CORNEA/EXTERNAL
`
`DISEASE
`
`into the
`Penetration of Cyclosporin
`Rabbit Cornea and Aqueous Humor
`after Topical Drop and Collagen
`Shield Administration
`
`Ayfer Kanpolat
`
`Figen Batioglu MD
`
`Merih Yilmaz MD
`
`Fatma Akbas MD
`
`We used commercially available 12-hour collagen shields to deliver
`cyclosporin CsA to the cornea and aqueous humor in rabbit eyes Six
`New Zealand white rabbits were divided into three groups The first
`group four eyes received 6mg of CsA in castor oil and the second group
`tour eyes received mg of CsA In olive oil applied as topical drops to
`rabbit eyes within 12 hours In the third group four eyes 12-hour
`collagen shields soaked in mg CsA in olive oil were applied to rabbit
`eyes The amount of CsA in corneal and aqueous samples from eyes
`treated with CsA castor oil and CsA olive oil were compared with each
`other and with collagen shield treated eyes CsA concentrations were
`mg CsA
`measured by radioimmunoassay After
`the total dose of
`percentage penetration was measured as follows CsA castor oil
`0.51% in aqueous and 20.75% in cornea CsA olive oilO.1 7% in aqueous
`and 11.1 3% in cornea and with collagen shieldsO.44% In aqueous and
`11.84% In cornea These results show that the CsA levels of castor oil
`drops were higher than those obtained with olive oil drops In eyes with
`collagen shields CsA levels were higher than olive oil drops but nearly
`equal to the castor oil drops Collagen shields may be useful as an ocular
`delivery system for CsA
`
`Introduction
`
`The successful
`treatment of many diseases of the eye
`depends upon adequate drug delivery An important develop
`ment in drug delivery has been the utilization of collagen shields
`Collagen shields have been used as bandage lenses after radial
`keratotomy and keratorefractive surgery to treat corneal abra
`sions and as an alternative therapy fordry eyes.2 The shields are
`made of porcine scleral collagen and are available in 12-24-and
`72-hour dissolution rates Their oxygen permeability is high
`After placement on the eye they become hydrated with tears
`take the shape of the cornea and are degraded by proteolytic
`enzymes in the tear film In addition to their capabilities of
`lubricating protecting and accelerating reepithelialization they
`are used as vehicles for enhancing the ocular penetration of
`various drugs.3 Recent
`investigations with collagen shields
`that delivery of gentamicin4 vancomycin4
`demonstrate
`
`tobramycin5 dexamethasone6 amphotericin B7 heparin8 and
`cyclosporin A9 to the eye are increased
`CsA is
`cyclic polypeptide of fungal
`Cyclosporin
`origin First reported to have immunosuppressive properties by
`in 197610 it has dramatically improved the prognosis for
`Borel
`solid organ transplantation because of its low myelotoxicity and
`lymphocyte function Systemic CsA
`suppression of specific
`has been used to suppress rejection after kidney transplantation
`as well as bone marrow heart and liver transplantations It has
`also been used to treat various autoimmune diseases
`such as
`
`uveitis psoriasis rheumatoid arthritis myasthenia gravis and
`diabetes mellitus type 1.10 It has been used with some success to
`treat patients with various ocular manifestations of systemic
`immune disease including Graves ophthalmopathy corneal
`peripheral melting syndrome Behcet disease and Sjogren
`syndrome Nussenblatt and colleagues suggested that
`
`topical
`
`Copyright 1994 Contact Len8 Association of Ophthalmologists
`All rIghts reserved
`
`119
`
`the CLAO journal
`
`April 1994
`
`Volume 20 Number
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`FAMY CARE - EXHIBIT 1018-0001
