PCT
`WORLD INTELLECfUAL PROPERTY ORGANIZATION
`Internadonai Bureau
`INTERNATIONAL APPLICATION PUBLISHED UNDER TIIE PATENT COOPERATION TREATY (PCT)
`WO 00n.8230
`(51) International Patent Classification 7:
`(11) International Publlcation Number:
`AOlN 37ß8, 57/00
`
`(43) International Publlcation Date:
`
`6 April 2000 (06.04.00)
`
`Al
`
`(21) International Appllcatlon Number:
`
`PCTIUS99/22199
`
`(22) International Fillng Date:
`
`24 September 1999 (24.09.99)
`
`(30) Priority Data:
`09/161,804
`
`28 September 1998 (28.09.98) US
`
`(71) Applicant: THE RESEARCH FOUNDATION OF STATE
`UNIVERSITY OF NEW YORK [USIUS]; P.O. Box 9,
`Albany, NY 12201-0009 (US).
`
`(72) Inventars: RAMAMURTIIY, Nungavarm, S.; 10 Lynam
`Court, Smithtown, NY 11787 (US). GOLUB, Lome, M.; 29
`Whitney Gate, Smithtown, NY 11787 (US). SORSA, Timo,
`A.; Lounaisvay1a 17, FIN-00200 He1sinkl (FI). TERONEN,
`Olli, P.; Kylanvanhimmankja 9B, FIN-00640 Helsinkl (FI).
`SALO, Tuu1a, A.; Fyysikontie 8, FIN-90570 Oulu (FI).
`
`(74) Agent: BARON, Ronald, J.; Hoffmann & Baron, LLP, 6900
`Jericho Tumpike, Syosset, NY 11791 (US).
`
`(81) Designated States: AE, AL, AM, AT, AU, AZ, BA, BB, BG,
`BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE,
`ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP,
`KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD,
`MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SO,
`SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ,
`VN, YU, ZA, ZW, ARIPO patent (GH, GM, KE, LS, MW,
`SD, SL, SZ, TZ, UG, ZW), Eurasian patent (AM, AZ, BY,
`KG, KZ, MD, RU, TJ, TM), European patent (AT, BE, CH,
`CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL,
`PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN,
`GW, ML, MR, NE, SN, TD, TG).
`
`Published
`With internationalsearch report.
`Before the expiration of the time Iimit for amending the
`c/aims and to be republished in the event of the receipt of
`amendments.
`
`(54) Title: COMBINATION OF BISPHOSPHONATE AND TETRACYCLINE
`
`4-r----------------------------------~
`
`b
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`3
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`2
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`TOOTH
`MOBILITY
`
`0-+---
`0
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`1=LPSALONE
`2= SALINE ALONE
`3• LPS + CMT -8 ( 1mg I DA V)
`
`4
`3
`2
`EXPERIMENTAL GROUPS
`4= LPS + CLODRONATE { 1mg I WEEK)
`5a LPS + COMBO (CMT-8 + CLOD)
`
`5
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`
`(57) Abstract
`
`Tissu<Hiestructive conditions related to excess proteinase activity in a blo1ogical system are treated or prevented by administering to
`the system a composition which comblnes a tetracycline and a bisphosphonate in synergistic proteinase lnhibiting amounts. The effecdveness
`of such compositions can be demonstrated in standard tests, for example by measuring tooth mobility.
`
`Dr. Reddy's Laboratories, Ud., et al.
`V.
`Galderma Laboratories, lnc.
`IPR2015-__ __
`Exhibit 1013
`
`Exh. 1013
`
`

`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`AL
`AM
`AT
`AU
`AZ
`BA
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`CI
`CM
`CN
`CU
`cz
`DE
`DK
`EE
`
`Albania
`Annenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Ben in
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Cöte d'Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Gennany
`Denmark
`Estonia
`
`ES
`FI
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`leeland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People's
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`Lesotho
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`The fonner Yugoslav
`Republic of Mace'donia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Po land
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`SI
`SK
`SN
`sz
`TD
`TG
`TJ
`TM
`TR
`TT
`UA
`UG
`us
`uz
`VN
`YU
`zw
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobaga
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`VietNam
`Yugoslavia
`Zimbabwe
`
`Exh. 1013
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`

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`PCT/US99/22199
`
`COMBINA TION OF BISPROSPRO NA TE AND TETRACYCLINE
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`This inventionwas made with government support under R37DE03987
`awarded by the National Institute ofDental Research (NIH). The government has
`certain rights in the invention.
