CORNEA/EXTERNAL DISEASE
`
`Penetration of Cyclosporin A into the
`Rabbit Cornea and Aqueous Humor
`after Topical Drop and Collagen
`Shield Administration
`
`Ayfer Kanpolat, M D
`
`Figen Batioglu, MD Merih Yilmaz, MD
`
`Fatma Akbas, MD
`
`We used commercially available 12-hour collagen shields to deliver
`cyclosporin A (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 6 mg of CsA in castor oil and the second group
`(four eyes) received 6
`mg of CsA
`in olive
`oil applied
`
`as to
`rabbit eyes within 12 hours. In the third group (four eyes) 12-hour
`rabbit
`collagen shields
`soaked
`
`in 6 mg CsA in olive oil were applied
`to
`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
`measured by radioimmunoassay. After the total dose of 6 mg CsA,
`percentage penetration was measured as follows: CsA castor oil—
`0.51 % in aqueous and 20.75% in cornea; CsA olive oil—0.17% in aqueous
`and 11.13% in cornea; and with collagen shields—0.44% in aqueous and
`11.84% in cornea. These
`results
`show
`that
`
`the 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
`equal to the castor oil drops. Collagen shields may be useful as an ocular
`delivery system
`for CsA.
`
`CsA
`
`topical
`
`levels
`
`of
`
`nearly
`
`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
`keratotomy and keratorefractive
`surgery,
`to
`treat
`sions, and as an alternative therapy for dry eyes.12 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
`demonstrate that delivery of gentamicin,4 vancomycin,4
`
`tobramycin,5 dexamethasone,6 amphotericin B,7 heparin,8 and
`cyclosporin A9 to the eye are increased.
`Cyclosporin A (CsA) is a cyclic polypeptide of fungal
`
`origin. First reported to have immunosuppressive properties by
`Borel in 1976,10 it has dramatically
`improved
`the
`prognosis
`after
`radial
`solid organ transplantation because of its low myelotoxicity and
`corneal
`abra­
`suppression of specific T
`lymphocyte
`function.
`has been used to suppress rejection after kidney transplantation,
`as well as bone marrow, heart, and liver transplantations.10 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 Grave's ophthalmopathy,10 corneal
`peripheral melting syndrome,11 Behcet's disease,10, and Sjogren's
`syndrome. Nussenblatt and
`
`colleagues10 suggested that topical
`
`Systemic
`
`for
`
`Contact
`Copyright 1994
`All rights
`reserved.
`
`
`
`Lens Association
`
`the CLAO
`of Ophthalmologists
`
`journal • April 1994 • Volume 20, Number 2
`119
`
`TEVA - EXHIBIT 1018
`
`

`
`therapy was effective only if the serum CsA levels entered the
`therapeutic range of 50 to 300 ng/mL.
`Delivery of therapeutic concentrations of CsA to the
`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 rejections,l2'13 Mooren' s
`ulcer, noninfiltrative marginal keratolysis,14 and herpetic stro­
`mal keratitis.15 Recently, investigations of CsA levels in the eye
`and various tissues after topical and systemic administrations
`have been made. Mosteller and coworkers16 investigated the
`systemic and ocular absorption of topically applied 10%
`cyclosporine ointment. Ben Ezra and colleagues17 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
`of commercially available collagen shields and CsA prepara­
`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.
`
`Materials and methods
`Six New Zealand white rabbits, male and female, 2,038 to
`3,220 g were divided into three groups.
`Eye drops: In group 1, the eye drops consisted of a
`commercially available intravenous form of CsA (castor oil) in
`a concentration of 50 mg CsA/mL (0.5 mg CsA/10|!L;
`Sandimmune, Sandoz Pharmaceuticals). In group 2, the eye
`drops consisted of the oral form of CsA (olive oil) in a concen­
`tration of 100 mg CsA/mL (1 mg CsA/10 |aL; Sandimmune,
`Sandoz Pharmaceuticals). Over 12 hours, a total of 6 mg CsA
`was administered as topical drops with a micropipette.
`Collagen shields:Four commercially available collage
`shields were used (12 hours [Bio-cor; Bausch & Lomb
`Pharmaceutics, Inc., Clearwater, FL]). For this purpose, 60 |iL
`(1 mg CsA/10 (iL) 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 2 hours,
`the total dose of 6 mg CsA was absorbed by the collagen shields.
`Experimental design:The six rabbits were divided into
`three groups. In group 1, four eyes of two rabbits received CsA
`castor oil applied with a micropipette (10 |iL [Socorex; Switzer­
`land]) at one drop (10 p.L) per hour for 12 doses for a total of 6
`mg Cs A. (Every 10 jxL drop contained 0.5 mg Cs A [ 12 x 0.5 mg
`= 6 mg CsA].) In the second group, four eyes of two rabbits
`
`TABLE I Cyclosporine concentrations in aqueous humor (ng/mL)
`Mean
`SD
`Range
`
`CsA, castor oil
`CsA, olive oil'
`Collagen shield
`
`30.625
`10.00
`26.475
`
`8.531
`0
`13.192
`
`+22.8 - +38.2
`0
`+14.7-+42.8
`
`'CsA, olive oil < 10 ng.
