Local Cyclosporine Therapy for Experimental
`
`Autoimmune Uveitis in Rats
`
`Robert B. Nussenblatt, MD; William J. Dinning, FRCS, MRCP; Leslie S. Fujikawa, MD;
`Chi-Chan Chan, MD; Alan G. Palestine, MD
`
`0 The use of locally applied cyclospo-
`rine was investigated in the retinal S-
`antigen-induced experimental
`autoim-
`mune uveitis (EAU) model in Lewis rats. A
`2% cyclosporine solution applied topical-
`ly four times a day for 14 days effectively
`prevented the expression of EAU. This
`treatment. however, produced circulating
`cyclosporine levels in the therapeutic
`range. Lower concentrations of cyclospo-
`rine applied topically did not produce
`therapeutic levels and were not capable
`ol reliably preventing disease. lntraocular
`levels of cyclosporine, measured by
`radioimmunoassay, were extremely low
`and outside the accepted therapeutic
`range.
`lntravitreal cyclosporine therapy
`appeared to protect eyes from EAU, with-
`out
`producing
`significant
`circulating
`cyCIosporine levels. These findings show
`that, In Its present form. cyclosporlne In
`oil is not an efficacious topical therapy.
`Therefore, a local cyclosporine prepara-
`tion with enhanced penetration into the
`globe may be a practical approach to
`therapy in the future.
`(Arch Ophthalmol
`1562)
`
`1985;103:1559-
`
`Experimental autoimmune uveitis
`(EAU) can be dependably induced
`by immunization of lower mammals
`and nonhuman primates with the ret-
`inal S antigen."2 This model has many
`characteristics
`similar
`to certain
`human uveitic conditions.2 The immu-
`nopathology, localized to the eye and,
`in some species, to the pineal gland,3
`appears
`to
`be T-cell mediated,‘
`although the susceptibility of various
`Accepted for publication June 10, 1986.
`From the Clinical Branch, National Eye Insti-
`tute, National Institutes of Health, Bethesda,
`Md.
`Reprint requests to National Eye Institute,
`National Institutes of Health, Bldg 10, Room
`10N222, Bethesda, MD 20205 (Dr Nussenblatt).
`
`inbred strains is related to the choroi-
`dal mast cell numbers.5 We have pre-
`viously reported the effective abroga—
`tion of the disease manifestations of
`this animal model with systemically
`administered cyclosporine,6 an agent
`with unique anti—T-cell characteris-
`tics.7 Further, we have also reported
`the beneficial effect of cyclosporine in
`the treatment of severe intermediate
`and posterior endogenous uveitis in
`patients who had failed to respond to
`corticosteroid
`and/or
`cytotoxic
`agents.8 The systemic administration
`of this agent is associated with well-
`recognized adverse reactions, with
`renal toxic eflects and hypertension
`being the most common serious com-
`plications noted in our patients.9 We
`present herein our experience in the
`topical and intracameral administra-
`tion of cyclosporine in Lewis rats, its
`effect on the development of experi-
`mental autoimmune uveitis, and its
`penetration into the eye.
`MATERIALS AND METHODS
`
`Female Lewis rats, each 6 weeks of age
`and weighing approximately 200 g, were
`used for this series of experiments. Ani-
`mals receiving topical and systemic medi-
`cations were immunized in both hind foot
`pads with a total of 50 ug of bovine S
`antigen, prepared as described elsewhere,”
`mixed with an equal portion of complete
`Freund's adjuvant augmented with H37
`Mycobacterium tuberculosis to a concen‘
`tration of 25 mg/mL. Animals receiving
`intracameral cyclosporine therapy were
`immunized with 30 pg of bovine S antigen
`prepared and mixed in the same fashion as
`above.
`
`Cyclosporine Therapy
`Topical Therapy.—A 2% cyclosporine
`solution in olive oil was the stock solution.
`Lower concentrations of the drug were
`obtained by diluting the stock solution
`
`with olive oil. Animals were treated topi-
`cally with 2%, 1%, 0.5%, and 0.2% cyclo-
`sporine. The frequency of administration
`and whether both eyes or only one eye were
`treated are mentioned in Table 1. For the
`determination of cyclosporine penetration
`into the eye, only one drop (50 uL) of the
`concentrations tested was placed onto the
`eye.
