ANNALS OF THE NEW YORK ACADEMY OF SCIENCES
`Volume 878
`
`INHIBITION OF MATRIX
`METALLOPROTEINASES
`THERAPEUTIC APPLICATIONS
`
`Edited by Robert A. Greenwald, Stanley Zucker,
`and Lorne M. Golub
`
`I
`
`The New York Academy of Sciences
`New York, New York
`1999
`
`Dr. Reddy's Laboratories, Ltd., et al.
`v.
`Galderma Laboratories, Inc.
`IPR2015-_
`Exhibit 1029
`Exh. 1029
`
`

`
`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 cover illustration was gener-
`ously provided by Drs. Biswanath De and Gien Mieling of Procter and Gamble Pharmaceuticals.
`
`Library of Congress Cataloging-in-Publicaiion Data
`Inhibition of matrix metalloproteinases : therapeutic applications
`I edited by Robert A. Greenwald, Stanley Zucker, and Lorne M. Golub.
`(Annals of the New York Academy of Sciences,
`p. cm. -
`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.
`Extracellular matrix proteins Congresses.
`I. Greenwald, Robert A.,
`II. Zucker, Stanley. m. Golub, Lorne M.
`1943-
`IV. Series.
`Qll .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. 1029
`
`

`
`Non-antimicrobial and Antimicrobial
`Tetracyclines Inhibit IL-6 Expression
`in Murine Osteobiasts
`
`KEITH L. KIRKWOOD,0•bJLORNE M. GOLUB,c AND PETER G. BRADFORDb,d,e
`
`0Department of Periodontics and hcenter for Molecular Mechanisms of Diseases
`and Aging, State University of New York at Buffalo, Buffalo, New York 14214, USA
`
`cDepartment of Oral Biolo gy and Pathology, State University of New York at Stony Brook,
`Stony Brook, New York 11794, USA
`
`dDepartment of Pharmacology and Toxicology and eDepartment of Oral Biology,
`State University ofNew York at Buffalo, Buffalo, New York 14214, USA
`
`IL-6 regulation in osteoblasts has bee~ the focus of several groups, since this cytok-
`ine appears to contribute toward the pathophysiological state of postmenopausal os-
`teopenia.1 IL-6 is the major cytokine regulated by sex steroids as compared to other
`cytokines, such as IL-1ß, IL-11, and TNF-a., that arenot regulated by sex steroids.2
`Previous results from our laboratory have indicated that intracellular calcium is a
`critical determinant of the osteoblast secretory capacity. 3.4 Since tetracycline is well
`known for its ability to bind divalent cations, such as calcium and zinc, and to affect
`intracellular calcium concentrations,5 we evaluated the abilities of doxycycline and
`chemically modified tetracyclines, which lack antimicrobial activity, to affect osteo-
`b last IL-6 secretion from MC3T3-EI osteoblastic cells:
`
`METHODS
`
`IL-6 Secretion Measurements
`
`MC3T3-El cell~ were cultured in 6-well tissue culture dishes under standard tis-
`sue culture conditions. Test cells were pretreated for 18-24 hr with doxycycline,
`CMTs (1-50 ~g), or vehicle (DMSO 10 ~g/ml). IL-1ß was added to culture media
`at physiological relevant concentrations (10-20 pM), and the cells continued to in-
`cubate for an additional18 hr. Time course experiments as well as dose response ex-
`periments with CMTs were conducted in the same manner. Mouse IL-6 secretion
`was measured by a capture ELISA kit (Quantikine, R&D Systems) following the
`manufacturer's protocol. Quantities of IL-6 were expressed in pg/ml of cultured su-
`pernatant.
`
`f Address for correspondence: Keith L. Kirk:wood, D.D.S. Ph.D., State University of New
`York at Buffalo, Department of Periodontics, 215 Squire Hall, 3435 Main Street, Buffalo, New
`York 14214-3000. Phone, 716/829-3845; fax, 716/837-7623; e-mail, klkirk@acsu.buffalo.edu
`
`667
`
`Exh. 1029
`
`

