-r
`
`Intraocular Penetration of Topically Applied
`Cyclosporine
`
`R.L Kaswan
`
`cell
`
`YCLOSPORINE CsA is
`potent
`immunosuppressive drug with specific
`inhibitory activity CsA appears to inter
`rupt
`cell activation at an early point by
`interfering with activation of
`intracellular
`mechanisms following antigen binding with
`resultant blockade
`cas
`of
`the lymphokine
`cade CsA has proven to be very beneficial
`in
`and certain autoim
`organ
`transplantation
`mune disorders in man.2 Recently systemi
`cally administered CsA has been found to be
`in Sjogrens syndrome3 and some
`beneficial
`forms of uveitis in both people and experimen
`tal species.4-2 Additionally topically applied
`CsA has been found to be useful
`in corneal
`allografts35 vernal
`
`keratoconjunctivitis6
`sicca7 immune me
`keratoconjunctivitis
`diated keratitist necrotizing scleritis19 and
`herpetic stromal keratitis.t2 These studies
`local ocular
`
`that
`
`immunosuppression
`suggest
`provides adequate therapy while avoiding the
`risks of generalized immunosuppression and
`renal toxicity
`topically applied CsA for
`The efficacy of
`external ocular disorders led us to conjecture
`that if CsA can reach therapeutic levels inside
`the eye following topical application it might
`be possible to treat
`intraocular diseases such
`
`From the Departments of Small Animal Medicine and
`Veterinary Pharmacology/Physiology
`College of Veteri
`nary Medicine University of Georgia Athens
`Supported
`in part by the National Eye Institute
`Institutes of Health R03 EY05720-Ol
`National
`The
`National Society for the Prevention of Blindness
`The
`University of Georgia Veterinary Medical Experimental
`Station 29-26-GR207-002 and Sandoz Pharmaceuticals
`Inc
`
`Kaswan DVM
`Address reprint requests to Renee
`MS Department of Small Animal Medicine College of
`Veterinary Medicine University of Georgia Athens GA
`30602
`1988 by Grune Stratton Inc
`
`0041-1345/88/2002-2121
`
`$03.00/U
`
`650
`
`as
`
`immune mediated uveitis with topical
`application Specific advantages to use of topi
`cal CsA as compared to corticosteroids would
`include lack of collagenase activation when
`immune suppression is indicated in eyes with
`corneas and potentially improved
`ulcerated
`immunosuppressive activity
`
`MATERIALS AND METHODS
`
`Rabbits
`
`rabbits
`
`female New Zealand White NZW
`We used 33 adult
`to 4kg divided into 11 groups of three rabbits
`Tetracycline
`g/gallon was added to the drinking water
`for prophylaxis of Pasteurella infection
`
`Treatment
`
`solution of 1% CsA radiolabeled with 3H specific
`activity of 8.5 zCi/mg kindly supplied by William
`Robinson Sandoz Pharmaceuticals Hanover NJ was
`prepared in olive oil Sigma St Louis Three rabbits
`containing olive oil without CsA as
`received
`eyedrops
`controls
`Varimetric positive
`displacement
`pipettor Labindustries Berkeley CA was used to
`ttL eyedrops This volume was chosen to avoid
`spillover since the tear volume in the rabbit
`
`negative
`
`deliver
`
`averages
`lead to greater
`
`pL.2
`
`Increased
`
`drop size does not
`confounds
`corneal
`hut
`penetration22
`topical absorption
`with recirculation following absorption of drugs through
`the nasolacrimal system Drops were applied to the dorsal
`limbus One drop was applied every
`corneoscleral
`15
`The total body dose per
`minutes for six applications.23
`rabbit was 0.84 mg CsA or 0.24 mg/kg The
`time
`from the last application until enucleation was
`interval
`varied from one to 72 hours Fig
`killed at each interval
`
`with three animals
`
`Sample Collection
`
`Rabbits were anesthetized with xylazine
`mg/kg
`IM Samples
`and ketamine 35 mg/kg intramuscularly
`of 200 ML of aqueous humor were drawn with
`30-gauge
`needle and tuberculin syringe inserted at
`limbus After heparinized cardiac
