.Touma/ of Periodontal Research 1983 18: 516-526
`
`Minocycline reduces gingival colla-
`genolytic activity during diabetes
`Preliminary observations and a proposed new mechanism of
`action
`
`L. M. GoLuB, H. M. Lim, G. LmmnR, A. NEMmoFF, T. F. McNAMARA, R. KAPLAN AND
`N. s. RAMAi\1UR11W
`Department of Oral Biology and Pathology, School of Dental Medicine, State University of New
`York at Stony Brook, Stony Brook, N.Y. and Department of Dentistry, Long Island Jewish-
`Hillside Medical Center, New Hyde Park, N.Y., USA
`
`Diabetes increases gingival colhtgenase activity, an effect that may be mediated by endogenous
`tissue changes and exacerbated by an overgrowth of Gram-negative organisms in the gingival
`crevice (sec Rarnamurthy & Golub 1983, McNamara et al. 1982). fn an attempt to reverse this
`collagenolytic abnormality, we administered an appropriate antibiotic, minocycline (a semi-
`synthetic tetracycline), to diabetic rats and humans. Adult male conventional or germfree rats
`were made diabetic with streptozotocin, and half of these ai1imals were administered minocy-
`cline (20 mg per day) by tube feeding for 3-4 weeks prior to sacrifice. The buccal gingiva, entire
`skins, and mandibles were dissected and tested for collagenolytic enzyme activity, collagen .
`content, and alveolar bone loss, respectively. ln a preliminary study, minocycline (200 mg per
`day) was administered for 7 days to an insulin-dependent diabetic adolescent human and an
`adult notl-diabetic human; the twin brother ofthc diabetic was treated with penicillin. Gingival
`fluid collagenase activity was measured (using [ 3H-methyl] collagen as substrate in a new
`microassay) in 8 periodontal pockets in each subject before and after antibiotic therapy.
`Examination of collagenase digestion products by SDS-polyacrylamide gel electrophoresis and
`fluorography was also carried out. In rats, minocycline treatment: (I) suppressed the abnonnally
`elevated collagenolytic enzyme activity in gingiva of diabetic rats, even under gemlfl·ee
`conditions; (2) inhibited PMN leukocyte collagenase activity in vitro, an effect that was reversed
`by the addition of calcium ions (penicillin-streptomycin had no eiTect on the activity of this
`enzyme); and (3) retarded the abnormal loss of skin collagen and alveolar bone in diabetic rats.
`In a preliminary study on humans, minocycline therapy reduced the collagenase activity of
`gingival crevicular f1uid, an effect not produced by penicillin.
`Our data suggests that (I) tetracycline therapy inhibits tissue collagenolytic enzyme activity by
`a mechanism at least in part unrelated lo its antibacterial efficacy, and (2) this mechanism may
`provide a new therapeutic approach for suppressing excessive collagen resorption which occurs
`during periodontal disease and which can occur during other pathologic conditions.
`
`(Accepted for pubficatio11 .June 21, 1983)
`
`Introduction
`We have found that diabetes in rats and
`humans increases tissue collagenase activity.
`
`Evidence for this effect was seen in extracts
`of gingiva and skin (Ramamurthy & Golub
`1983),
`in cultures of gingival
`tissue
`(Ramamurthy et al. 1974, Golub et al.
`
`Dr. Reddy's Laboratories, Ltd., et al.
`v.
`Galderma Laboratories, Inc.
`IPR2015-__
`Exhibit 1021
`
`Exh. 1021
`
`

`
`MINOCYCLINE REDUCES COLLAGENOLYTIC ACTIVITY 517
`
`1978a, Kaplan et al. 1982), and in gingival
`crevicular fluid (McNamara et al. 1979,
`Kaplan et al. 1982). Unusually severe
`periodontal disease, which occurs during
`diabetes in man (Finestone & Boorujy 1967,
`Cianciola et al. 1982) and experimental
`animals (Bissada, ShaiTer & Lazarow 1966),
`ref1ects accelerated collagen breakdown
`which could be mediated by the excessive
`collagenase generated during this systemic
`disease (sec Ramamurthy & Golub 1983).
`We have suggested that the overgrowth of
`Gram-negative organisms in the gingival
`crevice of the diabetic rat could, by genera-
`ting excessive endotoxin in the area, be the
`cause of the abnormally high collagenase
`levels in the gingiva (McN amara et al.
