Compositions and methods for treating invasive pathogens

Abstract

This disclosure provides compounds that inhibit the entry of intracellular pathogens into host cells. Furthermore, this disclosure relates to the use of such compounds in methods for the treatment and prevention of periodontitis or conditions or symptoms associated with periodontitis.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Application No. 63 / 472,968, filed on 14 June 2023, subject to "Compositions and Methods for Treating Invasive Pathogens," the entire contents of which are incorporated herein by reference.

[0002] This disclosure generally relates to methods for treating and / or preventing diseases and conditions, and more specifically to diseases and conditions associated with or characterized by invasive pathogens, such as those related to certain periodontal diseases. [Background technology]

[0003] The microbiome plays a crucial role in human health, participating in digestion and immune responses, and being linked to infections and diseases associated with gut microbiota abnormalities. Microbiome regulation, including the removal of keystone pathogens, is a potential therapeutic strategy for preventing or treating microbiome-related diseases. Porphyromonas gingivalis (Pg) is a Gram-negative bacterium that is the keystone pathogen of periodontitis, the sixth most common infectious disease in the world, and it is estimated that 5-20% of the population suffers from chronic periodontitis. Pg contributes to periodontitis by disrupting the homeostasis of periodontal tissues and microorganisms by transforming commensal microorganisms into pathogenic ones, leading to uncontrolled inflammation and tissue destruction.

[0004] Bacterial invasion is the process by which bacteria, such as Pg, actively invade non-phagocytic cells, such as epithelial cells. Generally, invading bacteria express surface invasins that interact with corresponding receptors on the surface of non-phagocytic cells. As a result, a series of signaling events are triggered in the host cell, including protein phosphorylation, cytoskeletal rearrangement, membrane stretching, and bacterial phagocytosis. After internal migration, bacteria can not only survive and proliferate in the cytoplasm but also spread to other host cells. The ability to invade host cells gives bacteria the ability to evade immune responses and antibiotic treatment, and to successfully infect. Intracellular bacteria are also known to contribute to the persistence of chronic infections, such as by functioning as opportunistic bacteria.

[0005] For example, Pg possesses a diverse array of effector molecules and systems that enable its survival in the oral cavity. One of Pg's characteristics is its ability to invade and survive in host cells such as fibroblasts, epithelial cells, and endothelial cells. Membrane proteins involved in Pg's attachment to and invasion of host cells include FimA, hemagglutinin A, and heat stress proteins. Pg strains lacking invasion ability may have their activity reduced to about one-tenth. Invasion ability is crucial for the establishment of Pg infection in the oral cavity. However, research on bacterial invasion inhibitors is limited.

[0006] High concentrations of porospermum glycerin (Pg) are observed in the periodontal tissues of patients with refractory periodontitis, and conventional periodontitis treatments, including scaling and root planing, are insufficient to prevent the re-emergence of pathogenic microorganisms. While systemic or topical antibiotics may be used adjunct to address severe or refractory periodontitis, recommended therapeutic doses fail to eliminate intracellular Pg, potentially allowing it to proliferate within cells and spread to adjacent cells. Indeed, the difficulty in removing Pg from the oral cavity stems, at least in part, from Pg's ability to invade host cells and the drug resistance of plaque, which helps Pg evade host defense systems and antibiotics. Furthermore, there are growing public health concerns regarding antibiotic use, including the disruption of symbiotic relationships between the host and commensal microbiome, and the emergence of bacterial resistance to antibiotics. Moreover, current treatment strategies often provide only temporary relief, and disease recurrence is common. Therefore, there is a need for therapeutic agents that can specifically remove intracellular Pg from the oral cavity. [Overview of the project]

[0007] In certain embodiments, the Disclosure provides compounds that inhibit the entry of one or more intracellular pathogens into host cells. These are useful, for example, in the treatment of periodontitis or periodontitis-related conditions or symptoms, in the prevention of periodontitis or periodontitis-related conditions or symptoms, in reducing the probability of developing periodontitis or periodontitis-related conditions or symptoms, in reducing the severity of periodontitis or periodontitis-related conditions or symptoms, or in combination thereof.

[0008] In a first embodiment, a pharmaceutical composition for inhibiting the entry of a pathogen into host cells is disclosed. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a protein kinase C beta (PKC-β) agonist and a pharmaceutically acceptable carrier, and the pharmaceutical composition is formulated for topical delivery to the gingival tissue of a subject. The PKC agonist may have a PKC-β agonist. In one embodiment, the PKC-β agonist comprises gunidimacrine or a pharmaceutically acceptable derivative thereof. In some embodiments, the therapeutically effective amount of the PKC-β agonist is sufficient to achieve a sub-nanomolecular concentration of gunidimacrine in the gingival tissue of the subject. The therapeutically effective amount of the PKC-β agonist may be sufficient to achieve a concentration of at least about 10 nM of gunidimacrine in the gingival tissue of the subject. In some embodiments, the therapeutically effective amount of the PKC-β agonist is sufficient to achieve a concentration of up to about 1000 nM of gunidimacrine in the gingival tissue of the subject. The therapeutically effective amount of the PKC-β agonist can be sufficient to achieve a concentration of gnidimacrine up to approximately 100 nM in the gingival tissue of the subject. In one embodiment, the therapeutically effective amount of the PKC-β agonist is sufficient to achieve a concentration of gnidimacrine up to approximately 20 nM in the gingival tissue of the subject.

[0009] In some specific embodiments, the subject is diagnosed with periodontitis, a periodontitis-related disease, or a periodontitis-related condition. In one embodiment, the pathogen comprises a bacterium. The bacterium may be Porphyromonas gingivalis.

[0010] In some embodiments, the pharmaceutical composition contains a therapeutically effective amount of antibiotic. The antibiotic may include gentamicin, metronidazole, or a combination thereof.

[0011] In one embodiment, the pharmaceutical composition comprises a toothpaste. The toothpaste may include a paste, gel, mouthwash, powder, tooth soap, or a combination thereof.

[0012] In a second embodiment, a method for treating or preventing bacterial invasion in non-phagocytic cells of a subject is disclosed. In some embodiments, the method comprises administering a therapeutically effective amount of a compound comprising a PKC-β agonist to the subject. The non-phagocytic cells may include epithelial cells, endothelial cells, fibroblasts, smooth muscle cells, or a combination thereof. The epithelial cells may include oral epithelial cells. In one embodiment, the oral epithelial cells include human oral keratinocytes (HOK). The endothelial cells may include aortic endothelial cells, cardiac endothelial cells, or a combination thereof. In some embodiments, the fibroblasts include gingival fibroblasts.

[0013] In some specific embodiments, the subject is suffering from a Porphyromonas gingivalis infection. The subject may be at risk of Porphyromonas gingivalis infection. In one embodiment, the PKC agonist comprises a PKC-β agonist. The PKC-β agonist may comprise gunidimacrine or a pharmaceutically acceptable derivative thereof.

[0014] In some embodiments, the therapeutically effective dose of the PKC-β agonist is sufficient to achieve a concentration of at least about 10 nM of gunidimacrine in the subject's gingival tissue. The therapeutically effective dose of the PKC-β agonist may be sufficient to achieve a concentration of up to about 1000 nM of gunidimacrine in the subject's gingival tissue. In one embodiment, the therapeutically effective dose of the PKC-β agonist is sufficient to achieve a concentration of about 20 nM of gunidimacrine in the subject's gingival tissue.

[0015] In some specific embodiments, the method further comprises administering a therapeutically effective dose of an antibiotic. The antibiotic may include gentamicin, metronidazole, or a combination thereof.

[0016] A third aspect of this disclosure comprises a method for treating or preventing periodontitis, periodontitis-related disease, or periodontitis-related symptoms in the gingival tissue of a subject where there is a need, comprising topically administering a therapeutically effective amount of a compound containing a PKC-β agonist to the gingival tissue of the subject. The periodontitis, periodontitis-related disease, or periodontitis-related symptoms may be bacterial-borne. In a particular embodiment, the periodontitis, periodontitis-related disease, or periodontitis-related symptoms are borne by Porphyromonas gingivalis.

[0017] In some specific embodiments of the method described above, the compound is administered to a toothpaste. The toothpaste includes a paste, gel, mouthwash, powder, tooth soap, or a combination thereof. The compound may contain an activator of PKC-β. In one embodiment, the compound includes gunidimacrine or a pharmaceutically acceptable derivative thereof.

[0018] In some specific embodiments, the gunigmacrin or its pharmaceutically acceptable derivative is administered to a compound containing at least 20 nM of gunigmacrin or its pharmaceutically acceptable derivative. The therapeutically effective amount of the compound can be administered together with a pharmaceutically acceptable carrier. In a specific embodiment, the compound contains gunigmacrin, and the therapeutically effective amount is sufficient to achieve a concentration of gunigmacrin up to about 1000 nM in the tissue of the subject. The therapeutically effective amount can be sufficient to achieve a concentration of gunigmacrin up to about 100 nM in the tissue of the subject. In an exemplary embodiment, the therapeutically effective amount is sufficient to achieve a concentration of gunigmacrin of about 20 nM in the tissue of the subject. The method can further include administering a therapeutically effective amount of an antibiotic.

[0019] In a fourth aspect, the disclosure relates to a method of reducing the likelihood of intracellular bacteria invading a host cell, the method including contacting the host cell with a therapeutically effective amount of a PKC-β agonist. The method can further include contacting the host cell with a therapeutically effective amount of an antibiotic. In one embodiment, the PKC-β agonist includes gunigmacrin or its pharmaceutically acceptable derivative.

[0020] In one embodiment, the therapeutically effective amount of the PKC-β agonist is about 20 nM of gunigmacrin or its pharmaceutically acceptable derivative. The bacterium can be Porphyromonas gingivalis. In some specific embodiments, the host cell includes epithelial cells, endothelial cells, fibroblasts, smooth muscle cells, or a combination thereof. The epithelial cells can include oral epithelial cells. In one embodiment, the oral epithelial cells include human oral keratinocytes (HOK). The endothelial cells can include aortic endothelial cells, cardiac endothelial cells, or a combination thereof. In one embodiment, the fibroblasts include gingival fibroblasts.

[0021] A fifth aspect of the present disclosure has a pharmaceutical composition for treating or preventing periodontitis, periodontitis-related diseases, or periodontitis-related symptoms in a subject, the pharmaceutical composition comprising a therapeutically effective amount of guinidimacrin or a pharmaceutically derivative thereof, a therapeutically effective amount of an antibiotic, and a pharmaceutically acceptable carrier, and the pharmaceutical composition being formulated for topical administration to the gingival tissue of the subject. In some embodiments, the therapeutically effective amount of the guinidimacrin or a pharmaceutically derivative thereof is sufficient to achieve a concentration of guinidimacrin in the gingival tissue of the subject from about 10 nM to about 100 nM. In other embodiments, the antibiotic comprises gentamicin, metronidazole, or a combination thereof. In still other embodiments, the guinidimacrin or a pharmaceutically derivative thereof is formulated into a dentifrice, and the dentifrice comprises a paste, a gel, a mouthwash, a powder, a tooth soap, or a combination thereof.

