LGM2605 for the mitigation of pathogen-associated gingival inflammation and periodontitis

Abstract

The present disclosure provides methods of using secoisolariciresinol diglucoside (SDG) (including stereoisomers thereof), for example, a synthetic SDG such as LGM2605, to treat periodontitis, for example, molar-incisor pattern periodontitis (MIPP), and / or pathogen-associated gingival inflammation in a subject.

Description

LGM2605 FOR THE MITIGATION OF PATHOGEN-ASSOCIATED GINGIVAL INFLAMMATION AND PERIODONTITISSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT.

[0001] This invention was made with government support under DE023071 awarded by National Institutes of Health. The government has certain rights in the invention.FIELD OF THE INVENTION

[0002] The present disclosure relates in general to the field of treatment for periodontitis and / or pathogen-associated gingival inflammation. In one embodiment, the present disclosure provides methods of using secoisolariciresinol diglucoside (SDG), for example, a synthetic secoisolariciresinol diglucoside (LGM2605), to treat periodontitis and / or pathogen-associated gingival inflammation.BACKGROUND OF THE INVENTION

[0003] Periodontal diseases encompass a group of multifactorial inflammatory conditions that affect the supporting structures of the teeth, including the gingiva, periodontal ligament, and alveolar bone. Among these, molar-incisor pattern periodontitis (MIPP), formerly known as localized aggressive periodontitis (LAP), stands out as a distinct and severe form of periodontal disease characterized by rapid tissue destruction without apparent poor oral hygiene and visible clinical inflammation. The etiology of MIPP has long intrigued researchers, as it is localized, and its aggressive nature suggests the involvement of microbial dysbiosis and uncontrolled host response.

[0004] One microbial factor that has garnered significant attention in the context of MIPP is Aggregatibacter actinomycetemcomitans (Ad). Among toxins that are produced by Aa, cytolethal distending toxin (Cdt) plays a significant role in distorting the host response. It acts as a phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase and causes PI-3K blockade, whichleads to a decrease in phagocytic response, exacerbates inflammation, and neutralizes the immune response. Also, there is a higher occurrence of Cdt expressing Aa in MIPP patient. This suggests a potential role of Cdt not only in the pathogenesis of MIPP but also in its clinical manifestation. However, the precise mechanisms through which Cdt contributes to MIPP remain incompletely understood and its role in alveolar bone loss underexplored.

[0005] Osteoclasts (OC), specialized bone-resorbing cells, play a pivotal role in maintaining the balance between bone formation and resorption within periodontal tissues. Dysregulation of osteoclast differentiation and activity can lead to pathological bone loss, a hallmark of periodontitis. Although conventional nuclear factor K(3 (RANKL) and Macrophage Colony-Stimulating Factor (M-CSF) regulate osteoclast differentiation, various inflammatory cytokines can also promote osteoclast differentiation. For instance, the pro-inflammatory cytokine, IL-ip, is a powerful stimulator of osteoclast differentiation and bone resorption by inducing RANKL expression. Similarly, IL-ip indirectly promotes TNF-a induced osteoclastogenesis by enhancing RANKL expression in stromal cells, and directly stimulating OC precursor differentiation under the control of p38 MAPK in the presence of sufficient RANKL. Sufficient RANKL levels are also needed for IL- la to activate the expression of OC markers such as TRAP, cathepsin K, matrix metallopeptidase 9 (MMP9), and nuclear factor of activated T-cells, cytoplasmic 1 (NFATcl). Additionally, IL- la can directly induce OC differentiation independently of RANKL by inducing microphthalmia transcription factor (MITF) in bone marrow macrophages (BMMs).

[0006] Furthermore, IL-6 positively modulates osteoclastogenesis through the induction of RANKL expression in osteoblasts (OBs) and stromal cells. In the IL-6 / IL-6R signaling pathway, the signal transducer and activator of transcription 3 (STAT3) is activated by JAKs, leading to OC marker expression. TNF-a, on the other hand, is a potent inducer of bone resorption and plays an important role in bone metabolism and inflammatory bone diseases. It directly induces the formation of TRAP+ multinucleated OCs from OC precursors in the presence of M-CSF and in the absence of RANKL by activating NF-KB signaling. TNF-a can also induce RANK expression in OC precursors and may accelerate RANKL-induced osteoclastogenesis through the activation of TRAF2 / 5 and MAPKs in TNFR1 -mediated signaling, leading to NF-KB and AP-1 activation. Additionally, TNF-a may indirectly affect osteoclastogenesis by inducing M-CSF and RANKL expression in stromal cells, OBs, and activated T cells.

[0007] The inflammatory markers previously mentioned, capable of impacting osteoclast formation, experience increased levels not just through Cdt intoxication but also due to Aa infection, potentially contributing to the generation of osteoclasts and subsequent bone resorption. Animal studies have confirmed that Aa triggers osteoclast formation, leading to subsequent bone loss. In humans, individuals testing positive for Aa exhibit notably greater periodontal bone loss compared to ria-negative individuals, highlighting Aa's significant influence on alveolar bone loss linked to periodontal disease. Consequently, Cdt and / or Aa may have a more intricate role in establishing an environment conducive to bone loss that contributes to MIPP pathogenesis.

[0008] Therapeutic strategies for addressing the hyper-responsive host response observed in MIPP patients remain limited. Sub-antimicrobial dose doxycycline, extensively studied for chronic periodontitis, acts as both an antibiotic and a host modulation agent. Steroidal and non-steroidal anti-inflammatory drugs, commonly used as host modulatory pharmacological agents, have limitations due to their side effects, restricting their prolonged usage. Consequently, there is a need to develop alternative agents to prevent and treat molar- incisor pattern periodontitis (MIPP).SUMMARY OF THE INVENTION

[0009] In one aspect, the present disclosure provides methods for treating periodontitis and / or pathogen-associated gingival inflammation in a subject in need thereof, comprising administering to said subject a composition comprising a therapeutically effective amount of secoisolariciresinol diglucoside (SDG), including stereoisomers thereof. In one embodiment, the periodontitis is molar-incisor pattern periodontitis (MIPP).

[0010] In another aspect, the present disclosure provides methods for reducing inflammation and / or pro-inflammatory cytokine secretion in periodontal tissue induced by Aggregatibacter actinomycetemcomitans or cytolethal distending toxin in a subject in need thereof, comprising administering to said subject a composition comprising a therapeutically effective amount of secoisolariciresinol diglucoside (SDG), including stereoisomers thereof.

[0011] In some embodiments for the methods disclosed herein, the composition comprising SDG is administered orally. In some embodiments for the methods disclosed herein, the composition comprising SDG is administered topically. In another embodiment, the composition comprisingSDG is formulated as a gel or an oral rinse. In another embodiment, the methods disclosed herein arc performed as an adjunctive treatment in conjunction with scaling and root planing in the subject.

[0012] In another aspect, the present disclosure provides pharmaceutical compositions for treating periodontitis and / or pathogen-associated gingival inflammation in a subject, the pharmaceutical composition comprising a therapeutically effective amount of secoisolariciresinol diglucoside (SDG), including stereoisomers thereof. In one embodiment, the pharmaceutical composition comprising SDG is formulated as a gel. In another embodiment, the pharmaceutical composition comprising SDG is formulated as an oral rinse.

[0013] In some embodiments for the methods and pharmaceutical compositions disclosed herein, the SDG is (S, S)-SDG. In another embodiment, the SDG is (?,7?)-SDG. In another embodiment, the SDG is a synthetic SDG. For example, the synthetic SDG is LGM2605. In another embodiment, the SDG is synthetic (S, S)-SDG. For example, the synthetic (S, S)-SDG is LGM1506. In another embodiment, the SDG is a racemic mixture of (?, / ?)-SDG and (?, / ?)-SDG.