`
`The invention relates to a combination of tetracyclines and bisphosphonates
`which act synergistically to inhibit, reduce, down-regulate and/or prevent degradation
`of connective tissue, basement membrane as weil as other factors in subjects
`susceptible to this type of tissue degradation.
`
`BACKGROUND OF THE INVENTION
`
`Proteolytic activity is responsible for darnage to connective tissues and
`basement membranes as a complication ofthe inflammatory and/or immune response
`and other disease processes, such as cancer cell invasion and metastasis. The
`inflammatory response contributes, for example, to the pathological changes in a
`number of acute and ehrenie processes involving diverse argans and tissues such as the
`lungs, bone, heart, joints, skin and periodontium, etc.
`
`The proteinases involved in these responses or disease processes include matrix
`metalloproteinase (NfMP's), M!v!P-like proteinases and related proteinases, serine
`proteinases and other proteinases. The .WIMP' s are zinc <rnd calcium-dependent for
`hydrolytic cleavage of substrate proteins and are secreted or released by a variety of
`hast cells ( e.g., polymorphonuclear neutrophils (P:MN' s), macrophages, bone cells,
`epithelium and fibroblasts). Certain other genetically distinct ~IP's called
`membrane-type MNIP' s 0-;IT -"NIMP' s) are cell membrane-bound; others are secreted
`into the extracellular matri.x (EC}v-1). With serine proteinases, the amino acid serine
`acts as a nucleophile for hydrolytic cleavage of substrate protein. Serine proteinases
`are released, e.g., by triggered leukocytes, more specifically by the azurophilic granules
`ofP~' s, and other cells including malignant tumor cells.
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`Exh. 1013
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`PCTJUS99/22199
`Several studies have shown that the expression and activities ofMNIPs are
`pathologically elevated over the body' s endogenaus anti-proteinase shield in a variety
`of diseases such as cancer metastasis, rheumatoid arthritis, multiple sclerosis,
`periodontitis, osteoporosis, osteosarcoma, osteomyelitis, bronchiectasis, ehrenie
`pulmonary obstructive disease, skin and eye diseases. Proteolytic enzymes, especially
`:MMPs, are believed to contribute to the tissue destruction darnage associated with
`these diseases.
`
`Same metalloproteinases (MMP' s) and their association with diseases are
`discussed by M.E. Ryan, et al., Curr. Op. Rheum., 1996, 8:238-247. More than twenty
`:MMP's have been identified and the nurober is growing. These include Interstitial
`Collagenases .MMP-1 (fibroblast-type), :M:MP-8 (polymorphonuclear leukocyte-
`PMNL- type or collagenase-2), MNfP-13 (collagenase-3); Gelatinases i'vThtfP-2 (72-kD
`gelatinase A) and MMP-9 (92-kD gelatinase B); Stromelysins i'vThtfP-3 (stromelysin-
`1 ), :MMP-1 0 (stromelysin -2), and MMP-7 (matrilysin or putative metalloproteinase
`(PUMP) -1); Membrane Type (MT-MMP's), MNfP-14 (NIT 1-Mi'vfP), MNfP-15 (MT2-
`:MMP), MNfP-16 (MT3-MrvfP); others are, for example, :MMP-11 (stromelysin -3),
`:NI.MP-12 (macrophage metalloelastase) and YIMP-20. Enamelysin (1--D.-fP-20) is
`described by Llano et al., Biochem. 1997, 36:15101-15108, and can also be expressed
`by human cancer cells such as squamous carcinoma cells of the human tongue
`indicating its potential contribution to cancer progression and invasion (Salo et al., J
`Dent. Res. 1998, 77:829, Abstr. No. 1978). Related proteinases include TACE's and
`AD Al\11' s fertilin or meltrin ( metalloproteinase/ disintegrin).
