`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
`
`APOTEX 1017, pg. 1
`
`
`
`Library of Congress Cataloging-in-Publication Data
`
`in experimental medic i ne and biology ; v.
`
`i ndex.
`
`basic science and
`Lacrimal gland, tear film, and dry eye syndromes 2
`clinical relevance I edited by David A. Sullivan, Darlene A. Dartt,
`and Michele A. Meneray .
`em.-- <Advances
`p .
`438.)
`"Proceedings of the Second International Conference 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
`ISBN 0-306-45812-8
`1. Lacrimal apparatus--Physiology--Congresses. 2. Tears-
`-Congresses. 3. Dry eye syndromes--Congresses.
`I . Sullivan , David
`D.
`II. Dartt, Darlene A.
`III. Meneray, Michele A.
`IV. Internat i onal Conference on the Lacrimal Gland, Tear Film, and
`Dry Eye Syndromes <2nd
`1996
`Southampton, Bermuda Is l ands)
`V. Series.
`[DNLM: 1. Lacrimal Apparatus--congresses. 2. Tears- - physiology-
`-congresses.
`3 . Dry Eye Syndromes--congresses.
`W1 Ad559 v. 438
`1998]
`1998
`QP188.T4L332
`612.8 ' 47--dc21
`DNLM / DLC
`for Library of Congress
`
`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 Di vision 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 or by any
`means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written
`permission from the Publisher
`
`Printed in the United States of America
`
`APOTEX 1017, pg. 2
`
`
`
`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(cid:173)
`munoreactivity. Allergan is currently evaluating the clinical efficacy of 0.05%-0.4% cy(cid:173)
`closporine emulsion for the treatment of immuno-inflammatory eye diseases, such as
`keratoconjunctivitis sicca, or dry eye syndrome. Topical ocular application of cyclospor(cid:173)
`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
`3
`surface inflammation and improve lacrimal gland secretion in dogs with KCS. 1
`-
`The aim of the present research was to determine the ocular tissue distribution of cy(cid:173)
`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(cid:173)
`sue drug concentrations and efficacy is important for optimizing delivery of pharma(cid:173)
`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 eta!., Plenum Press, New York, 1998
`
`1001
`
`APOTEX 1017, pg. 3
`
`
`
`1002
`
`A. Acheampong et al.
`
`111 dose of 0.2% 3H-cyclosporine formulation ( -1 mCi/ml) into the lower conjunctival cul(cid:173)
`de-sac of the left eye. Male beagle dogs (1 0-13 kg) received a 35 111 dose of 0.2% 3H-cy(cid:173)
`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(cid:173)
`periments were conducted according to USDA and Alle.rgan 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(cid:173)
`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(cid:173)
`ples were collected from all subjects at morning troughs after 1, 4, and 12 weeks of dos(cid:173)
`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 Gas 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% 3H-cy(cid:173)
`closporine emulsion. Significant cyclosporine concentrations (Cmax' -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. 4 The lacrimal gland Cmax was several-fold that ofblood (-1 ng-eq/g), espe(cid:173)
`cially in the dog.
`The ocular absorption and disposition of cyclosporine in rabbits and dogs were char(cid:173)
`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(cid:173)
`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(cid:173)
`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-
`
`APOTEX 1017, pg. 4
`
`
`
`Cyclosporine Distribution into the Conjunctiva
`
`1003
`
`,-..
`bJ)
`............
`0"'
`(I)
`I
`bJ)
`c
`'-"
`(I) c
`.......
`>-;
`0
`0..
`r:/1
`
`0 -u
`>--. u I ::c
`
`('<")
`
`100000
`
`10000
`
`1000
`
`100
`
`10
`
`1
`
`0.1
`
`tear
`0
`- i s - conj
`-D- cornea
`o
`sclera
`•
`lacrimal
`.-blood
`
`-
`
`0
`
`20
`
`40
`60
`Time (hour)
`
`80
`
`100
`
`Figure 1. Total radioactivity concentrations (mean ± SEM) in rabbit eyes and systemic blood.
`
`1000000
`
`100000
`
`10000
`
`1000
`
`100
`
`10
`
`1
`
`0.1
`
`o Tear
`- i s - Conjunctiva
`-D-Cornea
`Sclera
`0
`• Lacrimal
`• Blood
`
`0
`
`20
`
`40
`
`60
`
`80
`
`100
`
`Figure 2. Total radioactivity concentrations (mean values) in dog eyes and systemic blood.
`
`Time (hour)
`
`(I)
`I
`
`'-"
`
`,-..
`bJ)
`............ c::r
`bJ) c
`(I) c
`.......
`>-;
`0
`0..
`r:/1
`
`0 -u
`>--. u I ::c
`
`('<")
`
`APOTEX 1017, pg. 5
`
`
`
`1004
`
`A. Acheampong eta/.
`
`Table 1. Human blood trough and maximum
`cyclosporin A concentrations over 12 weeks
`
`Cyc1osporine
`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 to0.108
`<0.1 to0.157
`
`<0 ~ 1
`<0.1
`<0.1 to 0.144
`<0.1 to0.158
`
`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 Cmax tissue distribution was:
`tears > cornea > conjunctiva > lacrimal gland > blood.
`Systemic blood cyclosporine concentrations following topical application of cy(cid:173)
`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(cid:173)
`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. Stem 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, LamS, Angelov 0 , DingS . Ocular absorption of cyc1osporine
`from an aqueous emulsion: Comparison to other eyedrop formulations. ARVO Abstracts. Invest Ophthal(cid:173)
`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.
`
`APOTEX 1017, pg. 6