"NON-ANTIBIOTIC PROPERTIES OF TETRACYCLINES"
`
`Proceedings of a symposium held in
`Garden City, New York
`November 13-14, 1997
`
`Univ. of Minn.
`Bio-Medical
`Library
`
`Robert A. Greenwald
`Lome M. Golub
`Co-editors
`
`

`
`"NON-ANTIBIOTIC PROPERTIES OF TETRACYCLINES"
`
`Proceedings of a symposium held in
`Garden City, New York
`November 13-14, 1997
`
`Univ. of Minn.
`1 Bio-Medical
`Library
`
`Robert A. Greenwald
`Lome M. Golub
`Co-editors
`
`Exh. 1027
`
`

`
`TETRACYCLINES INHIBIT
`CONNECTIVE TISSUE BREAKDOWN
`BY MULTIPLE NON·ANTIMICROBIAL MECHANISMS
`
`L.M. GOLUB
`H.-M. LEE
`M.E.RYAN
`
`Department of Oral Biology and Pathology, School of Dental
`Meclicine, SUNY at Stony Brook, Stony Brook, NY
`w.v. GtANNOßiLE
`Departments of Perioclontology, Harvarcl School of Dental
`Medicine ancl Forsyth Dental Center, Boston, MA
`
`J.PAYNE
`
`Department of Su rg ical Sciences, School of Dentistry,
`University of Nebraska Meclical Center, Omaha, NE
`
`T. SORSA
`
`Department of Perioclontology ancl Meclical C hemistry,
`University of He lsi nki , Finlancl
`
`Adv Den! Res 12:12-26, November, /998
`
`Abstract-A seminal experiment involv ing a germ-ji·ee rat
`moclel of connective tissue breakdown (followecl soon thereafter
`by a series of in vitro stuclies) iclentified an unexpectecl non-
`antimicrobial prope1ty of tetracyclines (TCs). This ability of TCs
`to inhibit matrix metalloproteinases (MMPs) such as collagenase
`was found to reflect multiple clirect ancl inclirect mechanisms of
`action, ancl tobe therapeutically useful in a variety of dental (e. g.,
`adult perioclontitis) and meclical (e.g. , arthritis, osteoporosis,
`c<mcer) cliseases. The site on the TC molecule responsible for its
`MMP-inhibitory activity was iclentified which led to the
`development of a series of chemically moclitiecl non-antimicrobial
`analogs, callecl CMTs, which also have therapeutic potential but
`clo not appear to induce antibiotic sicle-effects. Longitudinal
`double-blind studies on humans with adult perioclontitis have
`eiemonstrared that a sub-antimicrobial close of cloxycycline
`Cpr~ v 10u s ly reported to suppress collagenase activity in the
`penodontal packet) is safe ancl effective ancl has recently been
`approvecl by the FDA as an adjunct to scaling ancl root planing.
`
`Key words: Non-antimicrobial tetracyclines, doxycycline,
`collagenase, matnx metalloproteinases, host moclulation.
`
`Pres ented at th e Workshop on th e "Non-Antib iotic
`Properlies of'Tetracyclines", held November 13- 14, 1997, in
`Garden City, New York, sponsored by the Long /stand Jewish
`Medtcal Center, CollaGenex Pharmaceuticals, Jnc., and the
`Nationalinstitute q/Dental Research (NIH).
`
`A s recently reviewecl, the early sc ie nce ( 1972-
`
`1982) that lecl to the cliscovery, by Golub ancl his
`"Stony Brook" g roup , of tetracycline's a nti -
`collagenolytic properties was originally clirected
`at exp lainin g how diabetes increases the seve rity of
`perioclontal di sease, and why cliabetics are often (not always)
`refractory to periodontal therapy (Golub et al., 1996). In their
`init ial stucl ies on thi s systemic disease/local disease
`interaction, they observed that alloxan-incluced type 1 (insulin-
`deficient) diabetes in the rat increased the urinary excretion
`of hyclroxyproline, an amino acid " marker" of col lagen and
`its clegraclation fragments, ancl reclucecl the solubility (at 4oC)
`of collagen in the g ing ival tissues (Ramamurthy et al., 1972).
`These a nd a series of subseq uent experime nts led to the
`concl usion that experimental cliabetes producecl a cascade of
`abnormalities in collagen metabolism in the perioclontal and
`other tissues, including: enhanced collagen breakdown (both
`intrac e llul arly following synthes is of the proc o llagen
`precursors, a nd extrace llularly after incorporation of the
`secreted and processed collagen into the matrix) ; suppressed
`intracellular collagen biosynthesis; and increased formation
`of intermo leCLilar covalent cross links in the extracellular
`matrix fibrils (premature "ag ing" of co llagen), the latter
`mecliated both enzymaticall y by lysyl ox iclase, and non-
`enzymatically following binding of g lucose to collage n to
`form aclvancecl glycosylation e nd -proclucts, or "AGEs"
`(Golub et al., 1978a,b; Schneir et al. , 1982; Ramamurthy et
`al. , 1983; Leung et al. , 1986; Ryan et al. , 1995). One oral
`manifestation of these abnormalities was the detect1on of
`f' c1· b t .. ·ats (Ram'JlllLJrthy et al.
