A novel modified-curcumin 2.24 resolves inflammation by promoting M2 macrophage polarization.

To assess resolving-like activity by a novel chemically-modified curcumin (CMC2.24) in a "two-hit" model of diabetes-associated periodontitis. Macrophages from rats were cultured in the presence/absence of either Lipopolysaccharide (LPS, 1st hit); or advanced-glycation-end products (AGE, 2nd hit); or both combined. CMC2.24 was added as treatment. The conditioned media were analyzed for MMP-9, cytokines (IL-1ß, IL-6, TNF-α), resolvins (RvD1, RvE1, lipoxin A4), and soluble receptor for AGE (sRAGE). The phenotypes of M1/M2 macrophage were analyzed by flow cytometry. Both LPS/AGE-alone, and two-combined, dramatically increased the secretion of MMP-9 by macrophages. CMC2.24 "normalized" the elevated levels of MMP-9 under all conditions. Moreover, CMC2.24 significantly reduced the secretion of IL-1ß and IL-6 with a fewer effects on TNF-α. Importantly, CMC2.24 increased RvD1 and sRAGE secretion by macrophages exposed to LPS/AGE; and both treatment groups exhibited increased M2 relative to M1 populations. Furthermore, scatter-diagram showed the macrophages gradually shifted from M1 towards M2 with CMC2.24-treated, whereas LPS/AGE-alone groups remained unchanged. CMC2.24 "normalized" cytokines and MMP-9, but also enhanced RvD1 and sRAGE in macrophages. Crucially, CMC2.24 appears to be a potent inhibitor of the pro-inflammatory M1 phenotype; and a promotor of the pro-resolving M2 phenotype, thus acting like a crucial "switch" to reduce inflammation.

Efficacy of a Novel Pleiotropic MMP-Inhibitor, CMC2.24, in a Long-Term Diabetes Rat Model with Severe Hyperglycemia-Induced Oral Bone Loss.

Purpose: CMC2.24, a novel 4-(phenylaminocarbonyl)-chemically-modified-curcumin, is a pleiotropic MMP-Inhibitor of various inflammatory/collagenolytic diseases including periodontitis. This compound has demonstrated efficacy in host modulation therapy along with improved resolution of inflammation in various study models. The objective of current study is to determine the efficacy of CMC2.24 in reducing the severity of diabetes, and its long-term role as an MMP-inhibitor, in a rat model. Methods: Twenty-one adult male Sprague-Dawley rats were randomly distributed into three groups: Normal (N), Diabetic (D) and Diabetic+CMC2.24 (D+2.24). All three groups were orally administered vehicle: carboxymethylcellulose alone (N, D), or CMC2.24 (D+2.24; 30mg/kg/day). Blood was collected at 2-months and 4-months' time-point. At completion, gingival tissue and peritoneal washes were collected/analyzed, and jaws examined for alveolar bone loss by micro-CT. Additionally, sodium hypochlorite(NaClO)-activation of human-recombinant (rh) MMP-9 and its inhibition by treatment with 10µM CMC2.24, Doxycycline, and Curcumin were evaluated. Results: CMC2.24 significantly reduced the levels of lower-molecular-weight active-MMP-9 in plasma. Similar trend of reduced active-MMP-9 was also observed in cell-free peritoneal and pooled gingival extracts. Thus, treatment substantially decreased conversion of pro- to actively destructive proteinase. Normalization of the pro-inflammatory cytokine (IL-1ß, resolvin-RvD1), and diabetes-induced osteoporosis was observed in presence of CMCM2.24. CMC2.24 also exhibited significant anti-oxidant activity by inhibiting the activation of MMP-9 to a lower-molecular-weight (82kDa) pathologically active form. All these systemic and local effects were observed in the absence of reduction in severity of hyperglycemia. Conclusion: CMC2.24 reduced activation of pathologic active-MMP-9, normalized diabetic osteoporosis, and promoted resolution of inflammation but had no effect on the hyperglycemia in diabetic rats. This study also highlights the role of MMP-9 as an early/sensitive biomarker in the absence of change in any other biochemical parameter. CMC2.24 also inhibited significant activation of pro-MMP-9 by NaOCl (oxidant) adding to known mechanisms by which this compound treats collagenolytic/inflammatory diseases including periodontitis.

