Hygromycin a for treatment of oral diseases and infections

Abstract

The present disclosure provides therapeutic agents e.g., hygromycin A for use in the treatment, prevention, or management of diseases. The diseases can be caused by an infection of a bacterium or a protozoan. The therapeutic agents of the disclosure can be used to inhibit the growth of the microbes or treat diseases described herein.

Description

Attorney Docket Number: 438919.001901 HYGROMYCIN A FOR TREATMENT OF ORAL DISEASES AND INFECTIONS STATEMENT REGARDING GOVERNMENT FUNDING

[0001] This invention was made with government support under grant R01DE030943 awarded bythe National Institute of Dental and Craniofacial Research / National Institutes of Health and RF1AG062496 awarded by the National institute of Health. The government has certain rights in the invention. PRIORITY

[0002] The present application claims the benefit of U.S. Provisional Application No. 63 / 592,394,entitled “HYGROMYCIN A FOR TREATMENT OF DISEASES AND INFECTIONS,” filed on October 23, 2023, and U.S. Provisional Application No.63 / 592,946 entitled “HYGROMYCIN A FOR TREATMENT OF BONE LOSS” filed on October 25, 2023, each of which applications are herein incorporated by reference in their entirety for all purposes. FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to hygromycin A compositions and methods of using thecompositions. In particular, methods and compositions related to the use of Hygromycin A for targeting diseases or disorders associated with an infection are provided. BACKGROUND

[0004] Treatment with broad-spectrum compounds comes at a considerable cost, disrupting the gutmicrobiome and selecting for resistance in off-target bacteria. The microbiome shapes the immune system during development and contributes to maintaining a healthy GI tract and preventing cardiovascular, mental health and autoimmune diseases. Accordingly, there is a need for new therapies which treat disease, without significantly disrupting the microbiomes. SUMMARY

[0005] The present disclosure provides a method of oral or periodontal disease by administeringhygromycin A. In some embodiments, the method includes applying a composition comprising hygromycin A to a surface of the subject’s mouth, lips and / or oral cavity. In some embodiments, the surface can be teeth, gums, tongue, palate, floor, roof, throat, pharynx, tonsils, cheeks, lips (interior and exterior), and epiglottis. In some embodiments, the composition reduces the number of one or more bacteria in the subject’s oral cavity. In some embodiments, the bacteria can be Treponema denticola, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Prevotella loescheii, , Porphyromonas endodontalis, Peptococcus anaerobius, Micros prevotii, Eubasterium limosum, Centipedia perio Centipedia periodontii, Selenomonas arterimidis, Fusobacterium periodonticum, Eubacterium spp., Bacteroides spp., Actinomyces viscosos, Streptococcus mutans, and / or Streptococcus sobrinus. In some embodiments, the composition is formulated as a mouthwash,Attorney Docket Number: 438919.001901 a toothpaste, a mouth and / or throat spray, a lozenge, a breath mint, a pen or tube with a brush, sponge, or nozzle applicator, a dissolvable strip, an adhesive tape for teeth, a toothpick, a chewing gum, a tongue scraper, a lip balm, a syringe, a nasal aspirator, or a dental floss. In some embodiments, the composition is administered as a single tier dosing regimen. In some embodiments, the single tier dosing regimen comprises a high dose of hygromycin A. In some embodiments, the single tier dosing comprises a low dose of hygromycin. In some embodiments, the high dose of hygromycin A is about 100 milligram per kg to 1500 milligram per kg body weight. In some embodiments, the high dose of hygromycin A is about 1,000 milligram per kg. In some embodiments, the low dose of hygromycin A is from 0.001 milligram per kg to 0.1 milligram per kg body weight. In some embodiments, the low dose is 0.01 milligram per kg body weight. In some embodiments, the composition is administered as a two-tiered dosing regimen, comprising a first high dose and one or more maintenance doses. In some embodiments, the first high dose is administered orally to the subject. In some embodiments, the one or more maintenance doses are applied to the surface of the subject’s mouth, lips and / or oral cavity. In some embodiments, the hygromycin A does not inhibit the growth of or kill at least one species of beneficial oral bacteria. In some embodiments, the first high dose is 100 milligram per kg to 1500 milligram per kg body weight. In some embodiments, the first high dose is 1,000 milligram per kg. In some embodiments, the one or more maintenance doses is from 0.001 milligram per kg to 0.1 milligram per kg body weight. In some embodiments, the one or more maintenance doses is 0.01 milligram per kg body weight.

[0006] In some embodiments, the present disclosure provides a method of treating an oral orperiodontal disease and an intestinal disease comprising applying a composition comprising hygromycin A to a surface of the subject’s mouth, lips and / or oral cavity. In some embodiments, the surface can be teeth, gums, tongue, palate, floor, roof, throat, pharynx, tonsils, cheeks, lips (interior and exterior), and epiglottis. In some embodiments, the composition reduces the number of one or more bacteria in the subject’s oral cavity and / or intestine of the subject. In some embodiments, the bacteria may be Treponema denticola, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Prevotella loescheii, , Porphyromonas endodontalis, Peptococcus anaerobius, Micros prevotii, Eubasterium limosum, Centipedia perio Centipedia periodontii, Selenomonas arterimidis, Fusobacterium periodonticum, Eubacterium spp., Bacteroides spp., Actinomyces viscosos, Streptococcus mutans, and / or Streptococcus sobrinus. In some embodiments, the composition may be formulated as a mouthwash, a toothpaste, a mouth and / or throat spray, a lozenge, a breath mint, a pen or tube with a brush, sponge, or nozzle applicator, a dissolvable strip, an adhesive tape for teeth, a toothpick, a chewing gum, a tongue scraper, a lip balm, a syringe, a nasal aspirator, or a dental floss.Attorney Docket Number: 438919.001901 In some embodiments, the intestinal disease is colorectal cancer. In some embodiments, the colorectal cancer is caused by Fusobacterium nucleatum.

[0007] In some embodiments, the present disclosure provides methods for treating or preventingperiodontal bone loss in a subject. Such methods can include administering an effective amount of hygromycin A to the subject. In some embodiments, the periodontal bone loss can be caused by Fusobacterium nucleatum infection. In some embodiments, the periodontal bone loss is in or around a molar tooth, an incisor tooth, a canine tooth, and / or a premolar tooth: In some embodiments, the periodontal bone loss is in or around a molar tooth. In some embodiments, the molar tooth is a second maxillary molar tooth. In some embodiments, the therapeutic agents described herein prevent or reduce Fusobacterium nucleatum colonization.

[0008] The present disclosure also provides methods of reducing periodontal inflammation. Insome embodiments, the methods can include administering an effective amount of hygromycin A to the subject., comprising administering an effective amount of hygromycin A to the subject. In someembodiments, the periodontal inflammation loss is caused by Fusobacterium nucleatum. In someembodiments, the periodontal inflammation comprises gingival inflammation. In some embodiments, the periodontal inflammation is associated with at least one pro-inflammatory cytokine. In someembodiments, WKH SUR&LQIODPPDWRU\ F\WRNLQH LV :<&*e DQG(RU B>9&b' :Q VRPH HPERGLPHQWV%DGPLQLVWHULQJ K\JURP\FLQ 5 GHFUHDVHV WKH OHYHOV RI :<&*e DQG(RU B>9&bBRIEF DESCRIPTION OF THE DRAWINGS

[0009] The novel features of the invention are set forth with particularity in the appended claims. Abetter understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0010] Figure 1A is a pictoral representation of the experimental set up. Figure 1B is a graphshowing the changes in the relative abundance of F. nucleatum across days. Figure 1C is a series ofalpha diversity graphs at different concentrations over the days.

[0011] Figure 2A is a pictoral representation of the experimental protocol. Figure 2B is a graphicalrepresentation of the Shannon diversity index, given as the representative of all tested alpha diversity indices. Figure 2C is graph of the principal coordinates analysis (PCoA) of the bray-Curtis dissimilarity. Figure 2D is a graph showing the comparison of the relative abundances of the most abundant 9 bacterial species between the control (vehicle) and hygromycin A treated groups.

[0012] Figure 3A is a representative images of maxillary alveolar unit morphology in the control(vehicle) and hygromycin A -treated groups. Figure 3B is a graph showing the morphometric analysis comparing alveolar bone loss between the two groups (n"="9 per group). Figure 3C is a series of graphsAttorney Docket Number: 438919.001901 of gingiva RT-PCR results for IL-1$ and tnf-# expressions in the vehicle group compared to the hygromycin A -treated group (n"="10 per group). DETAILED DESCRIPTION I. INTRODUCTION

[0013] With the advancement of microbial detection technologies, an increasing number ofpreviously overlooked microorganisms have been discovered to play important roles in human diseases. A paradigm shift has also been made in understanding the key role of the microbiome in health and disease. Hygromycin A

[0014] In the last two decades, the impact of antibiotics on gut microbiome has been studied. Broad-spectrum antibiotics such as ampicillin, doxycycline, amoxicillin and ceftriaxone have been shown to impact the gut microbiota, causing rapid and diminished levels of bacterial diversity and changes to the relative abundances of bacteria, leading to dysbiosis. The microbiome shapes the immune system during development and contributes to maintaining a healthy gastrointestinal tract and preventing cardiovascular, neurological and autoimmune diseases. Thus, there is a need in the art for therapeutic agents, new treatments and / or preventative measures against diseases associated with bacteria, that have minimal to no impact on host microbiome.

[0015] Hygromycin A is a fermentation-derived natural product, first isolated from Streptomyceshygroscopicus in 1953. Hygromycin A was considered a broad-spectrum antibiotic due to activity reported against certain acid-fast bacteria as well as certain gram-positive bacteria and gram-negative bacteria (see U.S. Patent 3,100,176). Hygromycin A only demonstrated modest effects against bacteria and was not pursued commercially.

[0016] It has been demonstrated that Hygromycin A is effective against Fusobacterium spp., whilenot affecting the beneficial bacteria of the oral cavity and / or gut. Examples of bacteria that are beneficial in the oral cavity and / or gut include, but are not limited to, Streptococcus oralis, Streptococcus parasanguinis, Bifidobacterium longum, Bacteroides nordii, Bacteroides cellulosilyticus, Streptococcus sanguinis, Parabacteroides merdae, Lactobacillus reuteri, Bacteroides fragilis, Blautia producta, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides eggerthii, Enterococcus faecalis, Enterobacter cloacae, and / or Bacteroides xylanisolvens. The present disclosure thus provides therapeutic agents that have the potential to target disease causing bacteria without affecting the microbiome. In an embodiment, the hygromycin A inhibits the growth of or kills the disease-causing bacteria but does not inhibit the growth of or kill at least one species of beneficial bacteria in the subject e.g., gut bacteria, vaginal bacteria or oral bacteria. Hygromycin A is a selective antibiotic targeting ribosomes in spirochetes. It was rediscovered while searching for targeted treatments against the Lyme diseaseAttorney Docket Number: 438919.001901 pathogen, Borrelia burgdorferi primarily efficacious against Treponema species at minimal doses, Hygromycin A also inhibits the growth of F. nucleatum without harming the gut’s commensal bacteria such as Streptococcus parasanguinis, Bifidobacterium longum, and Enterococcus faecalis. Overview of Fusobacterium nucleatum

[0017] With the advancement of microbial detection technologies, an increasing number ofpreviously overlooked microorganisms have been discovered to play important roles in human diseases. A paradigm shift has also been made in understanding the role of the microbiome in health and disease. Fusobacterium nucleatum (herein referred to as F. nucleatum), a Gram-negative anaerobe, one such emerging pathogen.

[0018] F. nucleatum is ubiquitous in the oral cavity. Under disease conditions, F. nucleatum has beendetected in extra-oral sites. F. nucleatum is a heterogeneous species with several proposed subspecies (ss), i.e., ss animalis, ss fusiforme, ss nucleatum, ss polymorphum, ss periodonticum and ss vincentii, whose prevalence in disease vary.

[0019] Fusobacterium nucleatum (F. nucleatum) is a Gram-negative obligate anaerobe bacterium inthe oral cavity and plays a role in several oral diseases, including periodontitis and gingivitis. Several studies have reported that the level of F. nucleatum is significantly elevated in human colorectal adenomas and carcinomas compared to that in adjacent normal tissue. A causal role for F. nucleatum in the pathogenesis of colorectal cancer has also been demonstrated. Recently, studies have also demonstrated a pathogenic role of Fusobacterium in the formation of endometriosis. Fusobacterium nucleatum is an invasive, adherent and pro-inflammatory anaerobic bacterium. It is common in dental plaque and there is a well-established association between F. nucleatum and periodontitis. Anecdotally, F. nucleatum has been implicated in cerebral abscesses and pericarditis and it is one of the Fusobacterium species implicated in Lemierre's syndrome, a rare form of thrombophlebitis. Various Fusobacteria, including F. nucleatum, have been implicated in acute appendicitis, where they have been found by immunohistochemistry (IHC) as epithelial and submucosal infiltrates that correlate positively with severity of disease. When isolated from human intestinal biopsy material, F. nucleatum has been found to be more readily culturable from patients with gastrointestinal (GI) disease than healthy controls, and the strains grown from inflamed biopsy tissue appeared to exhibit a more invasive phenotype.

[0020] Given the widespread role of F. nucleatum in disease, there remains a need to developtherapeutic agents that inhibit, reduce and / or kill F. nucleatum. The present disclosure provides therapeutic agents, such as, but not limited to, hygromycin A for treatment of diseases associated with F. nucleatum. Fusobacterium nucleatum in PeriodontitisAttorney Docket Number: 438919.001901

[0021] Antimicrobial resistance is increasing at an alarming rate worldwide. The inappropriate useof broad-spectrum antibiotics has led to the emergence of pan-resistant superbugs. Long-term treatments enable antibiotic-resistant species to colonize the spaces vacated by commensal flora, further complicating treatment outcomes. Thus, targeted antimicrobial therapies are needed, which offer promise by selectively inhibiting pathogens of interest without adversely affecting commensal species. These strategies have proven beneficial in various pathological processes, including periodontal diseases.

[0022] Fusobacterium spp. are opportunistic pathogens commonly found in the oral cavity andgastrointestinal tract, implicated in many oral and systemic diseases. Fusobacterium nucleatum, in particular, is prevalent in conditions such as periodontal disease, pulp infection, and oral cancer. Discovered as a part of the oral microbiome associated with periodontal diseases, the abundance of this species increases with the severity of periodontal inflammation. F. nucleatum is distinguished by various virulent characteristics, particularly its adhesion proteins. These proteins promote attachment to host cells and initial colonizing bacteria, thereby serving as a ‘bridging organism’ within dental plaque. F. nucleatum’s role is critical for supporting the adherence and proliferation of other periodontal pathogens by providing mechanical stability and metabolic benefits F. nucleatum can also induce the invasion of other pathogens by fostering epithelial–mesenchymal transformation in gingival epithelial cells and manipulating the local immune environment, favoring the progression of periodontal disease. F. nucleatum can survive in neutrophils, hamper the oxidative killing mechanism of these cells by inhibiting superoxide production and can use the host cells for dissemination between different organs. Systemic dispersion of oral F. nucleatum is linked with health issues, including colorectal cancer, adverse pregnancy outcomes, Alzheimer’s disease. The targeted elimination of F. nucleatum is a justified strategy to treat oral diseases while preventing the systemic pathologies associated with its trafficking to distant organs. Overview of treponematoses

[0023] The human treponematoses comprise venereal syphilis and the endemic treponematosesincluding yaws, bejel, and Pinta. The etiological agents of these diseases are Gram-negative bacteria that belong to the order Spirochaetales, family Spirochaetaceae, and genus Treponema. Syphilis, yaws, and bejel spirochetes were originally classified as separate species but are now considered to be subspecies of Treponema pallidum (T. pallidum subsp. pallidum, T. pallidum subsp. pertenue, and T. pallidum subsp. endemicum, respectively). The lack of availability of an isolate of the agent of pinta has precluded genetic analyses of this organism, and it retains its separate name, T. carateum.

[0024] All human treponematoses share similarities in pathogenesis and natural history. All aretransmitted by direct contact with infectious lesions and are chronic infections that manifest inAttorney Docket Number: 438919.001901 multiple stages involving the skin. All except pinta can progress to cause serious and destructive lesions of skin, bone, and cartilage. As in venereal syphilis, the clinical manifestations of the endemic treponematoses are commonly divided into an early stage (encompassing primary and secondary manifestations) and a late stage. Early-stage lesions are highly infectious and can persist for weeks to months, or even years, following appearance. Once the early manifestations spontaneously regress due to the host's immune response against the pathogen, the patient enters a state of latency that in many cases lasts for a lifetime. In a relatively small percentage of cases, however, the infection can progress from latency to tertiary disease, characterized by destruction of tissues.

[0025] Treponematoses have not yet been eradicated. Distinctive features have been identified interms of age of acquisition, mode of transmission, and capacity for invasion of the central nervous system. In 2012, the World Health Organization (WHO) set a goal to eradicate treponematoses.

[0026] Successful eradication requires therapeutic strategies targeting the causative Treponema.The present disclosure provides therapeutic agents for the treatment, prevention and / or management of Treponematoses. Overview of oral treponemes

[0027] Oral treponemes, along with over 600 other bacterial species, exist as part of apolymicrobial biofilm accreted to the tooth surface in the gingival crevice. Treponemes play a role in the etiology of several chronic diseases of humans including periodontal diseases including chronic periodontitis, acute necrotizing ulcerative gingivitis, endodontic infections and some acute dental abscesses. In addition, treponemes have been implicated in the development of chronic diseases of domestic animals, including periodontal diseases of dogs, bovine digital dermatitis of dairy cattle, and contagious ovine digital dermatitis.