`.
`.
`.
`osteoporos1s 111 the JHWS o
`Ja e IC '
`'
`.
`. •
`1973a), with type 1 collagen constituting 90% of the organ1c
`Only 1·ecently has the re latJOnshq) between
`·
`f b
`matnx o
`one.
`. ..
`osteoporosis (such as that seen in .estrogen-dehc1ent post-
`menopausal women as weil as lll diabetJcs) and accelerated
`periodontal breakdown begun to be c lanhecl (Ryan et al.,
`1995; Grodstein et al., 1996; Payne et al. , 1997).
`Although inclucing diabetes in the rat with either of two ß-
`cell-clestructive agents, alloxan and streptozotocin, produced
`th e comp lex seri es of co ll agen abnormalities described
`above, the observation whic h eventually lecl to the seminal
`tetracyclineexperimentwas o ur finding in the 1970's that
`g ingival tissues from hyperg lycem ic cliabetic rats produced
`dramatically elevated col lagenase activity compared w1th the
`low activity of thi s specific neutral proteinase generated by
`non-diabetic controls (Ramamurthy et al., 1973b; Golub et
`al. , 1978b). lnterstitial collagenase was the first-di scovered
`(early 1960 's ) of a family of ca ++_cJep e nclent zn++
`
`12
`
`~ I
`
`Exh. 1027
`
`

`
`VoL. /2
`
`13
`
`(---8~
`t Sub-gingival
`
`Gram-negative
`microorganisms
`)
`
`I f
`Altered
`host
`metabolism
`
`T ET/?,\ C YCUNES I N N/13 /T CoNNJ::C'TJVI:: T tssu E B REAKDOWN JJY Mut.Twu~· NoN -ANTIM JCROtJ/,\ 1. t\1/t:"('/-/A NISMS
`enclope ptidases (Gross and La i
`.
`c
`matri x metallopt·ote ·
`(M p .ete, 1962), no w called the
`mases MPs) Th
`res p o n s ibl e fo r d ecr .. ct·
`·
`ese MMPs are largely
`o ld tn <> th e V'lrl O
`.
`.
`connective tissues d
`.·
`"'
`'
`u s c o n s tttu e nt s of
`Llltng normal rem d I.
`.
`0 e mg and pathologtc
`(G
`tissue bre·tkdow
`reenwald et 1 1998
`n
`.
`•
`<
`· a .. ,
`; Konttmen et al. ,
`h
`.
`1998). Since colhne .
`<"' nase as Ion<> been tl
`b
`".1 .
`. 10Llo 1t to e the onl v
`.
`"'
`Proteinase 'lbl e t d
`o eg l a cl e th e tnple I
`I.
`I
`,
`molecttle under h siolo a· , ,
`. .
`- l e t ca c o II a ge n
`d Q . P Y
`o 1c condtttons (althou<>h recentl y
`A.
`Ut <>Jey f 1995) d
`tmes an
`0
`'
`'"
`emonstratecl that another M M p
`.
`gelatmase A , also possesses " t .· 1 h 1.
`"
`. .
`'
`11P e e tcase acttvtty) we
`·
`concludecl that the excess urin·try ex,. t.
`f
`' .
`cte ton o hyclroxyprolme
`•
`-
`•
`.
`<
`ex htbtted by dtabettc rats (see abo
`)
`. d
`.
`.·
`.
`'
`ve , an
`the unus ually
`severe pettodontdl breakdown exhibt.tecl b
`y man y (not all )
`.
`.
`.
`.
`clt a bett c p att e nts a ncl dtabe ti c rats
`.
`Iei b
`.
`.
`·, cou
`e re la te d to
`tncreased expresston ancl activity of col1 ·1cr
`(G 1 b
`o u et
`c
`. .
`, oenases
`al. , 1978b). Cut1 e ntl y at leas t tl11·ee c·
`11
`.
`,
`•
`o age na ses a re
`recogmzed, MMP- 1, MMP-8, ancl MMP- 13, althouoh MMP-
`"' ·
`·
`1 could not have been respo nsible fo1- OLl. b .
`1 o servat1 ons m
`.
`.
`.
`.
`·
`dwbettc rats, sn1ee thts colla<>enase 1·s 110t p ·od
`' b
`d
`.
`"'
`.
`1 ucec. y ro ent
`ttssues (Greenwalcl el al., 1998).
`
`t Endotoxin
`
`collagenase
`
`t Gingival
`t
`t Collagen
`
`b
`
`b
`
`Semina) tetracycline experiment
`·
`A s o utline cl in F iu
`I we
`.
`nex t qu es t10n e cl w he th e r the
`o ·
`'.
`elevatecl collagenase act1vity in tl1e u1.11 o 1·v·1 t' tl
`cl ' b
`·
`' o
`1e
`ta et1c rat
`o
`"'
`.