[0014] These and other aspects of the invention will be appreciated from the ensuing descriptions of the figures and detailed description of the invention.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0016] Figure 1 shows LGM2605 decreases Cdt-mediated upregulation of cytokine gene expression. Differentiated THP-1 macrophages were pre-treated with increasing doses of LGM2605 for 30 minutes and 100 ng / mL of Cdt were added for 2 hours to stimulate inflammation. RNA was extracted and quantitative real-time PCR was done for pro-IL-ip, IL-6, and TNF-oc. Data represent three independent experiments with technical triplicate. Statistical significance was determined using Student’s t-test. *, p-value <0.05, ***, p-value<0.005 Cdt only (0 uMLGM2605) vs different doses of LGM2605.

[0017] Figure 2 shows LGM2605 decreases Cdt induced pro-inflammatory cytokines. Differentiated THP-1 macrophages were pre-treated with increasing doses of LGM2605 for 30 minutes and different concentrations of Cdt were added for 5 hours to stimulate inflammation. Supernatant were collected and analyzed by ELISA specific for IL-10, IL-6, and TNF-a. Data represent 3 independent experiments with technical triplicate for ELISA, for n=9. Statistical significance was determined using Student’s t-test. *, p-value <0.05 OuM LGM2605 vs different dose of LGM2605 within same concentration Cdt treated group.

[0018] Figure 3 shows LGM2605 decreases Aa induced inflammation. Differentiated THP-1 macrophages were pre-treated with LGM2605 in increased doses (25, 50, lOOpM) for 30 minutes and inoculated with mid-log phase Aa (MOI of 1:10 and 1:100) for 4 hours to stimulate inflammation. Supernatant were collected and analyzed by ELISA specific for IL- 10, IL-6, and TNF-a. Data represent 3 independent experiments with technical triplicate for ELISA. Statistical significance was determined using Student’s t-test. *, p-value <0.05 OuM LGM2605 vs different dose of LGM2605 within same MOI group.

[0019] Figure 4 shows LGM2605 decreases Cdt induced pro-inflammatory cytokines in THP-lCasp4anc[ expression level in THP-lCaspl‘ and THP-1Casp4-. Differentiated THP-1 macrophages were pre-treated with 50pM and lOOpM LGM2605 for 30 minutes and 100 ng / mL of Cdt were added for 2 hours (for RNA expression, left panel - THP-lCaspland middle panel - THP-lCasp4) and for 5 hours (for IL- 10 secretion, right panel) to stimulate inflammation. RNA was extracted and quantitative real-time PCR was done, and supernatant was collected for pro-IL-10 expression and for IL- 10 secretion. Data represent three independent experiments with technical triplicate. Statistical significance was determined using Student’s t-test. ***, p-value <0.005 Cdt only (OuM LGM2605) vs 50pM and 100μM of LGM2605.

[0020] Figure 5 shows Cdt increases osteoclast differentiation. THP-1 monocytes were treated with high PMA concentration (100 ng / mL) for 2 days and osteoclast differentiation induced with RANKL and M-CSF (50 ng / mL each) for 6 days with replenish of differentiation media with or without Cdt every 2 days. TRAP staining was done at 8thday and TRAP+ cells were counted. Data represent 3 independent experiments. Statistical significance was determined using Student’s t-test. *, p-value <0.05 **, p-value <0.01 control (RANKL+M-CSF only) vs different concentrationCdt treated groups.

[0021] Figure 6 shows phosphatase-dependent increase in osteoclast differentiation induced by Cdt. THP- 1 monocytes underwent differentiation into macrophages using a high concentration of PMA (100 ng / mL). Osteoclast differentiation was initiated by exposing cells to RANKL and M-CSF (50 ng / mL each) for six days, with media replenishment every 48 hours. Cdt treatments (50 ng / mL (left panel) or 500 ng / mL (right panel)) for both CdtR117Aand CdtWTwere introduced concurrently with RANKL and M-CSF. At the endpoint, TRAP staining was conducted, and TRAP+ cells were quantified. The presented data are representative of three independent experiments. Statistical significance was determined using Student’s t-test. *, p-value <0.05 for the control (RANKL+M-CSF only) compared to Cdt (CdtR117Aand CdtWT) treated groups.

[0022] Figure 7 shows LGM2605 reduces Cdt-mediated increases in osteoclast differentiation. First, THP-1 monocytes were treated with high PMA concentration (100 ng / mL). Before RANKL and M-CSF (50 ng / mL each) induced osteoclast differentiation with or without Cdt treatment (50 ng / mL) for 6 days, THP-1 cells were pretreated with different concentrations of LGM2605 (25, 50, and lOOpM) for 30 minutes in media. Media with RANKL and M-CSF replenishment was done with and without Cdt and LGM2605 every 48 hours. TRAP staining was done at 6th day of differentiation and TRAP+ cells were counted for 3 independent experiments. Data represent 3 independent experiments with technical triplicate. Statistical significance was determined using Student’s t-test. *, p-value <0.05 **, p-value<0.01, Cdt treated vs Cdt+LGM2605 treated groups.

[0023] Figures 8A-8C show impact of Cdt and LGM2605 on osteoclast maturation. THP-1 cells underwent treatment with a high concentration of PMA (100 ng / mL) for two days before osteoclast differentiation using RANKL and M-CSF (50 ng / mL each) for six days. PMA-treated cells were pretreated with lOOpM LGM2605 for 30 minutes before differentiation and Cdt (50 ng / mL) was added with differentiation media. Media replenishment with RANKL and M-CSF, with or without Cdt and LGM2605, occurred every 48 hours. Immuno staining for TRAP, Cathepsin K (CTSK), and nucleus was performed, followed by multi-fluor confocal imaging as detailed in the Methods section. Figure 8A: Maximum intensity projection images depict control (only RANKL and M-CSF-induced differentiation), Cdt treatment (50 ng / mL), and Cdt (50 ng / mL) + LGM2605 (lOOpM) treatment for TRAP (left) and CTSK (right) in green, with nuclei in blue. Figure 8B:Quantification of TRAP (left) and CTSK (right) fluorescence intensity presented as a percentagerelative to the control. Results are expressed as mean + / - STDEV (five fields per condition) and compared using Student’s t-test. Statistical significance indicated by *, p-value<0.05 vs. other conditions. Figure 8C: Percent of 2 nuclei per cell (left) and 3 or more nuclei per cell (right) in each condition. The number of nuclei per cell was counted from confocal images and calculated as a percentage of whole population per condition. Graphs displaying cells with 2 nuclei per cell and 3 or more nuclei per cell were generated to represent maturation. Mean + / - STDEV (five fields per condition) and statistical comparisons using Student’s t-test are indicated. *p-value vs. other conditions.

[0024] Figures 9A-9C show LGM2605 mitigates Cdt-induced osteoclast differentiation markers. THP-1 monocytes were initially treated with a high concentration of PMA (100 ng / mL) for two days. Subsequently, osteoclast differentiation was induced by RANKL and M-CSF (50 ng / mL each) for six days, with or without concurrent treatment of Cdt (50 ng / mL) after pretreating PMA-treated THP-1 cells with lOOpM LGM2605 for 30 minutes. Media containing RANKL and M-CSF, with or without Cdt and LGM2605, were replenished every 48 hours. Lysates were collected and subjected to western blot analysis as described in the Methods section. Figures 9A-9B:Quantitative data depict the mean + / - STDEV of TRAP and CTSK levels (n-3), presented as a percentage relative to the untreated control and compared using Student’s t-test. *, p-value <0.05 compared to other conditions. Figure 9C: Representative immunoblot images demonstrating TRAP and CTSK staining, with GAPDH used as an internal normalization protein.