`blood
`samples were
`drawn the rabbits were killed with pentobarbital 325
`mg intracardiac
`Both eyes were enucleated
`injection
`and frozen Clean instruments were used for each rabbit
`Globes were dissected
`
`the corneal
`
`by the method of Abel and
`BoyleJ Briefly the globes were transected
`
`at
`
`the equa
`
`FAMY CARE - EXHIBIT 1011-0001
`
`

`
`INTRAOCULAR PENETRATION
`
`OF CYCLOSPORINE
`
`651
`
`9000
`
`0000
`
`7000
`
`6000
`
`5000
`
`4000
`
`0000
`
`2000
`
`000
`
`200
`
`150
`
`100
`
`Sb
`
`Anterior
`
`Sciera
`
`10
`
`20
`
`30
`
`40
`Time hours
`
`so
`
`so
`
`Time Sours
`
`Retina
`
`03i0Tirno4Lrs5bbb70
`
`00
`
`Aqueous
`
`10
`
`20
`
`30
`
`40
`Time hours
`
`50
`
`00
`
`70
`
`90
`
`90
`
`70
`
`00
`
`50
`
`40
`
`30
`
`20
`
`10
`
`11
`
`Time hours
`
`40
`Time hours
`
`zL of 1% 3HC5A in
`CsA content
`in various ocular tissues of the rabbit eye following six applications of
`Fig
`have been calculated from the specific activity of 3HCsA and expressed as nglg tissue or
`olive oil Concentrations
`ng/mL fluid Six eyes have been used for each individual point
`
`__
`
`FAMY CARE - EXHIBIT 1011-0002
`
`

`
`652
`
`R.L KASWAN
`
`In rabbits
`
`in three times its weight of
`
`tor The frozen vitreous was extruded and collected The
`iris and ciliary body choroid with retina
`anterior uvea
`lens cornea anterior selera and posterior sclera were
`dissected individually processed
`and assayed
`killed at two hours post-CsA treatment
`the lacrimal gland
`of the third eyelid was also removed and assayed Each
`tissue was digested
`ocular
`collagenase Sigma type
`in Michaelis barbital buffer.25
`Next 100 uL of collagenase buffer solution was added to
`each vitreous sample Tissue digests 250 pL whole blood
`and 100 tL aqueous samples were transferred to scintilla
`tion vials and mixed with 10 mL Scintiverse
`Blood
`samples were decolorized with BTS-450 Beckman
`Atlanta 30% H202 and glacial acetic acid.26 The disin
`tegrations were assayed as counts per minute cpm using
`Beckman LS 7000 liquid scintillation counter Counting
`assay was determined by H-number
`for each
`efficiency
`quench correction using commercial external standards
`for data conversion
`per minute
`to disintegrations
`DPM.26 Conversion to mg CsA was made
`following equation
`
`by the
`
`2.2
`
`06 DPM/pCi
`
`DPM of sample
`106 ng/mg
`8.5 MCi 3H/mg CsA
`ng CsA/g tissue
`
`tissue wt
`
`tissues were used for background
`Negative control
`traction of DPM for each tissue type
`Blood samples from rabbits from each time group were
`blind manner by Sandoz Research
`assayed in
`Institute
`
`sub
`
`tissue-burning technique
`to assay the vaporized
`using
`tridium from each
`blood sample because the tissue-
`for whole
`blood
`burning assay has higher sensitivity
`samples than can be attained with liquid scintillation.6
`
`RESULTS
`
`Figure
`
`illustrates the CsA content of the
`and ocular
`tissues Peak
`aqueous vitreous
`concentrations
`were reached within the first
`four hours in all tissues Tissue concentrations
`in excess of minimal therapeutic levels 50 to
`300 ng CsA/g tissue6 were achieved within
`one hour and maintained for at least 24 hours
`in the cornea anterior and posterior selera
`Levels of CsA were below therapeutic values
`within two hours in the retina vitreous and
`aqueous samples The average level of CsA in
`the laerimal gland of
`the third eyelid was
`2850 ng/g at two hours post-CsA treatment
`Iris and ciliary body CsA concentrations were
`therapy of uveitis6
`at equivocal
`levels
`for
`between 50 and 200 ng/g tissue Fig
`for all
`samples taken between one and 24 hours
`
`anterior
`
`The elimination rates of CsA in the cornea
`and anterior
`and posterior
`uvea
`sclera were determined as the slopes of
`terminal phase of
`the concentration