`could be ex plamed by a shift tow·1rd ·1 11101-e
`b. G
`anaero 1c
`ram-
`.
`.
`.
`.
`,
`'
`negatJ ve mJcrotlora 1n the <> inui v·ll c1·ev1'ce th
`·
`us uenerattno
`'
`'
`elevated enclotoxin Ievels which could penetrate th: cliabetes~
`damaged crev1cular epithelium/basement membrane into the
`subepithelial connective tissue and stimulate host cells, such
`as fibro bl as ts ancl/or macrophages , to produce excess ive
`collagenase (McN amara et a/. , 1982). Our alternative hypo-
`th es ts was th at clwbe tes tncreasec! g in g iv a l collage nase
`activity and, as a result, acceleratecl periodontal breakdown
`by a mechanism independent o i' micro bi al factors anc! clue
`onl y to altered host respo nses (e.g., Vlassara et al. 11 988 1
`proposed that elevatecl ex pr~ss ion o f MMPs by mac rophages
`in the chabet1C tt ssues was mcluced by bincling o l· AGEs to
`s urface rece pto r: o n th ese ce ll s) . T o ide ntify whe the r
`microbtal or host tactors were cau ati ve, Golub et al. ( 1983)
`re ported that treatment of the hyperglyce mic cl iabetic rats
`w ith min ocyc lin e, a se mi -sy nth e ti c te tracyc lin e (TC),
`" normalized" the excess collagenase acti vity generated by thc
`gingival ex plants in culture, even though the dru g c!id not
`affect the severi ty o f hyperglycemia. T o cletermine whether
`th e T C th e ra p y was sec undari/v re du c in g m a mmali a n
`coll agenase activity in the gingiva by .first suppressing the
`G ra m -negative mi c ro fl ora (the latte r wo uld pres uma bl y
`reduce bacterial enclotox in Ievels in the gingival crevice and,
`as a result, suppress gingival Collagenase acti vity: Fig. I), the
`in vestigators carried out a second ex periment, using ge nn -
`fr ee rats which we re re neiered cli abe ti c by stre ptozotoc in
`injecti on and treat d with a TC. to eliminate any microbial
`factors. To the amazement (ancl clelight) of the investi gators,
`min ocyc line tre atm e nt proc!u ced th e sam e re clu c ti o n in
`coll aoe nase acti vity in the ge rm-free rats, whi ch lac kecl a
`micr~'lora, just as it die! in conve ntional rats which exhibit a
`complex oral micro flora (Go lub er al. , 1983 . 1984) . These
`ancl a series o f subsequent expenments clearl y demonstratecl
`
`destruction
`
`Fig. 1- ExtJe rimenta/ diob etes induces e.rcess g in g i val
`collagenase acti vitv in tissue culture: mediared by microbial
`or hostjäc/ors ~
`
`that T Cs possess an un cx pec ted pro pc rt y and led us to
`propose the following : (i) that TCs can inhibit mammali an
`coll age nol ytic acti vit y by mechani sms indeJiendent o r the
`antimicrobial property o r these antibi otics, ancl (ii ) that this
`new ly cli scoverecl propert y o f these dru gs could provide a
`novel therapeutic approach to the treatment o f a variety o r
`dental (e.g. , peri oclontiti s, root caries, intra-oral ulcers, _and
`a rthriti s o f th e te mp o ro rn a ndibular j o int ) a nc! me dl c al
`di seases (e.g., the arthritides. different types of osteoporosis,
`cancer invas ion and metastasis) characteri zed by excessive
`collagen breakclown. A number of papers at thi s symposium
`adclressed both o r these working hypotheses.
`To begin to iclentify mechanisms, we examined the ellects
`o f T Cs in vilro Oll the co ll age nase ac ti v it y in ext ract s o r
`polymorphonuclear leukocytes (PMNLs) and in conditioned
`meclia ol· cultured macrophages (both types o r inrlammatory
`ce ll s we re iso lat e cl fr o m g lycoge n-indu cecl pe rit o nea l
`exudates of systemicall y normal adult rats) and founclthat: (i)
`the acldition of minocycline (but not other antibiolies such as
`penicillin, stre ptomycin, and metroniclazole) to thc incubation
`mi xture, in concentrations ran ging l'rom 2-25 J.LglmL (Ievels
`J'ound in the serum and GCF o r humans orall y ad mini sterccl
`this or other TCs), inhibitecl thc acti vity o r Collagenase rrom
`both cell ty pes in a dosc-dependent fas hion (Golub et al ..
`1983, 1994) ; ancl (ii ) adding inc reas ing co ncentrations o r
`C a++ to the inc ubatio n mi xture progress ive ly bloc kcd the
`ability of minocyc line to inhibit collage nasc acti vity in vitm
`(Golub et al ., 1983) . More recent in l'ilm stuclies showed that
`p.M concentrations of Z n++ (L. Yu el al .. 199 1: Lee er al ..
`1992) could produ ce the same ''bl ock" o f thi s anti -MMP
`
`Exh. 1027
`
`

`
`14
`
`GOLU/J ET AL.