`
`M1vfP's, i'v!NfP-like and related proteinases such as TACE's, ADAl'v1's, etc.,
`are involved in processing and modification of molecular phenomena such as tissue
`remodeling (Birkedal-Hansen, Current Opin. Ce!! Bio!. 1995, 7:728-735; JF
`Woessner, Jr., FASEB J 1991, 5:2145-215J, cytokine actions (S. Chandler et al., J
`Neuroimmunol. 1997, 72: 155-161), cell-cell fusion (RH van Huijsduijen, Gene 1998,
`206:273-282; Huovila et al., Curr. Opin. Ce!! Bio/. 1996, 8:692-699; Yagami-
`Hiromasa et al., Nature 1995, 377:662-656), angiogenesis, growth factor actions,
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`integrin and other adhesion factors and their receptor processings. See also, AC.
`Perry et al., Biochem. Biophys Acta 1994, 1207:134-137. The ADM1 enzymes are
`membrane proteins with A Disintegrin and Metalloproteinase Domain CN olfsberg et
`al., Dev. Bio/. 1995, 169:378-383). TACE is tumor necrosis factor converting
`enzyme.
`
`MN!P-like proteinases and related proteinases are metalloproteinases distinct
`from dassie M11P' s and can be involved in cellular processing of pro-TNF alpha
`(Tumor Necrosis Factor), cellular shedding of cytokine receptors, adhesion molecules,
`etc. as described by S. Chandler et al., J. Neuroimmunol. 1997, 72:155-161. MMP's
`and MMP-like and related enzymes, e.g., ADA.J.vf's, TACE's, etc., also mediate the
`release ofTNF alpha r.:Natanabe et al., Eur. J. Biochem. 1998,253: 576-582) and are
`involved in membrane-bound processing of TNF alpha by monocytes induced by
`bacterial-virulence factors. This event is mediated by membrane-bound
`metalloproteinases. Shapira et al., J. Period Res. 1997, 32:183-185.
`
`There is extensive evidence for the association between proteinases and a
`large number of disease processes. Microbial proteinases can act in concert with hast
`proteinases in the promotion oftissue destruction as seen in periodontium (Sorsa et al.,
`lnfect. Immun.1992, 60: 4491-4495). Recent studies indicate that a serine protease,
`i.e., elastase, may play a role in connective tissue breakdown and tissue invasion in the
`Dunning rat model of cancer invasion and metastases (prostate cancer) (Lowe and
`Isaacs, Cancer Res. 1984, 44:744-52). Also involved in irutiating the proteinase
`cascade that mediates tumor invasion and metastasis are trypsin and chymotrypsin-like
`activity (Sorsa et al., J. Bio/. Chem. 1997, 272:21067-21074). Serine proteinase is
`expressed in human cancers such as ovarian carcinoma and cholangiosarcoma (Sorsa
`et al., J. Bio/. Chem. 1997, 272:21067-21074).
`
`The role ofMMP's has been well-established in a great many disease states,
`e.g., tumor invasion and metastasis (Stetler-Stevenson et al., Annu. Rev. Cell Bio/.
`1993, 9:541-73; Tryggvason et al., Biochim. Biophys. Acta 1987, 907:191-217) and
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`bone and cartilage degradation ( Greenwald et al., Bone 1998, 22:33-38; Ryan et al.,
`Curr. Op. Rheumatol. 1996, 8;238-247). :MMP-20 is expressed by oral squamous cell
`carcinoma cells (Salo et al., J. Dent. Res. 1998, 77:829, Abstr. No. 1978).
`Bourguignon et al. (Mol. Bio/. Ce//. 1997, Si Supplement, Abstract 1603) describe the
`association of metalloproteinase with matrix degradation as being responsible for
`promoting lymphocyte infiltration that destroys insulin-producing pancreatic islet cells.
`Cytokines (TNF alpha) and :MMP's have also been implicated in the pathogenesis of
`multiple sclerosis (Liedtke et al., Ann. Neuro/. 1998, 44:35-46; Chandler et al., J.