`time
`curves using
`two compartmental
`visually fit
`model.27 The elimination half-life was calcu
`lated as 0.693 divided by the elimination rate
`and was found to be 33.5 hours 26.7 hours
`19.5 hours and 20.0 hours respectively
`Amongst
`20 blood
`samples
`assayed for
`by both liquid scintillation and
`radioactivity
`tissue burning techniques only one
`had significant
`than
`greater
`radioactivity
`three times baseline and it was identified by
`both methods The CsA content of this sample
`was 57 ng No radioactivity was detected in
`the remaining 10 samples which were assayed
`but
`not
`by
`liquid scintillation
`by
`tissue-
`
`the
`
`sample
`
`burning techniques
`The pigmented tissues and especially the
`whole blood samples were most affected
`by
`chemiluminescence The presence of biologic
`pigments
`red pigments
`causes
`especially
`level cpm values confounding the
`high control
`background activity The use of antioxidants
`and bleaching
`agents reduced this error but
`did not eliminate it making small amounts
`less than 50 ng/mL of CsA in the blood
`impossible to detect
`by liquid scintillation
`Use of
`the tissue-burning technique
`on por
`tions of
`confirmed that
`these samples
`no
`detectable CsA occurred
`in blood samples
`except one sample taken at
`hours posttreat
`ment
`
`DISCUSSION
`
`Two major questions must be determined
`before topical CsA would prove to have practi
`importance for uveitis Can CsA reach the
`cal
`target organ in therapeutic levels following
`reasonable therapeutic regimens Is the effect
`of CsA mediated locally in the eye or does
`the inhibition of
`intraocular
`inflammation
`
`require systemic immunosuppression
`CsA is an undecapeptide with molecular
`weight of 1202 The therapeutic range for
`is 200 to 600 ng/mL
`organ transplantation
`
`FAMY CARE - EXHIBIT 1011-0003
`
`

`
`INTRAOCULAR PENETRATION OF CYCLOSPORINE
`
`653
`
`the intraocular
`
`it
`
`serum but
`level speculated to
`be needed for control of uveitis is 50 to 300
`the key event is inhib
`ng/mL6 Alternately if
`iting the triggering of
`release
`lymphokine
`cells the required intraocu
`from activated
`to 199 ng/mL CsA
`lar dose would be only
`Compared to most ophthalmic medications
`CsA is
`large molecule but
`is highly
`lipophillic If compounds are lipid soluble and
`and conjunctival
`through the corneal
`pass
`size does not appreciably
`epithelium their
`Intraocular
`the
`alter
`rate of
`
`absorption of
`
`transport.28
`large molecular weight drugs
`inter
`
`occurs across the conjunctival-scleral
`the
`of passage
`face
`bypassing
`necessity
`through the aqueous humor.29 CsAs absence
`in the aqueous humor303 was therefore not
`discouraging because the aqueous is an inap
`for sampling of intra
`propriate compartment
`ocular drug absorption for those drugs that
`depend upon noncorneal absorption routes.29
`Since the total volume of CsA given to each
`in the present study was
`experimental animal
`zL 1% CsA in each eye the total
`doses of
`rabbit was 0.84 mg or 0.24
`body dose per
`mg/kg.2 The recommended systemic dose of
`CsA is 10 to 20 mg/kg2 or 60 to 120 times
`used
`than
`the doses
`in this study.2
`higher
`Following therapeutic dosage in people with
`levels were only 40% of
`uveitis intraocular
`serum levels.32 Bell and coworkers
`report
`aqueous levels of 12 to 49 ng/mL33 following
`topical or systemic administration to rabbits
`the RIA
`but
`the lower limit of sensitivity of
`assay used was 45 ng/mL.34
`the
`Based on
`absence of detectable CsA in 29 of 30 blood
`samples and the minimal
`total body doses
`used intraocular
`levels in this study probably
`were not due to blood redistribution
`Wiederholt and coworkers studied intraoc
`ular penetration of 1% CsA dissolved in castor
`reported their results in cpm rather
