Therapeutic potential of a novel semi-synthetic-sulfated-polysaccharide to suppress inflammatory mediators in P. gingivalis LPS stimulated human monocytes/macrophages.

BACKGROUND: Chronic periodontitis is associated with an increased risk for systemic conditions such as cardiovascular disease, diabetes, and osteoporosis. During chronic periodontitis, endotoxin (lipopolysaccharide, LPS) produced by P. gingivalis provokes monocyte accumulation and differentiation into macrophages and increased secretion of pro-inflammatory cytokines and matrix metalloproteases (MMPs). While normal levels of MMPs are important in cellular function, increased levels of cytokines and MMPs can cause connective tissue destruction. RESULTS: In the current study, we investigated the therapeutic capability of a novel semi-synthetic sulfated polysaccharide (SAGE) on the production of cytokines and MMPs by cultured human mononuclear cells and macrophages stimulated with endotoxin LPS produced by P. gingivalis, a periodontally-relevant cell culture model. Our research demonstrated SAGE inhibited the LPS induced synthesis of inflammatory mediators including TNF-α, IL-1ß, PGE2, and MMP-9 in this periodontal-relevant cell culture model. In addition, TLR-2 and TLR-4 levels were also reduced with the SAGE treatment. CONCLUSIONS: The therapeutic potential of this novel semi-synthetic sulfated polysaccharide compound may help to prevent tissue damage and bone loss in patients with periodontal disease or other inflammatory diseases.

Periodontal disease in acute coronary syndrome patients.

PURPOSE: This pilot study assessed the periodontal status and biomarkers of systemic inflammation in acute coronary syndrome (ACS) patients. METHODS: 15 ACS patients on statin (anti-cholesterol) therapy, were recruited into the study an average of 9 months after discharge from university hospital. Blood and mouthrinse samples were collected for analysis of inflammatory biomarkers including high sensitivity C-reactive protein (hsCRP), IL-6, IL-1ß, TNF-α, and MMP-9. Full-mouth periodontal examination, including pocket depth (PD), clinical attachment levels (CAL), bleeding on probing (BOP), and tooth mobility, was performed. RESULTS: When their periodontal status was assessed by CAL, 100% of these statin-treated ACS patients exhibited moderate (66.7%) to severe (33.3%) periodontal disease, which appears to be higher than the rate described for the general adult population (i.e., 47% for periodontitis). In addition, (1) their blood hsCRP levels ranged from 0.94 to 12.6 mg/L with a mean of 3.41 mg/L, which is considered high risk for cardiovascular disease (CVD) in spite of their statin therapy, and (2) the data demonstrated a positive correlation between severe periodontitis and elevated blood hsCRP levels (P< 0.05), consistent with systemic inflammation. CLINICAL SIGNIFICANCE: This pilot study provides preliminary data for future large-scale studies to define the relationship between ACS and chronic periodontitis, the underlying mechanisms, and the potential therapeutic efficacy of appropriate periodontal management to reduce the risk for cardiovascular disease.

A Modified Glycosaminoglycan, GM-0111, Inhibits Molecular Signaling Involved in Periodontitis.

BACKGROUND: Periodontitis is characterized by microbial infection, inflammation, tissue breakdown, and accelerated loss of alveolar bone matrix. Treatment targeting these multiple stages of the disease provides ways to treat or prevent periodontitis. Certain glycosaminoglycans (GAGs) block multiple inflammatory mediators as well as suppress bacterial growth, suggesting that these GAGs may be exploited as a therapeutic for periodontitis. METHODS: We investigated the effects of a synthetic GAG, GM-0111, on various molecular events associated with periodontitis: growth of Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) pathogenic bacteria associated with periodontitis; activation of pro-inflammatory signaling through TLR2 and TLR4 in mouse macrophage RAW 264.7 cells and heterologously expressed HEK 293 cells; osteoclast formation and bone matrix resorption in cultured mouse pre-osteoclasts. RESULTS: (1) GM-0111 suppressed the growth of P. gingivalis and A. actinomycetemcomitans even at 1% (w/v) solution. The antibacterial effects of GM-0111 were stronger than hyaluronic acid (HA) or xylitol in P. gingivalis at all concentrations and comparable to xylitol in A. actinomycetemcomitans at ≥2% (w/v) solution. We also observed that GM-0111 suppressed biofilm formation of P. gingivalis and these effects were much stronger than HA. (2) GM-0111 inhibited TLR-mediated pro-inflammatory cellular signaling both in macrophage and HEK 293 cells with higher selectivity for TLR2 than TLR4 (IC50 of 1-10 ng/mL vs. > 100 µg/mL, respectively). (3) GM-0111 blocked RANKL-induced osteoclast formation (as low as 300 ng/mL) and bone matrix resorption. While GM-0111 showed high affinity binding to RANKL, it did not interfere with RANKL/RANK/NF-κB signaling, suggesting that GM-0111 inhibits osteoclast formation by a RANKL-RANK-independent mechanism. CONCLUSIONS: We report that GM-0111 inhibits multiple molecular events involved in periodontitis, spanning from the early pro-inflammatory TLR signaling, to pathways activated at the later stage component of bone loss.

