Compositions including n-acetyl-d-glucosamine (glcnac) as a prebiotic agent against multiple oral diseases

Abstract

The present disclosure provides for compositions including GlcNAc and methods of treating or preventing oral diseases, and the like. In an aspect, GlcNAc can be used as an agent in a composition to treat and / or prevent oral diseases such as dental caries (or tooth decay), oral candidiasis (or oral thrush), periodontitis, peri-mucositis and peri-implantitis. Each of these diseases is a biofilm disease with a dysbiotic etiology.

Description

[0001] COMPOSITIONS INCLUDING N-ACETYL-D-GLUCOSAMINE (GIcNAc) AS A PREBIOTIC AGENT AGAINST MULTIPLE ORAL DISEASES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application entitled “COMPOSITIONS INCLUDING N-ACETYL-D-GLUCOSAMINE (GIcNAc) AS A PREBIOTIC AGENT AGAINST MULTIPLE ORAL DISEASES’’ and having serial number 63 / 665,399 filed on June 28, 2024, which is incorporated herein by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under Grant Nos. DE012236 and DE024782 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] Dental caries has significantly affected human health and is the most prevalent oral disease today. In the United States, it is estimated that greater than 50% of children aged 6 and above and greater than 90% of adults aged 20-64 years have experienced caries in their teeth. As adults retain their teeth later in life, the prevalence of caries has increased in this population, and the recession of the gums with aging increases susceptibility to root surface caries. Thus, there is a need for the development of novel and comprehensive treatment strategies for this preventable disease.

[0008] SUMMARY

[0009] The present disclosure provides for compositions including GIcNAc, pharmaceutical compositions including GIcNAc, methods of treating or preventing oral diseases, and the like. The present disclosure provides for a pharmaceutical composition comprising a therapeutically effective amount of the N-Acetyl-D-Glucosamine (GIcNAc) to treat an oral disease and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is a dentifrice, wherein oral disease is selected from: dental caries, oral candidiasis, periodontitis, peri-mucositis, peri-implantitis, or a combination thereof, and wherein the composition includes about 1 millimolar to 100 millimolar of GIcNAc.

[0010] The present disclosure provides for a composition comprising N-Acetyl-D- Glucosamine (GIcNAc).

[0011] The present disclosure provides for a pharmaceutical composition comprising a therapeutically effective amount of the composition, as described above and herein, to treat an oral disease. The present disclosure provides for method for treating an oral disease comprising contacting a biofilm or contacting a cell capable of forming a biofilm with a therapeutically effective amount of the composition or pharmaceutical composition, as described above and herein.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Aspects of the present disclosure can be better understood with reference to the following drawings. It is noted that the elements in the drawings are not necessarily to scale, with emphasis instead being placed upon clearly illustrating the principles of the embodiments. In the drawings, like reference numerals designate like or corresponding, but not necessarily the same, elements throughout the several views.

[0014] Fig. 1 is a schematic illustrating amino sugar metabolism by streptococci.

[0015] Figs 2A-2D illustrate graphs about GIcNAc reprograms metabolism by important oral bacteria. Figs. 2A-2B illustrate acetate and lactate levels in overnight cultures of S. gordonii DL1 and S. mutans UA159 supported with glucose (Glc) or GIcNAc. Fig. 2C illustrates H2O2levels in four commensal streptococci. Fig. 2D illustrates pH drop by S. mutans UA159 cultures conducted in a solution containing 50 mM of glucose or GIcNAc. Each experiment was repeated at least three times, with statistics calculated relative to glucose conditions (one-way ANOVA. *, P <0.05; **, P <0.01 ; ***, P <0.001 ; P <0.0001).

[0016] Figs. 3A and 3B illustrates figures regarding how GIcNAc reduces biofilm virulence. Fig. 3A illustrates dual-species biofilms formed using a clinical commensal S. gordonii BCC09 and S. mutans (One-way ANOVA; ***, P<0.001.). Fig. 3B illustrates the scanning electron microscopy showing significant tooth demineralization (arrows) after 30 days of treatment with CCS biofilms.

