CENTER FOR DRUG EVALUATION AND
`
`RESEARCH
`
`APPLICA TION NUMBER:
`
`21-023
`
`PHARMACOLOGY REVIEW! S!
`
`
`
`

`

`‘ Mbnaieogj
`
`
`
`Review and Evaluation of Pharmacology/Toxicology data
`
`Key Words: Topical Cyclosporine eye drop, Dry eye, Apoptosis, Acinar
`Epithelium, Laorimal gland
`
`Reviewer: Asoka Mukherjee
`
`Division of Anti—inflammatory, Analgesic and Ophthalmic Drug Products
`
`HFD-SSO
`
`Review Completion Date: June 2, 1999
`
`Review Number: One
`
`NDA: 21-023
`
`Serial Number, Date and Type of Submission: Serial
`Application under 505 [b)l, Feb 24, 1999
`
`# 1, Original NBA
`
`Information to the sponsor: Yes
`
`(
`
`I No
`
`(X 1
`
`Sponsor: Allergan lné. California 92623
`
`—~_....
`Manufacturer of Drug Substance:
`
`Ff—““—dd‘r"“““““‘“*~—-*—“-~»~.:?‘~\
`
`Drug: Cyclosporine ophthalmic emulsion
`
`Code Name: AGN 192371
`
`Generic Name: Cyclosporine Ophthalmic Emulsion, 0.05% preservatiVe free.
`
`Trade Name: RESTASIS (proposed)
`
`Chemical Name: CycloII(Eli(23,3R,4R)~3~hydroxy—4—methyl-2~{methylaminoi—G—
`octenoyl]—L—2aminobutyrylvN~methylglycyl—N—methyl—Lmleucyl~L—valyl—N—methyl—
`L—leucyl—L—alanyl—D-alanyl-N—methyl—L—leucyl-N—methyl—L-leucyl~N—methyl—L-
`valyli
`
`CA5 Registry Number: 059865~13~3
`
`MOIECUIBI FOUIIUla: C52 H1]; “11 012. Molecular Weight: 1202.6
`
`Structure:
`
`
`
`

`

`
`
`NTM32L023
`
`Relevant NDA:
`
`50-733,
`
`'" F'
`
`"‘—-‘—“”'
`
`I
`
`50—574 and 50-573
`
`/
`
`Relevant
`
`IND: 32,133 and c““
`
`Relevant DMF: DMF *"
`
`Type 1, DMF
`
`*”‘
`
`type III
`
`Drug Class:
`
`ImmunosuppreSsant and immunomodulator.
`
`Indication: ;
`
`dd_‘h___—___F_#Mh___"__“—‘"_m_—_—‘—“—**“-e‘h
`
`Clinical formulation:
`
`Cyclosporine, USP, 0.05% or 0.1% w/w
`Castor oil PhEur,
`‘
`Polysorbate 80 NF
`Carbomer 1342 NE
`Glycerine USP,
`Sodium hydroxyide USP, W
`Purified water USP,
`
`~__::)fl___.n
`
`<“*'__‘_“—*—-"
`
`Route of administration: Topical,
`
`instilled in eyes.
`
`Proposed Clinical use: The ophthalmic emulsion will be used topically for the
`treatment of KCS.
`
`The proposed dose is: Ophthalmic drops 0.05% Cyclosporine, one drop in each
`eye,
`twice a day approximately 12 hours apart
`
`Disclaimer: The sponsor submitted a letter of authorization from Novartis
`dated April 27, 1998 for cross—referencing all Cyclosporine INDs and NDAs.
`
`Introduction and drug history:
`
`is a cyclic polypeptide extracted from the fungus
`Cyclosporine (GSA)
`
`Beauveria nivea. It is an immunosuppressive agent approved for the treatment
`of the rejection of organ transplants,
`rheumatoid arthritis and psoriasis.
`Cyclosporine 0.2% ophthalmic ointment is approved for the treatment of
`chronic KCS in dogs. It CSA has also been investigated for several autoimmune
`and inflammatory diseases of the eye e.g. uveitis and Behcet's disease etc.
`The mode of action of CSA is through the inhibition of release of several
`cytokines e.g.
`IL—2,
`IL-3,
`IL-4,
`INF, GM—CSF and TNF. Most recently CSA has
`been shown to increase the transcription of TGF-fi. In the present NDA the
`sponsor submitted study reports for the efficacy and safety of CSA for the
`treatment of keratoconjunctivitis
`sicca (dry eye) with or without Sjogren
`syndrome; Dry eye conditions result from reduced secretion of the tear from
`acinar epithelial cells in the eye. Experimental studies shown that invasion
`of T lymphocytes in the secretory gland affect the function of acinar
`epithelial.
`
`
`
`