[0025] Figure 10 shows LGM2605 does not affect the cell viability. Differentiated THP-1 macrophages were treated with increasing dose of LGM2605 (25-400pM) for 24 hours and cell were detached, treated, and cell viability was recorded as described in Methods. Data represent 3 independent experiments with technical triplicate. Student’s t-test was done for a comparison.DETAILED DESCRIPTION OF THE INVENTION

[0026] The present disclosure explores the interplay between Aggregatibacter actinomycetemcomitans (Aa) cytolethal distending toxin (Cdt) and the host's inflammatory response in molar / incisor pattern periodontitis (MIPP). Cdt disrupts phosphatidylinositol-3,4,5-triphosphate (PIP3) signaling, influencing cytokine expression through canonical and non-canonical inflammasome activation as well as NF-KB activation, leading to inflammation in MIPP.Macrophages exposed to Cdt exhibited upregulation of pro-inflammatory genes and subsequent cytokine release, aggravating MIPP's hyper-inflammatory state. The present study investigated the therapeutic potential of LGM2605, known for its anti-inflammatory properties, and analyzed its ability to reduce pro-inflammatory gene expression and cytokine release in Cdt-exposed and Aa-inoculated macrophages. LGM2605's mechanism involves inhibiting NF-KB while activating Nrf2 and antioxidants. This small molecule therapeutic shows promise in mitigating Cdt-induced inflammation. The present study also further defines Cdt's impact on osteoclast differentiation in MIPP. Cdt promotes increased TRAP+ cells, indicating heightened osteoclast differentiation, specific to Cdt’s phosphatase activity. Cathepsin K levels rise during this process, reflecting changes in TRAP distribution between control and Cdt-treated cells. Exploring LGM2605's effect on Cdt-induced osteoclast differentiation, it was found that while all concentrations increased TRAP+ cells significantly, 50pM and lOOpM notably reduced TRAP+ cells compared to Cdt alone. In the presence of LGM2605 or upon LGM2605 treatment, immunocytochemistry revealed decreased TRAP intensity and cells with 3+ nuclei, while immunoblotting displayed reduced TRAP and Cathepsin K levels, suggesting LGM2605's potential to curb osteoclast differentiation and maturation by modulating inflammatory cytokines, possibly involving Nrf2 activation. In summary, the present study reveals the intricate connections between Cdt, inflammation, and osteoclast differentiation in MIPP, offering novel therapeutic possibilities for managing these conditions.

[0027] As used herein, the terms “comprise”, “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.

[0028] The term “consisting of” means “including and limited to”. The term “consisting essentially of’ means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0029] As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.

[0030] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can beused in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. Each literature reference or other citation referred to herein is incorporated herein by reference in its entirety.

[0031] In the description presented herein, each of the steps of the invention and variations thereof are described. This description is not intended to be limiting and changes in the components, sequence of steps, and other variations would be understood to be within the scope of the present invention.

[0032] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0033] In one aspect, the present disclosure provides a method for treating periodontitis and / or pathogen-associated gingival inflammation in a subject in need thereof, comprising administering to said subject a composition comprising a therapeutically effective amount of secoisolariciresinol diglucoside (SDG), including stereoisomers thereof, thereby treating the periodontitis and / or pathogen-associated gingival inflammation in said subject. In one embodiment, the periodontitis is molar-incisor pattern periodontitis (MIPP).

[0034] In one aspect, the present disclosure provides a method for reducing inflammation or pro-inflammatory cytokine secretion in periodontal tissue induced by Aggregatibacter actinomycetemcomitans or cytolethal distending toxin in a subject in need thereof, comprising administering to said subject a composition comprising a therapeutically effective amount of secoisolariciresinol diglucoside (SDG), including stereoisomers thereof, thereby reducing inflammation and / or pro -inflammatory cytokine secretion, respectively, in periodontal tissue in said subject.

[0035] In another aspect, the present disclosure provides pharmaceutical compositions for treatingperiodontitis and / or pathogen-associated gingival inflammation in a subject, the pharmaceutical composition comprising a therapeutically effective amount of secoisolariciresinol diglucoside (SDG), including stereoisomers thereof. In one embodiment, the pharmaceutical composition comprising SDG is formulated as a gel. In another embodiment, the pharmaceutical composition comprising SDG is formulated as an oral rinse.

[0036] Secoisolariciresinol diglucoside (SDG) can be isolated from natural sources or chemically synthesized. Due to complex extraction, purification and enrichment methods to isolate SDG from natural resources, in a preferred embodiment, SDG is chemically synthesized.

[0037] Techniques for synthesizing SDG, its stereoisomers and analogs are described in Mishra et al., Bioorganic & Medicinal Chemistry Letters (2013) 23:5325-5328 and in International Patent Publication No. WO2014 / 200964, which are hereby incorporated by reference in their entireties. For example, using the natural compounds vanillin and glucose, two enantiomers (their structures are depicted below) of SDG: SDG (S,S) and SDG (R,R), were successfully synthesized.

[0038] In one embodiment, the SDG administered in the methods described herein is SDG (S, S). In another embodiment, the SDG administered in the methods described herein is SDG (R, R). In another embodiment, the SDG administered in the methods described herein is a racemic mixture of SDG (S, S) and SDG (R, R).

[0039] LGM2605 is the synthetic lignan secoisolariciresinol diglucoside (SDG) with free radical scavenging, antioxidant, and anti-inflammatory properties in diverse inflammatory cell and mouse models. Synthesis of secoisolariciresinol diglucoside (LGM2605) is described in Mishra et al. (2013).

[0040] In some aspects, the present disclosure relates to a pharmaceutical composition. “Pharmaceutical composition” refers to an effective amount of an active ingredient, e.g., SDG, (S,S)-SDG and / or (R,R)-SDG, together with a pharmaceutically acceptable carrier or diluent. In some embodiments, the pharmaceutical composition comprises (5,5)-secoisolariciresinol diglucoside. Preferably, these compositions are pharmaceutical compositions comprising (5,5)-secoisolariciresinol diglucoside and at least one pharmaceutically acceptable excipient. In some embodiments, the compositions and pharmaceutical compositions may be prepared with substantially pure (S,S)-secoisolariciresinol diglucoside. In some embodiments, the compositions and pharmaceutical compositions have a diasteriomeric excess (DE) of at least 90% DE, preferably at least 95% DE, more preferably at least 98% DE, and even more preferably at least 99% DE and most preferably about 100% DE. The compositions and pharmaceutical compositions may also be prepared as mixture of the diasteriomeric forms of the compounds (e.g., as a racemic mixture or as a mixture with a ratio of 60:40, 70:30, 80:20 or 90:10 of (S,S)-SDG relative to (R,R)-SDG.

[0041] In some embodiments, the pharmaceutical composition comprises (R,R)-secoisolariciresinol diglucoside. Preferably, these compositions are pharmaceutical compositions comprising (R,R)-secoisolariciresinol diglucoside and at least one pharmaceutically acceptable excipient. In some embodiments, the compositions and pharmaceutical compositions may be prepared with substantially pure (R,R)-secoisolariciresinol diglucoside. In some embodiments, the compositions and pharmaceutical compositions have a diasteriomeric excess (DE) of at least 90% DE, preferably at least 95% DE, more preferably at least 98% DE, and even more preferably at least 99% DE and most preferably about 100% DE. The compositions and pharmaceutical compositions may also be prepared as mixture of the diasteriomeric forms of the compounds (e.g., as a racemic mixture or as a mixture with a ratio of 60:40, 70:30, 80:20 or 90:10 of (R,R)-SDG relative to (S,S)-SDG.

