v.
`Galderma Laboratories, Inc.
`IPR2015-__
`Exhibit 1022
`
`Exh. 1022
`
`

`
`A Non-antibacterial Chemically-modified Tetracycline
`Inhibits Mammalian Collagenase Activity
`
`L. M. GOLUB, T. F. McNAMARA, G. D'ANGEL0 1, R. A. GREENWALD2 , and N. S. RAMAMURTHY
`
`and Biophysics'. ~chao/ o.( Medicin~,
`Department of Oral Biology and Pathology, School of Dental Medicine, and Departn_wntof Physio~o~rtY
`State University o.f New York at Stony Brook, Stony Brook, New York 11794; and Department of Medlcme-, Long Island Jewzsh Med1cal Cente1.
`7
`New Hyde Park, New York 11042
`
`Tetracrclines (including the semi-synthetic analogues, minocycline and
`doxvc~·cline) are considered useful adjuncts in periodontal therapy
`bec~t;se they suppress Gram-negative periodontopathogens. Recently,
`these antibiotics were found to inhibit mammalian collagenase activ-
`ity, a property which may also be o.f therapeutic value. It has been
`suggested that the anti-collagenase properties of the tetracyclines are
`independent of their antibiotic efficacy. To advanc.e this hypothesis
`fitrther, we chemically converted tetracycline hydrochloride to its non-
`·antimicrobial analogue, de-dimethylaminotetracycline. This chemi-
`cally-modified tetracycline (CMT), although no longer an effective
`antibiotic, was found to inhibit the in vitro activity of coltagenase
`from partially pur{fied extracts of human rheumatoid synovial tissue
`and rachitic rat epiphysis. In a pre/imina!)' in vivo study, patholog-
`icallr-excessive collagenase in skin and gingiva was induced by ren-
`deri;lg adult male rats diabetic, and the oral administration of C.M_T
`to these rats significantly reduced the excessive collagenase acflvzty
`in both tissues. Moreover, CMT administration did not affect these-
`vere hyperglycemia in these rats but did prevent, at least in part, the
`diabetes-induced loss of body weight, skin weight, and skin collagen
`mass; these effects suggest a lack of toxicity in this animal model. A
`proposed clinical advantage of CMT over conventional tetracyclines,
`in the treatment of diseases characterized by excessive collagenolytic
`activit\", is the lack of development of antibiotic-resistant micro-or-
`ganisl;ls during prolonged use. Hm'>'ever, the consideration of clinical
`trials to support this hypothesis must await fwther laboratOI)' and
`extensive toxicity tests.
`
`J Dent Res 66(8):1310-1314, August, 1987
`
`Introduction.
`
`The therapeutic potential of the tetracycline antibiotics as ad-
`juncts in the treatment of periodontal diseases has traditionally
`been ascribed to two factors: (i) their ability to suppress micro-
`organisms such as Bacteroides gingiva/is and Actinobacillus
`actinomvcetemcomitans, which are believed to be periodon-
`topathogens (Slots and Rosling, 1983; Ciancio et a!., 1982;
`Lindhe, 1982; Lindhe eta!., 1983; Slots eta!., 1979; Kamman
`and Karl, 1982); and (ii) their concentration at the site of the
`lesion, within the gingival crevicular fluid, at levels much higher
`than those found in serum (Gordon et a!., 1981; Ciancio et
`a!., 1980). Recently, Golub et a!. (1983, 1984, 1985a, b) in-
`troduced a new concept concerning the therapeutic usefulness
`of the tetracyclines. They proposed that: (i) tetracyclines, but
`not other antibiotics, can inhibit the activity of collagenase (a
`specific collagenolytic metallo-neutral protease produced by
`host tissues which has repeatedly been implicated in periodon-
`tal destruction; see Birkedal-Hansen, 1980, for review); and
`(ii) this newly discovered property of the drugs could provide
`a novel approach to the treatment of diseases, such as peri-
`odontal disease, but also including certain medical· disorders
`
`Received for publication September 15, 1986
`Accepted for publication January 27, 1987
`This investigation was supported by USPHS Research Grant DE-
`03987 from the National Institute of Dental Research (NIH).