[0022] The above is a simplified summary for deepening a basic understanding of some aspects of the subject matter recited in the claims. This summary is not an extensive summary. Also, it is not intended to identify key or important elements or to limit the scope of the claims. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description presented herein.

[0023] Additional features and advantages can be ascertained from the following detailed description provided in connection with the following drawings.

Brief Description of the Drawings

[0024] [Figure 1] It is a diagram showing the chemical structure of guinidimacrin (GM) according to an embodiment of the present disclosure. [Figure 2A-2B]This document provides bar graphs illustrating the inhibition of Pg entry by GM and the removal of intracellular Pg. In Figure 2A, each bar graph represents the relative entry rate of Pg into human oral keratinocytes (HOKs) in the presence of different concentrations of GM. In Figure 2B, each bar graph represents the relative level of intracellular Pg in HOKs in the presence of either GM or antibiotics. Asterisks indicate the statistical significance of the entry rate or intracellular level between the presence or absence of GM (P<0.05; t-test). [Figure 3A-3E] This document provides confocal microscopy images demonstrating dose-dependent inhibition of Pg33277 vesicle entry into human oral keratinocytes (HOKs) by GM. HOKs were seeded in glass-bottom petri dishes and treated with PBS (control, see Figure 3A), treated with Pg vesicles generated over 30 minutes (see Figure 3B), treated with Pg vesicles in the presence of 5 nM GM (see Figure 3C), treated with Pg vesicles in the presence of 10 nM GM (see Figure 3D), or treated with Pg vesicles in the presence of 20 nM GM (see Figure 3E). HOKs were stained with polyclonal antibodies, followed by staining with anti-IgG containing Alex Fluor 546 (red) and DAPI (blue), and visualized under a confocal microscope. Scale bars in the panel represent 20 μm. [Figure 4] This graph compares Pg growth in the presence and absence of GM. Pg33277 is cultured in TSB with and without GM. OD600 was measured after 16 hours of incubation. Each bar represents the mean of three repeated samples, and the error bars indicate the standard deviation. [Figure 5]Western blot images used to analyze the effect of GM on PKC activation in activated human primary CD4 cells according to one embodiment are shown. Human primary CD4 cells activated with anti-CD3 / CD28 antibody (1 ug / ml) were cultured for 12 hours in the presence or absence of GM (1 nM). The cytoplasmic fraction of the cells was analyzed by 10% SDS PAGE, followed by Western blot analysis using a monoclonal antibody panel against PKC isozymes (Santa Cruz Biotech). [Figure 6] This graph shows luciferase levels as an indicator of cell viability in GM-treated HOK cells. Each bar represents the mean ± standard deviation of the luminescence level relative to the number of viable cells. The data are the mean values ​​from at least three independent experiments. [Figure 7] Confocal microscopy images of microtubule rearrangement in GM-induced HOK are shown. HOK was treated with GM (20 nM) for 0 minutes (left) or 30 minutes (right), stained with anti-α-tubulin antibody, Alex Fluor 546 anti-IgG antibody (red), and DAPI (blue), and observed by confocal microscopy. Scale bar is 20 μm. [Modes for carrying out the invention]

[0025] (A.Definition) Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as those generally understood by those skilled in the art in the field of this disclosure. Furthermore, terms defined in commonly used dictionaries, etc., should be interpreted to match their meaning in the context of this specification, and should not be interpreted in an idealized or overly formal sense unless expressly defined herein. For the sake of brevity or clarity, well-known functions or structures may not be described in detail.

[0026] The terms used herein are intended solely to describe specific embodiments and are not intended to limit them. The singular forms "a," "an," and "the" used herein also include the plural forms unless explicitly stated otherwise in the context.

[0027] The terms “first,” “second,” etc., used herein are used to describe various features or elements, but these features or elements should not be limited by these terms. These terms are used solely to distinguish one feature or element from another. Thus, the first feature or element discussed herein may be called the second feature or element, and similarly, the second feature or element discussed herein may be called the first feature or element, and these do not deviate from the teachings of this disclosure.

[0028] The phrase "consisting essentially of" means that, in addition to the elements described, other elements (such as steps, structures, components, and building blocks) may be included in the claims that do not adversely affect the operability to the intended purpose of the invention described in this disclosure. Importantly, this term excludes such other elements that adversely affect the operability to the intended purpose of the invention described in this disclosure, even if those elements could improve the operability of the invention claimed for other purposes.

[0029] The terms "about" and "approximately" generally refer to an acceptable degree of error or variation in a measured quantity, taking into account the nature or precision of the measurement. A typical degree of error or variation is within 20%, preferably within 10%, and more preferably within 5%, of a given value or range of values. In the case of biological systems, the term "about" refers to an acceptable standard deviation error, preferably not exceeding twice the given value. The numerical values ​​presented in this document are approximate unless otherwise specified, and the terms "about" or "approximately" mean that they can be inferred even if not explicitly stated.

[0030] The terms “administering” or “administration” include acts such as prescribing, dispensing, providing, or ingesting, so that the prescribed, dispensed, provided, or ingested substance actually comes into contact with the patient’s body outside or inside (or both). In each embodiment of this disclosure, the terms “administering” or “administration” include self-administration of a substance, self-administration, etc. In fact, it is specifically assumed that an act of administration is when a healthcare professional instructs or prescribes a subject or patient to ingest or self-administer a substance.

[0031] The terms “prevention,” “prevent,” and “preventing” as used herein refer to a series of measures (e.g., administration of a pharmaceutical composition) initiated before the onset of clinical symptoms of a disease state or condition in order to reduce the likelihood or severity of its occurrence. Such reduction of likelihood or severity does not need to be absolute to be useful.

[0032] The terms “treatment,” “treat,” and “treating” as used herein refer to a series of actions (e.g., administration of a pharmaceutical composition) initiated after the onset of clinical symptoms of a disease state or condition in order to eliminate or alleviate those clinical symptoms. Such treatment does not need to be absolute in order to be effective.

[0033] As used herein, the term “in need of treatment” refers to a situation in which a caregiver determines that a patient needs treatment or would benefit from treatment. This determination is based on a variety of factors within the caregiver’s professional scope, including knowledge that the patient is ill or likely to become ill as a result of a condition treatable by the methods or compositions of this disclosure.

[0034] As used herein, the term “in need of prevention” refers to a situation in which a caregiver determines that a patient needs prevention or would benefit from prevention. This determination is based on a variety of factors within the caregiver’s professional scope, including knowledge that the patient may become ill or be ill as a result of a condition preventable by the methods or compositions of this disclosure.

[0035] The terms “individual,” “subject,” or “patient” as used herein refer to any animal, including mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, primates, and mammals such as humans. These terms may specify male or female, or both, or they may exclude male or female.

[0036] The terms "comprise" and "include" as used herein are inclusive and non-exclusive and should be interpreted as "include / have, but not limited to." The permissible and optional terms used herein, such as "may," "can," and "some embodiments," are also inclusive and non-exclusive.

[0037] The expressions “inhibit,” “decrease,” and / or “reduce the likelihood of” (and similar terms) generally refer to actions that directly or indirectly reduce a function, activity, or behavior compared to its natural, expected, average, or current state. This is usually done in comparison to some baseline or expectation, i.e., relative. However, it is not always necessary to refer to a baseline or relative value. Such terms include completely inhibiting, completely reducing, or eliminating the likelihood of a function, activity, or behavior compared to its natural, expected, average, or current state.

[0038] The term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents, or encapsulants that do not cause significant irritation to humans or other vertebrates and do not impair the biological activity and properties of the administered compound. Such carriers include, but are not limited to, vehicles, adjuvants, surfactants, suspensions, emulsifiers, inert fillers, diluents, excipients, wetting agents, binders, lubricants, buffers, disintegrants, and carriers, as well as colorants and fragrances (collectively referred to here as carriers). Typically, pharmaceutically acceptable carriers are chemically inert to the active compound and do not have adverse side effects or toxicity under the conditions of use. Pharmaceutically acceptable carriers may include polymers and polymer matrices. The properties of a pharmaceutically acceptable carrier may vary depending on the specific dosage form and other properties of the composition used.

[0039] As used herein, the term "therapeutically effective amount" refers to the amount of a compound, either alone or as part of a pharmaceutical composition, that has a detectable effect in alleviating the symptoms, aspects, or characteristics of a disease state or condition. Such an effect does not need to be absolute to be beneficial.

[0040] As used herein, the term “prodrug” includes functional derivatives of the disclosed compounds that are readily converted in vivo to compounds of interest. Therefore, in the therapeutic methods of this disclosure, the term “administering” encompasses treating the various disease conditions / symptoms described using the specifically disclosed compounds, or prodrugs that are not specifically disclosed but are converted in vivo to specific compounds after administration to a patient. Conventional procedures for the selection and preparation of appropriate prodrug derivatives are described, for example, in “Design of Prodrugs” (Elsevier, 1985), edited by H. Bundgaard.

[0041] As used herein, the term “pharmaceutically acceptable salts” includes salts of active compounds prepared with relatively non-toxic acids or bases, depending on the specific substituents found in the compounds described herein. If the compounds of the present invention have relatively acidic functional groups, a base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the desired base in stock solution or a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium salts, potassium salts, calcium salts, ammonium salts, organic amino salts, magnesium salts, or similar salts. If the compounds of the present invention have relatively basic functional groups, an acid addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the desired acid in stock solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monobicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, monosulfate, hydroiodic acid, or phosphorous acid, as well as salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, oxalic acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, and methanesulfonic acid. Salts of amino acids such as alginic acid, and salts of organic acids such as glucuronic acid and galacturonic acid are also included (see, for example, Berge, SM et al., "Pharmaceutical Salts," "Journal of Pharmaceutical Science," 1977, 66, 1-19). The specific compounds of the present invention have both basic and acidic functional groups, and these compounds can be converted into either a base addition salt or an acid addition salt.

