T
`
`c
`
`Intraocular Penetration of Topically Applied
`Cyclosporine
`
`R.L. Kaswan
`
`MATERIALS AND METHODS
`
`YCLOSPORINE (CsA) is a potent
`as immune mediated uveitis with topical
`immunosuppressive
`drug
`with
`specific
`T
`application. Specific advantages
`to use
`of topi­
`cell inhibitory activity. CsA appears
`to
`inter­
`cal CsA as
`compared
`to corticosteroids would
`rupt T cell activation at an early point by
`include lack of collagenase activation when
`interfering with activation of intracellular
`immune suppression is indicated in eyes with
`mechanisms
`following
`antigen
`binding,
`with
`a
`ulcerated corneas, and potentially improved
`resultant blockade of the lymphokine cas­
`immunosuppressive
`activity.
`cade.1 CsA has proven to be very beneficial in
`organ transplantation and certain autoim­
`mune disorders in man.2 Recently, systemi-
`Rabbits
`cally administered CsA has been found to be
`We used 33 adult
`female New
`Zealand White (NZW)
`beneficial in Sjogren's syndrome3 and some
`rabbits (3
`4 kg), divided into
`to
`11 groups
`of
`three
`forms of uveitis
`in both people and
`experimen­
`Tetracycline 4
`g/gallon,
`to the drinking water
`was
`added
`tal species.4"12 Additionally, topically applied
`for prophylaxis of
`Pasteurella
`infection.
`CsA has been found to be useful in corneal
`Treatment
`allografts,
`vernal keratoconjunctivitis,16
`13-15
`keratoconjunctivitis sicca,17 immune me­
`A solution of 1% CsA radiolabeled with 3H specific
`activity of 8.5 ^Ci/mg (kindly supplied by William
`diated keratitis,18 necrotizing scleritis,19 and
`Robinson, Sandoz Pharmaceuticals, Hanover, NJ), was
`herpetic stromal keratitis.18,20 These studies
`prepared in olive oil (Sigma, St Louis). Three rabbits
`suggest that local ocular immunosuppression
`received eyedrops containing olive oil without CsA as
`provides adequate
`therapy while avoiding the
`negative controls. A Varimetric positive displacement
`risks of generalized immunosuppression and
`pipettor (Labindustries, Berkeley, CA) was used to
`deliver 7 ML eyedrops. This volume was chosen to avoid
`renal toxicity.
`spillover since the tear volume in the rabbit averages
`The efficacy of topically applied CsA for
`8 /iL.21
`Increased drop size does not lead to greater
`external ocular disorders
`led us to conjecture
`corneal penetration22 but confounds topical absorption
`that if CsA can reach
`therapeutic
`levels
`inside
`with recirculation following absorption of drugs through
`the eye following topical application,
`the nasolacrimal
`system.
`Drops
`to the dorsal
`were
`it might
`applied
`corneoscleral limbus. One drop was applied every 15
`be possible to treat intraocular diseases such
`minutes for six applications.23 The total body dose per
`rabbit was 0.84 mg CsA, or 0.24 mg/kg. The time
`interval from the last application until enucleation was
`Animal
`varied from one to 72 hours (Fig 1), with three animals
`of
`Veteri­
`killed at each
`interval.
`
`From the Departments
`of
`Medicine and
`Small
`Veterinary Pharmacology/Physiology,
`College
`nary Medicine, University
`of
`Georgia,
`Athens.
`Supported in part by the National Eye Institute,
`National Institutes of Health, R03 EY05720-01, The
`National Society for the Prevention of Blindness, The
`University
`of
`Georgia
`Veterinary Medical Experimental
`Station 29-26-GR207-002,
`and
`Sandoz
`Inc.
`Address
`reprint requests to Renee L. Kaswan, DVM,
`MS, Department of Small Animal Medicine, College of
`Veterinary Medicine,
`University
`of Georgia, Athens, GA
`30602.
`© 1988 by Grune &
`Stratton,
`Inc.
`0041-1345/88/2002-2l21$03MI0
`
`Sample Collection
`Rabbits were anesthetized with xylazine (5 mg/kg)
`and ketamine (35 mg/kg)
`intramuscularly IM. Samples
`Pharmaceuticals
`of 200
`/uL of aqueous humor were
`with a 30-gauge
`drawn
`needle and tuberculin syringe inserted at the corneal
`limbus. After heparinized cardiac blood samples were
`drawn, the rabbits were killed with pentobarbital (325
`mg) intracardiac injection. Both eyes were enucleated
`and frozen. Clean instruments were
`used
`for each rabbit.
`Globes were dissected by the method of Abel and
`Boyle.24 Briefly, the globes were transected at the equa-
`
`650
`
`rabbits.
`
` EXHIBIT 1011
`
`