`

`
`
`NDA 21—023
`
`
`
`The sponsor proposed that the local application of CSA would inhibit the T
`lymphocyte functions,
`reduce apoptosis of the epithelial,
`improve and restore
`the tear secretion.
`
`Pharmacology:
`
`The sponsor has submitted literature citations on the mechanism of actions of
`Cyclosporine and its effects in KCS in animal models. Summaries of some of
`these citations are presented in the review. However,
`the reviewer does not
`agree with the efficacy claims in the published papers for the indications
`not approved by the Agency.
`
`Mechanism of action:
`
`Page 011, vol 21:
`
`'
`
`The sponsor referred to several published papers on the immunosupPKESSiVe
`effect of Cyclosporine {CSA}. However,
`the review written by Borel et al.
`Adv. Pharmacology 35, 115 provided a detailed analysis of its mode of action.
`CSA inhibits both humoral and cell mediated immunity. It is effective in
`chronic immune mediated inflammatory conditions and inhibition of graft
`rejection. The effect of CSA has been demonstrated in several inflammatory
`conditions including autoimmune uveitis, psoriasis,
`idiopathic nephrotic
`syndrom and rheumatoid arthritis.
`I
`
`CSA inhibits the function of T- lymphocytes without affecting the function of
`phagocytes or hemopoietic stem cells. The mechanism of CSA involves
`inhibition of cytokine release from the helper T cells. CSA binds with
`cytosolic protein known as cyclophilin. The CSA-cyclophilin complex inhibits
`calcium calmodulin—dependent protein calcineurine. Inhibition of calcineurine
`phosphatase activity by CSA—cyclophilin complex contributes to the inhibition
`of the function transcription factors e.g. NEAT and NFKB. The inhibition of
`the nucleotide regulatory factors results in the down regulation of the
`cytokine gene expression. CSA also has antagonistic effect on prolactin.
`It is suggested that inhibition of prolactin contribute to the
`antiinflammatory effect of CSA.
`
`Drug activity related to proposed indication:
`
`Page 239, vol 21:
`
`The sponsor cited several published Papers on the effect of CSA in KCS in
`animals.
`
`1. Role of apoptosis in the pathogenesis of canine keratoconjunctivitis
`sicca: The effect of topical Cyclosporine A therapy. Gao et al. Cornea, 17
`(6}, 654-663, 1998.
`
`The authors stated that T lymphocytic infiltration was detected in the
`biopsies of lacrimal glands of dogs with spontaneous chronic idiopathic KCS.
`Apoptosis of the lymphocytes,
`lacrimal acinar epithelial and conjunctival
`Epithelial cells were evaluated in spontaneous idiopathic KCS in dogs. The
`role of CSA in the'apoptosis process has been discussed. Dogs that were
`clinically diagnosed with KCS were enrolled. Ten dogs with KCS were treated
`
`3
`
`
`
`

`

`
`
`NDA 21-023
`
`three dogs with KCS were treated with the
`with 0.2% CSA ophthalmic emulsion,
`vehicle and another four normal dogs were used as the baseline control.
`Treated animals were desed with one drop of CSA or the vehicle in each eye
`twice a day for 13 weeks. Lubricant eyedrops were applied at noon between the
`treatments. For the biopsy procedures, dogs were treated With atropine
`subcutaneously, anesthetized with i.v injections of Valium and Ketamine and
`treated With topical proparacaine. Doses of the treatment have not been
`mentioned. Biopsy specimens were obtained from the lacrimal glands and
`conjunctiva before and after 13 weeks of the treatment. Apoptotic cells were
`labelled and examined under light microscope. The apoptotic process was
`confirmed by DNA fragmentation analysis. Furthermore, monoclonal antibodies
`against fas-ligand, polyclonal antibodies against fas—ligand and monoclonal
`antibody against bcl—Z expressed proteins were used to show distribution of
`apoptotic receptor sites in the cell. Results indicated that the vehicle
`treated dogs had increase in the apoptosis of acinar, conjunctival epithelial
`cells and decrease in the apoptosis of the lymphocytes when compared to the
`normal dogs. The process was reversed after the treatment with CSA topically.
`It was concluded that suppression of apoptosis of lymphocytes and induction
`of apoptosis of epithelial cells of lacrimal glands and conjunctiva were the
`characteristic features of KCS in dogs.
`CSA treatment modulated the apoptic
`process.
`
`2. The sponsor has provided a review on KCS in dogs and the effect of BSA.
`The review was published by Kaswan and Salisbury, Vet clinics of North
`America, 20, 583, 1990. The review provided information covered by the
`citation described above.
`In addition,
`the review addressed the issue that
`the effect of CSA could be due to the irritant property of the drug. However,
`commonly known irritants are not effective in treating KCS. Therefore,
`the
`pharmacodynamic effect of CSA in KCS is not likely to be mediated by the|
`irritant effect of CSA. The article also discussed about the opportunistic
`infections in the cornea and conjunctiva. However,
`the risk may be similar to
`corticosteroids. The issue of bioavailability of CSA in the lacrimal gland
`was raised in the publication._However, data on the CSA level in the lacrimal
`gland have not been provided.
`
`Page 339! vol 21
`
`3. Characteristics of a canine model of KCS: effective treatment with topical
`Cylosporine. By Kaswan. Lacrimal gland,
`tear film and dry eye syndromes,
`Edited by D.A. Sullivan, Plenum Press, N.Y 1994.
`
`The review stated the mode of action of CSA for the treatment of KCS is as
`follows-
`
`a. Modulation of cytokine production in the lacrimal gland.
`b. Decreased recruitment of auto reactiVe lymphocytes from the conjunctiva to
`the lacrimal gland.
`c. A direct neurohormonal effect of CSA mediated through prolactin receptors
`identified on lacrimal acinar epithelium. Prolactin is considered to be a
`natural ligand for cyclophilin. However, role of prolactin receptor in the
`lacrimal gland.and its relationship to the effect of CSA is not clearly
`known.
`
`4
`
`