[0042] As used herein, the term “pathogen” refers to any factor capable of invading host cells and causing a pathological condition or disease in a subject. In each embodiment, the term “pathogen” refers to an organism. Pathogens may include bacteria, fungi, viruses, viroids, protists, prions, protozoa, or combinations thereof. Non-specific examples of bacteria include Porphyromonas gingivalis, Bartonella henselae, Francisella tularensis, Listeria monocytogenes, Salmonella typhi, Brucella spp., Legionella spp., Mycobacterium spp., Nocardia spp., Rhodococcus equi, Yersinia spp., Neisseria meningitidis, Chlamydia spp., Rickettsia spp., and Coxiella spp. Examples include (spp.). In one embodiment, the intracellular bacterium is Porphyromonas gingivalis. The pathogen can be any oral pathogen, regardless of its taxonomic group. In one embodiment, the pathogen is an intracellular parasitic fungus. Examples of intracellular parasitic fungi include, but are not limited to, Histoplasma capsulatum, Cryptococcus neoformans, and Pneumocystis jirovecii.Examples of protozoa include apicomplexans (e.g., Babesia spp., Plasmodium spp., Cryptosporidium parvum, Cyclospora cayetanensis, Isospora belli, and Toxoplasma gondii)) and trypanosomatids.

[0043] As used herein, “host cells” can include any cells that are susceptible to invasion by pathogens. Host cells can be present within the subject, obtained from the subject, or originate from the subject. Exemplary host cells include cells from various mammalian species, including humans and livestock. In each embodiment, the host cells include non-phagocytic cells. In each embodiment, the host includes non-phagocytic cells. Non-limiting examples of host cells include fibroblasts, epithelial cells, and endothelial cells. In each embodiment, the host cells are oral epithelial cells, gingival fibroblasts, aortic endothelial cells, cardiac endothelial cells, or vascular smooth muscle cells. These cells are vulnerable to various intracellular bacteria, including Porphyromonas gingivalis mentioned above. In a more specific embodiment, the host cells are cells derived from the subject's gingival tissue, selected from the group consisting of oral epithelial cells and gingival fibroblasts. In an even more specific embodiment, the host cells are oral keratinocytes, such as oral keratinocytes derived from the subject.

[0044] (B. Compound) This disclosure provides compounds, which may be referred to hereby as “active compounds,” that inhibit the entry of intracellular pathogens into host cells. In each embodiment, this disclosure comprises one or more active compounds that inhibit the entry of bacteria into one or more host cells of a subject. In certain embodiments, the active compound may inhibit the formation of reservoirs of one or more intracellular pathogens within host cells.

[0045] In each embodiment, the active compound comprises an inhibitor of any event that leads to bacterial phagocytosis of a host cell. In one embodiment, the active compound inhibits one or more invaders (which may be expressed on the surface of invading bacteria) from binding to relevant receptors on one or more non-phagocytic cells.

[0046] The active compound can inhibit one or more events, including protein phosphorylation, cytoskeletal rearrangement, membrane stretching, vesicle formation, and endocytosis, which lead to intracellular phagocytosis of pathogens. In each embodiment, the active compound inhibits one or more of these exemplary events, either in vitro or in vivo. This disclosure provides compounds that directly reduce one or more of these events, indirectly reduce one or more of these events, or a combination thereof. In each embodiment, the indirect modulation of one or more events is achieved by stimulating the activity of molecules that reduce these events, inhibiting the activity of molecules that increase these events, or a combination thereof. In each embodiment, the active compound interferes with receptor-mediated endocytosis of invasive pathogens. In each embodiment, the active compound includes a protein kinase C (PKC) activator. In one embodiment, the active compound includes a PKC-β agonist. In each embodiment, the active compound activates PKC-β in a dose-dependent manner.

[0047] The active compound may have downstream effects such as treating and preventing periodontitis or periodontitis-related conditions or symptoms in the subject. As mentioned above, Porphyromonas gingivalis (Pg) is a keystone pathogen of periodontitis, proliferating within the host cell and extracellular matrix, forming a reservoir of persistent infection. Therefore, without being bound by any theory, an active compound that targets Pg's entry into host cells may prevent or reverse the progression of periodontitis.

[0048] In each embodiment, the active compound comprises a diterpene. One specific example of the active compound is gnidimacrin (GM), a macrocyclic diterpenoid ester that can be isolated from Stellera chamaejasme. In each embodiment, the active compound has the chemical structure shown in Figure 1. As will be described in more detail in the examples described herein, the active compound can effectively inhibit Pg and its vesicles from entering human cells. In each embodiment, inhibition of Pg by the active compound is observed at low nanomolar concentrations of GM. In certain embodiments, Pg inhibition occurs when the concentration of the active compound is at least about 10 nM. For example, in some embodiments, Pg inhibition occurs when the concentration of the active compound is at least about 20 nM. Furthermore, this disclosure demonstrates that GM does not affect the viability of host cells. Taken together, the findings of this disclosure suggest that GM, with its potent inhibitory activity and low cytotoxicity, is an ideal candidate for the development of anti-Pg drugs.

[0049] Unless otherwise stated, all active compounds are to be interpreted as containing metabolites. Such metabolites may be produced in vivo or as a result of biochemical activity in vitro. It is also within the scope of this disclosure that any active compound may be limited to non-metabolites.

[0050] (C. Pharmaceutical Compositions) One embodiment includes a pharmaceutical composition having an active compound. Each disclosed composition may contain one or more active compounds in combination with a pharmaceutically acceptable carrier. For example, in one embodiment, this disclosure provides a pharmaceutical composition comprising a PKC-β agonist and a pharmaceutically acceptable carrier. In another embodiment, this disclosure provides a pharmaceutical composition comprising gunidimacrine and a pharmaceutically acceptable carrier. Examples of pharmaceutically acceptable carriers and formulation methods are described in "Remington: The Science and Practice of Pharmacy" (20th edition, Lippincott, Williams & Wilkins, edited by Daniel Limmer) and are generally well understood by those skilled in the art. To form a pharmaceutically acceptable composition suitable for administration, such compositions may contain a therapeutically effective amount of the active compound.

[0051] The pharmaceutical compositions of this disclosure can be used in the therapeutic and prophylactic methods of this disclosure. Such compositions are administered to a subject in an amount sufficient to deliver a therapeutically effective dose of the active compound in order to exert their effect in the therapeutic and prophylactic methods disclosed herein. The therapeutically effective dose may vary depending on various factors such as the subject's condition, weight, sex, and age. For example, some embodiments of the compositions of this disclosure use the median lethal dose (LD50) of the active compound. 50 Includes up to LD. 50 This can be confirmed using standard toxicological methods or by referring to past studies.

[0052] A therapeutically effective amount of a pharmaceutical composition can be formulated to achieve a desired concentration of the active compound in the gingival tissue of a subject. In some embodiments, the composition contains a therapeutically effective amount of the active compound sufficient to achieve an active compound concentration of at least about 1 nM in the gingival tissue of a subject. In some embodiments, the composition contains an amount of the active compound sufficient to achieve concentrations of up to about 10 μM, up to about 20 μM, up to about 30 μM, or up to about 40 μM in the gingival tissue of a subject. In one embodiment, the therapeutically effective amount of the active compound contains an amount sufficient to achieve an active compound concentration of at least about 20 nM in the gingival tissue of a subject. The composition may contain an amount of the active compound sufficient to achieve concentrations of about 5 nM to 1 μM in the gingival tissue of a subject. In each specific embodiment, the composition contains an amount of the active compound sufficient to achieve concentrations of at least about 10 nM, at least about 20 nM, at least about 30 nM, at least about 40 nM, at least about 50 nM, at least about 60 nM, at least about 70 nM, at least about 80 nM, at least about 90 nM, and at least about 100 nM in the subject's gingival tissue. In one specific embodiment, the pharmaceutical composition contains an amount of GM sufficient to achieve concentrations of at least about 5 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, or at least about 40 nM in the subject's gingival tissue. In each specific embodiment, the composition contains an amount of the active compound sufficient to achieve concentrations of up to 1 nM, up to 5 nM, up to 10 nM, up to 15 nM, up to 20 nM, up to 25 nM, up to 30 nM, up to 40 nM, up to 50 nM, up to 60 nM, up to 70 nM, up to 80 nM, up to 90 nM, and up to 100 nM in the subject's gingival tissue. In some embodiments, the composition contains an amount of the active compound sufficient to achieve concentrations below nanomolar. The concentration of the active compound in the composition can be less than about 1 nM.

[0053] In each embodiment, the active compound may be administered in combination with an antibiotic or a combination of two or more antibiotics. At least one antibiotic and the active compound may be provided as a single pharmaceutical composition or as separate pharmaceutical compositions or formulations. One embodiment includes a pharmaceutical composition containing a therapeutically effective amount of the active compound and a therapeutically effective amount of the antibiotic. In certain embodiments, the combined administration of a pharmaceutical composition containing the active compound and an antibiotic generally increases the therapeutic effect of the active compound. In other words, including an antibiotic in a pharmaceutical composition containing the active compound can produce a synergistic effect that more effectively inhibits the entry of pathogens into host cells. In some embodiments, a bidirectional synergistic effect is generally observed. That is, the combined use of two activators (antibiotics and the active compound) yields higher inhibitory activity than either activator alone.

[0054] In some embodiments, the amount of active compound in a pharmaceutical composition can be varied based on the amount of antibiotic administered to a subject. For example, in certain embodiments where the pharmaceutical composition contains an antibiotic, a lower concentration of the active compound may be required to achieve the same pathogen entry inhibitory effect as when the pharmaceutical composition contains only the active compound. Therefore, the therapeutically effective amount of the active compound in the pharmaceutical composition may be inversely proportional to the therapeutically effective amount of the antibiotic in the pharmaceutical composition.

[0055] In each embodiment, the therapeutically effective dose of the antibiotic is sufficient to eliminate extracellular bacteria at the target site of the subject. In one embodiment, the target site includes the oral cavity of the subject. The target site may also be the gingival tissue of the subject. In one embodiment, the therapeutically effective dose of the antibiotic is sufficient to achieve an antibiotic concentration of at least about 50 μg / ml in the gingival tissue of the subject. The therapeutically effective dose of the antibiotic can be sufficient to achieve an antibiotic concentration of up to about 1000 μg / ml in the gingival tissue of the subject. The therapeutically effective dose can be about 50 μg / ml, about 100 μg / ml, about 150 μg / ml, about 200 μg / ml, about 250 μg / ml, about 300 μg / ml, about 350 μg / ml, about 400 μg / ml, about 450 μg / ml, or about 500 μg / ml in the gingival tissue of the subject.

[0056] In one embodiment of the pharmaceutical composition, the active compound comprises a GM, and the pharmaceutical composition is configured to be administered in combination with an antibiotic. In each embodiment, the pharmaceutical composition comprises a therapeutically effective amount of GM and a therapeutically effective amount of antibiotic. In each particular embodiment, the antibiotic comprises any antibiotic capable of eliminating or reducing the development of invasive bacteria in host cells. In one embodiment, the antibiotic comprises any antibiotic capable of preventing invasive bacteria from entering host cells. The antibiotic may comprise any antibiotic capable of eliminating or reducing the development of oral bacteria in the gingival tissue of a subject. In each particular embodiment, the antibiotic comprises penicillin, streptomycin, gentamicin, metronidazole, or a combination thereof.

