Matrix metalloproteinases (MMPs) form a family of enzymes that mediate multiple functions both in the tissue destruction and immune responses related to periodontal inflammation. The expression and activity of MMPs in non-inflamed periodontium is low but is drastically enhanced to pathologically elevated levels due to the dental plaque and infection-induced periodontal inflammation. Soft and hard tissue destruction during periodontitis and peri-implantitis are thought to reflect a cascade of events involving bacterial virulence factors/enzymes, pro-inflammatory cytokines, reactive oxygen species and MMPs. However, recent studies suggest that MMPs can also exert anti-inflammatory effects in defence of the host by processing anti-inflammatory cytokines and chemokines, as well as by regulating apoptotic and immune responses. MMP-inhibitor (MMPI)-drugs, such as doxycycline, can be used as adjunctive medication to augment both the scaling and root planing-treatment of periodontitis locally and to reduce inflammation systematically. Furthermore, MMPs present in oral fluids (gingival crevicular fluid (GCF), peri-implant sulcular fluid (PISF), mouth-rinses and saliva) can be utilized to develop new non-invasive, chair/bed-side, point-of-care diagnostics for periodontitis and dental peri-implantitis.

A seminal experiment involving a germ-free rat model of connective tissue breakdown (followed soon thereafter by a series of in vitro studies) identified an unexpected non-antimicrobial property of tetracyclines (TCs). This ability of TCs to inhibit matrix metalloproteinases (MMPs) such as collagenase was found to reflect multiple direct and indirect mechanisms of action, and to be therapeutically useful in a variety of dental (e.g., adult periodontitis) and medical (e.g., arthritis, osteoporosis, cancer) diseases. The site on the TC molecule responsible for its MMP-inhibitory activity was identified which led to the development of a series of chemically modified non-antimicrobial analogs, called CMTs, which also have therapeutic potential but do not appear to induce antibiotic side-effects. Longitudinal double-blind studies on humans with adult periodontitis have demonstrated that a sub-antimicrobial dose of doxycycline (previously reported to suppress collagenase activity in the periodontal pocket) is safe and effective and has recently been approved by the FDA as an adjunct to scaling and root planing.

Tetracyclines have long been considered useful adjuncts in peridontal therapy based on their antimicrobial efficacy against putative periodontopathogens. However, recently these drugs were found to inhibit mammalian collagenases and several other matrix metalloproteinases (MMPs) by a mechanism independent of their antimicrobial activity. Evidence is presented that this property may be therapeutically useful in retarding pathologic connective tissue breakdown, including bone resorption. The experiments leading to this discovery are described and possible mechanisms are addressed, including the specificity of tetracyclines' anti-collagenase activity, the role of the drugs' metal ion (Zn2+, Ca2+)-binding capacity, and the site on the tetracycline molecule responsible for this nonantimicrobial property. Of extreme interest, the tetracycline molecule has been chemically modified in multiple ways, generating a new family of compounds called CMTs (chemically modified tetracyclines) that lack antimicrobial but still retain anti-collagenase activity. The first of these CMTs, 4-de-di-methylaminotetracycline, was found not to produce a major side-effect of antimicrobial tetracycline therapy--its administration to experimental animals did not result in the emergence of tetracycline-resistant microorganisms in the oral flora and gut. Numerous examples of the clinical potential of this non-antimicrobial property of tetracyclines in the treatment of periodontal and several medical diseases (e.g., sterile corneal ulcers, rheumatoid arthritis, skin bullous lesions, tumor-induced angiogenesis and metastasis) are discussed.

This is the first demonstration in human subjects of the simultaneous reduction of excessive MMP activity with concomitant reduction in levels of collagen degradation fragments. The findings are potentially applicable to a wide variety of human diseases characterized by excessive collagenase activity.

Doxycycline administered at subantimicrobial doses led to improvements in disease parameters, with no apparent side effects, and appears to have significant potential as an oral adjunctive therapy in the long-term management of adult periodontitis.