[0028] Periodontal disease, also called gum disease, is a common affliction among adults causedby oral bacteria growth. Whereas complex microflora exists in healthy gingival plaque posing little or no health risk, periodontal lesions can form and become dominated by proteolytic Gram-negative anaerobes and spirochetes, which are associated with severe and refractory periodontal conditions. Spirochetes disrupt intercellular junctions, invade underlying tissue, and give rise to destructive host responses in advanced periodontal disease. The genus Treponema includes more than 60 phylotypes of oral spirochetes, of which Treponema denticola is the most cultivable, and is implicated in the etiology of periodontal disease. Novel therapeutics and methods are needed to treat oral infections, including infections implicated in periodontal disease, and for treating Treponema infection.

[0029] Among treponemes, Treponema denticola (also referred to herein as T. denticola) is one ofthe most widely studied oral microbes. Treponema denticola is a Gram-negative, obligate anaerobic, motile and highly proteolytic spirochete bacterium. It is one of four species of oral spirochetes to beAttorney Docket Number: 438919.001901 reliably cultured, the others being Treponema pectinovorum, Treponema socranskii and Treponema vincentii. T. denticola dwells in a complex and diverse microbial community within the oral cavity and is highly specialized to survive in this environment.

[0030] Given the widespread role of oral microbes in disease, there remains a need to developtherapeutic agents that inhibit, reduce and / or kill oral microbes, particularly Treponema denticola. The present disclosure provides therapeutic agents, such as, but not limited to, hygromycin A for diseases associated with oral microbes. Overview of Lyme Disease

[0031] Lyme disease (Borreliosisis) caused by the bacterium Borrelia burgdorferi and rarely,Borrelia mayonii. It is transmitted to humans through the bite of infected blacklegged ticks. Symptoms include fever, headache, fatigue, and a characteristic skin rash called erythema migrans. If left untreated, infection can spread to joints, the heart, and the nervous system. Lyme disease is diagnosed based on symptoms, physical findings (e.g., rash), and the possibility of exposure to infected ticks. Lyme disease can be treated successfully with a few weeks of antibiotics. However, some patients can develop post treatment Lyme disease (PTLDS). II. THERAPEUTIC AGENTS

[0032] In some embodiments, the present disclosure provides therapeutic agents for the treatment ofdiseases. In some embodiments, the treatment of the disease in a subject can involve administering at least one therapeutic agent. Hygromycin

[0033] In some embodiments, the therapeutic agent can be hygromycin A. Hygromycin A (Hyg A)is a product of Streptomyces hygroscopicus first isolated in 1953. It has a unique structure consisting of a furanose, cinnamic acid and aminocyclitol moiety. It has a relatively broad antimicrobial spectrum. Hygromycin A has the structure shown here:Hygromycin A.

[0034] In some embodiments, the therapeutic agents can be analogs, derivatives, prodrugs orpharmaceutically acceptable salts of hygromycin A.

[0035] In some embodiments, the therapeutic agents can be any of the compounds described inU.S. patents US 6,245,745; US 6,313,100; US 6,342,497; US 6,492,342; US 6,562,792; US 6,867,230; or International Patent Publications WO1999057125A1, WO2000032616A1, WO2001030793A1,Attorney Docket Number: 438919.001901 WO2001030795A1, and / or WO2001092280A1 (the contents of each of which are herein incorporated by reference in their entirety). Macrolides

[0036] In some embodiments, the therapeutic agent can be a macrolide. As used herein, a“macrolide” refers to an antibiotic that contains a many-membered lactone ring to which one or more deoxy sugars are attached. Non-limiting examples of macrolides include, azithromycin, erythromycin, erythromycin estolate, erythromycin ethylsuccinate, erythromycin glucoheptonate, erythromycin lactobionate, erythromycin propionate, erythromycin stearate, clarithromycin, spiramycin, dirithromycine, josamycine, josamycine propionate, kitasamycine, midecamycine, miocamycine, oleandomycine phosphate, roxithromycine, spiramycine, spiramycine adipate, rovamycine, and / or clarithromycin. Probiotic compositions

[0037] In some embodiments, the therapeutic agent can be a probiotic composition. As used herein,the term "probiotic" refers to a microorganism (such as a bacteria or a yeast) having a beneficial effect on the general health of an animal or a human, or a beneficial effect on a specific health problem, disorder or disease, alleviating pain, symptoms or discomfort associated with those health problem, disorder or disease. Probiotic compositions may be designed to address several needs. The nature of the composition (the amount and type of bacteria present in the composition) will strongly influence its ability to address these specific needs.

[0038] In some embodiments, the probiotic compositions can include one or more genera of bacteriaand / or yeast. In some embodiments, the genera can be Lactobacillus, Gardnerella, Bifidobacterium, Streptococcus, Enterobacter, and / or Saccharomyces.

[0039] In some embodiments, the probiotic compositions can include one or more species of bacteriaand / or yeast such as, but not limited to, Lactobacillus rhamnosus, Lactobacillus acidophilus,Lactobacillus reuteri, , Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus gasseri,Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus helveticus,Lactobacillus casei, Lactobacillus paracasei, Streptococcus thermophilus, Saccharomyces boulardii,Bifdobacterium bifidum, Bifdobacterium breve, Bifdobacterium longum, Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium infantis, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, Bifidobacterium adolescentis, and / or Bifidobacterium angulatum.

[0040] In some embodiments, the probiotic compositions include microorganisms that are alive. Insome embodiments, the probiotic compositions include dead or inactivated microorganisms. In some embodiments, the probiotic compositions can include extracts, lysates, metabolic byproducts of the microorganisms.Attorney Docket Number: 438919.001901 Prebiotic compositions

[0041] In some embodiments, the therapeutic agents of the disclosure can include prebioticcompositions. As used herein, the term “prebiotic compositions” refers to non-digestible food ingredients which stimulate the growth and activity of beneficial microorganisms in the host e.g., humans or animals. In some embodiments, the prebiotic compositions include oligosaccharides, and / or sugar molecules of three to six chains and soluble fiber.

[0042] In some embodiments, the prebiotic compositions can include carbohydrates from fruits andvegetables. For example, prebiotic compositions can include carbohydrates from bananas, berries, asparagus, garlic, wheat, oatmeal, barley (and other whole grains), flaxseed, tomatoes, Jerusalem artichoke, onions and chicory, spinach, chard, kale, and legumes like lentils, kidney beans, chickpeas, navy beans, white beans and black beans.

[0043] In some embodiments, prebiotic compositions can include oligosaccharides such as, but notlimited to, fiber gums, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), inulins, isomalto-oligosaccharides, lactilol, lactosucrose, lactulose, oligofructose, pyrodextrins, soy oligosaccharides, transgalacto-oligosaccharides (TOS), and / or xylo-oligosaccharides. In some embodiments, the prebiotics include fructans, such as, but not limited to, inulin and fructo- oligosaccharide or oligofructose. In some embodiments, the prebiotic compositions can include GOS such as GOS with excess galactose at C3, C4 or C6 and / or GOS manufactured from lactose through enzymatic trans-glycosylation. The end product of GOS manufactured from lactose is mainly a mixtureRI WUL& WR SHQWDVDFFKDULGHV ZLWK JDODFWRVH LQ e #*c / $% e #*c,$% DQG e #*c-$ OLQNDJHV' :Q VRPHembodiments, the prebiotic compositions can include resistant starch. In some embodiments, the prebiotic compositions can include polydextrose and / or pectin.

[0044] In some embodiments, the prebiotic compositions can include non-carbohydrateoligosaccharides, such as but not limited to cocoa-derived flavanols. Combinations

[0045] In some embodiments, a combination of therapeutic agents can be utilized. The presentdisclosure provides two, three, four, five or more therapeutic agents in a combinatorial format. Combinations can be administered concurrently, sequentially and / or serially. In some embodiments, each therapeutic agent in a combination can be formulated as separate pharmaceutical formulations. In some embodiments, the therapeutic agents in a combination can be prepared as single pharmaceutical formulation.

[0046] In some embodiments, therapeutic modalities for enhancing or improving intratumor accessmay also be utilized. As a non-limiting example, the modality can be sonodynamic therapy (sonalasense).Attorney Docket Number: 438919.001901

[0047] In some embodiments, hygromycin A may be utilized in combination with chemotherapeuticagents (e.g.5 Fluorouracil), and / or immune checkpoint inhibitors.

[0048] In one embodiment, a combination of Hygromycin A and macrolides may be utilized. As anon-limiting example, the macrolide in the combination can be Azithromycin.

[0049] For purposes of the present disclosure, “in combination”, refers to providing two or moretherapeutic agents either separately or together, where the two therapeutic agents are administered as part of an appropriate dose regimen designed to obtain the benefit of the combination therapy. Thus, the two therapeutic agents can be administered either as part of the same pharmaceutical composition or in separate pharmaceutical compositions. The first therapeutic agent can be administered prior to, at the same time as, or subsequent to administration of the second therapeutic agent, or in some combination thereof. Where the one therapeutic agent is administered to the subject at repeated intervals, e.g., during a standard course of treatment, the second therapeutic agent can be administered prior to, at the same time as, or subsequent to, each administration of the first therapeutic agent, or some combination thereof, or at different intervals in relation to therapy with the first therapeutic agent, or in a single dose prior to, at any time during, or subsequent to the course of treatment with the first therapeutic agent.

[0050] Combinations of therapeutic agents of the present disclosure can include hygromycin A anda second therapeutic agent. In some embodiments, the second therapeutic agent can be a therapeutic agent utilized to treat a Fusobacterium nucleatum infection. As a non-limiting example, the second therapeutic agent can be a chemically modified transfer RNA (tsRNA) targeting F. nucleatum described in US Patent Publication 2023 / 0165889 (the contents of which are herein incorporated by reference in its entirety).

[0051] In some embodiments, the second therapeutic agent can be a prebiotic composition and / or aprobiotic composition.

[0052] Combinations of therapeutic agents of the present disclosure can include hygromycin A anda second therapeutic agent used to treat inflammation, destruction of connective tissues, periodontal ligament, and alveolar bone resorption, and ultimately tooth loss in chronic periodontal infection. In some embodiments, the second therapeutic agent can be enoxacin and / or a bisphosphonate derivative of enoxacin (bis-enoxacin).

[0053] Combinations of therapeutic agents of the present disclosure can include hygromycin A anda second therapeutic agent. In some embodiments, the second therapeutic agent can be a therapeutic agent utilized to treat treponematoses.

[0054] In some embodiments, the therapeutic agents can be combined with one or more agentsused in the treatment of syphilis. In some embodiments, the therapeutic agents of the present disclosure can be combined with penicillin.Attorney Docket Number: 438919.001901

[0055] In some embodiments, the therapeutic agents can be combined with one or more agentsused in the treatment of yaws. In some embodiments, the therapeutic agents of the present disclosure can be combined with azithromycin and / or benzathine benzylpenicillin.

[0056] In some embodiments, the therapeutic agents can be combined with one or more agentsused in the treatment of Bejel or Pinta. In some embodiments, the therapeutic agents of the present disclosure can be combined with doxycycline and / or benzathine benzylpenicillin.

[0057] In some embodiments, the therapeutic agents can be combined with one or more agentsused in the treatment of any of the disease, disorders or syndromes described herein.

[0058] In some embodiments, the therapeutic agents of the present disclosure e.g. hygromycin Acan be combined with macrolides and / or benzathine benzylpenicillin.

[0059] In some embodiments, the therapeutic agents of the present disclosure e.g. hygromycin Acan be combined with azithromycin and / or benzathine benzylpenicillin. III. METHODS OF USE

[0060] The present disclosure provides methods of use related to the therapeutic agents describedherein. In some embodiments, the methods can include a method of reducing the growth of bacteria e.g., Fusobacterium, Borrelia, Treponema, and / or any other spirochete. Therapeutic agents or combinations thereof targeting more than one type of bacteria, directly or indirectly through synergistic mechanisms are also contemplated. The present disclosure also contemplates methods of use wherein one or more bacteria in the ‘Red Complex’ are targeted, thereby targeting the other bacteria in the Red Complex. Non-limiting examples of bacteria in the Red Complex include Treponema denticola, Porphyromonas gingivalis, and Tannerella forsythia which can form a bacterial consortium.

[0061] In some embodiments, the therapeutic agents of the disclosure can be used to treat acuteinfections or chronic infections associated with the bacteria. In some embodiments, the therapeutic agents of the disclosure can be used to treat chronic diseases, long diseases or syndromes associated with infection. Oral and Periodontal disease

[0062] The therapeutic agents of the disclosure can exhibit potency against oral microbes, andtherefore have the potential to treat, and / or prevent an infection, or kill and / or inhibit the growth of an oral microbe. In some embodiments, the animal can be a human. In some embodiments, the spirochete infection can be treated and / or prevented, or the spirochete can be killed, or its growth is inhibited, through oral administration of the therapeutic agent of the disclosure. As a non-limiting example, the spirochete infection can be treated and / or prevented, or the spirochete can be killed, or its growth is inhibited through intravenous administration of the therapeutic agent of the disclosure.Attorney Docket Number: 438919.001901

[0063] In some embodiments, the present disclosure provides methods of enhancing and / ormaintaining the oral health of a mammalian subject, wherein a composition of the subject invention is applied to one or more surfaces of the subject’s mouth, lips or oral cavity. The one or more surfaces can include, for example, the subject’s teeth, gums, tongue, palate, floor, roof, throat, pharynx, tonsils, cheeks, lips (interior and exterior), epiglottis, and any of the tissues or passageways connected thereto. In some embodiments, the method further comprises rinsing the composition from the one or more surfaces of the mouth, lips or oral cavity. This can be performed with, for example, water or mouth wash. In preferred embodiments, the method can be used to treat and / or prevent plaque, biofilm and / or tartar formation; halitosis; oral infections / abscesses; mouth sores, including ulcers, cold sores and / or canker sores; stained teeth; thrush; oral cancers; gingivitis; tooth decay; periodontal disease; caries; and / or any other oral health condition caused by the presence of bacteria, fungi and / or viruses, such as, e.g., inflammatory conditions, tonsillitis, pharyngitis, laryngitis, glossitis, stomatitis and others.

[0064] In certain embodiments, the method can be used for reducing the number of undesirablemicroorganisms present in a subject’s oral cavity, thus allowing for an increase in beneficial and / or commensal microflora.

[0065] In some embodiments, the oral microbe can be a Treponema spp. , a Streptococcus spp. , aFusobacterium spp., and / or a Parvimonas spp.

[0066] In some embodiments, the hygromycin A does not inhibit the growth of or kill at least onespecies of beneficial oral bacteria.

[0067] In some embodiments, the therapeutic agents of the present disclosure can be used fortreating, preventing, protecting against and / or managing a disease caused by Treponema such as, but not limited to T. pallidum, T. denticola, T. vincentii, T. carateum, and / or T. phagedenis or clinical isolates or strains thereof.

[0068] In some embodiments, the therapeutic agents of the present disclosure can be used fortreating, preventing, protecting against and / or managing a disease caused by an oral microbe e.g., Treponema such as, but not limited to T. pallidum, T. denticola, T. vincentii, T. carateum, and / or T. phagedenis or clinical isolates or strains thereof.

[0069]

[0053] In some embodiments, the oral microbe can be Treponema denticola or a strain or aclinical isolate thereof, such as T. denticola (strain: ATCC35405), T. denticola (strain: ATCC35404), T. denticola (strain: ATCC33520), T. denticola (strain: ATCC33521), T. denticola (strain: OTK), T. denticola (strain: Hl-T), T. denticola (strain: H-22), T. denticola (strain: MYR-T), T. denticola (strain: US-Trep), T. denticola (strain: ASLM), T. denticola (strain: AL-2), T. denticola (strain: SP21), T. denticola (strain: SP23), T. denticola (strain: SP32), T. denticola (strain: SP33), T. denticola (strain: SP37), and / or T. denticola (strain: SP44).Attorney Docket Number: 438919.001901

[0070] In some embodiments, the therapeutic agents of the disclosure can be used to treat, preventor manage a disease caused by, or associated with an oral microbe such as, but not limited to, Fusobacterium nucleatum (e.g., any strain of . nucleatum animalis, ATCC 25586, ATCC551191, ATCC 10953, ATCC 23726, CTI-2, CTI-3, CTI-7, EAV0002, 7-1, FA2+, 7-33 Cl), Veillonella parvula (e.g., ATCC 10790), Actinomyces odontolyticus (e.g., ATCC 17982), Neisseria mucosa, Parvimonas micra (e.g., ATCC 33270), Porphyromonas gingivalis (e.g., ATCC 33277), Tannerella forsythia, Capnocytophaga, Peptostreptococcus, and / or Eikenella.

[0071] In some embodiments, oral microbes can include Porphyromonas gingivalis,Fusobacterium nucleatum, Prevotella intermedia, Prevotella loescheii, Treponema denticola (Treponema denticola), Porphyromonas endodontalis, Peptococcus anaerobius, Micros prevotii, Eubasterium limosum, Centipedia perio Centipedia periodontii, Selenomonas arterimidis, Fusobacterium periodonticum, Eubacterium spp., Bacteroides spp., Actinomyces viscosos, Streptococcus mutans, and / or Streptococcus sobrinus.

[0072] In some embodiments, administering a therapeutic agent described herein to a subjectinfected with an oral microbe or having a disease caused or compounded / exacerbated by the oral microbe, inhibits or reduces replication of the oral microbe by at least 20% to 25%, at least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, or up to at least 85% relative to a negative control as determined using an assay described herein or others known to one of skill in the art. In some embodiments, administering a therapeutic agent described herein to a subject (in some embodiments, an animal model) infected with an oral microbe inhibits or reduces replication of the infectious agent by at least 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 8 fold, 10 fold, 15 fold, 20 fold, or 2 to 5 fold, 2 to 10 fold, 5 to 10 fold, or 5 to 20 fold relative to a negative control as determined using an assay described herein or others known to one of skill in the art.

