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`ISSN 0015-5667
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`Nippon ganka kiyu = Fulin
`wphthalmolngica Japonica
`Bulletin of Japanese
`nphthalmolugy
`BML Stacks
`
`UNIVERSITY OF CQLIFURNIQ,
`- LIBRQRIES
`-,1
`
`1
`
`ALL 2004
`ARGENTUM PHARMACEUTICALS V. ALLERGAN
`IPR2016-01232
`
`

`
`The Eifect on the Cornea of Various Vehicles
`for Cyclosporin Eye Drops
`
`Rodolfo_M. Alba, Jr. Atsushi Kanai, Toshiyuki Takano,
`Chihiro Kobayashi and Akita Nakajima
`
`Department of Ophthalmology. Juntendo University. School of Medicine
`
`Kozo Kurihara
`
`Product Development Laboratories. Sankyo Co.
`
`Masaharu Fukami
`
`Biological Research Laboratories. Sankyo Co.
`
` ____
`
`We tested several solvents, possible vehicles for Cyclosporin, (CYA) as to which
`had the least corneal toxicity. They were: peanut oil, palm oil. polyoxyethylene castor
`oil, medium chain-length triglyceride emulsion (MCT) and alpha cyclo-dextrin (a-
`CD). The concentration of CYA in each vehicle was: 1% in peanut oil, palm oil and
`MCT; 0.1% in polyoxyethylene castor oil and 0.08% in a-CD. The drugs and normal
`saline, which served as control, were instilled to rat corneas at frequencies of 10x (every
`30 min.) and 5x. Light microscopy revealed that in the MCT, a-CD and peanut oil
`groups, corneal thickness approximated that in the controls.
`In the next phase. done
`on rabbit corneas, we instilled MCT (with and without CYA), a—CD and peanut oil 10x
`(every 30 min.). Normal saline was applied to the control eye. The Draize test, ultra-
`sonic pachymetry, light and electron microscopic examination indicated that, compared
`to the other vehicles, a-CD exhibited significant corneal toxicity as evidenced by edema,
`diminution of microvilli on the epithelium and epithelial craters. Radioimmunoassay
`of CYA levels in the cornea and aq. humor indicated that a-CD afforded the greatest
`CYA penetration of the cornea. We then tested 4- different concentrations of a-CD to
`determine the least toxic concentration. The concentrations were: 80, 40, 20 and
`10 mg./ml. of a-CD combined with 0.75, 0.25, 0.09 and 0.03 mg./ml. of CYA. They
`were applied to rabbit corneas 4x (every 2 hrs.). Histological and RIA studies indicate
`that 40.0 mg./ml a-CD with 0.25 mg./ml. CYA is an acceptable concentration.
`(Folia Ophthalmol. Jpn. 40: 902-908. 1989)
`
`
`¥$
`swiairaslzse : 113 sea.-rs;zar.7.<_:i:xes—rié' shirexscussatet-wag err
`Reprint requests to: Atsushi Kanar. M.D. Dept. of 0phthalrno|., Juntendo Univ. School of Med.
`3-1-3 Hongo, Bunkyo-ku. Tokyo 113. Japan
`
`2
`
`