Non-antibacterial tetracycline formulations: clinical applications in dentistry and medicine.

In 1983, it was first reported that tetracyclines (TCs) can modulate the host response, including (but not limited to) inhibition of pathologic matrix metalloproteinase (MMP) activity, and by mechanisms unrelated to the antibacterial properties of these drugs. Soon thereafter, strategies were developed to generate non-antibacterial formulations (subantimicrobial-dose doxycycline; SDD) and compositions (chemically modified tetracyclines; CMTs) of TCs as host-modulating drugs to treat periodontal and other inflammatory diseases. This review focuses on the history and rationale for the development of: (a) SDD which led to two government-approved medications, one for periodontitis and the other for acne/rosacea and (b) CMTs, which led to the identification of the active site of the drugs responsible for MMP inhibition and to studies demonstrating evidence of efficacy of the most potent of these, CMT-3, as an anti-angiogenesis agent in patients with the cancer, Kaposi's sarcoma, and as a potential treatment for a fatal lung disease (acute respiratory distress syndrome; ARDS). In addition, this review discusses a number of clinical studies, some up to 2 years' duration, demonstrating evidence of safety and efficacy of SDD formulations in humans with oral inflammatory diseases (periodontitis, pemphigoid) as well as medical diseases, including rheumatoid arthritis, post-menopausal osteopenia, type II diabetes, cardiovascular diseases, and a rare and fatal lung disease, lymphangioleiomyomatosis.

Non-antibacterial tetracyclines modulate mediators of periodontitis and atherosclerotic cardiovascular disease: a mechanistic link between local and systemic inflammation.

Periodontitis, one of the most common chronic inflammatory diseases afflicting man, is increasingly being recognized as a risk factor for atherosclerotic cardiovascular disease (ASCVD). Non-antimicrobial tetracyclines are known to have inhibitory effects on inflammatory mediators and effector molecules, including cytokines and matrix metalloproteinases (MMPs), associated with both diseases. In this paper, we discuss the evidence that doxycycline and related non-antibiotic chemically modified tetracyclines (e.g., CMT-3) can effectively reduce cytokine (TNF-α, IL-6, and MCP-1) production by human mononuclear inflammatory cells when stimulated either by endotoxin (LPS) or by a complex of C-reactive protein/oxidized LDL cholesterol relevant to the pathogenesis of periodontal disease and ASCVD, respectively. This inhibition by tetracycline compounds appears to be mediated at least in part by a suppression of the phosphorylation/activation of the NFκB cell signaling pathway. We are currently conducting clinical trials on patients who exhibit both diseases, and our preliminary data suggest that virtually all acute coronary syndrome (ACS) patients exhibit moderate-to-severe periodontitis, a higher incidence of this oral inflammatory disease than that seen in the population at large. In other studies, a non-antimicrobial formulation of doxycycline (SDD) has been found to dramatically reduce hsCRP, IL-6 and MMP-9 levels in plasma of ACS patients, and SDD has also been found to significantly increase serum levels of both cardio-protective HDL cholesterol and its core molecule apolipoprotein A-I in ASCVD-vulnerable patients with periodontitis. Our current research suggests that one mechanism involved may be the ability of SDD to inhibit MMP-mediated HDL loss by protecting apolipoprotein A-I from proteinase attack. These pleiotropic mechanisms of non-antimicrobial tetracyclines provide significant therapeutic potential to treat chronic inflammatory diseases including both periodontitis and ASCVD.

Subantimicrobial-dose doxycycline modulates gingival crevicular fluid biomarkers of periodontitis in postmenopausal osteopenic women.

