Hsi Ming Lee

A chemically modified nonantimicrobial tetracycline (CMT-8) inhibits gingival matrix metalloproteinases, periodontal breakdown, and extra-oral bone loss in ovariectomized rats.

ABSTRACT: Estrogen deficiency in the postmenopausal (PM) female is the major cause of osteoporosis and may contribute to increased periodontal disease, including alveolar bone loss, seen in these women. In the current study, an animal model of PM osteoporosis, the OVX adult female rat, was studied to determine: (i) the relationship between periodontal breakdown and skeletal bone loss, and (ii) the effect of CMT‐8 on gingival collagenase and bone loss. OVX rats were daily gavaged with CMT‐8 (1, 2, or 5 mg/rat) for 28 or 90 days; non‐OVX rats and those gavaged with vehicle alone served as controls. Elevated collagenase activity, assessed using [3H‐methyl] collagen as substrate in the presence or absence of APMA, was seen in the gingiva of the OVX rats, and CMT‐8 therapy suppressed this effect. Western blot revealed a similar pattern for MMP‐8 and MMP‐13 concentrations. The changes in the gingival collagenase activity paralleled changes in periodontal bone loss, which, in turn, reflected trabecular bone density changes. Preliminary studies on PM humans administered sub‐antimicrobial tetracycline as a matrix metalloproteinase inhibitor are under way.

Association of Gingival Crevicular Fluid Biomarkers During Periodontal Maintenance With Subsequent Progressive Periodontitis

BACKGROUNDnThe analysis of biomarkers in gingival crevicular fluid (GCF) may be helpful in forecasting patient vulnerability to future attachment loss. The purpose of this study is to correlate GCF biomarkers of inflammation and bone resorption with subsequent periodontal attachment and bone loss in a longitudinal trial of a matrix metalloproteinase (MMP) inhibitor.nnnMETHODSnGCF was collected from two periodontal pockets (mean +/- SD: 5.1 +/- 1.0 mm) at baseline and annually in postmenopausal females with moderate to advanced periodontitis undergoing periodontal maintenance every 3 to 4 months during a 2-year double-masked, placebo-controlled, randomized clinical trial of subantimicrobial dose doxycycline (SDD; 20 mg two times a day). Subjects were randomized to SDD (n = 64) or a placebo (n = 64). GCF was analyzed for the inflammation markers interleukin (IL)-1beta (using enzyme-linked immunosorbent assay), total collagenase activity (using hydrolysis of a synthetic octapeptide), and MMP-8 (using a Western blot) and the bone-resorption marker carboxyterminal telopeptide cross-link fragment of type I collagen (ICTP) (using a radioimmunoassay). Generalized estimating equations were used to associate these biomarkers, categorized into tertiles, with subsequent clinical attachment (using an automated disk probe) or interproximal bone loss (using radiography). Odds ratio (OR) values compared highest to lowest tertile groups.nnnRESULTSnIncreases in GCF IL-1beta and MMP-8 during the first year of periodontal maintenance were associated with increased odds of subsequent (year 2) periodontal attachment loss (OR = 1.67; P = 0.01 and OR = 1.50; P = 0.02, respectively) driven by the placebo group. Elevated baseline ICTP was also associated with increased odds of 1- and 2-year loss of alveolar bone density (OR = 1.98; P = 0.0001) in the placebo group, not the SDD group, and a loss of bone height (OR = 1.38; P = 0.06), again driven by the placebo group.nnnCONCLUSIONnThese data support the hypothesis that elevated GCF biomarkers of inflammation and bone resorption from a small number of moderate/deep sites have the potential to identify patients who are vulnerable to progressive periodontitis, and SDD may modify that risk.

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.

Characteristics of collagenase-2 from gingival crevicular fluid and peri-implant sulcular fluid in periodontitis and peri-implantitis patients: pilot study

Objective. To compare collagenase activity and collagenolytic matrix metalloproteinase (MMP) levels in gingival crevicular fluid (GCF) and in peri-implant sulcular fluid (PISF) in gingivitis (G), chronic periodontitis (CP), and peri-implantitis (PI) human subjects. Material and Methods. GCF and PISF were collected on filter paper strips, volume was determined, and samples were extracted in buffer containing general proteinase but not MMP inhibitors. Collagenase activity was measured using a DNP-synthetic octapeptide, and molecular and activation forms of collagenase-2 by Western immunoblotting. Results. GCF from CP and G sites exhibited elevated collagenase activity and flow, but collagenase concentrations expressed per µl were not significantly different between the healthy and G sites. Minimal fluid was obtained from healthy PISF, and collagenase concentration was the same or lower than in healthy GCF. Although PISF flow was 34% lower than GCF flow in CP subjects, collagenase concentration in CP and in PI sites was 78% and 971% greater, respectively, than in the appropriate healthy sites. Western immunoblot revealed MMP-8 in both PISF and GCF; fibroblast-type MMP-8 was not detected in healthy GCF and PISF. Immunoreactivity level and inactive and activated forms of PMN-type MMP-8 in GCF and PISF increased with the severity of periodontitis and peri-implantitis. Enhanced levels of fibroblast-type MMP-8 in active form were detected only in severe CP GCF and PI PISF. Conclusions. Peri-implantitis PISF contained higher collagenase-2 levels and activity than GCF from similar deep CP sites. GCF and PISF from severe CP and PI exhibited the highest activation of MMP-8 isoenzymes species (PMN and fibroblast-type).