`
`Am' DfNT Rn NovEMIIER !998
`
`property of TCs a s m~ concentrations of Ca++. These earl y
`stud1es w~re rap1dly foll owed by wide-ranging efforts by a
`11Limber of research teams to confirm this non-antim icrobial
`property of TCs, to elucidate the mechanisms involved, and
`to explore the immense therapeutic potential of this discovery
`(Golub et al., 1987, 199 1, 1997; Tamargo et al., 199 1; L. Yu
`et al., 199 1; Soto mayo r et al. , 1992; Co le et al., 1994 ;
`Masumori et al., 1994 ; Brandt, 1995b; Paeman et al., 1996;
`W illiams et a/., 1996; Greenwald et al., 1998).
`
`TCs PLEIOTROPIC THERAPEUTIC EFFECTS:
`ADDITIONAL NON-ANTIMICROBIAL
`MECHANISMS OF COLLAGENOLYTIC
`INHIBITION
`
`TC-me~iated EXTRACELLULAR anti-collagenolytic
`mechamsms
`In a series of experiments addressing mechanisms, in addition
`to the earl y approach whic h was directed at inhibiti o n of
`extracellular active forms of collagenase in vitro (see above),
`Golub and his colleagues in vestigated the specificity of the
`anti-collagenolytic activity of TCs and located the site on the
`TC moleet!l e respo ns ibl e fo r thi s host-modul ating, no n-
`antimicrobial property of the drug (Golub et a/., 1987, 199 1,
`1992). Regarding the former mechanism, TCs were found to
`directl y inhibit collagenases from a wide variety of human
`a nd a nim a l ce ll s, in c ludin g ( but no t limite d to) PMN
`le ukocy tes, m ac ro ph ages, os teo bl as ts, os teoc las ts,
`chondrocytes, and malignant melanoma cell s (Zucker et al. ,
`1985; Golub et al., 1987, 1992; Ramamurthy et al. , 1990).
`Other investi gato rs confirmed thi s effect using additi o nal
`human and animal sources of collagenase (Maehara et a/. ,
`1988; Burns et al., 1989; McCulloch et al. , 1990; Cole et al.,
`1994 ; Mas umo ri et al., 1994). lt is no w recog ni zed th at
`coll agenase-3 (MM P-1 3) is more sensiti ve to TC inhibition
`OC50 < l J.LM) than collagenase-2 (MMP-8; !C50 -
`30 J.LM)
`and that coll agenase- 1 (MMP- 1) is the least sensitive (IC50 >
`200 J.LM) to the inh ibition of enzyme activity by these drugs
`in vitro (Golub et al., 1995; Smith et al. , 1996; Greenwald et
`al., 1998), although the mechanisms are more complex than
`o ri g in al ly tho ug ht (see be low). O th e r MMPs- na me ly ,
`ge la tin ase A (MMP-2) a nd ge la tin ase B ( MMP-9) a nd
`macrophage metall oelastase (MMP- 12) and other ty pes of
`M M Ps- are also inhi bited by these drugs, whereas othe r
`classes of proteinases such as serine proteinases (e.g., PMN
`e las tase and plasm in ogen activator) a nd ac id prote inases
`(e.J:., cathepsins Band L) are not (G reenwald et a /., 1987;
`Go lub et a l. , 199 1, 1992; Pae m e n et a l ., 1996) . On e
`excepti o n to th is pa ttern o f p ro te in ase in h ibiti o n was
`identifi ed by S imon et al. (unpubli shecl res ul ts): A highl y
`lipophilic non-antimicrobial TC analog, 6-demethyl 6-deoxy
`4-de-cl imethyla mino te tracycl ine (now callecl CMT- 3; see
`below), but not other TCs, appears to inhibit PMN elastase, a
`serine prote inase, competiti vely.
`Regarding the site on the TC molecule responsible for its
`ant1-MMP actt v1ty, Golub et a/. ( 1987) producecl the first of
`the ir ch e mi ca ll y moclifi e cl T Cs, now ca ll ed C MTs, by
`re m ov tn g the cl tm e th y la min o g ro up fro m the car bo n-4
`
`positi o n [thi s side-chain is required for the a ntimicrobi al
`acti vity of TCs (Mitscher, 1978)] and found that the resttlting
`compound, 4-de-dimethyl amin~ TC, or CMT- l , had lilli.1 its
`antimi c ro bi a l pro perty but re ta in e d (ac tuall y s ho wed
`enhancecl) anticollage nase acti vity both in vitro and in vivo
`(Go lu b et al., 1987; Z. Yu et al., 199 1 ). C urre ntl y, I 0
`di ffe re nt no n-a ntimic ro bi al c hem ic a lly modi f ied TCs,
`namely, CMT- 1 through CMT- 1 0, have been generated, with
`diffe re nt s ide-c ha in s re m oved and/o r add ed to th e T C
`moleettle, and 9 of the I 0 compounds were fo und to be non-
`antibacterial MMP inhibitors (see reviews by Greenwald er
`al., 1998; Golub et al., 1992; Ryan et al., J996b). However,
`when the carbonyl oxygen at C- 11 and the hydroxyl group at
`C- 12 were repl aced by nitrogen atoms, thus producing the
`pyrazo le analog of TC (Valcavi et al., 1963), now call ed
`CMT-5, this compound was found to be the onl y CMT which
`lost its MMP inhibitory acti vity (Golub et al., 199 1 ). These
`stuclies de monstrated that thi s important (but not the only)
`Ca++ and Zn++ binding site (the ß-di ketone moiety at C- 1 I
`ancl C-12) was responsible, at least in part, for inhibiting the.
