480
`55.2
`529
`271
`Vitreous humor
`2.93
`22.8
`ND
`10.2
`87.0
`ND
`29.3 <146 ND 67.7 <395Optic nerve head ND
`
`
`
`
`
`
`
`
`
`
`
`
`
`ND: not determinable
`
`' Mean concentrations in tears at the first sampling time point of 20 minutes were 14,000
`and 41,000 ng-eqlg after the last dose of 0.05 and 0.1% CsA, respectively. Tissue concentrations
`were generally flat through 12 hours, with no pronounced Cmax. AUCo.12 in external tissues
`followed the rank order tears >> cornea > lower conjunctiva —- upper conjunctiva >> sclera, and
`in internal tissues followed the order iris—ciliary body >> choroid-retina > lens > vitreous humor
`> aqueous humor. Blood concentrations were <0.694 ng-eq/g and <1.88 ng-eq/g in 0.05%- and
`0. l%—treated animals, respectively.
`,
`Based on previously-reported single dose data, there was moderate accumulation in most
`tissues. Accumulation in lens, vitreous humor, and optic nerve head was 13- to 37-fold, although
`concentrations in these tissues remained less than 70 ng-eq/g. Except for lens, calculable
`elimination half-lives ranged from 25 to 57 hours.
`The results of this study indicate that CsA concentrations were substantial in ocular
`surface tissues, and have long elimination half-lives that are conducive to the twice-daily dosing
`regimen proposed for clinical ophthahnic use. Blood concentrations were below the quantitation
`limit, and support the systemic safety of ophthalmic CsA administration.
`
`KEY WORDS
`
`Cyclosporine, cyclosporin, albino rabbit, ocular distribution
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`PK-98-074
`D. Small
`Page 4 of 28
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`INTRODUCTION
`
`Cyclosporin A (CsA, AGN 192371) is an immunosuppressive drug widely used in organ
`transplantation, and is being developed for the treatment of Sj &'>gren’s and non-Sj6gren’s dry eye.
`Emulsion formulations of CsA have been assessed in previous ocular pharmacokinetic
`studies in albino rabbits (1-3). CsA is not metabolized in ocular tissues (1), indicating that ocular
`radioactivity after 3H-CsA administration consists of intact 3H-CsA. After instillation of a 0.2%
`3H-CsA emulsion to one eye of albino rabbits, the tissue concentration-time profiles were
`determined from 20 minutes to 96 hours postdose. CsA was detected in cornea, conjunctiva, and
`lacrimal gland, the target tissues for CsA action in Sj6gren’s Syndrome. In most ocular tissues
`peak concentrations were attained at 20 minutes to one hour postdose. and terminal half-lives
`ranged from 26 to 44 hours (2).
`Emulsion concentrations of 0.05%, 0.1% and 0.2% CsA were used in a previous Phase II
`clinical trial (4), and 0.05% and 0.1% emulsions were assessed in the two subsequent clinical
`trials 192371-002-00 and 192371-003-00. Formulations in these clinical trials were dosed twice-
`daily as eyedrops dispensed from unit dose containers.
`The current study was designed to investigate the absorption and disposition of CsA in
`blood and various ocular tissues under conditions that approximate those in clinical studies
`192371-002 and 192371-003. CsA emulsions of 0.05 and 0.1% were dosed bilaterally as 50 ul
`eyedrops twice-daily for 9‘/2 days, for a total of 19 doses per eye or 38 doses per rabbit. Fifty
`microliters were instilled because such volume is consistent with previous ocular
`pharmacokinetic studies in which the dosing volume was 50 pl and with the expected drop size
`from the unit dose containers that are being used in Phase 3 clinical trials. Based on a half-life of
`44 hours, 9'/2 days of dosing produced ocular concentrations that approximate steady state
`concentrations.
`
`MATERIALS AND METHODS
`
`Chemical and Reagents _
`Merocel’ Spon e Points (catalog #400115) were supplied by Merocel Corporation
`(Mystic, CT). Eutha- was purchased from Western Medical Supply (Vista, CA). Lens Plus°
`Sterile Saline was supplied by Allergan. Ultima Gold’ liquid scintillant was procured from
`Packard Instrument Company (Downers Grove, IL). All chemicals were reagent-grade or better;
`all solvents were I-IPLC-grade.
