`TEAR FILM, AND
`DRY EYE SYNDROMES2
`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
`
`TEVA - EXHIBIT 1008
`
`TEVA - EXHIBIT 1008
`
`
`
`Library of Congress Cataloging-in-Publication Data
`feEEEEEEEEEEEEe!
`
`tear film, and dry eye syndromes 2 : basic science and
`Lacrimal gland,
`clinical
`relevance / edited by David A. Sullivan, Darlene A. Dartt,
`and Michele A. Meneray.
`p.
`cm. -- (Advances
`
`in experimental medicine and biology ; v.
`
`438.)
`the Second International Conference on the
`"Proceedings of
`Lacrimal Gland, Tear Film, and Dry Eye Syndromes, held November
`16-19, 1996, at
`the Southhampton Princess Resort, Bermuda"--T.p.
`verso.
`§
`Includes bibliographical references and index.
`ISBN 0-306-45812-8
`2. Tears-—
`1. Lacrimal apparatus--Physiology--Congresses.
`I. Sullivan, David
`-Congresses.
`3. Dry eye syndromes--Congresses.
`D.
`II. Dartt, Dariene A.
`III. Meneray, Michele A.
`IV. International Conference on the Lacrimal Gland, Tear Film, and
`Dry Eye Syndromes
`(2nd : 1996 : Southampton, Bermuda Islands)
`V. Series.
`2. Tears--physiology-
`[DNLM:
`1. Lacrimal Apparatus--congresses.
`-congresses.
`3. Dry Eye Syndromes--congresses.
`w1 Ad559 v. 438
`1998 ]
`QP188.T4L332
`612.8'47--dc21
`DNLM/DLC
`for Library of Congress
`:
`
`1998
`
`98- 17987
`CIP
`
`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
`
`10987654321
`
`All rights reserved
`No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form orby 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
`
`inhibits T-lymphocyte-mediated im-
`Cyclosporine is an immune modulator that
`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 corn oil ointment
`(Schering-Plough), or 0.2% cyclosporine emulsion (Allergan), was found to reduce ocular
`surface inflammation and improvelacrimal gland secretion in dogs with KCS.'~
`The aim of the present research was to determine the ocular tissue distribution of cy-
`closporine in rabbits and dogs, and to comparetissue concentrations in rabbits, dogs, and
`humansafter topical administration. Determination of relationships between the oculartis-
`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 ofaction, and optimizing dosing regimen.
`
`2. METHODS
`
`2.1. Animal Studies
`
`[Mebmt -°H]-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
`
`
`
`1002
`
`A. Acheampongefal.
`
`ul dose of 0.2% °*H-cyclosporine formulation (~1 mCi/ml) into the lower conjunctival cul-
`de-sac ofthe left eye. Male beagle dogs (10-13 kg) received a 35 pl dose of 0.2% *H-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 oftissue, using the spe-
`cific activity of the dose formulation.
`
`2.2. Human Range-Finding Study
`
`One hundredsixty-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 lowerlimit of quantitation of the
`blood assay was 0.1 ng/ml.
`
`3. RESULTS AND DISCUSSION
`
`1 and 2 depict the time course of cyclosporine in tears, ocular surface tissues,
`Figs.
`and orbital lacrimal gland of rabbits and dogs after eyedropinstillation of 0.2% *H-cy-
`closporine emulsion. Significant cyclosporine concentrations (C,,,,, ~1000 ng/g) were
`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.* The lacrimal gland Ca, 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.
`| and 2). Similar ocular distribution char-
`acteristics were noted in previous rabbit and humanstudies.*”
`Table | showsless than 0.2 ng/ml blood concentrations in humans following multi-
`ple topicalinstillation 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 lowerthan 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 producesignificant concentrations in
`the cornea and conjunctiva to exert a local immunomodulatory effect. The ocular distribu-
`
`
`
`
`
`Cyclosporine Distribution into the Conjunctiva
`
`1003
`
`—©— tear
`—4— conj
`—{}— cornea
`
`—€— sclera
`—@— lacrimal
`—t— blood
`
`
`
`
`
`
`
`
`20
`
`40
`
`60
`
`80
`
`100
`
`Time (hour)
`
`Figure 1. Total radioactivity concentrations (mean + SEM) in rabbit eyes and systemic blood.
`
`J
`
`1000000 =
`100000 4
`
`10000
`
`100
`
`10
`
`1
`
`—o— Tear
`—A— Conjunctiva
`—}-— Cornea
`
`—— Sclera
`—@— Lacrimal
`—«— Blood
`
`
`
`
`
`
`
`
`
`
`
`
`—)
`
`
`
`
`
`
` 1000 3
`0.1
`
`
`
`H-Cyclosporine(ng-eq/g)3
`
`
`
`*H-Cyclosporine(ng-eq/g)
`
`20
`
`40
`
`60
`
`80
`
`100
`
`Figure 2. Total radioactivity concentrations (mean values) in dog eyes and systemicblood.
`
`Time (hour)
`
`
`
`
`
`1004
`
`A. Acheampongetal.
`
`Table 1. Humanblood trough and maximum
`cyclosporin A concentrations over 12 weeks
`
`Range of blood cyclosporine A
`concentration (ng/ml)
`Cyclosporine
` emulsion Trough level Maximumlevel
`
`
`0.05%
`<0.1
`<021
`0.1%
`<0.1 to 0.102
`<0.1
`0.2%
`<0.1 to 0.108
`<0.1 to 0.144
`0.4%
`<0.1 to 0.157
`<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:
`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 ofaction. The human blood cyclosporin A con-
`centrations were less than 0.2 ng/ml, much lowerthan 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. Theeffects of topical cyclosporine A (CsA) on dry eye dogs
`(KCS). ARVO Abstracts. /nvest OphthalmolVis 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. /nvest Ophthal-
`molVis Sci. 1996;37:S1026.
`5. Wiederholt M, Kossendrup D, Shulz W, Hoffman F. Pharmacokinetics of topical cyclosporin A in the rabbit
`eye. Invest OphthalmolVis Sci. 1986;27:5 19-524.
`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 humaneye.
`Br J Ophthalmol. 1990;74:350-352.
`
`
`
`