[0073] In some aspects, the therapeutic agent can be used to treat or prevent periodontal diseases.Periodontal diseases are mainly the result of infections and inflammation of the gums and bone that surround and support the teeth. In its early stage, called gingivitis, the gums can become swollen and red, and they can bleed. In its more serious form, called periodontitis, the gums can pull away from the tooth, bone can be lost, and the teeth can loosen or even fall out. In some embodiments, the therapeutic agents of the disclosure can be used to treat gingivitis. In some embodiments, the therapeutic agents of the disclosure can be used to treat periodontitis.

[0074] In some embodiments, the therapeutic agents of the disclosure can be used to treat, manageor prevent one or more symptoms associated with periodontal disease, such as, but not limited to, badAttorney Docket Number: 438919.001901 breath or bad taste, red or swollen gums, tender or bleeding gums, painful chewing, loose teeth, sensitive teeth, gums that have pulled away from teeth.

[0075] In some embodiments, the therapeutic agents of the disclosure can be used to treat, manageor prevent chronic periodontitis. Chronic periodontitis is a polymicrobial disease that results from the overgrowth of a limited number of bacterial species that are normal members of the oral microbiota. It is widely accepted that Treponema denticola, Porphyromonas gingivalis, and Tannerella forsythia form a bacterial consortium, often referred to as the ‘Red Complex’, that is strongly associated with the clinical progression of chronic periodontitis. The unifying features of the Red Complex bacteria are their extracellular proteolytic activity, their complex anaerobic fermentations of amino acids, production of toxic metabolites, and outer membrane (or sheath) vesicles.

[0076] In some embodiments, the therapeutic agents of the disclosure can be used to treat, prevent,or manage gingival disease. In some embodiments, the gingival disease can be caused by or correlated with the infection by or the presence of oral microbes.

[0077] In some embodiments, the therapeutic agents of the disclosure can be used to treat, prevent,or manage chronic periodontitis. In some embodiments, the chronic periodontitis can be caused by or correlated with the infection by or the presence of oral microbes.

[0078] In some embodiments, the therapeutic agents of the disclosure can be used to treat, prevent,or manage aggressive periodontitis. In some embodiments, the aggressive periodontitis can be caused by or correlated with the infection by or the presence of oral microbes.

[0079] In some embodiments, the therapeutic agents of the disclosure can be used to treat, prevent,or manage abscesses of the periodontium. In some embodiments, the abscesses of the periodontium can be caused by or correlated with the infection by or the presence of oral microbes.

[0080] In some embodiments, the therapeutic agents of the disclosure can be used to treat, prevent,or manage periodontitis associated with endodontic lesion. In some embodiments, the periodontitis associated with endodontic lesion of the periodontium can be caused by or correlated with the infection by or the presence of oral microbes.

[0081] In some embodiments, the therapeutic agents of the disclosure can be used to treat Stage Iof periodontitis characterized by 1-2 mm interdental clinical attachment loss. In some embodiments, the therapeutic agents of the disclosure can be used to treat Stage II of periodontitis characterized by 3-4 mm interdental clinical attachment loss. In some embodiments, the therapeutic agents of the disclosure can be used to treat Stage III or Stage IV of periodontitis characterized by > 5 mm interdental clinical attachment loss.

[0082] In some embodiments, the present disclosure provides methods for treating or preventingperiodontal or alveolar bone loss in a subject. Such methods can include administering an effectiveAttorney Docket Number: 438919.001901 amount of hygromycin A to the subject. In some embodiments, the periodontal bone loss can be caused by Fusobacterium nucleatum infection. In some embodiments, the periodontal bone loss is in or around a molar tooth, an incisor tooth, a canine tooth, and / or a premolar tooth: In some embodiments, the periodontal bone loss is in or around a molar tooth. In some embodiments, the molar tooth is a second maxillary molar tooth. In some embodiments, the therapeutic agents described herein prevent or reduce Fusobacterium nucleatum colonization.

[0083] The present disclosure also provides methods of reducing periodontal inflammation. Insome embodiments, the methods can include administering an effective amount of hygromycin A to the subject., comprising administering an effective amount of hygromycin A to the subject. In someembodiments, the periodontal inflammation loss is caused by Fusobacterium nucleatum. In someembodiments, the periodontal inflammation comprises gingival inflammation. In some embodiments, the periodontal inflammation is associated with at least one pro-inflammatory cytokine. In someembodiments, WKH SUR&LQIODPPDWRU\ F\WRNLQH LV :<&*e DQG(RU B>9&b' :Q VRPH HPERGLPHQWV%DGPLQLVWHULQJ K\JURP\FLQ 5 GHFUHDVHV WKH OHYHOV RI :<&*e DQG(RU B>9&b% E\ .&*)"% *)&+)"% +)&,)"%30-40%, 40-50%, 50-60%, 60-70% or more. Fusobacterium nucleatum associated diseases

[0084] In some embodiments, the therapeutic agents of the disclosure can be used to treat, prevent,or manage Fusobacterium nucleatum infections in subjects. F. nucleatum is an invasive bacterium that causes acute oral and gastrointestinal infections and can act as a pro-inflammatory agent. In some embodiments, the therapeutic agents of the present disclosure can be used to treat, prevent or manage Fusobacterium nucleatum associated diseases. As used herein, the term “Fusobacterium nucleatum associated diseases” can be used to refer to a disease or a condition that is caused by and / or associated with infection by one or more species of Fusobacterium nucleatum.

[0085] At least 6 different sub species of F. nucleatum have been described and include F. nucleatumanimalis, F. nucleatum fusiforme, F. nucleatum nucleatum, F. nucleatum polymorphum, F. nucleatum periodonticum and F. nucleatum vincentii. In some embodiments, the F. nucleatum can be a strain associated with a disease state. In some embodiments, the strain can be a F. nucleatum animalis subspecies strain such as, but not limited to, 7 / 1 (or 7_1), CRC 7 / 3 JVN3C1 (or CRC 7_3JVN3C1), and / or 218A8. In some embodiments, the strain can be a F. nucleatum vincentii subspecies strain such as, but not limited to, 215A9, CC53 and / or 3 / 1 / 36A2 (EAV018). In some embodiments, the strain can be a F. nucleatum nucleatum subspecies strain such as, but not limited to, ATCC25586T, 203L34 and / or 2 / 3 FMU 1 (2_3 FMU 1). In some embodiments, the strain can be a F. nucleatum polymorphum strain such as, but not limited to, 203L28, 203L29, and / or 13 / 3C (13_3C EAV005 or EAVG 005). In some embodiments, the strain can be a F. nucleatum fusiforme strain such as, but not limited to 203C15,Attorney Docket Number: 438919.001901 203L25, and / or 203L30. In some embodiments, the strain can be a F. nucleatum periodonticum strain, such as, but not limited to, 2 / 1 / 31 or (2_1_31 EAV015 or EAVG 015), 3 / 1 / 7B (3_1_7B or EAVG 011) and / or 209B32.

[0086] In some embodiments, the F. nucleatum infection is in the abdomen. In some embodiments,the F. nucleatum infection is in the pelvis. In some embodiments, the F. nucleatum infection is in the reproductive system, urinogenital system and / or the gastrointestinal system. In some embodiments, the F. nucleatum infection is in the endometrium. In some embodiments, the F. nucleatum infection is associated with inflammation. In some embodiments, the F. nucleatum is associated with the inflammation of the pelvis, e.g. uterus and more specifically the endometrial lining of the uterus.

[0087] In some embodiments, the present disclosure provides methods for inhibiting the growth ofFusobacterium species e.g., Fusobacterium nucleatum. The growth of the F. nucleatum species can be inhibited in vitro (e.g., in a cell, or a tissue sample from a subject) or in vivo in a subject. In some aspects, the growth of the F. nucleatum can be inhibited by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and / or 90%. In some embodiments, the growth of the bacteria is inhibited by 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%, 75- 85%, and / or 90-100%.

[0088] In some embodiments, administering a therapeutic agent described herein to a subject infectedwith a Fusobacterium or having a disease caused or compounded / exacerbated by Fusobacterium, inhibits or reduces replication of the Fusobacterium by at least 20% to 25%, at least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, or up to at least 85% relative to a negative control as determined using an assay described herein or others known to one of skill in the art. In some embodiments, administering a therapeutic agent described herein to a subject (in some embodiments, an animal model) infected with Fusobacterium inhibits or reduces replication of the infectious agent by at least 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 8 fold, 10 fold, 15 fold, 20 fold, or 2 to 5 fold, 2 to 10 fold, 5 to 10 fold, or 5 to 20 fold relative to a negative control as determined using an assay described herein or others known to one of skill in the art.

[0089] The present disclosure provides therapeutic agents and methods related to F. nucleatum andthe treatment, prevention and / or management of diseases associated with F. nucleatum.

[0090] In some embodiments, the present disclosure provides methods for inhibiting the growth ofFusobacterium nucleatum (F. nucleatum). Such methods can include contacting F. nucleatum with at least one therapeutic agent such that the growth of F. nucleatum is inhibited. The therapeutic agent canAttorney Docket Number: 438919.001901 be hygromycin A. Also provided herein are methods for inhibiting or reducing the growth of Fusobacterium nucleatum.

[0091] In some embodiment, the present disclosure provides therapeutic agents and methods fortreatment, prevention and / or management of diseases associated with F. nucleatum infection alone or in combination with Treponema infection e.g. Treponema denticola infection.

[0092] In one embodiment, the F. nucleatum associated diseases can be oral infection, cancer (e.g. amicrobiome influenced tumor such as colorectal cancer, oral squamous cell carcinoma, bladder cancer, breast cancer), endometriosis, neurodegenerative diseases (e.g. Alzheimer's disease, Parkinson's disease, prion disease, Amyotrophic lateral sclerosis, motor neuron disease, Huntington's disease, spinal muscular atrophy, and spinocerebellar ataxia) or a cardiovascular disease (e.g. atherosclerosis). Combinatorial treatment of oral and intestinal indications

[0093] In some embodiments, the therapeutic agents may be used to treat a combination of oral andintestinal indications. In some embodiments, the oral and / or periodontal disease may be caused by Treponema denticola and the intestinal disease can be colorectal cancer caused by Fusobacterium nucleatum. Spirochete infection and diseases

[0094] The therapeutic agents of the disclosure may exhibit potency against spirochetes, andtherefore have the potential to treat, and / or prevent a spirochete infection, or kill and / or inhibit the growth of a spirochete. In some embodiments, the spirochete is inside, or on the surface of an animal. In another exemplary embodiment, the animal is described herein. In another exemplary embodiment, the animal is a human.

[0095] In some embodiments, the may be of the Leptospirales order, the Brachyspirales order, theBrevinematales order, and the Spirochaetales order. In some embodiments, the spirochete may be of the Leptospiraceae family, the Brachyspiraceae family, the Brevinemataceae family, the Borreliaceae family, and the Spirochaetaceae family. In some embodiments, the spirochete may be of the Leptospiraceae family, the Brachyspiraceae family, the Brevinemataceae family, the Borreliaceae family, and the Spirochaetaceae family. In some embodiments, the therapeutic agents may be used to treat / kill or inhibit the Leptonema genus, the Leptospira genus, the Turneriella genus, the Brachyspira genus, the Brevinema genus, the Exilispira genus, the Borreliella genus, the Borrelia genus, the Cristispira pectinis genus, the Clevelandina reticulitermitidis genus, the Diplocalyx calotermitidis genus, the Hollandina pterotermitidis genus, the Pillotina calotermitidis genus, the Spironema culicis genus, the Spirochaeta genus, and the Treponema genus. In an exemplary embodiment, the spirochete is of the Treponema genus. In an exemplary embodiment, the spirochete is of the Leptospira genus. In an exemplary embodiment, the spirochete is of the Borrelia genus. In an exemplary embodiment, theAttorney Docket Number: 438919.001901 spirochete is of the Brachyspira genus. In an exemplary embodiment, the spirochete is Borrelia burgdorferi. In an exemplary embodiment, the spirochete is Borrelia mayonii. In an exemplary embodiment, the spirochete is Borrelia afzelii. In an exemplary embodiment, the spirochete is Borrelia garinii. In an exemplary embodiment, the spirochete is Borrelia recurrentis. In an exemplary embodiment, the spirochete is Treponema pallidum. In an exemplary embodiment, the spirochete is Brachyspira pilosicoli. In an exemplary embodiment, the spirochete is Brachyspira aalborgi.

[0096] In some embodiments, the therapeutic agents of the disclosure can be used to treat LymeDisease, Post Treatment Lyme Disease (PTLDS) or Leptospirosis. Infection & Disease or disorder associated with an infection

[0097] In some embodiments, the therapeutic agents described herein are useful in the treatment ofan infection or a disease or disorder associated with an infection. In some embodiments, the infection can be a bacterial infection or a protozoan infection. In some embodiments, the disease or disorder associated with an infection can be pneumonia, sinusitis, pharyngitis, tonsillitis, skin infections and / or otitis media.

[0098] Bacterial infections and protozoa infections and disorders related to such infections includebut are not limited to otitis media, sinusitis, bronchitis, tonsillitis, and mastoiditis related to infectionby Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Staphylococcusaureus, or Peptostreptococcus spp.: pharyngitis, rheumatic fever, and glomerulonephritis related to infection by Streptococcus pyogenes, Groups C and G Streptococci, Clostridium diphtheriae, or Actinobacillus; respiratory tract infections related to infection by Mycoplasma pneumoniae, Legionella pneumophila, Streptococcus pneumoniae, Haemophilus influenzae, or Chlamydia pneumoniae; uncomplicated skin and soft tissue infections, abscesses and osteomyelitis, and fever related to infection by Staphylococcus aureus, coagulase positive Staphylococci (e.g., S. epidermidis, S.hemolyticus, etc.), Streptococcus pyogenes Streptococcus agalactiae, Streptococcal groups C-F(minute-colony streptococci), Viridans streptococci, Corynebacterium minutissimum, Clostridium spp., or Bartonella henselae; uncomplicated acute urinary tract Infections related to infection by Staphylococcus saprophyticus or Enterococcus spp.; urethritis and cervicitis; and sexually transmitted diseases related to infection by Chlamydia trachomatis, Haemophilus ducrey, Treponema pallidum, Ureaplasma urealyticum, or Neisseria gonorrhea, toxin diseases related to infection by S. aureus (food poisoning and Toxic shock syndrome), or Groups A, B. and C Streptococci; ulcers related to infection by Helicobacter pylori: systemic febrile syndromes related to infection by Borrelia recurrentis; Lyme disease related to infection by Borrelia burgdorferi; conjunctivitis, keratitis, anddacryocystitis related to infection by Chlamydia trachomatis, Neisseria gonorrhoeae, S. aureus, S.pneumoniae, S. pyogenes, H. influenzae, or Listeria spp.; disseminated Mycobacterium aviumAttorney Docket Number: 438919.001901 complex (MAC) disease related to infection by Mycobacterium avium, or Mycobacterium intracellulare; gastroenteritis related to infection by Campylobacter jejuni; intestinal protozoa related to infection by Cryptosporidium spp.; odontogenic infection related to infection by Viridans streptococci; persistent cough related to infection by Bordetella pertussis; gas gangrene related toinfection by Clostridium perfringens, or Bacteroides spp.; and atherosclerosis related to infection byHelicobacter pylori or Chlamydia pneumoniae. Abdominal disease or disorders

[0099] In some embodiments, the therapeutic agents described herein are useful in the treatment ofan abdominal disease or disorder associated with F. nucleatum infection. In some embodiments, the abdominal disease or disorder is associated with the reproductive system, the urinogenital system and / or the gastrointestinal system.

[0100] In some embodiments, the abdominal disease is associated with an F. nucleatum infection ofthe reproductive system. In some embodiments, the F. nucleatum infection is in the female reproductive system. In some embodiments, the therapeutic agents of the disclosure are used to treat a disease or disorder such as, but not limited to, endometriosis, infertility, uterine fibroids, polycystic ovarian syndrome, menorrhagia, adenomyosis, primary dysmenorrhea, secondary dysmenorrhea, chronic pelvic pain syndrome, pelvic inflammatory disease (PID), vaginitis, cervicitis, endometritis, miscarriage, cervical cancer, vaginal dysbiosis, vaginal cancer, vulvar cancer, uterine cancer, ovarian cancer, adenomyosis, leiomyoma, salpingitis, and / or ovarian cyst.

[0101] In some embodiments, the therapeutic agents can be used to prevent adverse pregnancyoutcomes. For example, the therapeutic agents of the disclosure can be used to prevent preterm labor or still birth.

[0102] In some embodiments, the therapeutic agents can be used to treat, prevent or manage F.nucleatum infection associated with the urinogenital system.

[0103] In some embodiments, the therapeutic agents can be used to treat, prevent, or manageFusobacterium nucleatum associated gastrointestinal diseases or disorders such as, but not limited to colorectal cancer (CRC), inflammatory bowel disease (IBD) and appendicitis.

[0104] Cancers of the gastrointestinal tract represent a significant percentage of all cancer relateddeaths, and include gastric cancer, colorectal and esophageal cancers. Colorectal carcinoma (CRC) is the second leading cause of cancer deaths, responsible for approximately 655,000 deaths per year worldwide. CRC is also one of the first and best genetically characterized cancers in which specific somatic mutations on oncogenes and tumor suppressor genes associated with progression from adenomatous lesions (polyps) to invasive carcinoma have been identified. Inflammation has been recognized as a risk factor for CRC. In some embodiments, the therapeutic agents of the disclosure canAttorney Docket Number: 438919.001901 be used to treat, prevent, or manage colorectal cancer. In some embodiments, the therapeutic agents of the disclosure can be used to treat, prevent or manage Fusobacterium nucleatum associated colorectal cancer. The CRC can be caused by or linked to F. nucleatum infection.