The Maximum-Tolerated Dose and Pharmacokinetics of a Novel Chemically Modified Curcumin in Rats.

PURPOSE: CMC 2.24, a chemically modified curcumin, was developed as a novel, pleiotropic MMP-inhibitor to treat various inflammatory/collagenolytic diseases including periodontitis. To date, this compound has shown efficacy in vitro, in cell culture, and in vivo (oral administration) in mice, rats and dogs. In preparation for possible Phase I human clinical trials, the current study describes the maximum-tolerated-dose (MTD), pharmacokinetics (PK), and toxicology of CMC 2.24 in the rat model. METHODS: For the MTD study, 30 Sprague-Dawley rats were randomly distributed into 5 groups (3M/3F per group): Placebo (vehicle; carboxymethylcellulose) and CMC 2.24 at various doses (50, 100, 500, 1000 mg/kg/day), were administered once daily by oral gavage for 5 days. For the PK study, 24 rats were administered either Placebo or CMC 2.24 (100mg/kg/day) once daily for 28 days or only once (500 or 1000 mg/kg). Analysis of this test compound was done using LC/MS/MS for PK evaluation on blood samples drawn from rats at multiple time points. The animals were sacrificed after 5 or 28 days of treatment, and blood chemistry and serology were analyzed. Major organs (heart, lung, liver, kidney, spleen, intestine, brain) were histologically examined at necropsy. RESULTS: Orally administered, CMC 2.24 did not produce significant changes in body weight, food consumption or adverse events in the MTD and toxicology studies. Moreover, no obvious pathologic changes were observed based on histology, hematology, serum biochemistry, or necropsy compared to placebo-treated controls. The PK study demonstrated a peak-blood concentration (Cmax) at 45 mins after oral administration of 2.24 and a serum half-life of 10 hours. CONCLUSION: In conclusion, CMC 2.24, orally administered to rats once a day, appears to be safe and effective at a wide range of doses, consistent with efficacy previously demonstrated in studies on animal models of various collagenolytic diseases, such as periodontitis, diabetes and cancer.

A Novel Modified-Curcumin Promotes Resolvin-Like Activity and Reduces Bone Loss in Diabetes-Induced Experimental Periodontitis.

PURPOSE: Clinically, it is challenging to manage diabetic patients with periodontitis. Biochemically, both involve a wide range of inflammatory/collagenolytic conditions which exacerbate each other in a "bi-directional manner." However, standard treatments for this type of periodontitis rely on reducing the bacterial burden and less on controlling hyper-inflammation/excessive-collagenolysis. Thus, there is a crucial need for new therapeutic strategies to modulate this excessive host response and to promote enhanced resolution of inflammation. The aim of the current study is to evaluate the impact of a novel chemically-modified curcumin 2.24 (CMC2.24) on host inflammatory response in diabetic rats. METHODS: Type I diabetes was induced by streptozotocin injection; periodontal breakdown then results as a complication of uncontrolled hyperglycemia. Non-diabetic rats served as controls. CMC2.24, or the vehicle-alone, was administered by oral gavage daily for 3 weeks to the diabetics. Micro-CT was used to analyze morphometric changes and quantify bone loss. MMPs were analyzed by gelatin zymography. Cell function was examined by cell migration assay, and cytokines and resolvins were measured by ELISA. RESULTS: In this severe inflammatory disease model, administration of the pleiotropic CMC2.24 was found to normalize the excessive accumulation and impaired chemotactic activity of macrophages in peritoneal exudates, significantly decrease MMP-9 and pro-inflammatory cytokines to near normal levels, and markedly increase resolvin D1 (RvD1) levels in the thioglycolate-elicited peritoneal exudates (tPE). Similar effects on MMPs and RvD1 were observed in the non-elicited resident peritoneal washes (rPW). Regarding clinical relevance, CMC2.24 significantly inhibited the loss of alveolar bone height, volume and mineral density (ie, diabetes-induced periodontitis and osteoporosis). CONCLUSION: In conclusion, treating hyperglycemic diabetic rats with CMC2.24 (a tri-ketonic phenylaminocarbonyl curcumin) promotes the resolution of local and systemic inflammation, reduces bone loss, in addition to suppressing collagenolytic MMPs and pro-inflammatory cytokines, suggesting a novel therapeutic strategy for treating periodontitis complicated by other chronic diseases.