`extracellular acti vity of MMPs (intracellular mechanisms ol
`M M P reg ulati o n by T Cs a re desc ribed be low). Rece nt
`enzyme kinetic stuclies have inclicated that doxycycline, the
`most potent MMP inhi bitor of antimicrobial TCs (B urns ef
`a /., 1989 ; Golub et al., 1995), acts as a no n-competitive
`inhibi tor of collagenase (Sorsa et al. , 1994), whereas CMT-3,
`among the most potent collagenolytic inhibitors of the non-
`antimicrobi al TC anal ogs (Rifkin et al., 1994; Ryan et al. ,
`1996b; Greenwald et al., 1998), appears to exhibit this, plus
`more complex mechanisms of inhibition, at least for cancer
`cell MMP-2 (gelatinase A) and isa more potent inhibitor than
`doxycycline in vilro (Seftor et a/., 1998). ln this regard, (i)
`th e re la ti ve e ff icacy of T Cs a nd C MTs a ppea rs to be
`pos iti vely correlated to the Zn++ bincling e fficacy of these
`compounds (Us man et a/., 1996), and (ii) converting anti -
`microbial TCs, such as doxycyc line, to their nonantimicrobial
`analogs (such as C MT-8, the no n-a ntimi cro bi al analog of
`doxycyc lin e), a ppears to in c rease the MMP- inhi b ito ry
`effi cacy and some other therape utic, matrix-sparing, acti vities
`of these drugs (Paemen et a/., 1996; Zernic ke et a/., 1997;
`Greenwald et al., 1998; Ohyori et al., 1998).
`Numerous stuclies have found that TCs/CMTs can inhibit
`MMP acti vity and connecti ve tissue breakdown in vivo and in
`vitro in vari ous diseases in humans and experimental animals.
`As examples of in vitro efficacy, TCs and CMTs were found
`to inhibit bone resorptio n in both o rgan a ncl cell culture,
`rega rcll ess o f w he th e r t he reso rpti o n was inclu ce d by
`parathyroid hormo ne (PTH), PGE0 , or bacteri al endotoxi n
`(Golub et al. , 1984; Gomes et al. , (984; Ri fk in et a/., 1994).
`Doxycycline CDOXY) was the most potent MMP inhibitor of
`the an timicrobial TCs. Of the non-antimicrobial C MTs tested,
`CM T - 1 a nd -3 a nd C MTs-6, -7, a nd -8 we re effec ti ve
`inhibi to rs o f bo ne resorpti o n in c ul ture (C MT- 8 was the
`single most potent compound), whereas C MT-2, -4, and -5
`were not. (Note: CMT-2 and -4 did block PMN collagenase
`but no t b o ne ce ll co ll age nase, w he reas C M T-5 was
`ineffective as an inh ibitor in all acti ve col lagenolytic systems
`teste cl. ) As ot he r exa mpl es : min ocyc lin e a nd C MT - 1
`
`Exh. 1027
`
`

`
`T EHIACYCUNES I N!/1/IIT CONNIXTIV/o' T!SSU/o' B Rf',\ KDO\VN /J)' /v/ UI .T/1' /L NON-A NT/M/CIW/1/M . !vi!T //,\ NISMS
`
`15
`
`TABLE
`
`TETRACYCLINES (TCs) INHIBIT CONNECTIVE TISSUE BREAKDOWN: PLEIOTROPIC MECHA N ISMS
`
`(A) Mediated by extracellular mechani sms
`• Direct inhibition of active MMPs-dependent on Ca++ and Zn++ binding properties of TCs
`independent of cation-binding properlies of TCs
`• Inhibition of oxidative activation of Pro-MMPs-
`• TCs disrupt activation by promoting excessive proteolysis o f pro-MMPs into enzy maticall y-inacti ve fragmen ts-
`dependent (?) on cation binding of TCs
`• Inhibition of MMPs protects a 1-PI , thus INDIRECTLY 1 serine proteinase (e.g., PMNL elastase) activity
`(ß) Mediated by cellular regulation
`• TCs 1 cytokines, iNOS , PLA,, prostagtandin synthase
`• Effects on protein kinase C, c~1lmodu lin
`(C) Mediated by pro-anabolic effects
`• TCs i collagen production
`• TCs i osteoblast activity & bone formation
`
`inhibitecl malignant melanoma cell collagenase in vitro ancl
`blocked these cancer cells from lysing co-cultured host ce lls
`(Zucker et a/. , 1985), and these same (ancl other) TCs in vitro
`inhibitecl MMPs deri ved from inflamecl human gingiva and
`rheumatoid synovium (Golub et o/., 1987; Greenwald et ol ..