`1982). To test this hypothesis, we deter-
`mined (a) whether an antibiotic effective
`against oral Gram-negative organisms
`(minocycline; see Genco 1981) reduces gin-
`gival collagenase activity during diabetes;
`and (b) whether diabetes stimulates gingival
`col!agenase activity under germfree con-
`ditions. During the course of these investi-
`gations, minocycline (a semi-synthetic tetra-
`cycline) administered to rats was found to
`inhibit collagenase activity, at least in part,
`by a mechanism(s) unrelated to its antimi-
`crobial eiTicacy. This preliminary report de-
`scribes our initial studies using minocycline
`in diabetic rats and humans.
`
`Materials and Methods
`Anima/experiments
`Preparation of anima/s. In the experiment
`using conventional animals, five-month-old
`(mean weight, 445 g) male Sprague Dawley
`rats were made diabetic by LV. injection of
`streptozotocin (70 mg per kg body weight),
`after a 12 h fasting period, as described by
`us previously (Golub et al. 1978b, Golub et
`al. 1982). Age matched uninjected rats
`served as controls. Some of the diabetic rats
`were administered the tetracycline, minocy-
`
`cline (20 mg Minocin@ per day; Lederle
`Laboratories, Pearl River, N.Y.), by gavage
`on a duily basis beginning 3 days after
`diabeteswas induced. After a 25 day experi-
`mental period, a blood sample was taken for
`glucose analysis, and subgingival plaque
`samples were taken for assessment of the
`microflora in the region (McNamara et al.
`1982). The ~mimals were then sacrificed,
`gingival samples were ülken for tissue cul-
`ture, and the jaw bones dissected (sec
`below).
`In a second experiment, germfree rats
`(purchased from Taconic Farms, German~
`town, N.Y.) were maintained in inf1atable
`vinyl isolators (Standard Safety Equipment
`Co., Palatine, Illinois) until a weight of 360g
`± 15 was reached. At this time (6 weeks
`after purchase), diabetes was induced in
`some of the animals, as described above,
`and the control and diabetic animals main-
`tained under germfree conditions for an
`additional6 weeks prior to sacrifice. Half of
`the diabetic animals were administered
`Minocin@ (20 mgjday) by gavage for the last
`4 week:s of the protocol. Blood and tissues
`were then abtairred to assess gingival colla~
`genolytic enzyme activity and the loss of
`skin collagen and bone.
`The maintenance of gennfree conditions
`was tested by swabbing various surfaces of
`the isolators (and its contents) and the rat's
`oral cavity and fecal material with sterile
`cotton-tipped applicators. These were im-
`mediately placed into Brain Heart infusion
`broth (Difco Laboratories, Detroit, Mi-
`chigan) to detect the presence of aerobic
`organisms; separate samples were placed
`into pre-reduced PYG broth (Anaerobe
`Laboratory, Virginia Polytechnical Insti-
`tute) to detect anaerobes. All samples were
`incubated at 37°C for 7 days and examined
`daily for evidence (turbidity) of growth. The
`isolator was tested for sterility twice before
`the rats were introduced. Thereafter, the
`equipment and animals were monitared mi-
`
`Exh. 1021
`
`

`
`518
`
`GOLUB ET AL.
`
`crobiologically once per weck, including the
`day of sacrifice. At this time, the aerobic
`and anaerobic samples were Gram-stained
`and examined microscopically for the pre-
`sence of microorganisms.
`Assaying col/ageno/yOc enzyme activity.
`The techniques used to measure the colla-
`genolytic enzyme activity of gingival tissue
`in polymorphonuclear,
`in culture, and
`leukocytes (PMNLs), are those described by
`us previously (Golub et al. 1978a, Nieoll et
`al. 1981) with minor modifications. Briefly,
`the labial gingiva of maxillary incisors was
`excised, pre-incubated in Gey's media con-
`taining penicillin and Streptomycin, and dis-
`sected under aseptic conditions into frag-
`ments weighing 4-6 mg wet weight. Two
`fragments from each rat were cultured on a
`gel of reconstituted 14C-glycine
`labeled
`collagen fibrils (32,264 DPM per 0.5 ml gel).
`The acid-soluble rat skin collagen was pre-
`pared according to the method of Olimeher
`and Krane (1964); the animals were injected
`with 14C-glycine as describcd by us previ-
`ously (Golub et al. 1976). After incubation
`(3 days, 35°C, in a humid atmosphere of
`95% air and 5'% C0 2), the contents of cach
`culture dish wcrc centrifugcd (10,000 x g, 1
`h, 22 "C) and aliquots of the supernatant
`were analyzed for 14C-labelcd collagen frag-
`ments in a liquid scintillation spectrometer
`(In this system, 96-98% of the solubilized
`collagen is recovered as dialyzable break-
`down products, Golub et al. 1978a).
