Hygromycin A for the treatment of diseases and infections

Abstract

The present disclosure provides therapeutic agents, such as hygromycin A, for use in treating, preventing, or managing disease. The disease may be caused by a microorganism of the Fusobacterium or Treponema species. The therapeutic agents of the present disclosure may be used to inhibit the growth of the microorganisms described herein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 247,927, filed September 24, 2021; U.S. Provisional Application No. 63 / 247,928, filed September 24, 2021; and U.S. Provisional Application No. 63 / 247,929, filed September 24, 2021, the contents of each of which are incorporated by reference herein in their entirety. [Background technology]

[0002] Hygromycin A (also known as homomycin or totomycin) is a modified cinnamic acid antibiotic isolated from Streptomyces hygroscopicus. Initial investigations demonstrated that hygromycin A has broad-spectrum activity against gram-positive and gram-negative bacteria. Hygromycin acts by inhibiting ribosomal peptidyl transferase activity. Hygromycin A also blocks the binding of either chloramphenicol or lincomycin to the ribosome. Summary of the Invention [Means for solving the problem]

[0003] The present disclosure provides a therapeutic agent for inhibiting the growth of microorganisms. The therapeutic agent can be hygromycin A. The microorganism can be a Treponema species, a Streptococcus species, a Fusobacterium species, a Parvimonas species, or a Porphyromonas species. The Treponema species can be Treponema denticola, Treponema pallidum, or Treponema carateum. The Treponema pallidum can be Treponema pallidum pallidum, Treponema pallidum pertenue, or Treponema pallidum endemicum. The Fusobacterium species can be Fusobacterium nucleatum, including F. nucleatum animalis, F. nucleatum vincentii, F. nucleatum nucleatum, F. nucleatum polymorphum, F. nucleatum fusiforme, or F. nucleatum periodonticum. The Parvimonas species can be Parvimonas micra. The Porphyromonas species may be Porphyromonas gingivalis. In some embodiments, the concentration of hygromycin A is about 0.01 μg / ml to about 100 μg / ml. In some embodiments, the concentration of hygromycin A is about 0.01 μg / ml to about 1.0 μg / ml. In some embodiments, the concentration of hygromycin A may be 0.05 μg / ml. In some embodiments, the concentration of hygromycin A may be 0.1 μg / ml. In some embodiments, the concentration of hygromycin A may be 0.2 μg / ml. In some embodiments, the concentration of hygromycin A may be 0.5 μg / ml. In some embodiments, the concentration of hygromycin A may be 1.0 μg / ml. In some embodiments, the concentration of hygromycin A may be 2 μg / ml. In some embodiments, the concentration of hygromycin A may be 5 μg / ml.

[0004] The present disclosure provides therapeutic agents and methods related to oral microbes and the treatment, prevention, and / or management of diseases associated with oral microbes.

[0005] In some embodiments, the present disclosure provides methods for inhibiting the growth of oral microorganisms.

[0006] The present disclosure provides methods for inhibiting or reducing the growth of oral microorganisms in a subject. Such methods may include contacting the subject with at least one therapeutic agent, such as, but not limited to, hygromycin A.

[0007] The method of the present disclosure may include contacting oral microorganisms with at least one therapeutic agent. Contacting oral microorganisms with a therapeutic agent may inhibit the growth of oral microorganisms. In some embodiments, the therapeutic agent may be hygromycin A. The oral microorganism may be Treponema denticola, Fusobacterium nucleatum, Parvimonas micra, or Porphyromonas gingivalis.

[0008] In some embodiments, the concentration of hygromycin A is about 0.01 μg / ml to about 100 μg / ml. In some embodiments, the concentration of hygromycin A is about 0.01 μg / ml to about 1.0 μg / ml. In some embodiments, the concentration of hygromycin A can be 0.05 μg / ml. In some embodiments, the concentration of hygromycin A can be 0.1 μg / ml. In some embodiments, the concentration of hygromycin A can be 0.2 μg / ml. In some embodiments, the concentration of hygromycin A can be 0.5 μg / ml. In some embodiments, the concentration of hygromycin A can be 1.0 μg / ml. In some embodiments, the concentration of hygromycin A can be 2 μg / ml. In some embodiments, the concentration of hygromycin A can be 5 μg / ml.

[0009] Also provided herein is a method for treating or preventing a disease associated with oral microorganisms in a subject.The disease can be an oral disease, a systemic disease, a cancer, or a veterinary disease.In some embodiments, the oral disease can be periodontitis or gingivitis.In some embodiments, the disease can be cancer, such as colorectal cancer, gastric cancer, or esophageal cancer.

[0010] The present disclosure provides therapeutic agents and methods related to F. nucleatum, and the treatment, prevention, and / or management of diseases associated with F. nucleatum.

[0011] In some embodiments, the present disclosure provides a method for inhibiting the growth of Fusobacterium nucleatum (F. nucleatum). Such a method may include contacting F. nucleatum with at least one therapeutic agent such that the growth of F. nucleatum is inhibited. The therapeutic agent may be hygromycin A. Also provided herein is a method for inhibiting or reducing the growth of Fusobacterium nucleatum.

[0012] In some embodiments, the concentration of hygromycin A may be about 1 μg / ml to about 100 μg / ml. In some embodiments, the concentration of hygromycin A may be 5 μg / ml. In some embodiments, the concentration of hygromycin A may be 10 μg / ml. In some embodiments, the concentration of hygromycin A may be 20 μg / ml. In some embodiments, the concentration of hygromycin A may be 40 μg / ml.

[0013] F. nucleatum can be one or more subspecies, including F. nucleatum animalis, F. nucleatum vincentii, F. nucleatum nucleatum, F. nucleatum polymorphum, F. nucleatum fusiforme, or F. nucleatum periodonticum.

[0014] The present disclosure provides a method for treating or preventing a disease in a subject, comprising contacting the subject with at least one therapeutic agent. In some embodiments, the disease can be cancer, gastrointestinal disorder, or oral disease.

[0015] In some embodiments, the disease may be associated with F. nucleatum infection. In some embodiments, the disease may be cancer, such as colorectal cancer or oral cancer. In some embodiments, the disease may be a gastrointestinal disease, such as inflammatory bowel disease, Crohn's disease, ulcerative colitis, or colorectal cancer. In some embodiments, the disease may be an oral disease, such as periodontal disease. The periodontal disease may be localized aggressive periodontitis, generalized aggressive periodontitis, pulp necrosis, or apical periodontitis. In some embodiments, the disease may be oral cancer.

[0016] The present disclosure provides therapeutic agents and methods related to treponematoses, and the treatment, prevention, and / or management of diseases associated with Treponema pallidum infection.

[0017] Provided herein is a method for inhibiting the growth of Treponema by contacting Treponema with at least one therapeutic agent. Also provided herein is a method for reducing Treponema in a subject. Contacting Treponema of a subject with hygromycin A can inhibit the growth of Treponema. In some embodiments, the therapeutic agent can be hygromycin A. Treponema can be Treponema pallidum or Treponema carateum.

[0018] In some embodiments, the concentration of hygromycin A can be from about 0.01 μg / ml to about 100 μg / ml.

[0019] In some embodiments, the concentration of hygromycin A can be from about 0.01 μg / ml to about 10 μg / ml.

[0020] In some embodiments, the concentration of hygromycin A may be 0.06 μg / ml. In some embodiments, the concentration of hygromycin A may be 0.12 μg / ml. In some embodiments, the concentration of hygromycin A may be 0.24 μg / ml. In some embodiments, the concentration of hygromycin A may be 0.48 μg / ml. In some embodiments, the concentration of hygromycin A may be 0.96 μg / ml. In some embodiments, the concentration of hygromycin A may be 1.92 μg / ml. In some embodiments, the concentration of hygromycin A may be 3.84 μg / ml.

[0021] Treponema pallidum can be Treponema pallidum pallidum, Treponema pallidum pertenue, or Treponema pallidum endemicum.

[0022] The present disclosure also provides a method for treating or preventing treponematosis in a subject. Such a method may include contacting the subject with at least one therapeutic agent. The therapeutic agent may be hygromycin A. The treponematosis may be associated with Treponema pallidum or Treponema carateum infection. In some embodiments, the treponematosis may be syphilis. In some embodiments, the treponematosis may be yaws. In some embodiments, the treponematosis may be bejel. In some embodiments, the treponematosis may be pinta. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] I. Introduction As microbial detection technologies advance, an ever-increasing number of previously overlooked microorganisms are being discovered to play important roles in human disease. A paradigm shift is also occurring in our understanding of the key role of the microbiome in health and disease.

[0024] Hygromycin A In the last 20 years, the effect of antibiotics on gut microbiota has been investigated. It has been shown that broad-spectrum antibiotics such as ampicillin, doxycycline, amoxicillin, and ceftriaxone affect gut microbiota, causing rapid and reduced levels of microbial diversity and relative abundance changes of bacteria, leading to dysbiosis. Microbiota contributes to shaping the immune system during development, maintaining a healthy gastrointestinal tract, and preventing cardiovascular disease, neurological disease, and autoimmune disease. Therefore, there is a need in the art for therapeutic agents for bacteria-related diseases that have minimal to no effect on gut microbiota, as well as new treatments and / or prevention measures.

[0025] Hygromycin A is a fermentation-derived natural product that was first isolated from Streptomyces hygroscopicus in 1953. Hygromycin A was considered a broad-spectrum antibiotic due to its reported activity against certain acid-fast bacteria and certain gram-positive and gram-negative bacteria (see U.S. Patent No. 3,100,176). Hygromycin A exhibited only relatively little effect against bacteria and was not pursued commercially.

[0026] In this disclosure, the inventors demonstrate that hygromycin A is effective against certain oral treponemal Fusobacterium species and organisms that cause treponematosis, while not affecting beneficial bacteria in the oral cavity and / or intestine. Examples of beneficial bacteria in the oral cavity and / or intestine 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. Notably, oral treponema and Fusobacterium can cause disease in the mouth and gastrointestinal tract, where the importance of microflora has been previously demonstrated.Thus, the present disclosure provides a therapeutic agent that has the potential to target disease-causing bacteria without affecting the microflora.In one embodiment, hygromycin A inhibits or kills the growth of disease-causing bacteria, but does not inhibit or kill the growth of at least one species of beneficial oral or intestinal bacteria.

[0027] Overview of oral treponemes Along with over 600 other bacterial species, oral treponemes exist as part of a polymicrobial biofilm that fuses with the tooth surface in the gingival sulcus. Treponemae play a role in the pathogenesis of several chronic diseases in humans, including periodontal disease, including chronic periodontitis, acute necrotizing ulcerative gingivitis, endodontic infections, and some acute dental abscesses. In addition, treponemes are involved in the development of chronic diseases in livestock, including periodontal disease in dogs, bovine digital dermatitis in dairy cows, and contagious ovine digital dermatitis.

[0028] Periodontal disease, also called gum disease, is a common affliction among adults caused by oral bacterial growth. While a complex microflora resides in healthy gingival plaque that poses little or no health risk, periodontal lesions can be formed and dominated by proteolytic gram-negative anaerobic bacteria and spirochetes that are associated with severe and refractory periodontal pathology. In advanced periodontal disease, spirochetes disrupt intercellular junctions, invade underlying tissues, and produce a destructive host response. The genus Treponema includes more than 60 phylotypes of oral spirochetes, of which Treponema denticola is the most culturable and is involved in the pathogenesis of periodontal disease. Novel therapies and methods are needed to treat oral infections, including those involved in periodontal disease, and to treat Treponema infections.

[0029] Among treponema, Treponema denticola (also referred to herein as T. denticola) is one of the most widely investigated oral microorganisms. Treponema denticola is a gram-negative, obligately anaerobic, motile, and highly proteolytic spirochete bacterium. It is one of four oral spirochetes that can be reliably cultured; the others are Treponema pectinovorum, Treponema socranskii, and Treponema vincentii. T. denticola inhabits a complex and diverse microbial community in the oral cavity and is highly specialized to survive in this environment.

[0030] Given the broad role of oral microbes in disease, there remains a need to develop therapeutic 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.

[0031] Overview of Fusobacterium nucleatum With advances in microbial detection technology, an increasing number of previously overlooked microorganisms are being discovered to play important roles in human disease. A paradigm shift is also occurring in understanding the role of the microbiome in health and disease. Fusobacterium nucleatum (herein referred to as F. nucleatum), a Gram-negative anaerobic bacterium, is one such newly emerged pathogen.

[0032] F. nucleatum is ubiquitous in the oral cavity and absent or rarely detected elsewhere in the body under normal conditions. Under disease conditions, F. nucleatum has been detected in extraoral sites. F. nucleatum is a heterogeneous species with several proposed subspecies (ss), namely ss animalis, ss fusiforme, ss nucleatum, ss polymorphum, ss periodonticum, and ss vincentii, and its prevalence in disease varies.

[0033] Fusobacterium nucleatum (F. nucleatum) is a gram-negative obligate anaerobic bacterium in the oral cavity that plays a role in several oral diseases, including periodontitis and gingivitis. In recent years, several studies have reported that the levels of F. nucleatum are significantly elevated in human colorectal adenocarcinomas and cancers compared to those in adjacent normal tissues. A causal role of F. nucleatum in the pathogenesis of colorectal cancer has also been demonstrated. There remains a need to identify therapeutic agents that target F. nucleatum and / or treat F. nucleatum-associated diseases. Fusobacterium nucleatum is an aggressive, adhesive, and pro-inflammatory anaerobic bacterium. It is commonly found in dental plaque, and there is a well-established association between F. nucleatum and periodontitis. In isolated cases, F. nucleatum has been implicated in brain 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 positively correlate with disease severity. When isolated from human intestinal biopsies, F. nucleatum has been found to be more readily culturable from patients with gastrointestinal (GI) disease than from healthy controls, and strains grown from inflamed biopsy tissue appeared to exhibit a more aggressive phenotype.

