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`1
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`ALL 2004
`MYLAN PHARMACEUTICALS V. ALLERGAN
`IPR2016-01131
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`1
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`ALL 2004
`MYLAN PHARMACEUTICALS V. ALLERGAN
`IPR2016-01131
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`

`

`The Effect on the Cornea of Various Vehicles
`for Cyclosporin Eye Drops
`
`Rodolfo M. Alba, Jr. Atsushi Kanai, Toshiyuki Takano,
`Chihiro Kobayashi and Akira Nakajima
`
`Department of Ophthalmology, Juntendo University, School of Medicine
`
`Kozo Kurihara
`
`Product Development Laboratories, Sankyo Co.
`
`Masaharu Fukami
`
`Biological Research Laboratories, Sankyo Co.
`
`Wetested 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 peanutoil, 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 corneasat frequencies of 10x (every
`30 min.) and 5x. Light microscopy revealed that in the MCT, @-CD and peanutoil
`groups, corneal thickness approximated that in the controls.
`In the next phase, done
`on rabbit corneas, weinstilled MCT (with and without CYA), a-CD and peanutoil 10x
`(every 30 min.). Normal saline wasapplied to the control eye. The Draizetest, 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 CYAlevels in the cornea and aq. humorindicated 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)
`
`a
`
`
`
`RBIS113 BRARABI-1-3 WR KPEPORAERS Sit
`Reprint requests to: Atsushi Kanai. M.D. Dept. of Ophthalmol., Juntendo Univ, School of Med.
`3-1-3 Hongo, Bunkyo-ku. Tokyo 113, Japan
`
`2
`
`

`

`Vol,
`
`40, No.
`
`a
`
`903
`
`I.
`
`Introduction
`
`[mmunosuppressive drugs have contri-
`buted greatly to the success of corneal trans-
`plantation.
`Ideally,
`they should be able
`to traverse the cronea in sufficient amounts
`
`to prevent rejection from taking place and
`the dose should be low enough to avoid
`occurrenceof toxic side-effects.
`Ciclosporin, a cyclic undecapeptide of
`fungal origin, has been discovered to be a
`useful immunosuppressive compoundandis
`used widely in organ transplantation.
`In
`previous studies,'~*’ 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 studyis to investigate
`the effect on the cornea of various vehicles
`
`for Ciclosproin (CYA),
`
`Il. Materiais and Method
`
`We performed preliminary studies on
`rats by testing several 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
`(a-CD). We applied normal saline on the
`control eye. MCT Emulsion consists of
`glycine,
`lecithin, ethylene oxidepropylene
`oxide and NaCl. The concentration of
`CYAin each eye drop was: 1% in Peanut
`oil, Palm and MCT; 0.1% in Polyoxyethy-
`lene castor oil and 0.08% in a-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: MCT (with and without
`CYA), @-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 10x (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-
`dioimmunoassay. 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 +
`solutions of a-CD having different concen-
`trations following the previous procedure.
`The drugs were administered 4x (every 2
`hrs.), The
`corneas were examined by
`scanning
`electron microscopy. Corneal
`and aq. humor concentration of CYA was
`also determined by radioimmunoassay.
`
`Ill. Results
`
`Comparing the thickness of the rat cor-
`neas where the eye drops were admini-
`stered 10x and 5x 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 a-CD pro-
`duced the greatest amount of edema com-
`pared to the others (Table-1). This was
`(P<0.01).
`The
`statistically
`significant
`Draize test which is used to evaluate ocular
`lesions by assigning relative values to ocular
`
`3
`
`

`

` 0.08% CYA+ a-CD
`
`Fig.
`
`1
`
`Light micrograph of rat cornea after admi-
`nistrationof various vehicles with Ciclospo-
`rin. Frequency of administration was 10>
`(every 30 min.).
`
`0.1% CYA+Castor Oil
`
`
`
`Folia Ophthalmol. Jpn. 1989
`
`0.08%. CYA+«-CD}~
`
`~~
`
`0.1%. CYA+ Castor Oil
`
`Fig, 2. Light micrograph ofrat cornea after admi-
`nistration ofvarious vehicles with Ciclospo-
`rin. Frequencyof administration was5»
`
`Table 1 Pachymethry of rabbit cornea before and after administration of Ciclosporin
`with various vehicles
`Frequency : 10x (every 30 min.)
`
`drug
`
`;
`N
`
`before
`(mm)
`
`after
`(mm)
`
`difference a
`(mm)
`significance
`
`SN
`0,01—0,01
`0.334+0,01
`0.339+0.01
`3
`1% CYA+MCT
`p<0.01
`0.01-0,01
`0.366+0,05
`0,355+0,03
`3
`0.08% CYA+a-CD
`NS
`0.01 +0,03
`0.348+0,.03
`0,355+0.02
`3
`1% CYA+peanut oil
`
`
`
`
`8 0.358+0.02, 0.351=0,01(saline soln.)Control 0,01+0.01me
`NS : Not significant
`
`findings demonstrated that «-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.
`in the cor-
`We meausred the CYA level
`nea and aq. humor by means ofradioim-
`munoassay (Fig. 3).
`It revealed that
`the
`vehicle with the highest penetrating ability
`was a-CD andthe least was Peanut oil.
`The findings on gross examination was
`further elucidated with light microscopy.
`Whenwe compared the corneal thickness of
`
`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 numberof 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 wetested
`4 different concentrations of a-CD and
`they were 80, 40,20 and 10 mg./ml.
`com-
`
`4
`
`