`lntracameral Administration—Using the
`stock 2% cyclosporine solution, 40 pL (800
`ng) was injected intravitreally 11 days
`after S—antigen immunization. Other rats
`received intravitreal olive oil. This was
`performed using the operating room
`microscope for visualization and a 30-
`gauge needle.
`re—
`Systemic Administration—Animals
`ceived 10 mg/kg/day of cyclosporine intra-
`muscularly for 14 days. Cyclosporine pene~
`tration into the eye was evaluated by freez-
`ing the tested eyes for immunofiuorescent
`staining or for dissection of
`the inner
`contents in order to obtain cyclosporine
`levels. Cryostat sections, 4 pm thick, of
`eyes were washed with phosphate-buffered
`saline and incubated in a moist chamber
`for 30 minutes at room temperature with
`sheep anticyclosporine antibody (1:2) fol-
`lowed by fluoresceinated rabbit anti-sheep
`IgG (1:10). Washed sections were mounted
`in polyvinyl alcohol resin and viewed with
`an epi—illumination fluorescence micro—
`scope (Leitz). Eyes were frozen after enu-
`cleation at the level of the pars plana. and
`the intraocular contents (lens, vitreous,
`retina, and choroid) were then extruded.
`These were homogenized in 1 mL of 95%
`alcohol, spun at 2,000 rpm for ten minutes
`at 4 °C. The soluble fraction was then
`assayed for its cyclosporine content using a
`radioimmunoassay.
`
`Grading of Ocular
`Inflammatory Disease
`This was performed in a masked fashion
`using a modification of the grading system
`described by Wacker and associates,”
`which was for guinea pigs. In this system
`for the evaluation of posterior segment
`disease in the rat, the grading is as follows:
`
`Arch Ophthalmol—Vol 103. Oct 1985
`
`Cyclosporine for Uveitis—Nussenblali et al
`
`1559
`
`Downloaded From: http://archopht.jamanetwork.com/pdfaccess.ashx?urI=/data/journaIs/ophth/l7965/ by a Infotrieve Inc User on 06/19/2017
`1
`
`ALL 2077
`MYLAN PHARMACEUTICALS V. ALLERGAN
`|PR2016-01128
`
`1
`
`ALL 2077
`MYLAN PHARMACEUTICALS V. ALLERGAN
`IPR2016-01128
`
`

`

`Initiation and
`Duration of Treatment
`After S-Antigen
`Immunization
`
`Treatment Schedule
`and % of Cyclosporlne
`Solution
`
`" Tablé‘iLtV.—C‘yclossporineTherapy
`
`EAU‘
`
`‘
`
`Statistical
`Significancet
`
`e ililo .Traatmont
`0 I 8 (2 .95)§
`i can: em frosted‘iflthm Injection
`8/8 (0)
`‘ 7.6mm) #00 Treated-With Drops
`
`7/3 (1)
`
`4/6 (1.6)
`
`4/8 (2.75)
`4/8 (1.75)
`0/6 (2.9)
`
`olive oil
`
`None (control)
`
`V
`
`‘
`
`(Untreated
`
`L
`
`i
`
`_.
`'
`._‘Qr§up B: at} Only Treated Witnsbrops _
`
`.
`.
`t
`4/5 (1)
`4/5 (1‘)
`
`3/4 (2)
`3/4 (2)
`
`1/4 (2.7)
`1/4 (3.3)
`Days 7-14
`0/4 (3.0)
`0/4 (3.4)
`2/4 (2)
`2/4 (2)
`.
`.
`,
`arm 6: Intravitvoal Injection in' on Only '
`,
`.
`.
`4/4 (3.5)
`6/8 (0.75)
`3/3 (2.5)
`
`1 / 8 (2.6)
`
`Vitreous puncture wulh
`
`‘ Abbreviations are as follows: EAU, experimentat autoimmune uveitis; NS. not significant; IM, intramuscular; qd, every day; qid. four times a day; bid, twice a day; OD,
`right eye: 05. left eye; 0U. both eyes.