`
`I.
`
`Introduction
`
`lmmunosuppressive drugs have contri-
`buted greatly to the success of corneal trans-
`
`they should be able
`Ideally,
`plantation.
`to traverse the cronea in sufficient amounts
`
`to prevent rejection from taking place and
`the dose should be low enough to avoid
`occurrence of toxic side-efl'ects.
`
`Ciclosporin, a cyclic undecapeptide of
`fungal origin, has been discovered to be a
`useful immunosuppressive compound and is
`used widely in organ transplantation.
`In
`previous studies,"5’ it was shown that
`it
`can prolong survival of transplanted tissues
`by suppressing the function of T-lympho-
`cytes.
`It’s application in corneal
`trans-
`plantation has been rather limited due to
`it’s insolubility in water.
`The purpose of this study is to investigate
`the effect on the cornea of various vehicles
`
`for Ciclosproin (CYA).
`
`II. Materiais and Method
`
`We pcrlbrmed preliminary studies on
`rats by tcstingseveral solvents as possible
`vehicle for Ciclosporin. They were: Pea-
`nut oil, Palm oil, Polyoxyethylene Castor
`oil, Medium Chain Length Triglyceride
`Emulsion (MCT) and Alpha cyclo-dextrin
`(tr-CD). We applied normal saline on the
`control eye. MOT Emulsion consists of
`glycine,
`lecithin, ethylene oxidepropylene
`oxide and ';\'aCl. The concentration of
`
`l°6 in Peanut
`(IYA in each eye drop was:
`oil, Palm and MCT ; O.l?6 in Polyoxyethy—
`lene castor oil and 0.08% in ct-CD. These
`
`concentrations represented the maximum
`dose of CYA which the corneas did not
`
`opacity. The medications and normal sa-
`line were administered 10x (every 30 min.)
`and 5x a day. The rats were sacrificed
`with an overdose of anesthesia. After the
`
`corneas were harvested, they were examined
`under the light microscope and their thick-
`
`ness compared.
`Based on the results of the previous study,
`we did further experiments on rabbits. We
`tested 3 vehicles: M'CT (with and without
`CYA), ct-CD and Peanut oil. We omitted
`Polyoxyethylene castor oil and Palm oil.
`Normal saline was applied on the control
`eye. The drugs and normal saline were
`instilled 10::
`(every 30 min.). Ultrasonic
`pachymetry and the Draize test was per-
`formed before and after application of the
`eye drops. CYA concentration in the cor-
`nea and aq. humor was measured by ra-
`clioimmunoassay. Thirty minutes
`after
`the last eye drop, the rabbits were sacri-
`ficed with an overdose of anesthesia. After
`
`harvesting the corneas, one half was exa-
`mined under the light microscope and the
`other half with the electron microscope
`(both scanning and transmission).
`With the result of the study we tested 4-
`solutions of ct-CD having different concen-
`trations following the previous procedure.
`The drugs were administered 4.x (every 2
`hrs.l. The corneas were examined by
`scanning electron microscopy. Corneal
`and aq. humor concentration of CYA was
`also determined by radioimmunoassay.
`
`III. Results
`
`Comparing the thickness of the rat cor-
`neas where the eye drops were admini-
`stered lOx and 5:: showed that the conreas
`
`with MCT and Peaunt oil approximated
`the thickness of the control cornea (Fig.
`1,2). Edema was marked in the corneas
`where Palm oil and Polyoxyethylene cas-
`tor oil were administered.
`
`In the rabbit study ,Ultrasonic pachy-
`metry readings indicated that at-CD pro-
`duced the greatest amount of edema com-
`pared to the others (Table-1). This was
`statistically
`significant
`(P-(0.01). The
`Draize test which is used to evaluate ocular
`
`lesions by assigning relative values to ocular
`
`3
`
`

`
`Folia Ophthalmol. Jun. 1989
`
`'
`
`II
`1.09: CI] +Pill MI
`
`Fig. 1 Light micrograph of rat cornea after admi-
`nistration of various vehicles with Cic1ospo-
`rin. Frequency of administration was lO>.'
`(every 30 min.).
`
`‘A.
`
`fl.0l'n l2fl+n-Cl) " " "M"-'1 CVI+Clflll
`
`Fig. 2 Light micrograph of rat cornea after admi-
`nistration of various vehicles with Ciclospo-
`rin. Frequency of admin-istration was 5 :-‘ .
`
`drug
`
`N
`
`Tablel Pachymethry of rabbit cornea before and after administration of Ciclusporin
`with various vehicles
`Frequency 1 10x (every 30 min.)
` ??.—j
`before
`after
`difference
`mm“
`(mm)
`(mm)
`--o.m:o.o1
`0.3El4:0.0l
`0.339:D.0l
`1% CYA4.-MCT
`036610.05
`0.355:0.03
`0.0l:0.0l
`0.08% CYA+ar—CD
`—-o.m:o.o3
`0.34-8i0.03
`0.3551'0.02
`1% CYA-rpeanut oil
`-~o.o1:o.01
`0.35l:0.0l
`0.3581'0.02.
`Control (saline soln.)
`
`NS 3 Not significant
`
`significance
`SN
`p<0.0l
`NS
`
`findings demonstrated that a-CD did not
`casue any irritation and no macroscopic
`change was noted.
`In contrast, minimal
`irritation was evident
`in the eyes where
`MCT (with and without CYA) was admin-
`istered.
`We meausred the CYA level in the cor-
`
`nea and aq. humor by means of radioim-
`munoassay (Fig. 3).
`lt revealed that the
`vehicle with the highest penetrating ability
`was cc—CD and the least was Peanut oil.
`
`The findings on gross examination was
`further elucidated with light microscopy.
`
`the different samples. MCT approximated
`the thickness of the control cornea (Fig. 4).
`With the transmission electron microscope,
`
`the findings were unremarkable except for
`diminution in the number of microvillae on
`
`the epithelium associated with a-CD (Fig.
`5.)
`This was corroborated by the find-
`ings of the scanning electron microscope
`(Fig. 6).
`It showed that compared to the
`others,
`the cornea with a—CD had more
`epithelial craters.
`In the last phase of our study WI: tested
`4 different concentrations of 01-01) and
`
`When we compared the corneal thickness of
`
`they were 80, 40, 20 and 10 mg. ]ml.
`
`com-
`
`4
`
`