[0034] Given the broad role of F. nucleatum in disease, there remains a need to develop therapeutic agents that inhibit, reduce, and / or kill F. nucleatum. The present disclosure provides therapeutic agents, such as, but not limited to, hygromycin A, for the treatment of diseases associated with F. nucleatum.

[0035] Overview of Treponema Human treponematoses include venereal syphilis and endemic treponematoses including yaws, bejel, and pinta. The etiologic agents of these diseases are gram-negative bacteria belonging to the order Spirochaetales, family Spirochaetaceae, and genus Treponema. The 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 unavailability of isolates of the pinta agent has precluded genetic analysis of the organism, which retains its separate name, T. carateum.

[0036] All human treponematoses share similarities in pathogenesis and natural history. All are chronic infections transmitted by direct contact with infectious lesions and manifest in multiple cutaneous stages. All except pinta can progress to cause severe and destructive lesions of the skin, bone, and cartilage. As in venereal syphilis, the clinical signs of endemic treponematoses are generally divided into early stages (including primary and secondary signs) and late stages. Early stage lesions are highly infectious and may persist for weeks to months or even years after their appearance. Once the early signs spontaneously regress due to the host's immune response to the pathogen, the patient enters a state of latency, which in many cases lasts for life. However, in a relatively small number of cases, the infection can progress from latency to a tertiary disease characterized by tissue destruction.

[0037] Treponemases have not yet been eradicated. Unique characteristics have been identified in terms of age of acquisition, mode of transmission, and ability to invade the central nervous system. In 2012, the World Health Organization (WHO) set a goal to eliminate treponematoses. Successful eradication will require therapeutic strategies that target the causative Treponema. The present disclosure provides therapeutic agents for the treatment, prevention, and / or management of treponematoses.

[0038] II. Therapeutic Agents In some embodiments, the present disclosure provides a therapeutic agent for treating diseases, such as, but not limited to, oral microorganism-related diseases. In some embodiments, treating a disease in a subject can involve administering at least one therapeutic agent. In some embodiments, the therapeutic agent can be hygromycin A.

[0039] Hygromycin A (Hyg A) is a product of Streptomyces hygroscopicus that was first isolated in 1953. It has a unique structure consisting of a furanose, a cinnamic acid, and an aminocyclitol moiety. It has a relatively broad antimicrobial spectrum. Hygromycin A has the structure shown here:

[0040] [ka]

[0041] combination In some embodiments, combinations of therapeutic agents can be utilized. The present disclosure provides two, three, four, five or more therapeutic agents in combination form. The combination can be administered in parallel, sequentially, and / or continuously. In some embodiments, each therapeutic agent in the combination can be formulated as a separate pharmaceutical preparation. In some embodiments, the therapeutic agent in the combination can be prepared as a single pharmaceutical preparation.

[0042] For the purposes of this disclosure, "in combination" refers to providing two or more therapeutic agents separately or together, where the two therapeutic agents are administered as part of an appropriate dosing regimen designed to obtain the benefits of combined therapy. Thus, the two therapeutic agents can be administered as part of the same pharmaceutical composition or in separate pharmaceutical compositions. The first therapeutic agent can be administered before, simultaneously, or after the administration of the second therapeutic agent, or in any combination thereof. If one therapeutic agent is administered to a subject at repeated intervals, for example during a standard course of treatment, the second therapeutic agent can be administered before, simultaneously, or after each administration of the first therapeutic agent, or in any combination thereof, or at different intervals relative to the therapy with the first therapeutic agent, or in a single dose before, at any time during, or after the course of treatment with the first therapeutic agent.

[0043] The therapeutic combinations of the present disclosure may include hygromycin A and a second therapeutic agent. In some embodiments, the second therapeutic agent may be a therapeutic agent utilized to treat a Fusobacterium nucleatum infection.

[0044] The therapeutic combinations of the present disclosure may include hygromycin A and a second therapeutic agent used to treat inflammation in chronic periodontal infections, destruction of connective tissue, periodontal ligament, and alveolar bone resorption, and ultimately tooth loss. In some embodiments, the second therapeutic agent may be enoxacin and / or a bisphosphonate derivative of enoxacin (bis-enoxacin).

[0045] Therapeutic combinations of the present disclosure may include hygromycin A and a second therapeutic agent. In some embodiments, the second therapeutic agent may be a therapeutic agent utilized to treat treponematosis.

[0046] In some embodiments, the therapeutic agent may be combined with one or more agents used in the treatment of syphilis. In some embodiments, the therapeutic agent of the present disclosure may be combined with penicillin.

[0047] In some embodiments, the therapeutic agent may be combined with one or more agents used in the treatment of yaws. In some embodiments, the therapeutic agent of the present disclosure may be combined with azithromycin and / or benzathine benzylpenicillin.

[0048] In some embodiments, the therapeutic agent may be combined with one or more agents used in the treatment of bejel or pinta. In some embodiments, the therapeutic agent of the present disclosure may be combined with doxycycline and / or benzathine benzylpenicillin.

[0049] III. Method of Use The present disclosure provides methods of use related to the therapeutic agents described herein. In some embodiments, the methods may include methods of reducing the growth of bacteria, such as oral microorganisms. As used herein, the term "oral microorganisms" refers to any microorganism that lives in the oral cavity. In some embodiments, oral microorganisms may be present in the oral cavity of a healthy individual. In some embodiments, the presence of oral microorganisms in the oral cavity may be associated with a disease or pathological condition.

[0050] Oral and periodontal diseases The therapeutic agent of the present disclosure may exhibit efficacy against oral microorganisms and therefore has the potential to treat and / or prevent infections or kill and / or inhibit the growth of oral microorganisms. In some embodiments, the animal may be a human. In some embodiments, spirochete infections may be treated and / or prevented, or spirochetes may be killed or their growth inhibited, through oral administration of the therapeutic agent of the present disclosure. As a non-limiting example, spirochete infections may be treated and / or prevented, or spirochetes may be killed or their growth inhibited, through intravenous administration of the therapeutic agent of the present disclosure.

[0051] In some embodiments, the oral microorganisms may be Treponema species, Streptococcus species, Fusobacterium species, and / or Parvimonas species.

[0052] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, protect against, and / or manage 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.

[0053] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, protect against, and / or manage disease caused by oral microorganisms such as, but not limited to, Treponema, such as T. pallidum, T. denticola, T. vincentii, T. carateum, and / or T. phagedenis, or clinical isolates or strains thereof.

[0054] In some embodiments, the oral microorganism is T. denticola (strain: ATCC 35405), T. denticola (strain: ATCC 35404), T. denticola (strain: ATCC 33520), T. denti cola (strain: ATCC33521), T. denticola (strain: OTK), T. denticola (strain: H1-T), T. denticola (strain: H-22), T. denticola (strain: MYR-T), T. The strain may be Treponema denticola or a strain or clinical isolate thereof, such as 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).

[0055] In some embodiments, the therapeutic agent of the present disclosure is selected from the group consisting of Fusobacterium nucleatum (e.g., any strain of F. nucleatum animalis, ATCC 25586, ATCC5 51191, ATCC 10953, ATCC 23726, CTI-2, CTI-3, CTI-7, EAV0002, 7-1, FA2+, 7-33 C1), 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 The compositions may be used to treat, prevent, or manage diseases caused by or associated with oral microorganisms such as, but not limited to, forsythia, Capnocytophaga, Peptostreptococcus, and / or Eikenella.

[0056] In some embodiments, oral microorganisms may include Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Prevotella loescheii, Triponema denticola (Treponema denticola), Porphyromonas endodontalis, Peptococcus anaerobius, Micros prevotii, Eubasterium limosum, Centipedia perio Centipedia periodontii, Selenomonas arterimidis, Fusobacterium periodonticum, Eubacterium species, Bacteroides species, Actinomyces viscosos, Streptococcus mutans, and / or Streptococcus sobrinus.

[0057] In some embodiments, administering a therapeutic agent described herein to a subject infected with or having a disease caused or exacerbated / aggravated by an oral microorganism inhibits or reduces replication of the oral microorganism by at least 20%-25%, at least 25%-30%, at least 30%-35%, at least 35%-40%, at least 40%-45%, at least 45%-50%, at least 50%-55%, at least 55%-60%, at least 60%-65%, at least 65%-70%, at least 70%-75%, at least 75%-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 microorganism 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 2-5-fold, 2-10-fold, 5-10-fold, or 5-20-fold compared to a negative control, as determined using an assay described herein or others known to one of skill in the art.

[0058] In some aspects, the therapeutic agent can be used to treat or prevent periodontal disease. Periodontal disease is mainly the result of infection and inflammation of the gums and bone that surround and support the teeth. In its early stage, called gingivitis, the gums can swell, become red, and they can bleed. In its more severe form, called periodontitis, the gums can separate from the teeth, bone can be lost, and teeth can loosen or even fall out. In some embodiments, the therapeutic agent of the present disclosure can be used to treat gingivitis. In some embodiments, the therapeutic agent of the present disclosure can be used to treat periodontitis.

[0059] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, manage, or prevent one or more symptoms associated with periodontal disease, including, but not limited to, bad breath or unpleasant taste, red or swollen gums, weak or bleeding gums, painful chewing, loose teeth, sensitive teeth, gums that are pulling away from the teeth, etc.

[0060] In some embodiments, the therapeutic agent of the present disclosure can be used to treat, manage, or prevent chronic periodontitis. Chronic periodontitis is a polymicrobial disease caused by 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", which is strongly associated with the clinical progression of chronic periodontitis. The unifying characteristics of red complex bacteria are their extracellular proteolytic activity, their complex anaerobic fermentation of amino acids, the production of toxic metabolites, and outer membrane (or sheath) vesicles.

[0061] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage gum disease, which may be caused by or correlated with infection or the presence of oral microorganisms.

[0062] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage chronic periodontitis, which may be caused by or correlated with infection or the presence of oral microorganisms.

[0063] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage aggressive periodontitis, which may be caused by or correlated with infection by or the presence of oral microorganisms.

[0064] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage periodontal abscesses, which may be caused by or correlated with infection or the presence of oral microorganisms.

[0065] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage periodontitis associated with endodontic lesions. In some embodiments, periodontitis associated with endodontic lesions of the periodontium may be caused by or correlated with infection by or the presence of oral microorganisms.

[0066] In some embodiments, the therapeutic agents of the present disclosure may be used to treat stage I periodontitis, characterized by 1-2 mm of clinical attachment loss between the teeth. In some embodiments, the therapeutic agents of the present disclosure may be used to treat stage II periodontitis, characterized by 3-4 mm of clinical attachment loss between the teeth. In some embodiments, the therapeutic agents of the present disclosure may be used to treat stage III or stage IV periodontitis, characterized by >5 mm of clinical attachment loss between the teeth.

[0067] 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 μg / ml or more.

[0068] Fusobacterium nucleatum-associated diseases In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage a Fusobacterium nucleatum associated oral infection in a subject.

[0069] F.nucleatum is one of the most abundant species in the oral cavity, both in diseased and healthy individuals.In some embodiments, the therapeutic agent of the present disclosure can be used to treat, prevent or manage Fusobacterium nucleatum-associated periodontal disease, such as, but not limited to, gingivitis and progressive irreversible forms of periodontitis, including chronic periodontitis, localized aggressive periodontitis and generalized aggressive periodontitis.The therapeutic agent of the present disclosure can be used to treat, prevent or manage endodontic infections, such as pulp necrosis and apical periodontitis, which are frequently associated with F.nucleatum infection.

[0070] In one embodiment, the oral disease is oral cancer.

[0071] The prevalence of F. nucleatum correlates with disease severity, progression of inflammation, and increasing pocket depth. Among the five subspecies, ss fusiforme and ss vincentii are more frequently associated with healthy conditions, whereas ss nucleatum is more frequently associated with diseased conditions. In addition to periodontal sites, F. nucleatum has been detected in saliva and is increased in numbers in patients with gingivitis and periodontitis compared to healthy controls. Serum antibody titers against F. nucleatum have been reported to be elevated in affected patients.

[0072] F. nucleatum is an aggressive bacterium that causes acute oral and gastrointestinal infections and can act as a pro-inflammatory agent. In some embodiments, the therapeutic agent of the present disclosure can be used to treat, prevent, or manage Fusobacterium nucleatum-associated disease. As used herein, the term "Fusobacterium nucleatum-associated disease" can be used to refer to a disease or condition caused by and / or associated with infection with one or more species of Fusobacterium nucleatum.

[0073] In some embodiments, the F. nucleatum associated disease may be an inflammatory condition such as, but not limited to, sinusitis, endocarditis, septic arthritis, tonsillitis, and brain, skin, and / or liver abscesses.