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`

`
`the serum CsA levels entered the
`therapy was effective only if
`therapeutic range of 50 to 300 ng/mL
`therapeutic concentrations of CsA to the
`Delivery of
`cornea and the anterior chamber has been difficult
`to accomplish
`
`because of its poor solubility in water.9 Topical ophthalmic uses
`have included treatment of corneal graft rejections23 Mooren
`ulcer noninfiltrative marginal keratolysis4 and herpetic stro
`mal keratitis5 Recently investigations of CsA levels in the eye
`and various tissues after topical and systemic administrations
`have been made Mosteller and coworkers6 investigated the
`10%
`systemic and ocular absorption of
`topically applied
`cyclosporine ointment Ben Ezra and colleagues7 determined
`the tissue levels of CsA after oral intraperitoneal
`and intrave
`nous administrations Reidy and associates9 prepared collagen
`shields by mixing gelatinous collagen with crystalline CsA and
`compared them with topical eye drops
`The aim of this study was to investigate the effectiveness
`available collagen shields and CsA prepara
`of commercially
`tions and was performed to compare the penetration of CsA
`olive oil and castor oil forms administered as topical eye drops
`with each other and with commercially available collagen
`shields soaked in CsA olive oil
`
`in
`
`Materials and methods
`Six New Zealand white rabbits male and female 2038 to
`3220 were divided into three groups
`Eye drops In group
`the eye drops consisted of
`commercially available intravenous form of CsA castor oil
`of 50 mg CsA/mL 0.5 mg CsA/lOpL
`concentration
`Sandimmune Sandoz Pharmaceuticals
`In group
`the eye
`drops consisted of the oral form of CsA olive oil
`in concen
`tration of 100 mg CsA/mL
`mg CsAJ1O l.IL Sandimmune
`total of mg CsA
`Sandoz Pharmaceuticals Over 12 hours
`was administered as topical drops with micropipette
`Collagen shieldsFour commercially available collage
`shields were used 12 hours
`Lomb
`Bausch
`Pharmaceutics Inc Clearwater FL For this purpose 60 .tL
`mg CsAJ1O tL Sandimmune oral solution olive oil was
`dropped over collagen shields We used CsA olive oil because
`it was absorbed better than CsA castor oil Within 1.5 to hours
`the total dose of 6mg CsA was absorbed by the collagen shields
`Experimental designThe
`six rabbits were divided into
`four eyes of two rabbits received CsA
`three groups In group
`castor oil applied with micropipette 10 .tL
`Switzer
`land at one drop 10 1.tL per hour for 12 doses for
`
`mgCsA.Every 10tL drop contained 0.5 mgCsA mg
`mg CsA In the second group four eyes of two rabbits
`
`total of
`
`TABLE Cyclosporine concentrations
`
`in aqueous humor nglmL
`
`Mean
`
`30.625
`
`10.00
`
`26.475
`
`SD
`
`Range
`
`8.531
`
`22.8
`
`38.2
`
`13192
`
`14.7
`
`42.8
`
`CsA castor oil
`CsA olive oil
`
`Collagen shield
`
`GsA olive oil
`lOng
`SD
`standard deviation
`
`received CsA olive oil applied to the eyes with micropipette
`hours for 12 hours six times They also
`one drop every
`total of 6mg CsA every 10 j.iL drop contained mg
`received
`6.0 mg CsA In both groups the total dose of
`CsA
`mg
`CsA 6mg was equal to the amount of CsA delivered by
`collagen shield
`Four eyes of the two rabbits in the third group were treated
`with CsA olive oil soaked
`collagen shields Rabbits were
`injection of 0.5 mL 2% xylazine
`anesthetized by intramuscular
`hydrochloride Rompun Bayer and mL ketamine hydrochlo
`ride Ketalar Parke-Davis Collagen shields containing CsA
`were applied directly to the corneas and rehydrated with sterile
`saline The eyes were closed by lid sutures to ensure retention of
`the shields The shields remained in the rabbits eyes for 12