BACKGROUND: We recently demonstrated that a 2-year subantimicrobial-dose doxycycline (SDD) regimen (double-masked, placebo-controlled clinical trial) in postmenopausal (PM) women exhibiting mild systemic bone loss (osteopenia) and local bone loss (periodontitis) reduced the progression of periodontal attachment loss (intent-to-treat analysis) and the severity of gingival inflammation and alveolar bone loss (subgroups) without producing antibiotic side effects. We now describe SDD effects on biomarkers of collagen degradation and bone resorption in the gingival crevicular fluid (GCF) of the same vulnerable subjects. METHODS: GCF was collected from SDD- and placebo-treated PM subjects (n=64 each) at the baseline and 1- and 2-year appointments; the volume was determined; and the samples were analyzed for collagenase activity (using a synthetic peptide as substrate), relative levels of three genetically distinct collagenases (Western blot), a type-1 collagen breakdown product/bone resorption marker (a carboxyterminal telopeptide cross-link fragment of type I collagen [ICTP]; radioimmunoassay), and interleukin-1beta (enzyme-linked immunosorbent assay). Statistical analyses were performed using generalized estimating equations; primary analyses were intent-to-treat. RESULTS: Collagenase activity was significantly reduced by SDD treatment relative to placebo based on intent-to-treat (P=0.01). ICTP showed a similar pattern of change during SDD treatment, and GCF collagenase activity and ICTP were positively correlated at all time periods (P<0.001). Matrix metalloproteinase (MMP)-8 accounted for approximately 80% of total collagenase in GCF, with much less MMP-1 and -13, and SDD reduced the odds of elevated MMP-8 by 60% compared to placebo (P=0.006). CONCLUSION: These observations support the therapeutic potential of long-term SDD therapy to reduce periodontal collagen breakdown and alveolar bone resorption in PM women; effects on serum biomarkers of systemic bone loss in these subjects are being analyzed.

Combining host modulation and topical antimicrobial therapy in the management of moderate to severe periodontitis: a randomized multicenter trial.

BACKGROUND: Previous studies showed that host modulation therapy (HMT) or topical antimicrobial therapy (TAT) provided significant adjunctive benefits to scaling and root planing (SRP) in the treatment of chronic periodontitis (CP). The purpose of this study was to evaluate a combination therapy involving SRP, HMT, and TAT in the treatment of moderate to severe CP. METHODS: A 6-month, randomized, multicenter, placebo-controlled, examiner-masked study was undertaken to evaluate the clinical usefulness of a combination treatment of systemically delivered doxycycline hyclate (HMT; 20 mg, twice a day) plus locally delivered doxycycline hyclate gel (TAT; 10%, in pockets > or =5 mm) in combination with SRP versus SRP plus placebo. Clinical outcomes included mean changes in probing depth (PD), clinical attachment level (CAL), bleeding on probing (BOP), and gingival index (GI) at baseline and at 3 and 6 months. RESULTS: In 171 subjects, combination therapy provided significantly greater clinical benefits than control therapy for all clinical measures at 3 and 6 months. In moderate CP (PD of 4 to 6 mm), combination therapy provided significant benefits over control for PD (3 and 6 months: P <0.01), CAL (3 months: P <0.01; 6 months: P <0.03), BOP (3 months: P <0.02; 6 months: P <0.05), and GI (3 months: P <0.01; 6 months: P <0.03). In severe CP (PD > or =7 mm), combination therapy provided significant benefits over control for PD (3 and 6 months: P <0.01), CAL (3 months: P <0.01; 6 months: P <0.02), BOP (3 months: P <0.01; 6 months: P >0.05), and GI (3 months: P <0.01; 6 months: P <0.01). CONCLUSION: Combination therapy, including SRP, HMT, and TAT, provided significantly greater clinical benefits than SRP alone in the treatment of moderate to severe CP.

Efficacy of sub-antimicrobial dose doxycycline in post-menopausal women: clinical outcomes.

AIMS: To determine the clinical efficacy of a 2-year continuous sub-antimicrobial dose doxycycline (SDD; 20 mg bid) in post-menopausal, osteopenic, oestrogen-deficient women on periodontal maintenance. MATERIALS AND METHODS: One-hundred and twenty-eight subjects were randomized to SDD (n=64) or placebo (n=64). Clinical measurements were performed at posterior interproximal sites at baseline and every 6 months during this 2-year randomized, double-blind, placebo-controlled clinical trial with adjunctive, no-cost 3-4-month periodontal maintenance. Statistical analyses of secondary outcomes from this clinical trial used Generalized Estimating Equations in primarily intent-to-treat analyses. RESULTS: For the placebo group, 3.4% of the sites showed improvement in clinical attachment levels (CAL) and 2.7% had progressive loss in CAL; for the SDD group, 5.0% of the sites showed an improvement in CAL and 2.2% had progressive loss in CAL. This difference (2.1% of sites) was more favourable in the SDD group than in the placebo [odds ratio (OR)=0.81 [corrected] 95% confidence interval (CI): 0.67-0.97, p=0.03] in these well-maintained patients, whereas probing depths, bleeding on probing and supragingival plaque did not differ significantly between groups (p>0.2). However, in exploratory subgroup analysis of non-smokers, SDD showed reduced bleeding versus placebo (27%versus 33%; p=0.05). In protocol-adherent subjects, the odds of bleeding were 34% lower for SDD (p=0.05). CONCLUSIONS: Analyses of secondary outcomes of this clinical trial indicated that SDD may be of benefit in reducing progressive attachment loss in post-menopausal females; additional research is needed to confirm these findings. Protocol registered at (ClinicalTrials.gov). Identifier:NCT00066027.