[0074] At least six different subspecies of F. nucleatum have been described, including F. nucleatum animalis, F. nucleatum fusiforme, F. nucleatum nucleatum, F. nucleatum polymorphum, F. nucleatum periodonticum, and F. nucleatum vincentii. In some embodiments, F. nucleatum can be a strain associated with a disease state. In some embodiments, the strain can be an F. nucleatum animalis subspecies strain, such as, but not limited to, 7 / 1 (or 7_1), CRC 7 / 3JVN3C1 (or CRC 7_3JVN3C1), and / or 218A8. In some embodiments, the strain can be an F. nucleatum vincentii subspecies strain, such as, but not limited to, 215A9, CC53, and / or 3 / 1 / 36A2 (EAV018). In some embodiments, the strains may be F. nucleatum nucleatum subsp. strains, such as, but not limited to, ATCC25586T, 203L34, and / or 2 / 3 FMU 1 (2_3 FMU 1). In some embodiments, the strains may be F. nucleatum polymorphum strains, such as, but not limited to, 203L28, 203L29, and / or 13 / 3C (13_3C EAV005 or EAVG 005). In some embodiments, the strains may be F. nucleatum fusiforme strains, such as, but not limited to, 203C15, 203L25, and / or 203L30. In some embodiments, the strain may 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.

[0075] In some embodiments, the present disclosure provides a method for inhibiting the growth of Fusobacterium species, such as Fusobacterium nucleatum. The growth of F. nucleatum species can be inhibited in vitro (e.g., in cells or tissue samples from a subject) or in vivo in a subject. In some embodiments, the growth of F. nucleatum can be inhibited by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and / or 90%. In some embodiments, bacterial growth 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%.

[0076] In some embodiments, administering a therapeutic agent described herein to a subject infected with Fusobacterium or having a disease caused or exacerbated / aggravated by Fusobacterium inhibits or reduces replication of Fusobacterium by at least 20%-25%, at least 25%-30%, at least 30%-35%, at least 35%-40%, at least 40%-45%, at least 45%-50%, at least 50%-55%, at least 55%-60%, at least 60%-65%, at least 65%-70%, at least 70%-75%, at least 75%-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 2-5-fold, 2-10-fold, 5-10-fold, or 5-20-fold compared to a negative control, as determined using an assay described herein or others known to one of skill in the art.

[0077] The present disclosure provides therapeutic agents and methods related to F. nucleatum, and the treatment, prevention, and / or management of diseases associated with F. nucleatum.

[0078] In some embodiments, the present disclosure provides a method for inhibiting the growth of Fusobacterium nucleatum (F. nucleatum). Such a method may include contacting F. nucleatum with at least one therapeutic agent such that the growth of F. nucleatum is inhibited. The therapeutic agent may be hygromycin A. Also provided herein is a method for inhibiting or reducing the growth of Fusobacterium nucleatum.

[0079] In some embodiments, the concentration of hygromycin A may be about 1 μg / ml to about 100 μg / ml. In some embodiments, the concentration of hygromycin A may be 5 μg / ml. In some embodiments, the concentration of hygromycin A may be 10 μg / ml. In some embodiments, the concentration of hygromycin A may be 20 μg / ml. In some embodiments, the concentration of hygromycin A may be 40 μg / ml.

[0080] 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 μg / ml or more.

[0081] F. nucleatum can be one or more subspecies, including F. nucleatum animalis, F. nucleatum vincentii, F. nucleatum nucleatum, F. nucleatum polymorphum, F. nucleatum fusiforme, or F. nucleatum periodonticum.

[0082] The present disclosure provides a method for treating or preventing a disease in a subject, comprising contacting the subject with at least one therapeutic agent. In some embodiments, the disease can be cancer, gastrointestinal disorder, or oral disease.

[0083] In some embodiments, the disease may be associated with F. nucleatum infection. In some embodiments, the disease may be cancer, such as colorectal cancer or oral cancer. In some embodiments, the disease may be a gastrointestinal disease, such as inflammatory bowel disease, Crohn's disease, ulcerative colitis, or colorectal cancer. In some embodiments, the disease may be an oral disease, such as periodontal disease. The periodontal disease may be localized aggressive periodontitis, generalized aggressive periodontitis, pulp necrosis, or apical periodontitis. In some embodiments, the disease may be oral cancer.

[0084] 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 μg / ml or more.

[0085] systemic disease The presence of microbes and their metabolic by-products in the mouth can modulate immune responses beyond the oral cavity, thus promoting the development of systemic pathologies. A growing body of literature suggests that there is a link between oral microbes and systemic disease.

[0086] The therapeutic agents of the present disclosure may be used to treat, manage, or prevent systemic diseases associated with oral microbes, such as, but not limited to, cardiovascular disease, gastrointestinal disease, colorectal cancer, diabetes and insulin resistance, Alzheimer's disease, and respiratory infections and adverse pregnancy outcomes.

[0087] Oral microorganisms, such as oral pathogens, can directly or indirectly promote the development of non-oral diseases. Approximately 30 abundant species, mainly gram-negative anaerobic bacteria, in the oral cavity are known to produce endotoxins that can directly contribute to systemic diseases. In some cases, such as after surgical procedures, oral pathogens can also migrate into the bloodstream. Bacterial accumulation in teeth due to poor dental hygiene and / or environmental factors can induce a host inflammatory response that can lead to periodontitis and bone loss, but can also harm the organism systemically.

[0088] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage bacteremia. Tissue trauma, flossing, dental procedures, or even chewing can induce blood vessel breaks in proximity to plaque containing oral microorganisms, which can introduce oral microorganisms into the systemic bloodstream, resulting in bacteremia.

[0089] In some embodiments, the therapeutic agent of the present disclosure can be used to treat, prevent, or manage cardiovascular disease.In some embodiments, cardiovascular disease can be caused or correlated with infection or the presence of oral microorganisms.Bacterial DNA of species such as Treponema denticola, Actinobacillus actinomycetemcomitans, Tannerella forsythia, Eikenella corrodens, Fusobacterium nucleatum, and Campylobacter rectus has been identified in surgically collected atherosclerotic and atherosclerotic plaques.

[0090] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage respiratory infections. In some embodiments, respiratory infections may be caused by or correlated with infection or the presence of oral microorganisms. In some embodiments, the therapeutic agents of the present disclosure may be used to treat, manage, or prevent Lemierre's syndrome, which may be caused by Fusobacterium nucleatum and Fusobacterium necrophorum.

[0091] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage pneumonia, which may be caused by or correlated with infection by or the presence of oral microorganisms.

[0092] In some embodiments, the therapeutic agent of the present disclosure can be used to treat, prevent, or manage diabetes. In some embodiments, diabetes can be caused by or correlated with infection or the presence of oral microorganisms. Chronic infection during periodontitis can lead to exacerbated and dysregulated inflammatory response, which can lead to poor metabolic control of blood glucose and increased insulin requirements. Conversely, diabetes can also lead to various complications, such as poor wound healing, retinopathy, nephropathy, neuropathy, macrovascular disease, and periodontitis.

[0093] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage Alzheimer's disease, which may be caused or correlated with infection or the presence of oral microorganisms, such as Fusobacterium nucleatum.

[0094] 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 μg / ml or more.

[0095] cancer In some embodiments, the therapeutic agents of the present disclosure are used to treat, protect against, and / or manage cancer. In some embodiments, the therapeutic agents of the present disclosure are used to treat, prevent, or manage cancer associated with one or more oral microorganisms.

[0096] In some embodiments, the therapeutic agents of the present disclosure are used to treat, prevent, or manage cancer caused by Fusobacterium. In some embodiments, the cancer may be oral, colorectal, or esophageal cancer. As a non-limiting example, colorectal or esophageal cancer may be caused by Fusobacterium species.

[0097] The subject may have cancer, may be suspected to have cancer, or may have a predisposition to cancer.The therapeutic agent or its pharmaceutically acceptable salt can be administered to the subject as a treatment and maintenance for cancer in all patients.The effect of the therapeutic agent or preparation described herein on the proliferation of cancer cells can be detected by routine assays known in the art.The cancer cell lines that can be carried out such assays are well known to those skilled in the art.

[0098] Oral microbes, including Fusobacterium nucleatum, Porphyromonas gingivalis, Treponema denticola, Aggregatibacter actinomycetemcomitans, and Tannerella forsythia, have received increasing interest in the context of cancer pathogenesis. Periodontal pathogens may be associated with pancreatic and oral cancer. In some embodiments, the therapeutic agent of the present disclosure may be used to inhibit, reduce, or suppress the growth of one or more oral microbes associated with cancer. Oral microbes may contribute to carcinogenesis through various mechanisms, such as inhibiting apoptosis, activating cell proliferation, promoting cell invasion, inducing chronic inflammation, and producing carcinogens. As a non-limiting example, hygromycin A may be used to inhibit the immunosuppressive effects of Fusobacterium nucleatum in colorectal cancer by promoting macrophage polarization through a TLR4-dependent mechanism.

[0099] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, manage, or prevent colorectal, gastric, and / or esophageal cancer.

[0100] The present disclosure also provides a method of administering the therapeutic agent described herein to a subject. The subject may have cancer, may be suspected of having cancer, or may have a predisposition to cancer. The compound or its pharmaceutically acceptable salt is administered to the subject as a treatment and maintenance for cancer in all patients. The effect of the composition described herein on the proliferation of cancer cells can be detected by routine assays known in the art. Cancer cell lines that can be performed such assays are well known to those skilled in the art.

[0101] Cancers and related disorders that may be treated, protected against, or managed using the therapeutic agents and methods described herein include the following: leukemias, including but not limited to acute leukemia, acute lymphocytic leukemia, acute myeloid leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemic leukemia, and myelodysplastic syndromes, chronic leukemias such as, but not limited to, chronic myeloid (granular) leukemia, and chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; Hodgkin's disease and non-Hodgkin's disease. multiple myeloma, including but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; sarcoma of bone, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcoma, angiosarcoma (hemangiosarcoma sarcomas of bone and connective tissue, such as, but not limited to, sarcoma, fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, and synovial sarcoma; brain tumors, including, but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, non-glial tumors, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, and primary brain lymphoma; adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease, and inflammation breast cancer, including but not limited to pathological 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 tumors, and carcinoid or islet cell tumors; pituitary cancer, including but not limited to Cushing's disease, prolactin-secreting tumors, acromegaly, and diabetes insipidus;eye cancer, including but not limited to ocular melanomas such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancer, 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 cancer, including but not limited to squamous cell carcinoma and adenocarcinoma; uterine cancer, including but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancer, including but not limited to epithelial ovarian carcinoma, borderline tumors, germ cell tumors, and stromal tumors; esophageal cancer, including but not limited to squamous cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; adenocarcinoma, fungiform (polypoid), cancer of the stomach, including but not limited to ulcerative, superficial spreading, diffuse spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancer; rectal cancer; liver cancer, including but not limited to hepatocellular carcinoma and hepatoblastoma; gallbladder cancer, including but not limited to adenocarcinoma; cholangiocarcinoma, including but not limited to papillary, nodular, and diffuse; lung cancer, 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 cancer, including but not limited to germinal tumor, seminoma, undifferentiated, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk sac tumor); prostate cancer, including but not limited to adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancer; oral cancer, including but not limited to squamous cell carcinoma; basal carcinoma cancer); salivary gland cancer, including but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic carcinoma; pharyngeal cancer, including but not limited to squamous cell carcinoma and verrucous melanoma; skin cancer, including but not limited to basal cell carcinoma, squamous cell carcinoma, and melanoma, as well as superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, and acral lentigo melanoma; kidney cancer, including but not limited to renal cell carcinoma, renal carcinoma, adenocarcinoma, adenocarcinoma, adrenal tumor, fibrosarcoma, and transitional cell carcinoma (renal pelvis and / or ureter); Wilms' tumor;bladder cancer, including but not limited to transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, and carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendothelioma sarcoma, mesothelioma, synovoma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and papillary adenocarcinoma;

[0102] In some embodiments, the cancer is benign, such as polyps and benign lesions. In other embodiments, the cancer is metastatic. The compositions described herein can be used in the treatment of pre-malignant and malignant conditions. Pre-malignant conditions include hyperplasia, metaplasia, and dysplasia. The treatment of malignant conditions includes the treatment of primary and 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), B-cell acute lymphocytic leukemia, lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, or B-cell non-Hodgkin's lymphoma, rhabdomyosarcoma, neuroblastoma, Ewing's sarcoma, gastric cancer, liver cancer.

[0103] 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 μg / ml or more.

[0104] Gastrointestinal disorders In some embodiments, the therapeutic agents may be used to treat, prevent, or manage Fusobacterium nucleatum-associated gastrointestinal diseases or disorders, such as, but not limited to, colorectal cancer (CRC), inflammatory bowel disease (IBD), and appendicitis.

[0105] Cancers of the gastrointestinal tract account for a significant percentage of all cancer-related deaths, including gastric, colorectal, and esophageal cancers. Colorectal cancer (CRC) is the second most common 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, with specific somatic mutations on oncogenes and tumor suppressor genes identified that are associated with the progression from adenomatous lesions (polyps) to aggressive carcinomas. Inflammation has been recognized as a risk factor for CRC. In some embodiments, the therapeutic agents of the present disclosure can be used to treat, prevent, or manage colorectal cancer. In some embodiments, the therapeutic agents of the present disclosure can be used to treat, prevent, or manage Fusobacterium nucleatum-associated colorectal cancer. CRC can be caused or associated with F. nucleatum infection.