`
`Test Articles
`CsA (PSO# CSO-132R, PKDM Inventory #C-27) was supplied by Pharmaceutical
`Sciences Operation. [Mebmt-0-3H]-CsA (lot #'I‘RQ7478) was prepared by Amersham
`International (Buckinghamshire, England) with a molecular weight of 1,203 g/mole, a
`radiochernical purity of -98.8%, and a specific activity of 10.1 Ci/mmol (8.40 mCi/mg). It was
`supplied as an ethanol solution containing 1 mCi/0.119 mg/ml, and has been stored below -15°C
`within the Pharmacokinetic and Drug Metabolism Department (PKDM) as Crl7-19 since its
`‘receipt. The radiolabel is located in a metabolically stable position shown below by the asterisk.
`Formulation ingredients used to manufacture the emulsions were supplied as raw
`materials or stock solutions by PSO.
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`PK-98-074
`D. Small
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`Page 5 of 28
`
`ah
`
`(°“I)lG*¢'| I
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`3H-Cyclosporin A
`(asterisk denotes position of radiolabel)
`
`Formulation Manufacture
`Forty gram batches of 0.05% 3H-CsA emulsion (9054X-3H; lot #97P0l9-1) and 0.1%
`3H-CsA emulsion (8735X—3H; lot #97P0l9-2) were prepared by PKDM personnel using
`procedures tested and documented by the Product Develo ment Department (5, 6). The total
`amount of CsA in each 40 g batch of formulation was the H-CsA contained in 1.0 ml of the 1
`mCil0.l 19 mg/ml ethanolic solution and ~l9.9 or --39.9 mg of nonradiolabeled CsA in the 0.05
`or 0.1% formulations, respectively. Each emulsion contained the following:
`
`CsA USP + 3H-CsA
`Castor oil USP
`
`Polysorbate 80 NF
`Pemulen NF
`
`Glycerin NF
`NaOH
`Purified Water USP
`
`pH
`
`0.05 or 0.1% wlw
`1.25% w/w
`
`1.0% wlw
`0.05% wlw
`
`2.2% wlw
`pH adjustment
`~94% wlw
`
`7.2-7.6
`
`The formulations were white, homogeneous emulsions that were stable when stored in
`darkness at ambient temperature for up to six months (7). Each 50 pl drop of formulation was
`intended to contain 1.25 pCi of radioactivity and 0.025 or 0.050 mg of CsA. Formulation
`manufacture and usage was documented in the raw data.
`
`Formulation Analysis
`Formulations were analyzed three times during the study: before instillation of the first
`dose (predose), after all dosing of all animals sampled through 96 hours, but before the first dose
`given to animals sampled at 144 hours (interim), and after all dosing of the 144 hour animals
`(postdose). The following parameters were measured for both formulations during these
`analyses:
`
`CsA concentration. CsA concentration was detemiined using a published HPLC method with
`UV detection (8) that was used successfully in a previous study (3). Before and after the
`treatment period, the 0.05% formulation was diluted 1:100 and the 0.1% formulation was diluted
`to 1:200 with mobile phase. Each predose and postdose dilution was analyzed in triplicate.
`Standard curves of peak area vs CsA concentration were constructed with duplicate injections of
`CsA concentrations ranging from ~2 to ~10 pglml. Quality control samples of ~5 pglml were
`
`10
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`PK.-98-074
`D. Small
`
`Page 6 of 28
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`assayed in quadruplicate with the formulation dilutions in order to confirm reproducibility of the
`assay. The following chromatographic conditions were used:
`
`Mobile Phase:
`Pump:
`Flow Rate:
`
`Column:
`
`Acetonitrilezwater (68:32 v/V)
`Beckman Model 126 (San Ramon, CA)
`1.5 ml/min
`
`Beckman Ultrasphere ODS (4.6 mm x 15 cm, 5 pm)
`Bio-Rad column heater at ~70°C
`
`Injector:
`Radiochernical Detector:
`Cocktail:
`UV Detector:
`Data Processor:
`Run Time:
`Retention Time:
`
`Waters Wisp 717 Plus (Milford, MA)
`Packard Rarliomatic ISOTR
`Packard Flo-Scim‘ 111°, flow rate -4 ml/min
`Beckrnan 166 (Ramsey, NJ) at 214 nm
`Beckman System Gold version 8.1
`~15 min
`8-10 minutes
`
`Radiochemical purigg. Radiochemical purity was assessed during quantitation of formulation
`CsA concentrations using a radiochemical HPLC detector connected immediately following the
`UV detector. The radiochemical purity was defined as the area of the CsA peak divided by the
`combined area of all peaks reported during the run.