`Neuroimmuno/.1997, 72:155-71). MT1-:MMP has been found to act in the growth and
`spread ofbreast cancer cells (Li et al., Mol. Carcinog. 1998, 22:84-89).
`
`There are many other disorders in which extracellular protein
`degradation/destruction plays a prominent role. Examples of such diseases include
`osteoporosis, arthritides, acquired immune deficiency syndrome (AIDS), bums,
`wounds such as bed sores and varicose ulcers, fractures, trauma, gastric ulceration,
`skin diseases such as acne and psoriasis, lichenoid lesions, epidennolysis bullosa,
`aphthae (reactive oral ulcer), dental diseases such as periodontal diseases, peri-
`implantitis, jaw cysts and other periapical cysts, dental conditions which are the target
`of root canal treatment or endodontic treatment, related diseases, external and intrinsic
`root resorption, caries etc.
`
`The serine proteinases include human leukocyte elastase (HLE) and cathepsin
`G, and additional serine proteinases are involved in the cascade of pathways involved
`in connective tissue breakdown including but not limited to, plasmin, plasminogen
`activator, tumor-associated trypsins, etc.
`
`MN!P' s and serine proteinases can work in combinations to bring about
`destruction of most of the elements of the extracellular matrix and basement
`membranes. As examples ofthe major interaction between NfMP's and serine
`proteinases during tissue breakdown, 1) cathepsin G can activate NIMP-8; 2) the serine
`proteinase Human Leukocyte Elastase (HLE) can inactivate TThtiP's, the major
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`endogenaus Tissue Inhibitors ofMatrix Metalloproteinases, 3) .M:MP-8 and .M:MP-9
`can inactivate ct1 -Proteinase Inhibitor ( ct1 -PI), the major endogenaus inhibitor of
`human leukocyte elastase, (S.K. Mallya, et al., Annuals oj the New York Academy of
`Science, 1994, 732:303-314) and 4) tumor-associated-trypsin-2 can efficiently activate
`latent pro .M:MP's (Sorsa et al., J. Bio/. Chem. 1997, 272:21067-21074).
`
`Tetracyclines, including chemically modified tetracyclines, can inhibit .M:MP-
`mediated tissue breakdown in vitro andin vivo, in part by binding to metal ions
`(calcium or zinc) in the .M:MP molecular structure. See, e.g., R.F. Zernicke et al.,
`Journal oj Rheumatology, 1997, 24: 1324-31; T. Sorsa et al., Journal oj
`Rheumatology, 1998, 25:975-82; Golub et al., Adv. Dental Research 1998, in press.
`
`Certain tetracyclines have been shown to suppress matrix metalloproteinases
`independently oftetracycline antibiotic activity. U.S. Patent Nos. 5,459,135 to Golub
`et al., 5,321,017 to Golub et al., 5,308,839 to Golub et al., 5,258,371 to Golub et al.,
`4,935,412 to McNamara et al., 4,704,383 to McNamara et al., 4,666,897 to Golub et
`al., andRE 34,656 to Golub et al. describe the use ofnon-antimicrobial tetracyclines to
`treat tissue-destructive conditions, ehrenie inflammation, bone destruction, cancer and
`other conditions associated with excess activity of matrix metalloproteinases such as
`collagenases, gelatinase, and :MMP-12 (macrophage metalloelastase).
`
`U.S. Patent No. 5,773,430 to Sirnon et al. describes using hydrophobic
`tetracyclines to inhibit excess leukocyte elastase serine prdteinase activity in a
`biological system.
`
`Tetracyclines are a class of compounds which are particularly weil known for
`their early and spectacular success as antibiotics. Compounds such as tetracycline,
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`sporocycline, etc., are broad spectrum antibiotics, having utility against a wide variety
`ofbacterial and other microbes. The parent compound, tetracycline, has the following
`general structure:
`
`~(G13h
`OH
`
`OH
`OH
`The numbering system ofthe multiple ring nucleus is as follows:
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`Tetracycline, as well as the 5-0H (oxytetracycline, e.g., terramycin ™) and 7-
`Cl (chlorotetracycline, e.g. aureomycin™) derivatives, exist in nature, and are all well
`known antibiotics. Semisynthetic tetracyclines include, for example, doxycyline,
`minocycline and methacycline. The use of tetracycline antibiotics, while generally
`effeetive for treating infection, ean lead to undesirable side effeets. For example, the
`long-term administration of antibiotie tetracyclines can reduee or eliminate healthy
`flora, such as intestinal flora, and can lead to the produetion of antibiotie resistant
`organisms or the overgrowth of yeast and fungi. These signifieant disadvantages
`typieally preclude treatment regimens requiring ehrenie administration of these
`compounds.