`IJIO
`
`(e.g., non-infected corneal ulcers; Perry and Golub, 1985; Perry
`et al., 1986), which involve excessive collagen destruction.
`Tetracycline(s) appear to inhibit collagenase activity by a
`mechanism unrelated to the drug's antibacterial efficacy (see
`"Discussion"). In a recent preliminary report, McNamara et
`a!. (1986) described a minimal chemical modification of the
`tetracycline molecule (i.e., the removal of the dimethylamino
`group from the C4 position on the 'A' ring), which eliminated
`the drug's antibiotic efficacy both in vitro and in vivo. In the
`current study, we investigated the ability of this non-antimi-
`crobial chemically-modified tetracycline (CMT) to inhibit
`collagenase activity. The purpose was to determine, in a man-
`ner more direct than previously attempted, whether the anti-
`collagenase and antibiotic properties of the drug are unrelated,
`each being associated with a different part of the drug mole-
`cule. Of clinical relevance, the long-term use of such an agent
`would have the potential to inhibit collagen destruction during
`chronic disease without evoking a major antibiotic complica-
`tion, namely, the emergence of tetracycline-resistant micro-
`organisms.
`
`Materials and methods.
`Tetracycline (Sigma Chemical Co., St. Louis, MO) was
`chemically modified by removing the dimethylamino group
`from the C4 position of the 'A' ring of the molecule, since the
`normal a-orientation of this group is essential for the drug's
`antibiotic efficacy (Mitscher, 1978). The procedure was based
`on previously published techniques (McCormick et al., 1957;
`Boothe et a!., 1958). The details of the synthesis, chemical
`characterization (including the ultra-violet and infrared spectra
`and thin-layer chromatography Rf values), and loss of anti-
`bacterial activity against Bacillus cereus (the organism typi-
`cally used in standard bioassays for tetracyclines) and against
`several periodontopathogens (Fusobacterium nucleatum and B.
`gingiva/is) will be described elsewhere.
`In brief, tetracycline was reacted (six days, 22°C) in tetra-
`hydrofuran with iodomethane to produce the intermediate, tet-
`racycline methiodide. The solid tetracycline methiodide was
`precipitated, collected by vacuum filtration, washed with cold
`tetrahydrofuran and diethyl ether, and vacuum-dried at 50°C.
`The methiodide intermediate was found to have a melting point
`of 178-180°C, in good agreement with the findings of Mc-
`Cormick eta!. (1957). The intermediate was then dissolved in
`50% acetic acid containing powdered zinc (Zn) (17 mg/mL).
`The suspension was stirred for 30 minutes, the excess Zn was
`filtered off, and, after addition of cold (0-5°C) dilute HCl to
`the filtrate, a yellow precipitate formed. This compound, the
`chemically-modified tetracycline (CMT) or de-dimethylami-
`notetracycline, was washed and re-crystallized from an ethyl
`acetate-petroleum ether (BP 60-90°C) mixture and was found
`to have a melting point of 195-197°C, in agreement with pre-
`viously published data (see above).
`The CMT was administered by oral intubation on a daily
`basis to diabetic rats with pathologically-excessive collagenase
`
`Exh. 1022
`
`

`
`Vol. 66 No.8
`
`A NON-ANTIBACTERIAL TETRACYCLINE INHIBITS COLLAGENASE
`
`/3JJ
`
`activity, by means of techniques described previously (Golub
`eta/., 1983, 1984, 1985b). In the first of two studies, four
`groups of adult (body weight from 350 to 400 g each) male
`Sprague-Dawley rats were established as follows: non-diabetic
`control rats (C group; n = 4 rats), rats in which diabetes was
`induced by i. v. injection of streptozotocin (D group; n = 3);
`and two additional groups of diabetic rats which received either
`metronidazole (70 mg per day; D + met group; n = 5 rats)
`or CMT (20 mg per day; D + CMT group; n = 4 rats)
`throughout the experimental protocol. The regimen, 20 mg per
`day of CMT, was chosen for this initial set of studies because
`the same dose of minocycline, an antibiotically-active tetra-
`cycline, was successfully administered to diabetic rats in pre-
`vious experiments by Golub eta/. (1983 & 1985b). Moreover,
`in humans, metronidazole is administered at a level about 3.8
`times greater than the dosage prescribed for minocycline, and
`this dose, 70 mg metronidazole per day, was found to have no
`inhibitory effect on rat collagenase activity in vivo (Rama-
`murthy et a/., 1986).