`

`INTRAOCULAR PENETRATION OF CYCLOSPORINE
`
`651
`
`4500-
`
`4000-
`
`S 3500-
`^ 3000-
`o>
`^ 2500-
`
`X 2000-
`o>
`c 1500-
`
`1000-
`
`500-
`
`0
`
`90-
`
`8 0 -
`
`o 70
`
`< 6 0 -
`
`50-
`
`m
`^ 40-
`
`g ' S O -
`
`20-
`
`1 0 -
`
`Cornea
`
`Anterior
`Sclera
`
`10.000
`
`9.000-
`
`8,000-
`
`7,000-
`
`2
`£ 6.000-
`O)
`< 5.000-
`
`I 4,000-
`cn = 3,000-
`
`2,000-
`
`1.000-
`
`"20
`
`30
`
`40
`T i m e ( h o u r s )
`
`"io"
`
`60
`
`70*
`
`80
`
`0
`
`To
`
`~20
`
`lo"
`
`"40"
`T i m e ( h o u r s )
`
`50"
`
`60"
`
`70"
`
`80
`
`Aqueous
`
`Posterior
`Sclera
`
`1600-
`
`1400"
`2
`
`w 1200­
`
`000-
`
`O
`
`/\
`T 800-f
`
`600-
`
`400-
`
`200-
`
`0
`
`So"
`
`60
`
`I
`50
`
`60
`
`To"
`
`80
`
`0
`
`To
`
`To
`
`To
`
`To"
`T i m e ( h o u r s )
`
`i
`To"
`
`To
`
`To
`
`4o"
`T i m e ( h o u r s )
`
`200
`
`180-
`
`160-
`
`140
`
`£
`
`120-
`
`(3 100
`
`80-
`
`60-
`
`40-
`
`2 0 -
`
`0
`
`3
`
`50-
`
`%
`x 40-
`
`c* 30-
`
`20-
`
`1 0 -
`
`0
`
`Anterior
`Uvea
`
`Retina
`
`<l>
`
`<
`6
`X 200-
`a>
`
`150-
`
`100-
`
`50-
`
`To
`
`To
`
`30
`
`40
`T i m e ( h o u r s )
`
`To"
`
`60
`
`70
`
`0
`
`10
`
`i
`To
`
`30
`
`40
`T i m e ( h o u r s )
`
`To"
`
`60
`
`T"
`
`80
`
`Vitreous
`
`-i-
`
`1 0
`
`20
`
`30
`
`4:
`50
`
`40
`T i m e ( h o u r s )
`
`60
`
`70
`
`80
`
`Fig 1. CsA content in various ocular tissues of the rabbit eye following six applications of 7 ML of 1 % 3HCsA in
`olive oil. Concentrations have been calculated from the specific activity of 3HCsA and expressed as ng/g tissue or
`ng/mL fluid. Six eyes have been used for each individual point.
`
`