`
`Natural tetraeyclines may be modified without losing their antibiotic properties,
`although certain elements ofthe structure must be retained to do so. A dass of
`eompounds has been defined whieh are strueturally related to the antibiotie
`tetraeyclines, but whieh have had their antibiotie activity substantially or completely
`extinguished by ehemieal modifieation. The modifieations that may and may not be
`made to the basie tetraeycline strueture were reviewed by Mitseher, L.A., The
`Chemistry ojthe Tetracycline Antibiotics, Mareel Dekker, New York (1978), Ch. 6.
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`According to Mitscher, the modification at positions 5-9 of the tetracycline ring system
`can be made without causing the complete lass of antibiotic properties. However,
`changes to the basic structure of the ring system, or replacement of substituents at
`positions 1-4 or 10-12, generally lead to synthetic tetracyclines with substantially less,
`or essentially no, antibacterial activity.
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`PCT/US99/22199
`
`Chemically modified tetracyclines (CMT's) include, for example, 4-
`de( dimethylamino )tetracycline (CMT -1 ), tetracyclinonitrile (Cl\.1T -2), 6-demethyl-6-
`deoxy-4-de( dimethylamino )tetracycline ( CMT-3 ), 7 -chloro-4-
`de( dimethylamino )tetracycline ( CMT -4 ), tetracycline pyrazole ( CMT -5), 4-hydroxy-4-
`de( dimethylamino )tetracycline ( CMT -6), 4-de( dimethylamino )-12 et-deoxytetracycline
`(CMT -7), 6-deoxy-5 et-hydroxy-4-de( dimethylamino )tetracycline ( CMT -8), 4-
`de( dimethylamino )-12et-deoxyanhydrotetracycline (CMT -9), 4-
`de( dimethylamino )minocycline ( CMT -1 0).
`
`Further examples of tetracyclines modified for reduced antimicrobial activity
`include the 4-epimers of oxytetracycline and chlorotetracyline ( epi-oxytetracycline and
`epi-chlorotetracycline ).
`
`Bisphosphonates include a class of therapeutic preparations which have been
`used as bone resorption suppressants. U.S. Patent No. 5,652,227 to Teronen et al.
`describes using bisphosphonates to reduce degradation of connective tissue matrix
`protein components which results from excess metalloprot'einase activity. U.S. Patent
`No. 5,688,120 describes inhibiting alveolar bone resorption using iontophoretic
`delivery ofbisphosphonates to alveolar bone by administering bisphosphonate in a
`reservoir connected to gingival tissue and passing an electrical current therethrough.
`
`There has been no suggestion to use tetracyclines and bisphosphonates
`tagether in combination for the purpese of reducing, inhibiting, and down-regulating
`excess endogenaus proteinase activity and to reduce destruction of tissues, basement
`membrane and other factors.
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`It is an object of the invention to provide a combination of compounds to treat
`subjects susceptible to proteinase related tissue darnage and destruction.
`
`SUMMARY OF THE INVENTION
`
`A composition is provided for inhibiting, reducing, and down-regulating excess
`proteinases, thus treating or preventing proteinase-related connective tissue and
`basement membrane degradation in a biological system susceptible to structural and
`functional disturbances due to an excess of proteinase activity. The composition
`includes a tetracycline and a bisphosphonate. The inhibition involves reducing the
`amount and activity of proteinases and down-regulating the endogenaus production of
`the proteinases. The composition of the invention can treat or prevent diseases related
`to proteinase imbalance by downregulating, preventing, or reducing excess activity of
`~'s, serine proteinases, MMP-like and related enzymessuch as the tumor necrosis
`factor converting enzyme (TACE)-dependent tumor necrosis factor alpha (Th'Fct.)