`Thirty-seven days after induction of diabetes (drug therapy
`was initiated one day after streptozotocin administration), the
`rats were weighed (they had previously been weighed at time
`0, the day some were injected with streptozotocin, and at sev-
`eral time periods during the experimental protocol), and a blood
`sample was taken for glucose analysis. The animals were then
`killed, the entire skin was dissected and weighed, and samples
`(about 100 mg) of the tissue were analyzed for hydroxyproline
`(after hydrolysis in 6 mol!L HCl, 105°C, 24 hr) and for
`collagenase activity (see below) for each rat separately. The
`buccal gingivae from the maxillary jaws were dissected and
`pooled per group (individual rats did not yield sufficient gin-
`givae to assay for collagenase activity in the tissue extracts).
`As described in detail previously (Ramamurthy and Golub,
`1983; Golub eta!., 1985b), the tissues were minced and ex-
`tracted, the extracts partially purified by ammonium sulfate
`precipitation, and the collagenase activity in the extracts was
`measured with either 14C-gly labeled collagen or [3H-methyl]
`collagen used as substrate.
`The second study in this series was essentially the same as
`that described above, except that it was carried out for 21 rather
`than 37 days, and one of the diabetic groups was treated with
`minocycline (5 mg per day) instead of metronidazole. This
`lower-than-usual dose of minocycline had previously been found
`to be effective in a preliminary study (Ramamurthy et a!.,
`unpublished data). Each of the four groups in study no. 2
`(Controls, Diabetics, D + minocycline, and D + CMT) con-
`tained four rats.
`To determine whether CMT could directly inhibit a mam-
`malian collagenase in vitro, we performed two experiments.
`First, synovial tissue was obtained from knee joints of five
`adult humans with severe rheumatoid arthritis who required
`total joint implant surgery (Greenwald et a!., 1986). A 200-
`mg sample of each specimen was lyophilized, minced with
`scissors, ground with a mortar and pestle, extracted three times
`with petroleum ether for removal of lipids, and dried, and the
`collagenase activity was extracted as described by Ramamur-
`thy and Golub (1983). All of these steps were performed at
`4°C. The latent collagenase in the partially-purified extract was
`activated by brief pre-treatment with 4 f.Lmol!L trypsin ( 10 min,
`22°C), which was then inactivated with a five-fold molar ex-
`cess of soybean trypsin-inhibitor. The trypsin-activated syno-
`vial collagenase was incubated with 14C-gly-labeled collagen
`fibrils for 24 hr at 35°C (Ramamurthy and Golub, 1983) in the
`presence of 0, 2, 5, 10, and 20 f-lg/mL CMT or 0, 5, and 20
`f.Lg/mL minocycline. The radiolabeled collagen degradation
`products were separated from the undigested fibrils and counted
`in a liquid scintillation spectrometer as described previously.
`
`(Note: Trypsin degraded 7.3% ± 1.9 of the 14C-collagen sub-
`strate, confirming the expected resistance of .the collagen sub-
`strate to non-specific protease attack.) In a second experiment
`to evaluate the in vitro inhibition of mammalian collagenase,
`the tetracyclines, CMT or minocycline, were added to extracts
`of rachitic rat epiphyseal cartilage. Induction of rickets by a
`low.,phosphate, vitamin-D-deficient diet results in extremely
`·nigh collagenase activity in the growth plates of rachitic long
`bones (Dean et a!., 1985). Collagenase activity was measured
`in duplicate aliquots of rachitic cartilage extract with 14C-gly-
`labeled collagen fibrils after 48 hours of incubation as de-
`scribed above.
`The data were analyzed by analysis of variance, and the
`significance of the differences between the groups was ana-
`lyzed by Tukey's test (Snedecor and Cochran, 1967).
`
`Results.