`

`
`
`NIHK2L023
`
`The [eview also stated that CSA has a dose dependent side effect on hair
`growth and gingival hyperplasia.
`
`4. Are dry eyes a sign of testosterone deficiency in women. Mamalis et
`al.8ndocrine Soc. Intl. Mtg. 1996:849. Page 059, vol 22.
`
`the serum concentrations of hormone precursors were
`In a population study,
`determined from 26 women who had dry eyes and 19 matched control women.
`
`The mean and SEM data for parameters are shown in the following table. The
`severity of dry eyes was determined from a questionnaire using a 4-20 scale.
`
`Parameter
`
`'
`
`61.1 i 10.3
`
`Age
`Severity
`
`.
`
`Total Testosterone
`free Testosterone
`
`..
`
`Androstenedione
`PG E2
`Prolactin
`
`Patients
`(n=20)
`46.7 i1.l
`8.9 :t 0.5
`308.3 1 2.7
`
`25.6 i 2.4
`2.5 i 0.3
`1.5 i 0.2 fl
`
`61.8 i 6.0
`85.5 i 6.?
`
`The authors concluded that dry eyes in women resulted from testosterone
`deficiency. However,
`the testosterone levels between the patients and the
`control were not significantly different as shown in the above table.
`
`5. An article entitled “ Traffic of major histocompatibility complex class II
`molecules in rabbit lacrimal gland acinar cells”, Mircheff et al. Invest.
`Ophthalmology & Visual Science, 35, 3943, submitted to the RDA page 075, vol
`22.
`'
`
`The authors investigated the expression of NBC II molecules on acinar
`epithelial cells in the rabbit model. Using monoclonal antibodies,
`the
`authors showed that MHC II molecules are expressed in the cell surface of
`acinar cells. The MHC II molecule binds with the auto-antigen, presents to T
`lymphocytes and induces inflammatory changes. The article suggests that
`autoimmunity in the lacrimal gland contribute to the pathogenesis to dry eye
`syndrome.
`
`6. Hormonal support of lacrimal function, primary lacrimal deficiency,
`autoimmunity and peripheral tolerance in the lacrimal gland. Mircheff et
`al. Ocular Immunology and Inflammation, 4, 145, 1996. Page 84, vol 22:
`
`

