LACRIMAL GLAND,
`TEAR FILM, AND
`DRY EYE SYNDROMES 2
`Basic Science and Clinical Relevance
`
`Edited by
`David A. Sullivan
`Darlene A. Dartt
`The Schepens Eye Research Institute and
`Harvard Medical School
`Boston, Massachusetts
`
`and
`Michele A. Meneray
`Louisiana State University Medical Center
`New Orleans, Louisiana
`
`PLENUM PRESS • NEW YORK AND LONDON
`
` EXHIBIT 1008
`
`

`
`L i b r a r y of C o n g r e s s C a t a 1 o g1n g - 1 n - P u b 1 i c a t i o n D a t a
`
`i n s x p e rir a e n t a 1 m e d i c i n e a n d b i o l o g y ; v .
`
`L a c r i m a l g l a n d , t e a r f i l m , a n d d r y e y e s y n d r o m e s 2 : b a s i c s c i e n c e a n d
`c l i n i c a l r e l e v a n c e
`/ e d i t e d b y D a v i d A . S u l l i v a n , D a r l e n e A . D a r t t ,
`a n d M i c h e l e A . M e n e r a y .
`c m . — ( A d v a n c e s
`P •
`4 3 8 . )
`t h e S e c o n d I n t e r n a t i o n a l C o n f e r e n c e o n t h e
`" P r o c e e d i n g s o f
`L a c r i m a l G l a n d , T e a r F i l m , a n d D r y E y e S y n d r o m e s , h e l d N o v e m b e r
`1 6 - 1 9 , 1 9 9 6 , a t t h e S o u t h h a m p t o n P r i n c e s s R e s o r t , B e r m u d a " — T . p .
`v e r s o .
`I n c l u d e s b i b l i o g r a p h i c a l
`I S B N 0 - 3 0 6 - 4 5 8 1 2 - 8
`1 . L a c r i m a l a p p a r a t u s — P h y s i o l o g y — C o n g r e s s e s . 2 . T e a r s -
`- C o n g r e s s e s . 3 . D r y e y e s y n d r o m e s — C o n g r e s s e s .
`I , S u l l i v a n , D a v i d
`D .
`I I . D a r t t . D a r l e n e A .
`I I I . M e n e r a y , M i c h e l e A .
`I V . I n t e r n a t i o n a l C o n f e r e n c e o n t h e L a c r i m a l G l a n d , T e a r F i l m , a n d
`D r y E y e S y n d r o m e s
`( 2 n d
`; 1 9 9 6 : S o u t h a m p t o n , B e r m u d a I s l a n d s )
`V . S e r i e s .
`[ D N L M : 1 , L a c r i m a l A p p a r a t u s — c o n g r e s s e s . 2 . T e a r s — p h y s i o l o g y -
`- c o n g r e s s e s . 3 . D r y E y e S y n d r o m e s — c o n g r e s s e s .
`W 1 A d 5 5 9 v . 4 3 8
`1 9 9 8 1
`1 9 9 8
`Q P 1 8 8 . T 4 L 3 3 2
`6 1 2 . 8 ' 4 7 - - d c 2 1
`D N L M / D L C
`f o r L i b r a r y o f C o n g r e s s
`
`r e f e r e n c e s a n d
`
`i n d e x .
`
`9 8 - 1 7 9 8 7
`C I P
`
`Proceedings of the Second International Conference on the Lacrimal Gland, Tear Film, and Dry Eye
`Syndromes, held November 16- 19, 1996, at the Southampton Princess Resort, Bermuda
`
`ISBN 0-306-45812-8
`
`© 1998 Plenum Press, New York
`A Division of Plenum Publishing Corporation
`233 Spring Street, New York, N.Y. 10013
`
`http;//www.plenum.com
`
`1 0 9 8 7 6 5 4 3 2 1
`
`All rights reserved
`
`No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any
`means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written
`permission from the Publisher
`
`Printed in the United States of America
`
`