Active matrix metalloproteinase-8 (aMMP-8) point-of-care test (POCT) in the COVID-19 pandemic.

INTRODUCTION: Active matrix metalloproteinase (aMMP)-8 utilized in point-of-care testing (POCT) is regarded as a potential biomarker for periodontal and peri-implant diseases. Various host and microbial factors eventually influence the expression, degranulation, levels and activation of aMMP-8. The type of oral fluids (saliva, mouthrinse, gingival crevicular, and peri-implant sulcular fluids [GCF/PISF], respectively) affect the analysis. AREAS COVERED: With this background, we aimed to review here the recent studies on practical, inexpensive, noninvasive and quantitative mouthrinse and GCF/PISF chair-side POCT lateral flow aMMP-8 immunoassays (PerioSafe and ImplantSafe/ORALyzer) and how they help to detect, predict, monitor the course, treatment and prevention of periodontitis and peri-implantitis. The correlations of aMMP-8 POCT to other independent and catalytic activity assays of MMP-8 are also addressed. EXPERT OPINION: The mouthrinse aMMP-8 POCT can also detect prediabetes/diabetes and tissue destructive oral side-effects due to the head and neck cancers' radiotherapy. Chlorhexidine and doxycycline can inhibit collagenolytic human neutrophil and GCF aMMP-8. Furthermore, by a set of case-series we demonstrate the potential of mouthrinse aMMP-8 POCT to real-time/online detect periodontitis as a potential risk disease for coronavirus disease 2019 (COVID-19). The clinical interdisciplinary utilization of aMMP-8 POCT requires additional oral, medical, and interdisciplinary studies.

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.

An Unexplored Pharmacologic/Diagnostic Strategy for Peri-Implantitis: A Protocol Proposal.

Dental implants are widely utilized for the replacement of missing teeth and are increasingly being placed in patients with systemic diseases, as well as in those who are medically healthy. Furthermore, it is recognized that peri-implant mucositis and peri-implantitis are highly prevalent, affecting large numbers of patients with implants, and it is pertinent to consider whether there may be any systemic impact of these conditions, given that there are known links between periodontitis and a number of chronic inflammatory diseases. In this article, we propose that the potential systemic complications of peri-implant diseases should be investigated in future clinical research, together with studies to identify whether systemically-administered host modulation therapies (HMTs) may be of benefit in the treatment of peri-implant diseases. These "HMTs" may prove a useful adjunct to routinely employed debridement and disinfection protocols, as well as potentially being of benefit in reducing risks of systemic complications. We also consider the use of chair-side diagnostic tests for active matrix metalloproteinase-8 (aMMP-8) in the detection of peri-implant disease given the ability of such tests to detect active tissue breakdown associated with peri-implantitis and periodontitis before conventional clinical and radiographic measurements indicate pathologic changes. These novel diagnostic and therapeutic strategies are relevant to consider as they may improve the management of peri-implant disease (beyond local debridement procedures), especially in those patients in whom systemic inflammation might be of concern.

Chemically-Modified Curcumin 2.24: A Novel Systemic Therapy for Natural Periodontitis in Dogs.