`

`Vol.
`
`40, No. 5
`
`905
`
`bined 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
`
`singificant. The Draize test revealed that
`80 and 40 mg./ml. of a-CD was minimally
`irritating, 20 mg./ml. was practically non-
`
`Frequency : 10x (Every 30 min.)
`Cornea
`i
`Ag. humor
`(n=3)
`(n=3)
`
`fg gr)
`
`1.0% CYA+Peanut Of==—*1.0% CYA-+MCT
`
`
`\
`
`HFACYA+
`Peanut
`il
`
`O.OBCYA ICYA
`+ C0 +MCT
`
`OO8.CYA MCYA Vcr +
`+00 +MCT
`Peanut
`oil
`Rabbits sacrificed 30min. after last eye drop.
`Fig. 3. Pachymetry of rabbit cornea before and
`after administration of various vehicles with
`Ciclosporin.
`
`inevarh
`5.000 0
`See4
`
`
`
`2
`Fig. 4
`
`;
`CYAlevel determination in the cornea and
`aq. humorafter administration of various
`vehicles with Ciclosporin.
`
`Control {Saline Sol'n.)
`
`
`
`Peanut Oil
`1.0" CYA+
`1.0% CYA+ MCT
`
`,
`he
`ea
`tage
`
`
`
`Fig. 5 Light micrograph of rabbit cornea after administration of various
`vehicles with Ciclosporin. Frequency of administration was 10
`(every 30 min.).
`
`5
`
`

`

`0.08% CYA+a-CD ee
`ag ee
`
`fe-£0 :80mg/me /
`CYA: 8.75mg/ml
`
`1989
`
`E -CO: JOmp/mi
`
`be
`
` Folia Ophthalmol. Jpn,
`CYA: 0.03mg/ml
`
`Fig. 6 TEMofrabbit cornea after administration
`of various vehicles with Ciclosporin. Fre-
`quency of administration was 10% (every
`30min.). Samples of 0.08% CYA+a-CD
`showed diminution in the number of micr-
`ovillae on the epithelium (arrow).
`
`‘irritating and 10 mg./ml. was completely
`non-irritating. Scanning electron micro-
`scopy demonstrated few epithelial erosions
`associated with 80 and 40 mg./ml. of a@-
`CD (Fig. 7).
`We measured the concentration of CYA
`in the cornea and aq, humor by radioim-
`munoassay. The sample associated with
`80 mg./ml. of a-CD had the highest
`level
`and the sample with 10 mg./ml. of a-GD
`the lowest (Fig. 8). Measuring the CYA
`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
`
`Fig. 7
`
`SEMofrabbit cornea after administration
`of various vehicles with Ciclosporin. Fre-
`quency of administration-was 10* (every
`30 min.). Sample with 0.0896 CYA+a@-CD
`had moreepithelial craters (arrow) compa-
`red to the others.
`
`thereby negating it’s beneficial
`divisions
`effects. With the advent of newer drugs
`such as Ciclosporin, the success rate has in-
`creased. These new immunosuppressives
`are capable of selectively affecting certain
`subpopulation of immunocompetent cells
`thus
`preventing
`rejection
`from taking
`place.®
`The different routes of drug administra-
`tion have been previously discussed” and
`the topical routeis 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 efficacy of administrating Ciclosporin
`topically in preventing rejection from taking
`place."
`The objectives of our investigation are
`twofold. The first is to search for a suita-
`
`6
`
`

`

`Vol.
`
`40, No. 5
`
`Frequency: Every 2 hrs.
`
`(4x) (n=3)
`
`Cornea 1280+ 269
`
`0
`
`0.75 0.25 0.09 0.03 mg/me
`CYA
`aCD 80
`40
`20
`10 ng/me
`~ Rabbits sacrificed 30min. after last eye drop
`CYA concentration is maximum amount for
`solubility in «-CD
`
`Fig. 8
`
`SEM of rabbit cornea after administration
`of 4 different concentrations of a@-CD,
` Fr-
`equency of administration was 4% (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 new 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. Wetested several
`lipophilic vehicles on rat corneas in the
`initial phase of our study. These vehicles
`were: Medium Chain Length Triglyceride
`Emulsion
`(MCT), Alpha
`cyclo-dextrin
`(a-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 servedas control,
`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
`
`ficant corneal edema associated with Polyo-
`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:
`MCT (with and without CYA), e-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 a-GD caused a great amount ofcor-
`neal toxicity such as edema,
`loss of epithe-
`lial microvilli and epithelial erosion.
`‘The
`Draize test demonstrated that a-CD was
`notirritating 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.”~'’ 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 @-CD that would give the
`Jeast
`amount of corneal
`toxicity. We
`evaluated 4 different
`concentrations of
`
`a-CD following the previous procedure and
`criteria, Evaluation ofthe results suggests
`that 20 mg./ml. of a-CGD+0.09 mg./ml. of
`CYA is the acceptable concentration.
`In
`addition,
`the value we obtained is within
`the maintainance level of CYA for
`renal
`transplant
`patients
`(500-1500 ng. /gr.).’”
`This study, being preliminary in nature,
`sought
`to investigate the effects of various
`vehicles with Ciclosporin on the cornea.
`It still remains to be resolved whether this
`new combination would be effective as a
`
`topical drug to suppress rejection of corneal
`grafts,
`
`7
`
`

`

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