`fValues given are number of normal eyes/total number of eyes.
`#One-tailed Fisher's exact test performed. For systemic therapy and group A, treatment groups compared with untreated group. For groups B and C. comparisons are
`made between right and left eyes.
`§lnflarnn1atory index (see text).
`
`0, no evidence of inflammatory disease;
`trace (0.5+), architecture of retina grossly
`intact. Areas of focal destruction were 1+,
`focal areas of destruction with marked
`dropout of photoreceptors; 2+, small exu-
`dative retinal detachment with larger
`destruction, mild to moderate number of
`cells in vitreous; 3+, retinal architecture
`beginning to be lost, larger cxudative reti—
`nal detachment, moderate to large number
`of cells in vitreous; and 4+, total destruc—
`tion of retinal architecture.
`RESULTS
`
`The results of topical cyclosporine
`therapy can be seen in Table 1. All
`animals were killed 14 days after
`immunization. Good protection from
`the manifestation of EAU could be
`obtained by treating both eyes of rats
`four times a day for 14 days with a.
`topical solution containing 20 mg/mL
`of cyclosporine beginning on the day
`of immunization. Serum samples tak-
`en from these rats four hours after
`topical cyclosporine instillation, how-
`ever, dcmonstrated high circulating
`cyclosporine levels,
`the mean being
`285 ng/mL. A lower concentration of
`the
`topical
`cyclosporine
`solution
`(0.5%) was capable of protecting
`many eyes treated, if begun on the day
`of immunization, while the 2 mg/mL
`(0.2%) solution was less effective and
`a higher
`inflammatory index was
`noted. To further evaluate the poten-
`
`local effect of cyclosporine on
`tial
`EAU, some rats were treated topically
`only in the right eye. Animals receiv-
`ing drops to one eye appeared to have
`only partial protection. The animals
`receiving a 2% solution twice a day
`for 14 days only to the right eye had a
`mean cyclosporine plasma level of 108
`ng/mL. At lower concentrations pro-
`tection was not consistently ob-
`served.
`The efficacy of topical cyclosporine
`therapy appeared to be considerably
`less when begun seven days after S-
`antigen
`immunization
`if
`topical
`cyclosporine
`concentrations were
`those that did not result in consistent-
`ly detectable circulating levels. Intra-
`vitreal injection of 800 pg of cyclospo-
`rine 11 days after S-antigen immuni-
`zation appeared to alter the expres-
`sion of EAU (Table 1)
`in the eye
`receiving the cyclosporine. In a series
`of experiments in which cyclosporine
`was placed only in one eye, the treated
`eye manifested no disease or only
`minimum inflammatory changes with
`retention of the retinal architecture
`(Fig 1,
`left),
`in contrast with the
`untreated eye, where inflammation
`was seen (Fig 1, right). Mean circulat-
`ing plasma cyclosporine levels three
`hours after injection into the vitreous
`were 60 ng/mL, while at four hours
`the levels were low (26 i 8 ng/mL).
`
`Serum cyclosporine levels were not
`detectable at the time of death three
`days after instillation, however, sug-
`gesting that a local therapeutic effect
`had taken place.
`The intraocular contents of cyclo-
`sporine—treated eyes were evaluated
`for the presence of cyclosporine using
`two routes of administration (Table
`2). The topical application of one drop
`of cyclosporine at two concentrations
`led to levels in the intraocular con-
`tents of those eyes that were extreme-
`ly low, indeed at the level approaching
`the sensitivity of the radioimmunoas-
`say. Somewhat higher concentrations
`were noted when one drop of the 20
`mg/mL (2%) solution was used as
`opposed to the 2 mg/mL (0.2%) prepa-
`ration.
`A dose-response curve was obtained
`with the instillation of 80 or 800 pg of
`cyclosporine intracamerally. Poten-
`tially significant
`therapeutic levels
`were still found in the eye several
`days after the injection of the higher
`amount.