`SD = standard deviation
`
`received CsA olive oil applied to the eyes with a micropipette,
`one drop every 2 hours for 12 hours (six times). They also
`received a total of 6 mg CsA (every 10 |J.L drop contained 1 mg
`CsA [6 x 1 mg = 6.0 mg CsA]). In both groups, the total dose of
`CsA (6 mg) was equal to the amount of CsA delivered by a single
`collagen shield.
`Four eyes of the two rabbits in the third group were treated
`with CsA olive oil soaked collagen shields. Rabbits were
`anesthetized by intramuscular injection of 0.5 mL 2% xylazine
`hydrochloride (Rompun; Bayer) and 1 mLketamine 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.
`Twelve hours after application of either the drops or the
`collagen shields, the rabbits were killed with sodium pentobarbital
`injections given intravenously. The eight eyes to which the
`topical eye drops had been administered were rinsed with sterile
`saline. Aqueous humor was collected with a sterile 25 gauge
`needle attached to a 2 mL syringe. The anterior chamber was
`entered 1 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 a razor blade and trans­
`ferred to a 1 mL volume of methanol. They were left at 40C
`overnight and centrifuged at 2,000 revolutions per minute
`for 10 minutes.
`When the four eyes to which the collagen shields were
`applied were opened, no shields were found on the corneas.
`Small fragments of collagen, combined with mucus, were
`lodged in the fomices. 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 (RI A). The RIA
`laboratory used CYCLO Trac SP 125/RIA (INCSTAR Corp.,
`Stillwater, MN) and a gamma scintillation counter.
`
`Results
`In group 1, CsA castor oil treated eyes, the CsA concen­
`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 I); the
`concentrations in the cornea ranged between 865 ng/mL and
`1,622 ng/mL, with an average of 1,245 ng/mL (Table II). In
`group 2, the CsA concentrations in aqueous humor of eyes that
`received topical CsA olive oil drops were below levels that
`could be measured with RIA (< 10 ng/mL; Table I). The values
`obtained in the cornea ranged between 472 ng/mL and 855 ng/
`
`TABLE II Cyclosporine concentrations in cornea (ng/mL)
`Mean
`SD
`Range
`
`CsA, castor oil
`CsA, olive oil
`Collagen shield
`
`1245
`667.8
`710.5
`
`426.792
`157.559
`129.935
`
`+865-+1622
`+472 - +855
`+589 - +857
`
`the CLAO journal • April 1994 • Volume 20, Number 2
`120
`
`

`
`^ CsA (ng/ml)
`
`40 -
`
`30 '
`
`20 -
`
`10 -
`
`0
`0
`
`22^
`
`14.7
`10
`
`1
`
`23.J
`16.J
`
`10
`
`2
`
`36.2
`ai.i
`
`n>-
`
`3
`
`Eyes
`
`42.8
`
`10
`
`6
`
`%
`
`26
`
`2 0 • •
`
`1 8 - -
`
`1 0 - -
`
`0^=2;
`
`0 6
`
`20.76
`
`1113
`
`ii*b4
`
`I a i
`
`C0A (Castor oil)
`
`CsA (Olive oil)
`
`Collagen Shield
`
`Series 3
`Series 2
`Series 1
`CsAlCaator oil) CsA(Olive oil) Collagen Shield
`Olive olK<10 ng)
`Figure 1 Aqueous humor levels.
`
`Series 1
`Aqueous humor
`
`(•HI Series Z
`Cornea
`
`Figure 3 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
`1) and the cornea (Figure 2) were statistically higher in the group
`that received topically applied castor oil CsA (aqueous humor,
`P < 0.017; comea, P < 0.035, paired t test).
`In group 3, CsA applied by collagen shields, aqueous
`humor values ranged between 14.7 ng/mL and 42.8 ng/mL, with
`an average of 26.5 ng/mL (Table I), and the values of the comea
`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
`onstrated between these two groups (P > 0.316).
`When the results obtained by using collagen shields were
`compared with the results of topically applied CsA castor oil
`drops, the concentrations were nearly equal in aqueous humor
`(Figure 1). According to paired f-test, no significant difference
`could be demonstrated (P > 0.25), but the corneal concentrations
`were higher with topically applied CsA castor oil drops (Figure
`2), and this was significant {P <0.038).
`Figure 3 shows the penetration levels of CsA in aqueous
`
`CsA (ng/ml)
`
`2000
`
`1600 -
`
`1000 -
`
`600 -
`
`686
`
`680
`A f t
`
`0
`
`1
`
`BB
`Bjia
`6U
`
`2
`
`1807
`
`1682
`
`867
`
`3
`
`4
`
`6
`
`Eye
`
`Series 3
`" S e r i e s 2
`Series 1
`CaA(Caator oil) C8A<Ollve oil) Collagen Shield
`
`Figure 2 Comea levels.