[0057] Additional exemplary factors that may affect the therapeutically effective dose include, but are not limited to, the mode of administration, the site of administration, or a combination thereof. Pharmaceutical compositions can be formulated to be administered to a subject by any method known in the art. Exemplary forms of administration include subcutaneous, intravenous, topical, epithelial, oral, intraosseous, intramuscular, intranasal, mucosal absorption, and pulmonary administration. The compositions of this disclosure can be formulated to be administered to a subject once or multiple times. Furthermore, when the composition is administered to a subject multiple times, various dosing schedules can be used, such as once daily, once weekly, once monthly, or once year. The composition can also be formulated to be administered to a subject multiple times daily. The therapeutically effective dose and appropriate dosing schedule of the active compound can be determined by testing to obtain optimal activity while minimizing potential side effects. Furthermore, formulations for concomitant use with other drugs or for sequential administration may be desirable.

[0058] The compositions of this disclosure can be formulated for systemic administration, such as intravenous administration, or for topical administration, such as subcutaneous injection or absorption. They can also be formulated for administration by topical application, such as gel, fiber, paste, or cream.

[0059] The compositions of this disclosure can be formulated into a variety of dosage forms for administration. For example, the compositions can be in the form of tablets, capsules, sachets, lozenges, troches, pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups, ointments, creams, pastes, emulsions, or solutions for intravenous or injectable administration. Other dosage forms include transdermal administration such as patches and ointments. Furthermore, formulations suitable for delivery by nebulizer or metered-dose inhaler are also included. Any of the above dosage forms can be modified to be time-release and / or sustained-release formulations.

[0060] In this disclosure, pharmaceutical compositions may further include pharmaceutically acceptable carriers. Such carriers include, but are not limited to, vehicles, adjuvants, surfactants, suspensions, emulsifiers, inert fillers, diluents, excipients, wetting agents, binders, lubricants, buffers, disintegrants, and carriers, as well as auxiliary agents such as colorants and fragrances (collectively referred to here as carriers). Typically, pharmaceutically acceptable carriers are chemically inert to the active compound and do not have adverse side effects or toxicity under the conditions of use. Pharmaceutically acceptable carriers may include polymers and polymer matrices. The properties of pharmaceutically acceptable carriers may vary depending on the specific dosage form used and other properties of the composition.

[0061] For example, when an orally administered composition is a solid dosage form such as tablets, capsules, sachets, lozenges, troches, pills, powders, and granules, the active compound can be combined with an orally administered, non-toxic, and pharmaceutically acceptable inactive carrier, such as an inactive filler, a suitable binder, lubricant, disintegrant, and auxiliary agent. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose and beta-lactose, corn-derived sweeteners, natural and synthetic gums such as acacia gum, tragacanth gum, and sodium alginate, carboxymethylcellulose, polyethylene glycol, and wax. Lubricants used in these dosage forms include, but are not limited to, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, and sodium acetate. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, and xanthan gum. The tablets may contain lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and one or more of the other carriers described herein. The lozenge form may contain the active ingredient in a flavoring, which is typically sucrose and acacia or tragacanth, while the active ingredient may be contained in a pastille containing gelatin and glycerin, or in an inert base such as sucrose and acacia, or in an emulsion and gel containing the active ingredient in addition to carriers known in the art. In each embodiment, the orally administered pharmaceutical composition includes a push-fit capsule containing gelatin, and a soft-seal capsule containing gelatin and a plasticizer such as glycerol or sorbitol. In soft capsules, the pharmaceutical composition can be dissolved or suspended in a suitable liquid such as fatty oil, liquid paraffin, or liquid polyethylene glycol. Furthermore, stabilizers may be added.All formulations for oral administration are provided in doses suitable for the selected route of administration.

[0062] For buccal administration, the composition can take the form of tablets or lozenges prepared by conventional methods. Furthermore, toothpaste compositions can also be used for buccal administration.

[0063] Toothpaste is widely available to consumers. In fact, subjects are accustomed to using toothpaste, and many use it at least once a day as part of their oral hygiene routine. Furthermore, toothpaste is also effective in the local delivery of active ingredients to the gingival tissue or salivary glands.

[0064] Examples of toothpastes include pastes, gels, mouthwashes, powders, and toothpaste soaps. In some embodiments of the pharmaceutical composition, the toothpaste is a paste or gel comprising at least one of an abrasive, a surfactant, a humectant, and a thickener. Exemplary abrasives include, but are not limited to, hydrated silica, dicalcium phosphate dihydrate, calcium carbonate, sodium bicarbonate, calcium pyrophosphate, and alumina. Exemplary surfactants include, but are not limited to, sodium lauryl sulfate, sodium N-lauryl sarcosinate, Pluronic acid, and sodium lauryl sulfoacetate. Fluoride exemplifies a caries inhibitor. Exemplary calculus inhibitors include, but are not limited to, tetrasodium pyrophosphate, Gantrez® S-70, sodium tripolyphosphate, and methyl vinyl ether / maleic anhydride copolymer. Toothpaste may further contain one or more of the following: water, pH buffering agents, humectants (to prevent dryness and improve mouthfeel) such as glycerin, sorbitol, polypropylene glycol, xylitol, polyethylene glycol, etc., thickeners such as silica thickeners, sodium aluminum silicate, clay, gums such as sodium carboxymethylcellulose, cellulose ether, xanthan gum, carrageenan, sodium alginate, carbopole, etc., antimicrobial agents, flavoring agents such as water-insoluble essential oils, sweeteners such as saccharin, dextrose, revrose, cyclamate, aspartate, colorants, and binders that provide viscosity and shape.

[0065] When administered orally via mouthwash, the active compound may be combined with water and one or more alcohols (such as ethyl alcohol). The mouthwash may further contain one or more of the surfactants, tartar inhibitors, anticariogenic agents, buffers, humectants, antibacterial agents, fragrances, and colorants described herein.

[0066] In a specific embodiment, the toothpaste may be a powder containing any of the abrasives described herein. This powder may further contain any of the dry ingredients described herein that are suitable for toothpaste. In another specific embodiment, the toothpaste is a toothpaste soap containing one or more oils and water. The oil may be any oil known to be suitable for toothpaste soap, such as olive oil, coconut oil, essential oils, peppermint oil, or any combination thereof.

[0067] The pharmaceutical composition may be chewing gum. This gum may contain an active compound and gums such as butadiene-based synthetic rubber, birch bark tar, chicle, mastic gum, spruce gum, paraffin wax, tur resin, styrene-butadiene rubber, isobutylene-isoprene copolymer, and petroleum wax. The gum may be present in a concentration sufficient to provide the texture necessary to ensure the chewing sensation of the gum, and can be appropriately formulated by those skilled in the art. Flavorings, including those listed above, may also be added.

[0068] The compounds of this disclosure can be used in the form of toothpastes and gums, as well as in the form of oral liquid preparations such as tinctures, solutions, suspensions, elixirs, and syrups, dissolved in diluents such as water, saline, and alcohol. Furthermore, the oral liquid preparations may contain a suitable flavoring suspending or dispersing agent, such as synthetic gums or natural gums such as tragacanth gum, acacia gum, and methylcellulose. In addition, suitable colorants and other auxiliary agents may be incorporated into the mixture as desired or as needed. Other dispersing agents that can be used include glycerin.

[0069] For nasal inhalation administration, the pharmaceutical composition can be delivered in the form of an aerosol spray from a pressurized pack or nebulizer using a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve for quantitative dispensing. Capsules and cartridges, for example made of gelatin, for use in dispensers can be formulated containing a powder mixture of the compound and a suitable powder base such as lactose or starch.

[0070] The pharmaceutical compositions described herein can be formulated for parenteral administration, for example, by bolus injection or continuous infusion. Injectable formulations can be provided in unit dose forms, such as ampoules or multi-dose containers, and may contain preservatives as needed. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Pharmaceutical compositions for parenteral administration may include aqueous solutions of the pharmaceutical composition in water-soluble forms. Furthermore, suspensions of pharmaceutical compositions can be prepared as suitable oily or aqueous injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the pharmaceutical composition, enabling the preparation of concentrated solutions. Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that make the formulation isotonic with the patient's blood, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives.One or more compounds may be administered to a physiologically acceptable diluent such as a sterile liquid or liquid mixture, which may include water, physiological saline, aqueous dextrose and related sugar solutions, alcohols such as ethanol, isopropanol, or hexadecyl alcohol, glycols including propylene glycol or polyethylene glycol such as poly(ethylene glycol) 400, glycerol ketals such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides with or without pharmaceutically acceptable surfactants such as soap, oil or detergent, and pectin, carbomer, methylcellulose, hydroxypropyl methylcellulose, or carboxymethylcellulose, or emulsifiers, and other pharmaceutically adjuvants.

[0071] Oils that can be used in each specific embodiment of a pharmaceutical composition include petroleum, animal oils, vegetable oils, or synthetic oils. Oils can be useful in specific embodiments of a pharmaceutical composition in parenteral formulations, toothpastes, or combinations thereof. Specifically, non-limiting examples of oils include peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum oil, and mineral oil. Suitable fatty acids that can be used in a specific pharmaceutical composition include polyethylene sorbitan fatty acid esters such as sorbitan monooleate, and high molecular weight adducts of ethylene oxide and hydrophobic bases formed by the condensation of propylene oxide with propylene glycol, oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are non-limiting examples of suitable fatty acid esters. Soaps suitable for use in parenteral formulations include salts of aliphatic alkali metals, ammonium, and triethanolamine, and suitable detergents include (a) cationic detergents such as dimethyldialkylammonium halides and alkylpyridinium halides; (b) anionic detergents such as alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates; (c) nonionic detergents such as aliphatic amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers; (d) amphoteric detergents such as alkyl beta-aminopropionates and 2-alkylimidazoline quaternary ammonium salts; and (e) mixtures thereof.

[0072] Appropriate preservatives and buffers may be used in the formulation of certain pharmaceutical compositions. To minimize or completely eliminate irritation at the injection site, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of about 12 to about 17.

[0073] To prepare alcohol solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations, topical administration forms containing active compounds, such as ointments, creams, pastes, and emulsions, can be mixed with one or more carrier substances well known in the art, such as alcohol, aloe vera gel, allantoin, glycerin, vitamin A and E oils, mineral oil, and PPG2 myristylpropionate. Formulations with exfoliants or abrasives can also be used. Such topical administrations can be applied to patches, bandages, or dressings for transdermal delivery, or applied to bandages or dressings for direct delivery to wounds or skin injury sites.

[0074] Alternatively, the pharmaceutical composition may be in powder form for use with a suitable vehicle, such as a sterile, pyrogenic-free aqueous solution.

[0075] The formulations can also be incorporated into rectal compositions such as suppositories and retained enemas, using conventional suppository bases such as cocoa butter or other glycerides.

