Lorne M. Golub

Matrix metalloproteinases: Contribution to pathogenesis, diagnosis and treatment of periodontal inflammation

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.

Pharmacologic suppression of experimental abdominal aortic aneurysms: A comparison of doxycycline and four chemically modified tetracyclines

BACKGROUND Matrix metalloproteinases (MMPs) likely contribute to the degradation of medial elastin in abdominal aortic aneurysms (AAAs), and tetracycline antibiotics exhibit MMP-inhibiting properties. The purpose of this study was to compare the effects of doxycycline and several non-antibiotic chemically modified tetracyclines (CMTs) in a rat model of elastase-induced AAA. METHODS Fifty-two male Wistar rats underwent intraluminal perfusion of the abdominal aorta with porcine pancreatic elastase. The rats then were treated for 7 days with subcutaneous injections of saline solution, different doses of doxycycline, or 1 of 4 different CMTs. The aortic diameters were measured with microcalipers, and the fixed tissues were examined by means of light microscopy. Gelatin zymography was used to assess the MMP activity in the aortic tissue extracts. RESULTS The mean aortic diameter in the control group increased by 126% +/- 14% on day 7 (from 1.57 +/- 0.04 mm to 3.54 +/- 0.27 mm; P <.05), and 5 of 6 animals (83%) had AAAs. Doxycycline appeared to inhibit aortic dilatation in a dose-dependent manner, and AAAs did not develop in any animals. Half-maximal effects were observed at a dose of approximately 6 mg/kg/day, and maximal effects were noted at greater than 30 mg/kg/day. No AAAs were observed in the animals that were treated with CMTs at 15 mg/kg/day. Each of the following CMTs exhibited an efficacy that was similar to that of doxycycline (percent inhibition of aortic dilatation vs control; all P <.05): CMT-3 (47.6%), CMT-4 (38.9%), CMT-7 (47.6%), CMT-8 (54.0%), and doxycycline (51.6%). Tissues from saline solution-treated controls exhibited a transmural inflammatory response and marked destruction of the medial elastic lamellae. Tetracycline derivatives limited the disruption of medial elastin without appearing to alter either the inflammatory response or the rat aortic wall production of metallogelatinases. CONCLUSION Tetracycline derivatives suppress the development of AAAs after elastase-induced aortic injury in the rat. The aneurysm-suppressing effects of doxycycline appear to be dose-dependent and distinct from its antibiotic activities, and they coincide with the structural preservation of medial elastin fibers. Further studies are needed to explore the potential of MMP-inhibiting tetracyclines as a novel pharmacologic strategy for the suppression of aortic aneurysms.

Blocking Periodontal Disease Progression by Inhibiting Tissue-Destructive Enzymes: A Potential Therapeutic Role for Tetracyclines and Their Chemically-Modified Analogs

Tetracyclines (TCs) have wide therapeutic usage as antimicrobial agents; these drugs (e.g., minocycline, doxycycline) remain useful as adjuncts in periodontal therapy. However, TCs also have non-antimicrobial properties which appear to modulate host response. In that regard, TCs and their chemically-modified analogs (CMTs) have been shown to inhibit the activity of the matrix metalloproteinase (MMP), collagenase. The activity of this enzyme appears crucial in the destruction of the major structural protein of connective tissues, collagen. Such pathologic collagenolysis may be a common denominator in tissue destructive diseases such as rheumatoid and Osteoarthritis, diabetes mellitus, bullous dermatologic diseases, corneal ulcers, and periodontitis. The mechanisms by which TCs affect and, possibly, diminish bone resorption (a key event in the pathogenesis of periodontal and other diseases) are not yet understood. However, a number of possibilities remain open for investigation including the following: TCs may 1) directly inhibit the activity of extracellular collagenase and other MMPs such as gelatinase; 2) prevent the activation of its proenzyme by scavenging reactive oxygen species generated by other cell types (e.g. PMNs, osteoclasts); 3) inhibit the secretion of other collagenolytic enzymes (i.e. lysosomal cathepsins); and 4) directly affect other aspects of osteoclast structure and function. Several recent studies have also addressed the therapeutic potential of TCs and CMTs in periodontal disease. These drugs reduced excessive gingival collagenase activity and severity of periodontal breakdown in rats infected with Porphyromonas gingivalis and in diabetic rats. Furthermore, the latter drug (CMT) was not associated with the emergence of TC-resistant microorganisms. In human clinical trials, low-dose doxycycline therapy substantially reduced collagenase activity in the gingiva and GCF, and prevented the loss of attachment in adult periodontitis. Clearly, the non-antimicrobial properties of TCs have enormous medical and dental therapeutic potential since these drugs can inhibit the activity of MMPs and their degradation of non-osseous and osseous connective tissues. J Periodontol 1993; 64:819-827.