PURPOSE: To determine the effect of a pleiotropic MMP-inhibitor, a novel chemically-modified curcumin 2.24 (CMC2.24), on the clinical and biological measures of naturally-occurring periodontitis in the beagle dog. METHODS: Eight adult female dogs with generalized periodontitis were distributed into two groups: Placebo and Treatment (n=4/group). After a 1-hr full-mouth scaling and root planing (SRP) at time 0, placebo or CMC2.24 (10mg/kg) capsules were orally administered once/day for 3 months. Various clinical periodontal parameters (e.g., pocket depth, gingival index) were measured at different time periods (0, 1, 2 and 3 months), and gingival crevicular fluid (GCF) samples and gingival tissue biopsies (3-month) were analyzed for cytokines, MMPs and cell-signaling molecules. Standardized radiographs were taken at 0 and 3-month; in addition, peripheral blood monocytes/macrophages from these dogs at 3-month were cultured and analyzed for the pro-, activated-, and total-forms of both MMP-2 and MMP-9. RESULTS: CMC2.24 treatment significantly reduced gingival inflammation (gingival index, GCF flow), pocket depth (PD), and the numbers of pockets (PD≥4mm), compared to placebo. CMC2.24 also significantly reduced MMP-9 and MMP-2 (primarily in the activated-form) in gingival tissue, alveolar bone loss, and reduced GCF IL-1ß. Cell-signaling molecules, TLR-2 (but not TLR-4) and p38 MAPK, responded to CMC2.24 in a pattern consistent with reductions in inflammation and collagenolysis. In culture, CMC2.24 had no effect on pro-MMP-9 but essentially completely blocked the conversion of pro- to activated-MMP-9 in systemic blood-derived monocytes/macrophages from these dogs. CONCLUSION: In the beagle dog model of natural periodontitis, orally administered CMC2.24 (a novel triketonic phenylaminocarbonyl-curcumin) significantly decreased clinical measures of periodontitis as well as pro-inflammatory cytokines, MMPs, and cell-signaling molecules. These and previous studies, using other in vitro and in vivo models, support the clinical potential of CMC2.24 as a novel adjunct to SRP in the treatment of chronic periodontitis.

Periodontal therapeutics: Current host-modulation agents and future directions.

With the recognition in the 1960s and 1970s of the periodontopathic importance of the microbial biofilm and its specific anaerobic microorganisms, periodontitis was treated as an infectious disease (more recently, as a dysbiosis). Subsequently, in the 1980s, host-response mechanisms were identified as the mediators of the destruction of the collagen-rich periodontal tissues (gingiva, periodontal ligament, alveolar bone), and the periodontopathogens were now regarded as the "trigger" of the inflammatory/collagenolytic response that characterizes actively destructive periodontitis. Also at this time a new pharmacologic strategy emerged, entitled "host-modulation therapy", based on 2 major findings: (1) that the ability of tetracycline antibiotics to inhibit periodontal breakdown was due (in large part) to their previously unrecognized ability to inhibit the host-derived matrix metalloproteinases (notably, the collagenases, gelatinases, macrophage metalloelastase), and by mechanisms unrelated to the antimicrobial properties of these medications; and (2) that nonsteroidal anti-inflammatory drugs, such as flurbiprofen, again by nonantimicrobial mechanisms, could reduce the severity of periodontitis (however, the adverse effects of long-term therapy precluded their development as safe and effective host-modulatory agents). Additional mechanistic studies resulted in the development of novel nonantimicrobial formulations (Periostat® [now generic] and Oracea®) and compositions of tetracyclines (notably chemically modified tetracycline-3) as host-modulator drugs for periodontitis, arthritis, cardiovascular and pulmonary diseases, cancer, and, more recently, for local and systemic bone loss in postmenopausal women. Identification of the cation-binding active site in the tetraphenolic chemically modified tetracycline molecules drove the development of a new category of matrix metalloproteinase-inhibitor compounds, with a similar active site, the biphenolic chemically modified curcumins. A lead compound, chemically modified curcumin 2.24, has demonstrated safety and efficacy in vitro, in cell culture, and in vivo in mouse, rat, rabbit, and dog models of disease. In conclusion, novel host-modulation compounds have shown significant promise as adjuncts to traditional local therapy in the clinical management of periodontal disease; appear to reduce systemic complications of this all-too-common "inflammatory/collagenolytic" disease; and Oracea® is now commonly prescribed for inflammatory dermatologic diseases.

Osteoimmunology of Oral and Maxillofacial Diseases: Translational Applications Based on Biological Mechanisms.