[0076] The active compounds of this disclosure can also be formulated to be administered in the form of small monolayer vesicles, large monolayer vesicles, and liposome delivery systems such as antiemetics. The liposomes can be formed from various phospholipids such as cholesterol, stearylamines, or phosphatidylcholine. Such liposomes may also contain monoclonal antibodies for direct delivery of the liposomes to specific cell types or groups of cell types.

[0077] The compositions of this disclosure may further contain agents that improve the solubility, half-life, absorption, etc., of the active compound. Furthermore, the compositions of this disclosure may further contain agents that reduce undesirable side effects and / or toxicity of the active compound. Examples of such agents are described in various publications, such as "Remington: The Science and Practice of Pharmacy" (20th edition, Lippincott, Williams & Wilkins, edited by Daniel Limmer).

[0078] Embodiments of specific pharmaceutical compositions include an active compound conjugated with a soluble polymer as a target-directed drug carrier. Such polymers may include polyvinylpyrrolidone, pyran copolymers, polyhydroxypropyl methacryl-amidophenol, polyhydroxyethyl aspartamidophenol, or polyethylene oxide polylysine substituted with palmitoyl residues. Furthermore, the active compound of the present invention can be conjugated with biodegradable polymers useful for controlled drug release, such as polylactic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyran, polycyanoacrylates, and hydrogels consisting of crosslinked or amphiphilic block copolymers.

[0079] (D. Methods of treatment and prevention) This disclosure provides a method for treating or preventing a disease or condition related to or characterized by periodontitis in a subject requiring treatment, the method comprising administering to the subject a therapeutically effective amount of any of the active compounds or pharmaceutical compositions disclosed herein. A specific example of such active compounds is GM.

[0080] Examples of diseases associated with or characterized by periodontitis include periodontitis, arteriosclerosis, human immunodeficiency virus (HIV) infection, tooth loss, coronary artery disease, stroke, premature birth, low birth weight, poorly controlled diabetes, respiratory problems, rheumatoid arthritis, and asthma. In addition, several treatments are associated with xerostomia, which can lead to periodontitis. Such treatments include, but are not limited to, anticholinergics, sympathomimetic agents, topical radiotherapy, and chemotherapy. Anticholinergics that can cause xerostomia include, but are not limited to, atropine and its analogues (antimuscarinic agents), tricyclic antidepressants, serotonin reuptake inhibitors, antihistamines, antiemetics, and antipsychotics. Sympathomimetic agents associated with xerostomia include, but are not limited to, decongestants, bronchodilators, appetite suppressants, and amphetamines. Other therapeutic agents associated with xerostomia include, but are not limited to, lithium, omeprazole, oxybutynin, disopyramide, dideoxyinosine, didanosine, diuretics, and protease inhibitors. In some embodiments of the Method, subjects have previously received or are currently receiving medical treatment associated with xerostomia, including, but not limited to, any of the above-described treatments. Further embodiments of the Method include administering to subjects a combination of treatments associated with xerostomia and at least one of periodontitis, including, but not limited to, any of the treatments described herein.

[0081] A method of treatment or prevention includes administering to a subject an amount of an active compound sufficient to treat or prevent periodontitis or a condition or symptom associated with periodontitis. Furthermore, the method may include identifying subjects in need of such treatment or prevention. The method may also include administering to a subject a therapeutically effective amount of the active compound.

[0082] As described herein, the therapeutically effective dose can vary depending on various factors. Exemplary factors include the subject's condition, weight, sex, and age. For example, in some embodiments of this method, the median lethal dose (LD50) of the active compound is used. 50 This includes administration up to LD. 50 This can be confirmed using standard toxicological methods or by referring to past studies.

[0083] This method may include delivering an active compound at a desired concentration to the gingival tissue of a subject. In some embodiments of this method, the desired concentration is a therapeutically effective dose.

[0084] If, after administration of the active compound, the subject still has or is at risk of having periodontitis or a periodontitis-related condition or symptoms, an optional step of the method may be to continue administration of the active compound or pharmaceutical composition for an extended treatment period. In each embodiment, the extended treatment period is sufficient time to substantially eliminate periodontitis or a periodontitis-related condition. The extended treatment period may be up to approximately 6 months. The extended treatment period may be as short as approximately 1 day. In each embodiment, the extended treatment period is approximately 1 week, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 7 weeks, approximately 8 weeks, approximately 9 weeks, or approximately 10 weeks. The extended treatment period may be less than 1 week. In each embodiment, the extended treatment period is approximately 1 day, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, approximately 7 days, approximately 8 days, approximately 9 days, approximately 10 days, approximately 11 days, approximately 12 days, approximately 13 days, or approximately 14 days.

[0085] In one embodiment, the treatment or prevention method includes administering an active compound or pharmaceutical composition to a subject together with one antibiotic or a combination of two or more antibiotics. In one embodiment, at least one antibiotic and the active compound are provided to the subject as a single pharmaceutical composition. In an alternative embodiment, at least one antibiotic and the active compound are administered as separate pharmaceutical compositions or formulations. In one embodiment, the method includes administering to a subject a pharmaceutical composition containing a therapeutically effective amount of the active compound and a therapeutically effective amount of the antibiotic. In each particular embodiment, the combined administration of the antibiotic and the active compound generally enhances the therapeutic effect of the active compound. The combined administration of a pharmaceutical composition containing the active compound and the antibiotic yields a synergistic effect that enables more effective treatment or prevention of diseases or conditions associated with periodontitis. In some embodiments, when the pharmaceutical composition contains an antibiotic, a lower concentration of the active compound may yield the same therapeutic or preventive effect to the subject as when the pharmaceutical composition contains only the active compound. Therefore, the therapeutically effective amount of the active compound administered to the subject may be inversely proportional to the therapeutically effective amount of the antibiotic administered to the subject.

[0086] In one embodiment, a method for treating or preventing a disease or condition related to or characterized by periodontitis in a subject requiring treatment involves administering an active compound to the subject's gingival tissue. In such embodiments, delivery of the active compound to the subject's gingival tissue prevents intracellular invasion by at least one periodontal pathogen, such as Porphyromonas gingivalis. In various embodiments, targeted delivery of the active compound to the gingival tissue can prevent undesirable effects on other tissues or organs. In one embodiment, the method involves topically administering the active compound into the subject's oral cavity. In specific embodiments, this involves administering the active compound as a toothpaste or gum and topically administering it into the subject's oral cavity. The toothpaste or gum containing the active compound may be useful for treating or preventing periodontitis or a periodontitis-related condition or condition. Such formulations may further include additives (e.g., fluoride) to improve overall oral health. In each embodiment, the method involves administering the toothpaste or gum containing the active compound as part of the subject's established oral hygiene habits. Such treatment methods may be particularly useful for subjects who are already using similar formulations for routine oral hygiene.

[0087] In one embodiment, this method involves administering GM to a subject until Pg is eliminated from the subject's oral cavity.

[0088] The methods of administering the compositions described herein to subjects can vary. In one embodiment, the active compound or pharmaceutical composition is administered orally to the subject. For example, the active compound or pharmaceutical composition may be encapsulated or tableted as a solid oral dosage form. In one embodiment, the active compound or pharmaceutical composition is administered in the form of pills, tablets, capsules, or gel capsules.

[0089] The active compound or pharmaceutical composition may be provided in the form of a liquid, gel, or powder. For example, the active compound or pharmaceutical composition may be in the form of a powder suitable for mixing with water or other liquids. These formulations may be added to beverages or provided as pre-mixed ingredients in beverages. The active compound or pharmaceutical composition may be administered as an elixir or solution formulation. In another embodiment, the active compound or pharmaceutical composition may be administered in the form of a dietary supplement or food supplement. In yet another embodiment, the active compound or pharmaceutical composition may be administered in the form of a functional food, such as a protein bar. Furthermore, the compositions of the present invention may also be administered intravenously.

[0090] Preferred routes of administration include oral, rectal, transmucosal, transnasal, intestinal, or intramuscular, subcutaneous, intrathecal, and parenteral administration such as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection. Alternatively, the preparation may be administered locally rather than systemically.

[0091] The pharmaceutical composition can be manufactured by processes well known in the art, such as conventional mixing processes, dissolution processes, granulation processes, sugar-coated tablet manufacturing processes, liquefaction processes, emulsification processes, encapsulation processes, or freeze-drying processes. Therefore, the pharmaceutical composition used in the disclosed method facilitates the processing of the pharmaceutical composition into a pharmaceutically usable formulation, which can be formulated conventionally using one or more physiologically acceptable carriers, including excipients and adjuvants. The formulation method may depend on the intended route of administration of the pharmaceutical composition.

[0092] For example, when an active compound is injected, the pharmaceutical composition can be formulated by dissolving it in an aqueous solution, preferably a physiologically compatible buffer. Examples of physiologically compatible buffers include, but are not limited to, Hanks' solution, Ringer's solution, or saline buffer. For transmucosal administration, a specific penetrating agent suitable for transporting the pharmaceutical composition through a permeable barrier is used in the formulation. Such penetrating agents are generally known in the art.

[0093] For oral administration, compounds can be readily formulated by combining the pharmaceutical composition with a pharmaceutically acceptable carrier. Such carriers enable the formulation of compounds as tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral intake by subjects or patients. Pharmacological preparations for oral use can be manufactured using solid excipients, such as processing a granular mixture to obtain a tablet or sugar-coated tablet core, and optionally, after optionally grinding the resulting mixture and adding appropriate adjuvants as needed. Suitable excipients may include fillers. Exemplary, non-limiting fillers include, but are not limited to, sugars (including lactose, sucrose, mannitol, or sorbitol) and cellulose formulations containing physiologically acceptable polymers such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carbomethylcellulose, and polyvinylpyrrolidone (PVP), or combinations thereof. In each specific embodiment, a disintegrant containing cross-linked polyvinylpyrrolidone, agar, or a salt thereof such as alginic acid or sodium alginate may be added.

[0094] The core of a sugar-coated tablet can have a suitable coating. For this purpose, a concentrated sugar solution may be used to which, optionally, gum arabic, talc, polyvinylpyrrolidone, carbol gel, polyethylene glycol, titanium dioxide, lacquer solution, a suitable organic solvent or solvent mixture, or any combination thereof may be added. The coating of the tablet or sugar-coated tablet may have dyes or pigments added for identification or to characterize different dosage combinations of the pharmaceutical composition.