[0105] In some embodiments, the therapeutic agents of the disclosure can be used to treat, preventor manage IBD. In some embodiments, the therapeutic agents of the disclosure can be used to treat, prevent or manage Fusobacterium nucleatum associated IBD. IBD has been recognized as a risk factor for CRC. F. nucleatum strains isolated from inflamed tissues of the IBD patients are more invasive than those from the normal tissues. Several studies have reported association of F. nucleatum in appendicitis. Co-occurrence of F. nucleatum with other oral taxa has been observed.

[0106] In some embodiments, the therapeutic agents of the disclosure can be used to treat, preventor manage Crohn’s disease. In some embodiments, the therapeutic agents of the disclosure can be used to treat, prevent or manage Fusobacterium nucleatum associated Crohn’s disease.

[0107] In some embodiments, the therapeutic agents of the disclosure can be used to treat, preventor manage ulcerative colitis. In some embodiments, the therapeutic agents of the disclosure can be used to treat, prevent, or manage Fusobacterium nucleatum associated ulcerative colitis. Immune cell infection and integration

[0108] In some embodiments, therapeutic agents and / or methods of the disclosure are used in thetreatment, management or prevention of Fusobacterium nucleatum infection and / or integration into immune cells. In some embodiments, the immune cells can be neutrophils, macrophages, T cells, dendritic cells, B cells, natural killer cells, T helper cells, white blood cells, mast cells, cytotoxic T cells, Regulatory T cells, plasma cells, eosinophils, innate lymphoid cells, monocytes, basophils, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, memory B cells, gamma delta T cells, memory T cells, and / or effector cells. In some embodiments, the therapeutic agents of the disclosure prevent immune cell death induced by F. nucleatum infection. In some embodiments, the therapeutic agents of the disclosure can be used to prevent, treat or manage F. nucleatum infection of immune cells in the oral cavity. In some embodiments, the therapeutic agents of the disclosure can be used to prevent, treat or manage F. nucleatum infection of immune cells at extra-oral sites or sites other than the oral cavity of a subject. Inflammation

[0109] In some embodiments, the therapeutic agents and / or methods of the disclosure are used in thetreatment, management or prevention of inflammation or inflammatory diseases. In some embodiments, the inflammation or inflammatory disease may be associated with F. nucleatum infection. In some embodiments, the inflammatory disease may be inflammatory bowel disease, celiac disease, colitis, irritable bowel syndrome, intestinal hyperplasia, metabolic syndrome, obesity, diabetes, rheumatoidAttorney Docket Number: 438919.001901 arthritis, liver disease, hepatic steatosis, fatty liver disease, non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH). Cancer

[0110] In some embodiments, the therapeutic agents of the present disclosure are used for treating,protecting against, and / or managing cancer. In some embodiments, the therapeutic agents of the disclosure are used for treating, preventing or managing a cancer associated with one or more oral microbes. In some embodiments, the therapeutic agents of the disclosure are used for treating, preventing or managing metastasis of cancer.

[0111] In some embodiments, the therapeutic agents of the present disclosure are used for treatingand / or preventing metastasis.

[0112] In some embodiments, the therapeutic agents of the disclosure are used for treating,preventing or managing a cancer caused by Fusobacterium. In some embodiments, the cancer can be oral, colorectal or esophageal cancer. As a non-limiting example, the colorectal or esophageal cancer can be caused by Fusobacterium species.

[0113] The subject can have cancer, can be suspected of having cancer, or can have a predispositionto cancer. The therapeutic agents or pharmaceutically acceptable salts thereof can be administered to the subject as a treatment for cancer and maintenance in all patients. The effect of the therapeutic agents or formulations described herein on proliferation of cancer cells can be detected by routine assays known in the art. Cancer cell lines on which such assays can be performed are well known to those of skill in the art.

[0114] In some embodiments, the therapeutic agents of the disclosure can be used to treat, manageor prevent colorectal cancers, gastric cancer and / or esophageal cancers.

[0115] The present disclosure also provides methods of administering a therapeutic agent, asdescribed herein, to a subject. The subject can have cancer, can be suspected of having cancer, or can have a predisposition to cancer. The compounds or pharmaceutically acceptable salts thereof are administered to the subject as a treatment for cancer and maintenance in all patients. The effect of the compositions described herein on proliferation of cancer cells can be detected by routine assays known in the art. Cancer cell lines on which such assays can be performed are well known to those of skill in the art.

[0116] Cancers and related disorders that can be treated, protected against, or managed using thetherapeutic agents and methods described herein include, but are not limited to, the following: leukemias including, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocyticAttorney Docket Number: 438919.001901 (granulocytic) leukemia, and chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodgkin's disease, and non-Hodgkin's disease; multiple myelomas such as but not limited to smoldering multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenström's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas such as but not limited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, and synovial sarcoma; brain tumors including but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, and primary brain lymphoma; breast cancer including, but not limited to, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease, and inflammatory breast cancer; adrenal cancer, including but not limited to, pheochromocytoma and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer, including but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers including but not limited to, Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipidus; eye cancers including but not limited to, ocular melanoma such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers, including but not limited to, squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, including but not limited to, squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers including but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers including but not limited to, endometrial carcinoma and uterine sarcoma; ovarian cancers including but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancers including but not limited to, squamous cancer, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers including but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers; liver cancers including but not limited to hepatocellular carcinoma and hepatoblastoma; gallbladder cancers including but not limited to, adenocarcinoma;Attorney Docket Number: 438919.001901 cholangiocarcinomas including but not limited to, papillary, nodular, and diffuse; lung cancers including but not limited to, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; testicular cancers including but not limited to, germinal tumor, seminoma, anaplastic, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor); prostate cancers including but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers including but not limited to, oral squamous cell carcinoma; basal cancers; salivary gland cancers including but not limited to, adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharynx cancers including but not limited to, squamous cell cancer, and verrucous; skin cancers including but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, and superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma; kidney cancers including but not limited to, renal cell cancer, renal cancer, adenocarcinoma, hypernephroma, fibrosarcoma, and transitional cell cancer (renal pelvis and / or ureter); Wilms' tumor; bladder cancers including but not limited to, transitional cell carcinoma, squamous cell cancer, adenocarcinoma, and carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendothelioma sarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas.

[0117] In some embodiments, the cancer is benign, e.g., polyps and benign lesions. In otherembodiments, the cancer is metastatic. The compositions described herein can be used in the treatment of pre-malignant as well as malignant conditions. Pre-malignant conditions include hyperplasia, metaplasia, and dysplasia. Treatment of malignant conditions includes the treatment of primary as well as metastatic tumors. In some embodiments the cancer is melanoma, colon cancer, lung cancer, breast cancer, prostate cancer, cervical cancer, brain cancer, pancreatic cancer, or renal cancer, T-cell acute lymphocytic leukemia (ALL), a B-cell acute lymphocytic leukemia, a lymphoblastic leukemia, a B-cell chronic lymphocytic leukemia or a B-cell non-Hodgkin's lymphoma, rhabdomyosarcoma, neuroblastoma, Ewing sarcoma, gastric cancer, hepatoma. Treponematoses

[0118] The present disclosure provides therapeutic agents and methods related to treponematosesand the treatment, prevention and / or management of diseases associated with Treponema pallidum infection.

[0119] Provided herein are methods for inhibiting the growth of Treponema by contactingTreponema with at least one therapeutic agent. Also provided herein are methods of reducing Treponema in a subject. Contacting Treponema or the subject with hygromycin A can inhibit theAttorney Docket Number: 438919.001901 growth of Treponema. In some embodiments, the therapeutic agent can be hygromycin A. The Treponema can be Treponema pallidum or Treponema carateum.

[0120] In some embodiments, the concentration of hygromycin A can be from about 0.01 pg / ml toabout 100 pg / ml. In some embodiments, the concentration of hygromycin A can be about, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 pg / ml or more.

[0121] In some embodiments, the concentration of hygromycin A can be from about from about0.01 pg / ml to about 10 pg / ml.

[0122] In some embodiments, the concentration of hygromycin A can be 0.06 pg / ml. In someembodiments, the concentration of hygromycin A can be 0.12 pg / ml. In some embodiments, the concentration of hygromycin A can be 0.24 pg / ml. In some embodiments, the concentration of hygromycin A can be 0.48 pg / ml. In some embodiments, the concentration of hygromycin A can be 0.96 pg / ml. In some embodiments, the concentration of hygromycin A can be 1.92 pg / ml. In some embodiments, the concentration of hygromycin A can be 3.84 pg / ml.

[0123] The Treponema pallidum can be Treponema pallidum pallidum, Treponema pallidumpertenue, or Treponema pallidum endemicum.

[0124] The present disclosure also provides for a method for treating or preventing atreponematoses in a subject. Such methods can include contacting the subject with at least one therapeutic agent. The therapeutic agent can be hygromycin A. The treponematoses can be associated with Treponema pallidum or Treponema carateum infection. In some embodiments, the treponematoses can be syphilis. In some embodiments, the treponematoses can be yaws. In some embodiments, the treponematoses can be bejel. In some embodiments, the treponematoses can be pinta.

[0125] The present disclosure provides methods of use related to the therapeutic agents describedherein. In some embodiments, the methods can include a method of reducing the growth of bacteria such as Treponema pallidum.

[0126] In some embodiments, the therapeutic agents of the disclosure can be used to kill or inhibitthe growth of one or more sub species of Treponema pallidum. Subspecies of Treponema include, but are not limited to, Treponema pallidum pallidum, Treponema carateum, Treponema pallidum pertenue, and Treponema pallidum endemicum.

[0127] In some embodiments, the present disclosure provides methods for inhibiting the growth ofTreponema pallidum. The growth of the T. pallidum species can be inhibited in vitro (e.g., in a cell or a co-culture system, or a tissue sample from a subject) or in vivo in a subject. In some aspects, the growth of the T. pallidum can be inhibited by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%Attorney Docket Number: 438919.001901 and / or 90%. In some embodiments, the growth of the bacteria can be inhibited by 5-15%, 10-20%, 15- 25%, 20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70- 80%, 75-85%, and / or 90-100%.

[0128] In some embodiments, administering a therapeutic agent described herein to a subjectinfected with a Treponema pallidum or having a disease caused or compounded / exacerbated by Treponema pallidum, inhibits or reduces replication of Treponema pallidum by at least 20% to 25%, at least 25% to 30%, at least 30% to 35%, at least 35% to 40%, at least 40% to 45%, at least 45% to 50%, at least 50% to 55%, at least 55% to 60%, at least 60% to 65%, at least 65% to 70%, at least 70% to 75%, at least 75% to 80%, or up to at least 85% relative to a negative control as determined using an assay described herein or others known to one of skill in the art. In some embodiments, administering a therapeutic agent described herein to a subject (in some embodiments, an animal model) infected with Treponema pallidum inhibits or reduces replication of the infectious agent by at least 1.5 fold, 2 fold, 2.5 fold, 3 fold, 4 fold, 5 fold, 8 fold, 10 fold, 15 fold, 20 fold, or 2 to 5 fold, 2 to 10 fold, 5 to 10 fold, or 5 to 20 fold relative to a negative control as determined using an assay described herein or others known to one of skill in the art.

[0129] In some embodiments, the therapeutic agents of the disclosure can be used to treat, preventand / or manage venereal treponematoses or syphilis.

[0130] In some embodiments, the therapeutic agents of the disclosure can be used to treat, preventand / or manage endemic treponematoses or non-venereal treponematoses. Important differences between endemic treponematoses and syphilis relate to the target population, mode of transmission, and the tendency for systemic involvement. Endemic treponematoses predominantly affect children in the poor rural communities whereas syphilis is a universal disease. Endemic treponematoses can include bejel, yaws and pinta. Syphilis

[0131] In some embodiments, therapeutic agents of the disclosure can be used to treat Syphilis.Syphilis is a systemic disease caused by T. pallidum pallidum. The disease has been divided into stages on the basis of clinical findings, which guide treatment and follow-up. In some embodiments, the therapeutic agents of the disclosure can be used to treat primary syphilis. Primary syphilis classically presents as a single painless ulcer or chancre at the site of infection but can also present with multiple, atypical, or painful lesions. In some embodiments, the therapeutic agents of the present disclosure can be used to treat, prevent or manage secondary syphilis which is characterized by skin rash, mucocutaneous lesions, and lymphadenopathy. In some embodiments, the therapeutic agents of the disclosure can be used to treat, manage and / or prevent tertiary syphilis which can present with cardiac involvement, gummatous lesions, tabes dorsalis, and general paresis.Attorney Docket Number: 438919.001901

[0132] In some embodiments, the therapeutic agents of the disclosure can be used to treat latentinfections. Latent infections (i.e., those lacking clinical manifestations) are detected by serologic testing. Latent syphilis acquired within the preceding year is referred to as early latent syphilis; all other cases of latent syphilis are classified as late latent syphilis or latent syphilis of unknown duration.

[0133] In some embodiments, the therapeutic agents of the disclosure can be used to treat, preventor manage central nervous system (CNS) associated symptoms syphilis. T. pallidum can infect the CNS, at any stage of syphilis and result in neurosyphilis. Early neurologic clinical manifestations or syphilitic meningitis (e.g., cranial nerve dysfunction, meningitis, meningovascular syphilis, stroke, and acute altered mental status) are usually present within the first few months or years of infection. Late neurologic manifestations (e.g., tabes dorsalis and general paresis) occur 10 to >30 years after infection.

[0134] In some embodiments, the therapeutic agents of the disclosure can be used to treat, preventor manage ocular syphilis or otosyphilis. Infection of the visual system (ocular syphilis) or auditory system (otosyphilis) can occur at any stage of syphilis but is commonly identified during the early stages and can present with or without additional CNS involvement. Ocular syphilis often presents as panuveitis but can involve structures in both the anterior and posterior segment of the eye, including conjunctivitis, anterior uveitis, posterior interstitial keratitis, optic neuropathy, and retinal vasculitis. Ocular syphilis can result in permanent vision loss. Otosyphilis typically presents with cochleo- vestibular symptoms, including tinnitus, vertigo, and sensorineural hearing loss. Hearing loss can be unilateral or bilateral, have a sudden onset, and progress rapidly. Otosyphilis can result in permanent hearing loss.

[0135] In some embodiments, the concentration of hygromycin A can be about, 0.01, 0.02, 0.03,0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 pg / ml or more. Yaws

[0136] In some embodiments, therapeutic agents of the disclosure can be used to treat, prevent ormanage yaws. Transmission of yaws, caused by T. pallidum subsp. pertenue, occurs by direct skin contact with an infectious lesion and is facilitated by breaks in the skin of traumatic or other etiology (e.g., scratches or scabies). In some embodiments, the therapeutic agents of the disclosure can be used to treat one or more stages of the disease. The primary stage of the disease appears after a variable incubation period (approximately 21 days) as a solitary erythematous papule that can grow into a papilloma of 2 to 5 cm in diameter by peripheral extension or by coalescing with satellite papules. The lesion is not painful but can be pruritic, and it is typically covered by a crust that hides an ulcer withAttorney Docket Number: 438919.001901 raised dark margins and an erythematous moist center, overall resembling a raspberry (hence the African and French names). In some embodiments, the therapeutic agents of the disclosure can be used to treat, prevent and / or manage the primary lesion, which is often found on the lower extremities. The lesion is highly contagious and can persist for weeks or months before healing spontaneously, often leaving a hypopigmented or depressed area delimited by a dark border. At this stage, regional lymphadenopathy and arthralgia can also occur. In some embodiments, the therapeutic agents of the disclosure can be used to treat lymphadenopathy and / or arthralgia associated with yaws. In the majority of cases, the primary lesion heals spontaneously before the onset of yaws' secondary manifestations, whose appearance is due to the pathogen's systemic dissemination during early infection. In some embodiments, the therapeutic agents of the disclosure can be used to treat systemic dissemination associated with yaws. In some embodiments, the therapeutic agents of the disclosure can be used to treat, manage or prevent secondary manifestations associated with yaws. Secondary manifestations can include condylomata lata in moist crevices such as the axilla and groin and a measles-like eruption. Hyperkeratotic plaques on palms and soles are also common at this stage. In some embodiments, the therapeutic agents of the disclosure can be used to treat periostitis and osteitis which can affect the bones of the upper and lower limbs (tibia, fibula, and forearm) and the proximal phalanges of fingers and toes, resulting in bone pain and digital swelling. In some embodiments, the therapeutic agents of the disclosure can be used to treat latent disease. The secondary lesions heal spontaneously within weeks or months, and the patient enters the latent stage of the infection, which can be recognized only through serological tests and will, unless treated, last for a lifetime.

[0137] Recurrences (generally one or two) of secondary manifestations might be seen up to 5 yearsafter the initial infection, although relapses after 10 years have been reported.