The maxillofacial skeleton is highly dynamic and requires a constant equilibrium between the bone resorption and bone formation. The field of osteoimmunology explores the interactions between bone metabolism and the immune response, providing a context to study the complex cellular and molecular networks involved in oro-maxillofacial osteolytic diseases. In this review, we present a framework for understanding the potential mechanisms underlying the immuno-pathobiology in etiologically-diverse diseases that affect the oral and maxillofacial region and share bone destruction as their common clinical outcome. These otherwise different pathologies share similar inflammatory pathways mediated by central cellular players, such as macrophages, T and B cells, that promote the differentiation and activation of osteoclasts, ineffective or insufficient bone apposition by osteoblasts, and the continuous production of osteoclastogenic signals by immune and local stromal cells. We also present the potential translational applications of this knowledge based on the biological mechanisms involved in the inflammation-induced bone destruction. Such applications can be the development of immune-based therapies that promote bone healing/regeneration, the identification of host-derived inflammatory/collagenolytic biomarkers as diagnostics tools, the assessment of links between oral and systemic diseases; and the characterization of genetic polymorphisms in immune or bone-related genes that will help diagnosis of susceptible individuals.

Loss of alveolar bone density in postmenopausal, osteopenic women is associated with circulating levels of gelatinases.

OBJECTIVE: To determine whether circulating levels of two matrix metalloproteinases, MMP-2 and MMP-9, are associated with loss of alveolar bone density (ABD) or height (ABH), or with progression of periodontitis (relative clinical attachment level [RCAL]), among postmenopausal women with local and systemic bone loss. BACKGROUND: This study was planned as part of a 2-year randomized, double-blind, placebo-controlled, clinical trial examining efficacy/safety of subantimicrobial dose doxycycline (20 mg bid) in postmenopausal osteopenic women. This study examines whether serum levels of gelatinases are associated with local changes in the periodontium. METHODS: A sample of 113 women received periodontal maintenance for moderate to advanced chronic periodontitis and consented to analysis of stored serum biomarkers. Posterior vertical bitewings were taken, and serum collected, at baseline, one, and 2 years. ABD was determined by computer-assisted densitometric image analysis (CADIA), ABH by the Hausmann et al (1992, J Periodontol 63, 657) method, and RCAL by Florida Probe (every 6 months). MMPs were measured densitometrically on gelatin zymograms using denatured type I collagen as substrate and purified MMP-2 (72 kDa) and MMP-9 (92 kDa) as standards. Evidence of worsening in the periodontium at a tooth site was defined as a change from baseline of, for ABD, at least 14 densitometric units (for subcrestal locations) or 17 units (for crestal locations); of at least 0.4 mm for ABH; and of at least 1.5 mm for RCAL. Logistic regression models, while accounting for clustering, compared the odds of worsening in ABD, ABH, or RCAL, after 2 years of observation, between groups defined by baseline and concurrent levels of serum gelatinases. RESULTS: Changes in ABH and RCAL were not associated with circulating levels of MMP-2 or MMP-9. However, elevated odds of ABD loss over 24 months were associated, among smokers, with both baseline and concurrent levels of MMP-9 in the middle and highest tertile, and with concurrent levels of MMP-2 in the middle (but not the highest) tertile. Elevated odds of ABD loss were also associated, among women within 5 years of menopause, with baseline levels of MMP-2 in the highest tertile. CONCLUSION: Among postmenopausal osteopenic women, loss of ABD was associated, in smokers, with elevated circulating levels of MMP-9 and MMP-2. In those within 5 years of menopause, ABD loss was associated with elevated circulating levels of MMP-2.

Enhanced efficacy of chemically modified curcumin in experimental periodontitis: systemic implications.