`

`NDA 21—023
`
`The review covered the pathogenesis of dry eye diseases.
`
`Dry eye or KCS is the leading cause of patient visits to the eye care
`specialists other than the refractory correction. The clinical manifestation
`of dry eye ranged from contact lens intolerance to severe conjunctival
`inflammation and corneal damage. Lacrimal insufficiency could be immune
`related and non—immune related.
`
`The immune—related lacrimal insufficiency results from Sjogren's syndrome,
`graft vrs host disease, diffuse infiltrative lymphocytosis syndrome of HIV
`{DILS) and sarcoidosis.
`Inflammatory cells e.g. CD. positive T-lymphocytes,
`CDB positive T-lymphocytes, B—lymphocytes, granulomatous macrophages etc.
`infiltrate lacrimal glands in above conditions.
`Some of the immune mediators
`impair the ability of the lacrimal secretory epithelium to regenerate,
`
`Non—immune—mediated lacrimal insufficiencies are due to congenital alacrimia,
`sensory or secretomotor nerve damage, destruction of lacrimal secretory
`tissues and due to the side effects of number of drugs.
`The authors also indicated the role of estrogen and androgen deficiency in
`the pathogenesis of dry eye syndrome.
`
`7. Tear fluid influenceyon the ocular surface, Pflugfelder- S.C, Adv. Exp.
`Med. Bio, 438, 611+~6’17,1998.
`
`Page 217, vol 22:
`
`The review stated that factors secreted in the tear fluid by the lacrimal
`gland are classified into:
`
`lysozyme and IgA
`a- Antimicrobial factors e.g. lactoferrin,
`b. Growth regulating factors e.g. EGF, TGF-a, TGF—fi and vitamin A.
`
`Release of these factors from acinar cells is stimulated by cholinergic nerve
`stimulation and can be blocked by muscarinic antagonists.
`
`The rear fluid EGF concentrations decrease following induction of nasal—
`lacrimal-reflex. The review suggests that elaborate homeostatic mechanisms
`exist to maintain the tear fluid EGE‘ concentration within a_ range that may be
`optimal for the ocular surface epithelium.
`
`Human tear fluid inhibits TSP-[5 sensitive growth of mink lung epithelium.
`Therefore, TSP—fl is responsible for the antiproliferative activity of the tear
`fluid. Tear fluid maintains a low state of epithelial proliferation on the
`ocular surface under the normal condition. Under a disease condition as in
`
`KCS, reduction in the concentration or activity of TGF—fi in the tear fluid
`could lead to conjunctival epithelial proliferation e.g. squamous metaplasia
`of the conjunctival epithelium. The functions of normal tear fluids are to
`inhibit conjunctival epithelial proliferation,
`to promote terminal
`differentiation, stimulation of epithelial membrane mucus production and
`promotion of goblet cell differentiatiou.
`Ancillary Pharmacology studies: No ancillary pharmacology study reports have
`been provided.
`
`6
`
`

`

`Summary of Pharmacology:
`
`NDAZIfiZS
`
`KCS is associated with the immunological and non—immunological mechanisms.
`Increased infiltration of lymphocytes and degeneration of tear forming cells
`are the common causes of KCS. Expression of MHC II in the acinar epithelium
`and development of autoimmunity are contributory factors to the development
`of KCS. CSA ophthalmic emulsion is targeted for the suppression of the
`lymphocytic infiltration and autoimmunity of the lacrimal gland.
`
`CSA is a known immunosuppressant approved for organ transplantation, RA and
`psoriasis in humans. It inhibits several cytokines. Among other mechanisms,
`CSA also contributes to the efficacy in the inflammatory condition by
`displacing prolactin from lymphocytes. Presence of prolactin receptors has
`been shown in the lacrimal gland. However, its direct role in the tear
`secretion in KCS is not known.
`
`Effect of CSA in the KCS in dogs has been investigated. A published report
`suggests that CSA induces apoptosis of lymphocytes and inhibits apoptosis of
`acinar epithelial cells. The process contributes to the regeneration and
`differentiation of secretory epithelial cells in the lacrimal gland. Data
`from the literature also suggested that CSA induces growth of hair by its
`direct action in the hair follicle. The pharmacological effect can contribute
`to the growth of eyelashes.
`,
`
`The literature citations also indicated the role of endocrine hormones e.g.
`estrogen and testOSterone in the pathogenesis of KCS. As with many other
`autoimmune diseases,
`testesterone deficiency could contribute to KCS.
`However, CSA does not have a direct effect on the steroid hormone receptors.
`
`Tear fluids contain several growth factors among which TGF—fi is important
`because of its contribution to the immunosupression to the eye,
`differentiation and function of lacrimal epithelial cells. A recent
`publication on the mode of action of CSA indicates that CSA induces TGF—fi in
`
`vitro and in vivo (Hojo et al. Nature, 397, 530, 1999). TGFefl reduces
`epithelial proliferation in ocular tissues and contributes to the normal
`function of secretory cells in the eye.
`
`CSA inhibits cytokine and growth factors that contribute to its efficacy for
`the treatment of KCS. Results of long term deprivation of these factors on
`the growth, differentiation and function of acinar cells are unknown.
`Therefore, consequence of long term treatment of KCS with CSA needs to be
`monitored in the clinical studies.
`
`Safety pharmacology:
`
`No study report has been submitted in the NDA. However, approved package
`insert of CSA indicates hypertension and nephrotoxicity are the major side
`effects of CSA. Exact mechanism of the cardiovascular side effects is not
`known. Published literature indicated the contributions of endothelin and
`
`thromboxane in the pathogenesis of CSA induced kidney toxicity and
`hypertension. The package insert also indicated hepatotoxicity, convulsions,
`hirsutism and gum hyperplasia as the side effects of CSA.
`
`
`
`