Subantimicrobial-dose doxycycline treatment increases serum cholesterol efflux capacity from macrophages

ObjectiveSubantimicrobial-dose doxycycline (SDD) treatment has been reported to reduce the severity of chronic inflammation and to increase serum high-density lipoprotein cholesterol. In a double-blind, placebo-controlled clinical trial, we determined whether SDD affects the ability of serum to facilitate cholesterol removal from macrophages.MethodsForty-five postmenopausal osteopenic women with periodontitis were randomly assigned to take placebo (nxa0=xa026) or doxycycline hyclate (20xa0mg, nxa0=xa019) tablets twice daily for 2xa0years. Serum samples were collected at baseline, 1-, and 2-year appointments. The cholesterol efflux capacity of serum from cultured human macrophages (THP-1) was measured.ResultsSDD subjects demonstrated a significant increase in serum-mediated cholesterol efflux from macrophages at both time points compared to baseline (pxa0<xa00.04 for each). Mean cholesterol efflux levels over the first year of follow-up were 3.0xa0percentage points (unit change) higher among SDD subjects compared to placebo subjects (pxa0=xa00.010), while there was no significant difference in 2-year changes. There were no significant differences in the changes of apolipoprotein A-I, apolipoprotein A-II, or serum amyloid A levels between the groups.ConclusionsOur results suggest that SDD treatment may reduce the risk of cardiovascular disease in this patient group by increasing the cholesterol efflux capacity of serum.

A Novel Chemically Modified Curcumin “Normalizes” Wound-Healing in Rats with Experimentally Induced Type I Diabetes: Initial Studies

Introduction. Impaired wound-healing in diabetics can lead to life-threatening complications, such as limb amputation, associated in part with excessive matrix metalloproteinase- (MMP-) mediated degradation of collagen and other matrix constituents. In the current study, a novel triketonic chemically modified curcumin, CMC2.24, was tested for efficacy in healing of standardized skin wounds in streptozotocin-induced diabetic rats. Initially, CMC2.24 was daily applied topically at 1% or 3% concentrations or administered systemically (oral intubation; 30u2009mg/kg); controls received vehicle treatment only. Over 7 days, the diabetics exhibited impaired wound closure, assessed by gross and histologic measurements, compared to the nondiabetic controls. All drug treatments significantly improved wound closure with efficacy ratings as follows: 1% 2.24 > systemic 2.24 > 3% 2.24 with no effect on the severe hyperglycemia. In subsequent experiments, 1% CMC2.24 “normalized” wound-healing in the diabetics, whereas 1% curcumin was no more effective than 0.25% CMC2.24, and the latter remained 34% worse than normal. MMP-8 was increased 10-fold in the diabetic wounds and topically applied 1% (but not 0.25%) CMC2.24 significantly reduced this excessive collagenase-2; MMP-13/collagenase-3 did not show significant changes. Additional studies indicated efficacy of 1% CMC2.24 over more prolonged periods of time up to 30 days.

Chemically Modified Tetracycline Improves Contractility in Porcine Coronary Ischemia/Reperfusion Injury

Abstractu2002 Background: Reperfusion of ischemic myocardium has been implicated in extension of infarct size and deleterious clinical outcomes. Anti‐inflammatory agents reduce this reperfusion injury. Chemically modified tetracycline‐3 (CMT‐3) (Collagenex Pharmaceuticals, Newtown, PA, USA) lacks antimicrobial properties yet retains anti‐inflammatory activity. We examined infarct size and myocardial function in a porcine coronary artery occlusion/reperfusion model in CMT‐3‐treated and control animals. Methods: Yorkshire pigs (n = 8) underwent median sternotomy, pretreatment with heparin (300 U/kg and 67 U/kg/hr IV) and lidocaine (1 mg/kg IV) and were divided into two groups. Group one (n = 4) had the left anterior descending artery (LAD) occluded for 1 hour, after which it was reperfused for 2 hours. Group two (n = 4) had an identical protocol to group one except CMT‐3 (2 mg/kg IV) was administered prior to occlusion of the LAD. Results: Animals receiving CMT‐3 had significantly decreased infarct size in relation to the ventricular area‐at‐risk (AAR) (28 ± 9% vs. 64 ± 8%; p < 0.05). Myocardial contractile function was superior in the CMT‐3 treatment, indicated by a higher cardiac index (2.9 ± 0.3 vs. 2.0 ± 0.3 L/min/m2; p < 0.05) and stroke volume index (22 ± 2 vs. 17 ± 1 L/m2/beat; p < 0.05). Conclusions: CMT‐3 decreased infarct size in relation to the AAR resulting in relative preservation of contractility, suggesting CMT‐3 may improve outcomes during myocardial ischemia reperfusion.