[0042] The compositions described herein may include a “therapeutically effective amount.” A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time needed, to achieve the desired therapeutic result. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount is also one in which toxic or detrimental effects of the molecule are outweighed by the therapeutically beneficial effects.

[0043] As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, carriers, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0044] “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes an excipient that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable excipient” as used herein includes both one and more than one such excipient.

[0045] Pharmaceutical compositions can be administered to a subject by any suitable method known to a person skilled in the art, such as orally, topically, parenterally, transmucosally, transdermally, intramuscularly, intravenously, intra-dermally, subcutaneously, intra-peritoneally, intra- ventricularly, intra-cranially, intra-vaginally, intra-tumorally, or bucally. Controlled release may also be used by embedding the active ingredient in an appropriate polymer which may then be suitably administered. Coating a medical device or dental implant with the active ingredient is also covered.

[0046] In some embodiments, the pharmaceutical compositions are administered orally, and are thus formulated in a form suitable for oral administration, i.e., as a solid or a liquid preparation. Suitable solid oral formulations include tablets, capsules, pills, granules, pellets and the like. Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In some embodiments, the active ingredient is formulated in a capsule. In accordance with this embodiment, the compositions described herein comprise, in addition to the active compound and the inert carrier or diluent, drying agent, in addition to other excipients, as well as a gelatin capsule.

[0047] In some embodiments, suitable formulations include gel formulations. Suitable gel formulations are generally known in the art, for example, the gel formulation can be PERIOSEPT®. In other embodiments, the oral formulation is in the form of oral rinse.

[0048] In some embodiments, compositions for use in the methods provided herein are administered at a therapeutic dose once per day. In some embodiments, the compositions areadministered once every two days, twice a week, once a week, or once every two weeks.

[0049] In one embodiment, SDG (including (S,S)-SDG or (R,R)-SDG and mixtures thereof) may be administered at a dose of 0.1 ng / kg to 500 mg / kg. In another embodiment, SDG (including CS',5)-SDG or ( ’. / ?)-SDG and mixtures thereof) may be administered at a concentration of about 1 nanomolar (nM) to about 1 molar (M). In another embodiment, SDG (including (S,S)-SDG or (R,R)-SDG and mixtures thereof) may be administered at a concentration from about 25 μM to about 250 μM.

[0050] The treatment regimen with SDG (including (S,S)-SDG or (R,R)-SDG and mixtures thereof) may range from a single administration to several days, months, years, or indefinitely. In one embodiment, a treatment regimen with SDG (including (S,S)-SDG or (R,R)-SDG and mixtures thereof) comprises daily administration over one week. In another embodiment, a treatment regimen with SDG (including (S,S)-SDG or (R,R)-SDG and mixtures thereof) comprises daily administration over two weeks. In another embodiment, a treatment regimen with SDG (including (S,S)-SDG or (R,R)-SDG and mixtures thereof) comprises daily administration over three weeks. In another embodiment, a treatment regimen with SDG (including (S,S)-SDG or (R,R)-SDG and mixtures thereof) comprises daily administration over one month. In another embodiment, a treatment regimen with SDG (including (S,S)-SDG or (R,R)-SDG and mixtures thereof) comprises daily administration over two months. In another embodiment, a treatment regimen with SDG (including (S,S)-SDG or (R,R)-SDG and mixtures thereof) comprises daily administration over three months. In another embodiment, a treatment regimen with SDG (including (S,S)-SDG or (R,R)-SDG and mixtures thereof) comprises daily administration over six months.

[0051] As used herein, “treating” may refer to either therapeutic treatment or prophylactic or preventative measures, where the object is to prevent or lessen the targeted pathologic condition or disorder as described herein, or both. Therefore, compositions for use in the methods provided herein may be administered to a subject at risk of developing a pathologic condition or disorder and before said pathologic condition or disorder develops. In some cases, the compositions for use in the methods provided herein may be administered to a subject after a pathologic condition or disorder develops. Thus, treating a condition as described herein may refer to preventing, inhibiting, reversing, or suppressing the condition in a subject.

[0052] Furthermore, as used herein, the terms “treat” and “treatment” refer to therapeutictreatment, as well prophylactic or preventative measures, where the object is to prevent or slow down (lessen) an undesired physiological change associated with a disease or condition. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of the extent of a disease or condition, stabilization of a disease or condition (z.e., where the disease or condition does not worsen), delay or slowing of the progression of a disease or condition, amelioration or palliation of the disease or condition, and remission (whether partial or total) of the disease or condition, whether detectable or undetectable. Those in need of treatment include those already having a pathologic condition or disorder or those who are at risk of developing a pathologic condition or disorder.

[0053] As used herein, the term “maintaining” means to preserve or keep in a state or condition corresponding to absence of a disease or pathology and encompasses preventing a decline, lapse or cessation from that state or condition.

[0054] As used herein, the term “preventing” may refer to stopping, hindering, or suppressing a disease, disorder, or a symptom of a disease or disorder, through some action before the symptoms or consequences of the disease or disorder manifest themselves, or before a patient is exposed to conditions which may trigger the disease or disorder.

[0055] The term “subject” includes mammals, e.g., humans, companion animals (e.g., dogs, cats, birds, and the like), farm animals (e.g., cows, sheep, pigs, horses, fowl, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, birds, and the like). In addition to humans, the subject may include dogs, cats, pigs, cows, sheep, goats, horses, buffalo, ostriches, guinea pigs, rats, mice, birds (e.g., parakeets) and other wild, domesticated or commercially useful animals (e.g., chicken, geese, turkeys, fish). The term “subject” does not exclude an individual that is normal in all respects. The term “subject” includes, but is not limited to, a human in need of therapy for, or susceptible to, a condition or its sequelae.

[0056] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLE 1LGM2605; Alternative for Periodontitis Treatment via Host Response Modulation MATERIALS AND METHODS THP-1 Cell Derived Macrophages

[0057] The human acute monocytic leukemia cell line, THP-1, was obtained from ATCC (Manassas, VA); cells were maintained in RPMI1640-containing 10% FBS, 1 mM sodium pyruvate, 20μM 2-mercaptoethanol and 2% penicillin-streptomycin at 37°C with 5% CO2 in a humidified incubator. Macrophage differentiation was done on 35mm glass bottom dishes (MatTek; Ashland, MA), 12 wells cell culture plate (Thermo Scientific; Waltham MA), or 96 wells cell culture plate (Thermo Scientific; Waltham MA) by incubating cells in the presence of 50 ng / mL phorbol 12-myristate 13-acetate (PM A) for 48 hours at which time the cells were washed and incubated an additional 24 h in medium prior to use.Caspase-1 and -4 Deficient THP-1 cells

[0058] THP-1 cells with stable knockdown of caspase-1 (THP-lCaspl‘) were obtained from D. M. Ojcius and previously detailed (Shenker et al., Cell Microbiol. (2020) 22(7): p. el3194). Caspase-4 deficient cells (THP-lCasp4‘) were provided by P. R. Huang and generated similarly, employing lentiviral particles containing the sequence: 5”-GCAACGTATGGCAGGACAAAT-3” (SEQ ID: NO: 1). These modified cells were created via transduction of THP-1 cells with lentiviral particles, including nontarget controls, as outlined previously (Shenker et al.). Recent reports indicated a reduction of >95% in caspase-1 expression for THP-lCaspl" cells, while caspase-4 was undetectable via Western blot in THP- lCasp4‘ cells, showcasing no impact in the nontarget controls. Macrophage differentiation was achieved using a 12- well cell culture plate (Thermo Scientific; Waltham MA) by incubating cells in the presence of 50 ng / mL PMA for 48 hours, followed by a subsequent 24-hour incubation in medium before utilization.THP-1 Cell Derived Osteoclast