`staining of
`Immunofluorescence
`sections of eyes
`receiving topical
`cyclosporine demonstrates a clear
`pattern (Fig 2). The corneal epitheli—
`um appears to stain brightly within a
`few minutes of cyclosporine applica-
`tion. Staining of the internal struc-
`tures of the eye was insignificant.
`
`1560
`
`Arch OphthalmoI—Vol 103, Oct 1985
`
`Cyclosporine for Uveitis—Nussenblall et al
`
`Downloaded From: http://archopht.jamanetwork.com/pdfaccess.ashx?urI=/data/journaIs/ophth/l7965/ by a Infotricvc Inc User on 06/19/2017
`2
`
`2
`
`

`

`f:
`
`
`Fig 1_—Left, Right eye of Lewis rat that received 800 ug of cyclosporine intravitreally 1 1 days after S—antigen immunization. At 14
`days after immunization, retinal architecture is grossly intact. Only occasional inflammatory cells in anterior chamber,
`iris, and
`vitreous are visible. Right, Left eye of same rat as in Fig 1. left. This eye received no therapy. At 14 days, severe anterior and
`posterior inflammatory response is evident, with destruction of retinal architecture (X90).
`
`
`
`
`
`Table 2.—Ftat Vitreous Cyclosporine Leveis Alter Local Administration ,
`Cyclosporine Levels in Vitreous Alter Application,‘
`mg/mL
`
`
`/———A—--—
`Cyclosporlne
`98 hr
`
`1hr
`
`
`
`4 hr
`24 hr
`48 hr
`Administration
`197 7
`
`
`
`Topical
`2% solution
`
`
`33 3
`
`
`0 2% solution
`3
`
`
`390
`ND
`600 pg
`lntravitreal
`
`
`
`
`
`
`'Mean of at least four eyes per group.
`tND indicates not done.
`
`Faint staining of the iris, ciliary body,
`and retina was seen in some sections
`one to four hours after topical instilla—
`tion. No increase in the staining pat-
`tern, however, could be observed with
`time. Of
`interest was the intense
`staining of the posterior sclera.
`COMMENT
`
`We report herein that the effective
`control of EAU can be accomplished
`by the
`topical
`administration of
`cyclosporine in doses adequate to
`produce significant plasma levels. The
`systemic administration of cyclospo-
`rine effectively prevented the mani-
`festations of EAU, even after cells
`
`immunoreactive to the S antigen can
`be demonstrated.“ These animal stud-
`ies and human studies both suggest
`that the efi’erent arm of the immune
`system was particularly affected. The
`question as to whether local adminis-
`tration of cyclosporine would affect
`ocular inflammation raises not only
`practical but also theoretic considera-
`tions.
`Cyclosporine appears to interrupt
`T—cell activation at an early point at a
`state of antigen presentation.’ Dos
`Reis and Shevach,” in an in vitro
`model using guinea pigs, demonstrat-
`ed that cyclosporine appeared to block
`IL-2 production and responsiveness
`
`localization of
`Fig 2.——tmmunotluorescent
`cyclosporine in cornea 30 minutes after topi-
`cal application of 2% solution. Corneal epithe-
`lium stained brightly (X200).
`
`Arch Ophthalmol—Vol 103, Oct 1985
`
`Cyclosporine for Uveitiszussenblatt et al
`
`1561
`
`Downloaded From: http://archopht.jamanetwork.com/pdfaccess.ashx?urI=/data/journaIs/ophth/l7965/ by a Infotrieve Inc User on 06/19/2017
`3
`
`3
`
`

`

`and, depending on the stage of the T
`cell, appeared to directly block the
`induction of IL-2 receptors. The addi-
`tion of exogenous IL-2 was variable in
`its capacity to overcome cyclosporine
`inhibition, this depending on the T—
`cell stimulus as well as the stage of
`the differentiation. Further, Kauf-
`mann and colleagues,” employing
`hybridomas devoid of functional IL-2
`receptors
`in
`an in vitro system,
`obtained
`results
`suggesting
`that
`cyclosporine action on T cells could be
`through the interference of antigen
`binding to antigen receptors, with a
`resultant blockade of the lymphokine
`cascade. Further, it has been shown
`that T cells bearing IL—2 receptors are
`relatively immune from the effects of
`cyclosporine.7
`We surmised that effective control
`of severe ocular inflammation by sys-
`temically administered cyclosporine
`was due to a central effect of the
`agent, but primarily on the efferent
`arm of the immune response. We have
`reported the presence of intraocular T
`cells bearing the TAC (IL-2) recep-
`tor,” a sign of T-cell activation. We
`therefore suggested that in endoge-
`nous uveitis a rather rapid influx of
`immunoreactive cells into the eye was
`occurring, therefore making it possi-
`ble for cyclosporine to rapidly inter-
`rupt
`the cycle of
`immune recruit-
`ment.