`
`668
`
`ANNALS NEW YORK ACADEMY OF SCIENCES
`
`Northern Blot.Analysis of Osteoblast Gene Expression
`For detection of mRNA from control and CMT-treated MC3T3-EI osteoblastic
`cells, we employed Northem blot using methods as described previously.3 Rat IL-6
`cDNA clone was obtained as a gift from Dr. J. Goldie, McMaster University, Hamil-
`ton, Ontario. Dr. R. Franceschi, University of Michigan, kindly provided the mouse
`type l(al) procollagen cDNA probe. After hybridization with the appropriate
`probes, membranes were washed at high stringency, and the hybridized probe was
`quantitated using Bio-Rad's phosphoimager system and Molecular Analyst software
`version 1.5.
`
`RESULTS AND DISCUSSION
`
`Chemically Modified Tetracyclines Inhibit IL-6 Secretion in MC3T3-El Cells
`To determine if IL-6 secretion can be regulated by doxycycline or the other
`CMTs, IL-6 secretion from MC3T3-El c:ells was measured using a capture ELISA
`kit specific for mouse IL-6. Results shown in FIGURE IA indicate that CMT-8
`(10 jlg/ml) can inhibit IL-6 secretion from MC3T3-EI cells when stimulated by
`IL-lß (10 ng/ml). CMT-8 decreased IL-6 secretion by -49% compared to cells pre-
`
`1200
`
`1000
`
`800
`
`llr6
`(pg/011) 600
`
`400
`
`200
`
`0
`
`I
`
`-~ i
`~i
`i •• '*"
`';r,
`
`rf.t
`~
`-~
`\Ii
`'T ~
`
`• no agon1st
`Dll.rl
`
`BDOX 10
`lll Dox tonL-1
`BCMT-5 10
`
`•CMT-5/IL-1
`•cMT-8 10
`DCMT-8/IL-1
`
`400
`200
`o~~--~--P-~--~~~-
`0
`0.3
`1.0
`3.3
`10.0 33.3
`CMT-8
`(Jlglml)
`
`FIGURE 1. (Left) The effects of chemically modified tetracyclines on IL-lß-
`induced IL-6 secretion in osteoblasts. MC3T3-E1 cells were pretreated for 18 hours with
`doxycycline or CMTs at 10 J.Lg/rnl. Fresh mediawas added with the pretreatment condi-
`tions ± IL-1ß (12.5 ng/ml). Cells continued to incubate for an additiona118 hours, and
`cells supernatants were harvested. IL-6 secretion was measured using an IL-6 Quantikine
`Kit (R&D Systems). Quantitation of IL-6 was made using linear regression with a standard
`curve. Bach measurement was made in duplicate, and the mean of two different experi-
`ments was used for analysis with Standard deviations. The results are considered statisti-
`cally significant (p = 0.005). (Right) CMT-8 dose-response in IL-1ß treated MC3T3-El
`osteoblasts. MC3T3-E 1 cells were pretreated with CMT -8 at the following concentrations:
`0, 0.3, 1.0, 3.3, 10.0, and 30.0 J.Lg/ml. Cells were subsequently treated with IL-1ß (12.5 ng/
`ml) after a change in media and addition of pretreatment conditions. IL-6 measurements
`were made as described. Means of two separate experiments performed in duplicates ±
`standard deviations of the mean. The ICso was calculated to be 4.4 J.Lg/rnl.
`
`Exh. 1029
`
`