INTRODUCTION: Dental microbial biofilm initiates gingival inflammation, and its suppression is the current dominant strategy for treating periodontitis. However, the host response to the biofilm is largely responsible for the connective tissue breakdown including alveolar bone loss, which is mediated by proinflammatory cytokines and matrix metalloproteinases (MMPs). METHODS: The current study compared the efficacy of a novel host-modulation compound, a chemically modified curcumin (CMC 2.24), to that of its parent compound (natural curcumin), in both lipopolysaccharide (LPS) (a bacterial endotoxin)-induced cell culture and in vivo models of periodontitis. RESULTS: In cell culture, both CMC 2.24 and curcumin appeared similarly effective in suppressing LPS-induced cytokine (IL-1ß and TNF-α) secretion by mononuclear inflammatory cells; however, CMC 2.24 significantly reduced MMP-9 secretion by 78% (P<0.05) whereas curcumin was ineffective. In vivo, CMC 2.24 administration was more effective than curcumin in suppressing (a) IL-1ß in gingival tissue and (b) MMP-9 in both gingiva and plasma, the latter indicating a reduced severity of systemic inflammation. The difference in primary clinical outcome between the two treatments was that CMC 2.24 reduced the pathologically excessive alveolar bone loss, assessed morphometrically at multiple sites, by 80%-90% (P<0.01), whereas curcumin, surprisingly, either increased (P<0.05) or had no effect on alveolar bone loss at these sites. CONCLUSION: These data, plus that from previous studies, support the therapeutic potential of CMC 2.24 in the management of inflammatory periodontal disease and its ability to reduce the risk of associated systemic diseases. The current study also indicates that the MMP-9 inhibitor efficacy is associated with the ability of CMC 2.24 (but not curcumin) to inhibit alveolar bone loss in this rat model of periodontitis.

Dose-response assessment of chemically modified curcumin in experimental periodontitis.

BACKGROUND: CMC2.24, a novel tri-ketonic chemically modified compound based on natural di-ketonic curcumin, has been shown to reduce bone loss and inflammatory mediators in experimental periodontitis, however, a potential dose-response relationship was not determined. The purpose of this study was to assess the effects of different doses of CMC2.24 on inflammation and bone resorption in vivo and also to describe on the effects of CMC2.24 on macrophage response. METHODS: CMC2.24 was administered daily to animals for 28 days by oral gavage, at the following doses: 0 (control), 1, 3, 10, and 30 mg/kg of body weight. Experimental periodontitis was induced by injections of lipopolysaccharide (LPS) into the gingival tissues. Outcomes assessed were bone resorption, detection of tartrate-resistant acid phosphatase, and determination of gene expression. In vitro, macrophages (RAW264.7) were treated with different concentrations of CMC2.24: 1, 3, 10, and 30 µM and then subjected to different activation stimuli. Gene expression, phagocytic activity, production of reactive oxygen species (ROS) and cytokine production were evaluated. RESULTS: CMC2.24 inhibited bone resorption, osteoclastogenesis, and tumor necrosis factor (TNF)-α expression in vivo. These beneficial responses reached maximum levels at a dose of 1 mg/kg, i.e. no dose-dependent effect. In vitro, CMC2.24 reduced the production of TNF-α and interleukin-10, inhibited phagocytic activity and stimulated production of ROS. A dose-dependent effect was observed only for ROS production. CONCLUSION: Low doses of CMC2.24 (1 mg/kg/day) administered orally were sufficient to significantly inhibit alveolar bone resorption associated with the experimental periodontal disease; whereas in vitro macrophage inflammatory gene expression and phagocytosis were reduced, whereas production of ROS was stimulated.

Differential effects of natural Curcumin and chemically modified curcumin on inflammation and bone resorption in model of experimental periodontitis.

OBJECTIVE: The purpose of this study was to compare the effects of the oral administration of natural curcumin and a chemically modified curcumin (CMC2.24) on osteoclast-mediated bone resorption, apoptosis, and inflammation in a murine model of experimental periodontal disease. DESIGN: Fifty male rats were distributed among the following treatment groups: (i) 2% carboxymethylcellulose, (ii) CMC2.24 30 mg/kg body weight, (iii) Curcumin 100 mg/kg body weight and (iv) no treatment. Compounds were administered daily by oral intubation over a 15-day period of time. Periodontal disease was induced by injections of LPS (lipopolysaccharide) into the gingival tissues three times per week. Contralateral sides were injected with the same volume of PBS (phosphate buffered saline) vehicle. After 15 days, hemimaxillae and gingival tissues were harvested. Bone resorption was assessed by µCT (microcomputer tomography). Formalin-fixed, paraffin embedded histological sections were stained with haematoxylin/eosin (H/E) for the assessment of cellular infiltrate or subjected to immunohistochemistry for detecting TRAP (tartrate-resistant acid phosphatase)-positive cells and caspase-3. Apoptosis was assessed in the gingival tissues by DNA fragmentation. RESULTS: CMC2.24 and curcumin caused a significant reduction of the inflammatory cell infiltrate, however µCT analysis showed that only CMC2.24 reduced bone resorption and the number of TRAP-positive multinucleated cells (osteoclasts). Curcumin, but not CMC2.24, significantly reduced the number of apoptotic cells in the gingival tissues and of osteocytes in the alveolar bone crest. CONCLUSIONS: The results suggest that CMC2.24 and curcumin inhibit inflammation by different mechanisms, but only CMC2.24 was capable of reducing alveolar bone resorption in the LPS-induced model of periodontitis.