`

`652
`
`R.L. KASWAN
`
`tor. The frozen vitreous was extruded and collected. The
`anterior uvea (iris and ciliary body), choroid with retina,
`lens, cornea, anterior sclera, and posterior sclera were
`dissected, individually processed, and assayed. In rabbits
`killed at two hours post-CsA treatment the lacrimal gland
`of the third eyelid was also removed and assayed. Each
`ocular tissue was digested in three times its weight of
`collagenase (Sigma type I) in Michaelis, barbital buffer.25
`Next, 100
`of collagenase buffer solution was added to
`each vitreous sample. Tissue digests, 250
`whole blood,
`and 100 )xL aqueous samples were transferred to scintilla­
`tion vials and mixed with 10 mL Scintiverse Ia. Blood
`samples were decolorized with BTS-450 (Beckman,
`Atlanta), 30% H202, and glacial acetic acid.26 The disin­
`tegrations were assayed as counts per minute (cpm) using
`a Beckman LS 7000 liquid scintillation counter. Counting
`efficiency for each assay was determined by H-number
`quench correction using commercial external standards
`for data conversion to disintegrations per minute
`(DPM).26 Conversion to mg CsA was made by the
`following equation:
`
`DPM of sample x 106 ng/mg
`2.2 x 106DPM/yCi x 8.5^Ci3H/mgCsA x tissue wt(g)
`- ng CsA/g tissue
`
`Negative control tissues were used for background sub­
`traction of DPM for each tissue type.
`Blood samples from rabbits from each time group were
`assayed in a blind manner by Sandoz Research Institute
`using a tissue-burning technique to assay the vaporized
`tridium from each blood sample, because the tissue-
`burning assay has higher sensitivity for whole blood
`samples than can be attained with liquid scintillation.b
`
`RESULTS
`Figure 1 illustrates the CsA content of the
`aqueous, vitreous, and ocular tissues. Peak
`concentrations were reached within the first
`four hours in all tissues. Tissue concentrations
`in excess of minimal therapeutic levels (50 to
`300 ng CsA/g tissue)6 were achieved within
`one hour and maintained for at least 24 hours
`in the cornea, anterior, and posterior sclera.
`Levels of CsA were below therapeutic values
`within two hours in the retina, vitreous, and
`aqueous samples. The average level of CsA in
`the lacrimal gland of the third eyelid was
`2,850 ng/g at two hours post-CsA treatment.
`Iris and ciliary body CsA concentrations were
`at equivocal levels for therapy of uveitis,6
`between 50 and 200 ng/g tissue, (Fig 1) for all
`samples taken between one and 24 hours.
`
`The elimination rates of CsA in the cornea,
`anterior uvea, and anterior and posterior
`sclera were determined as the slopes of the
`terminal phase of the concentration v time
`curves using a visually fit two compartmental
`model.27 The elimination half-life was calcu­
`lated as 0.693 divided by the elimination rate
`and was found to be 33.5 hours, 26.7 hours,
`19.5 hours, and 20.0 hours, respectively.
`Amongst 20 blood samples assayed for
`radioactivity by both liquid scintillation and
`tissue burning techniques, only one sample
`had significant radioactivity (greater than
`three times baseline) and it was identified by
`both methods. The CsA content of this sample
`was 57 ng. No radioactivity was detected in
`the remaining 10 samples, which were assayed
`by liquid scintillation but not by tissue-
`burning techniques.
`The pigmented tissues and especially the
`whole blood samples were most affected by
`chemiluminescence. The presence of biologic
`pigments, especially red pigments, causes
`high control level cpm values, confounding the
`background activity. The use of antioxidants
`and bleaching agents reduced this error but
`did not eliminate it, making small amounts
`(less than 50 ng/mL) of CsA in the blood
`impossible to detect by liquid scintillation.
`Use of the tissue-burning technique on por­
`tions of these samples confirmed that no
`detectable CsA occurred in blood samples
`except one sample taken at 8 hours posttreat-
`ment.
`
`DISCUSSION
`Two major questions must be determined
`before topical CsA would prove to have practi­
`cal importance for uveitis. Can CsA reach the
`target organ in therapeutic levels following
`reasonable therapeutic regimens? Is the effect
`of CsA mediated locally, in the eye, or does
`the inhibition of intraocular inflammation
`require systemic immunosuppression?
`CsA is an undecapeptide with a molecular
`weight of 1,202. The therapeutic range for
`organ transplantation is 200 to 600 ng/mL
`
`I
`
`I
`
`i
`i
`
`