`

`
`
`NINK2L023
`
`Borel et. a1. Adv. Pharmacology, 35, 115, 1996, also suggested that the
`stimulation of hair growth (hypertrichosis)
`is a common side effect of CSA
`obserVed in humans and animals. The effect of the Immunosuppressive on hair
`growth may be unrelated to immunosuppression.
`
`Studies reviewed in the submission:
`
`1. A six—month ocular and systemic toxicity study with a two—month recovery
`period in New Zealand white rabbits. Report
`# 1793—2936-6. Page 001, vol
`15.
`(see page 21)
`
`2. A fifty—two week ocular and systemic studv of Cyclosporine in dogs with an
`eight —week recovery period. Report # " 985—126. Page 001, vol 17.
`(see
`page 10)
`
`3. A six-month interim toxicokinetic report: Pharmacokinetic analysis of
`Cyclosporine A in dog blood for study # fl—q 985—126, 52~week ocular and
`systemic study of Cyclosporine in dogs with an 8 week recovery period.
`Report PK-96—001, page 27, vol 19. Also submitted in NBA page 138, vol 18
`(see page 17)
`
`4. The blood to plasma concentration ratio of JH—Cyclosporine-A in mouse, rat,
`rabbit, dog and human in vitro. Report
`# PK—94-108, Page 187, vol 18. {see
`page 47)
`
`.. Ocular pharmacokinetics of Cyclosporine after a single eye drop
`instillation of a 0.2% 3H-cyclosporine ophthalmic emulsion into albino
`rabbit eyes. Study # PK—95—010, page 257, vol 18.
`(see page 42)
`
`Investigation of ocular metabolism of Cyclosporine after a single drop
`instillation of a 0.2% 3H cyclosporine ophthalmic emulsion into albino
`rabbit eyes. Study # PK-95—011, page 289, vol 18. {see page 58)
`
`7. Pharmacokinetic kinetic analysis of Cyclosporine A in rabbit blood for
`study # 1793—2936—6 “ AGN 192371-Cyclosporine ophthalmic emulsion: A six"
`month ocular and systemic toxicity study with a two month recovery period
`in New Zealand white rabbits”. Study # PK—95—066, page 361, vol 18.
`(see
`page 37)
`
`8. The effect of oil globule size on ocular absorption of 3HWCyclosporine
`after topical instillation of three 0.2% JH—cyclosporine oil—in—water
`emulsions into rabbit eyes. Report # PK-95-074, page 001, vol 19.
`( see
`page 60)
`
`Dose proportionality of ocular tissue JH-Cyclosporine concentrations after
`a single dose administration of 0.05%, 0.2% and 0.4% JH-Cyclosporine
`emulsions into rabbit eyes. Report PK-96-011, Page 075, vol 19.
`(see page
`48)
`
`10. Ocular absorption and disposition in beagle dogs following multiple
`ocular doses of 0.2% 3H Cyclesporine emulsion. Report PK—96-016, page 100, vol
`19.
`(see page 49)
`
`
`
`

`

`NDAZIQZJ
`
`11.
`
`Ocular absorption and disposition in beagle dogs following single
`ocular doses of 0.2% 3H Cyclosporine emulsion. Report PK—96e017, page 194,
`vol 19.
`(see page 55)
`
`12.
`
`Pharmacokinetic analysis of Cyclosporine in dog blood for 52—week
`ocular systemic study of Cyclosporine in dogs with an 8—week recovery
`period. Report PKH96—023, page 298, vol 19.
`(see page 17}
`
`13.
`
`Ocular Cyclosporine distribution during 9 H days of dosing of 0.05% and
`0.1% JH-Cyclosporine A emulsions to albino rabbits. Report PK-98—074, vol
`328, vol 19.
`(see page 46)
`
`14.
`
`‘"‘“““‘*““‘"‘~”‘“W————v
`Cyclosporine a
`PA—1998*010, page 002, vol 20.
`(see page 38)
`
`special study. Report #
`
`15.
`
`randomized, vehicle—controlled parallel—
`A multicenter, double masked,
`group study of the safety and efficacy of Cyclosporine 0.05% and 0.1%
`ophthalmic emulsions used twice daily for up to one year in patients with
`moderate to severe Keratoconjunctivitis Sicca. Clinical report # 192371“
`002, page 240, vol 20. {see page 41)
`
`Studies not reviewed in the submission:
`
`WW‘
`
`- my.“ »a_...r_....v.......,
`
`
`__
`
`WWWhW‘I -
`
`
`
`
`
`

`

`flmmw'
`
`"wag-6’'uW=
`
`,
`
`,
`
`NTMAZLfiZJ
`
`w- ._W
`
`WW gflquWsnmwfl 6..., _, 1--
`
`
`
`Eur-go.“;. ,u
`
`.5mrdmwfimmmmfldm‘ 1-» -
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`7
`2 z—VJEWEWMPWm124-Hr‘_:w ,
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`;w,.-52K‘-W
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`
`
`
`General Comments: Nil
`
`Study Title:
`
`A fifty—two week ocular and systemic toxicity study of Cyclosporine in dogs
`with an eight—week recovery period.
`
`Study 3:
`
`«“153, 985-126.
`
`Page 001,v01 17.
`
`Conducting Laboratorv and Location:
`7 A...“
`—-
`man—
`-———-——n—‘”""
`
`,
`
`!,
`
`fl—-—"“"——“*-*-““_“"“—-*—”“——**
`
`Date of the study initiation: March 9, 1995
`
`10
`
`