`1987), fro m human lung cancer cell s (Zucker et al .. 1989),
`from cliabetic rat skin ancl gingiva (Golub et al .. 1983. 1987).
`from rachiti c rat cartilage (Greenwalcl et al., 19 88 ), from
`osteo-arthritic human cartilage (L. Yu et al ., 1991 ), from
`ulceratecl rabbit cornea (Seedor et al. , 1987; Burn s et al. ,
`1989), and fro m human ging ival fluid (Golub et al .. 1983,
`1991; Maehara e t al. , 19 88; M c Culloch e t al ., 1990) .
`Moreover, whe n TCs or CMT-1 were aclmini stered p.o. in
`vi vo , they were founcl to recluce patho log icall y e xcessive
`collagenase (ancl gelatinase) activity, ancl/or to inhibit matri x
`breakclown, in the gingival tissue ancl GCF of humans (TCs
`onl y) wit h adult pe rioclontiti s (Golub et al. , 1983, 1995 ,
`1990a) , in s ynovia l tissu e ancl fluid of human s with
`rh eumatoicl arthriti s (Greenwalcl et al. , 19 87), in skin and
`ging iva of rats w ith diabetes (Golu b et al .. 1983 , 1987), in
`cartilage of dogs with osteo-arthritis (ßrandt, 1995b) and rats
`with rickets (Gree nwalcl et al. , 1988), and in humans and
`rabbits with sterile cornealulcers <Seedor et al., 1987) . (Note :
`CMTs have not ye t been approved for hum an use by the
`FDA , although the National Cance r In stitute has rece ntly
`initiated preliminary stuclies, using CMT-3. on humans with
`cancer .) M o re re c e nt stucli es ha ve d e mon s trate d th e
`therapeutic potential of TCs' anti-MMP acti vity in in vi vo
`and cell cu lture models of cancer invasion , metastasis, ancl
`ang ioge nes is (Zuc ker et al .. 1985 : Tamargo et a/. , 1991 ;
`Sotomayor et a/., 1992; Masumori et a/ .. 1994; Lokeshwar et
`a/., 1997 ; Seftor et al. , 1998).
`A s s ummari zecl in th e Tab le. e ve n th e ex trace llular
`mechani sms (intracellular are cliscussed in a later section of
`thi s paper) of TC inhibition of rnatri x clegraclation are now
`recog ni 7.e cl to be p le iotropic ; additional ex ampl es no w
`follow:
`
`TC effects on pro-MMP activation
`The initial ex periments on this mechanism were carriecl out
`
`b y m e an s of an anim a l m o d e l o f mi c robiall y inclu ce d
`p e ri o dontal bre akd ow n. In bri ef, thi s m o d e l in vo lv e d
`infecting either germ-free or pathogen-reduced rats with the
`human pe ri od on t opa l ho ge n. Po rph v romona s g in g i va I is
`(P.g.) . which results in clevated MMP (collage nase or MMP-
`8 and gelatinase or MMP-9) acti vity in the g ingival ti ssues
`(as ex pected. MMP-1 was not t'ound in the rat ti ssues) and
`sevcre al veolar bone resorption ; in vivo admi ni stration or
`either doxycycline or C MT- 1 by claily oral gavage reduccd all
`o f these ·' mark c rs" of pe riodontal di sc ase lo es se ntially
`normal Ieve ls (Chang et al. , 1994; G o lu b et al .. 1994 a) .
`Rece nt ex pe rim e nt s idc ntifi ed a two-wee k "window" ot'
`di sease activity. characlcri zcd by e lcvated Ievels of acti ve
`collagenase. aclive gelatinase, and rapid alveolar bone loss in
`thi s animal model o r microbiall y induced period<Jntal di sease
`(Ryan et al. , 1997). Howcver, the initial scries of stuclics with
`thi s mode l found that. at the end o f a seven-wee k in l'il'o
`protocol , the elevated collagenase (and gc latinase) activity in
`th e g ing iva of th e P. g . - i~t'ec t ed rals~ was present as th e
`inacti ve or late nt form (pro-coll agenase) . These pro- MMPs
`required in vitro acti vation either by APMA or oxidatively by
`hypochlora us acid (HOC!) I in vivo acti vation or pro-MMPs
`is thought tobe primarily mediated by limi ted proteolysis by
`ne ut ral prote inases s uch as plasmin . o r tumor-assoc iated
`trypsin (TAT-2), or even other MMPs such as stromelysin
`(Sorsa e r al .. 1997 ) ; ho we ve r, Sorsa e t a/. ( 1992 ) ha ve
`propo se d that le uk ocy te -type pro - MMPs , MMP -8 and
`MMP-9, are primaril y acti vatcd by reacti ve oxygen spec ies
`(ROS), e.g .. HOCI. produced by inflammatory cell s l. Based
`on the sequence o f aclcling thc fo llow ing reagent s- namely.