Clinical and Biochemical Results of the Metalloproteinase Inhibition with Subantimicrobial Doses of Doxycycline to Prevent Acute Coronary Syndromes (MIDAS) Pilot Trial

Background—Vulnerable plaque demonstrates intense inflammation in which macrophages secrete matrix metalloproteinases (MMPs) that degrade the fibrous cap, ultimately leading to rupture, in situ thrombosis, and an associated clinical event. Thus, inhibition of MMP activity or more general suppression of vascular inflammation are attractive targets for interventions intended to reduce plaque rupture. We hypothesized that subantimicrobial doses of doxycycline (SDD) (20 mg twice daily) would benefit patients with coronary artery disease by reducing inflammation and MMP activity and thus possibly prevent coronary plaque rupture events. Methods and Results—We conducted a prospective, randomized, double-blind, placebo-controlled pilot study of 6 months of SDD or placebo treatment to reduce inflammation and prevent plaque rupture events. A total of 50 patients were enrolled, of whom 24 were randomized to placebo and 26 to SDD. At 6 months, there was no difference in the composite endpoint of sudden death, fatal myocardial infarction (MI), non-fatal MI, or troponin-positive unstable angina in SDD compared with placebo-treated patients (8.4% versus 0%, P =0.491). Biochemical markers of inflammation were assessed in plasma at study entry and after 6 months of therapy in 30 patients. In SDD-treated patients, high-sensitivity C-reactive protein (CRP) was reduced by 46% from 4.8±0.6 &mgr;g/mL to 2.6±0.4 &mgr;g/mL (P =0.007), whereas CRP was not significantly reduced in placebo patients. Interleukin (IL)-6 decreased from 22.1±3.7 pg/mL at baseline to 14.7±1.8 pg/mL at 6 months in SDD-treated patients (P =0.025) but did not decrease significantly in placebo-treated patients. On zymography, pro-MMP-9 activity was reduced 50% by SDD therapy (P =0.011), whereas it was unchanged by placebo treatment. Conclusion—SDD appears to exert potentially beneficial effects on inflammation that could promote plaque stability. These findings should be investigated in a larger study.

Inhibition of cell proliferation, invasion, tumor growth and metastasis by an oral non-antimicrobial tetracycline analog (COL-3) in a metastatic prostate cancer model

Antibiotic forms of tetracycline exhibit antitumor activity in some tumor models. However, their low in vivo efficacy and associated morbidity limit their long‐term application in cancer therapy. This report appraises the efficacy of doxycycline (DC) and non‐antimicrobial, chemically modified tetracyclines (CMTs) against prostate cancer. Both DC and several CMTs inhibited prostate tumor cell proliferation in vitro. Some of the CMTs were significantly more potent than DC. One of the CMTs, 6‐deoxy, 6‐demethyl, 4‐de‐dimethylamino tetracycline (CMT‐3, COL‐3), was the most potent inhibitor (50% inhibition dose [GI50] ≤ 5.0 μg/ml). Exposure of tumor cells to CMT‐3 induced both apoptosis and necrosis. Mitochondrial depolarization and increased levels of reactive hydroxyl radicals were also observed in cells treated with CMT‐3. Cell cycle arrest at the G0/G1 compartment was observed in CMT‐3‐ and DC‐treated cells. DC and CMTs also inhibited the invasive potential of the tumor cells in vitro, from 10% (CMT‐6) to >90% (CMT‐3). CMT‐3 and DC decreased matrix metalloproteinase (MMP)‐2, tissue inhibitor of MMP (TIMP)‐1 and TIMP‐2 secretion in treated cultures and inhibited activity of secreted MMPs, CMT‐3 was a stronger inhibitor. Daily oral gavage of DC and CMT‐3 inhibited tumor growth and metastasis in the Dunning MAT LyLu rat prostate tumor. Decreases in tumor growth (27–35%) and lung metastases were observed (28.9 ± 15.4 sites/animal [CMT‐3‐treated] versus 43.6 ± 18.8 sites/animal [DC‐treated] versus 59.5 ± 13.9 [control]; p < 0.01]. A delay in tumor growth (27 ± 9.3%, p < 0.05), reduction in metastases (58 ± 8%) and decrease in tumor incidences (55 ± 9%, CMT‐3‐treated) were also observed, when rats were predosed for 7 days. No significant drug‐induced morbidity was observed in any of the animals. These results, along with a recently concluded clinical trial, suggest a potential use of CMT‐3 as an oral, nontoxic drug to treat metastatic prostate and other cancers.