[0106] In some embodiments, the therapeutic agent of the present disclosure can be used to treat, prevent, or manage IBD. In some embodiments, the therapeutic agent of the present disclosure can be used to treat, prevent, or manage Fusobacterium nucleatum-associated IBD. IBD is recognized as a risk factor for CRC. F. nucleatum strains isolated from inflamed tissues of IBD patients are more aggressive than those from normal tissues. Several studies have reported the association of F. nucleatum in appendicitis. Co-occurrence of F. nucleatum with other oral taxa has been observed.

[0107] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage Crohn's disease. In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage Fusobacterium nucleatum associated Crohn's disease.

[0108] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage ulcerative colitis. In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage Fusobacterium nucleatum-associated ulcerative colitis.

[0109] 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 μg / ml or more.

[0110] Veterinary Diseases In some embodiments, the Therapeutic Agents may be used in the treatment, prevention, or management of one or more diseases in animals (also referred to herein as veterinary diseases). In some embodiments, the veterinary diseases may be caused by bacteria such as, but not limited to, Treponema, Fusobacterium, Bacteroides, Campylobacter, Mycoplasma, and Porphyromonas.

[0111] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage digital dermatitis (DD). DD is a foot disease that causes lameness in cattle. It is characterized by an inflammatory dermatitis of the digital skin that is most commonly found on the plantar aspect of the interdigital clefts. In some embodiments, the therapeutic agents may be used to inhibit the growth of bacterial species associated with DD. As a non-limiting example, DD may be caused by Treponema species. In some embodiments, Treponema may be Treponema phagedenis, T. medium, and T. pedis.

[0112] In some embodiments, the therapeutic agents may be used in the treatment, prevention, or management of one or more periodontal diseases in dogs.

[0113] In some embodiments, the therapeutic agents of the present disclosure may be used in the treatment, prevention, or management of Treponem-associated hoof disease. Treponem-associated hoof disease (TAHD) in elk is a debilitating and progressive condition that shares many similarities with bovine digital dermatitis and contagious ovine digital dermatitis.

[0114] 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 μg / ml or more.

[0115] Treponema The present disclosure provides therapeutic agents and methods related to treponematoses, and the treatment, prevention, and / or management of diseases associated with Treponema pallidum infection.

[0116] Provided herein is a method for inhibiting the growth of Treponema by contacting Treponema with at least one therapeutic agent. Also provided herein is a method for reducing Treponema in a subject. Contacting Treponema or a subject with hygromycin A can inhibit the growth of Treponema. In some embodiments, the therapeutic agent can be hygromycin A. Treponema can be Treponema pallidum or Treponema carateum.

[0117] In some embodiments, the concentration of hygromycin A can be about 0.01 μg / ml to about 100 μg / 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 μg / ml or more.

[0118] In some embodiments, the concentration of hygromycin A can be from about 0.01 μg / ml to about 10 μg / ml.

[0119] In some embodiments, the concentration of hygromycin A may be 0.06 μg / ml. In some embodiments, the concentration of hygromycin A may be 0.12 μg / ml. In some embodiments, the concentration of hygromycin A may be 0.24 μg / ml. In some embodiments, the concentration of hygromycin A may be 0.48 μg / ml. In some embodiments, the concentration of hygromycin A may be 0.96 μg / ml. In some embodiments, the concentration of hygromycin A may be 1.92 μg / ml. In some embodiments, the concentration of hygromycin A may be 3.84 μg / ml.

[0120] Treponema pallidum can be Treponema pallidum pallidum, Treponema pallidum pertenue, or Treponema pallidum endemicum.

[0121] The present disclosure also provides a method for treating or preventing treponematosis in a subject. Such a method may include contacting the subject with at least one therapeutic agent. The therapeutic agent may be hygromycin A. The treponematosis may be associated with Treponema pallidum or Treponema carateum infection. In some embodiments, the treponematosis may be syphilis. In some embodiments, the treponematosis may be yaws. In some embodiments, the treponematosis may be bejel. In some embodiments, the treponematosis may be pinta.

[0122] The present disclosure provides methods of use related to the therapeutic agents described herein. In some embodiments, the methods may include methods of reducing the growth of bacteria such as Treponema pallidum.

[0123] In some embodiments, the therapeutic agents of the present disclosure may be used to kill or inhibit the growth of one or more subspecies of Treponema pallidum, including but not limited to Treponema pallidum pallidum, Treponema carateum, Treponema pallidum pertenue, and Treponema pallidum endemicum.

[0124] In some embodiments, the present disclosure provides a method for inhibiting the growth of Treponema pallidum. The growth of T. pallidum species may be inhibited in vitro (e.g., in a cell or co-culture system, or in a tissue sample from a subject) or in vivo in a subject. In some embodiments, the growth of T. pallidum may be inhibited by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and / or 90%. In some embodiments, the growth of the bacteria may 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%.

[0125] In some embodiments, administering a therapeutic agent described herein to a subject infected with Treponema pallidum or having a disease caused or exacerbated / aggravated by Treponema pallidum inhibits or reduces the replication of Treponema pallidum by at least 20%-25%, at least 25%-30%, at least 30%-35%, at least 35%-40%, at least 40%-45%, at least 45%-50%, at least 50%-55%, at least 55%-60%, at least 60%-65%, at least 65%-70%, at least 70%-75%, at least 75%-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 2-5-fold, 2-10-fold, 5-10-fold, or 5-20-fold compared to a negative control, as determined using an assay described herein or others known to one of skill in the art.

[0126] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, and / or manage venereal treponematosis or syphilis.

[0127] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, and / or manage endemic or non-venereal treponematoses. Key differences between endemic treponematoses and syphilis relate to the target population, mode of transmission, and propensity for systemic involvement. Endemic treponematoses primarily affect children in poor rural areas, while syphilis is a ubiquitous disease. Endemic treponematoses may include bejel, yaws, and pinta.

[0128] syphilis In some embodiments, the therapeutic agent of the present disclosure can be used to treat syphilis. Syphilis is a systemic disease caused by T. pallidum pallidum. The disease is divided into stages based on clinical findings that guide treatment and follow-up. In some embodiments, the therapeutic agent of the present disclosure can be used to treat primary syphilis. Primary syphilis classically appears as a single painless ulcer or chancre at the site of infection, but may also present with multiple, atypical, or painful lesions. In some embodiments, the therapeutic agent 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 agent of the present disclosure can be used to treat, manage, and / or prevent tertiary syphilis, which may present with cardiac symptoms, gummatous lesions, tabes dorsalis, and progressive paralysis.

[0129] In some embodiments, the therapeutic agent of the present disclosure can be used to treat latent infection. Latent infection (i.e., lacking clinical signs) is detected by serological testing. Latent syphilis acquired within the previous year is called early latent syphilis; all other cases of latent syphilis are classified as late latent syphilis or latent syphilis of unknown duration.

[0130] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage central nervous system (CNS)-related symptoms of syphilis. T. pallidum may infect the CNS at any stage of syphilis, resulting in neurosyphilis. Early neurological clinical signs or syphilitic meningitis (e.g., cranial nerve dysfunction, meningitis, meningovascular syphilis, stroke, and acutely altered mental status) usually appear within the first few months or years of infection. Late neurological signs (e.g., tabes dorsalis and progressive paralysis) occur 10 to >30 years after infection.

[0131] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage ocular or otosyphilis. Infection of the visual system (ocular syphilis) or auditory system (otosyphilis) may occur at any stage of syphilis, but is generally identified during the early stages and may or may not present additional CNS symptoms. Ocular syphilis often presents as a pan-uveitis, but may affect structures in both the anterior and posterior segments of the eye, including conjunctivitis, anterior uveitis, posterior interstitial keratitis, optic neuropathy, and retinal vasculitis. Ocular syphilis may result in permanent vision loss. Otosyphilis typically presents with cochlear-vestibular symptoms, including tinnitus, vertigo, and sensorineural hearing loss. Hearing loss may be unilateral or bilateral, may have a sudden onset, and may progress rapidly. Otosyphilis may result in permanent hearing loss.

[0132] 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 μg / ml or more.

[0133] Yaws In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage yaws. The spread of yaws caused by T. pallidum subsp. pertenue occurs by direct skin contact with infectious lesions and is facilitated by breaks in the skin of traumatic or other etiology (e.g., abrasions or scabies). In some embodiments, the therapeutic agents of the present disclosure may be used to treat one or more stages of the disease. The first stage of the disease appears after a variable incubation period (approximately 21 days) as a solitary erythematous papule that may grow into a papilloma 2-5 cm in diameter by peripheral extension or by coalescing with satellite papules. The lesion is non-painful but may be pruritic, and it is typically covered by a crust that conceals an ulcer with a raised dark border and an erythematous moist center, giving it an overall resemblance to a raspberry (hence the African and French names). In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, and / or manage primary lesions, which are often found on the lower extremities. The lesions are highly contagious and may persist for weeks or months before spontaneously healing, often leaving a hypopigmented or sunken area bounded by a dark border. At this stage, local lymphadenopathy and joint pain may also occur. In some embodiments, the therapeutic agents of the present disclosure may be used to treat lymphadenopathy and / or joint pain associated with yaws. In the majority of cases, the primary lesions spontaneously heal before the development of secondary signs of yaws, whose appearance is due to systemic dissemination of the pathogen during the initial infection. In some embodiments, the therapeutic agents of the present disclosure may be used to treat systemic dissemination associated with yaws. In some embodiments, the therapeutic agents of the present disclosure may be used to treat, manage, or prevent secondary signs associated with yaws. Secondary signs may include flat condyloma in moist spaces such as the axillae and groin, as well as a morbilliform rash. Keratinized plaques on the palms and soles are also common at this stage.In some embodiments, the therapeutic agents of the present disclosure may be used to treat periostitis and osteitis, which may affect the bones of the upper and lower limbs (tibia, fibula, and forearm) and the proximal phalanges of the fingers and toes, resulting in bone pain and swelling of the toes. In some embodiments, the therapeutic agents of the present disclosure may be used to treat latent disease. Secondary lesions heal spontaneously within weeks or months, and the patient enters a latent stage of infection that can only be recognized by serological testing and will last a lifetime unless treated. Recurrence of secondary manifestations (generally once or twice) may be seen up to five years after the initial infection, although relapses after 10 years have been reported.

[0134] In some embodiments, the therapeutic agent of the present disclosure can be used to treat, prevent, or manage tertiary yaws. Approximately 10% of untreated patients will develop tertiary yaws, which is characterized by subcutaneous gummatous nodules, chronic periostitis that can cause obvious curvature of the tibia (i.e., sabre shin), and a destructive process that leads to a saddle nose and perforation / rupture of the palate and nasal septum (i.e., gangos). Bilateral hypertrophic periostitis of the paranasal sinuses maxillary and nasal bridge causes the clinical sign known as goundou.

[0135] Bejel In some embodiments, the therapeutic agent of the present disclosure may be used to treat bejel, which is the Arabic name for endemic (or non-venereal) syphilis caused by T. pallidum subspecies endemicum. In some embodiments, the therapeutic agent of the present disclosure may be used to treat acute infections observed in children aged 2-15 years in dry, arid climates. Although the mode of transmission has not been thoroughly investigated, it is believed to occur by mucous membrane and skin contact, or by sharing eating utensils or drinking vessels.

[0136] In contrast to other treponematoses, the primary lesion of bejel often goes unnoticed. However, when seen, it appears as a small and painless mucosal papule or ulcer developing in the oral cavity or nasopharynx. In some embodiments, the therapeutic agents of the present disclosure may be used to treat the primary and / or secondary lesions associated with bejel. The secondary lesions are very similar to those of venereal syphilis and may appear as mucosal patches on the oral mucosa, tonsils, tongue, lips, and nasopharynx. Fissicular papules in the labial commissures (angular stomatitis seen in yaws patients), nonpruritic skin rash, generalized lymphadenopathy, and laryngitis are common signs. Secondary skin lesions include condylomata lata in intertrigonoid body areas, corresponding to those in yaws and syphilis. Maculopapular or papular-scaling lesions, as well as nonpruritic generalized papular skin eruptions, may be observed in a minority of patients with bejel. As in yaws, osteitis and periostitis of the long bones and hands may occur, causing nocturnal bone pain. Secondary signs heal within 6-9 months, and the disease goes into latent state. In some embodiments, the therapeutic agents of the present disclosure may be used to treat the third stage of bejel. The third stage may appear earlier than for yaws (6 months to several years), but is characterized by gummatous lesions of the skin, mucous membranes, and bone that may progress to destructive ulcers, as in yaws. The skin lesions eventually resolve, leaving characteristic hypopigmented scars surrounded by hyperpigmentation.

[0137] 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 μg / ml or more.

[0138] Pinta In some embodiments, the therapeutic agents of the present disclosure may 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 present disclosure may be used to treat pinta caused by Treponema carateum, which is considered the mildest form of treponematosis in that its lesions are limited to the skin with no systemic symptoms or evidence of vertical transmission. The disease is found locally in tropical Central and South America. Because laboratory strains of this pathogen are not currently available for investigation, T. carateum is the least characterized agent of human treponematosis and remains classified independently from 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) with chronic skin lesions are thought to be the primary carriers of the disease. Pinta also appears to be transmitted from their mothers to young children by close contact. In some embodiments, the therapeutic agents of the present disclosure may be used to treat early or late stages of Pinta. Primary lesions appear as papules or erythematous-scaly plaques on exposed parts of the body after an incubation period of one week to two months. Satellite lesions may be present. Over time, the papules increase in size and coalesce to form plaques with pale centers. After several months, many of the plaques become pale or acquire a pale bluish / grayish pigmentation with a more prominent color in the center of the lesion. Primary lesions may heal, leaving a slightly pigmented or pale area, or may persist for several years and become indistinguishable from secondary lesions. Local lymphadenopathy is common at this stage. After several months or years, small disseminated secondary lesions (called pintids) may appear in the form of scaly papules that may again expand and coalesce in psoriasiform plaques. These plaques may be hypo- or hyperpigmented, as well as erythematous or peeling.Different types of pinecone may be present simultaneously in the same individual. This stage usually lasts for 2-4 years, during which time some plaques will heal and others will persist and expand. The later stage usually develops 2-4 years after the initial infection and is characterized by the appearance of pigmentary changes, skin atrophy, and hyperkeratosis.