`To test the effect of physiologic Ievels of
`minocycline (the concentration in human
`serum during a therapeutic regimcn is ap-
`proximately 2 pg/ml, that in the gingival
`crevicular fluid is 4-5 times greater; Ciancio,
`Muther & McMullen 1980) on collagenase
`activity in vitro, we collccted peritoneal exu-
`date PMNLs from 15 male control rats 4 h
`after I.P. injection of 0.15% glycogen. An
`extract of PMNLs (in 0.05 M Tris-0.2 M
`NaCI-5 mM CaC1 2 ) was prepared, 250 1t1
`14C-gly
`aliquots were
`incubated with
`
`labeled collagen fibrils (12,720 DPM per 50
`pl gel) for 14 h at 35°C (in the presence or
`abscnce of minocycline, EDTA, or added
`CaCI 2), and collagenolytic activity was
`measured as described by us previously
`(Nicoll et al. 1981).
`M easuring the lass f~j' alveolar bone and
`skin collagen. Alveolar bone loss was as-
`sessed in the right and left mandibles of the
`conventional and germfree rats (control and
`diabetic) using a modification of a previ-
`ously described technique (Stralfors, Thil-
`ander & Bergenholtz 1967, Heijl et al. 1980).
`The mandibles were defleshed (autoclaving
`followed by soaking in 2 N NaOH for 2-·3 h)
`and dried, and the distance ( expressed as
`units; 1 unit = 0.05 mm) between
`the
`cemento-enamel junction (CEJ) and the
`crest of the alveolar bone was measured in
`the long axis of the root surface at 10
`specified sites on lingual surfaces of the 3
`molar teeth (see Crawford, Taubman &
`Smith 1978) using an eye-piece micrometer
`in a dissecting microscope (20 x magnifi-
`cation).
`Because uncontrolled diabetes in the rat
`results in thc massive resorption of skin
`collagen (Schneir & Golub 1981) associated
`with increased collagenase activity in this
`tissue (Ramamurthy & Golub 1983), we
`also measured the collagen content of entire
`skins from control and diabetic rats (both
`germfree and conventional), including those
`treated with minocycline. The techniques
`used were described by Schneir and Golub
`(1981).
`
`Initial human study
`Gingival crevicular fluid (GCF) samples
`were collected on filter paper strips inserted
`into 8 interproximal pockets of selected
`teeth in the maxillary arch of two 19 year-
`old twin brothers with juvenile-onset (insu-
`lin-dependent) diabetes mellitus, as de-
`scribed by us previously (Golub et al. 1976).
`Fluid volume was immediately determined
`
`Exh. 1021
`
`

`
`MINOCYCLINE REDUCES COLLAGENOLYTIC ACTIVITY 519
`
`on a moditled Periotron (model 6000
`[Harco
`Electronics
`Ltd., Winnipeg,
`Canada]; see Hinrichs & Bandt 1983). The
`filter strips were stored frozen for I h in
`plastic microfuge tubes then incubated with
`10 ttl CI-I-methyl] collagen (20,675 DPM),
`prepared as described by Bhatnagar and
`Decker ( 1981 ), and 70 pl buffer (50 mM
`Tris-HCI, pH 7.8, containing 0.2 M NaCl
`and 5 mM CaCI2). The GCF samples (or
`reagent blanks or trypsin controls) were
`incubated at 27oC (Birkedal-Hansen &
`Demo 1981) for 18 h wi th gentle shaking.
`The rcaction was stoppcd and undigcsted
`collagen precipitated by adding 10 pl 0.1 M
`phenanthroline in dioxane/water (1: 1, v/v),
`10 pl of non-radioactive methylated catTier
`collagen (2 mg/ml) in 50 mM Tris-HCI
`buffer containing 1 M NaCI (pH 7.0), and
`100 ttl dioxane (Bhatnagar & Decker 1981 ).
`After mixing,
`the radiolabeled collagen
`degradation products were collected by m-
`tration and counted in a liquid scintillation
`spectrometer (Golub et al. I 976). Using this
`technique, the reaction blank (no enzyme or
`GCF added) released about I 0% of the
`total substrate counts, 2~-20 ng bacterial
`Collagenase produced a linear increase in the
`release of radioactive counts from the 3H-
`collagen substrate (the 20 ng Ievel of enzyme
`degraded 60% of the collagen substrate),
`and 50 ng trypsin released less than I 0/(, of
`the counts above blank values. GCF colla-
`genase activity was expressed as units of
`equivalent activity of vertebrate collagenase
`(obtained from New England Nuclear, Cat.
`No. NEK-016); 1 unit was operationally
`defined as the amount of enzyme that de-
`grades 1 pg of collagen per hour at 2JüC.