[0059] THP-1 cells were differentiated into macrophages on either 6, 12, or 24 well cell culture plates (Thermo Scientific; Waltham MA) by incubating cells in the presence of 100 ng / mL PMA for 2 days. Cells were then incubated with 50 ng / mL of RANKL and M-CSF for 6 days for osteoclast differentiation. Cells were replenished with fresh media + RANKL and M-CSF (PeproTech, Cranbury NJ) with and without other treatments (Cdt and LGM2605) every 48 hours.LGM2605

[0060] Synthetic SDG (referred to as LGM2605 in the literature) was generated as previously described (Dhingra et al., Int J Mol Sci. (2021) 22(11)). Briefly, secoisolariciresinol diglucosides (S, S)-SDG (the major isomer in whole grain flaxseed) and (R, R)-SDG (the minor isomer in whole grain flaxseed) were synthesized from vanillin via secoisolariciresinol and glucosyl donor (perbenzoyl-protected trichloacetimidate under the influence of TMSOTf) through a concise route involving chromatographic separation of diastereomeric diglucoside derivatives (Chemveda Life Sciences Inc., Hyderabad, India). Absorbance spectra of 36mM LGM2605 measured using a SpectraMax M5 fluorescent plate reader (Molecular Devices, San Jose, CA, USA).Expression and Purification of Cdt, CdtB Mutants And Cdt Holotoxin

[0061] Construction and expression of the plasmid containing the cdt genes for the holotoxin (pUCAacdtABChis) have previously been reported. The plasmid was constructed so that cdt genes were under control of the lac promoter and transformed into E. coli DH5a. Cultures of transformed E. coli were grown in 1L LB broth and induced with 0.1 mM of isopropyl β-D-1-thiogalactopyranoside for 2 h; bacterial cells were harvested, washed, and resuspended in 50 mM of Tris (pH 8.0). The cells were frozen overnight, thawed, and sonicated. The histidine-tagged peptide holotoxin was isolated by nickel affinity chromatography as previously described.Treatment with LGM2605

[0062] For Macrophage Studies: Differentiated THP-1 macrophages were pretreated with different concentrations of LGM2605 (50-200μM) in media for 30 min prior to Cdt (50-500 ng / mL) stress induction or Aa (MOI of 1:10 or 1:100).

[0063] For Osteoclast Studies: After 48 hours of PMA treatment (high concentrations: 100 ng / mL) to induce differentiation, THP-1 with were pretreated with different concentrations of LGM2605 (50-200μM) in media before RANKL and M-CSF (50 ng / mL each) induced osteoclast differentiation with or without Cdt treatment in different concentration. Media and LGM2605 replenishment were done with and without Cdt treatment every 48 hours.RNA Isolation And Gene Expression Analysis

[0064] Total RNA was isolated using a RNeasy Plus Mini Kit and quantitative polymerase chain reaction (qPCR) analysis was performed, as previously described (Dhingra et al). Total RNA was quantified using a NanoDrop 2000 apparatus (Thermo Fisher Scientific, Waltham, MA, USA).Reverse transcription of RNA to cDNA was then performed on a Veriti® Thermal Cycler using the High Capacity RNA-to-cDNA Kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). qPCR was performed using individual TaqMan® Probe-Based Gene Expression Assays (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). Individual TaqMan gene expression assays were selected (Table below). qPCR was performed using 50 ng of cDNA per reaction well on an Applied Biosystems QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). Gene expression data are presented normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). All data were calibrated to the control, untreated samples (THP-1 macrophage) according to the A ACT method as previously described (Dhingra et al).Bacteria And Growth Curve

[0065] A.actinomycetemcomitans strains, D7S-SA (wild type Arz) and D7S-SA CHE001 (Cdt deficient Aa mutant), were obtained as described previously (Kim et al., Front Cell Infect. Microbiol. (2023) 13: p. 1220089). Each strain was plated on AAGM agar which consisted of 20g of BBL trypticase soy agar (BD; Sparks, MD); 3g of yeast extract (ThermoFisher) supplemented with 0.4% sodium bicarbonate and 0.8% dextrose. After bacteria were grown on plates for 24 or 48 hours in the incubator with 10% CO2 at 37°C, they were inoculated in 10 mL of AAGM broth until OD600 close to 0.2. Bacteria were plated from different dilutions at various time points on AAGM agar plates and incubated for 24 hours in the incubator with 10% CO2 at 37 °C. OD600 was measured using DU 650 Spectrophotometer (Beckman Coulter, Indianapolis IN) for each time point.Tartrate-Resistant Acid Phosphatase (TRAP) Staining

[0066] On the 6thday of differentiation, the supernatants were removed and discarded. The cells were washed three times with PBS, and fixation solution (citrate buffer containing 60% acetone and 10% methanol) was added and left at room temperature for 5 min. After fixation, cells were stained using a TRACP & ALP Double-stain Kit (MK300; Takara Bio Inc., Kusatsu, Shiga, Japan)following the manufacturing protocol and TRAP positive cells were counted by using Nikon TMS-F inverted microscope.Western Blot Analysis

[0067] Cells were treated with Cdts and solubilized in 20mM Tris-HCl buffer (pH7.5) containing 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate, and protease inhibitor cocktail (ThermoFisher Scientific; Waltham, MA). Samples (15μg) were separated on 4-12% SDS-PAGE and then transferred to PVDF membranes. The membrane was blocked with BLOTTO and then incubated with primary antibodies overnight (18hrs) at 4°C. Membranes were washed and incubated with secondary antibodies conjugated to horseradish peroxidase. Western blots were developed using chemiluminescence and analyzed by digital densitometry (LiCor Biosciences; Lincoln, NE). Each protein was normalized to either actin or GAPDH.Cytokine Analysis

[0068] Cytokine production was measured in the culture supernatants from THP cells challenged as described above in the presence or absence of LGM2605 at the concentrations indicated in the Figure legends. Culture supernatants collected at the time points indicated in the figure legends were analyzed by ELISA for IL-1β, IL-6, and TNF-α (DuoSet Elisa Kit; R& D Systems) commercially available kits according to the manufacturer’s instructions. In each instance, the amount of cytokine present in the supernatant was determined using a standard curve.Confocal Microscopy

[0069] The cells were fixed in 4% PFA for 15mins, washed three times, and permeabilized / blocked in blocking solution containing 1% BSA, 1% normal donkey serum and 0.1% saponin in PBS at room temperature for 1 hour. Next, incubation with the primary antibody listed in the antibody table with concentration o / n at 4°C in antibody solution (PBS with 1% BSA), washed in PBS three times, and secondary antibody (1:1,000) with nuclear staining (Hoechst 33342, 1:10,000). Samples were imaged using a Nikon AIR laser scanning confocal microscope with a PLAN APO VC 60x water (NA 1.2) objective. Intensity of 488nm was measured and averaged with number of cells in field. Number of nuclei per cell was also counted to determined maturation of osteoclast.Cell Viability[00701 THP-1 cells were differentiated into macrophages by PMA (50 ng / mL) and treated for 24 hours with LGM2605 with increasing concentration (25-400pM). After the treatment, cells were lifted with 0.05% trypsin-EDTA and treated with Vi-CELL XR reagents (Beckman Coulter) according to the manufacture protocol. Cell viability is processed with Vi-CELL XR Cell Viability Analyzer (Beckman Coulter) in experimental triplicate with technical triplicate.RESULTS LGM2605 Treatment Down Regulates Expression of Inflammatory Cytokines

[0071] Macrophages exposure to Cdt results in decreased PIP3 and reduced phosphorylation of both GSK3P and Akt, leading to an increase in GSK3P kinase activity. GSK3P, in turn, influences the association of the p65 subunit with the common co-activator (CBP), thereby influencing the expression of pro-inflammatory cytokines by modifying NF-KB activation. To determine if Cdt treatment modulates cytokine expression, differentiated THP-1 macrophages were treated with Cdt (100 ng / mL) for 2 hours and the expression of pro-inflammatory genes (proIL-ip, IL-6, and TNF-a) was assessed using quantitative real-time PCR (Figure 1). There was a 57-fold increase in expression of pro- IL-ip, a 283-fold increase in IL-6, and a 39-fold increase in TNF-a mRNA expression when compared to untreated THP-1 macrophages. These findings align with previous studies examining Cdt-mediated inflammatory mechanism.