[0139] 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 μg / ml or more.

[0140] Other indications In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage Fusobacterium nucleatum-associated infections and abscesses, including infections of the head and neck (Lemierre's syndrome, acute and chronic mastoiditis, chronic otitis and sinusitis, tonsillitis, peritonsillar and retropharyngeal abscesses, postanginal cervical lymphadenitis, periodontitis), brain, lungs, abdomen, pelvis, bones, joints, and blood.

[0141] In some embodiments, the therapeutic agent of the present disclosure can be used to treat, prevent, or manage cardiovascular disease. F. nucleatum is involved in cardiovascular disease (CVD). It is frequently detected in atherosclerotic plaque and is one of the most common periodontal pathogens detected in ruptured cerebral aneurysms.

[0142] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage cerebral aneurysms. In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage Fusobacterium nucleatum-associated cerebral aneurysms.

[0143] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage Lemierre's syndrome. In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage Fusobacterium nucleatum-associated Lemierre's syndrome.

[0144] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage respiratory infections. In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage Fusobacterium nucleatum associated respiratory infections.

[0145] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage organ abscesses. In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage Fusobacterium nucleatum-associated organ abscesses.

[0146] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage rheumatoid arthritis. In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage Fusobacterium nucleatum-associated rheumatoid arthritis.

[0147] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage Alzheimer's disease. In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage Fusobacterium nucleatum associated Alzheimer's disease.

[0148] Co-infections and comorbidities In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage a subject with an additional bacterial infection. In some embodiments, the subject may have a disease associated with Tannerella forsythia, Porphyromonas gingivalis, and Streptococcus.

[0149] In some embodiments, the therapeutic agents of the present disclosure may be used to treat, prevent, or manage subjects with comorbidities. By way of non-limiting example, the comorbidity may be diabetes (type 1 or type 2), heart disease, and / or hypertension, SARS-CoV-2 infection (e.g., COVID-19).

[0150] IV. Formulation In some embodiments, the therapeutic agent may be administered to a human, human patient, or subject. For purposes of this disclosure, the phrase "active ingredient" generally refers to the therapeutic agent to be delivered as described herein.

[0151] Although the description of the formulations provided herein is primarily directed to formulations suitable for administration to humans, it will be understood by those skilled in the art that such therapeutic agents are generally suitable for administration to any other animal, for example, non-human animals, such as non-human mammals. The modification of formulations suitable for administration to humans to make therapeutic agents suitable for administration to various animals is well understood, and a veterinary pharmacologist of ordinary skill can design and / or perform such modifications using only routine experimentation, if at all. The subjects to which the formulations are intended to be administered 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.

[0152] The formulations of the therapeutic agents described herein can be prepared by any method known or hereafter developed in the field of pharmacology.In general, such preparation methods include 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 desired, dividing, shaping, and / or packaging the product into the desired single or multiple dosage units.

[0153] The formulation according to 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, "unit dose" is a discrete amount of pharmaceutical composition that contains a predetermined amount of active ingredient.The amount of active ingredient is generally equal to the dosage of active ingredient that would be administered to a subject, and / or a convenient fraction of such dosage, such as, for example, a half or a third of such dosage.

[0154] The relative amounts of active ingredient, pharma- ceutically acceptable excipient, and / or any additional ingredients in a formulation according to the present disclosure will vary depending on the identity, size, and / or condition of the subject being treated, as well as depending further on the route by which the formulation is to be administered. By way of example, the formulation may comprise from 0.1% to 100%, e.g., from .5 to 50%, 1 to 30%, 5 to 80%, or in some embodiments at least 20%, at least 40%, at least 60%, or at least 80% (w / w) active ingredient.

[0155] The therapeutic agent of the present disclosure may be formulated with one or more excipients to (1) increase stability; (2) allow sustained or delayed release; (3) modify biodistribution; (4) modify the release profile of the therapeutic agent in vivo.Non-limiting examples of excipients include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonicity agents, thickening or emulsifying agents, and preservatives.The excipients of the present disclosure may also include, but are not limited to, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, hyaluronidase, nanoparticle mimics, and combinations thereof.

[0156] In some embodiments, the pharmaceutical compositions or formulations of the present disclosure may be adapted 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 for incorporating therapeutic agents into pharmaceutical preparations are widely known in the art. Determining the appropriate prescribed dosage of a pharmacologically active compound to include in a pharmaceutical formulation to achieve a desired biological outcome is within the level of skill of the artisan. Pharmaceutical formulations may include excipients such as, but not limited to, binders, coatings, disintegrants, fillers, diluents, flavorings, colorings, lubricants, glidants, preservatives, adsorbents, sweeteners, conjugated linoleic acid (CLA), gelatin, beeswax, purified water, glycerol, any type of oil, including but not limited to fish oil or soybean oil, etc. The therapeutic agent and / or pharmaceutical formulation may include suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as, for example, polyethylene glycols. It will be further understood by those skilled in the art that the term also encompasses those therapeutic 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.

[0157] Pharmaceutical formulations according to the present disclosure can be prepared, packaged, and / or sold in bulk, as single unit doses, and / or as multiple single unit doses.As used herein, "unit dose" refers to a discrete amount of pharmaceutical formulation that contains a predetermined amount of therapeutic agent or other compound.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 a dosage, including but not limited to a half or a third of such a dosage.

[0158] In some embodiments, the therapeutic agents of the present disclosure may be formulated as toothpaste, mouthwash, gum massage cream, edible film, or dental floss.

[0159] Excipients Formulation, as used herein, may additionally include pharma- ceutically acceptable excipients, including any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, appropriate for the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st ed., AR 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 their preparation. As long as any conventional excipient vehicle is not incompatible with the substance or its derivatives, such as by producing any undesirable biological effects or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition, its use is contemplated within the scope of this disclosure.

[0160] In some embodiments, the pharma- ceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipient is approved for human use and for veterinary use. In some embodiments, the excipient is approved by the U.S. Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0161] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants, and / or oils. Such excipients may be optionally included in the pharmaceutical composition.

[0162] 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, corn starch, powdered sugar, and the like, and / or combinations thereof.

[0163] 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), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and the like, and / or combinations thereof.

[0164] Exemplary surfactants and / or emulsifiers include natural emulsifiers (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, glyceryl ...monostearate), ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, 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® 100], sorbitan monostearate [SPAN® 110], sorbitan monostearate [SPAN® 120], sorbitan monostearate [SPAN® 130], sorbitan monostearate [SPAN® 140], sorbitan monostearate [SPAN® 150], sorbitan monostearate [SPAN® 160], sorbitan monostearate [SPAN® 170], sorbitan monostearate [SPAN® 180], sorbitan monostearate [SPAN® 190], sorbitan monostearate [SPAN® 20 ... 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, and the like, and / or combinations thereof.

[0165] Exemplary binders include, but are not limited to, starches (e.g., corn starch and starch paste); 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 husk, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabinogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohols; and the like; and combinations thereof.

[0166] Exemplary preservatives may 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, butylparaben, methylparaben, ethylparaben, propylparaben, 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 hydroxyanisole (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®.

[0167] Exemplary buffering agents include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, 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, calcium hydrogen phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dipotassium phosphate, potassium dihydrogen phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixture, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and the like, and / or combinations thereof.

[0168] Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and the like, and combinations thereof.

[0169] Exemplary oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, juniper, chamomile, canola, caraway, carnauba wax, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cottonseed, emu, eucalyptus, evening primrose, fish, linseed, geraniol, gourd, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, maize, and the like. Exemplary oils include, but are not limited to, cadamia nuts, mallow, mango seeds, meadowfoam seeds, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seeds, pumpkin seeds, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savory, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, camellia, vetiver, walnut, and wheat germ oil. 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.

[0170] 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 discretion of the formulator.

[0171] V. Medication and Administration In some embodiments, the therapeutic agent and / or pharmaceutical formulation comprising the therapeutic agent may be administered according to one or more routes of administration. In some embodiments, administration may be enteral (into the intestine), transdermal, intravenous bolus, intralesional (introduced directly into or into a localized lesion), intrapulmonary (into the lungs or its bronchi), diagnostic, intraocular (into the eye), transtympanic (across or through the tympanic cavity), intravesical infusion, sublingual, nasogastric (through the nose and into the stomach), spinal, intrachondral (into cartilage), insufflation (breathing through the nose), rectal, intravascular (into one or more blood vessels), buccal (towards the cheek), dental (into one or more teeth), intratesticular (into the testicles), intratympanic (into the aurus media), transdermal, intrathoracic (into the chest), submucosal, dermal, epidermal (application on the skin), intracoronal, intraperitone ... intracornal), intramedullary (into the medullary cavity of a bone), intraabdominal, epidural (into the dura), intramuscular (into a muscle), intralymphatic (into lymphatic fluid), iontophoresis (by electric current that moves ions of soluble salts into body tissues), subcutaneous (under the skin), intragastric (into the stomach), intranasal (through the nose), vaginal, intravenous infusion, intrasinus, intraprostatic (into the prostate), soft tissue, intradural (into or under the dura), subconjunctival, oral (through the mouth), peridural, parenteral, intraduodenal (into the duodenum), intracapsular (into the cisterna magna cerebellomedularis), periodontal, periarticular, biliary perfusion, intracoronary (into a coronary artery), intrathecal (into the cerebrospinal fluid at any level of the cerebrospinal axis), intrameningeal (into the meninges), intracavernous injection (into a pathological cavity), intracavitary (into the base of the penis), intrabiliary, subarachnoid, intracapsular, ureteral (into the ureter), intratendinous (into a tendon), auricular (at or via the ear), intracardiac (into the heart), enema, intraepidermal (into the epidermis), intraventricular (into the ventricle), intramyocardial (into the myocardium), intraductal (into the tubules of an organ), vaginal, sublabial, intracorporus cavernosum (corpus cavernosum of the penis)into the expansile spaces of the skin (cavenosa), intradermal (into the skin itself), intravitreal (through the eye), perineural, cardiac perfusion, irrigation (bathing or flushing an open wound or body cavity), ear drops, intratracheal, intraosseous injection (into the bone marrow), caudal anesthesia, intrauterine, transtracheal (through the wall of the trachea), intra-articular, intracorneal (into the cornea), intracervical, extracorporeal, intraspinal (into the spinal column), transmucosal (diffusion through mucous membranes), topical, photopheresis, oropharyngeal (directly into the mouth and pharynx), occlusive dressing technique (a local route of administration that is then covered by a dressing that closes the area), transplacental (through or across the placenta), intrapericardial (into the pericardium), intraarterial (into an artery), interstitial, intracerebral (into the cerebrum), intraventricular (into the ventricles), intrapleural (into the pleura) ), infiltration, intrabronchial, intranasal (into the nose or periorbital sinuses), intraductal (into the ducts of a gland), intracoccygeal (into the cauda equina), nerve block, retrobulbar (behind the pons or behind the eyeball), intravenous (into a vein), intra-amniotic, conjunctival, intrasynovial (into the synovial cavity of a joint), gastrointestinal, intraluminal (into the lumen of a tube), electroosmosis, intraileal (into the distal part of the small intestine), intraesophageal (into the esophagus), extra-amniotic administration, hemodialysis , intragingival (into the gums), intratumoral (into a tumor), ophthalmic (onto the conjunctiva), laryngeal (directly onto the larynx), urethral (into the urethra), intravaginal, intraperitoneal (instillation or injection into the peritoneum), respiratory (into the airways by inhalation orally or nasally for localized or systemic effect), intradiscal (into the intervertebral disc), ophthalmic (into the external eye), and / or intraovarian (into the ovary).

[0172] In some embodiments, the therapeutic agent and / or pharmaceutical formulation comprising the therapeutic agent is administered intra-articularly, extracorporeally, intrabronchially, intracervically, intrasinusially, intratracheally, intraintestinally, epidurally, intraabdominally, intrabiliary, intrasynovially, oropharyngeally, interstitially, intracardiacly, intrachondrally, intracoccygeally, intracavernosally, intracerebrally, intracavernosally, intracavitaryly, intracorneally, intracapsularly, intracranially, intradermally, intralesionally, intratympanically, intragingivally, intraocularly, intradiscally, intraductally, intraduodenally, ophthalmologically, intradurally, intraepidermally, intraesophageally, nasogastricly, nasally, laryngeally, intraventricularly, intragastricly, intrahepatically, intraluminally, intranidally ... It may be administered intrauterine, intravesicular, intralymphatic, intramammary, intramedullary, intranasal, intrathecal, intranodal, intraovarian, intraperitoneal, intrapleural, intraprostatic, intraluminal, intraspinal, intrasynovial, intratendinous, intratesticular, subconjunctival, intraventricular, superficial, intravenous, retrobulbar, periarticular, intrathoracic, subarachnoid, intraductal, periodontal, transtympanic, transtracheal, intratumoral, vaginal, urethral, ​​intrauterine, oral, gastrointestinal, parenteral, sublingual, ureteral, transdermal, peridural, transmucosal, perineural, transdermal, rectal, soft tissue, intra-arterial, subcutaneous, topical, extra-amniotic, ear drop, or intravesical.