[0095] (E. Methods to prevent invasion into host cells) Here, methods are also provided for reducing the likelihood of invasion of host cells by invasive pathogens. In each embodiment, the method involves contacting host cells with one of the above active compounds at an effective concentration. Contact can be performed in vitro or in vivo. In various embodiments, the methods disclosed herein can be applied to prevent intracellular invasion by bacteria, fungi, viruses, protists, or combinations thereof. Non-limiting examples of bacteria include Porphyromonas gingivalis, Bartonella hensele, Tularemia bacillus, Listeria monocytogenes, Salmonella typhi, Brucella species, Legionella species, Mycobacterium species, Nocardia species, Rhodococcus equii, Yersinia species, Neisseria meningitidis, Chlamydia species, Rickettsia species, and Coxiella species. Examples of intracellular parasitic fungi include, but are not limited to, Histoplasma capsulatum, Cryptococcus neoformans, and Pneumocystis irovecii. Examples of protozoa include apicomplexes (e.g., Babesia, Plasmodium, Cryptosporidium parvum, Cyclospora kaietanensis, Isosporabelli, and Toxoplasma gondii) and trypanosomes. In some embodiments of this method, intracellular bacteria are oral pathogens, regardless of their taxonomic group.

[0096] The effective concentration is any concentration at which the active compound exerts an effect of inhibiting cellular invasion by an invasive pathogen. In one embodiment, the effective concentration is the therapeutically effective amount of the active compound. The effective concentration may be the amount of the active compound that achieves a desired concentration in the subject's gingival tissue. In some embodiments, the effective concentration of the active compound is sufficient to achieve an active compound concentration of at least about 1 nM in the subject's gingival tissue. In some embodiments, the effective concentration of the active compound is sufficient to achieve a concentration of up to about 10 μM in the subject's gingival tissue. In one embodiment, the effective concentration of the active compound is sufficient to achieve an active compound concentration of at least about 20 nM in the subject's gingival tissue. The effective concentration of the active compound can be sufficient to achieve an active compound concentration between about 5 nM and 1 μM in the subject's gingival tissue. In each specific embodiment, the effective concentration of the active compound is sufficient to achieve at least one of the following concentrations in the subject's gingival tissue: about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, and about 100 nM. In a particular embodiment, the effective concentration of GM is sufficient to achieve a concentration of at least about 5 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, or at least about 40 nM in the gingival tissue.

[0097] (F. Screening Methods) A method is provided for screening candidate factors that have activity in the treatment or prevention of periodontitis or a disease or symptom related to periodontitis, the method comprising exposing multiple host cells to the candidate factor in the presence of intracellular bacteria and measuring the infection rate of the multiple host cells by the intracellular bacteria. The bacteria and host cells may be any of those disclosed herein.

[0098] Candidate factors can be any type of factor, including physical, chemical, genetic, biochemical, or ecological factors. Examples of physical factors include temperature, oxidation potential, pressure, and radiation. Examples of chemical factors include osmotic potential, inorganic compound concentration, organic compound concentration, pH, and salinity. Examples of biochemical factors include nutrient concentration, prion, and toxin concentration. Examples of ecological factors include population density, intrinsic growth rate, environmental carrying capacity, predation, parasitism, and growth rate. Examples of genetic factors include genotype, mutation, episome, transposable elements, viruses, and viroids. However, since the purpose of this assay is to identify previously unknown therapeutic or preventive factor candidates, it is important to emphasize that any factor to which cells may be exposed can be a candidate factor.

[0099] The method for measuring the infection rate can be any method known in the art. In a specific embodiment of this method, the infection rate is measured by staining a mixture of host cells and bacteria and observing the stained mixture under a microscope. Fluorescent dyes can be used to enhance the signal when observed with an epifluorescence microscope. The use of fluorescent dyes also makes it possible to measure the number of intracellular bacteria using fluorescence assays (for example, after separating host cells from free bacteria). In a specific embodiment, the infection rate is measured by permeabilizing host cells, immunostaining the sample with a fluorescent probe, obtaining a confocal microscope image of the immunostained sample, counting the total number of host cells by direct counting, and counting the number of host cells containing intracellular bacteria by direct counting. Such direct counting methods may include counting a certain number of cells in the microscope field of view and extrapolating the result to the entire sample. However, any method can be employed for direct counting.

[0100] Some embodiments of this method are intended to specifically screen for factors that reduce the rate of pathogen entry into host cells. In such embodiments, the factors may be non-antibiotic factors (i.e., factors that are not necessarily toxic to bacteria). Also, such embodiments of this method include counting the total number of bacteria after exposure to the factors in order to distinguish between factors that have antibiotic activity and factors that have anti-invasion activity.

[0101] In some embodiments of this method, the host cells are gingival tissue cells. Gingival tissue cells may be oral epithelial cells or gingival fibroblasts. Exemplarily, gingival tissue cells include those derived from various mammalian species, including humans and livestock. In more specific embodiments of this method, the host cells are oral keratinocytes, such as oral keratinocytes derived from humans or livestock. Furthermore, although each example related to Pg inhibition is provided here, those skilled in the art will understand that Pg can be used as a model for the development of therapeutic agents against other invasive pathogens.

[0102] Known statistical methods can be used to detect a decrease in the infection rate of samples exposed to the factor compared to a benchmark value. The benchmark value can be established using parallel-controlled studies or obtained from literature describing past research results. However, such statistical methods are not always necessary if the effect of the factor is clear.

[0103] (G. Examples) (1. Example 1: Activity of gnidimacrin (GM), its derivatives and analogs against bacteria in relation to human cell invasion) The microbiome plays a crucial role in human health, participating in digestion and immune responses within our bodies, as well as being linked to infectious diseases and dysbiotic disorders. 1,2Microbiota modification, including the removal of keystone pathogens, has been considered a therapeutic strategy for the prevention and treatment of microbiome-related diseases. Porphyromonas gingivalis (Pg) is considered a keystone pathogen of periodontitis, as it transforms commensal microorganisms into pathogenic ones, inducing a breakdown of periodontal tissue and microbial homeostasis, leading to uncontrolled inflammation and tissue destruction. Therefore, inhibition and removal of Pg is an attractive strategy for the prevention and treatment of chronic periodontitis. However, despite considerable effort, the development of therapeutic agents that selectively target Pg, particularly intracellular Pg, has been limited. The difficulty in removing Pg from the oral cavity stems from Pg's ability to invade host cells and the drug resistance of plaque, which is its ability to evade host defense systems and antibiotic infection. Pg proliferates within the host cell and extracellular matrix, resulting in the formation of a reservoir of persistent infection. Therefore, identifying inhibitors that target intracellular Pg would be a rational approach to halting the progression of periodontitis.

[0104] Bacterial invasion is the process by which bacteria actively invade non-phagocytic cells, such as epithelial cells. Generally, invading bacteria express surface invasins that interact with corresponding receptors on the surface of non-phagocytic cells. This triggers a series of signaling events in the host cell, including protein phosphorylation, cytoskeletal rearrangement, membrane elongation, and bacterial phagocytosis. After internalization, bacteria can not only survive and proliferate within the cytoplasm but also spread to other host cells. The ability to invade host cells helps bacteria evade immune responses and antibiotic treatment, thus enabling successful infection. Intracellular bacteria are also known to contribute to the persistence of chronic infections by acting as opportunity bacteria. 3 While the mechanisms by which bacteria invade host cells are well understood, research on inhibiting bacterial invasion is limited.

[0105] One example of bacterial invasion is Porphyromonas gingivalis (Pg), the keystone pathogen of chronic periodontitis. This disease is the sixth most common infectious disease in the world. 4 It is estimated that 5-20% of the population suffers from chronic periodontitis.5,6 P. gingivalis has diverse effector molecules and systems that enable its survival in the oral cavity. 7-10 One of the important characteristics of P. gingivalis is its ability to invade and survive in host cells such as fibroblasts, epithelial cells, and endothelial cells. 11 Several membrane proteins involved in the attachment and invasion of P. gingivalis to host cells have been identified, such as FimA, hemagglutinin A, and heat stress protein. 12-15 A P. gingivalis strain lacking invasive ability had a reduced invasive activity by about one-tenth 16 and could not induce alveolar bone loss in notobiotic rats observed with wild-type P. gingivalis strains. 17 Obviously, the invasive ability is important for the establishment of P. gingivalis infection in the oral cavity, and the inhibition of P. gingivalis invasion may be a new treatment strategy to prevent the formation of P. gingivalis reservoirs. Furthermore, P. gingivalis can be used as a model of invasive bacterial pathogens and has become a paradigm for the development of therapeutic agents against other invasive bacterial pathogens. The role of bacterial invasion in the pathogenicity of P. gingivalis has been studied by microbiologists, but research on bacterial invasion inhibitors is limited.

[0106] As disclosed herein, the natural compound guniperin (GM) effectively inhibits the invasion of P. gingivalis and its vesicles into primary cultured human oral keratinocytes (HOK) at a low nanomolar concentration (20 nM). GM also does not affect the survival rate of host cells. These findings suggest that GM is an ideal candidate for anti-P. gingivalis drug development due to its potent inhibitory activity and low cytotoxicity.

[0107] Regarding the treatment of chronic periodontitis, conventional periodontitis treatments including scaling and root planing are still the gold standard for chronic periodontitis. However, as shown in past studies, high concentrations of P. gingivalis have been observed in the periodontal tissues of patients with refractory periodontitis 18,19 The conventional treatment methods cannot prevent the reappearance of pathogenic microbiota. Systemic or local administration of antibiotics may be used adjunctively for severe and refractory periodontitis symptoms 20-22However, the recommended therapeutic dose is insufficient to kill intracellular Pg that can proliferate within cells and potentially spread to neighboring cells. 23 Furthermore, the use of antibiotics disrupts the symbiotic relationship between the host and the normal bacterial flora. 24 Public health concerns are growing, including the emergence of antibiotic resistance in oral bacteria. Furthermore, these treatment strategies often provide only temporary relief, and the pathogens are prone to re-emergence. 27 Recurrences, which are thought to be caused by the same factors, are also common. 25,26 Therefore, novel therapeutic agents that can specifically remove intracellular Pg are attractive, and the combination of Pg entry inhibitors and antibiotic treatment is thought to increase the likelihood of removing Pg from the oral cavity. Accordingly, this study focuses on elucidating the mechanism of Pg entry inhibition by GM and developing more practical and efficient candidate drugs for periodontal disease treatment.

[0108] Regarding the scientific premise, this case study is based on evidence that current treatments for periodontitis are only partially effective, requiring lifelong periodontal treatment and maintenance, which is also costly. Intracellular PG survives under mechanical therapy of periodontal tissue and is resistant to antibiotic therapy. By selectively targeting keystone pathogens of periodontal disease such as PG, an effective adjunctive therapy can be achieved to completely eliminate PG from host cells. Equally important is the possibility that the identification of anti-invasive compounds could open new avenues for the prevention and treatment of infections caused by other invasive bacteria.