`
`KIRKWOOD et al.: CMT-8 INHIBITS IL-6 EXPRESSION
`
`669
`
`treated with vehicle. CMT-5 decreased IL-6 secretion by -13%. Doxycycline de-
`creased IL-1ß-induced.IL-6 secretion by -33%. The results shown are the means of
`two separate experiments performed in duplicate. Standard deviations are shown;
`these results are considered statistically significant (p = 0.005). Inhibition of IL-1 ß-
`stimulated IL-6 secretion occurred in a dose-dependent manner with pharmacologi-
`cally relevant concentrations of this drug (1-10 ~g/ml) reducing IL-6 by about 50%
`as shown in FIGURE 1A. Pretreatment with CMT-8 was reduced to 8 hours in these
`experiments. FIGURE 1B illustrates the dose-response effect with decreasing
`amounts of IL-6 secretion when increasing the amount. of CMT-8. The IC50 for.
`CMT-8 inhibition of IL-1 ß-induced IL-6 secretion was experimentally determined
`to be 4.4 ~g/ml.
`
`CMT-8 Decreases Steady State IL-6 mRNA Levels in MC3T3-El Cells
`Northern blot analysiswas used to determine IL-6 mRNA expression in IL-1ß-
`treated cells with or without pretreatment with doxycycline or CMTs. IL-6 mRNA
`species of 1.2-2.4 kb were detected in MC3T3-EI cells (FIGURE 2). As shown previ-
`ously in these cells, IL-1ß increases steady state mRNA levels. When cells are pre-
`treated with doxycycline. or CMTs or treated after IL-1ß Stimulation (data not
`shown), CMT-8 decreases the IL-6 mRNA steady state levels in these cells. In con-
`trast, neither doxycline nor CMT-5 were able to decrease IL-6 mRNA steady state
`levels (FIG. 2). When levels of GAPDH were used to normalize the data, an approx-
`imate 50% reduction in IL-6 mRNA with CMT-8 treatment is seen compared to
`
`No tx
`
`IL-lß CMT-8 CMT-8 Dox
`
`CMT·S
`
`IL-6
`
`Type 1 (al)
`Procollagen
`
`GAPDH
`
`FIGURE 2. MC3T3-E1 cells were cultured in the presence or absence of CMTs of
`doxycycline and then exposed to IL-1 ß (12.5 ng/rnl) for 18 hours. Total RNA was purified,
`separated on agarase gels, blotted onto nylon membranes, and suquentially hybridized
`with [32-P]-labeled cDNA prob es specific for rat IL-6, mause type 1 ( a1) procollagen, and
`ratGAPDH
`
`Exh. 1029
`
`

`
`ANNALS NEW YORK ACADEMY OF SCIENCES
`670
`IL-1ß treated cells (n = 2). Additionally, CMT-8 had no effect on constitutive type I
`procollagen a1(I) gene expression in MC3T3-E1 cells. IL- 1ß decreased type 1 pro-
`collagen a1(I) gene expression by 54% in these cells. This effect was somewhat in-
`hibited by CMT-8 (34%) and to a greater extent by doxycycline (76%).
`· The results of the present study suggest an additional mechariism of CMTs, inhi-
`bition of IL-6 gene expression, and secretion from osteoblasts. Our data indicate that
`IL-1ß-stimulated expression and secretion ofiL-6 in MC3T3-E1 osteoblasts can be
`inhibited by CMT-8 in a dose-dependent manner, and the effect of CMT-8 is consis-
`tent with decrease of IL-6 gene expression. These results support the role for chem-
`ically modified tetracyclines to provide anabolic effects in hone: In this paper,
`CMT-8 and, to a lesser extent, doxycycline inhibited the potentially catabolic effects
`of IL-1 ß, suggesting a novel molecular mechanism of these drugs with the treatment
`of metabolic hone diseases, such as osteoporosis.
`
`REFERENCES
`
`1. HOROWITZ, M.C. 1993. Cytokines and estrogen in hone: antiosteoporotic effects. Sci-
`ence 260: 626-627.
`2. MANOLAGUS, S.C. 1995. Role of cytokines in hone resorption. Bone 17: 63S-67S.
`3. K.lRKWOOD, K., R. DZIAK & P. BRADFORD. 1996. lnositol trisphosphate receptor gene
`expression and hormonal regulation in osteoblast-like celllines and primary osteo-
`blastic cell cultures. J. Bone Miner. Res. 11: 1877.
`4. KIRKWOOD, K., M. DRAGON, K. HoMICK & P. BRADFORD. 1997. Cloning and charac-
`terization of the type I inositol 1,4,5-trisphosphate receptor gene promoter: regula-
`tion by 17 ~-estradiol in osteoblast. J. Biol. Chem. 272: 22425-22431.
`5. DONAHUE, H.J., K. liJIMA, M.S. GOLIGORSKY, C.T. RUBIN & B.R. RIFKIN. 1992. Reg-
`ulation of cytoplasmic calcium concentration in tetracycline-treated osteoclasts. J.
`Bone Miner. Res. 7: 1313.
`6. LITTLEWOOD, A.J., J. RUSSELL, G.R. HARVEY, D.E. HUGHES, R.G.G. RUSSELL &
`M. GoWEN. 1991. The modulation of the expression of IL-6 and its receptor in
`human osteoblasts. Endocrinology 129: 1513-1520.
`
`Exh. 1029