[0138] In some embodiments, the therapeutic agents of the disclosure can be used to treat, preventor manage tertiary yaws. Approximately 10% of untreated patients will develop tertiary yaws, characterized by subcutaneous gummatous nodules, chronic periostitis that can cause apparent bowing of the tibia (i.e., saber shin), and destructive processes leading to saddle nose and perforation / collapse of the palate and nasal septum (i.e., gangosa). Bilateral hypertrophic periostitis of the paranasal maxilla and nasal bridge causes the clinical manifestation known as goundou. Bejel

[0139] In some embodiments, the therapeutic agents of the disclosure can be used to treat Bejel.Bejel is the Arabic name for endemic (or nonvenereal) syphilis caused by T. pallidum subsp. endemicum. In some embodiments, the therapeutic agents of the disclosure can be used to treat acute infection, which is observed in in children between 2 and 15 years of age in dry, arid climates.Attorney Docket Number: 438919.001901

[0140] Although the mode of transmission has not been adequately studied, it is believed to occurthrough mucosal and skin contact or the sharing of eating utensils or drinking vessels.

[0141] In contrast to the other treponematoses, bejel's primary lesion is often unobserved. Whenseen, however, it appears as a small and painless mucous papule or ulcer that develops in the oral cavity or nasopharynx. In some embodiments, the therapeutic agents of the disclosure can be used to treat primary and / or secondary lesions associated with bejel. Secondary lesions are very similar to those of venereal syphilis and can manifest as mucous patches on the oral mucosa, tonsils, tongue, lips, and nasopharynx. Split papules at the labial commissures (angular stomatitis, as in yaws patients), non-itchy skin eruptions, generalized lymphadenopathy, and laryngitis are common manifestations. Secondary skin lesions include condylomata lata in intertriginous body areas, comparable to those in yaws and syphilis. Maculopapular or papulosquamous lesions, as well as a nonpruritic generalized papular rash, can be observed in a minority of patients with bejel. As in yaws, osteitis and periostitis of the long bones and hands can occur, causing nocturnal bone pain. Secondary manifestations heal in 6 to 9 months, and the disease enters latency. In some embodiments, the therapeutic agents of the present disclosure can be used to treat tertiary stage of bejel. The tertiary stage might manifest earlier than for yaws (6 months to several years) but, as in yaws, is characterized by gummatous lesions of the skin, mucosa, and bone that can progress to destructive ulcers. Skin lesions resolve in time and leave characteristic depigmented scars surrounded by hyperpigmentation.

[0142] In some embodiments, the concentration of hygromycin A can be about, 0.01, 0.02, 0.03,0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 pg / ml or more. Pinta

[0143] In some embodiments, the therapeutic agents of the present disclosure can be used to treat,prevent and / or manage Pinta (also known as mal de pinto, enfermedad azul and carate or cute). In some embodiments, the therapeutic agents of the disclosure can be used to treat Pinta caused by Treponema carateum, and is regarded as the mildest of the treponematoses, in that its lesions are limited to the skin and there is no evidence of systemic involvement or vertical transmission. The disease is found focally in tropical Central and South America. Because no laboratory strain of this pathogen is currently available for studies, T. carateum is the least characterized of the agents of the human treponematoses and is still classified independently from the other T. pallidum subspecies, whose genetic and antigenic relatedness has been experimentally demonstrated. The mechanism of transmission is unknown, although repeated skin-to-skin contact appears to be the most plausible. Young adults (<15 years old) with chronic skin lesions are considered to be the disease's main reservoir. Pinta also appears to be transmitted to infants from their mothers by close contact. In someAttorney Docket Number: 438919.001901 embodiments, the therapeutic agents of the disclosure can be used to treat early or late stages of the Pinta. The primary lesion manifests as a papule or an erythemato-squamous plaque on exposed parts of the body after an incubation period of 1 week to 2 months. Satellite lesions can be present. With time, the papules increase in size and coalesce to form patches with a pale center. After months, many of the patches become hypochromic or acquire a light-blue / grayish pigmentation, with the color more marked at the center of the lesion. The initial lesions can either heal, leaving a slightly pigmented or hypochromic area, or persist for years and become indistinguishable from the secondary lesions. Regional lymphadenopathy is common at this stage. After months or years, small disseminated secondary lesions (called pintids) can appear in the form of scaly papules that will again enlarge and coalesce in psoriasiform plaques. These plaques might become hypo- or hyperchromic, as well as erythematous or desquamative. Different types of pintids might be present at the same time in the same individual. This stage usually lasts 2 to 4 years, during which some patches will heal, and others will persist and enlarge. The late stage usually develops 2 to 4 years after initial infection and is characterized by the appearance of pigmentary changes, skin atrophy, and hyperkeratosis.

[0144] In some embodiments, the concentration of hygromycin A can be about, 0.01, 0.02, 0.03,0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 pg / ml or more. IV. FORMULATIONS

[0145] In some embodiments, therapeutic agents can be administered to humans, human patients orsubjects. For the purposes of the present disclosure, the phrase “active ingredient” generally refers to the therapeutic agents to be delivered as described herein.

[0146] Although the descriptions of formulations provided herein are principally directed toformulations which are suitable for administration to humans, it will be understood by the skilled artisan that such therapeutic agents are generally suitable for administration to any other animal, e.g., to non- human animals, e.g., non-human mammals. Modification of formulations suitable for administration to humans in order to render the therapeutic agents suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the formulations is contemplated include, but are not limited to, humans and / or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and / or turkeys.

[0147] Formulations of the therapeutic agents described herein can be prepared by any methodknown or hereafter developed in the art of pharmacology. In general, such preparatory methods includeAttorney Docket Number: 438919.001901 the step of bringing the active ingredient into association with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, dividing, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0148] A formulation in accordance with the disclosure can be prepared, packaged, and / or sold inbulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the therapeutic agents is generally equal to the dosage of the active ingredient which would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0149] Relative amounts of the therapeutic agents, the pharmaceutically acceptable excipient, and / orany additional ingredients in a formulation in accordance with the disclosure will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the formulation is to be administered. By way of example, the formulation can include between 0.1% and 100%, e.g., between .5 and 50%, between 1-30%, between 5-80%, or, in some embodiments, at least 20%, at least 40%, at least 60%, or at least 80% (w / w) of the therapeutic agents.

[0150] The therapeutic agents of the present disclosure can be formulated using one or moreexcipients to: (1) increase stability; (2) permit the sustained or delayed release; (3) alter the biodistribution; (4) alter the release profile of the therapeutic agents in vivo. Non-limiting examples of the excipients include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, and preservatives. Excipients of the present disclosure can also include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, hyaluronidase, nanoparticle mimics and combinations thereof.

[0151] In some embodiments, pharmaceutical compositions or formulations of the disclosure can beadapted to deliver a prescribed dosage of one or more therapeutic agents to a cell, a group of cells, an organ or tissue, an animal or a human. Methods of incorporating therapeutic agents into pharmaceutical preparations are widely known in the art. The determination of an appropriate prescribed dosage of a pharmacologically active compound to include in a pharmaceutical formulation in order to achieve a desired biological outcome is within the skill level of an ordinary practitioner of the art. The pharmaceutical formulation can include excipients, such as without limitation, binders, coating, disintegrants, fillers, diluents, flavors, colors, lubricants, glidants, preservatives, sorbents, sweeteners, conjugated linoleic acid (CLA), gelatin, beeswax, purified water, glycerol, any type of oil, including, without limitation, fish oil or soybean oil, or the like. Therapeutic agents and / or pharmaceutical formulations can comprise suitable solid or gel phase carriers or excipients. Examples of such carriersAttorney Docket Number: 438919.001901 or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as, e.g., polyethylene glycols. It will further be appreciated by an ordinary practitioner of the art that the term also encompasses those therapeutic agents and / or pharmaceutical formulations that contain an admixture of two or more pharmacologically active compounds, such compounds being administered, for example, as a combination therapy.

[0152] A pharmaceutical formulation in accordance with the present disclosure can be prepared,packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical formulation comprising a predetermined amount of therapeutic agent or other compounds. The amount of therapeutic agent can generally be equal to the dosage of therapeutic agent administered to a subject and / or a convenient fraction of such dosage including, but not limited to, one-half or one-third of such a dosage.

[0153] In some embodiments, the therapeutic agents of the disclosure can be formulated as atoothpaste, a mouth wash, a gum massage cream, an edible film, or floss. Oral formulations

[0154] In some embodiments, the therapeutic agents can be formulated as, e.g., a suspension, anemulsion, a hydrogel, a paste, a multiphase solution, a vesicular dispersion, a liquid, a gel, or a powder, for use in, e.g., a toothpaste, a mouthwash, a mouth and / or throat spray, a lozenge, a breath mint, a pen or tube with a brush, sponge, or nozzle applicator, a dissolvable strip, an adhesive tape for the teeth, a toothpick, a chewing gum, a tongue scraper, a lip balm, a syringe, a nasal aspirator, a dental floss and / or any other oral care product known in the art.

[0155] In some embodiments, the present disclosure provides for oral Formulations can additionallycomprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington’s The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.Attorney Docket Number: 438919.001901

[0156] In some embodiments, any of the therapeutic agents, compositions, and formulationsdescribed in International Patent Publication WO2023049849 may be useful in the present disclosure (the contents of which are herein incorporated by reference in its entirety). Excipients

[0157] Formulations can additionally comprise a pharmaceutically acceptable excipient, which, asused herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington’s The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.

[0158] In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0159] Pharmaceutically acceptable excipients used in the manufacture of pharmaceuticalcompositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Such excipients can optionally be included in pharmaceutical compositions.

[0160] In some embodiments, the sugar alcohols may be used as excipients to enhance access oftherapeutic agents to the microenvironment of the infection. Non-limiting example of sugar alcohols include xylitol, ad / or allulose.

[0161] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate,calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and / or combinations thereof.Attorney Docket Number: 438919.001901

[0162] Exemplary granulating and / or dispersing agents include, but are not limited to, potato starch,corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, etc., and / or combinations thereof.

[0163] Exemplary surface active agents and / or emulsifiers include, but are not limited to, naturalemulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEENn®60], polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ®45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether [BRIJ®30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER®188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and / or combinations thereof.

[0164] Exemplary binding agents include, but are not limited to, starch (e.g., cornstarch and starchpaste); gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystallineAttorney Docket Number: 438919.001901 cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; etc.; and combinations thereof.

[0165] Exemplary preservatives can include, but are not limited to, antioxidants, chelating agents,antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and / or phenylethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL®115, GERMABEN®II, NEOLONE™, KATHON™, and / or EUXYL®.

[0166] Exemplary buffering agents include, but are not limited to, citrate buffer solutions, acetatebuffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate,Attorney Docket Number: 438919.001901 sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer’s solution, ethyl alcohol, etc., and / or combinations thereof.

[0167] Exemplary lubricating agents include, but are not limited to, magnesium stearate, calciumstearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.

[0168] Exemplary oils include, but are not limited to, almond, apricot kernel, avocado, babassu,bergamot, black current seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and / or combinations thereof.

[0169] Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents,sweetening, flavoring, and / or perfuming agents can be present in the composition, according to the judgment of the formulator.

[0170] In some embodiments, therapeutic agents can be formulated for applications of a topical type,preferably in the form of a solution, suspension, powder for gynecological suspensions, vaginal suppositories, vaginal capsules, vaginal douches, vaginal creams or enemas.

[0171] In some embodiment, the therapeutic agents, e.g., probiotic compositions are preparedextemporaneously by mixing the composition with water or with a saline solution. In some embodiments, probiotic compositions described herein may contain one or more antioxidants. Examples of suitable antioxidants include, but are not limited to, vitamin C and E. Vitamin C, when incorporated in the formulation in the form of ascorbic acid, serves as a preservative and also helps to reduce vaginal pH upon administration of the formulation. Vitamin E may act as a preservative and may also have an anti-inflammatory effect on the vaginal tissues. The probiotic compositions or formulations thereof can also contain various other components, in addition to those described above. Non-limiting examples of such additional components are disintegrants, preservatives, dyes, fragrances or natural extracts, suchAttorney Docket Number: 438919.001901 as plant extracts. Some examples of additional ingredients are guar gum, citric acid and / or aloe vera extract in powdered form. V. DOSING AND ADMINISTRATION

[0172] In some embodiments, therapeutic agents and / or pharmaceutical formulations that includetherapeutic agents can be administered according to one or more administration routes. In some embodiments, administration is enteral (into the intestine), transdermal, intravenous bolus, intralesional (within or introduced directly to a localized lesion), intrapulmonary (within the lungs or its bronchi), diagnostic, intraocular (within the eye), transtympanic (across or through the tympanic cavity), intravesical infusion, sublingual, nasogastric (through the nose and into the stomach), spinal, intracartilaginous (within a cartilage), insufflation (snorting), rectal, intravascular (within a vessel or vessels), buccal (directed toward the cheek), dental (to a tooth or teeth), intratesticular (within the testicle), intratympanic (within the aurus media), percutaneous, intrathoracic (within the thorax), submucosal, cutaneous, epicutaneous (application onto the skin), dental intracornal, intramedullary (within the marrow cavity of a bone), intra-abdominal, epidural (into the dura matter), intramuscular (into a muscle), intralymphatic (within the lymph), iontophoresis (by means of electric current where ions of soluble salts migrate into the tissues of the body), subcutaneous (under the skin), intragastric (within the stomach), nasal administration (through the nose), transvaginal, intravenous drip, endosinusial, intraprostatic (within the prostate gland), soft tissue, intradural (within or beneath the dura), subconjunctival, oral (by way of the mouth), peridural, parenteral, intraduodenal (within the duodenum), intracisternal (within the cisterna magna cerebellomedularis), periodontal, periarticular, biliary perfusion, intracoronary (within the coronary arteries), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrameningeal (within the meninges), intracavernous injection (into a pathologic cavity) intracavitary (into the base of the penis), intrabiliary, subarachnoid, intrabursal, ureteral (to the ureter), intratendinous (within a tendon), auricular (in or by way of the ear), intracardiac (into the heart), enema, intraepidermal (to the epidermis), intraventricular (within a ventricle), intramyocardial (within the myocardium), intratubular (within the tubules of an organ), vaginal, sublabial, intracorporus cavernosum (within the dilatable spaces of the corporus cavernosa of the penis), intradermal (into the skin itself), intravitreal (through the eye), perineural, cardiac perfusion, irrigation (to bathe or flush open wounds or body cavities), in ear drops, endotracheal, intraosseous infusion (into the bone marrow), caudal block, intrauterine, transtracheal (through the wall of the trachea), intra-articular, intracorneal (within the cornea), endocervical, extracorporeal, intraspinal (within the vertebral column), transmucosal (diffusion through a mucous membrane), topical, photopheresis, oropharyngeal (directly to the mouth and pharynx), occlusive dressing technique (topical route administration which is then covered by a dressing which occludes the area), transplacentalAttorney Docket Number: 438919.001901 (through or across the placenta), intrapericardial (within the pericardium), intraarterial (into an artery), interstitial, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), intrapleural (within the pleura), infiltration, intrabronchial, intrasinal (within the nasal or periorbital sinuses), intraductal (within a duct of a gland), intracaudal (within the cauda equine), nerve block, retrobulbar (behind the pons or behind the eyeball), intravenous (into a vein), intra-amniotic, conjunctival, intrasynovial (within the synovial cavity of a joint), gastroenteral, intraluminal (within a lumen of a tube), electro-osmosis, intraileal (within the distal portion of the small intestine), intraesophageal (to the esophagus), extra-amniotic administration, hemodialysis, intragingival (within the gingivae), intratumor (within a tumor), eye drops (onto the conjunctiva), laryngeal (directly upon the larynx), urethral (to the urethra), intravaginal administration, intraperitoneal (infusion or injection into the peritoneum), respiratory (within the respiratory tract by inhaling orally or nasally for local or systemic effect), intradiscal (within a disc), ophthalmic (to the external eye), and / or intraovarian (within the ovary).

[0173] In some embodiments, the administration can take place through any route. The therapeuticagents can be administered via the topical route, via the vaginal, intrauterine or intratubal route. In some embodiments, the therapeutic agents can be administered via the intrauterine route through injection by means of an intrauterine catheter or a transcervical catheter. In some embodiments, the therapeutic agents can be administered into the tubes of the uterus (salpinges). The therapeutic agents can be administered via the intratubal route through injection by means of an intratubal catheter.

[0174] In some embodiments, therapeutic agents and / or pharmaceutical formulations that includetherapeutic agents can be administered by intraarticular administration, extracorporeal administration, intrabronchial administration, endocervical administration, endosinusial administration, endotracheal administration, enteral administration, epidural administration, intra-abdominal administration, intrabiliary administration, intrabursal administration, oropharyngeal administration, interstitial administration, intracardiac administration, intracartilaginous administration, intracaudal administration, intracavernous administration, intracerebral administration, intracorporus cavernosum, intracavitary administration, intracorneal administration, intracisternal administration, cranial administration, intracranial administration, intradermal administration, intralesional administration, intratympanic administration, intragingival administration, intraocular administration, intradiscal administration, intraductal administration, intraduodenal administration, ophthalmic administration, intradural administration, intraepidermal administration, intraesophageal administration, nasogastric administration, nasal administration, laryngeal administration, intraventricular administration, intragastric administration, intrahepatic administration, intraluminal administration, intravitreal administration, intravesicular administration, intralymphatic administration, intramammaryAttorney Docket Number: 438919.001901 administration, intramedullary administration, intrasinal administration, intrameningeal administration, intranodal administration, intraovarian administration, intraperitoneal administration, intrapleural administration, intraprostatic administration, intraluminal administration, intraspinal administration, intrasynovial administration, intratendinous administration, intratesticular administration, subconjunctival administration, intracerebroventricular administration, epicutaneous administration, intravenous administration, retrobulbar administration, periarticular administration, intrathoracic administration, subarachnoid administration, intratubular administration, periodontal administration, transtympanic administration, transtracheal administration, intratumor administration, vaginal administration, urethral administration, intrauterine administration, oral administration, gastroenteral administration, parenteral administration, sublingual administration, ureteral administration, percutaneous administration, peridural administration, transmucosal administration, perineural administration, transdermal administration, rectal administration, soft tissue administration, intraarterial administration, subcutaneous administration, topical administration, extra-amniotic administration, ear drops, or intravesical infusion.