A novel tricarbonylmethane agent (CMC2.24) reduces human pancreatic tumor growth in mice by targeting Ras.

Pancreatic Cancer (PC) is a deadly disease in need of new therapeutic options. We recently developed a novel tricarbonylmethane agent (CMC2.24) as a therapeutic agent for PC, and evaluated its efficacy in preclinical models of PC. CMC2.24 inhibited the growth of various human PC cell lines in a concentration and time-dependent manner. Normal human pancreatic epithelial cells were resistant to CMC2.24, indicating selectivity. CMC2.24 reduced the growth of subcutaneous and orthotopic PC xenografts in mice by up to 65% (P < 0.02), and the growth of a human patient-derived tumor xenograft by 47.5% (P < 0.03 vs vehicle control). Mechanistically, CMC2.24 inhibited the Ras-RAF-MEK-ERK pathway. Based on Ras Pull-Down Assays, CMC2.24 inhibited Ras-GTP, the active form of Ras, in MIA PaCa-2 cells and in pancreatic acinar explants isolated from Kras mutant mice, by 90.3% and 89.1%, respectively (P < 0.01, for both). The inhibition of active Ras led to an inhibition of c-RAF, MEK, and ERK phosphorylation by 93%, 91%, and 87%, respectively (P < 0.02, for all) in PC xenografts. Furthermore, c-RAF overexpression partially rescued MIA PaCa-2 cells from the cell growth inhibition by CMC2.24. In addition, downstream of ERK, CMC2.24 inhibited STAT3 phosphorylation levels at the serine 727 residue, enhanced the levels of superoxide anion in mitochondria, and induced intrinsic apoptosis as shown by the release of cytochrome c from the mitochondria to the cytosol and the further cleavage of caspase 9 in PC cells. In conclusion, CMC2.24, a potential Ras inhibitor, is an efficacious agent for PC treatment in preclinical models, deserving further evaluation.

A Chemically Modified Curcumin (CMC 2.24) Inhibits Nuclear Factor κB Activation and Inflammatory Bone Loss in Murine Models of LPS-Induced Experimental Periodontitis and Diabetes-Associated Natural Periodontitis.

The purpose of this study was to assess the effect of a novel chemically modified curcumin (CMC 2.24) on NF-κB and MAPK signaling and inflammatory cytokine production in two experimental models of periodontal disease in rats. Experimental model I: Periodontitis was induced by repeated injections of LPS into the gingiva (3×/week, 3 weeks); control rats received vehicle injections. CMC 2.24, or the vehicle, was administered by daily oral gavage for 4 weeks. Experimental model II: Diabetes was induced in adult male rats by streptozotocin injection; periodontal breakdown then results as a complication of uncontrolled hyperglycemia. Non-diabetic rats served as controls. CMC 2.24, or the vehicle, was administered by oral gavage daily for 3 weeks to the diabetics. Hemimaxillae and gingival tissues were harvested, and bone loss was assessed radiographically. Gingival tissues were pooled according to the experimental conditions and processed for the analysis of matrix metalloproteinases (MMPs) and bone-resorptive cytokines. Activation of p38 MAPK and NF-κB signaling pathways was assessed by western blot. Both LPS and diabetes induced an inflammatory process in the gingival tissues associated with excessive alveolar bone resorption and increased activation of p65 (NF-κB) and p38 MAPK. In both models, the administration of CMC 2.24 produced a marked reduction of inflammatory cytokines and MMPs in the gingival tissues, decreased bone loss, and decreased activation of p65 (NF-κB) and p38 MAPK. Inhibition of these cell signaling pathways by this novel tri-ketonic curcuminoid (natural curcumin is di-ketonic) may play a role in its therapeutic efficacy in locally and systemically associated periodontitis.

