ANNALS OF THE NEW YORK ACADEMY OF SCIENCES
`Volume 878
`
`INHIBITION OF MATRIX
`METALLOPROTEINASES
`THERAPEUTIC APPLICATIONS
`
`Edited by Ro_bert A. Greenwald, Stanley Zucker,
`and Lorne M. Golub
`
`The New York Academy of Sciences
`New York, New York
`1999
`
`Dr. Reddy's Laboratories, Ltd., et al.
`v.
`Galderma Laboratories, Inc.
`IPR2015-_
`Exhibit 1028
`
`Exh. 1028
`
`

`
`Copyright© 1999 by the New York Academy of Sciences. All rights reserved. Under the provi-
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`
`The cover of the paper-bound edition of this volume shows the catalytic domain of a crystal struc-
`ture of truncated MMP-3 bound to an MMP inhibitor. The inhibitor shown is PGE-116611, the
`chemical name of which is: (2R)-isobutyl-(3S)-[N-hydroxycarboxamido]-6-hydroxyhexanoic
`acid amide of (1N)-2-[methoxyethyl]-caprolactam-(3S)-amine. The coverillustrationwas gener-
`ously provided by Drs. Biswanath De and Gien Mieling of Procter and Gamble Pharmaceuticals.
`
`Library of Congress Cataloging-in-Publication Data
`Inhibition of matrix metalloproteinases : therapeutic applications
`I edited by Robert A. Greenwald, Stanley Zucker, and Lorne M. Golub.
`p.
`cm. -
`(Annals of the New York Academy of Sciences,
`0077-8923 ; v. 878)
`Includes bibliographical references and index.
`ISBN 1-57331-180-4 (cloth: alk. paper)
`ISBN 1-57331-181-2 (pbk: alk. paper)
`1. Metalloproteinases-Inhibitors-Therapeutic use Congresses. 2.
`I. Greenwald, Robert A.,
`Extracellular matrix proteins Congresses.
`II. Zucker, Stanley.
`111. Golub, Lorne M.
`1943-
`IV. Series.
`Q11 .N5 vol. 878 RM666.M512
`500 s-dc21
`[615' .35]
`
`99-30122
`CIP
`
`K-M Research/PCP
`Printed in the United States of America
`ISBN 1-57331-180-4 (cloth)
`ISBN 1-57331-181-2 (paper)
`ISSN 0077-8923
`
`Exh. 1028
`
`

`
`Tetracycline Derivative CMT-3 Inhibits
`Cytokine Production, Degranulation, and
`Proliferation in Cultured Mouse
`and Human Mast Cells
`
`KARI K. BKLUNDa,c AND TIMO SORSAb
`alnstitute of Biomedicine, Department of Medical Chemistry and
`Department of lnternal Medicine, Helsinki University Hospital, Helsinki, Finland
`bDepartment of Dentistry, Helsinki University, Helsinki, Finland
`
`INTRODUCTION
`
`Activated mast cells (MC) produce a wide variety of inftammatory mediators
`such as histamine, eicosanoids, proteases, and several cytokines. Recent findings
`emphasize the pathogenetic role of MC not only in allergic diseases, hut also in dis-
`eases associated with ehrenie inftammation, such as connective tissue diseases.1
`Tetracyclines are commonly used antihiotics that have therapeutic properties oth-
`er than those related to their antimicrobial activity. They have heen shown to he po-
`tent inhihitors of collagenases and to have heneficial antiinftammatory effects in
`rheumatoid arthritis.2•3 Chemically modified tetracyclines (CMT) are tetracycline
`derivatives that do not have the antimicrohial activity hut have retained their other
`properties. We decided to study the effect of three CMTs (CMT-1, CMT-3, CMT-5)
`on key functions of mast cells to find out whether CMTs could also have antiallergic
`properties.
`
`MATERIALSAND METHODS
`
`Cell Culture
`Mouse hone marrow-derived mast cells (mBMMC) were generated hy culturing
`hone marrow cells of BALB/c mice (animal facilities of Helsinki University) for 2-
`5 weeks in enriched medium supplemented with 50% WEHI-3 cell (line TIB-68;
`ATCC, Rockville, MD) conditioned medium as a source of IL-3. Human mast cell
`line (HMC)-1 cells4 were cultured in Iscove' s medium supplemented with 10% FCS.
`
`Activation of MC
`mBMMC were activated with calcium ionofore A23187 (5 mM, Sigma), and su-
`pematants and cells were analyzed for ß-hexosaminidase activity. HMC-1 cells were
`
`c Address for correspondence: Kari Eklund, Institute of Biomedicine, Department of Medical
`Chemistry, University of Helsinki, Siltavuorenpenger 10, 00170 Helsink.i, Finland.
`
`689
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`Exh. 1028
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`