[0173] The therapeutic and / or pharmaceutical formulations of the present disclosure may be administered orally, although any suitable route of administration may be employed to provide a subject with an effective dosage of the drug of the chemical composition described herein. For example, oral, rectal, topical, parenteral, ocular, pulmonary, nasal, etc. may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, etc. In certain embodiments, it may be advantageous to administer the compositions described herein orally.

[0174] The therapeutic agent and / or pharmaceutical preparation of the present disclosure can be administered in a conventional manner by any route in which 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, or vaginally, by inhalation, by depot injection, or by implantation.Thus, the mode of administration of the composition of the present disclosure (alone or in combination with other pharmaceuticals) can be, but is not limited to, sublingual, injection material (including short-acting, depot, implant, and pellet forms that are injected subcutaneously or intramuscularly), or by using transdermal forms such as vaginal cream, suppository, pessary, vaginal ring, rectal suppository, intrauterine device, and patch and cream.

[0175] For inhalation or intranasal administration, pharmaceutical formulations can be delivered in the form of aerosol spray presentation from a pressurized pack or nebulizer. Compounds can also be delivered in the form of creams, liquids, sprays, powders, or suppositories. Metered doses of formulations can be provided from a reservoir of formulations. In addition, pre-measured dosages can be provided, for example, suppository forms can be provided with pre-measured dosages for insertion into the nose. A kit can be provided that includes a prepared dosage form and instructions for use to administer the dosage.

[0176] Suitable topical formulations for use in this embodiment may also include transdermal devices, aerosols, creams, ointments, lotions, dusting powders, gels, and the like.

[0177] dosage The therapeutic agents and / or pharmaceutical formulations described herein may be administered to a subject using any amount and any route of administration effective to treat 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.

[0178] In some embodiments, the therapeutic agents described herein may be provided to a subject at a concentration of about 0.01 μg / ml to about 100 μg / ml. In some embodiments, the therapeutic agents may be provided to a subject at a concentration of about 0.01 to about 0.05 μg / ml, about 0.05 μg / ml to about 1.0 μg / ml, about 1.0 μg / ml to about 10 μg / ml, about 10 μg / ml to about 50 μg / ml, or about 50 μg / ml to about 100 μg / ml. In some embodiments, the therapeutic agents described herein may be provided to a subject at a concentration of about 1 μg / ml to about 5 μg / ml, about 5 μg / ml to about 10 μg / ml, about 10 μg / ml to about 20 μg / ml, about 20 μg / ml to about 30 μg / ml, about 30 μg / ml to about 40 μg / ml, about 40 μg / ml to about 50 μg / ml, about 50 μg / ml to about 60 μg / ml, about 60 μg / ml to about 70 μg / ml, about 70 μg / ml to about 80 μg / ml, about 80 μg / ml to about 90 μg / ml, or about 90 μg / ml to about 100 μg / ml.

[0179] 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 μg / ml or more.

[0180] In some embodiments, the therapeutic agents described herein may be provided to a subject at a concentration of 0.05 μg / ml.

[0181] In some embodiments, the therapeutic agents described herein may be provided to a subject at a concentration of 0.06 μg / ml. In some embodiments, the therapeutic agents described herein may be provided to a subject at a concentration of 0.12 μg / ml. In some embodiments, the therapeutic agents described herein may be provided to a subject at a concentration of 0.24 μg / ml. In some embodiments, the therapeutic agents described herein may be provided to a subject at a concentration of 0.48 μg / ml. In some embodiments, the therapeutic agents described herein may be provided to a subject at a concentration of 0.96 μg / ml. In some embodiments, the therapeutic agents described herein may be provided to a subject at a concentration of 1.92 μg / ml. In some embodiments, the therapeutic agents described herein may be provided to a subject at a concentration of 3.84 μg / ml. In some embodiments, the therapeutic agents described herein may be provided to a subject at a concentration of 0.1 μg / ml. In some embodiments, the therapeutic agents described herein may be provided to a subject at a concentration of 0.2 μg / ml. In some embodiments, the therapeutic agents described herein may be provided to a subject at a concentration of 0.5 μg / ml. In some embodiments, the therapeutic agents described herein may be provided to a subject at a concentration of 1.0 μg / ml. In some embodiments, the therapeutic agents described herein may be provided to a subject at a concentration of 2.0 μg / ml. In some embodiments, the therapeutic agents described herein may be provided to a subject at a concentration of 5.0 μg / ml. In some embodiments, the therapeutic agents described herein may be provided to a subject at a concentration of 10 μg / ml. In some embodiments, the therapeutic agents described herein may be provided to a subject at a concentration of 20 μg / ml. In some embodiments, the therapeutic agents described herein may be provided to a subject at a concentration of 40 μg / ml.

[0182] In some embodiments, the therapeutic agent and / or pharmaceutical formulation of the present disclosure is provided in one or more doses and administered to the subject one or more times. Some therapeutic agents and / or pharmaceutical formulations are provided in only a single administration. Some therapeutic agents and / or pharmaceutical formulations are administered according to a dosing schedule that includes two or more administrations. Each administration can be the same dose or can be different from the previous and / or subsequent doses. In some embodiments, the subject is provided with an initial dose that is higher than the subsequent doses (referred to herein as a "loading dose"). In some embodiments, the dose is decreased over the course of administration.In some embodiments, the dosing schedule is about every 2 hours to about every 10 hours, about every 4 hours to about every 20 hours, about every 6 hours to about every 30 hours, about every 8 hours to about every 40 hours, about every 10 hours to about every 50 hours, about every 12 hours to about every 60 hours, about every 14 hours to about every 70 hours, about every 16 hours to about every 80 hours, about every 18 hours to about every 90 hours, about every 20 hours to about every 100 hours, about every 22 hours to about every 120 hours, about every 24 hours to about every 132 hours, about every 30 hours to about every 144 hours, about every 36 hours to about every 18 hours, about every 18 hours to about every 100 hours, about every 22 hours to about every 120 hours, about every 24 hours to about every 132 hours, about every 30 hours to about every 144 hours, about every 18 hours to about every 18 ... every ~ about every 156 hours, every 48 hours ~ about every 168 hours, every 2 days ~ about every 10 days, every 4 days ~ about every 15 days, every 6 days ~ about every 20 days, every 8 days ~ about every 25 days, every 10 days ~ about every 30 days, every 12 days ~ about every 35 days, every 14 days ~ about every 40 days, every 16 days ~ about every 45 days, every 18 days ~ about every 50 days, every 20 days ~ about every 55 days, every 22 days ~ about every 60 days, every 24 days ~ about every 65 days, every 30 days ~ about every 70 days, every 2 weeks ~ about every 8 weeks, every 3 weeks ~ about every 12 weeks , approximately every 4 weeks to approximately every 16 weeks, approximately every 5 weeks to approximately every 20 weeks, approximately every 6 weeks to approximately every 24 weeks, approximately every 7 weeks to approximately every 28 weeks, approximately every 8 weeks to approximately every 32 weeks, approximately every 9 weeks to approximately every 36 weeks, approximately every 10 weeks to approximately every 40 weeks, approximately every 11 weeks to approximately every 44 weeks, approximately every 12 weeks to approximately every 48 weeks, approximately every 14 weeks to approximately every 52 weeks, approximately every 16 weeks to approximately every 56 weeks, approximately every 20 weeks to approximately every 60 weeks, approximately every 2 months to approximately 6 months, approximately every 3 months to approximately 12 months, approximately every 4 months to approximately 18 months, approximately every 5 months The present invention includes administration of a pharmaceutical formulation at intervals of about every 24 months to about every 24 months, about every 6 months to about every 30 months, about every 7 months to about every 36 months, about every 8 months to about every 42 months, about every 9 months to about every 48 months, about every 10 months to about every 54 months, about every 11 months to about every 60 months, about every 12 months to about every 66 months, about 2 years to about 5 years, about 3 years to about 10 years, about 4 years to about 15 years, about 5 years to about 20 years, about 6 years to about 25 years, about 7 years to about 30 years, about 8 years to about 35 years, about 9 years to about 40 years, about 10 years to about 45 years, about 15 years to about 50 years, or more than every 50 years.

[0183] The desired dosage may be delivered for a duration of about 5 days to 365 days, about 5 days to 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.

[0184] In some embodiments, the desired dosage of the formulations described herein can be administered once a day or multiple times a day.For example, a treatment regimen can include administering a dosage level sufficient to deliver 10mg / kg body weight twice a day, 20mg / kg body weight twice a day, 50mg / kg body weight once a day, 10mg / kg body weight three times a day, 20mg / kg body weight four times a day, or 50mg / kg body weight twice a day.

[0185] Pulse Dosing In some embodiments, the subject may be administered pulse dosing of the therapeutic agent and / or pharmaceutical formulation of the present disclosure. As used herein, "pulse" refers to multiple dosing at time intervals separated. Generally, upon administration of the first dosing, bacterial growth may be inhibited, delayed, and / or bacteria may be killed. After the first dosing, bacterial levels may increase; a second dosing may be initiated. Thus, eradication of bacteria may be achieved by several rounds of pulse dosing. As a non-limiting example, a pulse dosing schedule may include a first round of dosing that includes administration of the pharmaceutical formulation for 5 days, allotting a period of recovery of about 24 hours, followed by a second round of dosing. Additional rounds of dosing (e.g., a third and fourth round of dosing) of 5 days each may be incorporated after a 24-hour recovery period between each round of dosing. In some embodiments, the pulse dosing schedule is about every 2 hours to about every 10 hours, about every 4 hours to about every 20 hours, about every 6 hours to about every 30 hours, about every 8 hours to about every 40 hours, about every 10 hours to about every 50 hours, about every 12 hours to about every 60 hours, about every 14 hours to about every 70 hours, about every 16 hours to about every 80 hours, about every 18 hours to about every 90 hours, about every 20 hours to about every 100 hours, about every 22 hours to about every 12 hours to about every 12 hours to about every 14 hours to about every 12 hours to about every 14 hours to about every 12 hours to about every 12 hours to about every 14 ... This includes administration of the pharmaceutical formulation every hour to about every 120 hours, about every 24 hours to about every 132 hours, about every 30 hours to about every 144 hours, about every 36 hours to about every 156 hours, about every 48 hours to about every 168 hours, about every 2 days to about every 10 days, about every 4 days to about every 15 days, about every 6 days to about every 20 days, about every 8 days to about every 25 days, about every 10 days to about every 30 days, about every 12 days to about every 35 days, or about every 14 days to about every 40 days.

[0186] VI.Definition Administer: The terms "administer", "administration", "administering", and the like, when used in conjunction with a therapeutic agent, refer to the delivery of the therapeutic agent to a subject, whereby the therapeutic agent has a positive impact, i.e., has a therapeutic effect on the subject or tissue or organ to which it is targeted. The therapeutic agents described herein may be administered alone or in combination (concurrently or sequentially) and / or with other pharmaceutical agents. For example, the therapeutic agent may be administered in combination with a vaccine, an antibiotic, an antiviral agent, an anticancer agent, or an anti-neoplastic agent, or in combination with other therapies, such as herbal remedies, acupuncture, naturopathy, etc.

[0187] Effective amount: As used herein, the term "effective amount" generally refers to the amount of a therapeutic agent administered to reduce, prevent, or inhibit a disease. The amount will vary for each compound and depending on known factors related to the item or use to which the therapeutic agent is applied.

[0188] Modulation: The term "modulation" refers to the upregulation (ie, activation or stimulation), downregulation (ie, inhibition or suppression) of a response, or the two, in combination or separately.

[0189] Pharmaceutically acceptable: As used herein, the term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that are within the scope of sound medical judgment and suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio, in accordance with guidelines from agencies such as the U.S. Food and Drug Administration. As used herein, "pharmaceutical acceptable carrier" refers to all components of a pharmaceutical formulation that facilitates delivery of the composition in vivo. Pharmaceutically acceptable carriers include, but are not limited to, diluents, preservatives, binders, lubricants, disintegrants, swelling agents, fillers, stabilizers, and combinations thereof.

[0190] Subject: "Subject" may include a human subject for medical purposes, such as for the treatment of an existing disease, disorder, or condition, or for prophylaxis to prevent the onset of a disease, disorder, or condition, or an animal subject for medical, veterinary, or development purposes. Suitable animal subjects include mammals, including, but not limited to, primates, such as humans, monkeys, apes, gibbons, chimpanzees, orangutans, macaques, etc.; cattle, such as cattle, bulls, etc.; sheep, such as sheep; goats, such as goats; swine, such as pigs, adult pigs, etc.; equines, such as horses, donkeys, zebras, etc.; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, etc.; and rodents, including mice, rats, guinea pigs, etc. The animal may be a transgenic animal. In some embodiments, the subject is a human, including, but not limited to, fetuses, neonates, infants, juveniles, and adult subjects. Furthermore, a "subject" may include a patient suffering from or suspected of suffering from a disease, disorder, or condition. Thus, the terms "subject" and "patient" are used interchangeably herein. A subject also includes an animal disease model (e.g., a rat or mouse used in an experiment, etc.).