[0017] Figs. 4A and 4B illustrates nano-CT showing caries (Fig. 4A, arrows) and abundance of S. mutans (Srn) and C. albicans (Ca) (Fig. 4B) in a rat caries model. The dashed arrow highlights the lesion with discontinued enamel. Pups were infected for 5 consecutive days, then rested for 2 days before treatment. Group SmCa-GIcNAc was treated by swabbing once daily with 20% GIcNAc; groups Sm, SmCa were swabbed with plain BHI. The treatment lasted for three weeks (*P<0.05, **<0.01 ; one-way ANOVA).

[0018] Figs 5A-5D illustrates microbial composition of plaque-derived microcosm biofilms. Plaque sample from early childhood caries (ECC) carious dentin containing both S. mutans and C. albicans was diluted 1 :100 in biofilm medium with 25% saliva with the addition of glucose, sucrose, or GIcNAc (n = 3). After 48 h, biofilms were collected and subjected to whole-genome sequencing. Fig. 5A illustrates the top 15 species of bacteria in all conditions tested. Fig. 5B illustrates a PCA plot and Fig. 5C and Fig. 5D illustrate the relative abundance of C. albicans and S. mutans, respectively. Figs. 6A-6G illustrate the safety of hydrogel microparticles. Fig. 6A illustrates a LDH assay, and live / dead microscope images of IMR90 cells with concentration of PEG-peptide microparticles at (Fig. 6B) 0 mg / mL, (Fig. 6C) 0.1 mg / mL, (Fig. 6D) 0.5 mg / mL, (Fig. 6E) 1 mg / mL, (Fig. 6F) 1.5 mg / mL and (Fig. 6G) 2 mg / mL.

[0019] DETAILED DESCIPTION

[0020] The present disclosure provides for compositions including GIcNAc and methods of treating or preventing oral diseases, and the like.

[0021] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0022] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit (unless the context clearly dictates otherwise), between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0024] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0025] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature. The following description and examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is in bar or psi. Standard temperature and pressure are defined as 25 °C and 1 bar.

[0026] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible. Different stereochemistry is also possible, such as products of cis or trans orientation around a carbon-carbon double bond or syn or anti addition could be both possible even if only one is drawn in an embodiment.

[0027] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a support” includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

[0028] Definitions

[0029] The terms “administering” and “administration” as used herein refer to introducing a composition (e.g., N-Acetyl-D-Glucosamine (GIcNAc)) of the present disclosure into a subject. As used herein, “administering” can refer to an administration that is oral or topical, or other using a device (e.g., dental appliance, orthodontic retainer (removable), denture, aligner, mouthguard, glide) that administers, either actively or passively the composition to the oral cavity.

[0030] The term "composition" as used herein refers to a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. Such a term in relation to a pharmaceutical composition is intended to encompass a product comprising the active ingredient(s), and the inert ingredient(s) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation, or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present disclosure encompass any composition made by admixing a compound of the present disclosure and a pharmaceutically acceptable carrier.

[0031] When a compound of the present disclosure is used contemporaneously with one or more other drugs, a pharmaceutical composition containing such other drugs in addition to the compound of the present disclosure is contemplated. Accordingly, the pharmaceutical compositions of the present disclosure include those that also contain one or more other active ingredients, in addition to a compound of the present disclosure. The weight ratio of the compound of the present disclosure to the second active ingredient may be varied and will depend upon the effective dose of each ingredient. Generally, an effective dose of each will be used. Thus, for example, but not intended to be limiting, when a compound of the present disclosure is combined with another agent, the weight ratio of the compound of the present disclosure to the other agent will generally range from about 1000: 1 to about 1 : 1000, preferably about 200: 1 to about 1 :200. Combinations of a compound of the present disclosure and other active ingredients will generally also be within the aforementioned range, but in each case, an effective dose of each active ingredient should be used. In such combinations the compound of the present disclosure and other active agents may be administered separately or in conjunction. In addition, the administration of one element may be prior to, concurrent to, or subsequent to the administration of other agent(s).

[0032] A composition of the disclosure can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Various delivery systems are known and can be used to administer a composition of the disclosure, e.g. hydrogels, encapsulation in liposomes, microparticles, microcapsules, and the like.