`oil but
`universally comparable term.26 The
`corneal half-life in both studies is remarkably
`long 34 hours in this study and 52 hours in
`Wiederholts.35 Our
`findings do not support
`the intraocular
`
`than in
`
`their conclusion
`
`that
`
`levels
`
`occur due to blood redistribution to both eyes
`following CsA administration to either eye.35
`The concentration of CsA reached in the
`present study was well above therapeutic lev
`els in the cornea and in anterior and posterior
`sclera but the intraocular CsA levels achieved
`low The
`uvea
`approached
`were
`anterior
`levels with levels
`hypothesized therapeutic
`ranging from 50 to 200 ng/gm An assay of
`state pharmaeokinetics with chronic
`steady
`for five elimination half-lifes
`administration
`eyes may be
`and
`from inflamed
`assays
`required to determine whether or not higher
`therapeutic levels can be achieved in the ante
`and retina under
`rior uvea
`typical clinical
`
`cell
`
`conditions
`Where is the target site If CsA works by
`entering the eye and blocking intraocular
`cells responding to ocular antigens within the
`inflamed eye then local administration can be
`advocated
`If however ocular antigens
`are
`disseminated to lymphatic organs where
`systemic CsA therapy
`occurs
`activation
`would be mandated
`Topical CsA had demonstrated efficacy in
`immune-mediated diseases of the cornea and
`sclera including experimental corneal graft
`transplantation35 vernal keratoeonjunetivi
`sicca7 chronic im
`tis6 keratoconjunctivitis
`mune mediated keratitis8 neerotizing scleri
`tis9 and
`stromal
`herpetic
`experimental
`keratitis.82 In dogs with keratoconjunctivitis
`sicca topical CsA ameliorated the chronic
`keratitis and increased the average Schirmer
`mm/mini7
`These studies
`by
`tear
`test
`immune suppres
`that
`local
`strongly suggest
`immune-mediated
`sion is adequate to control
`disorders with CsA providing
`the CsA
`the target tissue
`reaches therapeutic levels at
`The
`data confirm
`present pharmacokinetic
`that CsA levels are very high in the lacrimal
`the third eyelid cornea and sciera
`gland of
`following topical dosage
`The question of whether or not uveitis can
`be controlled with topical CsA has been more
`elusive The intraocular dosage of CsA may
`inflammation at all
`not be sufficient to control
`
`FAMY CARE - EXHIBIT 1011-0004
`
`

`
`654
`
`R.L KASWAN
`
`areas of
`
`antigens
`
`cell
`
`the globe The current
`investigation
`and previous kinetic work has determined the
`elimination half-life but has not addressed
`state intraocular
`the steady
`concentrations
`one would anticipate during multiple chronic
`dosage
`Previous uveitis studies that used intrasub
`ject positive control eyes may have inadver
`immune reaction
`tently triggered an afferent
`in the positive control eye causing
`immune response with an efferent
`response
`occurring in both the control and treated eye.6
`Studies that treated both eyes with CsA and
`used an extrasubject positive control were
`Two studies
`more successful.6
`have exam
`for topical CsA in experi
`ined the potential
`mental uveitis When Nussenblatt
`and
`coworkers6 tested CsA in
`limited number of
`rats with experimental autoimmune uveitis
`they could not separate systemic effects from
`topical effects because the ophthalmic drops
`used were so large 50 1iL and the subjects so
`small 200
`rats that
`serum
`therapeutic
`levels were reached via nasolacrimal absorp
`tion Considering the mechanism of CsA
`activity the problem of differentiating
`the
`effects of systemic absorption of topical drug
`was not well addressed by unilateral
`drug
`dosing CsA prevents initiation of the immune
`recognition of
`response by blocking
`and thereafter production of
`inter
`which
`leukin
`the
`activates
`cascading