[0191] Therapeutic Agent: As used herein, the term "therapeutic agent" refers to any substance used to restore or promote the health and / or well-being of a subject and / or to treat, prevent, ameliorate, cure, or diagnose a disease, disorder, or condition.

[0192] Treatment or Treating: The terms "treatment", "treating" and the like refer to an intervention performed with the intent of 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.

[0193] Preventing: As used herein, the term "preventing" refers to an intervention performed to reduce the chance of acquiring a disorder, disease, or condition, or a symptom associated with a disorder, disease, or condition.

[0194] Details of one or more embodiments of the present disclosure are set forth in the accompanying description 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, preferred materials and methods are described herein. Other features, objects, and advantages of the present disclosure will be apparent from the description. In the description, the singular form also includes the plural form unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present disclosure belongs. In case of conflict, the present description will control.

[0195] The present disclosure is further illustrated by the following non-limiting examples. EXAMPLES

[0196] Example 1 Experimental Overview: Assaying Treponema denticola Sensitivity to Hygromycin A (Hyg A) Hyg A sensitivity as determined by both minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for a representative group of T. denticola strains, including both "laboratory" and "clinical isolate" strains. The strains tested have levels of characterization ranging from substantial to essentially none. For control and comparison purposes, the Hyg A sensitivity of several oral bacterial species, representing a wide range of bacterial phyla present in the oral microbiome, was tested in parallel. Although essentially all oral microorganisms are naturally occurring "commensal organisms," several species are more highly associated with disease (dental caries, gingivitis, periodontitis), especially when present at elevated levels compared to those commonly found in healthy conditions. In this study, the strains tested included Fusobacterium nucleatum (ATCC 25586), Parvimonas micra (ATCC 33270), and Porphyromonas gingivalis (ATCC 33277). The MICs and MBCs of Hyg A for selected non-Treponema oral bacterial species were determined according to the Clinical and Laboratory Standards Institute (CLSI) criteria described by Wiegand et al., 2008 ("Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances." Nat Protoc 3:163-75.10.1038 / nprot.2007.521, incorporated herein by reference), with the modifications documented below. The strains were grown at 37°C in standard tryptone yeast extract medium with supplements required for growth of the individual species and under appropriate atmospheric conditions (anaerobic or 5% CO2).

[0197] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of hygromycin A (Hyg A) against Treponema denticola strains were determined. T. denticola was grown at 37°C in an anaerobic chamber (Coy Laboratory Products, Grass Lake, MI) in an atmosphere consisting of nitrogen, hydrogen, and carbon dioxide (85:10:5 ratio). Under these conditions, T. denticola generation time in liquid medium is approximately 12 hours. T. denticola was grown in deep 96-well polystyrene plates (ThermoScientific 278606; well volume 0.9 ml) with lids sealed with parafilm, as compared to growth in polystyrene tubes. T. denticola was grown to late logarithmic phase under anaerobic conditions (4 days) in TYGVS medium containing 2.5% heat-inactivated serum, then diluted 1:20 in 5 ml of fresh complete TYGVS containing a wide range of Hyg A concentrations and monitored for up to 6 days under anaerobic conditions to determine the MIC. A range of Hyg A concentrations from 0.01 μg / ml to 1.0 μg / ml was selected for the initial investigation. Controls were included in all experiments; specifically, no antimicrobial (negative control) and erythromycin (40 μg / ml; positive control). All experiments were performed with triplicate samples, and each experiment was performed twice.

[0198] Example 2 MIC of hygromycin A against oral microorganisms As used herein, minimum inhibitory concentration (MIC) refers to the optical density at 600 nm (OD 600 This refers to the lowest concentration of antimicrobial that inhibits visible growth compared to the negative control, as indicated by a difference (p≦0.05) between the positive and negative controls.

[0199] Phenol red was used to monitor T. denticola growth. T. denticola cultures were diluted 1:20 in TYGVS medium containing phenol red (50 μg / ml) and a wide range of concentrations of Hyg A. Initial concentrations tested were 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, and 5 μg / ml. Kanamycin (100 μg / ml) was included as a positive control. Initial experiments with T. denticola 35405 were performed in duplicate with and without phenol red to ensure that visible growth in the broth corresponded with a phenol red color change from yellow to red (not shown). Assays were performed in 96-well polystyrene plates with a total volume of 0.5 ml per well. Plates were sealed to prevent evaporation and incubated anaerobically for up to 14 days. Stable results were obtained for Treponema strains in 7-10 days. Incubation conditions and times were varied depending on the species. For example, S. salivarius was grown in 5% CO2 atmosphere for 24 hours or 4 days. Prolonged incubation resulted in an increase in the MIC for this species from 10 μg / ml to 20 μg / ml.

[0200] After incubation, the plates were photographed. To quantify the results, 0.1 ml of culture supernatant was transferred to a flat-bottom 96-well plate and the A562 / A630 ratio was calculated for each well by scanning in a Varioskan Flash plate reader (Thermo Scientific). Higher A562 / A630 ratio values ​​indicate growth inhibition.

[0201] Preparation of cultures for antibiotic testing T. denticola cultures were grown from frozen stocks in TYGVS broth medium. Bacteria other than T. denticola were grown from frozen stocks on Brucella blood agar supplemented with hemin and vitamin K (ThermoScientific BD297848). Cell integrity and morphology were verified by microscopy (400× dark field). OD 600The OD was measured for all cultures and the culture volume required for 1 / 20 volume deep well plate cultures was calculated (900 μl final volume per well). For each set of triplicate samples, the OD 600 OD of 0.45 culture is 1 / 10 of the final volume (i.e., OD of 1.8 ml). 600 = 0.45 culture + 16.2 ml medium), a final volume of 18 ml [medium + culture] was prepared and adjusted appropriately. 450 μl of antibiotic-containing indicator medium (containing 100 μg / ml phenol red) was added to the appropriate wells of a deep 96-well plate. After incubation, the absorbance was measured using a 100 μl / well plate. 562 and A 630 was measured.

[0202] P. gingivalis cannot grow at acidic pH and therefore no phenol red color change could be detected. Therefore, growth was monitored and OD 600 MICs were determined both by change in value and by dark field microscopy, as well as by comparing cells / field at the time of inoculation and after several days of incubation.

[0203] Example 3 Determining the MBC of Hygromycin A against T. denticola and other oral microorganisms For MBC determination, 0.1 ml bacterial samples were removed from tubes with antimicrobial concentrations equivalent to and higher than the MIC and cultured in TYGVS broth medium in the absence of antimicrobial or serially diluted and plated on TYGVS-Noble agar plates. To isolate individual colonies and / or determine CFU, T. denticola was plated at appropriate dilutions in low melting agar formulations and incubated for 1-2 weeks until subsurface colonies formed. MBC was defined as the lowest concentration of Hyg A that killed at least 99.9% of bacteria in a given time. An MBC / MIC ratio of >4 indicates bacteriostatic effect, while an MBC / MIC ratio of <4 indicates bactericidal effect (see Pankey et al., 2004. Clin Infect Dis 38:864-70.10.1086 / 381972; the contents of which are incorporated herein by reference in their entirety).

[0204] For T denticola, a single 24-well plate was prepared with 2ml / well of TYGVS 0.8% Noble agar + 5% HIHS, supplemented with rifampicin 4ug / ml, fosfomicin 100ug / ml. 5μl culture from the bottom of each well of the deep 96-well MIC plate was stuck onto the solid medium of the 24-well plate. Plates were incubated (37°C, anaerobic) for at least one week and the presence or absence of growth was recorded. For P.micra, S.salivarius, F.nucleatum, and P.gingivalis, 3-5μl of culture was spotted from each triplicate well onto Brucella blood agar plates + hemin and vitamin K at concentrations at, above, and / or below the MIC. Cultures were incubated at 37°C (anaerobic or 5% CO2 as appropriate) and the presence or absence of growth was recorded.

[0205] Table 1 below summarizes the MIC and MBC results, as well as the MIC to MBC ratios.

[0206] [Table 1]

[0207] This study revealed that MIC values ​​for Treponema denticola strains were fairly consistent within the range of 0.5 μg / ml (0.2–1.0 μg / ml). The type strain (ATCC 35405) was the most sensitive (MIC = 0.2 μg / ml). Another ATCC strain (35404) was less sensitive (0.5–5 μg / ml). Hyg A sensitivity of low-passage clinical isolates ranged from 0.2 μg / ml (strain ASLM) to 1 μg / ml (strain SP44). There was no obvious bias between established laboratory strains and relatively low-passage clinical isolates. Interestingly, ATCC 35404 was the most resistant strain to Hyg A (MIC and MBC = 5 μg / ml). The MBC / MIC ratio for all T. denticola strains averaged 2.3, suggesting that the effect of Hyg A was bactericidal.

[0208] Most of the other oral bacterial species tested were significantly more resistant to the effects of Hyg A than T. denticola. In general, Gram-positive strains were more resistant to Hyg A than Gram-negative strains. Fusobacterium nucleatum ATCC 25586 was the most sensitive at approximately 1 μg / ml (MIC and MBC). Overall, Streptococcus salivarius ATCC13419 was the most resistant at 10 μg / ml (MIC and MBC). Parvimonas micra ATCC 33270 had a relatively low MIC value of 2 μg / ml, but its MBC value was >10 μg / ml. Relatively little is known about the metabolism of this Gram-positive anaerobic organism. These data suggest that Hyg A was bacteriostatic against P. micra and bactericidal against the other strains tested.

[0209] Example 4 Kirby-Bauer diffusion assay for Fusobacterium nucleatum strains Three representatives of six subspecies of Fusobacterium nucleatum (F. nucleatum) for a total of 18 strains were tested in the experiment. The subspecies and strains are listed in Table 2.

[0210] [Table 2]

[0211] All strains were verified by 16S V3 / V6 sequence analysis prior to use. The methods described herein were carried out in an anaerobic chamber at 37° C. All media and plates were degassed prior to use.

[0212] Hygromycin A (Hyg A) disks were prepared by solubilizing Hyg A in distilled water and sterile filtering. Clindamycin was used as a positive control. Defined amounts of Hygromycin A (40, 20, 10, or 5 micrograms), clindamycin (2 micrograms), or sterile distilled water were absorbed onto Kirby-Bauer disks in a biosafety cabinet.

[0213] Overnight cultures were grown in Wilkins-Chalgren (WC) broth and back-diluted 1:10 in fresh broth. When the OD600 reading reached 0.5, 300 μL of log-phase culture was plated onto WC agar plates. Plates were sectioned into quadrants and a Kirby-Bauer (KB) disk impregnated with a defined amount of hygromycin A was placed in each quadrant. Plates were thus prepared in triplicate, with four amounts of hygromycin A being 40, 20, 10, and 5 micrograms. The same volume of culture was spread onto a fourth plate that was sectioned into six sections for three positive and three negative controls. A KB disk with water served as the negative control and a KB disk impregnated with 2 micrograms of clindamycin served as the positive control. Plates were incubated for 24-48 hours and the zones of inhibition of growth were measured. Where inhibition of a strain was too great to measure (i.e., greater than the plate would allow), a maximum value of 42 mm was assigned to those replicates. Table 3, Table 4, Table 5, Table 6, Table 7, and Table 8 show the average size of the zone of inhibition for each of the variants tested using increasing concentrations of hygromycin A, with the standard error of the mean presented in parentheses.

[0214] [Table 3]

[0215] [Table 4]

[0216] [Table 5]

[0217] [Table 6]

[0218] [Table 7]

[0219] [Table 8]

[0220] The amount of hygromycin A was plotted against the size of the zone of inhibition for each line tested. The results are shown in Table 9. In Table 9, N / A indicates R 2 The fully resistant lines, for which no R value was determined, are shown (CRC 7_3JVN3C1 and 215A9). A line of best fit was applied to each plot. Linear regression, a statistical tool used to compare data to the line of best fit, was then performed to determine the best fit line and R 2 A value of 1 indicates a perfect fit, and lower values ​​indicate a less than perfect fit. The lowest R 2 The value was 0.72, with most values ​​being considerably higher. These data indicate that the relationship between hygromycin A resistance and strain sensitivity is linear over the range of hygromycin A concentrations.

[0221] [Table 9]

[0222] Three strains were tested from each subspecies. Among the strains tested, a trend was observed in which F. nucleatum vincentii, F. nucleatum polymorphum, and F. nucleatum periodonticum strains were more resistant to hygromycin A compared to other F. nucleatum strains. Meanwhile, F. nucleatum nucleatum and F. nucleatum fusiforme showed a trend toward being more sensitive. The resistance of F. nucleatum animalis strain CRC 7_3JVN3C1 and F. nucleatum vincentii strain 215A9 among all strains tested was unexpected considering the sensitivity of F. nucleatum strain 25586 to hygromycin A shown in Example 3.

[0223] Example 5 Susceptibility of Treponema pallidum pallidum The efficacy of hygromycin A against Treponema pallidum pallidum strains was tested by co-cultivating the pathogen with rabbit epithelial cells.

[0224] SS14, and the Chicago strain of T. pallidum were obtained from frozen stocks of treponemes propagated intratesticularly (IT) in New Zealand white rabbits (Oryctolagus cuniculus) as previously reported by Baker-Zander SA et al. (J Immunol. 1988;141(12):4363-9; the contents of which are incorporated herein by reference in their entirety).