`Four additional GCF samples were col-
`lected from other maxillary pockets of the
`same diabetic twin brothers on filter paper
`strips, their volume determined (see above),
`and collagenase activity assessed using the
`same procedure described above but with a
`pl
`(10
`of eH-
`different
`substrate
`
`propionate] collagen, NET-660 [New En-
`gland Nuclear Corp., Boston, MA.],
`302,000 DPM). Halfofthe 12 GCF samples
`per subject were activated by pretreatment
`with 1.04 pM trypsin followed by the ad-
`dition of a 5-fold molar excess of soybean
`trypsin inhibitor (Hassell 1982). Sampies
`were selected to be treated or not treated
`with trypsin by matehing them for Gingival
`Index (Löe & Silness 1967), GCF flow, and
`for pocket depth; the clinical parameters
`(GI, PD) were measured immediately after
`GCF collection. All measurements were car-
`ried out
`immediately before antibiotic
`coverage and after 7 days of treatment with
`either penicillin G (1 g per day; patient P.D.)
`or minocycline (200 mg per day; patient
`K.D.).
`The same protocol using minocycline
`therapy was carried out on a 41-year-old
`non-diabetic male (R.R.), in 12 selected sites
`in the maxillary arch (01=1.7±0.2; PD
`=4.2 mm±0.3), and GCF collagenolytic
`activity was measured as described above.
`This patient exhibited deeper periodontal
`pockets than the 19 year-old diabetic twin
`brothers. Two additional GCF samples
`were collected from the same 4 mm pocket,
`in this patient, before and after minocycline
`therapy. These samples were incubated with
`10 pl 3H-collagen for 18 h (22"C) then
`thermally denatured, and the collagen sub-
`units and Collagenase digestion products
`were separated by SDS-polyacrylamide gel
`electrophoresis (Nicoll et al. 1981 ). The gels
`were processed for fluorography (sec Sodek,
`to
`Hurum & Feng 1981) and exposed
`Kodak XAR-5 film for 5 days.
`
`Results
`Anima! Experiments
`The conventional and germfree control rats
`all exhibited relatively low blood glucose
`concentrations ranging from 85 to 186 mg%
`(mean=141 mg%±14). As expected, the
`
`Exh. 1021
`
`

`
`520
`
`14
`
`GOLUB
`
`ET AL.
`
`--;)
`
`LJ..) u
`E .g
`J5 ·c:
`2
`Cl) u c:
`0
`17;
`0
`
`1.1'1
`1.1'1
`0
`_J
`Cl)
`c:
`0
`CQ
`
`14
`
`(b}
`
`6
`
`(o)
`
`"C 60
`.. ~
`.;?:'o
`:~ _g40
`c=
`<(
`~ ~
`i =§ 20
`g~
`8 ~ 0"'---::~~~::-!-~"---="='--:-..J.-­
`Control Diabetes Diabet. +
`minocycl.
`
`Cl.l
`
`I
`
`Fig. 1. The adminlstration of rnlnocycllne to conven-
`tlonal dlabetlc rats: effect on (a) collagenolytic activlty
`of glngiva in tissue culture, and (b) alveolar bone lass.
`Each value is the mean ± S.E.M. for 3-5 rats per group.
`
`diabetic conventional and germfree rats,
`with or without minocycline therapy, were
`severely hyperglycemic; blood
`glucose
`values ranged from 310 to 832 mg%, with a
`mean of 503 mg% ± 42. The germfrce rats
`and isolators showed negative results for
`aerobic and anaerobic oultures throughout
`the experimental period, and treatment of
`the conventional diabetic rats with minocy-
`cline eliminated detectable signs of a Gram-
`negative microflora in the gingival crevices.
`The gingiva from the conventional and
`gennfree control rats produced minimal col-
`lagenolytic activity in tissue culture; Iess
`than 10'% of the 14C-coltagen fibrHs were
`degraded after 3 days in culture (Figs. la
`and 2a). In contrast, coltagenolytic activity
`was marked1y increased in gingiva from the
`
`80
`
`(o)
`
`-c
`Cl)
`.~2 60
`>
`0'1
`·..;: Cl)
`(.) "0
`c::: 40
`<I:
`;;o
`..g~
`~ 8 20
`g:i
`0 u 0
`u ~ ~------~------._ __ ._ __ .__
`Control Diabetes Diabet. +
`minocycl.
`Flg. 2. The administration of rnlnocycline to germfree
`diabetic rats; effect on (a) collagenolytic activity or
`gingiva ln tis~ue culture, and (b) alveolar bone loss.
`Each vaiue is the mean±S.E.M. of 4-5 rats per group.
`
`1200
`
`200
`
`\Germ·lreel
`
`0o
`~
`s
`5
`4
`2
`l
`Time lweeks ofler slreplozolocin odmin.)