`hours
`
`single
`
`to
`
`ferred to
`
`Twelve hours after application of either the drops or the
`collagen shields the rabbits were killed with sodium pentobarbital
`The eight eyes
`to which the
`injections given intravenously
`topical eye drops had been administered were rinsed with sterile
`saline Aqueous humor was collected with
`sterile 25 gauge
`mL syringe The anterior chamber was
`needle attached
`entered mm away from the limbal margin of the cornea and
`0.2 to 0.5 mL of aqueous humor was aspirated The corneas were
`removed by 8.5 mm trephine After rinsing them with sterile
`saline the corneas were minced with
`razor blade and trans
`mL volume of methanol They were left at 4C
`at 2000 revolutions per minute
`overnight and centrifuged
`for 10 minutes
`When the four eyes to which the collagen shields were
`applied were opened no shields were found on the comeas
`combined with mucus were
`Small
`fragments of collagen
`lodged in the fornices Eyes were rinsed with sterile saline and
`samples were obtained in the same manner as previously de
`scribed
`CsA concentrations in all samples were evaluated by
`monoclonal antibody based radioimmunoassay RIA The RIA
`laboratory used CYCLO Trac SP 125/RIA INCSTAR Corp
`Stillwater MN and
`gamma scintillation counter
`
`Results
`
`CsA castor oil
`treated eyes the CsA concen
`In group
`tration in the aqueous humor ranged between 22.8 ng/mL and
`38.2 ng/mL with an average of 30.6 ng/mL Table
`the
`concentrations in the cornea ranged between 865 ng/mL and
`1622 ng/mL with an average of 1245 ng/mL Table II In
`the CsA concentrations in aqueous humor of eyes that
`group
`received topical CsA olive oil drops were below levels that
`could be measured with RIA
`10 ng/mL Table
`The values
`obtained in the cornea ranged between 472 ng/mL and 855 ng/
`
`TABLE II Cyclosporine concentrations
`
`in cornea ng/mL
`
`CsA castor oil
`CsA olive oil
`
`Collagen shield
`
`Mean
`
`1245
`
`667.8
`
`710.5
`
`SD
`
`426.792
`
`157.559
`
`129.935
`
`Range
`
`865
`472
`589
`
`1622
`855
`857
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`the CLAO journal
`
`April 1994
`
`Volume 20 Number2
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`120
`
`FAMY CARE - EXHIBIT 1018-0002
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`

`
`CsA ng/ml
`
`40
`
`30
`
`20
`
`10
`
`14.7
`
`10
`
`42.8
`
`382
`
`25
`
`20
`
`15
`
`10
`
`Eyes
`
`Series
`
`Series
`
`Series
`
`C8ACastor oil CsAOilve oil Collagen Shield
`Olive 011410 ng
`
`Figure
`
`Aqueous humor
`
`levels
`
`Series
`Aqueous humor
`
`Series
`Cornea
`
`Figure
`
`Aqueous humor and cornea levels
`
`mL with an average of 667.7 ng/mL of CsA Table II Thus
`after 12 hours the concentrations in the aqueous humor Figure
`and the cornea Figure
`were statistically higher in the group
`that received topically applied castor oil CsA aqueous humor
`0.035 paired test
`0.0 17 cornea
`CsA applied by collagen shields aqueous
`In group
`humor values ranged between 14.7 ng/mL and 42.8 ng/mL with
`an average of 26.5 ng/mL Table
`and the values of the cornea
`ranged between 589 ng/mL and 857 ng/mL with an average of
`710.5 ng/mL Table II Whereas the mean aqueous humor
`concentrations of CsA in eyes treated with collagen shields were
`significantly greater than the mean concentrations of CsA in
`eyes treated with topically applied CsA olive oil drops no
`significant difference in corneal concentrations could be dem
`0.316
`these two groups
`onstrated between
`When the results obtained by using collagen shields were
`compared with the results of topically applied CsA castor oil
`in aqueous humor
`drops the concentrations were nearly equal