`

`INTRAOCULAR PENETRATION OF CYCLOSPORINE
`
`653
`
`serum, but the intraocular level speculated to
`be needed for control of uveitis is 50 to 300
`ng/mL.6 Alternately, if the key event is inhib­
`iting the triggering of lymphokine release
`from activated T cells, the required intraocu­
`lar dose would be only 5 to 199 ng/mL CsA.
`Compared to most ophthalmic medications,
`CsA is a large molecule, but it is highly
`lipophillic. If compounds are lipid soluble and
`pass through the corneal and conjunctival
`epithelium their size does not appreciably
`transport.28 Intraocular
`alter the rate of
`absorption of large molecular weight drugs
`occurs across the conjunctival-scleral inter­
`face, bypassing the necessity of passage
`through the aqueous humor.29 CsA's absence
`in the aqueous humor30,31 was therefore not
`discouraging, because the aqueous is an inap­
`propriate compartment for sampling of intra­
`ocular drug absorption for those drugs that
`depend upon noncorneal absorption routes.29
`Since the total volume of CsA given to each
`experimental animal in the present study was
`6 doses of 7 fxL 1% CsA in each eye, the total
`body dose per rabbit was 0.84 mg, or 0.24
`mg/kg.2 The recommended systemic dose of
`CsA is 10 to 20 mg/kg2 or 60 to 120 times
`higher than the doses used in this study.2
`Following therapeutic dosage in people with
`uveitis, intraocular levels were only 40% of
`serum levels.32 Bell and coworkers report
`aqueous levels of 12 to 49 ng/mL33 following
`topical or systemic administration to rabbits,
`but the lower limit of sensitivity of the RIA
`assay used was 45 ng/mL.34 Based on the
`absence of detectable CsA in 29 of 30 blood
`samples and the minimal total body doses
`used, intraocular levels in this study probably
`were not due to blood redistribution.
`Wiederholt and coworkers studied intraoc­
`ular penetration of 1% CsA dissolved in castor
`oil, but reported their results in cpm, rather
`than in a universally comparable term.26 The
`corneal half-life in both studies is remarkably
`long, 34 hours in this study and 52 hours in
`Wiederholt's.35 Our findings do not support
`their conclusion that the intraocular levels
`
`occur due to blood redistribution to both eyes
`following CsA administration to either eye.35
`The concentration of CsA reached in the
`present study was well above therapeutic lev­
`els in the cornea, and in anterior and posterior
`sclera, but the intraocular CsA levels achieved
`were low. The anterior uvea approached
`hypothesized therapeutic levels with levels
`ranging from 50 to 200 ng/gm. An assay of
`steady state pharmacokinetics with chronic
`administration for five elimination half-lifes
`and assays from inflamed eyes may be
`required to determine whether or not higher
`therapeutic levels can be achieved in the ante­
`rior uvea and retina under typical clinical
`conditions.
`Where is the target site? If CsA works by
`entering the eye and blocking intraocular T
`cells responding to ocular antigens within the
`inflamed eye, then local administration can be
`advocated. If, however, ocular antigens are
`disseminated to lymphatic organs where T cell
`activation occurs, systemic CsA therapy
`would be mandated.
`Topical CsA had demonstrated efficacy in
`immune-mediated diseases of the cornea and
`sclera, including experimental corneal graft
`13-15 vernal keratoconjunctivi-
`transplantation,
`tis,16 keratoconjunctivitis sicca,17 chronic im­
`mune mediated keratitis,18 necrotizing scleri-
`tis,19 and experimental herpetic stromal
`keratitis.18,20 In dogs with keratoconjunctivitis
`sicca, topical CsA ameliorated the chronic
`keratitis and increased the average Schirmer
`tear test by 9 mm/min.17 These studies
`strongly suggest that local immune suppres­
`sion is adequate to control immune-mediated
`disorders with CsA, providing the CsA
`reaches therapeutic levels at the target tissue.
`The present pharmacokinetic data confirm
`that CsA levels are very high in the lacrimal
`gland of the third eyelid, cornea, and sclera
`following topical dosage.
`The question of whether or not uveitis can
`be controlled with topical CsA has been more
`elusive. The intraocular dosage of CsA may
`not be sufficient to control inflammation at all
`
`