`Flg. 3. The effect ot streptozotocln-diabetes on sKin
`collagen Ieveis in germfree rats (see bar graphs at 6
`weel<s after streptozotocin adminlstratlon) ancl con-
`vent!onal rats (see bar graphs at 8 weeks); half ot the
`diabetic germtree and conventional rats were admlni-
`stered rnlnocycllne on a dally basls (see Materlais and
`Methods). C = contro\
`rats; D = dlabetic
`rats;
`D + M""' minocycHne treated diabetlc rats. Each va!ue
`is the mean±S.E.M. of 4-5 rats per group, The line
`graphs for the C and D conventlonal rats were adapted
`1rom Scline\r ano Golub 19ßi.
`
`Exh. 1021
`
`

`
`MINOCYCLINE REDUCES COLLAGENOLYTIC ACTIVITY
`
`521
`
`diabctic rats, under both conventional (Fig.
`la) and gcnnfree (Fig. 2a) conditions. Ad-
`tninistering minocycline orally on a daily
`basis reduced thc gingivai coiiagenolytic
`activity in both diabetic conventional and
`germfree rats by 62% and 70%, respectively
`(Figs. la and 2a).
`Diabetes in the conventional rats pro-
`duced a slight, but significant, increase
`(p < 0.05) in bone loss in thc interproximal
`region, compared to controls; this effect was
`complctely reversed by tetracycline admini-
`stration (Fig. lb). The patterns ofchange on
`the lingual surfaces of the mandiblcs werc
`similar; however, these effects werc not stat-
`istically significant. In the germfrec rats, no
`differences in bone Ievels could be detected
`bctween the three groups (Fig. 2b); however,
`significant changes were seen in skin (Fig.
`3). Diabetes in the germfree rats (6 wecks
`after streptozotocin administration) resul-
`ted in a 48% reduction in skin collagen
`content compared to the germfree controls
`(p < 0.01 ), and minocycline trcatment of the
`germfree diabetics increased the collagen
`Ievels
`in
`this
`tissue by 45% (p <0.05).
`Similar patterns of change in skin co1lagen
`content were seen in conventional control,
`diabetic, and minocycline-treated diabetic
`rats (Fig. 3).
`
`As expected, the collagenolytic activity in
`the PMNL extract was complete1y inhibited
`by EDTA (mammalian collagenase is a
`calcium eiependent neutral protease; SeHers
`& Murpby 1981), and the collagen substrate
`was not susceptible to non-specific proteoly-
`sis by trypsin (Table 1 ). Minocycline, in
`concentrations approximating those found
`in human serum and gingival crevicular
`fluid (Ciancio ct al. I 980), produced ahnost
`complete inhibition of PMNL collagenase
`activity, an efTect which was reversed by
`increasing concentrations of calcium (Table
`1 ). Othcr antibiotics (penicillin- Streptomy-
`cin) had no detectable efTecl on collagenase.
`
`Initial human study
`The adolescent diabetic twin (K.D.) was
`treated with minocycline (Fig. 4). Prior to
`treatment, he demonstrated a greater sever-
`ity or gingival inflammation (a significantly
`towards in-
`higher GI and a tendency
`creased GCF t1ow) but similar pocket depth
`compared to the other diabetic twin (P.D.)
`who was treated with penicillin. Prior to
`antibiotic therapy, K.D. also exhibited in-
`creased active collagenase and total col-
`lagenase (the latter measured after trypsin
`pretreatment) activities compared to P.D.
`using either radiolabeled collagen substrate
`
`Table 1
`The effect of minocycline on rat PMNL collagenolytic activity in vitro
`----·-··--·----------··----····-··------~--"-----···--···
`Collagenolytlc Activity:*
`% 1'C-collagen gel lysed
`
`lncubation Mixture
`
`85.5
`1. PMNL extract (control)**
`3.1
`2. PMNL extract+EDTA (54 mM)
`2.6
`3. Trypsin (1 JL9)
`88.7
`4. Bacterial collagenase (250 JIQ)
`12.4
`5. PMNL extract+ Minocin'1~ (5 pQ/ml)
`8,3
`6. PMNL extract+ Minocin°'' (20 pg/ml)
`38.5
`7. Above, (6.), + CaCI 2 (10 mM)
`82.1
`8. Above, (6.), +CaCI 2 (50 mM)
`90.3
`9. Above, (1.), +Penicillin-Streptomycin (20 f!gjml)
`* Each value is the mean ot dupllcate analysls alter bacl<ground actlvity (3.5%) was subtracted.
`** Extract obtalned trom 38 x 106 cells/ml.