`According to paired -test no significant difference
`Figure
`could be demonstrated 0.25 but the corneal concentrations
`were higher with topically applied CsA castor oil drops Figure
`0.038
`and this was significant
`shows the penetration levels of CsA in aqueous
`
`Figure
`
`CsA ng/ml
`
`1607
`
`1622
`
`2000
`
`1500
`
`1000
`
`SOD
`
`Eye
`
`Series
`
`4-- Series
`
`Series
`
`C8ACaator oil CaAOlive oil Collagen Shield
`
`Figure
`
`Cornea levels
`
`humor and cornea with topical castor oil olive oil drops and
`olive oil soaked collagen shields as
`percentage of the total dose
`of mg
`
`Discussion
`The results of this study indicate that
`in rabbits topically
`applied CsA castor oil drops resulted in higher concentrations of
`the drug in the aqueous humor and cornea than when CsA olive
`oil drops were used An additional
`finding is that
`collagen
`shield soaked in CsA olive oil form is an equally effective means
`of delivering CsA to the cornea and aqueous humor as topically
`applied castor oil drops
`The topical application of CsA to the eye has been studied
`the risk of systemic toxicity while
`to reduce
`in an attempt
`maximizing its local therapeutic potential Because of the hy
`drophobic nature of the corneal epithelium CsA would be
`expected to penetrate it easily Conversely the stroma should be
`is hydrophilic By using
`quite impermeable to the drug because it
`2% solution of CsA castor oil Wiederholdt and colleagues8
`layers of rabbit eyes
`evaluated its penetration into the different
`Following single dose of 10 IL of 2%CsA concentration of
`900 to 1400 ng/mL was detected in the cornea at
`hours of
`which 67% was found in the epithelium 25% in the stroma and
`8% in the endothelium Concentrations in the remaining tissues
`45 ng/mL.8 Kaswan9
`lens vitreous uvea retina were
`measured the absorption of topically administered 1% CsA in
`olive oil After the total dose of 0.84mg was given tissue levels
`cornea anterior and posterior sclera greater than 50 ng/mL
`were achieved within hour but lower levels were obtained in
`the retina vitreous ciliary body iris and aqueous humor
`In our study the penetration of topically applied CsA
`castor oil and olive oil drops were compared with each other
`total dose of mg of topically applied CsA castor oil
`After
`0.51% was found in aqueous humor and 20.75% in the cornea
`compared with 0.17% in aqueous and 11.13% in the cornea with
`topical CsA olive oil drops Thus topical drops prepared using
`CsA in castor oil are more effective than CsA in olive oil
`Reidy and coworkers9 prepared collagen shields by mix
`ing gelatinous collagen with crystalline CsA as
`means of
`delivering CsA to the cornea and aqueous humor in rabbit eyes
`
`the CLAO journal
`
`April 1994
`
`Volume 20 Number
`
`121
`
`FAMY CARE - EXHIBIT 1018-0003
`
`

`
`Schwartz SD Harrison SA Engstrom RE Jr et al Collagen shield
`delivery of Amphotericin
`Murray TG Stern WH Chin DH et al Collagen shield heparin delivery
`fibrin Arch Ophthalmol 1990 108104-
`of postoperative
`
`Am Ophhalmol 1990
`
`for prevention
`106
`Reidy Ji Gebhardt BM Kaufman HE The collagen shielda new vehicle
`to the eye Cornea 1990 196-199
`for delivery of cyclosporin
`10 Belin MW Bouchard CS Phillips TM Update on topical cyclosporin
`immunology and pharmacology Cornea 1990 184-
`Abackground
`194
`II Kruit PJ Cyclosporine
`
`treatment
`
`in four cases with comeal melting
`
`syndrome Transplant Proc 1988 4170-172
`Br Ophtlzalmol l982
`
`12 Hunter PA Garner Wilhelmus KR et alCorneal graft rejectiona new
`rabbit model and cyclosporin
`13 Foets
`Wanderweren
`Missoten
`
`survival of
`
`et al Prolonged
`allogeniccornealgraftsinrabbits treated with topically applied cyclosporin