`

`654
`
`R.L. KASWAN
`
`areas of the globe. The current investigation
`and previous kinetic work has determined the
`elimination half-life, but has not addressed
`the steady state intraocular concentrations
`one would anticipate during multiple chronic
`dosage.
`Previous uveitis studies that used intrasub-
`ject positive control eyes may have inadver­
`tently triggered an afferent immune reaction
`in the positive control eye, causing a systemic
`immune response with an efferent response
`occurring in both the control and treated eye.6
`Studies that treated both eyes with CsA and
`used an extrasubject positive control were
`more successful.6,10 Two studies have exam­
`ined the potential for topical CsA in experi­
`mental uveitis. When Nussenblatt and
`coworkers6 tested CsA in a limited number of
`rats with experimental autoimmune uveitis,
`they could not separate systemic effects from
`topical effects because the ophthalmic drops
`used were so large (50 ^L), and the subjects so
`small (200 g rats), that therapeutic serum
`levels were reached via nasolacrimal absorp­
`tion. Considering the mechanism of CsA
`activity, the problem of differentiating the
`effects of systemic absorption of topical drug
`was not well addressed by unilateral drug
`dosing. CsA prevents initiation of the immune
`response by blocking T cell recognition of
`antigens and thereafter production of inter-
`leukin 2, which activates the cascading
`immune response. Antigenic recognition and
`lymphocyte activation within the untreated
`eye would be expected to trigger a systemic
`immune response36 affecting both eyes; there­
`fore, unilateral treatment designs would likely
`fail. Re-evaluation of Nussenblatt's work
`
`demonstrates that bilateral topical therapy
`was effective in preventing experimental
`autoimmune uveitis (EAU) at concentrations
`of 2%, 0.5%, 0.2%, but unilateral treatment
`did not show a significant beneficial effect in
`the eye treated with 2%, 0.5%, and 0.2% CsA.6
`This result was attributed to the fact that rats
`receiving bilateral drops got more volume of
`CsA, but actually, the rats receiving unila­
`teral 2% CsA had more total CsA than rats
`with bilateral 0.5% or 0.2% treatments. An
`alternate explanation is that it is the intrasub-
`ject positive control eye that instigates EAU
`in the CsA-treated eye,37 and that bilateral
`CsA is effective in EAU. Based on the small
`sample size (two rats per treatment group)
`and the ambiguity of results, further investi­
`gation into use of topical CsA for uveitis may
`be warranted.
`Advocacy of topical CsA for intraocular
`disease still awaits proof positive of intraocu­
`lar penetration. What is most encouraging
`from our data is that CsA does reach very
`high levels in the cornea, sclera, and lacrimal
`gland of the rabbit. Considering the results
`from topical trials of CsA in corneal trans­
`plantation,
`13-15 herpes stromal keratitis,18,20
`keratoconjunctivitis sicca,17 and chronic im­
`mune mediated keratitis,18 it is predictable
`that other immune-mediated disorders of the
`cornea, conjunctiva, and sclera may also
`respond positively to topical CsA.
`
`ACKNOWLEDGMENT
`The author thanks Susan Gardner for her expert advice
`on ocular pharmacokinetics, Antoinette Jernigan for
`assistance with the pharmacokinetic determinations, and
`William Robinson for furnishing 3HCsA and for perform­
`ing corroborative assays.
`
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`

`

`INTRAOCULAR PENETRATION OF CYCLOSPORINE
`
`655
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`