Doxycycline and Chemically Modified Tetracyclines Inhibit Gelatinase A (MMP‐2) Gene Expression in Human Skin Keratinocytes

The mechanism of tetracycline-induced inhibition of matrix metalloproteinases (MMP) was studied by measuring the MMP secretion and MMP-2 mRNA levels in unkeratinizing periodontal ligament epithelial cells and skin keratinocytes cultured in the presence of doxycycline or chemically modified tetracyclines (CMT) lacking antimicrobial activity. Doxycycline, CMT-1, and CMT-8 exerted a direct dose-dependent inhibition of porcine periodontal ligament epithelial cell medium MMP activity as assayed by gelatin enzymography. Both the 92-kDa (MMP-9) and 72-kDa (MMP-2) gelatinases were inhibited by the tetracyclines added to the conditioned medium. Culturing the cells in the presence of the tetracyclines required considerably smaller concentrations to reduce the secreted MMP activity. The drugs were not toxic to the epithelial cells at concentrations from 4 to 250 micrograms/mL up to 24 h of culture. Tetracycline effects on the MMP-2 mRNA levels were studied in human skin keratinocytes using Northern hybridization analysis with a specific cDNA probe. A marked inhibition in the MMP-2 gene expression was observed by 6 h with 5 micrograms/mL of doxycycline, CMT-1 or CMT-8. Doxycycline inhibition was somewhat stronger than the two other tetracyclines. After 24 h of culture with 50 micrograms/mL of the drugs, the total RNA levels also decreased by 33 to 40%. The 72-kDa gelatinase activity in culture medium of the keratinocytes followed roughly the pattern of inhibition of the gene expression. We conclude that doxycycline and the chemically modified tetracyclines, in addition to inhibiting the MMP activity may also reduce the enzyme expression at the transcriptional level.

Matrix metalloproteinase inhibitor prevents acute lung injury after cardiopulmonary bypass.

BACKGROUND Acute lung injury (ALI) after cardiopulmonary bypass (CPB) results from sequential priming and activation of neutrophils. Activated neutrophils release neutral serine, elastase, and matrix metalloproteinases (MMPs) and oxygen radical species, which damage alveolar-capillary basement membranes and the extracellular matrix, resulting in an ALI clinically defined as adult respiratory distress syndrome (ARDS). We hypothesized that treatment with a potent MMP and elastase inhibitor, a chemically modified tetracycline (CMT-3), would prevent ALI in our sequential insult model of ALI after CPB. METHODS AND RESULTS Anesthetized Yorkshire pigs were randomized to 1 of 5 groups: control (n=3); CPB (n=5), femoral-femoral hypothermic bypass for 1 hour; LPS (n=7), sham bypass followed by infusion of low-dose Escherichia coli lipopolysaccharide (LPS; 1 microgram/kg); CPB+LPS (n=6), both insults; and CPB+LPS+CMT-3 (n=5), both insults plus intravenous CMT-3 dosed to obtain a 25-micromol/L blood concentration. CPB+LPS caused severe lung injury, as demonstrated by a significant fall in PaO(2) and an increase in intrapulmonary shunt compared with all groups (P<0.05). These changes were associated with significant pulmonary infiltration of neutrophils and an increase in elastase and MMP-9 activity. CONCLUSIONS All pathological changes typical of ALI after CPB were prevented by CMT-3. Prevention of lung dysfunction followed an attenuation of both elastase and MMP-2 activity. This study suggests that strategies to combat ARDS should target terminal neutrophil effectors.

Scientific Basis of a Matrix Metalloproteinase‐8 Specific Chair‐side Test for Monitoring Periodontal and Peri‐implant Health and Disease

ABSTRACT: 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‐im‐plant 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.

Tetracyclines inhibit parathyroid hormone-induced bone resorption in organ culture

Several tetracyclines (minocycline, doxycycline, tetracycline), in levels approximating physiologic concentrations, were found to inhibit parathyroid hormone-induced bone resorption in organ culture; the specificity of this effect was demonstrated by comparison with other (non-tetracycline) types of antibiotics. The ability of tetracyclines to inhibit bone resorption is consistent with the recent proposal by Golub et al2. that these antibiotics can inhibit mammalian collagenolytic enzymes by a mechanism unrelated to the drugs antibacterial efficacy, a property which could be therapeutically useful in diseases characterized by excessive collagen breakdown.

Potential of tetracyclines to modify cartilage breakdown in osteoarthritis.

For several decades, it has been recognized that an imbalance between activated matrix metalloproteinases, generated locally by both infiltrating and resident cells, and their endogenous inhibitors may play a role in the pathologic breakdown of the joint extracellular matrix in osteoarthritis. This understanding has stimulated the search for a number of synthetic matrix metalloproteinase inhibitors that could serve as potential therapeutic agents. Tetracycline analogues are currently on the threshold of approval as anti-matrix metalloproteinases for another extracellular matrix-destructive disease, periodontitis, and this application could be extended to osteoarthritis and rheumatoid arthritis therapy. In this regard, specially formulated low-dose regimens of a commercially available tetracycline, doxycycline, have been used in long-term clinical trials and were found to reduce extracellular matrix breakdown, including bone loss, in adult periodontitis. Matrix metalloproteinase inhibition by tetracycline analogues is now recognized as complex, and multiple mechanisms have been proposed. A series of recently discovered nonantimicrobial chemically modified tetracyclines are potent inhibitors of several classes of matrix metalloproteinases, preventing collagen breakdown and bone loss in a variety of animal models, although these analogues have not yet been approved for human use. Various tetracyclines have reduced the severity of osteoarthritis in animal models, indicating therapeutic potential for this class of compounds in the future.