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`690
`
`ANNALS NEW YORK ACADEMY OF SCffiNCES
`
`activated with a combination of phorbol 12-myristate 13-acetate (PMA) 50 ng/ml
`(Sigma) and calcium ionoforeA23187 0.5 J.LM, and cytokines were analyzed 24 hr
`after activation of the cells using a commercial ELISA method (R&D Systems,
`London, England). The viability of the cells was assessed by counting the trypan
`blue excluding cells 24 hr after activation. Viability was not affected by the CMTs
`in the concentrations used.
`
`RESULTS AND DISCUSSION
`
`A clear dose-dependent inhibition of TNF-a and to a lesser degree of IL-8 pro-
`duction was observed in HMC-1 cells by CMT-3 but not by CMT-1 or CMT-5
`(FIG. 1). In the presence of 25 J!M CMT-3 75% and 40% inhibition of TNF-a and
`IL-8 production, respectively, was observed. The effect of CMT-3 on mBMMC de-
`. granulation, as revealed by the granule-associated ß-hexosaminidase release, is
`shown in FIGURE 2. A clear inhibition of calcium ionofore-induced degranulation
`was observed in the presence of 25 J.LM and 50 J.LM CMT-3. No clear effect on de-
`granulation could be observed by the CMT-1 or CMT-5 (data not shown).
`The reason for the inhibition of degranulation and cytokine production by CMT-3
`is not clear at present. Both 'serine and metalloproteinases have been implicated in
`the degranulation of mast cells. Tetracyclines are known also to have metal chelating
`properties, which could perhaps account for the inhibitory effect of CMT-3. To con-
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`a e 1: 0
`
`(,)
`
`'ö
`~ e..
`c
`0
`~
`
`:I " e a.
`as .c a.
`'i u. z t-
`
`FIGURE 1. The effect of CMT -3
`on TNF-a production in HMC-1 cells.
`(1 x 106/ml) were activated
`Cells
`with PMA (50ng/ml) and calcium ion-
`ofore A12387 (0.5 ~M). CMT-3 was
`added 1 hr before the activators. Sim-
`ilar results were obtained in a repli-
`cate experiment.
`
`0
`
`5
`
`25
`
`50
`
`CMT-3(uM)
`
`Exh. 1028
`
`

`
`EKLUND & SORSA: CMT-3 INHIBITS CYTOKINE PRODUCTION
`
`691
`
`1::' s ::1 iii
`u e
`i
`~
`GI
`l'd
`~
`~
`~
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`0
`
`5
`
`25
`
`50
`
`CMT-3 (uM)
`
`FIGURE 2. Degranulation of mause bone marrow-derived mast cells (1 x 106/ml) in
`the presence of CMT ~3. Degranulation was induced with calcium ionofore (A12387, 5 J..LM),
`and ß~hexosaminidase activity of the cells and supernatants was analyzed 20 min later. Sim-
`ilar results were obtained in a replicate experiment.
`
`clude, CMT-3 inhibits very efficiently several key functions of MC and could there-
`fore have potential use in the treatment of mast cell-related diseases such as variou~
`allergic diseases.
`
`REFERENCES
`1. ARNASON, J. & D.G. MALONE. 1995. Role of mast cells in arthritis. Chem. Immunol.
`62: 204-238.
`2. 2. KLOPPENBURG, M., F.C. BREEDVELD, J.P. TERWIEL, C. MALLEE & B.A.C.
`DIJKMANS. 1994. Minocycline in active rheumatoid arthritis: a double~blind placebo~
`controlled trial. Arthritis Rheum. 37: 629-636.
`3. TILLEY, B.C., G.S. ALARCON, S.P. HEYSE, D.E. TRENTHAM et al, 1995. Minocycline in
`rheumatoid arthritis: a 48-week, double blind, placebo controlled trial. Ann. Intern.
`Med. 122: 81-89.
`4. ßUTTERFIELD, J.H., D. WEILER, G. DEWALD & G.J. GLEICH. 1988. Establishment of an
`immature mast cellline from a patient with mast cellleukemia. Leuk. Res. 2: 345.
`
`Exh. 1028