`
`humor and cornea with topical castor oil, olive oil drops, and
`olive oil soaked collagen shields as a percentage of the total dose
`of 6 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 comea than when CsA olive
`oil drops were used. An additional finding is that a collagen
`shield soaked in CsA olive oil form is an equally effective means
`of delivering CsA to the comea and aqueous humor as topically
`applied castor oil drops.
`The topical application of CsA to the eye has been studied
`in an attempt to reduce the risk of systemic toxicity while
`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
`quite impermeable to the drug because it is hydrophilic. By using
`a 2% solution of CsA (castor oil), Wiederholdt and colleagues'8
`evaluated its penetration into the different layers of rabbit eyes.
`Following a single dose of 10 jiL of 2% CsA, a concentration of
`900 to 1,400 ng/mL was detected in the comea at 6 hours, of
`which 67% was found in the epithelium, 25% in the stroma, and
`8% in the endothelium. Concentrations in the remaining tissues
`(lens, vitreous, uvea, retina) were < 45 ng/mL.18 Kaswan19
`measured the absorption of topically administered 1% CsA in
`olive oil. After the total dose of 0.84 mg was given, tissue levels
`(comea, anterior and posterior sclera) greater than 50 ng/mL
`were achieved within 1 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.
`After a total dose of 6 mg of topically applied CsA castor oil,
`0.51% was found in aqueous humor and 20.75% in the comea,
`compared with 0.17% in aqueous and 11.13% in the comea 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 a means of
`delivering CsA to the comea and aqueous humor in rabbit eyes.
`
`the CLAO journal • April 1994 • Volume 20, Number 2
`121
`
`

`
`They found that both the corneal and aqueous humor concentra­
`tions 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.
`Commercially available collagen shields can be utilized
`as a means of delivery system for CsA and in addition to
`commercially available oral and intravenous forms of CsA
`dispensed as topical drops. Our study is the first to show the
`efficacy of using commercially available collagen shields as a
`means of delivering CsA.
`The application of CsA in a collagen shield delivery
`system is easy to apply to patients with diseases such as
`Mooren's ulcer, corneal peripheral keratolysis, herpetic stromal
`keratitis, and corneal allograft reactions, thus minimizing sys­
`temic toxicity and maximizing local penetration.
`
`Acknowledgment
`Ms. Adalet YUziibenli for help in the research laboratory and Ms. F. GUI
`Cakmak for statistical consultation. Collagen shields were supplied by Bausch
`and Lomb Pharmaceuticals.
`
`References
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`4. Phinney RB, Schwartz SD, Lee DA, et al:Collagen-shield drug delivery
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`7. Schwartz SD, Harrison, SA, Engstrom RE Jr., et al: Collagen shield
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`11. Kruit PJ: Cyciosporine A treatment in four cases with corneal melting
`syndrome. Transplant Proc 1988;20(Suppl. 4): 170-172.
`12. Hunter PA, Gamer A, Wilhelmus KR, et al:Comeal graft rejections new
`rabbit model and cyclosporin A. Br J Ophthalmol 1982;66:292-302.
`13. Foets B, Missoten L, Wandenveren P, et al: Prolonged survival of
`allogenic corneal grafts in rabbits treated with topically applied cyclosporin
`A:systemic absorption and local immunosuppressive effect. Br J
`Ophthalmol 1985;69:600-603.
`14. Nussenblatt RB, Palestine AG:Cyclosporine immunology, pharmacol­
`ogy and therapeutic uses. Surv Ophthalmol 1986;31:159-169.
`15. Colling J, Chastel C, Bonissent JF: Keratitis herpetiques
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`1986;15:1245.
`16. Mosteller MW, Gebhardt BM, et akPenetration of topical cyciosporine
`into the rabbit cornea, aqueous humor and serum. Arch Ophthalmol
`1985;103:101-102.
`17. Ben Ezra D, Maftzir G: Ocular penetration of cyclosporin A in the rat eye.
`Arch Ophthalmol 1990;108:584-587.
`18. Wiederholdt M, Kossendrup D, Schulz, et al:Pharmacokinetics of topical
`cyclosporin A in the rabbit eye. Invest Ophthalmol Vis Sci 1986;27:519-
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`
`From the Faculty of Medicine, Department of Ophthalmology, University of
`Ankara, Ankara, Turkey.
`This work was presented in part at the 22nd ECLSO Congress, 1992,
`Rome, Italy.
`Correspondence and reprint requests to: Doc. Dr. Ayfer Kanpolat,
`Bogaz sok. 24/10,06700 GOP, Ankara, Turkey.
`Accepted for publication November 23, 1993.
`
`ERRATUM The article "Deposition of Ciprofloxacin, Prednisolone Phosphate, and Prednisolone Acetate in SeeQuence Disposable
`Contact Lenses," which appeared in our July 1993 issue {CLAO J 1993;19:166-168), failed to indicate a legal name change for one of
`the article's co-authors. The correct spelling of the second author's name is Ameet K. Goyal, MD. The editors apologize for this error.
`
`the CLAO journal • April 1994 • Volume 20, Number 2
`122