[0175] Therapeutic agents and / or pharmaceutical formulations of the present disclosure can beadministered orally but any suitable route of administration can be employed for providing a subject with an effective dosage of drugs of the therapeutic agents described herein. For example, oral, rectal, topical, parenteral, ocular, pulmonary, nasal, and the like can be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols. In some embodiments, the therapeutic agents can be administered intravaginally as vaginal tablets, vaginal vegetarian (cellulose) capsules, vaginal cream, vaginal gel, vaginal ointment, vaginal suppositories, vaginal lotion, intrauterine devices, vaginal tampons or pessaries and the like.

[0176] In some embodiments, the therapeutic agents can be applied to the subject. As used herein,“applying” can refer to any of the following exemplary modes of application: using a toothbrush to scrub and / or polish the teeth and / or gums with the therapeutic agents; using a brush or sponge, or a pen fitted with a brush or sponge, to paint or spread the composition onto a surface, e.g., the subject’s teeth and gums; adhering temporary strips or tapes having the composition thereon to the teeth for a certain number of minutes, e.g., for 20 to 60 minutes; rinsing and / or swishing the composition inside the mouth (e.g., as a mouthwash); spraying or misting the therapeutic agents into the mouth; dissolving a strip, lozenge or mint on the tongue or in the cheek; placing the therapeutic agents into a tooth mold or mouth guard and soaking the teeth in the therapeutic agents; flossing or picking in between the teeth with a floss or toothpick impregnated or coated with the therapeutic agents; scraping the tongue with a tongue scraper coated with the therapeutic agents; using the finger or tube to spread the therapeutic agents inside or on the lips (e.g., as a mouth sore treatment or lip balm); spraying the therapeutic agents into theAttorney Docket Number: 438919.001901 nostrils and to the pharynx (e.g., using a nasal aerator); and / or any other mode of applying dental and / or oral treatments to the surfaces of a subject’s mouth, lips and / or oral cavity.

[0177] Therapeutic agents and / or pharmaceutical formulations of the present disclosure can beadministered in the conventional manner by any route where they are active. Administration can be systemic, parenteral, topical, or oral. For example, administration can be, but is not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral, buccal, or ocular routes, or intravaginally, by inhalation, by depot injections, or by implants. Thus, modes of administration of the composition of the present disclosure (either alone or in combination with other pharmaceuticals) can be, but are not limited to, sublingual, injectable (including short-acting, depot, implant and pellet forms injected subcutaneously or intramuscularly), or by use of vaginal creams, suppositories, pessaries, vaginal rings, rectal suppositories, intrauterine devices, and transdermal forms such as patches and creams.

[0178] For administration by inhalation or intranasal, pharmaceutical formulation can be deliveredin the form of an aerosol spray presentation from pressurized packs or nebulizers. The compounds can also be delivered in the form of a cream, liquid, spray, powder, or suppository. A metered dose of the formulation can be provided from a reservoir of the formulation. In addition, predetermined dosages can be provided, for example, suppository forms can be provided for insertion into the nose having a predetermined dosage. Kits can be provided, where prepared dosage forms and instructions for administering the dosages are included.

[0179] Suitable topical formulations for use in the present embodiments can also include transdermaldevices, aerosols, creams, ointments, lotions, dusting powders, gels, and the like. Dosing

[0180] Therapeutic agents and / or pharmaceutical formulations described herein can be administeredto a subject using any amount and any route of administration effective treating a disease, disorder, and / or condition. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular formulation, its mode of administration, its mode of activity, and the like.

[0181] In some embodiments, the therapeutic agents described herein can be provided to theVXEMHFW DW D FRQFHQWUDWLRQ RI IURP DERXW )')* gJ(PO WR DERXW *)) gJ(PO' :Q VRPH HPERGLPHQWV% WKHtherapeutic agents can be provided to the subject at a concentration of from about 0.01 to about)'). gJ(PO% IURP DERXW )'). gJ(PO WR DERXW *') gJ(PO% IURP DERXW *') gJ(PO WR DERXW *) gJ(PO%from about 10 gJ(PO WR DERXW .) gJ(PO% IURP DERXW .) gJ(PO WR DERXW *)) gJ(PO' :Q VRPHembodiments, the therapeutic agents described herein can be provided to the subject at aFRQFHQWUDWLRQ RI IURP DERXW * gJ(PO WR DERXW . gJ(PO% IURP DERXW . gJ(PO WR DERXW *) gJ(PO% fromAttorney Docket Number: 438919.001901DERXW *) gJ(PO WR DERXW +) gJ(PO% IURP DERXW +) gJ(PO WR DERXW ,) gJ(PO% IURP DERXW ,) gJ(PO WRDERXW -) gJ(PO% IURP DERXW -) gJ(PO WR DERXW .) gJ(PO% IURP DERXW .) gJ(PO WR DERXW / ) gJ(PO%IURP DERXW / ) gJ(PO WR DERXW 0) gJ(PO% IURP DERXW 0) gJ(PO WR DERXW 1) gJ(PO% from about 80gJ(PO WR DERXW 2) gJ(PO% IURP DERXW 2) gJ(PO WR DERXW *)) gJ(PO'

[0182] In some embodiments, the concentration of hygromycin A can be about, 0.01, 0.02, 0.03,0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1, 2, 3, 4, 5, 6, 7, 8,2% *)% *.% +)% +.% ,)% ,.% -)% -.% .)% ..% / )% / .% 0)% 0.% 1)% 1.% 2)% 2.% *)) gJ(PO RU PRUH'

[0183] In some embodiments, the composition is administered as a single tier dosing regimen. Insome embodiments, the single tier dosing regimen comprises a high dose of hygromycin A. In some embodiments, the single tier dosing comprises a low dose of hygromycin. In some embodiments, the composition is administered as a two-tiered dosing regimen, comprising a first high dose and one or more maintenance doses. In some embodiments, the first high dose is administered orally to the subject. In some embodiments, the one or more maintenance doses are applied to the surface of the subject’s mouth, lips and / or oral cavity. In some embodiments, the hygromycin A does not inhibit the growth of or kill at least one species of beneficial oral bacteria. In some embodiments, the high dose or the first high dose of hygromycin A is about 100 milligram per kg to 1500 milligram per kg body weight. In some embodiments, the high dose or the first high dose of hygromycin A is about 1,000 milligram per kg. In some embodiments, the low dose or the one or more maintenance dose of hygromycin A is from 0.001 milligram per kg to 0.1 milligram per kg body weight. In some embodiments, the low dose or the one or more maintenance dose is 0.01 milligram per kg body weight.. In some embodiments, the high dose or the first high dose is from 100 milligram per kg to 1500 milligram per kg body weight, from 150 milligram per kg to 1500 milligram per kg body weight, from 200 milligram per kg to 1500 milligram per kg body weight, from 250 milligram per kg to 1500 milligram per kg body weight, from 300 milligram per kg to 1500 milligram per kg body weight, from 350 milligram per kg to 1500 milligram per kg body weight, from 400 milligram per kg to 1500 milligram per kg body weight, from 450 milligram per kg to 1500 milligram per kg body weight, from 500 milligram per kg to 1500 milligram per kg body weight, from 550 milligram per kg to 1500 milligram per kg body weight, from 600 milligram per kg to 1500 milligram per kg body weight, from 650 milligram per kg to 1500 milligram per kg body weight, from 700 milligram per kg to 1500 milligram per kg body weight, from 750 milligram per kg to 1500 milligram per kg body weight, from 800 milligram per kg to 1500 milligram per kg body weight, from 850 milligram per kg to 1500 milligram per kg body weight, from 900 milligram per kg to 1500 milligram per kg body weight, from 950 milligram per kg to 1500 milligram per kg body weight, from 1000 milligram per kg to 1500 milligram per kg body weight, from 1050 milligram per kg to 1500 milligram per kg body weight,Attorney Docket Number: 438919.001901 from 1100 milligram per kg to 1500 milligram per kg body weight, from 1150 milligram per kg to 1500 milligram per kg body weight, from 1200 milligram per kg to 1500 milligram per kg body weight, from 1250 milligram per kg to 1500 milligram per kg body weight, from 1300 milligram per kg to 1500 milligram per kg body weight, from 1350 milligram per kg to 1500 milligram per kg body weight, from 1400 milligram per kg to 1500 milligram per kg body weight, from 1450 milligram per kg to 1500 milligram per kg body weight, from 1500 milligram per kg to 1500 milligram per kg body weight, from 100 milligram per kg to 150 milligram per kg body weight, from 100 milligram per kg to 200 milligram per kg body weight, from 100 milligram per kg to 250 milligram per kg body weight, from 100 milligram per kg to 300 milligram per kg body weight, from 100 milligram per kg to 350 milligram per kg body weight, from 100 milligram per kg to 400 milligram per kg body weight, from 100 milligram per kg to 450 milligram per kg body weight, from 100 milligram per kg to 500 milligram per kg body weight, from 100 milligram per kg to 550 milligram per kg body weight, from 100 milligram per kg to 600 milligram per kg body weight, from 100 milligram per kg to 650 milligram per kg body weight, from 100 milligram per kg to 700 milligram per kg body weight, from 100 milligram per kg to 750 milligram per kg body weight, from 100 milligram per kg to 800 milligram per kg body weight, from 100 milligram per kg to 850 milligram per kg body weight, from 100 milligram per kg to 900 milligram per kg body weight, from 100 milligram per kg to 950 milligram per kg body weight, from 100 milligram per kg to 1000 milligram per kg body weight, from 100 milligram per kg to 1050 milligram per kg body weight, from 100 milligram per kg to 1100 milligram per kg body weight, from 100 milligram per kg to 1150 milligram per kg body weight, from 100 milligram per kg to 1200 milligram per kg body weight, from 100 milligram per kg to 1250 milligram per kg body weight, from 100 milligram per kg to 1300 milligram per kg body weight, from 100 milligram per kg to 1350 milligram per kg body weight, from 100 milligram per kg to 1400 milligram per kg body weight, or from 100 milligram per kg to 1450 milligram per kg body weight. In some embodiments, the low dose or the one or more maintenance dose is 0.001 milligram per kg to 0.01 milligram per kg body weight ,0.002 milligram per kg to 0.02 milligram per kg body weight ,0.003 milligram per kg to 0.03 milligram per kg body weight ,0.004 milligram per kg to 0.04 milligram per kg body weight ,0.005 milligram per kg to 0.05 milligram per kg body weight ,0.006 milligram per kg to 0.06 milligram per kg body weight ,0.007 milligram per kg to 0.07 milligram per kg body weight ,0.008 milligram per kg to 0.08 milligram per kg body weight ,0.009 milligram per kg to 0.09 milligram per kg body weight ,0.01 milligram per kg to 0.1 milligram per kg body weight ,0.02 milligram per kg to 0.2 milligram per kg body weight ,0.03 milligram per kg to 0.3 milligram per kg body weight ,0.04 milligram per kg to 0.4 milligram per kg body weight ,0.05 milligram per kg to 0.5 milligram per kg body weight ,0.06 milligram per kg to 0.6 milligram per kg body weight ,0.07Attorney Docket Number: 438919.001901 milligram per kg to 0.7 milligram per kg body weight ,0.08 milligram per kg to 0.8 milligram per kg body weight ,0.09 milligram per kg to 0.9 milligram per kg body weight , or 0.1 milligram per kg to 1 milligram per kg body weight .

[0184] In some embodiments the therapeutic agents described herein can be provided to theVXEMHFW DW D FRQFHQWUDWLRQ RI )'). gJ(PO'

[0185] In some embodiments the therapeutic agents described herein can be provided to the subjectDW D FRQFHQWUDWLRQ RI )') / gJ(PO' :Q VRPH HPERGLPHQWV WKH WKHUDSHXWLF DJHQWV GHVFULEHG KHUHLQ FDQ EHSURYLGHG WR WKH VXEMHFW DW D FRQFHQWUDWLRQ RI )'*+ gJ(PO' :Q VRPH HPERGLPHQWV WKH WKHUDSHXWLF DJHQWVGHVFULEHG KHUHLQ FDQ EH SURYLGHG WR WKH VXEMHFW DW D FRQFHQWUDWLRQ RI )'+- gJ(PO' :Q VRPH HPERGLPHQWVWKH WKHUDSHXWLF DJHQWV GHVFULEHG KHUHLQ FDQ EH SURYLGHG WR WKH VXEMHFW DW D FRQFHQWUDWLRQ RI )'-1 gJ(PO'In some embodiments the therapeutic agents described herein can be provided to the subject at aFRQFHQWUDWLRQ RI )'2 / gJ(PO' :Q VRPH HPERGLPHQWV WKH WKHUDSHXWLF DJHQWV GHVFULEHG KHUHLQ FDQ EHSURYLGHG WR WKH VXEMHFW DW D FRQFHQWUDWLRQ RI *'2+ gJ(PO' :Q VRPH HPERGLPHQWV WKH WKHUDSHXWLF DJHQWVGHVFULEHG KHUHLQ FDQ EH SURYLGHG WR WKH VXEMHFW DW D FRQFHQWUDWLRQ RI ,'1- gJ(PO' In some embodimentsthe therapeutic agents described herein can be provided to the subject at a concentration of 0.1gJ(PO' :Q VRPH HPERGLPHQWV WKH WKHUDSHXWLF DJHQWV GHVFULEHG KHUHLQ FDQ EH SURYLGHG WR WKH VXEMHFWDW D FRQFHQWUDWLRQ RI )'+ gJ(PO' :Q VRPH HPERGLPHQWV WKH WKHUDSHXWLF DJHQWV GHVFULEHG KHUHLQ FDQEH SURYLGHG WR WKH VXEMHFW DW D FRQFHQWUDWLRQ RI )'. gJ(PO' :Q VRPH HPERGLPHQWV WKH WKHUDSHXWLFDJHQWV GHVFULEHG KHUHLQ FDQ EH SURYLGHG WR WKH VXEMHFW DW D FRQFHQWUDWLRQ RI *') gJ(PO' :Q VRPHembodiments the therapeutic agents described herein can be provided to the subject at aFRQFHQWUDWLRQ RI +') gJ(PO' :Q VRPH HPERGLPHQWV WKH WKHUDSHXWLF DJHQWV GHVFULEHG KHUHLQ FDQ EHSURYLGHG WR WKH VXEMHFW DW D FRQFHQWUDWLRQ RI .') gJ(PO' In some embodiments the therapeutic agentsGHVFULEHG KHUHLQ FDQ EH SURYLGHG WR WKH VXEMHFW DW D FRQFHQWUDWLRQ RI *) gJ(PO' :Q VRPH HPERGLPHQWVWKH WKHUDSHXWLF DJHQWV GHVFULEHG KHUHLQ FDQ EH SURYLGHG WR WKH VXEMHFW DW D FRQFHQWUDWLRQ RI +) gJ(PO' :Qsome embodiments the therapeutic agents described herein can be provided to the subject at aFRQFHQWUDWLRQ RI -) gJ(PO'

[0186] In some embodiments, the probiotic compositions can be administered at a dose of about 1x103,1 x 104, 1 x 105, 1x106, 1 x 107, 1x 108, 1x109, 1x 1010or more colony forming units (CFU) of one or more microorganisms.

[0187] In some embodiments, therapeutic agents and / or pharmaceutical formulations of the presentdisclosure are provided in one or more doses and are administered one or more times to subjects. Some therapeutic agents and / or pharmaceutical formulations are provided in only a single administration. Some therapeutic agents and / or pharmaceutical formulations are provided according to a dosing schedule that include two or more administrations. Each administration can be at the same dose or canAttorney Docket Number: 438919.001901 be different from a previous and / or subsequent dose. In some embodiments, subjects are provided an initial dose that is higher than subsequent doses (referred to herein as a “loading dose”). In some embodiments, doses are decreased over the course of administration. In some embodiments, dosing schedules include pharmaceutical formulation administration from about every 2 hours to about every 10 hours, from about every 4 hours to about every 20 hours, from about every 6 hours to about every 30 hours, from about every 8 hours to about every 40 hours, from about every 10 hours to about every 50 hours, from about every 12 hours to about every 60 hours, from about every 14 hours to about every 70 hours, from about every 16 hours to about every 80 hours, from about every 18 hours to about every 90 hours, from about every 20 hours to about every 100 hours, from about every 22 hours to about every 120 hours, from about every 24 hours to about every 132 hours, from about every 30 hours to about every 144 hours, from about every 36 hours to about every 156 hours, from about every 48 hours to about every 168 hours, from about every 2 days to about every 10 days, from about every 4 days to about every 15 days, from about every 6 days to about every 20 days, from about every 8 days to about every 25 days, from about every 10 days to about every 30 days, from about every 12 days to about every 35 days, from about every 14 days to about every 40 days, from about every 16 days to about every 45 days, from about every 18 days to about every 50 days, from about every 20 days to about every 55 days, from about every 22 days to about every 60 days, from about every 24 days to about every 65 days, from about every 30 days to about every 70 days, from about every 2 weeks to about every 8 weeks, from about every 3 weeks to about every 12 weeks, from about every 4 weeks to about every 16 weeks, from about every 5 weeks to about every 20 weeks, from about every 6 weeks to about every 24 weeks, from about every 7 weeks to about every 28 weeks, from about every 8 weeks to about every 32 weeks, from about every 9 weeks to about every 36 weeks, from about every 10 weeks to about every 40 weeks, from about every 11 weeks to about every 44 weeks, from about every 12 weeks to about every 48 weeks, from about every 14 weeks to about every 52 weeks, from about every 16 weeks to about every 56 weeks, from about every 20 weeks to about every 60 weeks, from about every 2 months to about every 6 months, from about every 3 months to about every 12 months, from about every 4 months to about every 18 months, from about every 5 months to about every 24 months, from about every 6 months to about every 30 months, from about every 7 months to about every 36 months, from about every 8 months to about every 42 months, from about every 9 months to about every 48 months, from about every 10 months to about every 54 months, from about every 11 months to about every 60 months, from about every 12 months to about every 66 months, from about 2 years to about 5 years, from about 3 years to about 10 years, from about 4 years to about 15 years, from about 5 years to about 20 years, from about 6 years to about 25 years, from about 7 years to about 30 years, from about 8 yearsAttorney Docket Number: 438919.001901 to about 35 years, from about 9 years to about 40 years, from about 10 years to about 45 years, from about 15 years to about 50 years, or more than every 50 years.