`
`Exh. 1021
`
`

`
`GOLUB ET AL .
`
`522
`
`0
`
`I
`O
`
`. :~·r "I" ~::~·r ·r ~:liDIDT· ,..t
`
`~
`40
`O
`
`PO
`
`KD
`
`PO
`
`KO
`
`0
`a..2
`O
`
`P
`
`KO
`
`Trrp•l•
`.,_.~=-~~._./-.<••hol
`
`~ Actlve Enzyme
`0 Trypsin·Aclived Enzyme
`
`Flg. 4. The treatment of two juvenile-diabetic twin
`brothers with peniclllin (patient P.D.) or with minocy-
`cllne (patient K.D.): effect on gingival dlsease and on
`GCF collagenase activity. The pockets measured for
`actlve collagenase or for trypsin-actlvated collagenase
`were selected so that the two groups of pockets
`showed simllar values for GI, PD and GCF flow. Eight
`GCF samples from each subject were assayed for
`collagenase using ['H-methyl] collagen substrate (see
`A). Four other samples from each subject were as-
`sayed Using the eH-propionate] collagen substrate
`(see insert; B).
`
`for the assay (Fig. 4, A and B). Treatment
`wit.h penicillin, reduced the Gram-positive
`but increased the Gram-negative organisms
`in the pockets and had little or no effect on
`GI, GCF f1ow, PD, or on active collagenase;
`in fact, total enzyme activity appeared to
`increase, at least with the [ 3H-methyl] colla-
`gen as substrate (Fig. 4). In contrast,
`minocycline produced signiticant reductions
`in GI, GCF flow, active and total Collage-
`nase, but no decrease in PD after the 7 days
`of treatment (Fig. 4). These pockets showed
`a marked reduction in Gram-negative and a
`relatively small number of Gram-positive
`organisms at this time. Similar to the effect
`on K.D., treatment of the non-diabetic
`(R.R.) with minocycline reduced active col-
`lagenase by 86%, trypsin-activated Collage-
`nase by 59%, GCF f1ow by 71%, GI by
`26 1Yo, whereas pocket depth was essentially
`
`2
`
`3
`
`4
`
`5
`
`~A-f3-
`o<{ ~
`o<.A(
`
`Flg. 5. Fluorography of "H-collagen and its degradation
`products alter SDS-polyacrylamide gel electropho-
`resis. Tracks, i, 2 and 3 show the collagen before
`incubation and after an i8 hour lncubatlon (22°C) with
`i or 2 Jtg verlebrate (tadpole tail) collagenase, respec-
`tively. The last two tracks show collagen and lts
`digestion products after lncubation with a GCF sample
`from the same 4 mm periodontal pocl<et, in patient
`R.R., before (track 4) and after (track 5) minocycline
`therapy.
`
`unchanged (data not shown). The collagen
`digestion fragments produced by the GCF is
`shown on the i1uorograph (Fig. 5). The
`GCF dcavage of the radiolabeled collagen
`generated aA (a 3/4 fragment of the collagen
`molecule) and an (a 1/4 fragment) digestion
`products characteristically produced by ver~
`tebrate collagenase (track 4), and minocy~
`cline therapy (1) reduced the breakdown of
`the intact collagen (note the increased den-
`sity of the rx and ß subunits in track 5
`compared to track 4) and (2) inhibited the
`production of the aA and an collagen diges~
`tion fragments (compare tracks 4 & 5).
`Densitometric analysis of the fluorographs
`demonstrated that minocycline therapy re-
`duced the percentage of the total radio~
`
`Exh. 1021
`
`

`
`M I N 0 C Y C L I N E R E D U C E S C 0 L L A G E N 0 L Y T I C A C T I V I T Y 523
`
`labeled collagenaus material present as de-
`gradatim1 products ( data not shown).
`
`Discussion
`A series of experiments are currently being
`conducted to answer the following question:
`Does diabetes increase gingival collagenase
`activity endogenously, through endocrine-
`mediated alterations in tissue metabolism,
`or exogenously, by producing an over-
`growth of Gram-negative organisms in the
`gingival crevice which increases endotoxin
`levels in the gingiva tlms stimulating colla-
`genase production (McNamara ct al. 1982,
`Ramamurthy & Golub 1983). To date, our
`data indicates that the collagenase defect is,
`at least in part, endogenously mediated.
`Insulin treatmcnt of diabetic rats, which
`reduces the severe pyperglycemia, decreases
`the abnormally high collagenolytic activity
`tissue
`in culture
`produced by gingival
`(Ramamurthy et al. 1974). However, the
`most convincing evidence for an endogen-
`aus mechanism, independent of bacterial
`factors, is the observation that diabetes
`produces an increase in gingival collagenase
`activity under germfree conditions (Lehrer
`et al. 1981 ). This efTect was confirmed in the
`present study.