`

`
`
`PULKZL£23
`
`GLP Compliance: The study was conducted in compliance with GLP regulation.
`
`QA report: Yes
`
`Methods:
`
`Dosing:
`
`Species/Strain
`
`
`
`Dog, Beagle
`
`
`
`(kg)
`Weight
`6.3—10.9 M, 5.8-
`9.1 F
`
`
`
`
`Satellite group used for recovery:
`
`Two animals per sex/group were sacrificed after 8 weeks of recovery period
`for groups 1,3 and 4. However, only one male was allowed to undergo 8 weeks
`of recovery in the group 2.
`
`
`
`
`
`
`
`Dosage groups in administered units:
`
`1, Control
`
`2, Low
`
`c... m
`
`0, Placebo for
`0.4% "
`0.1
`
`
`
`631853—631853
`
`631865-631870
`
`631859—
`6631864
`631871—
`
`
`
`Drops/day
`
`3
`
`0.2
`
`831877—631882
`
`high
`
`
`Route,
`
`form, volume:
`
`631883—
`
`.631808
`
`
`631895-
`631889-631894
`
`631900
`
`
`
`
`
`
`The drug product or the vehicle was administered as ocular drops in the left
`eye. The right eye served as the untreated control. The control and the high
`dose groups received six applications at 2-hour intervals. The remaining
`animals received three applications at 3—hour intervals. Animals were treated
`until the day before the terminal sacrifice. Each drop was approximately 40 uL
`in volume.
`
`Drug, Lot #,
`
`formulation and vehicle:
`
`Cyclosporine ophthalmic emulsion (ASH 192371)
`
` Concentration
`
`1. 8746X-106193
`
`Placebo, pH 7:4
`
`O
`2. B735X—10718
`
`
`.1%, pH 7.4, analyzed concentration 0.097%
`3.‘8734X-10813
`
`
`.2%, pH 7.3, analyzed concentration 0.192%
`
`4. 8734x-106218
`
`
`
`
`5. BT33X-106228
`
`
`6. 8733X—10814
`.
`on 7.2, anal zed concentration 0.374%
`
`
`
`COC nth-L 0909 n.
`
`ll
`
`
`
`

`

`W
`
`hHthlfiZS
`
`Formulations of the drug product and vehicle for the study have not been
`provided in the report. However, chemistry review dated'May 21, 1999, page 45
`provided the preclinical formulations (see attachment}.
`
`Observations and times:
`
`Clinical signs:
`
`All dogs were observed twice daily for mortality and moribund conditions at
`least six hours between each observation. Each dog was observed once daily
`for the evidence of toxicity or side effects.
`A thorough physical
`examination was conducted for each dog once a week.
`
`Body weights:
`
`Weekly body weights of the animals were recorded prior to the treatment,
`during weeks 1—14 of the treatment and once every fourwweek thereafter.
`However,
`the body weight was recorded at three—week interval between weeks
`50—53.
`
`Food Consumption:
`
`Food consumption was recorded weekly for 1—13 weeks and once every four week
`from weeks 13— 49. Food consumption was recorded once between weeks 49— 52.
`
`Ophthalmic Examination:
`
`Ocular discomfort and irritation were examined and scored before the first
`
`dose and before last dose on each day for the first week. Ocular discomfort
`was examined once weekly thereafter. Indirect ophthalmoscopic examinations
`were performed on days -8, 27, 90, 170, 268, 364 and 420, respectively (page
`060, vol 17). Slit lamp examinations were conducted prior to the treatment,
`during weeks 4, 13, 25, 39, 52 and 60 (page 063, vol 17). Surviving animals
`were examined by slit lamp on week 62. One percent Mydriacyl was applied in
`both eyes before the examination. Fluorescein stain was used for each
`examination after week 4.
`
`Blood pressure and ECG:
`
`Indirect blood pressure and ECG were recorded prior to initiation of the
`treatment, on weeks 26 and 52.
`
`Hematology:
`
`Blood samples were collected from the jugular vein. Animals were fasted
`overnight prior to all sample collections. Following parameters were
`determined prior to the treatment and during weeks 13, 26, 39, 52. Samples
`were collected in week 62 from animals allotted for recovery.
`
`Activated partial thromboplastin time, corrected leukocyte count,
`differential leukocyte count and cell morphology, erythrocyte count,
`hematocrit, hemoglobin,
`leukocyte count, mean cell hemoglobin, mean cell
`hemoglobin concentration, mean cell volume, platelet count and prothrombin
`time.
`
`12
`
`
`
`
`
`