`C MT- 1, excess C a++ (50 mM in stead of the tl s ua l 5 mM
`concentration in the buller). and HOC I-
`to th c incubation
`mi xture containi ng extract of gingiva t'rom thc P.g.-infccted
`rat s. plu s the racli olabe lcd coll age n substralc , Golub et o/.
`( 1994a) t'ouncl th at th e T C anal og inhibit cd lwth ac tiv e
`collagenasc (convcrtecl from thc pro-form by APM A) and thc
`o x idat i ve con ve rs ion or pro -collagc nase i nt (l l he ac t i vc
`e nzyme by HOC !. Based on these ex perime nt s and on thc
`fo llow ing in vitro Observations. it was pro posed th at these
`two anti-MMP fun c tion s ot' C MTs (and oth e r T Cs) may
`
`Exh. 1027
`
`

`
`- - ----------------
`
`-
`
`16
`
`kDa
`m 0 [ 20o-
`CM
`92 -
`72 -
`
`2
`
`3
`
`4
`
`5
`
`75
`~
`c::_.
`"""Cl
`<J.)
`"""Cl
`CU 50
`Ci:>
`<J.)
`"""Cl
`c
`~ 25
`w CO
`S:
`
`~
`
`0
`
`4M
`2M
`-1M OM
`'--- Sc ___, '-- L D D --'
`
`Fig . 2-Gelatinase in whole-mouth GCF (collected by
`mouthrinse technique) _fi-om Jour adult periodontitis patients
`I mo cifter scaling and prophylaxis (Sc) and cifier a four~
`month regim.en of LDD: ejf"ects on molecular species (top)
`and activity (bollom) of this matrix metalloproteinase. (top)
`Enzyrnograph ofwhole-mouth GCF was carried out with the
`use o/ denalured type I collagen (gelati11.; I mg/mL) co-
`polymerized with 10 % SDS-po lya cry lamide and
`electrophoresed at 3 m Altane for 45 min. A.fier overnight
`incubation, the gels were stained with Coonwssie 8/ue, and
`the lytic zones in each lane shmv the dUferent types of
`gelotinase. Lane I shows macropfwge-conditioned media (m
`0 CM) containing 92-kDa gelatinase (MMP-9) and its
`homodimer (Golub et al. , 1995), plus 72-kDa gelatinase
`(MMP-2) obtainedfi·om Tripie Point Biologics (Foresl
`Grove, OR). Lanes 2-5 show predominantly 92 kDa and its
`h.omodimer, plus an in terrnediate band, presum.ably
`reflecting a hererodimer of MMP-9 complexed to lipocalin
`or TIMPs (Golub et a l. , 1995). Lan es 4 & 5 show the
`different species of MMP-9, as in. Iones 2 & 3, plus an
`additional smaller-molecular- weight band seen afier 2 & 4
`months ' treatment with LDD. ( bottom) Gelatinolytic activity
`(assessed with. [3Hj methyl gelcllin used CIS substrate) of
`whole-mouth GCF in the swne patients and at the sam.e time
`p eriods CIS shown for zymography Iones 2 -5, above.
`Gelatinolytic activity was measured bef'ore treatm.ent (- 1M)
`and I mo afier subjects received .fitll-mouth scaling and
`prophylaxis (OM). Then, at firne = OM, a/1 subjects were
`adrninistered LDD every day ./(Jr 4 mos, and gelatinolytic
`activity was measured after 2 mos (2M) and 4 nws (4M) of
`drug th e rap y . Each value represe nt s the 111 ea n of 4
`determinations ± SEM. The reduction i11. gelotinase activity
`due to LDD therapy was statistically significant (p < 0.05)
`cornpored with the time = OM va!ue.
`
`C OLUB ET A L.
`
`;\ov DENT RES NOI'WßER /998
`
`reside in different parts of the drug moleCLtle: When CMT-1
`and excess Ca++ were added to the partially purified gingival
`extract together with HOCI, CMT- I was still able to inhibit
`the oxidative activation of the g ingival pro-collagenase.
`However, when the pro-collagenase was activated by HOC!
`prior to the addition of CMT- 1 and Ca++, the excess meta!
`ion preve nted the dru g from blockin g a lready -active
`coll agenase, as described previously. Consistent with these
`studies o n gingival collagenase, (i) the addition of excess
`Ca++ (mM) or Zn (fLM) clid not prevent TCs from inhibiting
`the oxidative activation of pro-collagenase sec reted by
`osteoblasts in culture (Ramamurthy et a/., 1993), and (ii)
`CMT-5, the pyrazole analog of TC, which is the only one of
`I 0 CMTs to have lost the ability to inhibit already-active
`collagenase, presumably due to the Ioss of the Ca++ ancl Zn++_
`binding site at the cm·bon- I I and carbo n-12 positions ,
`retained its ability to prevent pro-collagenase activation by
`HOC! (Sorsa et al., 1998).