`immunosuppressive effect Br
`Asystemic
`absorption and local
`
`Ophthalmol l985
`
`14 Nussenblatt RB Palestine AGCyclosporine immunology pharmacol
`uses Sun Ophthalmol 1986 1159-169
`ogy and therapeutic
`iF Keratitis herpetiques
`15 CoIling
`Bonissent
`Chastel
`collyre Presse Med
`stromalestraitement par
`Ia cyclosporine
`1986 1245
`16 Mosteller MW Gebhardt BM et alPenetration of topical cyclosporine
`1985
`into the rabbit cornea aqueous humor and serum Arch Ophthalmol
`
`17 Ben Ezra Maftzir
`Ocular penetration of cyclosporin
`Arch Ophthalmol 1990 108584-587
`18 Wiederholdt
`Schulz et aIPharmacokinetics of topical
`Kossendrup
`in the rabbit eye Invest Ophihalinol Vis Sci 986 19-
`Transplant Proc 1988 2650-655
`
`cyclosporin
`524
`19 Kaswan RLIntraocular
`
`penetration
`
`of topically applied
`
`cyclosporine
`
`in the rat eye
`
`They found that both the corneal and aqueous humor concentra
`lions of CsA achieved with the shield delivery system were
`higher than those obtained with topical CsA olive oil drops In
`our study 12-hour collagen shields were soaked in CsA olive oil
`and applied to the eyes CsA levels in the aqueous humor were
`0.44% of the total dose and 11.84% of the total dose was found
`
`in the cornea
`
`as
`
`Commercially available collagen shields can be utilized
`means of delivery system for CsA and in addition to
`available oral and intravenous forms of CsA
`commercially
`to show the
`dispensed as topical drops Our study is the first
`efficacy of using commercially available collagen shields as
`means of delivering CsA
`The application of CsA in
`collagen shield delivery
`such as
`to apply to patients with diseases
`system is easy
`Moorens ulcer corneal peripheral keratolysis herpetic stromal
`keratitis and corneal allogralt reactions thus minimizing sys
`temic toxicity and maximizing local penetration
`
`Acknowledgment
`Ms Adalet YtIzUbenli
`for help in the research laboratory and Ms
`GUI
`Cakmak for statistical consultation Collagen shields were supplied by Bausch
`and Lomb Pharmaceuticals
`
`References
`Ros FE TijIJW Faber JAJ Bandage lensescollagen shields vs hydrogel
`
`lens CL4OJ 1991
`
`Shaker GJ Ruffini
`Arora
`et al Soluble
`disks
`collagen
`treatment of dry eye syndromes CLAO 1989 15298-304
`JV et alEffect of collagen
`Ruffini
`shields on corneal
`.11 Aquavella
`epithelization following penetrating
`keratoplasty Ophthahnic
`
`for
`
`the
`
`Surg
`
`1989
`Phinney RB Schwartz SD Lee DA et alCollagen-shield drug delivery
`and vancomycin Arch Ophthalmol 1988 1061599-1604
`of gentamicin
`SR Rootman DS Hill JM et al Collagen shield drug
`Unterman
`Cataract Refract Surg 1988
`deliverytherapeutic concentrations
`of tobramycin in the rabbit cornea
`and aqueous humor
`Hwang DG Stem WH Hwang PH et al Collagen shield enhancement
`of topical dexamethasone penetration.Arch Ophthalnol 1989 1071375-
`1380
`
`From the Faculty of Medicine Department of Ophthalmology University of
`Ankara Ankara Turkey
`This work was presented in part at
`Rome Italy
`requests to Doc Dr Ayfer Kanpolat
`and reprint
`Correspondence
`06700 GOP Ankara Turkey
`Bogaz sok 24/10
`Accepted for publication Novenber 23 1993
`
`the 22nd ECLSO Congress 1992
`
`Contact Lenses which appeared in our July 1993 issue CLAO 1993 failed to indicate
`
`ERRATUM The article Deposition of Ciprofloxacin Prednisolone Phosphate and Prednisolone Acetate in SeeQuence
`Disposable
`legal name change for one of
`Goyal MD The editors apologize for this error
`
`the articles co-authors The correct spelling of the second authors name is Ameet
`
`the CLAO journal
`
`April 1994
`
`Volume 20 Number
`
`122
`
`FAMY CARE - EXHIBIT 1018-0004