[0072] Whether LGM2605 could mitigate Cdt-dependent enhancement of cytokine gene expression was examined next. Initially, THP-1 macrophages were pre-treated with LGM2605 (50pM and lOOpM) for 30 minutes before exposure to Cdt (100 ng / mL for 2 hours). Subsequent analysis via quantitative real-time PCR (Figure 1) evaluated the expression of pro-IL-ip, IL-6, and TNF-a. interestingly, IL-ip displayed a significant reduction to 52-fold (at 50pM LGM2605) and 43-fold (at lOOpM LGM2605) (p<0.05) compared to macrophages treated solely with Cdt (57-fold). Similarly, IL-6 exhibited a significant reduction to 44-fold (at 50pM LGM2605) and 33.7-fold (at lOOpM LGM2605) (p<0.05) compared to Cdt-treated macrophages without LGM2605 (283-fold). Finally, TNF-a levels decreased significantly to 15-fold (at 50pM LGM2605) and 16-fold (at lOOpM LGM2605) (p<0.05) when compared to the macrophages treated only with Cdt (39-fold).LGM2605 Diminishes Cdt Induced Pro-Inflammatory Cytokine Release or Secretion

[0073] The impact of LGM2605 treatment on cytokine secretion was further investigated. Differentiated THP-1 macrophages were pretreated with varying doses (25, 50, lOOpM) of LGM2605 for 30 minutes, followed by the addition of increasing concentrations of Cdt (25, 50, 100, 200, 500 ng / mL) for a 5-hour period to promote cytokine release. The supernatants were collected, and levels of IL-ip, IL-6, and TNF-a were determined by ELISA. Cdt treatment led to an increase in the release of pro-inflammatory cytokines at all concentrations studied (Figure 2, LGM2605 OpM). Conversely, LGM2605 treatment reduced cytokine release when compared to Cdt exposure alone. Notably, for IL-ip, samples treated with 100 ng / mL Cdt showed a substantial decrease in IL-ip secreted when cells were pretreated with LGM2605 at all concentrations (25pM, 50pM, and lOOpM). The most significant reduction in IL-ip release (40%) was observed in samples treated with 100 ng / mL Cdt in combination with lOOpM LGM2605 (172±6 pg / mL compared to 107+6 pg / mL). In the case of IL-6, all Cdt-treated samples exhibited a notable decrease in cytokine levels when treated with 50pM or lOOpM LGM2605. The most substantial reduction in IL-6 released (40%) was observed in samples treated with 500 ng / mL Cdt in combination with lOOpM LGM2605 (577+45 pg / mL compared to 352+22 pg / mL). For TNF-a, samples treated with 200-500 ng / mL Cdt in combination with 50pM and lOOpM LGM2605 concentrations exhibited a substantial decrease. The most pronounced reduction (40%) was noted in samples treated with 100 ng / mL Cdt in combination with lOOpM LGM2605 (1593+36 pg / mL compared to 985±38 pg / mL).LGM2605 Diminishes Aa-Induced Inflammation

[0074] The preceding studies analyzed the impact of LGM2605 on Cdt-mediated cytokine expression and release. To gauge the preventive potential of LGM2605 against MIPP pathogenesis, whether LGM2605 treatment could yield similar effects when applied to Aa was examined. Differentiated THP-1 macrophages were pre-treated with various doses of LGM2605 (25, 50, lOOpM) for 30 minutes and subsequently exposed to Aa (at MOI of 1:10 and 1:100) for a4-hour to incite an inflammatory response. This inoculation effectively induced inflammation, resulting in elevated levels of IL-ip (1:10 - 158±13 pg / mL, 1:100 - 551±19 pg / mL), IL-6 (1:10 -92±4 pg / mL, 1:100 -244±14 pg / mL), and TNF-a(l: 10 - 347±10 pg / mL, 1:100 - 4119±94 pg / mL).

[0075] Consistent with the earlier experiment, the administration of LGM2605 significantly diminished Lz- induced pro-inflammatory response (Figure 3). Regarding IL-ip, Aa at an MOI of 1:10 exhibited a significant decrease when treated with LGM2605 at 50pM and lOOpM, while at an MOI of 1:100, a significant decrease was observed only with LGM2605 at lOOpM. The most substantial reduction (50%) was noted in Aa at an MOI of 1:10 when combined with lOOpM LGM2605 (551±19 pg / mL compared to 73±13 pg / mL). For IL-6, Aa at an MOI of 1:10 demonstrated a significant decrease when treated with LGM2605 at 25 pM and 50pM, whereas at an MOI of 1:100, a significant decrease was observed with all concentrations of LGM2605. The most notable reduction (20%) occurred in Aa at an MOI of 1:10 when treated with lOOpM LGM2605 (244+14 pg / mL compared to 193+14 pg / mL). Concerning TNF-a, both Aa at MOI of 1:10 and 1:100 exhibited a significant decrease with all LGM2605 concentrations. The most pronounced reduction (27%) was observed in Aa at an MOI of 1:100 when combined with lOOpM LGM2605 (4119+94 pg / mL compared to 2990+74 pg / mL).LGM2605 Diminished IL-1 / 3 Release via Cannonical-lnflammasome Activation Pathway (Caspase 1) and Decreased IL- 1 / 3 Expression in Both THP-1-caspland THP-1-casp4THP-1 Cells

[0076] Previous studies documented that both caspase- 1 and caspase-4 are activated in Cdt-treated macrophages resulting in the release of IL-ip. To determine if LGM2605 affects the expression level of pro-IL-ip in caspase 1 (THP-1-caspl) or caspase 4 deficient (THP-1-casp4) THP-1 cells, the deficient cells were pretreated with LGM2605 50pM and lOOuM Cdt 100 ng / mL, and the expression of pro-IL-ip was assessed using quantitative real-time PCR (Figure 4, left and middle panels). IL-ip exhibited 1-fold (50uM LGM2605) and 0.9-fold (lOOuM LGM2605) increase compared to the control with significant (p<0.05) decrease compared to only Cdt treated macrophage (3.1-fold, OuM LGM2605) with THP-1-caspl. For THP-1-casp4, IL-ip exhibited 2.7-fold (50uM LGM2605) and 1.4-fold (lOOuM LGM2605) increase compared to the control with significant (p<0.05) decrease compared to only Cdt treated macrophage (4.7-fold, OuM LGM2605). The effect of LGM2605 on IL-ip release in THP-l‘caspland THP-l‘casp4was also tested. The deficient cells pretreated with LGM2605 with 50pM and lOOpM, Cdt 100 ng / mL wassubsequently applied for 5 hours and IL-1 P release was assessed by ELISA (Figure 4, right panel). While THP-l‘casplshowed undetectable level of IL- ip for all (data not shown), THP-1-casp4showed 108±13 pg / mL with Cdt 100 ng / mL treated while LGM2605 significantly reduced IL-ip release at both 50p. M (30+4 pg / mL, 70% reduction compared to Cdt only) and lOOuM (25+5 pg / mL, 77% reduction compared to Cdt only).Cdt Phosphatase Activity Induced Pro-Inflammatory Environment Leads to Increase in Osteoclast Differentiation