`Jn addition to scavenging ROS, thus preventing pro-MMP
`activation, TCs appear to interact directly with pro-MMPs as
`weil, thus affecting other mechanisms of activation. In thi s
`reg arcl , Brandt ancl hi s gro up st udi ed th e effect of
`doxycycline on recombinant human pro-MMP-8 and detectecl
`two different mechanisms of inhibition (Branclt, 1995a; Smith
`et a/., 1996). As reviewecl by Ryan et al. ( 1996b), when
`doxycycline was added in vitro after the activation of pro-
`MMP-8 (activation was achievecl either by Iimited proteolysis
`or by the organomercurial agent, APMA), approx imately 30
`fLM of the dru g was required to inhibit the Co llage nase
`activity by 50%, an IC50 simil ar to that clescribecl by others
`(Sorsa et al. , 1994; Golub et a/. , 1995). In contrast, when
`doxycycline was added during activation, the IC50 droppecl to
`5- I 2 fLM. Based on Western blot analysis of the different
`molecular species of MMP- 8, Brandt and hi s colleag ues
`(Smith er a/., I 996) proposed that doxycycline binds to the
`pro-MMP (possibly complexing with Ca++ in the enzyme),
`th us altering the enzyme 's conformation a nd res ulting in
`excess ive degradation of the proteinase precursor, during
`activation, to small-molecular-weight, enzymatically inactive
`fragments. Thi s mechanism of MMP inactivation, although
`attractive, remains controversial, since Sorsa et al. ( 1994)
`were unable to cletect any frag mentation of wi Id-type Pro-
`MMP-8 during activation by APMA in the prese nc e o f
`doxycyc line. Of extreme interest, this mechanism may extend
`to other MMPs, since MMP- 13 was recently found to be
`inhibitecl by very Iow concentrations ( < I fLM) of several
`CMTs (CMT-3, CMT-7, and especially CMT-8) as weil as
`I 0 fLM) in vitro whe n the TCs were
`doxycycline (IC50 -
`added together with APMA cluring activation (Greenwald et
`al. , I 998). However, when the drugs were added qfier pro-
`MMP- I 3 was APMA-activated, thi s coll age nase was found
`tobe relatively resistant to TC inhibition (Lindy et al., 1997).
`Th e re leva nce of th is mec hani s m to hum a n di sease is
`suggestecl by preliminary data clescribed Iater in thi s paper:
`When ad ult perioclontitis patients were ad ministered a four-
`month regimen of non-antimicrobial Iow-dose cloxycyc line,
`MMP activity in whole-mouth gingival crevicular fluid (GCF)
`samples was reduced coi ncident with the appearance, at the
`
`Exh. 1027
`
`

`
`VOL./2
`
`T ETHA CYCUNt:s I NII//.J/'1' CoNNEcnvJ:.: TJssuJ:.· ßuf.~\ KIJOIIIN Ul' M u LTII'LL NoN-,\ NTJMJCNOHf,\1. A1t:cn\NJSI\IS
`
`17
`
`two- ·mct f'
`.
`MM ' . our-month time periods, of small-molecular-we1ght
`p fragments and reduced pro-MMP Ievels (see Fig. 2).
`
`;es lndirectly inhibit serine proteinases: a 1-PI protection
`11 Vtfro, cell culture, and in vivo approaches have all been
`u(Ssect to determine the effects of TCs o n a 1-PI inactivation
`ors
`L
`a et al., 1993; Golub et al., l 994a; Crout et al. , 1996;
`ee et al. , 1997 ) . The reaso ns? a 1- PI (a lso cal led a 1-
`proten1 ase inhibitor or a 1-ant itryps in ) is th e m ajor
`endogena us inhi bitor of another class of ti ssue-destructive
`enzymes, the serine proteinases (particularl y PMN leukocyte
`ela_stase), and a 1-PI is known to be inactivated, e. g., during
`lnO a mmatory diseases, both by reacti ve oxygen species
`(ROS) and by MMPs including collagenase (Carp and Janoff,
`1979; Michaeliset al., 1990; Desrocl1ers et al., 1992).
`When the animal model of periodontitis described above
`Was used, in addition to the detection of severe alveolar bone
`loss and elevated MMPs in the gingiva- namel y, collagenase
`and gelatinase (presumabl y MMP-8, MMP- 13, and MMP-9,
`Slnce MMP- 1 is not fou ncl in roclent tissues and MMP-2 was
`e las ta se
`not found to be e levatecl in thi s expe riment)-
`activ ity was also seen to be elevated in extracts o f g ingiva
`from these P. gingivalis (P.g.)-infected rats (Golu b et al ..
`l994a). This ging ival elastase activity was characterizecl as a
`serine prote in ase, not a me tall o pro te in ase , basecl o n its
`response to a series of proteinase inhibitors such as PMSF,
`EDTA, l , I 0-phenanthroline, and CMT-1 , ancl when the P. g.-
`infected rats were treated in vivo by da ily oral gavage w ith
`CMT- 1, the excess ive e lastase act ivity ( li ke that o f the
`MMPs) was recluced to esse nti a ll y norm a l Ie ve ls . S ince
`g ingival extracts from the untreated P.g. -infectecl rats were
`fo