`activation, and membrane proteins with a disintegrin and metalloproteinase domain
`(ADA..Vf's).
`
`The degradation treated according to the invention can involve hard and soft
`tissues including connective tissue and basement membranes. The degradation can be
`associated with conditions such as bone resorption, cartilage destruction or destruction
`of soft tissues, and tissue invasion and metastasis by malignant cells,. The structural
`and functional disturbances include as non-limiting exampfes, lass of teeth due to
`periodontal breakdown and fracture of skeleton due to excess bone destruction, for
`example, during osteoporosis. Excess proteinase is inhibited by a reduction in the
`amount and activity of proteinase and downregulating of the proteinase production.
`Down-regulating means blocking the gene expression and secretion of the proteinase,
`i.e., decreasing the synthesis andrelease ofthe enzyme protein. The therapy can also
`bleck the activity or activation of the proteinases independent of an effect on enzyme
`synthesis.
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`The composition can be in the form of a phannaceutical or cosmetic
`preparation and therefore the composition can be included with a phannaceutical or
`cosmetic preparation or carrier.
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`The composition preferably comprises a combination of tetracycline and
`bisphosphonate in synergistic amounts for inhibiting excess proteinase activity so that
`the combination exhibits synergy in the efficiency of inhibiting, reducing and down-
`regulating of proteinases involved in tissue breakdown. This means that the
`combination is more effective than either tetracycline alone or bisphosphonate alone
`and the efficiency of the combination is generally greater than that expected by adding
`the two effects.
`
`In a method for inhibiting and/or reducing the activity of, and down-regulating
`excess proteinases and related breakdown of connective tissue, basement membranes
`and other factors reflecting functional and structural disturbances in a biological system
`susceptible to this tissue breakdown, a composition which includes a combination of
`tetracycline and bisphosphonate is administered to the system in proteinase inhibiting,
`reducing and/or down-regulating amounts.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`FIG. l isabar graph illustrating Tooth Mobility results in Example 3;
`FIG. 2 isabar graph illustrating Aveolar Bane Loss results in Example 3;
`FIG. 3 isabar graph illustrating Effect on Gingival Collagenase activity in
`Example 3;
`FIG. 4 is a bar _graph illustrating Effect on Gingival Gelatinase activity in
`Example 3;
`FIG. 5 is a bar graph illustrating the Inhibition of Osteoclast Gelatinase in
`Example 4;
`FIG. 6 is a bar graph illustrating the Inhibition of Cancer Cell :Nligration in
`Example 5;
`
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`FIG. 7 is another bar graph illustrating the Inhibition of Cancer Cell Migration
`by another combination in Example 5;
`FIG. 8 is a bar graph illustrating the Inhibition of Casein Degradation by MT 1 -
`MMP (MMP-14) inExample 6.
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`DETAll..ED DESCRIPTION OF THE INVENTION
`
`The invention includes a composition and method for inhibiting tissue
`destructive conditions associated with excess production and activity of connective
`tissue and basement membrane degrading proteinases, e.g., metalloproteinases,
`metalloproteinase-like and related enzymes, serine proteinases and other proteinases,
`as well as microbial, viral and fungal proteinases. Application ofthe composition of
`the invention to a subject in need of treatment inhibits or prevents breakdown of
`connective tissue, basement membranes, and other disease processes.
`
`The tissue destructive conditions which can be treated and/or prevented
`with the present invention result from excess proteinase activity of metalloproteinases,
`metalloproteinase-like proteinases and related proteinases, serine proteinases or
`combinations of these enzymes, as well as microbial, viral and fungal proteinases.