Tetracyclines are now recognized to have non-antimicrobial properties with therapeutic potential--for example, these agents can inhibit pathologic collagenolysis by blocking mammalian collagenases and other matrix-degrading metalloproteinases. In the current study, adult human subjects with moderate chronic periodontitis were administered specially formulated capsules of doxycycline, containing lower-than-usual amounts of this semi-synthetic tetracycline, on a daily basis for 2 weeks prior to a full-thickness flap procedure; control subjects were administered placebo capsules. The gingiva excised during this surgical procedure were extracted, the extracts partially purified and analyzed for collagenase activity using [3H-methyl] collagen as substrate and the techniques of SDS-PAGE/fluorography or liquid scintillation spectrometry. In the absence of any drug pre-treatment, or after a 2-wk regimen of placebo capsules, the gingival extracts exhibited pathologically-excessive mammalian collagenase activity. The 2-wk regimen of low-dose doxycycline capsules reduced this activity by approximately 60-80% (p less than 0.05 and less than 0.01, respectively); in vitro exposure of the gingival extract to doxycycline also inhibited its collagenase activity. Collagenase activity in the crevicular fluid of periodontal pockets of an additional group of subjects was also significantly reduced, as was the severity of inflammation at the same gingival sites. The results suggest that a regimen of low-dose doxycycline capsules may provide a safe (other studies indicate that this regimen may not induce tetracycline resistance in the subgingival plaque) and effective adjunct to instrumentation therapy in the management of pathologic collagenolysis in the periodontal patient. However, further studies are necessary to confirm this hypothesis.

Matrix metalloproteinases (MMPs), especially collagenase-2 (MMP-8), are key mediators of irreversible tissue destruction associated with periodontitis and peri-implantitis. MMP-8 is known to exist in elevated amounts and in active form in the gingival crevicular fluid (GCF) and peri-implant sulcular fluid (PISF) from progressing periodontitis and peri-implantitis lesions and sites, respectively. (Sorsa et al. Ann. N.Y. Acad. Sci. 737: 112-131 [1994]; Teronen et al. J. Dent. Res. 76: 1529-1537 [1997]). We have developed monoclonal antibodies to MMP-8 (Hanemaaijer et al. J. Biol. Chem. 272: 31504-31509 [1997]) that can be used in a chair-side dipstick test to monitor the course and treatment of periodontitis and peri-implantitis. Monoclonal and polyclonal antibody tests for MMP-8 coincided with the classical functional collagenase activity test from GCF and PISF (Sorsa et al. J. Periodont. Res. 22: 386-393 [1988]) in periodontal and peri-implant health and disease. In future a chair-side functional and/or immunological MMP-test can be useful to diagnose and monitor periodontal and peri-implant disease and health.

Doxycycline inhibits neutrophil (PMN)-type matrix metalloproteinases in human adult periodontitis gingiva. J Clin Periodontol 1995; 22: 100^109. © Mutiksgaard, i995.Abstract We previousiy reported tliat low-dose doxycycline (DOXY) therapy reduces host-derived coliagenase activity in gingival tissue of aduit periodontitis (AP) patients. However, it was not ciear wiiether this in vivo effect was direct or indirect. In the present study, iniiamed human gingival tissue, obtained from AP patients during periodontal surgery, was extracted and the extracts partia!!y purified by (NH4)2SO4 precipitation. The extracts were then ana!yzed for coUagenase activity using SDS-PAGE/iluofography/iaser densitometry, and for geiatinase activity using type I geiatin zymography as weii as a new quantitative assay using biotinyiated type I geiatin as substrate. DOXY was added to the incubation mixture at a fina! concentration of 0-!000 ^M. The concentration of DOXY required to inhibit 50% of the gingiva! tissue coiiagenase (IC50) was found to be i6-18 fiM in the presence or absence of i.2 mM APMA (an optimai organomercurial activator of latent procoiiagenases); this IC50 for DOXY was simiiar to that exhibited for coiiagenase or matrix metaiioproteinase (MMP)-8 from polymorphonuclear leukocytes (PMNs) and from gingival crevicuiar fluid (GCF) of AP patients. Of interest. Porphyromonas gingivalis collagenase was also inhibited by similar DOXY levels (IC5o=15 /^M), however the collagenase activity observed in the gingival tissue extracts was found to be of mammalian not bacterial origin based on the production of the specific a'^ (3/4) and a^ (i/4) coliagen degradation fragments. In contrast, the inhibition of coiiagenase purified from cuiture media of human gingival fibroblasts (MMP-1) required much greater DOXY levels (IC5o=280 //M). The predominant molecular forms of gelatinolytic activity present in the AP patients gingivai tissue extracts were found to closeiy correspond to the 92 kD PMN-type geiatinase (MMP-9) although small quantities of 72 kD fibrobiast-type geiatinase (MMP-2), and some other iow moiecuiar weight geiatinases, were also detected. The IC50 of DOXY versus gingivai tissue gelatinoiytic activity was estimated at 30-50 //M measured using either type I geiatin zymography or the biotinylated type I gelatin assay. We conclude that MMPs in inflamed gingival tissue of AP patients, iike those in GCE, originate primarily from infiltrating PMNs rather than resident gingival cells (fibroblasts and epitheha! ce!!s) or monocyte/macrophages, and that their patho!ogica!!y-elevated tissue-degrading activities can be directly inhibited by pharmaco!ogic !eve!s of doxycyc!ine.