`
`Vol.
`
`'10, No. 5
`
`bincd with 0.75, 0.25, 0.09 and 0.03 mg./‘
`ml. of CYA respectively. Ultrasonic pach—
`ymetry indicated that the difference in the
`corneal
`thickness before and after appli-
`cation of the drugs were not statistically
`
`Framnwlnr (Evan aouil.)
`Gama
`;
`An. Inna
`[I=3l
`M
`tII=3|
`
`singificant. The Draize test fcveahgd that
`80 and 40 mg./ml. of ct-CD was minimally
`irritating, 20 mg./‘ml. was practically non-
`
`unrtn MM I',m+
`o-.41! mar
`Peanut
`an
`mum sacrificed Jllmin. after last are he
`
`I-.m+
`Ill‘-fll Mu
`+.-all +m:r Fun:
`oil
`
`Fig. 3 Pachymetry of rabbit cornea before and
`after administration ol'var_ious vehicles with
`Ciclosporin.
`
`Fig. 4 CYA lcvcl determination in the cornea and
`aq. humor after administration of various
`vehicles with Ciclosporin.
`
`Control [Salim Su|‘n.|
`
`Fig. 5 Light micrograph of rabbit cornea after administration of various
`vehicles with Ciclosporin. Frequency of administration was 10 X
`(every 30 min.).
`
`5
`
`

`
`Folia Ophthalmol. Jpn. 1969
`
`mflluflflfl
`Gil :IlIlI'I_
`
`4
`12685 2BFu
`
`Fig. 6 TEM of rabbit cornea after administration
`of various vehicles with Giclosporin. Fre-
`quency of administration was lD>< (every
`30 min.). Samples of 0.03% CYA+at-CD
`showed diminution in the number of micr-
`ovillae on the epithelium (arrow).
`
`Fig. 7 SEM of rabbit cornea after administration
`of various vehicles with Ciclosporin. Fru-
`quency of administration‘-was IOX (every
`30 min.). Sample with 0.08% CYA +41-Cl)
`had more epithelial craters (arrow) compa-
`red to the others.
`
`‘irritating and 10 mg./ml. was completely
`non—irritating. Scanning electron micro-
`scopy demonstrated few epithelial erosions
`associated with 80 and 40mg./ml. of a-
`GD (Fig. 7).
`We measured the concentration of CYA
`
`in the cornea and aq, humor by radioim-
`munoassay. The sample associated with
`80 mg. fml. of a-CD had the highest
`level
`and the sample with 10 mg.,(ml. of a-CD
`the lowest (Fig. 8). Measuring the (JYA
`level
`in the aq. humor gave unreliable
`values and were disregarded, We attri-
`buted this to inadequate procedure.
`
`IV. Discussion
`
`Despite the introduction of immunosup-
`pressives 30 years ago, graft rejection has
`remained a problem in corneal transplan-
`tation. The first generation immunosup-
`pressives indiscriminately blocked all cell
`
`thereby negating it's beneficial
`divisions
`effects. With the advent of newer drugs
`such as Ciclosporin, the success rate has in-
`creased. These new imrnunosuppressives
`are capable of selectively affecting certain
`subpopulation of immunocompetent cells
`rejection
`from
`thus
`preventing
`taking
`' place."
`The different routes of drug administra-
`tion havc been previously discussed" and
`the topical route is still the preferred means
`of instilling the drug in the anterior seg-
`ment of the eye. Aside from minimizing
`the occurrence of systemic reactions,“'°’ it
`encourages patient compliance with the
`drug schedule. Previous studies attest to
`the efiicacy of administrating Ciclosporin
`topically in preventing rejection from taking
`p]ace.'°"‘”
`The objectives of our investigation are
`twofold. The first is to search for a suita-
`
`6
`
`