`These conditions include, e. g., tissue invasion by malignant cells, bone resorption,
`cartilage destruction, destruction of soft tissues ( e.g., skin, tendons, Iigaments, blood
`vessel walls, etc. ), as weil as tumor spread and cancer metastasis to both soft and hard
`tissues, and bronchiectasis, ehrenie destructive and obstruC:tive lung disease, asthma,
`and other lung diseases. Mammalian diseases such as periodontitis, osteoarthritis,
`rheumatoid arthritis, reactive and other arthritides, cancer invasion and metastasis,
`osteomyelitis, osteoporosis, osteosarcoma, and other bone diseases can be
`advantageously prevented and/or treated. While it is not intended tobe bound by
`theory, the treatment may be effective at least in part because both the Cv1T' s and the
`bisphosphonates are bone-seeking pharmacologic agents. The combination also can be
`used to treat tissue-destructive diseases in pets ( cats, dogs, etc.) and I arge mammals
`such as horses and other mammals.
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`Particular tissue and basement membrane destructive conditions treated
`according to the invention include as non-limiting examples, bone diseases such as
`osteoclast-mediated bone resorption, and disorders involving cellular passage through
`basement membranes such as cancer metastases and lymphocyte infiltration, e.g., in the
`islets ofLangerhans related to the onset ofType-1 diabetes.
`
`The composition of the invention can be linked to pharmaceutical preparations
`containing molecules that target sites such as tumor tissue, metastasis, and/or
`vasculature for delivery thereto. Examples ofthese molecules are homing peptides
`ry.l. Arap et al., Science 1998, 279:377-380).
`
`A need for treatment can be estimated from or based on various clinical,
`radiological and biochemical parameters of disease severity. One example for arthritis
`diseases includes clinical signs ofjoint pain and weakness, x-ray evidence ofbone and
`cartilage destruction and detection of elevated levels of collagen crosslink (pyridinoline
`and deoxypyridinoline) fragments in serum and urine indicating increased bone and
`cartilage collagen breakdown. A diagnosis of periodontitis and peri-implantitis
`includes, e.g., clinical evidence (e.g., increased depth ofperiodontal pockets; loss of
`periodontal and peri-implant attachment), microbiological, biochemical, immunological
`and/or molecular biology evidence of periodontal tissue breakdown.
`
`Connective tissue forms the extracellular matrix which connects and supports
`other tissues in all parts of the body. Connective tissue indudes collagenaus ( white
`fibers in skin, tendon, bone, cartilage, etc. made up of coiled protein fibrils ), elastic
`(yellow fibers of albuminoid scleroprotein), mucous, reticular (net-like), osseous
`(bone), and cartilagenous ( chondrocytes embedded in ehendrin and including hyaline
`( clear ), elastic or fibrocartilage) and sometimes blood vessels/ components ( endothelial
`cells, e.g., which proliferate at a site ofinflammation). Connective tissue may be
`further classified as loose (areolar) and dense (more fibrous).
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`The basement membrane is a membrane of modified eonneetive tissue beneath
`epithelial tissue, as of a gland eontaining acini or special secreting portions. The
`basement membrane is a complex structure eomprised oftype IV collagen, heparan
`sulfate proteoglycan and laminins, whieh attaches the epithelium to the underlying
`connective tissue. After basement membrane destruction to overcome extracellular
`matrix barriers, specifie cleavage oflaminin-5 by gelatinases (Giannelli et al., Science
`1997, 277:225-228) is required for inflammatory and malignant eell migration. The
`present eombination oftetraeycline and bisphosphonate inhibits this also.
`
`In one embodiment, the eomposition ofthe invention is used to treat .Ml\.fP-
`dependent conditions. MMP-dependent eonditions include, for example, wounds,
`bums, fractures, lesions, trauma, ulcers, eaneer and metastasis progression in
`eonnective tissues and bone; other conditions include periodontitis, gingivitis, peri-
`implantitis, jaw eysts, internaland exrernal root resorption, caries, AIDS, eorneal
`ulceration, gastric uleeration, aphthous uleers, aene, psoriasis, loosening of hip
`prosthesis, osteomyelitis, osteoporosis, tissue remodeling, angiogenesis, arthritides
`(rheumatoid, reactive and osteo arthritis), lung diseases (bronehieetasis and ehrenie
`obstructive pulmonary diseases and other lung diseases).
`
`T etraeyclines in combination with bisphosphonate have been found to inhibit
`the produetion andlor aetivity ofendogenaus proteinases in a biological system ..