`

`
`
`Serum Chemistry:
`
`Blood samples were taken during the same time as indicated in the hematology
`section for the assay of following parameters:
`
`Alanine aminotransferase, albumin, albumin/globulin ratio, alkaline
`phosphatase, aspartate aminotransferase, gamma glutamyltransferase, globulin,
`glucose,
`total bilirubin, total protein and urea nitrogen.
`
`Urine analysis:
`
`Urine samples were collected in clean containers placed under the drainage
`opening of each cage. Samples were collected prior to the treatment, and
`during weeks 13, 26, 39, 52.
`Samples were collected on week 60 from animals
`allotted for recovery. Following parameters were examined.
`
`Appearance/color, glucose, ketones, occult blood, protein and specific
`gravity.
`
`Toxicokinetics:
`
`Blood samples were collected from first three males and females in each group
`on day 7 and week 49 at following time points: prior to the first daily dose,
`1, 5, 7, 9, 11,12,24 hours after the first daily dose. Also blood samples
`were collected (for the trough level) prior to the first daily dose on one
`day during weeks 12 and 26.
`"” “'“
`
`mfi
`. The samples were shipped to Allergan for the analysis.
`
`Gross pathology:
`
`After at least 52 weeks of the treatment, first four animals/sex fgroup were
`weighed and anesthetized with sodium thiopental and exsanguinated. Remaining
`animals were anesthetized and exsanguinated after 8 weeks of the recovery. At
`necropsy,
`the carcass, all external body orifices and visceral organs were
`examined. One male # 631869 from group 2 was sacrificed in the moribund
`condition during week 33.
`
`Organ weights:
`
`Following organs were weighed after removing the fat and other tissues.
`
`Adrenals, epididymides, heart, ovaries, spleen, liver, kidneys,
`testes,
`thymus,
`thyroids, brain, pituitary gland.
`
`lungs,
`
`HistOpathology:
`
`Histology
`_l_lu_mfiwlmnmwwflh~“*ce
`Following tissues were preserved in
`sections were prepared by a standard method. Microscopic examinations were
`performed for all tissues at all doses of CSA and placebo treated animals.
`Sections were stained with ,rm stain.
`
`l3
`
`
`
`

`

`
`
`
`
`ZNDA21$23
`
`Adrenals, aorta, sternum bone marrow, brain with brainstem, medulla, pons,
`cerebellar cortex and cerebral cortex, cervical spinal cord, colon, cecum,
`rectum, duodenum,
`jejunum,
`ileum, esophagus, eyes with optic nerves,
`femur
`including articular surface, heart, kidneys, liver with gall bladder,
`lumbar
`spinal cord,
`lungs and trachea, mammary gland, mesenteric lymph node, mid—
`thoracic spinal cord, ovaries, pancreas, pituitary, prostate, salivary
`thigh
`glands, sciatic nerve, skin, spleen, stomach, testes with epididymedes,
`musculature,
`thymus,
`thyroid,
`trachea, urinary bladder, uterus, vagina and
`cerVix.
`
`Results:
`
`Clinical signs:
`
`there
`There was no CSA treatment related clinical signs observed. However,
`were incidences of lacrimation both in the placebo treated and Cyclosporine
`(CSA)
`treated eyes. Number of animals that showed lacrimation are shown in
`the following table.
`
`"U |-' DJ 0 ('DU‘ 0
`
`O )—| up
`
`
`
`
`
`
`
`Right
`(untreated)
`Left
`
`(treated)
`
`The data suggest that the lacrimation in the left eye could be placebo
`related. The untreated eye did not show lacrimation except male dogs in the
`placebo, 0.2% and 0.4% groups.
`
`Male # 31869 treated with 0.1% CSA was sacrificed on week 33 for humane
`reasons.
`
`Body weight:
`
`in treated animals during
`There was a reduction in the body weight gain (kg)
`weeks 1 to 52. However, it was not statistically significant and considered
`to be incidental.
`
`Group 1
`(placebo)
`
`
`
`Group 2 (0.1%)
`
`Group 3 ( 0. 2%)
`
`
`
`Group 4
`
`(0.4%)
`
`
`
`____
`
`
`
`
`
`1.6
`1.7
`2.0
`2. 3
`1.4
`1 4
`1.1
`1.7
`
`
`Food consumption:
`
`Mean food consumption (kg) and the standard deviation of the mean are shown
`in the following table.
`
`14
`
`
`
`