`
`144
`
`CYCLOSPORINE DISTRIBUTION INTO THE
`CONJUNCTIVA, CORNEA, LACRIMAL GLAND,
`AND SYSTEMIC BLOOD FOLLOWING
`TOPICAL DOSING OF CYCLOSPORINE TO
`RABBIT, DOG, AND HUMAN EYES
`
`Andrew Acheampong, Martha Shackleton, Steve Lam, Patrick Rudewicz,
`and Diane Tang-Liu
`
`Allergan
`Irvine, California
`
`1. INTRODUCTION
`
`Cyclosporine is an immune modulator that inhibits T-lymphocyte-mediated im-
`munoreactivity. Allergan is currently evaluating the clinical efficacy of 0.05%-0.4% cy­
`closporine emulsion for the treatment of immuno-inflammatory eye diseases, such as
`keratoconjunctivitis sicca, or dry eye syndrome. Topical ocular application of cyclospor­
`ine, formulated as 2% cyclosporine in olive oil, 0.2% cyclosporine in com oil ointment
`(Schering-Plough), or 0.2% cyclosporine emulsion (Allergan), was found to reduce ocular
`1-3
`surface inflammation and improve lacrimal gland secretion in dogs with KCS.
`The aim of the present research was to detennine the ocular tissue distribution of cy­
`closporine in rabbits and dogs, and to compare tissue concentrations in rabbits, dogs, and
`humans after topical administration. Determination of relationships between the ocular tis­
`sue drug concentrations and efficacy is important for optimizing delivery of pharma­
`cologically active concentrations in the target ocular surface tissues, providing support to
`the local mechanism of action, and optimizing dosing regimen.
`
`2. METHODS
`
`2.1. Animal Studies
`
`[Mebmt -3H]-cyclosporin-A was prepared by Amersham (UK) with radiochemical
`purity greater than 98%. Female New Zealand white rabbits (2—3 kg) received a single 50
`
`Lacrimal Gland, Tear Film, and Dry Eye Syndromes 2
`edited by Sullivan et a/.. Plenum Press, New York, 1998
`
`1001
`
`f
`
`

`
`1002
`
`A. Acheampong et al.
`
`|il dose of 0.2% 3H-cyclosporine formulation (~1 mCi/ml) into the lower conjunctival cul-
`de-sac of the left eye. Male beagle dogs (10-13 kg) received a 35 p.! dose of 0.2% 3H-cy-
`closporine emulsion (~1 mCi/ml) into the lower conjunctival cul-de-sac, twice daily for 7
`days. Ocular tissues and systemic blood were also collected at selected time points over a
`96-h period postdose. Two dogs or four rabbits were used per time point. The rabbit ex­
`periments were conducted according to USDA and Allergan ACUC guidelines. The dog
`study was conducted at Huntingdon Life Sciences. Tissue radioactivity concentrations
`were expressed as ng equivalents (eq) of cyclosporine per gram of tissue, using the spe­
`cific activity of the dose formulation.
`
`2.2. Human Range-Finding Study
`
`One hundred sixty-two human subjects with KCS received an eyedrop of vehicle or
`0.05%, 0.1%, 0.2%, or 0.4% cyclosporine emulsion twice daily for 12 weeks. Blood sam­
`ples were collected from all subjects at morning troughs after 1, 4, and 12 weeks of dos­
`ing. In addition, blood samples were collected from selected subjects at 1,2, and 4 h after
`the last dose at week 12. Cyclosporin A (CsA) concentrations in blood samples were
`measured by a validated liquid chromatography-tandem mass spectrometry (LC/MS/MS)
`method with Cyclosporin G as the internal standard. The lower limit of quantitation of the
`blood assay was 0.1 ng/ml.
`
`3. RESULTS AND DISCUSSION
`
`Figs. 1 and 2 depict the time course of cyclosporine in tears, ocular surface tissues,
`and orbital lacrimal gland of rabbits and dogs after eyedrop instillation of 0.2% ^-cy­
`closporine emulsion. Significant cyclosporine concentrations (C
`~1000 ng/g) were
`max'
`found in the conjunctiva and cornea, the target tissues for CsA reduction of ocular surface
`inflammation. The 0.2% emulsion provided approximately 7-fold higher cyclosporine
`concentrations in the rabbit cornea and conjunctiva than those for 0.2% cyclosporine in
`pure castor oil.4 The lacrimal gland Cmax was several-fold that of blood (~1 ng-eq/g), espe­
`cially in the dog.
`The ocular absorption and disposition of cyclosporine in rabbits and dogs were char­
`acterized by rapid absorption into ocular and extraocular tissues, reservoir effect of the
`cornea, relatively low intraocular tissue concentrations, and a long terminal elimination
`half-life of 20-44 h in most ocular tissues (Figs. 1 and 2). Similar ocular distribution char­
`acteristics were noted in previous rabbit and human studies.4-7
`Table 1 shows less than 0.2 ng/ml blood concentrations in humans following multi­
`ple topical instillation of 0.05%, 0.1%, 0.2%, and 0.4% cyclosporine ophthalmic emulsion
`over a 12-week period of dosing. The systemic blood CsA concentrations in humans after
`topical CsA doses of the emulsions were much lower than the blood trough concentrations
`of 20-100 ng/ml used for monitoring the safety of patients receiving systemic cyclospor­
`ine therapy.
`
`4. CONCLUSIONS
`
`Topically applied cyclosporine emulsion can produce significant concentrations in
`the cornea and conjunctiva to exert a local immunomodulatory effect. The ocular distribu-
`
`