The immunoglobulin A1 proteinase from Streptococcus pneumoniae is inhibited by tetracycline compounds.

Streptococcus pneumoniae produces a zinc-dependent proteinase that cleaves human immunoglobulin (Ig) A1 in the hinge region. This metalloproteinase is hypothesized to act as a virulence factor by allowing S. pneumoniae to evade the protection provided by IgA1, thus enhancing its ability to colonize the human nasopharyngeal region. No biologically compatible inhibitors of this enzyme have been identified. We determined that doxycycline and a chemically modified tetracycline inhibit this enzyme in vitro at low micromolar concentrations.

Subantimicrobial dose doxycycline efficacy as a matrix metalloproteinase inhibitor in chronic periodontitis patients is enhanced when combined with a non-steroidal anti-inflammatory drug.

BACKGROUND: Administration of subantimicrobial dose doxycycline (SDD) to chronic periodontitis (CP) patients has repeatedly been found to reduce mammalian collagenase and other matrix metalloproteinase (MMP) activity in gingival tissues and crevicular fluid, in association with clinical efficacy, without the emergence of antibiotic-resistant bacteria either orally or extra-orally. More recently, SDD adjunctive to repeated mechanical debridement resulted in dramatic clinical improvement in patients (>50% smokers) with generalized aggressive periodontitis. As an additional pharmacologic approach, non-steroidal anti-inflammatory drugs (NSAIDs) can reduce gingival inflammation and alveolar bone resorption, at least under experimental conditions. In the current study, we determined the effect of administering a combination (combination) of these two host-modulating drugs (SDD plus low-dose NSAID) to CP patients, on selected neutral proteinases in gingiva, enzymes believed to mediate periodontal breakdown. Earlier preliminary studies in humans with bullous pemphigoid, which is also associated with excessive levels of host-derived proteinases including MMPs, indicated improved clinical efficacy of combination therapy. METHODS: Nineteen CP patients, scheduled for mucoperiosteal flap surgery bilaterally in the maxillary arch, were randomly distributed into three experimental groups administered either 1) low-dose flurbiprofen (LDF) alone, 50 mg q.d.; 2) SDD (20 mg b.i.d.) alone; or 3) a combination of SDD plus LDF (combination). The gingival tissues were biopsied during surgery from right and left maxillary posterior sextants, before and after a 3-week regimen of medication, respectively. The tissues were then extracted, the extracts partially purified, then analyzed for the endogenous proteinase inhibitor, alpha1-PI, and its breakdown product, and for host-derived matrix metalloproteinases (i.e., collagenases, gelatinases) and neutrophil elastase activities. RESULTS: Short-term therapy with SDD alone produced a significant reduction and LDF alone produced no reduction in host-derived neutral proteinases. However, the combination therapy produced a statistically significant synergistic reduction of collagenase, gelatinase, and serpinolytic (alpha1-PI degrading) activities (69%, 69%, and 75% reductions, respectively) and a lesser reduction of the serine proteinase, elastase (46%). CONCLUSIONS: Consistent with previous studies on animal models of chronic destructive disease (e.g., rheumatoid arthritis), the SDD and NSAID combination therapy synergistically suppressed MMP and other neutral proteinases in the gingiva of CP patients. A mechanism, suggested by earlier animal studies, involves the NSAID, in the combination regimen, increasing the uptake of the tetracycline-based MMP inhibitor in the inflammatory lesion, thus synergistically enhancing the efficacy of this medication.

Cloning and expression of a pectate lyase from the oral spirochete Treponema pectinovorum ATCC 33768.

The pelA gene, encoding a pectate lyase, from Treponema pectinovorum ATCC 33768 was isolated by heterologous expression of a cosmid library in Escherichia coli. In vitro transposon mutagenesis identified an open reading frame of 1293 bp capable of encoding a protein of 430 amino acids with a predicted amino-terminal signal sequence of 21 amino acids. Analysis of the amino acid sequence suggested that it is a member of the polysaccharide lyase family 10 of which all characterized members show pectate lyase activity. An amino-terminal His-tagged recombinant form of PelA was expressed and purified from E. coli. The recombinant enzyme has characteristics common to other bacterial pectate lyases such as an alkaline pH optimum, dependence on calcium ions for activity, and inhibition by zinc ions.