`Other experiments suggest that exogen-
`aus mechanisms also play a role and may
`exacerbate the underlying abnormality in
`collagenolytic
`enzyme
`activity during
`diabetes. McNamara et al. (1982) observed
`a shift in the gingival crevicular microflora,
`to one that was predominantly Gram-
`negative, only 3-4 days after inducing
`diabetes in the rat which persisted during 12
`weeks of experimentation. More recently,
`it was (Golub, McNamara and Rama-
`murthy, unpublished results) found that
`mono-infecting gennfree diabetic rats with
`one of these Gram-negative organisms in-
`creased gingival collagenase activity in cul-
`
`ture above the abnormally high 1evels (com-
`pared to non-diabetic controls) produced by
`gingiva from strictly germfree diabetic rats.
`Mono-infection with
`a Gram-positive
`organism had no effect on collagenase
`activity.
`In an attempt to clarify the relative im-
`portance of the bacterial factors in the
`collagenase abnonnality, we treated the
`conventional dia betic rats with a tetracy-
`cline (Minocin®) which concentrates in the
`gingival crevice and suppresses oral Gram-
`negative organisms (Ciancio et al. 1980).
`Gingival collagenolytic activity was signifi-
`cantly reduced by this therapy even though
`this group still showed more enzyme activity
`than the control rats. Of extreme interest,
`we also found that: (1) minocycline pro-
`duced an almest identical effect in germfree
`diabetic rats (gingival collagenolytic activity
`was reduced by 70% in these animals com-
`pared to a 62% reduction in the conven-
`tional diabetic rats); (2) minocycline parti-
`ally blocked skin collagen resorption (a
`characteristic of severe diabetes in the rat;
`Schneir and Golub 1981. Connective tissue
`abnormalities in skin have also been ob-
`served in diabetic humans; Maczar et al.
`1976) in germfree and conventional diabetic
`rats; and (3) minocycline (but not non-
`tetracycline antibiotics) inhibited PMNL
`collagenase activity in vitro. Thus, the drug
`appears, from these initial expet:iments, to
`be inhibiting collagenase activity, at least in
`part, by a mechanism other than its activity
`as an antibacterial agent. We propose the
`following explanation: Mammalian Collage-
`nases are calcium-dependent metallopro-
`teases. Other cations (zinc) also play a role
`in the sta bility, conforma tion, and hydroly-
`tic activity of the enzyme (Seltzer, Jeffrey &
`Eisen 1977, Berman 1980, Cawston &
`Murphy 1981). Tetracyclines are known to
`chelate multivalent metal cations (Neidle,
`Kroeger & Y agula 1980) and through this
`property could inhibit excessive collagenase
`
`Exh. 1021
`
`

`
`524
`
`GOLUB ET AL.
`
`activity in tissues. In fact, adding calcium to
`leukocyte collagenase in vitro completely
`overcame the inhibitory effect of minocy~
`cline (see Table 1).
`In recent years, tetracycline has been ad-
`vocated in the treatment of periodontal
`diseases, including chronic periodontitis in
`the adult (Fasciano & Fazio 1981) and more
`often,
`für rapidly progressing juvenile
`periodontitis (Slots et al. 1979, Genco, Cian~
`ciola & Rosling 1981 ). Its therapeutic effi~
`cacy has been attributed solely to the drug's
`antimicrobial activity particularly against
`specific Gram-negative organisms believed
`to be the cause of these diseases (Genco
`1981). Recently, Williams et al. (1981) de-
`scribed a
`significant
`improvement
`in
`periodontal disease in dogs on long~term
`tetracycline therapy, an effect that did not
`appear to correlate with expected shifts in
`the crevicular microflora. Similar clinical
`changes were observed in a limited number
`of human subjects (Williams, personal
`communica tion).
`Our initial studies on non-diabetic and
`diabetic humans indicate that the reduced
`production of gingival collagenolytic en-
`zymes observed in rats is also seen in the
`GCF of humans. This is consistent with our
`earlier observations using relatively long-
`term tetracycline (doxycycline) therapy on a
`juvenile-onset diabetic
`adolescent girl
`(McNamara et al. 1979, Golub & Schneir
`1983). Thus, the inhibition of collagenase by
`tetracyclines ( or by other chemotherapeutic
`agents that chelate cations?) may provide a
`new therapeutic approach for suppressing
`excessive
`collagen
`resorption
`during
`periodontal disease or during other patho-
`logic conditions. Clearly, further investiga-
`tion is required. Accordingly, we are now
`expanding our studies (a) to compare the
`effect of different types of tetracycline,
`which have different calcium binding char-
`acteristics, on collagenase activity in oral
`and extra-oral tissues, (b) to investigate the
`
`possibility of using non-antibiotic calcium~
`binding compounds to achieve reduced tis-
`sue collagenolytic activity, and (c) to
`determine whether tetracyclines are capable
`of directly inhibiting bone resorption in
`tissue culture.