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; 30 mg/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.

Non-antibacterial tetracycline formulations: host-modulators in the treatment of periodontitis and relevant systemic diseases.

Traditionally, the dental profession has primarily treated periodontitis using a mechanical/surgical, rather than a pharmaceutical, approach. However, based on experiments several decades ago which demonstrated that tetracyclines, unexpectedly, inhibit collagen- and bone-destructive mammalian-derived enzymes (e.g. the collagenases), and through non-antibiotic mechanisms, the concept of host-modulation therapy (HMT) was developed. Accordingly, two drug-development strategies evolved: (i) the development of non-antimicrobial formulations of doxycycline; and (ii) the chemical modification of tetracyclines to eliminate their antibiotic activity but retain (or even enhance) their anti-collagenase properties. Regarding the latter, these chemically modified tetracyclines (CMTs) showed efficacy in vitro, in animal models of periodontal (and relevant systemic) disease, and in preliminary clinical trials on patients with Kaposi's sarcoma (however, at the high doses used, photosensitivity was a significant side-effect). In the first strategy, subantimicrobial-dose doxycycline (SDD) demonstrated safety and efficacy in human clinical trials and was approved by the U S Food and Drug Administration (U S FDA) and in other countries for the treatment of periodontitis (20 mg, twice daily, i.e. once every 12 hours) adjunctive to scaling and root planing, and for chronic inflammatory skin diseases (40-mg sustained-release 'beads'). SDD also showed efficacy in patients with systemic diseases relevant to periodontitis, including diabetes mellitus and arthritis, and in postmenopausal women with local and systemic bone loss. Importantly, long-term administration of SDD, of up to 2 years, in clinical trials did not produce antibiotic side-effects. SDD (and in the future, new HMTs, such as low-dose CMT-3, resolvins and chemically modified curcumins) may shift the paradigm of periodontal therapy from a predominantly surgical approach to the greater use of medicinal/pharmacologic strategies, ultimately to benefit larger numbers of patients.

DIFFERENTIAL SUSCEPTIBILITY OF HUMAN SP-B GENETIC VARIANTS ON LUNG INJURY CAUSED BY BACTERIAL PNEUMONIA AND THE EFFECT OF A CHEMICALLY MODIFIED CURCUMIN.

Staphylococcus aureus is a common cause of nosocomial pneumonia frequently resulting in acute respiratory distress syndrome (ARDS). Surfactant protein B (SP-B) gene expresses two proteins involved in lowering surface tension and host defense. Genotyping studies demonstrate a significant association between human SP-B genetic variants and ARDS. Curcumins have been shown to attenuate host inflammation in many sepsis models. Our hypothesis is that functional differences of SP-B variants and treatment with curcumin (CMC2.24) modulate lung injury in bacterial pneumonia. Humanized transgenic mice, expressing either SP-B T or C allele without mouse SP-B gene, were used. Bioluminescent labeled S. aureus Xen 36 (50 µL) was injected intratracheally to cause pneumonia. Infected mice received daily CMC2.24 (40 mg/kg) or vehicle alone by oral gavage. Dynamic changes of bacteria were monitored using in vivo imaging system. Histological, cellular, and molecular indices of lung injury were studied in infected mice 48 h after infection. In vivo imaging analysis revealed total flux (bacterial number) was higher in the lung of infected SP-B-C mice compared with infected SP-B-T mice (P < 0.05). Infected SP-B-C mice demonstrated increased mortality, lung injury, apoptosis, and NF-κB expression compared with infected SP-B-T mice. Compared with controls, CMC2.24 treatment significantly reduced the following: mortality, total bacterial flux and lung tissue apoptosis, inflammatory cells, NF-κB expression (P < 0.05), and MMPs-2, -9, -12 activities (P < 0.05). We conclude that mice with SP-B-C allele are more susceptible to S. aureus pneumonia than mice with SP-B-T allele, and that CMC2.24 attenuates lung injury thus reducing mortality.