`
`The preferred tetraeyclines are 4-de( dimethylamin6}tetracycline (CMT -1 ), 6-
`demethyl-6-deoxy-4-de( dimethylamino )tetraeycline (CMT -3 ), 6-deoxy-5-alpha-
`hydroxy-4-de( dimethylamino )tetracycline (CMT -8), also, doxycycline, minocycline,
`lymecycline and combinations of the tetracyclines.
`
`Bisphosphonates are compounds related to inorganie pyrophosphonic acid and
`are eommereially available or ean be prepared aeeording to known rnethods. The
`bisphosphonates useful herein include as non-limiting examples, e.g., alendronate ((4-
`amino-1- hydroxybutylidene) bisphosphonie aeid), clodronate ( diehloromethane
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`diphosphonic acid), etidronate ( ( 1-hydroxyethylidene) disphosphanic acid) and
`parnidronate ( (3-arnino-1- hydroxypropylidene) bisphosphonic acid); also risedronate
`([-hydroxy-2-(3-pyridinyl)ethylidene] bisphosphonic acid), tiludronate, i.e., tiludronic
`acid ([(4-chlorophenyl) thio] methylene] bisphosphonic acid) and zolendronate.
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`Others include [1-hydroxy-3- (methylpentylamino)propylidene]bis-phosphonate
`(BM21. 095 5), [( cycloheptylarnino) methylene] bisphosphonate (YM175), 1-hydroxy-
`3 -( 1-pyrrolidinyl)-propylidene ]bisphosphonate (EB-1 0 53), [ 1-hydroxy-2-( 1H-
`imidozol-1-yl) ethylidene] bisphosphonate (CGP 42'446) and (1-hydroxy-2-imidazo-
`[1,2-a] pyridin-3-yl-ethylidene) bisphosphonate (YM: 529).
`
`Bisphosphonates are comprehensively described by H. Fleisch, Endocr. Rev.,
`1998, 19(1):80-100; see also, H. Fleisch, Bisphosphonates in Bone Disease: From the
`Labaratory to the Patient, 1997, 3rd Edition. The Parthenon Publishing Group, New
`Y ork and London.
`
`The preferred bisphosphonates are alendronate, clodronate ( clodrinate ),
`etidronate, parnidronate, medronate, nedrinate, tiludronate, zolendronate and
`combinations thereof.
`
`The amount of each campeund in the composition of the invention including
`tetracycline and bisphosphonate for use in a specified case will vary depending on the
`particular composition formulated, the mode of applicatiod, the subject, the site to
`which the composition is administered, the degradative condition being treated or
`prevented, and the mode of administration. Dosages will be deterrnined using
`conventional consideration, e.g., by customary comparison ofthe differential activities
`ofthe formulations and ofa known agent, e.g., by means ofan appropriate
`conventional pharmacologic protocol. Typical doses for human use include 10-1000
`mg/day tetracyclinein combination with 20-2000 mg/day bisphosphonate depending
`upon type ofbisphosphonate and raute of administration. The amounts ofthe
`tetracycline and the bisphosphonates useful in the invention are amounts which in
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`combination result in an inhibition ofthe activity and/or secretion and synthesis of
`excess proteinase in a system or subject susceptible to excess proteinases. These
`amounts are advantageously as much as ten-fold less than amounts which are optimal
`or needed when each campeund is used alone, thereby significantly reducing the
`possibility of side-effects caused by higher doses if the compounds were to be taken
`individually, e.g., when using individual compounds, 10-30J.1.M is needed; tagether 2.0-
`1 0. 5 J.LM is needed.
`
`F or oral administration, the composition of the invention may be formulated
`in the form oftablets, capsules, elixirs, suspensions, solutions, or the like. For
`parenteral administration, the composition may be formulated into injectable forms
`such as solutions or suspensions, e.g., for intramuscular injection. For topical
`application, the composition may be applied directly or incorporated with a delivery
`system such as a carrier or substrate, e.g. a polmer, or formulation into a cream,
`ointment, aerosol, membranes., etc.
`
`The activity of combinations of various tetracyclines a