[0188] The desired dosage can be delivered for a duration of about 5 days to 365 days, about 5 daysto 300 days, about 5 days to 300 days, about 5 days to 250 days, about 5 days to 200 days, about 5 days to 100 days, about 5 days to 60 days, about days to 30 days, about 5 days to 14 days, or about 3 days to 7 days, preferably about 21 days to 28 days.

[0189] In some embodiments, the desired dosage of the formulations described herein can beadministered once daily or multiple times in a day. For example, a treatment regimen can include administering a dosage level sufficient to deliver 10 mg / kg body weight twice daily, 20 mg / kg body weight twice daily, 50 mg / kg body weight once daily, 10 mg / kg body weight three times daily, 20 mg / kg body weight four times daily, or 50 mg / kg body weight twice daily. VI. DEFINITIONS

[0190] Administer: The terms “administer,” “administration,” “administering,” and the like, whenused in conjunction with a therapeutic agent means to deliver a therapeutic agent to a subject whereby the therapeutic agent positively impacts, i.e., has a therapeutic effect on, the subject or the tissue or the organ to which it is targeted. The therapeutic agents described herein can be administered either alone or in combination (concurrently or serially) and / or with other pharmaceuticals. For example, the therapeutic agents can be administered in combination with vaccines, antibiotics, antiviral agents, anti- cancer or anti-neoplastic agents, or in combination with other treatment modalities such as herbal therapy, acupuncture, naturopathy, etc.

[0191] Effective Amount: The term "effective amount" as used herein generally refers to an amountof the therapeutic agent that is administered to decrease, prevent or inhibit the disease. The amount will vary for each compound and upon known factors related to the item or use to which the therapeutic agent is applied.

[0192] Pharmaceutically acceptable: The term “pharmaceutically acceptable”, as used herein, refersto compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio, in accordance with the guidelines of agencies such as the U.S. Food and Drug Administration. A “pharmaceutically acceptable carrier”, as used herein, refers to all components of a pharmaceutical formulation that facilitate the delivery of the composition in vivo. Pharmaceutically acceptable carriers include, but are not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.Attorney Docket Number: 438919.001901

[0193] Subject: A "subject" can include a human subject for medical purposes, such as for thetreatment of an existing disease, disorder, condition or the prophylactic for preventing the onset of a disease, disorder, or condition or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, gibbons, chimpanzees, orangutans, macaques and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, guinea pigs, and the like. An animal can be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a "subject" can include a patient afflicted with or suspected of being afflicted with a disease, disorder, or condition. Thus, the terms "subject" and "patient" are used interchangeably herein. Subjects also include animal disease models (e.g., rats or mice used in experiments, and the like).

[0194] Therapeutic agent: As used herein, the term “therapeutic agent” refers to any substance usedto restore or promote the health and / or wellbeing of a subject and / or to treat, prevent, alleviate, cure, or diagnose a disease, disorder, or condition.

[0195] Treatment or Treating: The terms "treatment," "treating," and the like, refer to an interventionperformed with the intention altering the pathology or symptoms of a disorder. In some embodiments, the treatment is for therapeutic treatment. Those in need of treatment can include those already with the disorder. In some embodiments, the treatment is for experimental treatment.

[0196] Preventing: The term “preventing” as used herein, refers to an intervention performed todecrease the chance of getting a disorder, disease or condition or a symptom associated with a disorder, disease or a condition.

[0197] The details of one or more embodiments of the disclosure are set forth in the accompanyingdescription below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred materials and methods are now described. Other features, objects and advantages of the disclosure will be apparent from the description. In the description, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the case of conflict, the present description will control.

[0198] The present disclosure is further illustrated by the following non-limiting examples.Attorney Docket Number: 438919.001901 Equivalents and Scope

[0199] Those skilled in the art will recognize or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments in accordance with the disclosure described herein. The scope of the present disclosure is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

[0200] In the claims, articles such as “a,” “an,” and ““they”” can mean one or more than one unlessindicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. As used in the description herein and throughout the claims that follow, the meaning of “a”, “an”, and “the” includes plural reference as well as the singular reference unless the context clearly dictates otherwise. The term “about” in association with a numerical value means that the value varies up or down by 5%. For example, for a value of about 100, means 95 to 105 (or any value between 95 and 105).

[0201] It is also noted that the term “comprising” is intended to be open and permits but does notrequire the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of” is thus also encompassed and disclosed. All patents, patent applications, and other scientific or technical writings referred to anywhere herein are incorporated by reference herein in their entirety. The embodiments illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are specifically or not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising," "consisting essentially of," and "consisting of" can be replaced with either of the other two terms, while retaining their ordinary meanings. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claims. Thus, it should be understood that although the present methods and compositions have been specifically disclosed by embodiments and optional features, modifications and variations of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of the compositions and methods as defined by the description and the appended claims.Attorney Docket Number: 438919.001901

[0202] Where ranges are given, endpoints are included. Furthermore, it is to be understood thatunless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0203] In addition, it is to be understood that any particular embodiment of the present disclosurethat falls within the prior art can be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they can be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the disclosure (e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[0204] It is to be understood that the words which have been used are words of description ratherthan limitation, and that changes can be made within the purview of the appended claims without departing from the true scope and spirit of the disclosure in its broader aspects.

[0205] While the present disclosure has been described at some length and with some particularitywith respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the disclosure.

[0206] The compositions and methods are more particularly described below, and the Examples setforth herein are intended as illustrative only, as numerous modifications and variations therein will be apparent to those skilled in the art. The terms used in the specification generally have their ordinary meanings in the art, within the context of the compositions and methods described herein, and in the specific context where each term is used. Some terms have been more specifically defined herein to provide additional guidance to the practitioner regarding the description of the compositions and methods.

[0207] Any single term, single element, single phrase, group of terms, group of phrases, or group ofelements described herein can each be specifically excluded from the claims.

[0208] Whenever a range is given in the specification, for example, a temperature range, a timerange, a composition, or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the aspects herein. It will be understood that any elements orAttorney Docket Number: 438919.001901 steps that are included in the description herein can be excluded from the claimed compositions or methods.

[0209] In addition, where features or aspects of the compositions and methods are described in termsof Markush groups or other grouping of alternatives, those skilled in the art will recognize that the compositions and methods are also thereby described in terms of any individual member or subgroup of members of the Markush group or other group. References 1. Talebi Bezmin Abadi A, Rizvanov AA, Haertlé T, et al.World health organization report: current crisis ofantibiotic resistance. BioNanoscience. 2019;9(4):778–788. doi: 10.1007 / s12668-019- 00658-4 2. Al-Haroni M, Skaug N, Bakken V, et al. Proteomic analysis of ampicillin-resistant oral Fusobacterium nucleatum. Oral Microbiol Immunol. 2008;23(1):36–42. doi: 10.1111 / j.1399- 302X.2007.00387.x 3. Nyfors S, Könönen E, Syrjänen R, et al. Emergence of penicillin resistance among Fusobacterium nucleatum populations of commensal oral flora during early childhood. J Antimicrob Chemother.2003;51(1):107–112. doi: 10.1093 / jac / dkg022 4. Elashiry M, Morandini AC, Cornelius Timothius CJ,et al. Selective antimicrobial therapies for periodontitis: win the “battle and the war. Int J Mol Sci. 2021;22(12):6459. doi: 10.3390 / ijms22126459 5. Chen Y, Huang Z, Tang Z, et al. More than just a periodontal pathogen –the research progress on Fusobacterium nucleatum. Front Cell InfectMicrobiol. 2022;12:815318. doi: 10.3389 / fcimb.2022.815318 6. Brennan CA, Garrett WS. Fusobacterium nucleatum —symbiont, opportunist and oncobacterium. Nat RevMicrobiol.2019;17(3):156–166. doi: 10.1038 / s41579-018-0129-6 7. Yang NY, Zhang Q, Li JL, et al. Progression of periodontal inflammation in adolescents is associated with an increased number of porphyromonas gingivalis, Prevotella intermedia, tannerella forsythensis, and Fusobacterium nucleatum. Int J Paediatr Dent.2014;24(3):226–233. doi: 10.1111 / ipd.12065 8. Lima BP, Shi W, Lux R. 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Wildblueberry (Vaccinium angustifolium Ait.) polyphenols target Fusobacterium nucleatum and the host inflammatory response: potential innovative molecules for treating periodontal diseases. J Agric Food Chem.2015;63(31):6999–7008. doi: 10.1021 / acs.jafc.5b01525 42. Yang M, Dong PT, Cen L, et al. Targeting Fusobacterium nucleatum through chemical modifications of host-derived transfer RNA fragments. Isme J.2023;17(6):880–890. doi: 10.1038 / s41396-023-01398 43. Lee S, Haraga H, Satoh T, et al. Effect of periodontitis induced by fusobacterium nucleatum on the microbiota of the gut and surrounding organs. Odontology. 2024;112(1):177–184. doi: 10.1007 / s10266-023-00827-8

[0044] de Molon RS, Park CH, Jin Q, et al. Characterization of ligature- induced experimental periodontitis. Microsc Res Tech. 2018;81(12):1412–1421. doi: 10.1002 / jemt.23101.Attorney Docket Number: 438919.001901 44. Yakar, N., Unlu, O., Cen, L., Hasturk, H., Chen, T., Shi, W., Kantarci, A. (2024). Targeted elimination of Fusobacterium nucleatum alleviates periodontitis. Journal of Oral Microbiology, 16(1). https: / / doi.org / 10.1080 / 20002297.2024.2388900

[0210] The following are provided for exemplification purposes only and are not intended to limitthe scope of the embodiments described in broad terms above. EXAMPLES EXAMPLE 1: Material and Methods

[0211] This example provides the materials and methods for the data described in Example 2.Efficacy of hygromycin A on a multispecies microbial community

[0212] To assess the impact of on a human multispecies microbiome, an in vitro multispeciescommunity from the subgingival plaque of a patient with clinically diagnosed periodontitis was established. The patients were enrolled at the Center for Clinical and Translational Research under the ADA Forsyth IRB protocol number IRB #16–08 and were provided informed consent before sampling. Subgingival plaque samples were collected using Gracey curettes from a 6"mm pocket depth and quickly suspended in 350"µl of PBS supplemented with 20% glycerol. Fifty microliters of the frozen stock of each subgingival plaque suspension were inoculated into 2"ml pre-reduced SHIPHGLXP% DQG WKH UHVW RI WKH VXVSHQVLRQ ZDV VWRUHG DW `1)]7 IRU ODWHU XVH' BKH FXOWXUHV ZHUH LQFXEDWHGovernight at 37°C in a microaerophilic environment (2% oxygen, 5% carbon dioxide, balanced with nitrogen). The microbial profiles of the resultant communities in the SHI medium were determined by 16S rRNA V1V3 amplicon sequencing. The plaque sample that gave rise to a community with the highest abundance of Fusobacteria spp. was chosen to establish an in vitro multispecies community and test the efficacy of in eliminating Fusobacteria spp. Briefly, 50"µl of the frozen stock of the subgingival plaque suspension was inoculated into a 2"ml pre-reduced SHI medium. The culture wasincubated at 37°C in a microaerophilic environment. The MIC for on F. nucleatum is 1"%g / ml. After24-h incubation, the culture was then diluted at a ratio of 1:10 in fresh SHI medium, and the resultant diluted culture was used to set up 2 treatment groups with different concentrations of 1"%g / ml (~2"%M) and 5"%g / ml (~10"%M), respectively. The was added to 2"ml of diluted culture for each treatment group to final concentrations of 1"%g / ml and 5"%g / ml. A vehicle-treated control group was also set up. The cultures were then incubated in a microaerophilic environment and passaged at 24-h intervals for 48 h at a 1:10 dilution into a fresh medium containing the respective concentrations of hygromycin A.

[0213] Five hundred microliters of the bacterial culture were collected every 24 h. The cells wereharvested by centrifugation at 17,000"×"g for 10"min, and genomic DNA was extracted using the MasterPure complete DNA Purification Kit (Epicentre) according to the manufacturer protocol. DNAAttorney Docket Number: 438919.001901 concentration was determined with the Nanodrop 2000 spectrophotometer (Thermo Scientific), and samples were stored at &80°C until 16S rRNA gene sequencing to profile the bacterial communities.

[0214] Sequencing was performed on the Illumina MiSeq platform, and bioinformatic processingwas conducted by Zymo Research Corporation (CA, USA) and the ADA Forsyth Oral Microbiome Core. Taxonomic assignment utilized a comprehensive set of 16S rRNA reference sequences, including MOMD v0.1, HOMD v15.2, HOMD 16S rRNA RefSeq Extended v1.1, GreenGenes (GG), and the NCBI 16S rRNA reference sequence collection. The microbial community composition was analyzed using QIIME 2 software, with alpha diversity assessed by the Kruskal–Wallis H test, and ANCOM-BC was employed for the differential abundance analysis. Mouse periodontitis model

[0215] A mouse periodontitis model was used to test the efficacy of targeted elimination of F.nucleatum in vivo and prevent F. nucleatum-induced periodontal disease. Twenty 8-week-old C57BL / 6 mice were randomly assigned to treatment and control (vehicle) groups. The mice were housed in a specific pathogen-free environment, with a 12:12-hour light / dark cycle at 24"±"0.5°C and 40–70% relative humidity. The Institutional Animal Care and Use Committee (IACUC) of the ADA Forsyth Institute reviewed and approved the experimental protocols.

[0216] Overnight cultures of F. nucleatum (ATCC 23726) were adjusted to the turbidity of a 0.5McFarland standard. As previously detailed, silk ligatures immersed in the culture for 1 min were used to induce periodontitis by application around the maxillary second molars. Starting the day after ligature placement, was administered to the test group mice via oral gavage at 200"mg / kg daily, at 3"mg / ml, while the control group received the vehicle (distilled water). Both the antibiotic and vehiclewere administered twice daily. One hundred microliters of F. nucleatum suspension was administeredto both groups in the interval between two antibiotic administrations. One week later, the animals were euthanized. Ligatures were collected for microbial analyses; maxillae were harvested to measure alveolar bone loss, and the keratinized gingiva surrounding the teeth was excised to measure theH[SUHVVLRQ RI SUR&LQIODPPDWRU\ F\WRNLQHV :<&*e DQG B>9&b'Recovery of F. nucleatum from ligatures and microbial profiling of the oral microbiome

[0217] The content of the ligature was extracted into 150"µl of PRAS dilution blank (AnaerobeSystems) with 30 min of shaking on ice. Subsequently, 10"µl of this solution was inoculated ontoblood agar plates and incubated anaerobically for 48 h to determine the presence of cultivable F.nucleatum. After the incubation period, 2"ml of sterile phosphate-buffered saline (PBS) was added to the culture plates, and the colonies were dispersed within the PBS using a sterile loop. The suspension was then collected, and DNA extraction using the boiling method followed by real-time PCR analysis. The Fusobacterium adhesion A (FadA) gene, which is the most significant virulence factor identifiedAttorney Docket Number: 438919.001901 as highly conserved in oral Fusobacterium, was used to quantify F. nucleatum. All PCR tests were also validated with 16S rRNA primer. The presence of F. nucleatum was confirmed by real-time PCR with SYBR Green and species-specific primers with StepOne™ Real-Time PCR System (Applied Biosystems™). The PCR reaction was conducted using an initial cycle of 95°C for 10"min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 30 s. DNA from the ligature content of five samples from each of the vehicle and groups were isolated using the MasterPure™ kit (Biosearch Technologies) and subjected to 16S rRNA sequencing to assess microbial diversity and relative abundance, as detailed above. Alveolar bone loss assessment

[0218] Bone morphometric analysis was used to evaluate the severity of alveolar bone loss amongthe groups. After euthanasia and ligature removal, the maxillae were subjected to defleshing by dermestid beetles for 4–5"days. Subsequently, the samples were immersed in 5% hydrogen peroxide for 8 h to ensure cleaning, followed by rinsing with water. To enhance contrast, the samples were stained for 10 s using methylene blue (1% solution in water), allowing clear differentiation between bone and dental structures before the morphometric analysis.