`As shown in Table 1, streptozotocin-induced diabetes in-
`creased the blood glucose concentration about 5-8-fold, com-
`pared with control values (p<0.01), and there was no significant
`difference (p>0.05) in this parameter between the four groups
`of diabetics (D, D + metronidazole, D + CMT, D + mino-
`cycline).
`As expected from previous studies (Golub et al., 1983; Schneir
`and Golub, 1981), induction of a severely hyperglycemic, di-
`abetic state in the rat produced both a dramatic increase in skin
`collagenase activity (skin tissue from the control group pro-
`duced no detectable collagenolytic activity above trypsin ac-
`tivity values) and about a 50-60% loss of skin weight (studies
`no. I and 2; Table 1). These differences between the C and D
`groups were statistically significant (p<0.01). Treating the di-
`abetic rats with metronidazole produced no significant effect
`on any of the parameters in skin, including skin weight, col-
`lagen content, and collagenase activity (study no. 1). In con-
`trast, CMT treatment reduced the pathologically-excessive
`collagenase activity by 55% in both studies no. 1 and 2, an
`effect found to be statistically significant (p<0.01). CMT
`administration also increased skin weight in the diabetic rats
`by 60-88% (studies 1 and 2), compared with the values seen
`in the untreated diabetics (p<O. 01). These anti -catabolic ef-
`fects of CMT on connective tissue metabolism were not as-
`sociated with a significant effect on the severity of the
`hyperglycemic state (Table 1). The changes in skin collagen
`mass appeared to follow the same pattern as skin weight. Al-
`though these changes in skin hydroxyproline were found to be
`statistically significant in study no. 2, this was not the case for
`study no. 1. In previous studies where the same animal model
`was used, diabetes produced a significant loss of skin collagen
`(Schneir and Golub, 1981; Golub eta!., 1983). The tetracy-
`cline antibiotic, minocycline, had essentially the same effect
`on the skin parameters as did CMT (study no. 2).
`Parallel changes were seen in the pools of gingival tissue
`from four of these groups of rats. The collagenase activities
`(expressed as the% [3H-methyl]-collagen degraded) in the gin-
`givae from the control, diabetic, diabetic + metronidazole,
`and diabetic + CMT rats were 0%, 97.8%, 93.9%, and 53.0%,
`respectively.
`As shown in the Fig., the control rats gained weight during
`the 37 -day protocol (about a 19% increase, compared with time
`0 values; study no. 1), while both the untreated diabetics and
`the diabetics treated with metronidazole lost 21% of their orig-
`inal weight. CMT (and minocycline; see study no. 2) treatment
`prevented the loss of body weight seen in the other two groups
`of diabetic rats. Similar changes in the control and diabetic
`. groups were seen in study no. 2 and, once again, tetracycline
`
`Exh. 1022
`
`Exh. 1022
`
`

`
`1312
`
`GOLUB et al.
`
`J Dent Res August 1987
`
`TABLE 1
`STREPTOZOTOCIN-INDUCED DIABETES IN THE RAT STIMULATES COLLAGENASE ACTIVITY IN SKIN: EFFECT OF ORAL
`OF
`OR METRONIDAZOLE*
`Skin Collagen
`Content
`(mg Hyp/total skin)
`
`·Blood
`Glucose
`(mg/dL)
`
`Skin
`Weight
`(g)
`
`Skin Collagenase Activity:
`14C-Collagen lysed(%)
`
`Experimental
`Group
`Study No. l:
`Controls
`Diabetics (D)
`D + metronidazole
`D + CMT
`Study No.2:
`6A
`9.4 ± 0.3A
`168A
`1650
`±
`113 ±
`Controls
`552 ± 388
`81 B.a
`38.1 ± 2.18
`660 ±
`Diabetics (D)
`548 ± 308
`J.QC
`1130 ± 69B.b
`19.2 ±
`D + minocycline
`1040 ± 64B.b
`17.2 ± O.lc
`553 ± 318
`D + CMT
`.
`.
`. .
`.
`.
`. .
`,
`*Each value represents the mean ± S.E.
`A.B.C. p < O.Ol; a.b.c. p < 0.05. Group values with different symbols (e.g., A vs. B.) are stgmftcantly different at the levels mdtcated.