[0077] Pro-inflammatory cytokines can also promote osteoclast differentiation via autocrine / paracrine mechanisms. Macrophage exposure to Cdt results in PI-3K blockade leading to increased cytokine release as shown above and in previously published studies and increase TRAP activity was observed with murine macrophage based osteoclast. Thus, whether Cdt can increase osteoclast differentiation in human cell line was determined. First, THP-1 monocytes were differentiated into macrophage using high dose PMA (100 ng / mL) in media for 48 hours with osteoclast differentiation subsequently induced with RANKL and M-CSF (50 ng / mL each) in media for 6 days as described herein. Increasing concentrations of Cdt were added in osteoclast differentiation media and replenished every 2 days. TRAP staining was done on the 6thday of differentiation and the number of TRAP+ cells were counted to analyze differentiated osteoclasts as distinct from macrophage. An increasing trend in TRAP+ cells was observed upon increase in Cdt concentration. At 50 ng / mL, there was a two-fold statistically significant increase in TRAP+ cells (Figure 5).

[0078] Next, to determine whether the increase in osteoclast differentiation is dependent on CdtB phosphatase activity leading to depletion of PI(3,4,5)P3, the phosphatase inactive Cdt mutant (CdtR117A) and wild type Cdt (CdtWT) were utilized. Cells are initially treated with high PMA (100 ng / mL) for two days and differentiated to osteoclast with RANKL and M-CSF for 6 days. On 6thday of differentiation, TRAP + cells were counted. There was significant increase in TRAP+ cells for both 50 ng / mL and 500 ng / mL CdtWTtreated cells by 170% and 200%, respectively (Figure 6). There was no significant increase in TRAP+ cells upon treatment with CdtR117A(Cdt 50 ng / mL: 107% and Cdt 500 ng / mL: 109%).Cdt Mediated Increase in Osteoclast Differentiation Can Be Mitigated by LGM2605

[0079] In this next set of experiments, the hypothesis that LGM2605 diminishes osteoclastdifferentiation as showed above (Figures 2-3) that it can reduce pro-inflammatory cytokine release was tested. THP-1 cells were pretreated with LGM2605 after high PMA (100 ng / mL) treatment and added during osteoclast differentiation, 6 days, with RANKL and M-SCF. Cdt 50 ng / mL was added for Cdt treatment and Cdt+LGM2605 treatment. TRAP staining revealed that there was a significant decrease (p-value < 0.05 and <0.01, respectively) in TRAP activity when LGM2605 50pM (79% of Cdt only) and lOOpM (66% of Cdt only) was applied concurrent with 50 ng / mL Cdt treatment during differentiation (Figure 7).

[0080] To further study the effect of Cdt and LGM2605 on osteoclast maturation, levels of osteoclast markers, TRAP and Cathepsin K, and number of nuclei were examined. Multi-fluor confocal imaging (Figure 8) revealed a significant increase in TRAP staining (1295% of control) and Cathepsin K immunoreactivity (272% of control) when 50 ng / mL Cdt was added during differentiation. The addition of lOOpM LGM2605 mitigated the effect of Cdt as indicated by a significant decrease in TRAP level (820% of control) and Cathepsin K level (185% of control). Osteoclast maturation is determined by multi-nucleation, therefore the number of nuclei per cell was counted. While both Cdt only group (42% of cells) and Cdt+LGM2605 group (42.2% of cells) had significant increase in two nuclei per cell compared to control (17% cells), three or more nuclei was significantly less in Cdt+LGM2605 group (2.8% of cells) and control group (1.2% of cells) compared to Cdt only group (6.9% of cells). Protein levels of osteoclast markers were also analyzed by Western blot (Figure 9). TRAP levels were highest with Cdt alone (control group), there was a significant decrease in the Cdt+LGM2605 group (55% of control) compared to Cdt only group (77% of control). Cathepsin K revealed that Cdt only group had the highest level (150% of control) compared to Cdt+LGM2605 group (89% of control) and control group.LGM2605 Does Not Affect Cell Viability.

[0081] Lastly, to determine if LGM2605 was cytotoxic, THP-1 derived macrophages were treated with increasing dose of LGM2605 (25, 50, 100, 200, and 400p. M) for 24 hours. Cells were detached and cell viability assessed. The Cdt treated cells remained viable up to 400uM LGM2605 (Figure 10).DISCUSSION

[0082] The present study probes the intricate interplay between cytolethal distending toxin (Cdt), produced by Aggregatibacter actinomycetemcomitans (Aa), and the host's inflammatory response,specifically exploring its role in molar / incisor pattern periodontitis (MIPP). Previous studies have indicated that Cdt's phosphatase activity disrupts phosphatidylinositol-3,4,5-triphosphate (PIP3) signaling, impacting the phosphorylation of GSK3P and Akt and modulating pro-inflammatory cytokine expression through NF-KB activation. When treating macrophages with Cdt, a significant upregulation of pro-inflammatory genes and corresponding cytokine release were observed, including pro-IL-ip, IL-6, and TNF-a. This evidence strongly supports the notion that Cdt actively contributes to the development of the hyper-inflammatory environment associated with MIPP, ultimately exacerbating the disease's progression.

[0083] Moreover, the present study highlights a potential solution to mitigate the inflammatory response triggered by Cdt. The application of LGM2605, a bioactive lignan known for its antiinflammatory properties, was explored. It notably reduced the expression of pro-inflammatory genes in Cdt-exposed macrophages. Demonstrating further efficacy, LGM2605 effectively curtailed the release of cytokines IL-ip, IL-6, and TNF-a in macrophages exposed to Cdt and inoculated with Aa, showcasing its capacity to modulate the host's inflammatory response (Figures 2-3). These results echo previous studies involving LGM2605 in asbestos and radiation triggered lung disease, age-related retinal epithelium disease, reactive oxygen species involved cardiovascular disease, and neuronal inflammatory disease. On a molecular level, one aspect of LGM2605’s action is to inhibit NF-KB nuclear translocation while activating downstream targets, alongside augmenting Nrf2 activity and the antioxidant response. Given the exacerbated inflammation induced by Cdt and Aa infection in MIPP, LGM2605 emerges as a promising therapeutic agent to counteract the detrimental effects observed, aligning with its success in managing similar pathological conditions.

[0084] In the present investigation, it was found that Cdt impacts osteoclast differentiation, a crucial aspect of MIPP pathogenesis. The present study revealed that Cdt promotes the increase in TRAP+ cells, indicating a heightened differentiation of osteoclasts, as illustrated in Figure 5. This aligns with existing studies suggesting that an environment rich in pro-inflammatory cytokines can facilitate osteoclast differentiation. In the present case, Cdt-mediated release of inflammatory cytokines stimulates this differentiation process. Crucially, it is established that this effect of Cdt relies on its phosphatase activity. When cells were treated with CdtR117A, a phosphatase-inactive form of Cdt, there was no significant increase in TRAP+ cells, confirming the dependency of this effect on phosphatase activity. The result was consistent across concentrations of low (50 ng / mL)and high (500 ng / mL) concentrations (Figure 6). Beyond the TRAP+ assay, osteoclast maturation was assessed using TRAP and Cathepsin K via immunoblotting and immunocytochemistry, in addition to evaluating the number of nuclei per cell.