`The effectiveness of intracameral
`cyclosporine
`administration would
`suggest that the final activation of the
`recruited T cells may be a local (ocu—
`lar) phenomenon. This could explain
`the profound inflammatory ocular
`response one can observe with little
`evidence of a systemic one in many
`uveitic conditions. Another potential
`mechanism may be
`cyclosporine's
`effect on ocular tissue involved in the
`localization to the eye of the immune
`response. It has now been demon—
`strated that the localized expression
`of Ia on vascular endothelial cells is
`associated with T-cell—mediated dis—
`orders, such as experimental allergic
`encephalomyelitis." Since y-interfer-
`on is the most effective natural stimu-
`lant for Ia expression, it is possible
`that cyclosporine’s prevention of the
`release of lymphokines from T cells
`might alter the continued expression
`of Ia on the ocular vascular endotheli-
`um,
`thereby interrupting the pro-
`posed “homing” mechanism of immu-
`
`noreactive cells to a site of inflamma-
`tion.
`staining
`immunofluorescent
`The
`deserves
`comment. This
`pattern
`that
`cyclosporine
`quickly
`showed
`coated the corneal epithelium. The
`presence of cyclosporine intraocularly
`after
`topical application, however,
`was minimal at best. Weak staining of
`the iris and retina could be seen on
`occasion, but this was not the case for
`all eyes. This observation was sup-
`ported by the use of the radioimmuno—
`assay, which also failed to detect sig-
`nificant intraocular levels of cyclospo-
`rine. We do know from our human
`experience that cyclosporine can be
`detected in the eye after systemic
`administration,” but these observa-
`tions were made in eyes with a nonins
`tact blood-aqueous barrier. It may be
`that the lipophilic structure of cyclo-
`sporine does not permit
`it
`ready
`access into the eye. Our findings sup-
`port those observed by Mosteller and
`colleagues.‘6 They noted that cyclospo-
`rine collected in high concentrations
`in the
`rabbit
`cornea, while
`low
`amounts were measured in the aque-
`ous humor. This lack of penetration
`would explain our inability to reliably
`protect rats from EAU with topical
`cyclosporine therapy.
`As we have noted, topical therapy
`seemed predictably effective only if
`Serum cyclosporine
`levels
`entered
`what
`is considered the therapeutic
`range of 50 to 300 ng/mL. Although
`this observation would support
`the
`notion that the action of cyclosporine
`is central, the subsequent finding that
`intracameral cyclospOrine effectively
`protected the eye in which it was
`injected raises the possibility of a
`peripheral effect as well. The levels in
`the eye receiving cyclosporine indi’
`cated a
`therapeutically acceptable
`range. Therefore,
`locally
`applied
`cyclosporine may potentially be useful
`in certain human uveitic conditions,
`particularly those endogenous disor-
`ders with no systemic associations.
`The need for greater drug penetration
`into the eye, however, still has to be
`addressed. It may be that a change in
`the vehicle, such as to liposomes, may
`permit a higher concentration of
`cyclosporine to enter the eye. The
`development of this methodology will
`permit the testing of many theoretic
`considerations and will also be of
`practical import.
`
`The cyclosporine and radioimmunoassay used
`in this investigation were supplied by Sandoz,
`Ltd, Basel, Switzerland.
`
`References
`
`1. Nussenblatt RB, Gery I, Wacker WB: Exper-
`imental autoimmune uveitis: Cellular immune
`responsiveness.