[0109] As disclosed herein, GM exhibits a potential therapeutic mechanism for efficiently removing intracellular Pg. Conventional treatments for periodontitis remain the gold standard. However, they fail to prevent the re-emergence of pathogenic microbial communities, as evidenced by the presence of high levels of Pg in the periodontal tissues of patients with refractory periodontitis. 18,19Currently, ARESTIN® (minocycline hydrochloride) is used clinically as an adjunct treatment to ensure the removal of periodontal pocket bacteria. However, concerns regarding such treatments include the potential to induce antibiotic resistance, nonspecific action, and the inability to eliminate intracellular bacteria. Ultimately, conventional antibiotic treatments can lead to overgrowth of periodontitis-related bacteria after killing the majority of oral bacteria. The ability of Pg to invade and colonize poses a challenge to conventional periodontitis treatments. GM could be an ideal anti-Pg agent, effectively inhibiting Pg invasion while possessing low toxicity and high water solubility. One of the advantages of GM disclosed herein is its potential use in the development of agents that remove intracellular Pg by inhibiting bacterial invasion and re-invasion of host cells.

[0110] (Preliminary results) Gunidimacrin (GM) is a diterpene compound that can be isolated from Stellera chamaejasme.

[0111] Regarding the inhibition of Pg cell invasion by GM, the ability of Pg to invade oral epithelial cells was measured using the described antibiotic defense assay. 28 Human primary oral keratinocyte (HOK, 5×10 4Cells (ScienCell® Research Laboratories) were seeded in 24-well plates. After culturing with Pg 33277 for 1 hour, HOK was cultured for a further 4 hours in the presence of antibiotics (gentamicin (300 μg / ml) and metronidazole (200 μg / ml)) to remove extracellular bacteria. Next, HOK was lysed, and the migrated bacteria were spread onto TSB blood agar. The medium was cultured anaerobically at 37°C for 7 days, and the CFU of Pg was measured. As shown in Figure 2A, GM significantly reduced the entry of Pg cells into HOK in a dose-dependent manner. In the presence of 40 nM GM, more than 95% of Pg were inhibited from entering HOK. Furthermore, the role of GM in Pg re-entry was tested. After exposing Pg cells to GM for 1 hour and then treating with antibiotics for 4 hours, HOK was cultured continuously for 24 hours in the presence of GM (20 nM), antibiotics, or both GM and antibiotics. Intracellular Pg was measured as described. 28 As expected, intracellular Pg cells entered and exited the HOK. Once outside the HOK, Pg re-entry was inhibited by GM, and extracellular Pg cells were killed by antibiotics (see Figure 2B). As can be seen, the combination of GM and antibiotics showed a synergistic effect, removing approximately 90% of the intracellular Pg.

[0112] Regarding the inhibitory activity and efficiency of GM in invading Pg vesicles, it has been previously reported that Pg vesicles can efficiently invade oral epithelial cells. 29 Recently, the role of GM in Pg vesicle entry was investigated using confocal microscopy. GM inhibited Pg vesicle entry into HOK in a dose-dependent manner (see Figure 3). GM almost completely inhibited vesicle entry at 20 nM. Previously, two compounds, Alop1 and dynasore, were also reported to inhibit vesicle entry at 30 μM. 30 These results indicate that GM is a more potent and efficient Pg vesicle entry inhibitor than previously studied compounds.

[0113] The effects of GM on bacterial growth and host cell viability were investigated, specifically the role of GM in the growth of Pg 33277 in Trypticase soy broth (TSB). Bacterial growth in TSB was measured by optical density at 600 nm. As shown in Figure 4, when GM was added up to 200 μM, the growth rate of Pg was not significantly affected compared to when GM was not added.

[0114] Regarding the selective activation of (protein kinase C-β)PKC-β by GM, it has been previously shown that GM is a PKC-β activator with potent bifacial activity against HIV-1 replication. 31,32 In short, to determine whether GM is a selective PKC agonist, anti-CD3 / CD28 (1 ug / ml) activated human primary CD4 cells were cultured for 12 hours in or without GM (1 nM). The cytoplasmic fraction of the cells was analyzed by 10% SDS-PAGE, followed by Western blotting using a monoclonal antibody panel against PKC isozymes (Santa Cruz Biotech). PKC activation is expected to reduce PKC levels due to the translocation of the enzyme from the cytoplasm to the membrane. As shown in Figure 5, GM significantly reduced cytoplasmic PKC-β1 and β2 levels without affecting the other six PKC isozymes. This result suggests that GM is a selective PKC-β agonist. Without being bound by any theory, it is expected that PKC-β selective activity will also be observed in HOK.

[0115] To investigate the potential cytotoxic effects of GM, HOK cells were cultured for 48 hours in the presence of GM at concentrations of 0-1,000 nM, and the number of viable cells in the culture medium was measured using Promega® CellTiter-Glo®. As shown in Figure 6, even when HOK cells were exposed to a high concentration of GM (1 μM, approximately 50 times the concentration that can inhibit Pg entry) for 48 hours, there was no effect on cell viability compared to cells not exposed to GM.

[0116] Regarding the effects of GM on the cytoskeleton of oral epithelial cells, cytoskeletal dynamics are involved in bacterial invasion. To investigate whether GM affects the cytoskeletal structure of epithelial cells, HOK cells were treated with GM, immunostained with monoantibodies for α-tubulin or actin, and observed under a confocal microscope. Thirty minutes after GM treatment, microtubule rearrangement and formation of microtubule shells directly beneath the cell membrane were observed in HOK cells (see Figure 7). However, no significant actin remodeling was observed after treatment (data not shown).

[0117] In summary, 1. Gunidimacrin and its derivatives and analogs inhibit the entry of Porphyromonas gingivalis into human cells. While not bound by any theory, gunidimacrin may also inhibit the entry of other oral bacteria into human cells, including but not limited to one or more of Treponema denticola, Fusobacterium nucleatum, and Tannerella forsythia. 2. Gunidimaclin can be used alone or in combination with antibiotics to remove Porphyromonas gingivalis from the oral cavity. 3. Gnidimaclin could be a future treatment for periodontitis and potentially open up new avenues for the prevention and treatment of infections caused by invasive bacteria.

[0118] (References in this embodiment) 1. Libertucci, J. & Young, VB, “The role of the microbiota in infectious diseases”, Nature microbiology 4, 35-45 (2019). 2.Hajishengallis,G.&Lamont,R.J.Polymicrobial,「communities in periodontal disease:Their quasi-organismal nature and dialogue with the host」,Periodontology 2000(2021). 3.Cornejo,E.,Schlaermann,P.&Mukherjee,S.,「How to rewire the host cell:A home improvement guide for intracellular bacteria」,The Journal of cell biology 216,3931-3948(2017). 4.Kassebaum,N.J.,et al.,「Global burden of severe periodontitis in 1990-2010:a systematic review and meta-regression」,Journal of dental research 93,1045-1053(2014). 5.Burt,B.,「Research,S.&Therapy Committee of the American Academy of,P.Position paper:epidemiology of periodontal diseases」,Journal of periodontology 76,1406-1419(2005). 6.Petersen,P.E &Ogawa,H.「The global burden of periodontal disease:towards integration with chronic disease prevention and control」.Periodontology 2000 60,15-39(2012). 7.Hajishengallis,G.,McIntosh,M.L.,Nishiyama,S.I.&Yoshimura,「F.Mechanism and implications of CXCR4-mediated integrin activation by Porphyromonas gingivalis」,Molecular oral microbiology 28,239-249(2013). 8.Lamont,R.J.&Jenkinson,「H.F. Life below the gum line: pathogenic mechanisms of Porphyromonas gingivalis」,Microbiology and molecular biology reviews:MMBR 62,1244-1263(1998). 9.Lamont,R.J.&Jenkinson,「H.F. Subgingival colonization by Porphyromonas gingivalis」,Oral microbiology and immunology 15,341-349(2000). 10.Xie,H.,「Biogenesis and function of Porphyromonas gingivalis outer membrane vesicles.」,Future microbiology 10,1517-1527(2015). 11.Tribble,G.D.&Lamont,R.J.,「Bacterial invasion of epithelial cells and spreading in periodontal tissue」.,Periodontology 2000 52,68-83(2010). 12.Belanger,M.,Kozarov,E.,Song,H.,Whitlock,J.&Progulske-Fox,「A. Both the unique and repeat regions of the Porphyromonas gingivalis hemagglutin A are involved in adhesion and invasion of host cells」.,Anaerobe 18,128-134(2012). 13.Zhang,W.,Ju,J.,Rigney,T.&Tribble,G.D.「Fimbriae of Porphyromonas gingivalis are important for initial invasion of osteoblasts, but not for inhibition of their differentiation and mineralization」.,Journal of periodontology 82,909-916(2011). 14.Yilmaz,O.,Watanabe,K.&Lamont,R.J.「Involvement of integrins in fimbriae-mediated binding and invasion by Porphyromonas gingivalis」.,Cellular microbiology 4,305-314(2002). 15. Yuan,L.,Rodrigues,P.H.,Belanger,M.,Dunn,W.A.,Jr.&Progulske-Fox,A.,「Porphyromonas gingivalis htrA is involved in cellular invasion and in vivo survival」.,Microbiology 154,1161-1169(2008). 16.Mantri,C.K.,Chen,C.,Dong,X.,Goodwin,J.S.&Xie,H.,「Porphyromonas gingivalis-mediated Epithelial Cell Entry of HIV-1」.,Journal of dental research 93,794-800(2014). 17.Malek,R.,etal.,「Inactivation of the Porphyromonas gingivalis fimA gene blocks periodontal damage in gnotobiotic rats」.,Journal of bacteriology 176,1052-1059(1994). 18.Colombo,A.P.,et al.,「Impact of periodontal therapy on the subgingival microbiota of severe periodontitis:comparison between good responders and individuals with refractory periodontitis using the human oral microbe identification microarray」.,Journal of periodontology 83,1279-1287(2012). 19.Colombo,A.P.,et al.,「Comparisons of subgingival microbial profiles of refractory periodontitis, severe periodontitis,and periodontal health using the human oral microbe identification microarray」.,Journal of periodontology 80,1421-1432(2009). 20.Walters,J.&Lai,P.C.,「Should Antibiotics Be Prescribed to Treat Chronic Periodontitis?」,Dental clinics of North America 59,919-933(2015). 21.Santos,R.S.,etal.,「The use of systemic antibiotics in the treatment of refractory periodontitis:A systematic review」.,Journal of the American Dental Association 147,577-585(2016). 22.Jepsen,K.&Jepsen,S.,「Antibiotics / antimicrobials:systemic and local administration in the therapy of mild to moderately advanced periodontitis」.,Periodontology 2000 71,82-112(2016). 23. Yilmaz,O.,Verbeke,P.,Lamont,R.J.&Ojcius,D.M.,「Intercellular spreading of Porphyromonas gingivalis infection in primary gingival epithelial cells」.,Infection and immunity 74,703-710(2006). 24.Blaser,M.J.&Falkow,S.,「What are the consequences of the disappearing human microbiota?」,Nature reviews. Microbiology 7,887-894(2009). 25.Fisher,S.,et al.,「Progression of periodontal disease in a maintenance population of smokers and non-smokers:a 3-year longitudinal study」.,Journal of periodontology 79,461-468(2008). 26.Claffey,N.,Polyzois,I.&Ziaka,P.,「An overview of nonsurgical and surgical therapy」.,Periodontology 2000 36,35-44(2004). 27.Uzel,N.G.,et al.,「Microbial shifts during dental biofilm re-development in the absence of oral hygiene in periodontal health and disease」.,Journal of clinical periodontology 38,612-620(2011). 28.Ho,M.H.,Lamont,R.J.&Xie,H.,「A novel peptidic inhibitor derived from Streptococcus cristatus ArcA attenuates virulence potential of Porphyromonas gingivalis」.,Scientific reports 7,16217(2017). 29.Mantri,C.K.,et al.,「Fimbriae-mediated outer membrane vesicle production and invasion of Porphyromonas gingivalis」.,MicrobiologyOpen 4,53-65(2015). 30. Ho, MH, etal., “Two Small Molecules Block Oral Epithelial Cell Invasion by Porphyromons gingivalis”., PloS one 11, e0149618 (2016). 31. Huang, L., et al., “Picomolar dichotomous activity of gnidimacrin against HIV-1”., PloS one 6, e26677 (2011). 32. Lai, W., et al., “Gnidimacrin, a Potent Anti-HIV Diterpene, Can Eliminate Latent HIV-1 Ex Vivo by Activation of Protein Kinase C beta”., Journal of medicinal chemistry 58, 8638-8646 (2015).