[0085] TRAP activity and levels have traditionally served as markers for osteoclast differentiation. However, it's crucial to note that while TRAP is expressed and secreted by various monohistiocytic lineage cells, it does not precisely define differentiation of cell. This was evident in the present study as well. While immunoblot analysis did not reveal a statistical difference in protein levels between the control and Cdt- treated osteoclasts (Figure 9), immunocytochemistry results using confocal microscopy showed significant differences. Specifically, the control group displayed a stronger intensity localized in a granular pattern with faint diffusion in the basolateral side of the cell. In contrast, the Cdt-treated cells exhibited strong intensity across the entire basolateral side of the cell.

[0086] This difference in TRAP distribution aligns with its distinct localization in macrophages and osteoclasts. In macrophages, TRAP localizes within lysosomes, while in osteoclasts, it resides in vesicles that fuse with transcytotic vesicles. These transcytotic vesicles facilitate the transportation of bone matrix degradation products to the basolateral surface, where they are secreted. Given the treatment approach involving THP-1 cells with a high dose of PMA before osteoclast differentiation, a common step for THP-1 monocytes to differentiate into macrophages, it is possible that the observed difference in TRAP distribution between control and Cdt-treated THP-1 cells indicates a faster differentiation and maturation of Cdt-treated THP-1 cells into osteoclasts.

[0087] Cathepsin K is primarily secreted by activated osteoclasts to facilitate the degradation of collagen and other matrix proteins during bone resorption. While TRAP activity can be detected in pre-osteoclastic cells, Cathepsin K serves as a marker indicative of activated osteoclasts possessing the capacity to degrade bone tissue. In the present study, both immunoblot and immunocytochemistry analyses revealed an increase in Cathepsin K levels when cells were treated with Cdt during differentiation compare to control (Figures 8-9). Additionally, osteoclast maturation was assessed by examining the number of nuclei per cell. Although osteoclasts that fail to multinucleate can retain certain osteoclast characteristics, express markers like TRAP and Cathepsin K, and maintain limited bone resorption ability, multinucleation is a hallmark of the latephase of osteoclast differentiation. In the present study, maturation was distinguished by categorizing cells with 2 nuclei per cell from those with 3 or more nuclei per cell. The Cdt-treated osteoclasts showed a significantly higher population in both categories, indicating a further progression in the maturation process. The collective findings suggest that the Cdt-triggered inflammatory response disrupts the balance of osteo-immuno homeostasis, fostering bone loss — a defining characteristic of periodontitis.

[0088] Following the effective response of LGM2605 against the inflammatory reaction induced by Cdt and Aa in macrophages, its potential to counteract the Cdt-induced rise in osteoclast differentiation and maturation was explored. In the TRAP+ assay, while all concentrations of LGM2605 treatment exhibited a significant increase compared to the control, LGM2605 at 50 M and 100 M notably reduced the number of TRAP+ cells compared to conditions treated with Cdt alone (Figure 7). In the maturation study conducted through immunocytochemistry, although it did not exhibit a significant decrease in Cathepsin K levels or the population of cells with 2 nuclei per cell, it did reveal a decrease in TRAP intensity and the population of cells with 3 or more nuclei per cell (Figure 8). Additionally, the maturation study employing immunoblotting demonstrated a notable decrease in both TRAP and Cathepsin K levels compared to conditions treated solely with Cdt (Figure 9). LGM2605 possibly mitigated inflammatory cytokines, resulting in the suppression of osteoclast differentiation and maturation. This effect might also involve the activation of Nrf2. Extensive studies on primary cells or cell lines have demonstrated that Nrf2 hyperactivation inhibits osteoclast differentiation, whereas its inhibition yields contrasting effects. RANKL's binding to RANK triggers crucial intracellular signaling cascades through TRAF-6, leading to ROS generation crucial for osteoclast formation. Nrf2's regulation of oxidative stress could be a primary mechanism influencing osteoclast differentiation. In this study, LGM2605 demonstrated its ability to mitigate this effect, introducing a novel approach to managing bone loss in MIPP.

[0089] In conclusion, the present study has unveiled the intricate relationship between Cdt, the host's inflammatory response, and osteoclast differentiation, offering significant insights into MIPP's pathogenesis. The potential therapeutic role of LGM2605 presents new approaches to managing MIPP and other inflammatory conditions, holding promise for patients affected by these diseases.

[0090] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

What is claimed is:

1. A method for treating periodontitis and / or pathogen-associated gingival inflammation in a subject in need thereof, comprising administering to said subject a composition comprising a therapeutically effective amount of secoisolariciresinol diglucoside (SDG).

2. The method of claim 1, wherein said periodontitis is molar- incisor pattern periodontitis (MIPP).

3. The method of claim 1 or claim 2, wherein said SDG is (S, S)-SDG.

4. The method of claim or claim 2, wherein said SDG is (7?,7?)-SDG.

5. The method of claim 1 or claim 2, wherein said SDG is a synthetic SDG.

6. The method of claim 5, wherein said SDG is LGM2605.

7. The method of claim 5, wherein said SDG is LGM1506.

8. The method of claim 1 or claim 2, wherein said SDG is a racemic mixture of (R. R)SDG and (?,7?)-SDG.

9. The method of any one of claims 1-8, wherein said composition is administered orally.

10. The method of any one of claims 1-8, wherein said composition is administered topically.

11. The method of any one of claims 1-8, wherein said composition is formulated as a gel or an oral rinse.

12. The method of any one of claims 1-11, wherein the method is performed as an adjunctive treatment in conjunction with scaling and root planing in the subject.

13. A method for reducing inflammation and / or pro-inflammatory cytokine secretion in periodontal tissue induced by Aggregatibacter actinomycetemcomitans or cytolethal distending toxin in a subject in need thereof, comprising administering to said subject a composition comprising a therapeutically effective amount of secoisolariciresinol diglucoside (SDG).

14. The method of claim 13, wherein the subject has periodontitis and / or pathogen-associated gingival inflammation.

15. The method of claim 14, wherein the periodontitis is molar-incisor pattern periodontitis(MIPP).

16. The method of any one of claims 13-15, wherein said SDG is (S, S)-SDG.

17. The method of any one of claims 13-15, wherein said SDG is (R, R)-SDG.

18. The method of any one of claims 13-15, wherein said SDG is a synthetic SDG.

19. The method of claim 18, wherein said SDG is LGM2605.

20. The method of claim 15, wherein said SDG is LGM150621. The method of any one of claims 13-15, wherein said SDG is a racemic mixture of (R, R)-SDG and (R, R)-SDG.

22. The method of any one of claims 13-21, wherein said composition is administered orally.

23. The method of any one of claims 13-21, wherein said composition is administered topically.

24. The method of any one of claims 13-21, wherein said composition is formulated as a gel or an oral rinse.

25. The method of any one of claims 13-24, wherein the method is performed as an adjunctive treatment in conjunction with scaling and root planing in the subject.

26. A pharmaceutical composition for treating periodontitis and / or pathogen-associated gingival inflammation in a subject, the pharmaceutical composition comprising a therapeutically effective amount of secoisolariciresinol diglucoside (SDG).

27. The pharmaceutical composition of claim 26, wherein said composition is formulated as a gel.

28. The pharmaceutical composition of claim 26, wherein said composition is formulated as an oral rinse.

29. The pharmaceutical composition of any one of claims 26-28, wherein said SDG is (S, S)- SDG.

30. The pharmaceutical composition of any one of claims 26-28, wherein said SDG is (R, R)- SDG.

31. The pharmaceutical composition of any one of claims 26-28, wherein said SDG is asynthetic SDG.

32. The pharmaceutical composition of claim 31, wherein said SDG is LGM2605.

33. The pharmaceutical composition of claim 31, wherein said SDG is LGM150634. The pharmaceutical composition of any one of claims 26-28, wherein said SDG is a racemic mixture of (R, R)-SDG and (R, R)-SDG.

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