`
`Radioactivity concentratigm. Fifty microliters of formulation were diluted 1:100 with methanol,
`after which 50 ul of the dilution were added to 10 ml of scintillation cocktail and analyzed by
`liquid scintillation counting. Each 50 pl of the 1:100 dilution contained ~27,750 dpm.
`
`Animals
`
`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 have been approved by Allergan's Animal Care
`and Use Committee (AACUC), and are described in approved AACUC protocol #76 located in
`Allergan's Laboratory Animal Sciences Department.
`Female albino rabbits were used because of the availability of uniform strain, ease of
`housing, ease of handling, size, relatively low cost, availability of information concerning ocular
`bioavailability of ophthalmic drugs, and because they’ve been used in previous ocular
`cyclosporine studies. A previous study of ocular CsA distribution after acute instillation of a
`CsA 0.2% emulsion found no difference in ocular tissue distribution between male and female
`albino rabbits (2), and therefore males were not included in order to minimize the number of
`animals used. All rabbits were purchased from Covance Research Products (Denver, PA) and
`were quarantined for at least seven days prior to the start of the study. Animals were individually
`identified by ear tag, and only those that appear healthy were used.
`Rabbits were housed individually in stainless steel cages in a room dedicated to the
`housing of small animals. The temperature and humidity ranges for this room were 16-22°C and
`30-70%, respectively. The light-dark cycle was ~12 hours light (~6 am—~6 pm); ~12 hours dark
`(~6 pm-6 am). Animals were given ~l45 grams of Purina Certified Rabbit Chow’ per day and
`were allowed tap water purified by reverse osmosis ad libitum. Analysis of water is performed
`on a regular basis, and management is unaware of any contaminants in the water that would have
`interfered with the goals of the study.
`A total of 42 rabbits was used.- The 40 rabbits used for predose and postdose sampling
`times were stratified by body weight between treatment groups in order to
`bias, and
`
`1 1
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`PK-98-074
`D. Small
`
`Page 7 of 28
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`weighed 1.92-2.51 kg upon instillation of the first dose. All carcasses were considered
`nonradioactive for purposes of disposal.
`
`Experimental
`
`A summary of pertinent study design parameters is shown in Table I.
`
`fitratificatign. Rabbits were divided into two groups of 20 and one group of two that remained
`undosed and served as bioanalytical controls. Rabbits that were used for the predose sampling
`time and those through 96 hours were born within one week of June 28, 1997 and weighed 1.80-
`2.39 kg on September 30, 1997, the day on which they were weighed for assignment to treatment
`groups. Animals used as undosed bioanalytical controls and those sampled at 144 hours were
`acquired later but were comparable in weight and age to those used in the first segment. In order
`to
`bias, rabbits to be dosed with CsA were stratified by body weight between the two
`CsA treatment groups.
`
`Dosing. All rabbits except controls were dosed bilaterally, twice daily, for nine days, followed
`by a single dose on the morning of day 10. Doses were generally instilled once in the morning
`and once in the late afternoon or evemng. One treatment group of 20 was given 0.05% CsA, and
`the other treatment group of 20 was given 0.1% CsA.
`Doses were administered by gently pulling the lower eyelid away from the eye and
`instilling 50 ul of the fonnulation into the lower cul-de-sac with a calibrated positive
`displacement pipette. Formulations were stirred or agitated before aliquoting each dose in order
`to counteract the effect of any settling that might have occurred. After dosing, the eyelids were
`hand-held closed for approximately five seconds, after which the eyelids were released. Dosing
`was bilateral because such a regimen required half the number of animals that would have been
`needed were dosing unilateral, previous studies indicate that transfer of radioactivity from the
`dosed eye to the untreated eye after ocular 3H-CsA administration is negligible (2), and dosing in
`clinical use will be bilateral for most patients.
`
`Sampling. Timing for sampling began when the morning dose on day 10 was instilled into the
`cul-de-sac. Immediately before this dose (0 hour) and 0.33, 1, 3, 6, 12, 24, 48, 96, and 144 hours
`after this dose, at least 1 ml of blood was taken from an ear artery of each animal, immediately
`after which the animal was euthanized by intravenous injection of Eutha-6°. Blood was
`transferred immediately upon sampling to a refrigerator maintained at 2-8°C.