`
`ISA
`103 ±
`850 ± 608
`638 ± so8
`713 ± 628
`
`4.7A.a
`±
`63.7
`30.7 ± 2.38
`25.9 ± 3.58
`49.1 ± 3.2A.b
`
`3.3A
`62.1 ±
`27.8 ± 3.38
`43.0 ± 7.2A
`52.5 ± 6.8A
`
`1160
`±
`617 ±
`654 ±
`850 ±
`
`127
`65
`95
`64
`
`5.2 ± 0.9A
`47.3 ± 0.38
`46.0 ± 3.QB
`20.9 ± 3.2c
`
`550
`
`500
`
`2450
`
`-.c
`
`C'
`"Q) 400
`3=
`
`550
`
`!Study 21
`
`500
`
`450
`
`400
`
`350
`
`' \
`0+ min
`\
`\!----1---·1
`0/
`
`ol 0
`
`I
`
`10
`
`I
`
`20
`
`I
`
`I
`
`I
`10
`
`I
`
`20
`
`300
`of
`30
`40
`0
`(days)
`Time
`Sequential changes in body weight during the 37-day (study
`Fig. -
`no. l) and 21-day (study no. 2) experimental protocols. C = non-diabetic
`control rats; D = untreated diabetics; D +met, D +min, D + CMT
`diabetics treated on a daily basis with metronidazole, minocycline, and
`chemically-modified tetracycline, respectively. Each value represents the
`mean ± S.E.
`
`(this time minocycline as well as CMT) prevented body weight
`loss.
`In the first in vitro study, the activated collagenase in the
`partially purified extracts of human synovial tissue degraded
`about 23% of the 14C-collagen fibril substrate (Table 2). Add-
`ing CMT to the reaction mixture in concentrations ranging
`from 2-20 j.Lg/mL decreased the collagenase activity by 35-
`94%, respectively- effects that were statistically significant.
`At the two concentrations tested (5 and 20 j.Lg/mL), minocyc-
`line inhibited collagenase activity to the same extent as did
`CMT. In the second in vitro study, a comparable effect was
`also noted with the rachitic cartilage extract and these two
`tetracyclines (Table 3).
`
`Discussion.
`Tetracyclines are broad-spectrum antibiotics which, through
`their ability to inhibit bacterial protein synthesis, can suppress
`periodontopathic organisms in the oral cavity. Numerous clin-
`
`ical studies (see "Introduction") as well as case reports (Fas-
`ciano and Fazio, 1981; Moskow, 1986) support the usefulness
`of tetracyclines as adjuncts in the treatment of periodontal dis-
`eases. A recent series of studies addressed the additional ability
`of these drugs to inhibit the activity of mammalian collagen-
`ases and the likelihood that this newly discovered property
`contributes significantly to their clinical efficacy in the man-
`agement of periodontal and perhaps other diseases as well (Golub
`eta!., 1983, 1984, 1985a,b; Gomes eta!., 1984; Zucker et
`a!., 1985; Perry and Golub, 1985; Seedor et al., 1985; Green-
`wald eta!., 1986, 1987).
`In the present study, CMT decreased collagenolytic activity
`in skin and gingivae when administered to diabetic rats in vivo,
`and when incubated with mammalian connective tissue colla-
`genases in vitro. We have reported elsewhere that CMT, when
`added to a reaction mixture containing [3H-methyl] collagen
`and synovial fluid from a patient with severe rheumatoid ar-
`thritis, was found to inhibit both the breakdown and loss of a-
`collagen components and the generation of a Adigestion prod-
`ucts (assessed by a combination of SDS-polyacrylamide gel
`electrophoresis and fluorography; Greenwald et a!., 1987).
`Consistent with our previous reports (Ramamurthy eta!., 1986),
`metronidazole (a non-tetracycline antibiotic also used clinically
`as an adjunct in periodontal therapy; Loesche et a!., 1981) had
`no inhibitory effect on the collagenase activity in rat skin and
`gingiva, whereas minocycline (an antibiotic tetracycline re-
`cently shown to be effective in reducing the severity of peri-
`odontal disease; Ciancio et a!., 1980 and 1982) seemed to be
`equally as effective as CMT in inhibiting collagenase activity
`in vivo and in vitro.