[0219] The prepared specimens were mounted and imaged from both buccal and palatal sites in theleft and right maxillae at 10X magnification with an inverted microscope (Axiovert 200, Zeiss, Thornwood, NY, USA) using AxioVision 4.8 software. Measurements of the area between the alveolar bone crest and the cementoenamel junction for each of the three maxillary molars were performed using ImageJ Software, and the results were expressed in square millimeters. Analysis of inflammatory gene expression in gingiva

[0220] Gingival samples, stored in RNA stabilization solution (RNAlater, ThermoFisherScientific) until processing, underwent mRNA extraction using a commercially available kit (RNeasy, Qiagen). From each sample, 1"%g of RNA was converted to cDNA using a reverse transcription kit (Applied Biosystems™). PCR reactions utilized gene-specific oligonucleotide probes for murine IL-1$ (Mm00434228_m1, ThermoFisher Scientific) and TNF-# (Mm00443258_m1, ThermoFisher Scientific), with 5"%l of TaqMan Universal Master Mix® and 0.5"%l of the specific probe, adjusted to a final volume of 9"%l with water. The reaction included 1"%l of cDNA and followed these conditions: an incubation step at 95°C for 20"sec, then 40 cycles of 1"sec at 95°C and 20"sec at 60°C using the real- time PCR instrument (StepOnePlus™, Applied Biosystems). Data was evaluated using the comparative ""Ct method, with mouse GAPDH (435292E, Applied Biosystems™) as the endogenous control. The relative expression of individual samples compared to the mean of the Vehicle group was calculatedE\ +C&dd7W% UHSRUWHG DV IROG FKDQJH'Attorney Docket Number: 438919.001901 Statistical analysis

[0221] Morphometric analysis data were analyzed using GraphPad Prism Software v9.10. TheShapiro – Wilk test verified the normality of the data distribution. An unpaired t-test compared twogroups. Data are presented as mean"±"SEM, with p"<"0.05 denoting significance.EXAMPLE 2: Results and Discussion Selective elimination of Fusobacterium species by low-dose hygromycin A without significantly affecting microbial diversity

[0222] In vitro multispecies community derived from the subgingival plaque of a periodontitispatient exhibited a diverse bacterial population. Fusobacteria spp., particularly F. nucleatum and F. periodonticum, were abundant, constituting over 25% of the microbial community. While the relative abundance of Fusobacteria spp. remained relatively stable over 2"days in the control culture,hygromycin A effectively reduced Fusobacterium spp. at both tested concentrations (p"<"0.05). With1"%g / ml (~2"%M) of hygromycin A, there was a marked reduction in the relative abundanceof Fusobacteria to 3.5% by day one and 0.08% by day two. At a higher concentration of 5"%g / ml(~10"%M), the reduction was 8.3% on day one and 0.2% by day two.

[0223] The lower dose of hygromycin A (1"%g / ml) did not significantly alter the alpha diversity ofthe microbial community compared with the non-treatment control. Although not statistically significant, a higher dose of hygromycin A (5"%g / ml) treatment resulted in a more drastic reduction in alpha diversity at day 2 compared to the non-treatment group, as reflected by a more notable decrease in observed species, Shannon and Simpson indices (Figure 1). In Figure 1, efficacy of hygromycin A on the multispecies microbial community in an in vitro model is shown. At a 1"µg / ml concentration,hygromycin A reduced the relative abundance of F. nucleatum without significantly altering the alphadiversity. Increasing the concentration to 5"µg / ml yielded no additional benefit in bacterial reduction.Figure 1A shows the experimental set up. Figure 1B shows the changes in the relative abundance of F.nucleatum across days. Figure 1C shows the alpha diversity graphs at different concentrations over the days. Suppression of Fusobacterium growth in ligature-induced periodontitis model by hygromycin A

[0224] F. nucleatum colonies were detectable in every sample from the control group. In contrast,samples from the treatment group showed no F. nucleatum colonies (Table 1). Table 1 shows the presence of cultivable F. nucleatum in ligature following vehicle or hygromycin A treatment. Detection of F. nucleatum was performed using species-specific primers polymerase chain reaction (PCR) on a mixed sample of all colonies that grew following 48 h of anaerobic incubation. TABLE 1: F. NUCLEATUM IN CULTURE OF LIGATURESAttorney Docket Number: 438919.001901 F. nucleatum Vehicle Hygromycin A (n=10) (n=10) Undetectable 0 / 10 10 / 10 Detectable 10 / 10 0 / 10

[0225] 16S rRNA sequencing analysis revealed a decrease in alpha diversity within the oralmicrobiota of the treatment group; however, this reduction did not result in a statistically significant difference in the mean alpha diversity in observed features, Shannon or Simpson indices (p">"0.05).Furthermore, the relative abundance of F. nucleatum was reduced to a mean of 3.8% in the treatmentgroup, compared to 16.7% in the control group (adjusted p (q)"="0.001). Mammalicoccus lentus, amember of the mouse commensal microenvironment, showed a significantly higher relative abundance in the treatment groups (q"<"0.001) (Figure 2). Figure 2 demonstrates the microbial community analysis using 16S rRNA sequencing. hygromycin A treatment reduced the relative abundance of F. nucleatum without a significant change in alpha diversity, but a significant community shift was demonstrated by beta diversity. Figure 2A is the experimental protocol, Figure 2B is a representation of the Shannon diversity index, given as the representative of all tested alpha diversity indices. Figure 2B showed no difference between treatment and vehicle groups (n"="5 per group). Figure 2C is the principal coordinates analysis (PCoA) of the bray-Curtis dissimilarity, demonstrating significant differences in community structure between the vehicle and hygromycin A groups (p"="0.02) (n"="5 per group). Figure 2D is a comparison of the relative abundances of the most abundant 9 bacterial species (Streptococcus thoraltensis, Enterococcus faecalis, Mammaliicoccus lentus, Acinetobacter radioresistens, Fusobacterium nucleatum, Faecalibaculum rodentium, Mammaliicoccus sciuri, Lactobacillus johnsonii, and a Staphylococcus species ambiguously identified as S. argenteus, S. aureus, or S. roterodami) between the control (vehicle) and hygromycin A treated groups. Alleviation of alveolar bone loss and pro-inflammatory cytokine expression through hygromycin A treatment

[0226] Morphometric analysis showed that hygromycin A treatment alleviated alveolar bone loss.Specifically, the average bone loss in the hygromycin A group was measured at 0.14"±"0.02"mm2, compared to 0.18"±"0.03"mm2in the vehicle group, with a statistically significant difference (p"="0.016). Additionally, there was a significant decrease in TNF-# expression within the hygromycin A group (p"="0.018). Expression of IL-1$ was also reduced in the hygromycin A treated group compared to the vehicle-treated controls (p"="0.052) (Figure 3).Attorney Docket Number: 438919.001901

[0227] Figure 3 shows the efficacy of hygromycin A in the mouse model of ligature-inducedperiodontitis. hygromycin A effectively eradicated cultivable F. nucleatum from the ligature site and significantly mitigated alveolar bone loss in a mouse model of ligature-induced periodontitis. Figure 3A is a representative images of maxillary alveolar unit morphology in the control (vehicle) and hygromycin A -treated groups. Figure 3B is a morphometric analysis comparing alveolar bone loss between the two groups (n"="9 per group). Figure 3C are gingiva RT-PCR results demonstrated higher IL-1$ and tnf-# expressions in the vehicle group compared to the hygromycin A -treated group (n"="10 per group). P-values were calculated using the Mann-Whitney test. Discussion

[0228] The findings disclosed herein indicate that hygromycin A administration effectivelyeradicated F. nucleatum in vitro and in vivo, thereby reducing gingival inflammation and alveolar bone loss in a mouse model of ligature and F. nucleatum-induced periodontitis. This study was the first to evaluate the efficacy of a targeted antibiotic, hygromycin A, treatment against oral F. nucleatum.

[0229] F. nucleatum elicits a robust immune response in periodontal tissues, inducing theexpression of inflammatory cytokines from gingival fibroblasts and antimicrobial peptides from epithelial cells. The pathogenicity of F. nucleatum is further demonstrated through its interactions with other periodontal pathogens. It enhances the invasiveness of P. gingivalis acts synergistically with Tannerella forsythia. A synergistic relationship was also reported between F. nucleatum and Treponema denticola. In this interaction, T. denticola suppressed the immune responses against F. nucleatum . Hygromycin A was reported to eradicate T. denticola strains at low doses, a process facilitated by the uptake of the compound by Treponema sp. through their nucleoside transporter. The ability of the compound to effectively disrupt the bidirectional interaction between T. denticola and F. nucleatum further underscores its potential value in the management of periodontal disease.

[0230] Targeting the elimination of F. nucleatum is crucial not only for oral health but also toprevent its extraoral impacts. As an oral bacterium, F. nucleatum can colonize distant organs and is associated with severe pathologies. It disseminates to non-oral sites via the bloodstream. Recent research demonstrated that neutrophils may play a crucial role in this dissemination, acting as a ‘Trojan horse’ for carrying the bacterium to distant sites. Thus, F. nucleatum poses a significant risk for oral-systemic disease connection, and its targeted elimination is critical to disrupting this link to prevent systemic complications associated with oral diseases. To this end, F. nucleatum has been identified in tumor tissue in colorectal cancer, with its abundance linked to increased tumor growth and a poorer prognosis. Antibiotic treatment targeting F. nucleatum has been correlated with reduced tumor growth. Additionally, periodontal therapy has decreased fecal levels of F. nucleatum. Therefore,Attorney Docket Number: 438919.001901 therapies designed to eradicate oral Fusobacteria would not only treat intraoral conditions, such as periodontitis and halitosis but also reduce the risk of systemic diseases such as colorectal cancer.

[0231] Emerging therapeutic approaches against this bacterium cover vaccines, plant extracts,probiotics, bacteriophages, and engineered bioactive molecules, including modified transfer-RNA derived small RNA (tsRNA). Although many of these proposed strategies are still in the in vitro testing stage, in vivo evaluation in animal models shows promising results in reducing alveolar bone loss. For example, gold nanoclusters have been shown to reduce inflammation and alveolar bone loss by eradicating F. nucleatum from dental plaque. Additionally, probiotic treatment against F. nucleatum with Akkermansia municiphila has been observed to moderate the periodontal inflammation in a mouse model. hygromycin A is emerging as a viable alternative to these various targeted modalities, owing to its ease of application and effectiveness at very low doses.

[0232] A mouse model to study F. nucleatum-induced periodontitis was employed, as itsapplication has been previously reported to cause bone loss in the alveolar bone or abscesses in murine models. Ligature placement around the maxillary molars of mice is known to result in significant bone loss within 7"days. Silk ligatures for their retentive properties when applying F. nucleatum. The antibiotic dose was selected based on the minimum concentration (MIC) necessary to prevent bacterial growth in mice, as identified in the prior study on this compound. This dose of hygromycin A, administered intraorally, was highly effective against F. nucleatum while decreasing the inflammatory response and alveolar bone loss. This compelling observation supports the targeted elimination of a specific pathogenic bacterium, which demonstrates the potency of these novel antibiotic strategies in therapeutic regimens designed to treat infectious diseases.

[0233] Increased doses of antibiotics may lead to more intense selection pressure on mutations thatconfer resistance, potentially resulting in hygromycin A-resistant Fusobacterium spp. Additionally, a higher dose of hygromycin A could inhibit the growth of other species that are less sensitive to lower dosages. Both scenarios may have contributed to the increased relative abundance of Fusobacterium observed with high-dose hygromycin A treatment in the in vitro microbial community. As a result, the community changes affected by hygromycin A treatment positively impacted disease progression in the mouse model.

[0234] Considering the adverse effects of commonly used broad-spectrum antimicrobial agents inadjunctive periodontal therapy, hygromycin A may offer a promising treatment for periodontal disease. Thus, this study aimed to evaluate the impact of FP-100 on the multispecies microbial community associated with periodontitis and to assess its potential to prevent periodontal inflammation and destruction in an F. nucleatum-induced periodontitis model in mice.

Claims

Attorney Docket Number: 438919.001901 CLAIMS What is claimed is:

1. A method of treating an oral or periodontal disease comprising administering acomposition comprising hygromycin to a subject in need thereof.

2. The method of claim 1, comprising applying the composition comprising hygromycin A toa surface of the subject’s mouth, lips and / or oral cavity.

3. The method of claim 2, wherein the surface is selected from teeth, gums, tongue, palate,floor, roof, throat, pharynx, tonsils, cheeks, lips (interior and exterior), and epiglottis.

4. The method of any one of claims 1-3, wherein the composition reduces the number of oneor more bacteria in the subject’s oral cavity.

5. The method of claim 3, wherein the bacteria are Fusobacterium nucleatum, Treponemadenticola, Porphyromonas gingivalis, Prevotella intermedia, Prevotella loescheii, , Porphyromonas endodontalis, Peptococcus anaerobius, Micros prevotii, Eubasterium limosum, Centipedia perio Centipedia periodontii, Selenomonas arterimidis, Fusobacterium periodonticum, Eubacterium spp., Bacteroides spp., Actinomyces viscosos, Streptococcus mutans, and / or Streptococcus sobrinus.

6. The method of claim 2, wherein the composition is formulated as a mouthwash, atoothpaste, a mouth and / or throat spray, a lozenge, a breath mint, a pen or tube with a brush, sponge, or nozzle applicator, a dissolvable strip, an adhesive tape for teeth, a toothpick, a chewing gum, a tongue scraper, a lip balm, a syringe, a nasal aspirator, or a dental floss.

7. The method of any one of claims 1-5, wherein the composition is administered as a singletier dosing regimen.

8. The method of any one of claims 1-6, wherein the single tier dosing regimen comprises ahigh dose of hygromycin A, wherein the high dose is 100 milligram per kg to 1500 milligram per kg body weight 9. The method of any one of claims 1-8, wherein the high dose is 1,000 milligram per kg.Attorney Docket Number: 438919.001901 10. The method of any one of claims 1-7, wherein the single tier dosing comprises a low doseof hygromycin, wherein the low dose is from 0.001 milligram per kg to 0.1 milligram per kg body weight.

11. The method of any one of claims 1-10, wherein the low dose is 0.01 milligram per kg bodyweight.

12. The method of any one of claims 1-7, wherein the composition is administered as two-tiered dosing regimen, comprising a first high dose and one or more maintenance doses.

13. The method of claim 12, wherein the first high dose is administered orally to the subject.

14. The method of claim 12, wherein the one or more maintenance doses are applied to thesurface of the subject’s mouth, lips and / or oral cavity.

15. The method of any one of claims 1- 14, wherein the hygromycin A does not inhibit thegrowth of or kill at least one species of beneficial oral bacteria.

16. The method of claim 12, wherein the first high dose is 100 milligram per kg to 1500milligram per kg body weight 17. The method of claim 16, wherein the first high dose is 1,000 milligram per kg.

18. The method of claim 12, wherein the one or more maintenance doses is from 0.001milligram per kg to 0.1 milligram per kg body weight.

19. The method of claim 18, wherein the one or more maintenance doses is 0.01 milligram perkg body weight.

20. A method of treating an oral or periodontal disease and an intestinal disease comprisingapplying a composition comprising hygromycin A to a surface of the subject’s mouth, lips and / or oral cavity.

21. The method of claim 20, wherein the surface is selected from teeth, gums, tongue, palate,floor, roof, throat, pharynx, tonsils, cheeks, lips (interior and exterior), and epiglottis.

22. The method of claim 21, wherein the composition reduces the number of one or morebacteria in the subject’s oral cavity and / or intestine of the subject.Attorney Docket Number: 438919.001901 23. The method of claim 22, wherein the bacteria are Treponema denticola, Fusobacteriumnucleatum, Porphyromonas gingivalis, Prevotella intermedia, Prevotella loescheii, , Porphyromonas endodontalis, Peptococcus anaerobius, Micros prevotii, Eubasterium limosum, Centipedia perio Centipedia periodontii, Selenomonas arterimidis, Fusobacterium periodonticum, Eubacterium spp., Bacteroides spp., Actinomyces viscosos, Streptococcus mutans, and / or Streptococcus sobrinus.

24. The method of claim 20, wherein the composition is formulated as a mouthwash, atoothpaste, a mouth and / or throat spray, a lozenge, a breath mint, a pen or tube with a brush, sponge, or nozzle applicator, a dissolvable strip, an adhesive tape for teeth, a toothpick, a chewing gum, a tongue scraper, a lip balm, a syringe, a nasal aspirator, or a dental floss.

25. The method of claim 20, wherein the intestinal disease is colorectal cancer.

26. The method of claim 20, wherein the colorectal cancer is caused by Fusobacteriumnucleatum.

27. A method of treating or preventing periodontal or alveolar bone loss in a subject,comprising administering an effective amount of hygromycin A to the subject.

28. The method of claim 27, wherein the periodontal bone loss is caused by Fusobacteriumspp. infection.

29. The method of claim 28, wherein the Fusobacterium spp. is Fusobacterium nucleatumand / or Fusobacterium periodonticum.

30. The method of claim 29, wherein the hygromycin A does not inhibit the growth of or kill atleast one species of beneficial oral bacteria.

31. The method of any one of claims 27-30, wherein the periodontal or alveolar bone loss is inor around a molar tooth, an incisor tooth, a canine tooth, and / or a premolar tooth:

32. The method of claim 31, wherein the molar tooth is a second maxillary molar tooth.

33. The method of any one of claims 27-32, wherein the hygromycin A reduces or preventsFusobacterium nucleatum colonization.Attorney Docket Number: 438919.001901 34. A method of reducing periodontal inflammation, comprising administering an effectiveamount of hygromycin A to the subject.

35. The method of claim 34, wherein the periodontal inflammation loss is caused byFusobacterium nucleatum.

36. The method of claim 35, comprising gingival inflammation.

37. The method of claim 34, wherein the hygromycin A does not inhibit the growth of or kill atleast one species of beneficial oral bacteria.

38. The method of claim 34, wherein the periodontal inflammation is associated with at leastone pro-inflammatory cytokine, ZKHUHLQ WKH SUR&LQIODPPDWRU\ F\WRNLQH LV :<&*e DQG(RU B>9&b% DQG ZKHUHLQ DGPLQLVWHULQJK\JURP\FLQ 5 GHFUHDVHV WKH OHYHOV RI :<&*e DQG(RU B>9&b'

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