`Invest Ophthalmol Vis Sci
`1980;19:686-690.
`2. Nussenblatt RB, Kuwabara T, deMonasterio
`FM, et al: S—antigen uveitis in primates: A new
`model
`for human disease. Arr]? Ophthalmol
`1981;99:1090-1092.
`3. Mochizuki M, Nussenblatt RB, Charley J, et
`a]: Involvement of the pineal gland in rats with
`experimental autoimmune uveitis. Invest Oph-
`lemol Vis Sci 1983;24:1333-1338.
`4. Salinas-Carmona MC, Nussenblatt RB,
`Gary 1: Experimental autoimmune uveitis in the
`athymis nude rat. Br J Immunol 1982;12:480-
`484.
`5. Mochizuki M, Kuwabara T, Chan CC, et al: A
`correlation between susceptibility to experimen—
`tal autoimmune uveitis and choroidal mast cells.
`J Immunol 1984;133:1699-1701.
`6. Nussenbiatt RB, Rodrigues MM, Wacker
`WB, et a1; Cyclosporin A: Inhibition of experi-
`mental autoimmune uveitis in Lewis rats. J Clin
`Invest 1981;67:1228—1231.
`7. Bore] JF, Lafierty KJ: Cyclosporine: Specu~
`lotion about its mechanism of action. Transplant
`Proc 1983;15:1881-1885.
`8. Nussenblatt RB, Palestine AG, Chan CC:
`Cyclosporin A therapy in the treatment of intra-
`ocular inflammatory disease resistant to system-
`ic corticosteroids and cytotoxic agents, Am J
`Uphtlmlmol 1983;96:275—282.
`9. Palestine AG, Nussenblatt RB, Chan CC:
`Side effects of systemic cyclosporine in nontrans»
`plant patients. Am J Med 1984;77:652-656.
`10. Wacker WB, Donoso LA, Kalsow CM, et al:
`Experimental allergic uveitis: Isolation, charac»
`terization, and localization of a soluble uveito-
`pathogenic antigen from bovine retina. Jimmy.—
`nol 1977;119:1949-1958.
`11. Dos Reis GA, Shevach EM: Effect of
`cyclosporin A on T cell function in vitro: The
`mechanism of suppression of T cell proliferation
`depends on the nature of the T cell stimulus as
`well as the differentiation state of the respond-
`ing T cell. J Immunol 1982;102:2360—2367.
`12. Kaufmann Y, Chang AE, Robb RJ, Et al:
`Mechanism of action of cyclosporin A: Inhibition
`of lymphokine secretion studied with antigen,
`stimulated T cell hybridomas. J Immzmol
`1984;133:3107-3111.
`’
`13. Nussenblatt RB. Palestine AG, El-Saied M,
`et al: Long-term antigen-specific and non-specif-
`ic T-cell lines and clones in uveitis. Curr Fly? Res
`1984;23:299—305.
`14. Sobel RA, Calvin RE: The immunopatholo-
`gy of experimental allergic encephalomyelitis
`(EAE):
`III. Differential
`in situ expression of
`strain 13 la on endothelial and inflammatory
`cells of (strain 2 X strain 13)F, guinea pigs With
`EAE. J Immunol 1984;134:2333-2337.
`15. Palestine AG, Nussenblatt RB, Chan CC:
`Cyclosporine penetration into the anterior cham—
`ber and ccrebrospinal fluid. Am J Ophthalmol
`1985391210211.
`16. Mosteller MW, Gebhardt BM, Hamilton
`AM, et al: Penetration of topical cyclosporine
`into the rabbit cornea, aqueous humor, and
`serum. Arch Ophthalmol 19851032101402.
`
`1562
`
`Arch OphthalmoI—Vol 103. Oct 1985
`
`Cyclosporine for Uveiiis—Nussenblafl et al
`
`Downloaded From: http://archopht.jamanetwork.com/pdfaccess.ashx?urI=/data/jnurnaIs/ophtll/l7965/ by a Infotrieve Inc User on 06/19/2017
`4
`
`4
`
`