`The mechanism by which tetracyclines inhibit collagenase
`appears to be independent of their antibiotic activity. The evi-
`dence includes the following facts: (i) These drugs inhibit
`collagenase activity and· collagen breakdown in germ-free as
`well as conventional rats (Golub et a!., 1983; Golub et a!.,
`1985b ); (ii) their administratio~ in vivo inhibits collagenase
`activity and/or collagen destruction in non-infected tissues of
`humans (Perry and Golub, 1985; Greenwald eta!., 1986), as
`well as in experimental animals (Golub eta!., 1983; Golub et
`a!., 1985b; Seedor eta!., 1985); (iii) these drugs directly in-
`hibit mammalian collagenolytic activity in vitro (Golub et al.,
`1983; Golub et al., 1984; Gomes eta!., 1984; Golub et al.,
`1985a,b; Zucker eta!., 1985); and (iv) their administration to
`humans in low doses substantially reduces the collagenase ac-
`tivity in the fluid of the periodontal pocket without producing
`a detectable effect on the subgingival microflora (Golub eta!.,
`1985a; Golub et a!., 1987). The present study provides direct
`
`Exh. 1022
`
`

`
`Vol. 66 No.8
`
`A NON-ANTIBACTERIAL TETRACYCLINE !NH!B!TS COLLAGENASE
`
`1313
`
`TABLE 2
`A CHEMICALLY-MODIFIED TETRACYCLINE (CMT) INHIBITS COLLAGENASE ACTIVITY FROM HUMAN RHEUMATOID SYNOVIUM IN
`VITRO: A COMPARISON WITH THE
`'
`% Inhibition of
`Collagenase activity by
`
`1 . Synovial collagenase
`alone (0 j.Lg/mL drug)
`2.Collagenase +
`2 j.Lg/mL drug
`3. Collagenase +
`5 j.Lg/mL drug
`4. Collagenase +
`10 j.Lg/mL drug
`5. Collagenase +
`
`CMT
`
`22.6 ± 2.2
`
`15.0 ± 2.2a
`
`10.1 ± 1.6b
`
`4.7
`
`.~
`
`22.6 ± 2.2
`
`11.3 ± 2.1b
`
`CMT
`
`0
`
`35
`
`57
`
`78
`
`0
`
`52
`
`1.0 ± 0.4b
`1.3 ± O.Sb
`94
`96
`*Each value represents the mean value ± S.E. for five patients with severe rheumatoid arthritis except for incubation no. 4, which is the value for one
`patient only.
`ap< 0.05, 1 VS. 2.
`bp<0.01, 1 vs. 3 and 1 vs. 5.
`
`TABLE 3
`AND MINOCYCLINE ON COLLAGENASE
`
`IN EXTRACTS OF RACHITIC RAT CARTILAGE
`% Inhibition of
`Collagenase activity by
`
`Femoral (FEM) cartilage
`extract + Oj.Lg/mL drug
`Tibial (TIB) cartilage
`extract + Oj.Lg/mL drug
`FEM + 3j.Lg/mL drug
`TIB + 3j.Lg/mL drug
`FEM + 1 Oj.Lg/mL drug
`TIB + IOj.Lg/mL drug
`FEM + 20j.Lg/mL drug
`
`CMT
`
`40.4
`
`51.4
`30.3
`32.6
`23.7
`
`8.8
`
`*Each value represents the mean of duplicate analyses.
`
`evidence that the antibiotic and anti-collagenase properties of
`tetracycline reside in different parts of the molecule, since
`chemically modifying the drug to eliminate its anti-bacterial
`efficacy (Mitscher, 1978; McNamara eta/., 1986) did notre-
`duce its anti-collagenase activity. Golub et a!. (1983) sug-
`gested that tetracycline's inhibitory effect on collagenase might
`relate to the drug's well-known ability to bind metal ions (Ca2 +
`and Zn2 +; see Ross and Picozzi, 1985), since this enzyme is
`dependent on these cations to maintain its normal conformation
`and hydrolytic activity (Berman, 1980). The current study sup-
`ports this proposal by demonstrating that the removal of the
`dimethylamino group at C4 , which eliminates the drug's an-
`tibacterial efficacy, does not affect its anti-collagenase prop-
`erties, presumably because the metal-binding carbonyl and
`hydroxyl groups, on the opposite side of the tetracycline mol-
`ecule, are unaffected by the chemical modification (future studies
`will compare the Ca2 + and Zn2 + binding capacity of tetracy-
`cline and CMT). This proposal is supported by the following:
`(i) Minocycline (an antibacterial tetracycline which retains the
`dimethylamino group) and CMT (a non-antibacterial tetracy-
`cline lacking this group at C4) appear to be equally effective
`as inhibitors of mammalian collagenase in vivo and in vitro
`(see Tables 1-3); (ii) consistent with these results, Golub eta!.
