LACRIMAL GLAND,
`TEAR FILM, AND
`DRY EYE SYNDROMES 2
`
`Basic Science and Clinical Relevance
`
`Edited by
`
`David A. Sullivan
`
`Darlene A. Dartt
`
`The Schepens E e Research Institute and
`Harvard Medic
`chool
`Boston, Massachusetts
`
`and
`
`Michele A. Meneray
`Louisiana State University Medical Center
`New Orleans, Louisiana
`
`PLENUM PRESS 0 NEW YORK AND LONDON
`
`TEVA - EXHIBIT 1008
`
`TEVA - EXHIBIT 1008
`
`

`
`Library of Congress Cataloging—1n—Pub1icatiun Data
`
`
`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.)
`"Proceedings of the Second International Confierence on the
`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, Darlene A.
`III. Meneray, Michele A.
`IV. International Conference on the Lacrimal Gland, Tear Film. and
`Dry Eye Syndromes
`(2nd : 1998 : Southampton, Bermuda Islands)
`V. Series.
`2. Tears-—physiology—
`[DNLM:
`1. Lacrimal Apparatus—*congresses.
`—congresses.
`3. Dry Eye Syndromes-—congresses.
`W1 Ad559 v. 438
`19931
`0P188.T4L332
`812.8'47—-dc2i
`DNLM/DLC
`98-17987
`For Library of Congress
`CIP
`~
`
`
`1998
`
`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, NY. 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 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
`
`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 improve lacrimal 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 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 al., Plenum Press, New York, 1998
`
`1001
`
`
`
`

`
`
`
`l002
`
`A. Acheampong et al.
`
`111 dose of 0.2% 3H-cyclosporine formulation (~l mCi/ml) into the lower conjunctival cul-
`de-sac of the left eye. Male beagle dogs (10-13 kg) received a 35 pl dose of 0.2% 3H-cy-
`closporine emulsion (~l mCi/ml) into the lower conjunctiva] 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
`
`1 and 2 depict the time course of cyclosporine in tears, ocular surface tissues,
`Figs.
`and orbital lacrimal gland of rabbits and dogs after eyedrop instillation of 0.2% 3H-cy-
`closporine emulsion. Significant cyclosporine concentrations (Cmax, ~l000 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.4 The lacrimal gland Cm“ was several-fold that of blood (~l 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.“
`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
`
`—e— tear
`
`
`
`
`-—fiA— conj
`——j— cornea
`
`—e— sclera
`
`%— lacrimal
`
`—~A- blood
`
`
`
`100000
`
`3?
`D"
`
`10000
`
`1000
`
`100
`
`10
`
`1
`
`9 §
`
`§
`8
`-,2:
`
`O 8
`
`‘
`.2
`
`O 5
`
`?
`£1:
`
`
`
`
`
`
`
`0.1
`
`I
`
`60
`
`80
`
`Time (hour)
`
`Figure 1. Total radioactivity concentrations (mean :1: SEM) in rabbit eyes and systemic blood.
`
`
`
`—% Tear
`
`—Ar— Conjunctiva
`—I:— Cornea
`
`—<>— Sclera
`
`—¢— Lacrimal
`
`—A—~ Blood
`
`
`
`1 000000
`
`35
`
`100000
`
`10000
`
`1000
`
`E ‘
`
`1’
`00
`
`EO
`
`100
`10
`
`-2
`§ _
`»%
`E?
`F
`1
`
`
`20
`
`40
`
`60
`
`80
`
`Figure 2. Total radioactivity concentrations (mean values) in dog eyes and systemic blood.
`
`Time (hour)
`
`
`
`
`

`
`
`
`1004
`
`A. Acheampong et al.
`
`Table 1. Human blood trough and maximum
`cyclosporin A concentrations over 12 weeks
`
`Range of blood cyclosporine A
`concentration (ng/ml)
`
`Cyclosporine
`Maximum level
`Trough level
`emulsion
`<0:l
`<O.l
`0.05%
`<0.1
`<O.l to 0.102
`0.1%
`<0.1 to 0.144
`<O.l to 0.108
`0.2%
`
`
`<0.1 to 0.1570.4% <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 Cm“ 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 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
`
`l. 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. Stem ME, Gelber TA, Gao J, Ghosn CR. The effects of topical cyclosporine A (CsA) on dry eye dogs
`(KCS). ARVO Abstracts. Invest Ophthalmol Vls Sci. 1996;37:Sl026.
`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 Vts Sci. 199637181026.
`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):65(}—655.
`7. Ben Ezra D, Mafzir G, de Courten C, Timonen P. Ocular penetration of cyclosporin A. III. The human eye.
`BrJ Ophthalmol. 1990;74:350-352.