`immune response Antigenic recognition and
`lymphocyte activation within the untreated
`eye would be expected to trigger
`systemic
`immune response36 affecting both eyes there
`fore unilateral treatment designs would likely
`of Nussenblatts work
`fail Re-evaluation
`
`systemic
`
`treatment
`
`in
`
`topical
`
`therapy
`
`experimental
`
`demonstrates that bilateral
`was effective
`in preventing
`autoimmune uveitis EAU at concentrations
`of 2% 0.5% 0.2% but unilateral
`did not show
`significant beneficial effect
`the eye treated with 2% 0.5% and 0.2% CsA.6
`This result was attributed to the fact
`that rats
`receiving bilateral drops got more volume of
`CsA but actually the rats receiving unila
`teral 2% CsA had more total CsA than rats
`with bilateral 0.5% or 0.2% treatments An
`is the intrasub
`alternate explanation is that it
`instigates EAU
`ject positive control eye that
`in the CsA-treated eye37 and that bilateral
`CsA is effective in EAU Based on the small
`sample size two rats per
`treatment group
`and the ambiguity of results further investi
`gation into use of topical CsA for uveitis may
`be warranted
`
`of
`
`topical CsA for
`Advocacy
`intraocular
`disease still awaits proof positive of intraocu
`lar penetration What
`is most encouraging
`from our data is that CsA does reach very
`high levels in the cornea selera and lacrimal
`gland of
`the rabbit Considering the results
`trials of CsA in corneal
`from topical
`trans
`plantation35
`stromal
`herpes
`keratitis182
`sicea7 and chronic im
`keratoconjunctivitis
`mune mediated keratitis15 it
`is predictable
`that other immune-mediated disorders of the
`cornea
`and sclera may also
`conjunctiva
`respond positively to topical CsA
`
`ACKNOWLEDGMENT
`
`The author thanks Susan Gardner for her expert advice
`on ocular pharmacokinetics
`Antoinette
`Jernigan for
`assistance with the pharmacokinetic determinations
`and
`William Robinson for furnishing 3HCsA and for perform
`ing corroborative assays
`
`REFERENCES
`In Schindler ed Ciclosporin
`Borel iF Ryffel
`disease New York Springer-Verlag
`in autoimmune
`1985 p24
`Kahan BD Cyclosporine Biological Activity and
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`
`Leuenberger PM Mieschcr PA KIm Mbl Augen
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`
`Nussenblatt RB Palestine AG Chan CC Ocular
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`Nussenblatt RB Salinos-Carmona
`Waksman
`BH et al mt Arch Allergy AppI
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`Nussenblatt RB Dinning Wi Fujikawa LS et al
`Arch Ophthalmol 1031559 1985
`Nussenblatt RB Rodriques MM Salinas-Carmona
`MC et al Arch Ophthalmol 1001146 1982
`
`FAMY CARE - EXHIBIT 1011-0005
`
`

`
`INTRAOCULAR PENETRATION OF CYCLOSPORIN
`
`655
`
`Am
`
`24 Abel
`
`Boyle GL Invest Ophthatmol 15216
`
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`
`Martin CL Trans Am Cot
`
`et
`
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`
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`York Marcel Delker 1982
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`1985
`30 Kaswan RL Tackett RL Martin CL et at Invest
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`1984
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`
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`
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`
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`
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`
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`
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`10 1278 1986
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`
`et
`
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`
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`
`FAMY CARE - EXHIBIT 1011-0006