`Tears were sampled using Merocel° sponge points immediately following euthanasia,
`after which eyes were rinsed with approximately 1 ml of Lens Plus° sterile saline solution. The
`solid tissues lacrimal gland, upper and lower bulbar conjunctivae, sclera, cornea, iris-ciliary
`body, lens, vitreous humor, choroid-retina, and optic nerve head were dissected from the eye,
`weighed, and then stored at ambient temperature until analysis. Aqueous humor was aspirated
`using a tuberculin syringe and transferred to a glass vial for later analysis. Unpowdered gloves
`were worn during sampling in order to avoid transferring powder to tear sampling sponges, vials,
`or ocular tissues.
`
`To avoid contamination of tissues with radioactivity, dissection instruments were rinsed
`in ethanol, then wiped with clean gauze or tissue, between each tissue removal. All fluid
`samples were stored capped at ambient temperature, and all solid tissue samples were left to air
`dry at ambient temperature, before quantitation of radioactivity.
`
`Tissue Bioanalysis
`In duplicate, 200 [.11 samples of blood were transferred to combustion cones with pads.
`All samples were allowed to air dry at least overnight before oxidation. All blood and tissue
`samples were oxidized in a Packard Model 307 Oximate 80 Oxidizer, after which dpm were
`
`12
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`PK-98-074
`D. Small
`Page 8 of 28
`
`quantified in a Packard Model Tri—Carb 2700TR liquid scintillation counter. Measured dpm
`were reported without adjustment for oxidizer efficiency since efficiency was >97%.
`
`Data Analysis
`Tissue weights were recorded using Software Wedge Program version 2.0 (9) into
`Microsoft’ Excel‘ version 5.0 (Microsoft Corporation, Redmond, WA). Calculations were
`performed using Microsoft’ Excel’ version 5.0 and plotted using KaleidaGraph version 2.1.3
`(Synergy Software, Reading, PA). Final concentrations were reported to three significant figures
`in all tissues except tears, the sample weights for which required rounding to two significant
`figures.
`A lower limit of quantitation (LOQ) was calculated for each tissue. Concentrations in
`units of ng—eq/g or ml were calculated in each undosed tissue sample by dividing dpm by tissue
`weight, then multiplying by the mean of pre- and postdose ng-eq/dpm derived from formulation
`analyses. The mean concentration and standard deviation (SD) for each tissue were calculated,
`and for each tissue the LOQ was defined as the mean undosed tissue concentration plus two
`standard deviations.
`
`Concentrations in units of ng-eq/g were calculated in each dosed tissue sample by
`dividing dpm by tissue weight, then multiplying by the mean formulation ng-eq/dpm derived
`from formulation analyses. Mean and SD were calculated within each tissue and formulation
`using standard methods (10).
`Pharrnacokinetic parameters were calculated using noncompartmental methods (ll).
`Within each formulation and tissue, the maximum mean concentration (Cam) and time taken to
`reach Cmax (tmax) were identified by inspection of the data. ‘The areas under the concentration-
`time curves from zero to 12 hours (AUCo_12) and from zero to the last quantifiable time point
`(AUCo.aaso were calculated using the validated macro AUCRANDOM version 1.01 (12). The
`area under the concentration-time curve from zero to infinity (AUCo.¢,) was calculated as AUCO.
`.1”; + C13,;/k, where C1”; is the concentration at the last quantifiable sampling time and k is the
`absolute value of the slope derived by regressing the linear terminal portion of the natural
`Ev->‘gar'it/l1r(‘m of mean concentrations vs time. The corresponding half-life was estimated as
`.
`Concentrations and pharmacokinetic parameters calculated in albino rabbits were
`compared with those previously reported after single dosing (3). For each tissue an accumulation
`index was calculated as °" ‘°
`/ Day 1 Cmax, where Day 10 Cmax is the maximum concentration
`after the last dose and Day 1 Cmax is the dose-normalized maximum concentration previously
`reported after a single dose of 0.2% CsA emulsion. This method of calculation deviates from
`that described in the study protocol, and was changed because the report of ocular concentrations
`after a single dose doesn't include calculations of AUCo.12.
`
`n 2)
`
`Data and Report Handling and Storage
`All data were compiled into Allergan laboratory logbooks. These notebooks and the final
`report will be stored in A1lergan’s R&D Records Management.
`
`PROTOCOL DEVIATIONS
`
`0
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`0
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`Samples collected nominally at 144 horns were actually collected at 147 hours. This
`error was discovered after all data analysis and verificafion had been completed.