`
`Vol.
`
`4!), No. 5
`
`Fvmucv: rm am. my tn=:n
`
`1 Zflfli 265
`
`III
`
`I175 I125 I109 0.03 mg/mt
`[Wfi
`B0
`40
`20 W mglmfi
`at-BI]
`-mum uerlllutl aunt. am! In in ten
`CY! cnuclntralul is llllhltlll flllltl 10!
`SOUND! h u-CD
`
`Fig. 8 SEM of rabbit cornea after administration
`of 4 different concentrations ol'a-CD. Fr-
`equency of administration was 42 (every
`2 hrs.).
`
`ble vehicle which when combined with Ci-
`
`closporin would cause the least amount of
`corneal toxicity and the second is to deter-
`mine whether this ncw combination would
`
`be effective in suppressing the rejection
`phenomenon. This paper will address the
`first objective.
`A major obstacle in the application of
`Ciclosporin in ophthalmic preparations is
`it's insolubility in water. We tested several
`lipophilic vehicles on rat corneas in the
`initial phase of our study. These vehicles
`were: Medium Chain Length Triglyceride
`Emulsion MCZT). Alpha
`cyclo—dextrin
`(at-CD), Palm oil, Peaunt oil and Polyoxye-
`thylene Castor oil. The test drugs were ap-
`plied at fixed intervals on one eye of rats
`while normal saline, which served ascontrol,
`was applied on the contralateral eye. After
`the corneas were harvested they were exa-
`mined histologically. The result of the
`examination indicated that there was signi-
`
`907
`
`Ftcant corneal edema associated with Polyc-
`xyethylene Castor oil and Palm oil com-
`pared to the others. Thus,
`in the next
`phase of our study, we limited our
`test
`vehicles
`to three drugs and they were:
`MUT (with and without CYA}, ct-CD and
`Peanut oil. They were instilled on rabbit
`eyes at regular intervals while normal sa-
`line was applied to the control eye. Several
`procedures were also performed in conjun-
`ction with this phase. They were: ultra-
`sonic pachymetry, Draize test, histological
`examination under light and electron mi-
`croscopy and radioimmunoassay (RIA}.
`The results showed that compared to the
`others at-CD caused a great amount of cor-
`neal toxicity such as edema,
`loss of epithe-
`lial microvilli and epithelial erosion. The
`Draize test demonstrated that ct-CD was
`
`not irritating unlike the others.
`We measured the CYA level in the cor-
`
`nea and aq. humor in order to assess the
`ability of the drug to traverse the cornea
`in substantial amounts.‘5‘”’ The assay in-
`dicated that a-CD had the highest level of
`concentration and Peanut oil the least.
`
`With these facts in mind, we embarked
`on the third phase of our investigation and
`that was to determine the acceptable con-
`centration of a-CD that would give the
`least
`amount of corneal
`toxicity. We
`evaluated 4- different
`concentrations
`of‘
`
`at-GD following the previous procedure and
`criteria. Evaluation of the results suggests
`that 20 mg.,lml. of ct-CD +0.09 mg.,lml. of
`CYA is the acceptable concentration.
`In
`addition,
`the value we obtained is within
`the maimainance level of CYA lbr renal
`
`(5100-1500 ng.,lgr.}.“"
`patients
`transplant
`This study, being preliminary in nature,
`sought
`to investigate the efiects of various
`vehicles with Cliclosporin on the cornea.
`It still remains to be resolved whether this
`new combination would be eliective as a
`
`topical drug to suppress rejection of corneal
`grafts.
`
`7
`
`

`
`References
`
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`8!.
`'Wiederhnlt, M.: Loitalc Be-
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`'
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`
`8