`( 1983, 1985b) reported that the in vivo administration of mino-
`cycline (20 mg per day) produced about a 60% reduction in
`the pathologically-excessive collagenase activity in diabetic rat
`tissues (skin, gingivae). The administration of the same dose
`of CMT to diabetic rats resulted in a similar reduction of
`
`40.4
`
`51.4
`
`19.9
`
`13.7
`
`CMT
`
`0
`
`0
`25.0
`36.5
`41.3
`
`78.2
`
`0
`
`0
`
`61.3
`
`73.3
`
`collagenase activity in the same tissues in the current study;
`and (iii) the role of cations in the anti-collagenase effectiveness
`of tetracyclines, including chemically-modified analogues, is
`suggested by the observation that excess Ca2 + can overcome
`the inhibition of the enzyme by the drugs in vitro (Golub et
`al., 1983; Zucker et al., 1985).
`The clinical potential of the anti-collagenase properties of
`tetracyclines is now being considered in the management of a
`number of disease processes involving collagen catabolism (see
`Sheridan, 1984). These include the healing of non-infected
`corneal ulcers (Perry and Golub, 1985; Perry et a!., 1986;
`Seed or et a!., 1985), the inhibition of collagenase in the joint
`tissue and fluid of patients with rheumatoid arthritis (Green-
`wald et a!., 1986, 1987), the reduction of collagenolytic ac-
`tivity generated by melanoma tumor cells (Zucker et a!., 1985),
`the inhibition of excessive bone resorption induced either by
`systemic (Gomes eta!., 1984) or local factors (Golub et a!.,
`1984), and, of course, the reduction of collagenolysis in the
`soft as well as hard tissues during periodontal disease (Golub
`et a!., 1983, 1984, 1985a,b). Consistent with this approach,
`Simonson eta!. (1986) recently demonstrated that minocycline
`and doxycycline (both tetracyclines), administered in vivo to
`rachitic rats, can suppress epiphyseal cartilage collagenase ac-
`tivity and restore some parameters of abnormal bone metabo-
`lism toward normal. However, it is recognized that other steps
`in the breakdown of connective tissues, in addition to the ex-
`tracellular activity of collagenase, may be inhibited by tetra-
`cyclines. Possibilities include (but are not limited to) the
`
`Exh. 1022
`
`

`
`1314
`
`GOLUB eta/.
`
`J Dent Res August 1987
`
`inhibition of other metalloproteinases, such as macrophage
`elastase, and these are currently under consideration.
`A major disadvantage of the long-term use of tetracyclines
`(as with any antibiotic), even though clinically effective in
`reducing the severity of periodontal disease, is the possible
`overgrowth of antibiotic-resistant bacteria (Kornman and Karl,
`1982). A potential advantage of the therapeutic use of CMT
`is its lack of antibiotic activity, which should prevent the above-
`described complication without impairing the drug's anti-
`catabolic efficacy. This hypothesis remains to be tested. The
`current data are encouraging, since the chemically-modified
`tetracycline, when administered to diabetic rats, retarded the
`loss of body and skin-tissue weight and the apparent break-
`down of skin collagen. However, the consideration of clinical
`trials must await extensive toxicity testing and further labora-
`tory studies.
`
`Acknowledgments.
`The authors wish to thank Hsi-Ming Lee, Anna Maria Vidal,
`and Susan A .. Moak for their ex.cellent technical assistance,
`and Pat Calia for typing the manuscript.
`
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