`
`Acknowledgements
`This study was supported by grants from
`the Kroc Foundation and from the Na-
`tional Institute of Dental Research (grant
`no. DE- 03987). The authors thank Ms. N.
`Manivannan for her excellent technical and
`secretarial assistance.
`
`References
`Berman, M. B. 1980. Collagenase and corneal
`ulceration. In: Collagenase in Normaland Path-
`o/ogical C01mective Tissues, eds: D. E. Woolley
`and J. M. Evanson, pp. 141-174. N.Y.: John
`Wiley and Sons, Ltd.
`Bhatnagar, R. & Decker, K. 1981. A collagenase
`assay using eH-methyl] collagen. Journal of
`Biochemical and Biophysica/ Methods 5: 147-
`152.
`Birkedal-Hansen, H. & Dano, K. 1981. A sensi-
`tive collagenase assay using [ 3H] collagen
`labeled by reaction with pyridoxal phosphate
`and [ 3H] borohydride. Analytical Biochemistry
`115: 18-26.
`Bissada, N. F., Shaffer, E. M. & Lazarow, A. A.
`1966. Effect of alloxan diabetes and local
`irritating factors on the periodontal structures
`of lhe rat. Periodontics 4: 223-231.
`Cawston, T. E. & Murphy, G. 1981. Mammalian
`collagenases. In: .Methods in Enzymology, Vol.
`80, pp. 711-722, Academic Press.
`Ciancio, S. G. 1976. Tetracyclinesand periodon-
`tal therapy. Journal of Periodontology 43: 155-
`159.
`Ciancio, S. G., Mather, M. L. & McMullen, J. A.
`1980. An evaluation of minocycline in patients
`with periodontal disease. Journal o.f Periodon-
`tology 51: 531-534.
`Cianciola, L. J., Park, B. H., Bruck, E., Mosov-
`ich, L. & Genco, R. J. 1982. Prevalence of
`periodontal disease
`in
`insulin dependent
`diabetes mellitus. Journal oftlze American Den-
`tal Assodalion 104: 653-660.
`Crawford, J. M., Taubman, M. A. & Smith, D. J.
`
`Exh. 1021
`
`

`
`M I N 0 C Y C L I N E R E D U C E S C 0 L LAG E N 0 L Y T I C A C T I V I T Y 525
`
`1978. The natural history of periodontai bonc
`loss in germfree and !,ll10tobiotic rats infcct·ed
`with periodontopathic microorganisms. Jow··
`naf of Periodomal Research 13: 316-325.
`Fasciano, R. W. & Fazio, R. C. 1981. Periodental
`regeneration with
`long
`term
`tetracyclinc
`thcrapy. Quintessence /ntemational, Oclober,
`number 10: 1081-1088.
`Finestone, A. J. & Boort1jy, S. R. 1967. Diabetes
`mellitus and pcriodontal disease. Diabete/16:
`336-340.
`Genco, R. J. 1981. Antibiotics in the treatment of
`Journal o(
`human
`periodontal
`disease.
`Periodon!o/ogy 52: 545-·558.
`·
`Genco, R. J ., Cianciola, L. J. & Rosling, B. 1981.
`Treatment of Jocalizcd juvenile periodontitis.
`Journal of Dmtal Research 60: special issue A,
`abslract 872.
`Glimcher, M. J. & Krane, S. M. 1964. The
`incorporation of radioactive inorganic ortho·
`phosphate by collagen fibrils in vitro. Bioche-
`mistry 3: 195-202.
`Golub, L. M., Schneir, M. & Ramamurthy, N. S.
`1978a. Enhanced collagcnasc activity in diabe-
`tic rat gingiva. Journal of Dental Research 51:
`520-525.
`Go!ub, L. M., Greenwald, R. A., Zebrowski, E. J.
`& Ramamurthy, N. S. 1978b. The etTcct of
`experimental diabctes on the molecular charac-
`teristics of rat tail tcndon collagen. Biochimica
`et Biophysica Acta 534: 73-81.
`Golub, L. M., Nicoll, G. A., Iacono, V. J. &
`Ramamurthy, N. S. 1982. In vivo crevicular
`Ieukocytc responsc to a chemotactic challengc:
`inhibition by experimental diabetes. Infection
`and lmmunity 37: 1013