`

`
`
`hflhk2L023
`
`Group 1
`
`M
`
`M
`
`Group 2 (0.1%)
`
`Group 3 (0.2%)
`
`Group 4
`
`(0.4%)
`
`
`
`
`
`_— _
`
`
`
`
`
`
`
`
`52.1i 5.3
`
`41.0i 4-4
`
`46.4 i 7.4
`
`40.414.8
`
`40.6i5.8
`
`M
`
`45.9 i6.0
`
`35.71 2.0
`
`AbOVe table suggest that male and female dogs in group 4 had a reduced food
`consumption compared to the placebo group. However, it was not statistically
`significant and considered to be incidental.
`
`Ophthalmic Examination:
`
`Total number of positive conjunctival discharge scores in the left eye
`(treated eye)
`for the entire study period is shown in the table below.
`Figures in parentheses represent number of dogs that showed positive score.
`
`
`
`Placebo
`M
`F
`
`
`
`
`
`51 (3)
`121(5)
`83(3)
`178(6)
`
`/,
`
`Prior to l5
`dose
`After last
`dose
`
`
`
`
`0.1% CSA
`M
`F
`
`5(2)
`47(3)
`33(4)
`110(6)
`
`
`
`
`
`_
`
`0.4% CSA
`M
`F.
`
`105 (5)
`93(4)
`199(6)
`201(6)
`
`
`
`
`The score was given in 0—3 scale. D= no discharge, 1: any amount different
`from normal, 2= discharge with moistening of the lids and hair, 3: discharge
`with moistening of the lid, hair and considerable area around the eye.
`
`Above data suggest that the conjunctival discharge was noted in the placebo
`and CSA treated animals. The conjunctival discharge increased at 0.4% CSA
`compared to the placebo treatment. The composite score for the untreated eye
`is not provided in the result.
`
`Number of animals affected by conjunctival redness in the left eye is shown
`below. These data were taken from page 57 and 59, vol 17.
`3
`
`Left eye
`
`Predose, Left
`eY
`
`Post Dose,
`
`The redness was observed in all CSA treated animals. The.prevalence of
`redness was greater in the 0.4% group. However,
`incidences in the placebo
`treated animals were smaller than the treated groups.
`
`15
`
`

`

`
`
`J.l
`
`DHMKZL£23
`
`Indirect ophthalmoscopy and slit lamp examinations did not show compound
`related change in the cornea,
`lens and retina. Mild unilateral
`epiphora/lacrimation was observed in the male dogs in the treated eye. The
`incidences are shown below. Total number of dogs in each group was 6.
`
` Placebo
`
`M F
`
`1
`
`Lacrimation/
`
`Electrocardiography:
`
`There was no abnormality in the ECG. Mean arterial blood pressure did'not
`change significantly during the treatment period. Group mean Values for heart
`rate were decreased at 0.4% CSA in the male animals. Female dogs did not show
`similar change in the heart rate. However, biological significance for the
`change is unknown. The data are shown in the following table.
`
`Heart rate (b/min)
`
`in male dogs.
`
`
`
`
`
`
`
`
`
`————
`
`
`
`
`
`
`
`
`
`
`p< 0.05
`
`Hematology:
`
`
`
`
`
`
`
`
`
`Male dogs did not show treatment-related changes in the hematology
`parameters. Female dogs showed statistically significant reduction in the
`RBC, hemoglobin and hematocrit values at 0.4% dose on week 52. The changes
`were considered to be incidental due to a small magnitude. The data are shown
`in the following table.
`
`
`
`
`
`———
`Placebo,‘(CSA
`6.54
`15.6
`.
`0%)
`
`_3_
`
`
`
`
`
` 43.8
`
`
`14.4*
`
`
`
`
`*P<0.05
`
`Clinical Chemistry:
`
`There was no treatment—related change in the blood (serum) chemistry.
`
`Urine analysis:
`
`There were no treatment related-changes in the urine chemistry.
`
`16
`
`
`
`

`

`
`
`bunxzpnzs
`
`Organ weights:
`
`There was no significant change in the organ weight.
`
`Gross Pathology:
`
`There was no treatment-related change in the gross pathology of the treated
`dogs.
`
`Histopathology:
`
`There was no treatment related histopathology changes observed in the study.
`Animal that was sacrificed for humane reasons on week 33 (M31869)
`showed
`severe multiple abscesses in the abdominal cavity. Lymphocytic infiltration
`in the conjunctiva (minimall of the left and right eyes was also observed.
`The animal showed uterine horn although it was a male dog. Generalized sepsis
`was reported to be the cause of deterioration of the health condition. The
`blood samples from the animal were not collected for the determination of the
`systemic exposure to CSA.
`
`Toxicokinetics for oneeyear toxicity study in beagle dogs:
`
`rage 124, vol 18:
`
`Blood samples Here collected and shipped to Allergen Pharmaceuticals for
`assay of CSA levels in the blood.
`The study protocol has been described in
`page 13. The assay was conducted at Allergen Pharmaceuticals during Oct 96 to
`Sept 95 and Aug 96 to Sept 96. Blood Cyclosporine levels were determined by
`validated liquid chromatographic technique.
`
`PK