[0119] (H. Conclusion) It should be understood that any element of the disclosed embodiments may be embodied by a single structure, a single step, a single substance, etc. Similarly, any element of the disclosed embodiments may be embodied by multiple structures, steps, substances, etc.

[0120] The above descriptions illustrate and illustrate the processes, machinery, products, composition of materials, and other teachings of this disclosure. Furthermore, while this disclosure illustrates and illustrates only specific embodiments of the disclosed processes, machinery, products, composition of materials, and other teachings, it should be understood that, as stated above, the teachings of this disclosure are usable in a variety of other combinations, modifications, and environments, and can be modified or altered within the scope of the teachings presented herein according to the skills and / or knowledge of a person with ordinary skill and / or knowledge in the relevant art. The embodiments described above are intended to illustrate the best known forms for carrying out the processes, machinery, products, compositions, and other teachings of this disclosure, and further intended to enable a person skilled in the art to utilize the teachings of this disclosure in such embodiments or other embodiments with various modifications necessary for a particular use or application. Accordingly, the processes, machinery, products, compositions, and other teachings of this disclosure are not limited to the embodiments and examples disclosed herein. Section headings in this specification are provided solely for consistency with the implications of 37 CFR §1.77 or to indicate an organizational order. These headings are not intended to limit or characterize the inventions described herein.

Claims

1. A pharmaceutical composition for inhibiting the entry of a pathogen into host cells, comprising a therapeutically effective amount of a protein kinase C-beta (PKC-β) agonist and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is formulated for topical delivery to the gingival tissue of a subject.

2. The pharmaceutical composition according to claim 1, wherein the PKC-β agonist comprises gunidimacrine or a pharmaceutically acceptable derivative thereof.

3. The pharmaceutical composition according to claim 2, wherein the therapeutically effective amount of the PKC-β agonist is sufficient to achieve a sub-nanomolecular concentration of gunidimacrine in the gingival tissue of the subject.

4. The pharmaceutical composition according to claim 2, wherein the therapeutically effective amount of the PKC-β agonist is sufficient to achieve a concentration of gnidimacrine up to approximately 1000 nM in the gingival tissue of the subject.

5. The pharmaceutical composition according to claim 2, wherein the therapeutically effective amount of the PKC-β agonist is sufficient to achieve a concentration of gnidimacrine up to approximately 100 nM in the gingival tissue of the subject.

6. The pharmaceutical composition according to claim 2, wherein the therapeutically effective amount of the PKC-β agonist is sufficient to achieve a concentration of approximately 20 nM of gunidimacrine in the gingival tissue of the subject.

7. The pharmaceutical composition according to claim 1, wherein the subject has been diagnosed with periodontitis, a periodontitis-related disease, or a periodontitis-related symptom.

8. The pharmaceutical composition according to claim 1, wherein the pathogen includes bacteria.

9. The pharmaceutical composition according to claim 8, wherein the bacterium is Porphyromonas gingivalis.

10. The pharmaceutical composition according to claim 1, further comprising a therapeutically effective amount of antibiotic.

11. The pharmaceutical composition according to claim 10, wherein the antibiotic comprises gentamicin, metronidazole, or a combination thereof.

12. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is formulated into a toothpaste.

13. The pharmaceutical composition according to claim 12, wherein the toothpaste comprises a paste, gel, mouthwash, powder, tooth soap, or a combination thereof.

14. A method for treating or preventing bacterial invasion in non-phagocytic cells of a subject, comprising administering a therapeutically effective amount of a compound containing a PKC-β agonist to the subject.

15. The method according to claim 14, wherein the non-phagocytic cells include epithelial cells, endothelial cells, fibroblasts, smooth muscle cells, or a combination thereof.

16. The method according to claim 15, wherein the epithelial cells include oral epithelial cells.

17. The method according to claim 16, wherein the oral epithelial cells include human oral keratinocytes (HOK).

18. The method according to claim 15, wherein the endothelial cells include aortic endothelial cells, cardiac endothelial cells, or a combination thereof.

19. The method according to claim 15, wherein the fibroblasts include gingival fibroblasts.

20. The method according to claim 14, wherein the subject is suffering from a Porphyromonas gingivalis infection.

21. The method according to claim 14, wherein the subject is at risk of Porphyromonas gingivalis infection.

22. The method according to claim 14, wherein the PKC-β agonist comprises gunidimacrine or a pharmaceutically acceptable derivative thereof.

23. The method according to claim 22, wherein the therapeutically effective amount of the PKC-β agonist is sufficient to achieve a sub-nanomolecular concentration of gunidimacrin in the gingival tissue of the subject.

24. The method according to claim 22, wherein the therapeutically effective amount of the PKC-β agonist is sufficient to achieve a concentration of gnidimacrine up to approximately 1000 nM in the gingival tissue of the subject.

25. The method according to claim 22, wherein the therapeutically effective amount of the PKC-β agonist is sufficient to achieve a concentration of approximately 20 nM of gunidimacrine in the gingival tissue of the subject.

26. The method according to claim 14, further comprising administering a therapeutically effective dose of antibiotic.

27. The method according to claim 26, wherein the antibiotic comprises gentamicin, metronidazole, or a combination thereof.

28. A method for treating or preventing periodontitis, periodontitis-related disease, or periodontitis-related symptoms in the gingival tissue of a subject in need, comprising topically administering a therapeutically effective amount of a compound containing a PKC-β agonist to the gingival tissue of the subject.

29. The method according to claim 28, wherein the periodontitis, periodontitis-related disease, or periodontitis-related symptoms are bacterially transmitted.

30. The method according to claim 29, wherein the periodontitis, periodontitis-related disease, or periodontitis-related symptoms are transmitted by Porphyromonas gingivalis.

31. The method according to claim 28, wherein the compound is administered to a toothpaste.

32. The method according to claim 31, wherein the toothpaste comprises a paste, gel, mouthwash, powder, tooth soap, or a combination thereof.

33. The method according to claim 32, wherein the compound comprises gunidimacrine or a pharmaceutically acceptable derivative thereof.

34. The method according to claim 33, wherein the gunidimacrine or a pharmaceutically acceptable derivative thereof is administered to a compound containing at least 20 nM of gunidimacrine or a pharmaceutically acceptable derivative thereof.

35. The method according to claim 28, wherein the therapeutically effective amount of the compound is administered together with a pharmaceutically acceptable carrier.

36. The aforementioned compound comprises gunidimacrin, The therapeutically effective dose is sufficient to achieve a concentration of gunidimacrine up to approximately 1000 nM in the subject's tissue. The method according to claim 28.

37. The aforementioned compound comprises gunidimacrin, The therapeutically effective dose is sufficient to achieve a concentration of gunidimaclin up to approximately 100 nM in the subject's tissue. The method according to claim 28.

38. The aforementioned compound comprises gunidimacrin, The therapeutically effective dose is sufficient to achieve a concentration of approximately 20 nM of gunidimacrine in the subject's tissue. The method according to claim 28.

39. The method according to claim 28, further comprising administering a therapeutically effective amount of antibiotic.

40. A method for reducing the possibility of intracellular bacteria invading host cells, comprising contacting the host cells with a therapeutically effective amount of PKC-β agonist.

41. The method according to claim 40, further comprising contacting the host cells with a therapeutically effective amount of antibiotic.

42. The method according to claim 40, wherein the PKC-β agonist comprises gunidimacrine or a pharmaceutically acceptable derivative thereof.

43. The method according to claim 42, wherein the therapeutically effective amount of the PKC-β agonist is about 20 nM of gunidimacrine or a pharmaceutically acceptable derivative thereof.

44. The method according to claim 40, wherein the bacterium is Porphyromonas gingivalis.

45. The method according to claim 40, wherein the host cells include epithelial cells, endothelial cells, fibroblasts, smooth muscle cells, or a combination thereof.

46. The method according to claim 45, wherein the epithelial cells include oral epithelial cells.

47. The method according to claim 46, wherein the oral epithelial cells include human oral keratinocytes (HOK).

48. The method according to claim 45, wherein the endothelial cells include aortic endothelial cells, cardiac endothelial cells, or a combination thereof.

49. The method according to claim 45, wherein the fibroblasts include gingival fibroblasts.

50. A pharmaceutical composition for treating or preventing periodontitis, periodontitis-related diseases, or periodontitis-related symptoms in a subject, A therapeutically effective amount of gunidimacrine or a pharmaceutically effective derivative thereof, A therapeutically effective dose of antibiotics, Pharmacologically acceptable carriers, Includes, The pharmaceutical composition is formulated for topical administration to the gingival tissue of the subject.

51. The pharmaceutical composition according to claim 50, wherein the therapeutically effective amount of gunidimacrine or its pharmaceutically effective derivative is sufficient to achieve a concentration of gunidimacrine in the gingival tissue of the subject ranging from about 10 nM to about 100 nM.

52. The pharmaceutical composition according to claim 50, wherein the antibiotic comprises gentamicin, metronidazole, or a combination thereof.

53. The aforementioned gunidimacrin or its pharmaceutical derivatives are formulated into toothpaste, The toothpaste includes a paste, gel, mouthwash, powder, tooth soap, or a combination thereof. The pharmaceutical composition according to claim 50.

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