`
`Cyclosporine Distribution into the Conjunctiva
`
`1003
`
`tJQ
`cr
`(U
`bJQ
`c
`OJ
`G
`
`• J-H o cx
`
`.
`
`CJ
`
`u
`
`CO
`
`o
`
`100000
`
`10000
`
`1000
`
`100
`
`10
`
`[ " =
`5 teg~A
`
`0.1
`
`0
`
`T
`20
`
`O— tear
`conj
`—•— cornea
`—O— sclera
`lacrimal
`^— blood
`
`o—
`
`40
`60
`Time (hour)
`
`T
`80
`
`1
`100
`
`Figure 1. Total radioactivity concentrations (mean ± SEM) in rabbit eyes and systemic blood.
`
`1000000 i
`100000 1
`
`10000 1
`1000 j
`100
`
`10 1
`
`o.i
`
`0
`
`cr <u
`b0 c
`0)
`C3
`o
`O)
`o
`o
`>> u
`ffi
`cn
`
`-©— Tear
`Conjunctiva
`—•— Cornea
`—O— Sclera
`Lacrimal
`Blood
`
`e
`
`•A
`
`-D—
`
`o
`
`20
`
`T
`40
`
`T
`60
`
`80
`
`100
`
`Time (hour)
`Figure 2. Total radioactivity concentrations (mean values) in dog eyes and systemic blood.
`
`P
`
`1
`
`

`
`1004
`
`A. Acheampong et al.
`
`Table 1. Human blood trough and maximum
`cyclosporin A concentrations over 12 weeks
`
`Cyclosporine
`emulsion
`0.05%
`0. 1 %
`0.2%
`0.4%
`
`Range of blood cyclosporine A
`concentration (ng/ml)
`Trough level
`Maximum level
`
`< 0 . 1
`<0.1 to 0.102
`<0.1 to 0.108
`<0.1 to 0.157
`
`<0.1
`<0.1
`<0.1 to 0.144
`<0.1 to 0.158
`
`tion of cyclosporine after topical application of CsA emulsion was generally similar in
`rabbit and dog. In the rabbits dosed with 0.2% emulsion, the C
`tissue distribution was:
`max
`tears > cornea > conjunctiva > lacrimal gland > blood.
`Systemic blood cyclosporine concentrations following topical application of cy­
`closporine emulsion were very low in rabbits, dogs, and humans, obviating concerns about
`systemic toxicity or systemic mechanism of action. The human blood cyclosporin A con­
`centrations were less than 0.2 ng/ml, much lower than the blood trough concentrations of
`20-100 ng/ml used for monitoring the safety of patients receiving systemic cyclosporine
`therapy.
`
`REFERENCES
`
`1. Kaswan RL. Spontaneous canine keratoconjunctivitis, a useful model for human keratoconjunctivits sicca:
`Treatment with cyclosporine eyedrops. Arch Ophthalmol. 1989;107:1210—1216.
`2. The 0.2% cyclosporine ointment is marketed by Schering-Plough for treatment of keratoconjunctivitis
`sicca in dogs.
`3. Stern ME, Gelber TA, Gao J, Ghosn CR. The effects of topical cyclosporine A (CsA) on dry eye dogs
`(KCS). ARVO Abstracts. Invest Ophthalmol Vis Sci. 1996;37:S1026.
`4. Acheampong A, Tang-Liu D, Shackleton M, Lam S, Angelov O, Ding S. Ocular absorption of cyclosporine
`from an aqueous emulsion: Comparison to other eyedrop formulations, ARVO Abstracts. Invest Ophthal­
`mol Vis Sci. 1996;37:S1026.
`5. Wiederholt M, Kossendrup D, Shulz W, Hoffman F. Pharmacokinetics of topical cyclosporin A in the rabbit
`eye. Invest Ophthalmol Vis Sci. 1986;27:519—524.
`6. Kaswan RL. Intraocular penetration of topically applied cyclosporine. Transplant Proc. 1988;20 (Suppl
`2):650-655.
`7. Ben Ezra D, Mafzir G, de Courten C, Timonen P. Ocular penetration of cyclosporin A. III. The human eye.
`Br J Ophthalmol. 1990;74:350-352.