[0225] A T. pallidum culture system was set up to perform susceptibility testing and was modified from the culture system previously described by Edmondson et al. (mBio. 2018;9(3). Epub 2018 / 06 / 28; the contents of which are incorporated herein by reference). After passaging the treponemes in 6-well culture plates (Corning Inc, Corning, NY), the cells were subcultured into two 96-well cell culture plates to allocate a total of eight replicates for each antibiotic concentration of interest to be tested. The day before inoculation, three 96-well cell culture plates were inoculated with 3 × 10 641 cells per well in 150 μL of culture medium. 3 Rabbit Sf1Ep cells were seeded on the 96-well plates. The plates were then incubated overnight in a Hera Cell 150 incubator (Thermo Fisher Scientific) in 5% CO2 atmosphere to encourage cell attachment to the plate surface. On the same day, TpCM-2 medium was prepared as described by Edmondson et al. (mBio. 2018;9(3).Epub 2018 / 06 / 28). TpCM-2 medium was equilibrated overnight at 34°C in a microaerobic environment consisting of 1.5% O2, 3.5% CO2, and 95% N2 supplied as a three-gas mix (Praxair, Danbury, CT) in a three-gas incubator (Hera Cell VIOS 160i, Thermo Fisher). The next day, cell culture medium was removed from the 96-well plates and the cells were rinsed with equilibrated TpCM-2 medium. Each well was then filled with 150 μL of equilibrated TpCM-2 medium and the plates were transferred to a 3-gas incubator to equilibrate in a microaerobic atmosphere for at least 3 h. To prepare treponemal inoculum for the 96-well plates, Sf1Ep cells inoculated with T. pallidum the previous week were trypsinized to allow release and counting of spirochetes. Treponemae were counted using dark-field microscopy with a Leica DM2500 LED microscope (Leica, Wetzlar, Germany) and counted at 2.0 × 10 in TpCM-2. 4 The culture was diluted to 3 × 10 T. pallidum cells / ml. 3To obtain a single-cell treponemal inoculum, 150 μL was added to each well of a 96-well plate. Two antibiotics were tested in this study: various concentrations of hygromycin A (0.03, 0.06, 0.12, 0.24, 0.48, 0.96, 1.92, 3.84 μg / mL), and 60 ng / ml of penicillin G (a known treponemal-killing antibiotic). Each concentration was tested in eight replicates. To minimize volumetric variation, each antibiotic solution was added from a 100× concentrated stock to achieve the final concentration to be tested. No antibiotic wells as well as solvent-only wells were also included as controls. After treponemal seeding and antibiotic addition, the culture plates were incubated at 34° C. for 7 days in a 3-gas incubator.

[0226] After a 7-day incubation period, two of the four plates were processed for DNA extraction, while the remaining two plates were used to seed the no-antibiotic control plates prepared the previous day as described above. For DNA extraction, the plates were removed from the incubator and the culture medium was removed from each well with a multichannel pipette and discarded. The cells were not trypsinized but directly mixed with 200 μL of genomic lysis buffer for DNA extraction (Zymo Research, Irvine, CA) and incubated at room temperature for 30 minutes to complete cell lysis. After cell lysis, the plates were frozen at -20°C until extraction.

[0227] To purify DNA, 96-well plates were thawed at 56°C in a dry incubator and quickly spun to remove condensation on the well lids. DNA was extracted using the Quick DNA-96 kit (Zymo Research) according to the manufacturer's protocol. DNA was eluted in 100 μl of molecular grade water (Sigma-Aldrich) and stored at -20°C until amplification. Two other 96-well plates were processed and inoculated with new ones containing Sf1Ep cells in culture medium but without antibiotics to confirm the treponemal activity of the test drugs in each lineage tested. Culture medium was removed from the third 96-well plate and 20 μl of trypsin was added to detach Sp1Ep and treponemes. Half of the resulting suspension was inoculated into a new 96-well plate without antibiotics (prepared as described above), which was then incubated for another 7 days. After one week, the antibiotic-free plate was processed to extract DNA and evaluate the treponemal load by qPCR.

[0228] Following DNA extraction, treponemal load was assessed for each sample in triplicate using a qPCR approach targeting the tp0574 gene (also called T47) as previously described (see Giacani L, et al., Infect Immun. 2007;75(1):104-12; the contents of which are incorporated herein by reference in their entirety). A 313 bp amplicon was generated by qPCR using an 89°C melting temperature. Briefly, an absolute quantification protocol using external standards was used to quantitate tp0574 gene copy numbers at the time of sample harvest. Amplification was run on a QuantStudio 5 thermal cycler (Thermo Fisher Scientific) and results were analyzed using the instrument software. Data was imported into Prism 8 (GraphPad Software, San Diego, CA).

[0229] Cytotoxicity assays were performed according to the Cell Proliferation Reagent WST-1 protocol (supplied; Roche, Basel, Switzerland). Absorbance was measured after 1 h of incubation with the reagent. No significant differences were found between day 7 cells incubated without antibiotics and day 7 cells incubated with increasing concentrations of hygromycin A. The WST-1 assay showed that hygromycin A was non-toxic to SF1Ep cells at any of the concentrations tested. This result supports the conclusion that the outcome of the susceptibility assay was solely due to the effect of the drug on treponemal cells.

[0230] Susceptibility assays support that hygromycin A was effective against the Chicago strain up to 120 ng / ml (0.12 μg / ml). Significantly higher growth (p<0.05) was detected at 0.06 μg / ml when compared to higher concentrations of hygromycin A. No significant differences were found between 0.12 μg / ml and higher concentrations of hygromycin A. Analysis was performed using ANOVA with significance set at p<0.05. Data are shown in Table 10.

[0231] Table 11 shows the effect of hygromycin A on Chicago strains plated on no antibiotic control plates. These studies confirmed the treponemal activity of hygromycin A at 0.12 μg / ml and above. At these concentrations, hygromycin A was as effective as penicillin G (treponemalicidal at 60 ng / ml). As expected, based on the results shown in Table 10, growth was detected at 0.06 μg / ml. In Tables 10 and 11, Ctrl means control sample, avg means average, and Pen means penicillin.

[0232] [Table 10]

[0233] [Table 11]

[0234] Susceptibility assays using the SS14 line also supported the efficacy of hygromycin A against SS14 up to 1.92 μg / ml. Growth was detected, albeit minimally, at all lower concentrations. No significant differences were found between 1.92 μg / ml and higher concentrations of hygromycin A. Analysis was performed using ANOVA with significance set at p<0.05. Data are shown in Table 12.

[0235] SS14 strains plated on no antibiotic control plates confirmed the treponemal activity of hygromycin A at 0.12 μg / ml and above. At these concentrations, hygromycin A was as effective as penicillin G (treponemalicidal at 60 ng / ml). The results are shown in Table 13. As expected based on the results shown in Table 12, growth was detected at 0.06 μg / ml. In Tables 12 and 13, Ctrl refers to control samples, avg refers to average value, and Pen refers to penicillin.

[0236] [Table 12]

[0237] [Table 13]

[0238] Taken together, these data indicate that hygromycin A at concentrations of 0.12 μg / ml and above has a treponemal effect.

[0239] Equivalents and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the present 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.

[0240] In the claims, articles such as "a," "an," and "the" may mean one or more than one, unless the context indicates or makes clear to the contrary. 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, a claim or description containing "or" between one or more members of the group is considered to be satisfied, unless the context indicates or makes clear to the contrary. The present 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 present disclosure includes embodiments in which more than one or all 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 meanings of "a," "an," and "the" include plural references as well as singular references, unless the context clearly dictates otherwise. The term "about" in connection with a numerical value means that the value varies above or below 5%. For example, with respect to a value of about 100, it means 95 to 105 (or any value between 95 and 105).

[0241] It is also noted that the term "comprising" is intended to be open, allowing but not requiring the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of" is therefore also included and disclosed. All patents, patent applications, and other scientific or technical documents mentioned anywhere in this specification are incorporated herein by reference in their entirety. The embodiments illustratively described herein may be suitably practiced without any element or elements, limitation or limitations specifically disclosed or not specifically disclosed herein. Thus, for example, in any instance herein, any of the terms "comprising", "essentially consisting of" and "consisting of" may be replaced with either of the other two terms while retaining their ordinary meaning. The terms and expressions employed are used as terms of description and not of limitation, and in the use of such terms and expressions, there is no intention to exclude any equivalent of the characteristics shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the claims. Thus, while the present methods and compositions have been specifically disclosed by embodiment and optional features, it should be understood that modifications and variations of the concepts disclosed herein may be employed by those skilled in the art, and that such modifications and variations are believed to be within the scope of the compositions and methods as defined by this description and the appended claims.

[0242] Where ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or otherwise apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges should be understood to contemplate any specific value or subrange within the described range in various embodiments of the present disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0243] In addition, it should be understood that any particular embodiment of the present disclosure that is within the scope of the prior art may be expressly excluded from any one or more of the claims. Such embodiments may be excluded even if the exclusion is not expressly set forth herein, since they are deemed to be known to those skilled in the art. Any particular embodiment of the composition of the present disclosure (e.g., any antibiotic, treatment, or active ingredient; any method of production; any method of use, etc.) may be excluded from any one or more of the claims for any reason, whether related to the existence of prior art or not.

[0244] It is to be understood that the words which have been used are words of description rather than of limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the present disclosure in its broader aspects.

[0245] While the present disclosure has been described at some length and in some detail with respect to certain described embodiments, it is not intended that it should be limited to any such details or embodiments or to any particular embodiment, but rather it should be viewed with reference to the appended claims in order to provide the broadest possible interpretation of such claims in view of the prior art, and therefore in order to effectively encompass the intended scope of the present disclosure.

[0246] The compositions and methods are described in more detail below, and the examples set forth herein are intended to be merely illustrative, since numerous modifications and variations therein will be apparent to those skilled in the art.Terms used herein generally have their ordinary meanings in the art within the context of the compositions and methods described herein, and in the specific context in which each term is used.Some terms are more specifically defined herein to provide practitioners with additional guidance regarding the description of compositions and methods.

[0247] Any single term, single element, single phrase, group of terms, group of phrases, or group of elements described herein may each be specifically excluded from the claims.

[0248] Whenever a range is given herein, such as a temperature range, time range, composition, or concentration range, all intermediate ranges and subranges and all individual values ​​included in the given range are intended to be included in the present disclosure. It will be understood that any subrange or individual value in a range or subrange included in the description herein may be excluded from the embodiments herein. It will be understood that any element or step included in the description herein may be excluded from the claimed composition or method.

[0249] In addition, where features or aspects of compositions and methods are described in terms of a Markush group or other grouping of options, those of skill in the art will recognize that the compositions and methods are also thereby described in terms of any individual members or subgroups of members of the Markush group or other group.

[0250] The following are provided for illustrative purposes only and are not intended to limit the scope of the embodiments broadly described above.

Claims

1. 1. A pharmaceutical composition comprising hygromycin A for use in a method for treating an oral infection of Treponema denticola, Fusobacterium nucleatum, Parvimonas micra, or Porphyromonas gingivalis in a subject.

2. A pharmaceutical composition comprising hygromycin A for use in a method for treating a Fusobacterium nucleatum infection in a subject.

3. A pharmaceutical composition comprising hygromycin A for use in a method for treating a pre-malignant condition, cancer, gastrointestinal disorder, or oral disease in a subject.

4. A pharmaceutical composition comprising hygromycin A for use in a method for treating a treponematosis infection in a subject.

5. 10. The pharmaceutical composition of claim 1, wherein hygromycin A does not inhibit the growth of or kill at least one species of beneficial oral or intestinal bacteria.

6. The pharmaceutical composition according to any one of claims 1 to 4, wherein the concentration of hygromycin A is from about 0.01 µg / ml to about 100 µg / ml.

7. 7. The pharmaceutical composition of claim 6, wherein the concentration of hygromycin A is from about 0.01 μg / ml to about 1.0 μg / ml.

8. The pharmaceutical composition of claim 2, wherein the subject has an oral disease, a systemic disease, cancer, or a veterinary disease.

9. The pharmaceutical composition according to claim 3 or 8, wherein the oral disease is periodontitis or gingivitis, and the periodontitis may be localized aggressive periodontitis, generalized aggressive periodontitis, pulp necrosis, or apical periodontitis.

10. 10. The pharmaceutical composition of claim 3 or 8, wherein the cancer is colorectal cancer, gastric cancer, oral cancer, or esophageal cancer, and the cancer may be metastatic.

11. 5. The pharmaceutical composition of any one of claims 1 to 4, wherein hygromycin A is administered to a subject by oral or parenteral route, which may be subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, ocular, or inhalation route.

12. 3. The pharmaceutical composition of claim 2, wherein the F. nucleatum is one or more of F. nucleatum animalis, F. nucleatum vincentii, F. nucleatum nucleatum, F. nucleatum polymorphum, F. nucleatum fusiforme, or F. nucleatum periodonticum.

13. 4. The pharmaceutical composition of claim 3, wherein the gastrointestinal disorder is inflammatory bowel disease, Crohn's disease, ulcerative colitis, or colorectal cancer.

14. 5. The pharmaceutical composition of claim 4, wherein the treponematosis infection is caused by a Treponema pallidum or Treponema carateum infection, and the treponematosis may be syphilis, yaws, bejel, or pinta.

15. The pharmaceutical composition of claim 3, wherein the premalignant condition is hyperplasia, metaplasia, or dysplasia.