`Considering samples collected at 147 hours to have been collected at 144 hours produces
`an error of 53.7% in estimates of half-life in the five tissues in which radioactivity was
`quantifiable at 147 hours. This error is pharmacokinetically and clinically insignificant.
`
`Some dosing intervals deviated from the 12 :l: 2 hour dosing interval described in the
`protocol. Given the results of the study, these deviations had no effect on the
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`establishment of steady state drug concentrations over the course of the treatment period,
`and therefore did not affect results.
`
`RESULTS AND DISCUSSION
`
`Formulations
`CsA concentrations, radioactivity concentrations, and radiochemical purities quantified
`during each analysis are listed in Table ll. Both formulations were milky emulsions of low
`apparent viscosity. Assayed CsA concentrations from each analysis deviated less than 20% from
`nominal concentrations, with the single exception of the postdose 0.1% formulation in which the
`assayed concentration was only 66% that of nominal. The reason for this anomaly is unknown.
`Triplicate aliquots of this formulation yielded consistent concentrations, which eliminates a
`single pipetting error as the cause of the low mean. The lower CsA concentration was
`unaccompanied by a lower radioactivity concentration, which eliminates a maladjusted pipette as
`the cause. The same standard curve was used to quantify both postdose formulations, and
`produced results for tile 0.05% emulsion that were consistent with the nominal concentration and
`with previous analyses, so the low concentration can't be explained by an error in the standard
`curve used to quantify that bioanalytical run.
`Insufficient formulation remained for reliable reanalysis, and therefore a decision to
`include or exclude this datum in the calculation of formulation concentration was based on
`existing analyses. Based on the following considerations, the CsA concentration in the postdose
`0.1% formulation was considered unreliable and was not included in the calculation of the
`overall mean CsA concentration:
`
`' 0
`
`0
`
`0
`
`0
`
`Only one sampling time in one treatment group was affected, which minimized the
`impact of omitting this postdose analysis on the overall study results and conclusions.
`Including this datum would substantially increase the significance of this analysis since it
`would affect tissue concentrations in animals sacrificed earlier than 144 hr, a time interval
`bracketed by acceptable formulation analyses.
`
`The 144 hour data in the 0.1% treatment group were consistent with expectations based
`on data from the 0.05% treatment group. in which the formulation was acceptable
`through 144 hours.
`
`CsA in castor oil emulsions is known to be stable at ambient temperature for at least six
`months, which is sufficient to support the in-life portion of this study (7).
`
`The 0.05% formulation was stable during the period of dosing.
`
`Radiochemical concentrations were stable in both formulations, and radiochemical
`purities were measured as 100%.
`
`Animals
`
`All animals appeared to be healthy and completed the study with no apparent discomfort
`or other adverse event. Body weights (mean :t SD) of animals in the 0.05% and 0.1% treatment
`groups were 2.19 1: 0.15 (N=20) and 2.23 :t 0.15 kg (N=20), respectively, upon instillation of the
`first dose. Body weights of all dosed animals were 2.21 :1: 0.15 kg (N=40) upon instillation of the
`first dose. Based on mean assayed formulation concentrations and these body weights, mean
`instilled dpses were 0.0431 and 0.0960 mglkg/day for 0.05 and 0.1%-treated animals,
`
`respective y.
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`Bioanalysis
`Oxidizer recovery was >97% for all bioanalytical runs. LOQ's derived from blank tissues
`are listed in Table III". Quantitation limits in the 0.1% group were about 170% higher than those
`in the 0.05% group and reflect the difference in specific activities between the two formulations.
`There were no notable unexpected events during any of the bioanalyses.
`
`Tissue Concentrations and Pharmacokinetic Parameters
`Tissue masses are listed in Table III. Tissue radioactivity concentrations in animals
`treated with 0.05 and 0.1% CsA emulsions are listed in Tables IV and V, respectively. The
`concentration-time profiles resulting from these concentrations are depicted in Figures 1-6.
`Mean tissue masses differed by 11% or less between treatment groups, except for tears for which
`the mean mass from 0.1%—treated animals was 53% higher than that from 0.05%-treated animals
`(0.0l07 vs 0.0070 g, respectively). Since tear sampling occurred after sacrifice, this difference
`likely doesn't indicate sampling of reflexive tears from the 0.1%-treated animals, which would
`have diluted actual concentrations and resulted in underestimated tear concentrations in that
`
`group.
`
`Concentrations in sur