Potato dextrose agar antifungal susceptibility testing for yeasts and molds: evaluation of phosphate effect on antifungal activity of CMT-3.

The broth macrodilution method (BMM) for antifungal susceptibility testing, approved by the National Committee for Clinical Laboratory Standards (NCCLS), was found to have deficiencies in testing of the antifungal activity of a new type of antifungal agent, a nonantibacterial chemically modified tetracycline (CMT-3). The high content of phosphate in the medium was found to greatly increase the MICs of CMT-3. To avoid the interference of phosphate in the test, a new method using potato dextrose agar (PDA) as a culture medium was developed. Eight strains of fungi, including five American Type Culture Collection strains and three clinical isolates, were used to determine the MICs of amphotericin B and itraconazole with both the BMM and the PDA methods. The MICs of the two antifungal agents determined with the PDA method showed 99% agreement with those determined with the BMM method within 1 log(2) dilution. Similarly, the overall reproducibility of the MICs with the PDA method was above 97%. Three other antifungal agents, fluconazole, ketoconazole, and CMT-3, were also tested in parallel against yeasts and molds with both the BMM and the PDA methods. The MICs of fluconazole and ketoconazole determined with the PDA method showed 100% agreement within 1 log(2) dilution of those obtained with the BMM method. However, the MICs of CMT-3 determined with the BMM method were as high as 128 times those determined with the PDA method. The effect of phosphate on the antifungal activity of CMT-3 was evaluated by adding Na2HPO4 to PDA in the new method. It was found that the MIC of CMT-3 against a Penicillium sp. increased from 0.5 microg/ml (control) to 2.0 microg/ml when the added phosphate was used at a concentration of 0.8 mg/ml, indicating a strong interference of Na2HPO4 with the antifungal activity of CMT-3. Except for fluconazole, all the other antifungal agents demonstrated clear end points among the yeasts and molds tested. Nevertheless, with its high reproducibility, good agreement with NCCLS proposed MIC ranges, and lack of interference of phosphate, the PDA method shows promise as a useful assay for antifungal susceptibility testing and screening for new antifungal agents, especially for drugs that may be affected by high (supraphysiologic) phosphate concentrations.

A chemically modified tetracycline (CMT-3) is a new antifungal agent.

Several chemically modified tetracycline analogs (CMTs), which were chemically modified to eliminate their antibacterial efficacy, were unexpectedly found to have antifungal properties. Of 10 CMTs screened in vitro, all exhibited antifungal activities, although their efficacies varied. Among these compounds, CMT-315, -3, and -308 were found to be the most potent as antifungal agents. The MICs of CMT-3 against 47 strains of fungi in vitro were determined by using amphotericin B (AMB) and doxycycline as positive and negative controls, respectively. The MICs of CMT-3 were generally found to be between 0.25 and 8.00 microg/ml, a range that approximates the blood levels of this drug when administrated orally to humans. Of all the yeast species tested to date, Candida albicans showed the greatest sensitivity to CMT-3. The filamentous species most susceptible to CMT-3 were found to be Epidermophyton floccosum, Microsporum gypseum, Pseudallescheria boydii, a Penicillium sp., Scedosporium apiospermum, a Tricothecium sp., and Trichophyton rubrum. Growth inhibition of C. albicans by CMT-3, determined by a turbidity assay, indicated a 50% inhibitory concentration of 1 microg/ml. Thirty-nine strains, including 20 yeasts and 19 molds, were used to measure viability (the ability to grow after treatment with a drug) inhibition by CMT-3 and AMB. CMT-3 exhibited fungicidal activity against most of these fungi, especially the filamentous fungi. Eighty-four percent (16 of 19) of the filamentous fungi tested showed more than 90% inhibition of viability by CMT-3. In contrast, AMB showed fungicidal activity against all yeasts tested. However, most of the filamentous fungi (16 of 19) showed less than 50% inhibition of viability by AMB, indicating that AMB is fungistatic against most of these filamentous fungi. To begin to identify the sites in fungal cells affected by CMT-3, C. albicans and a Penicillium sp. were incubated with the compound at 35 degrees C, and then the fluorescence of CMT-3 was observed by confocal laser scanning electron microscopy. CMT-3 appeared to have widespread intracellular distribution throughout C. albicans and the Penicillium sp. The mechanisms of the antifungal activity of CMT-3 are now being explored.