[0033] A therapeutic composition of the disclosure may comprise a carrier, such as one or more of a hydrogel, a polymer, carbohydrate, peptide or derivative thereof, which may be directly or indirectly covalently attached to the compound. A carrier may be substituted with substituents described herein including without limitation one or more alkyl, amino, nitro, halogen, thiol, thioalkyl, sulfate, sulfonyl, sulfinyl, sulfoxide, hydroxyl groups. In aspects of the disclosure the carrier is an amino acid including alanine, glycine, praline, methionine, serine, threonine, asparagine, alanyl-alanyl, prolyl-methionyl, or glycyl-glycyl. A carrier can also include a molecule that targets a compound of the disclosure to a particular tissue or organ.

[0034] Compounds of the present disclosure may be formulated into a pharmaceutical composition for administration to a subject by appropriate methods known in the art. Pharmaceutical compositions of the present disclosure or fractions thereof comprise suitable pharmaceutically acceptable carriers, excipients, and vehicles selected based on the intended form of administration, and consistent with conventional pharmaceutical practices. Suitable pharmaceutical carriers, excipients, and vehicles are described in the standard text, Remington: The Science and Practice of Pharmacy (21. sup. st Edition. 2005, University of the Sciences in Philadelphia (Editor), Mack Publishing Company), and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999. By way of example for oral administration in the form of a capsule or tablet, the active components can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as lactose, starch, sucrose, methyl cellulose, magnesium stearate, glucose, calcium sulfate, dicalcium phosphate, mannitol, sorbitol, and the like. For oral administration in a liquid form, the chug components may be combined with any oral, non-toxic, pharmaceutically, acceptable inert carrier such as ethanol, glycerol, water, and the like. Suitable binders (e.g., gelatin, starch, corn sweeteners, natural sugars including glucose; natural and synthetic gums, and waxes), lubricants (e.g. sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride), disintegrating agents (e.g. starch, methyl cellulose, agar, bentonite, and xanthan gum), flavoring agents, and coloring agents may also be combined in the compositions or components thereof. Compositions as described herein can further comprise wetting or emulsifying agents, or pH buffering agents.

[0035] The terms "subject", "individual", or "patient" as used herein are used interchangeably and refer to an animal preferably a warm-blooded animal such as a mammal. Mammal includes without limitation any members of the Mammalia. A mammal, as a subject or patient in the present disclosure, can be from the family of Primates, Carnivora, Proboscidea, Perissodactyla, Artiodactyla, Rodentia, and Lagomorpha. In a particular embodiment, the mammal is a human. In other embodiments, animals can be treated; the animals can be vertebrates, including both birds and mammals. In aspects of the disclosure, the terms include domestic animals bred for food or as pets, including equines, bovines, sheep, poultry, fish, porcines, canines, felines, and zoo animals, goats, apes (e.g. gorilla or chimpanzee), and rodents such as rats and mice.

[0036] The term "pharmaceutically acceptable carrier" as used herein refers to a diluent, adjuvant, excipient, or vehicle with which a probe of the disclosure is administered, and which is approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. When administered to a patient, the probe and pharmaceutically acceptable carriers can be sterile. Water is a useful carrier when the probe is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as glucose, lactose, sucrose, glycerol monostearate, sodium chloride, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0037] The present compositions advantageously may take the form of solutions, emulsion, sustained-release formulations, or any other form suitable for use. Pharmaceutically acceptable carriers may also include a dentifrice.

[0038] The term “dentifrice” or “dentifrice composition” refers to products used for purposes of administering therapeutic agent(s) to the oral cavity of a subject, during which time they are retained in the oral cavity for a time sufficient to allow for contact with substantially all surfaces of the teeth and / or oral tissues. A dentifrice composition may be in the form of a paste, powder, liquid, mouthwash, mouth rinse, chewing gum, tablet, cream, dental strips, gels, flosses, and the like.

[0039] The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Additionally, the term “pharmaceutically acceptable” describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.

[0040] As used herein, the terms “effective amount” and “amount effective” refer to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition or prevention of a disease or condition (e.g., biofilms, dental caries) or enhance and / or tune the immune system of the subject to the desirable responses (e.g., to Streptococcus mutans (S. mutans)). For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms or prevention of a disease or condition (e.g., biofilms, dental caries) and / or tune the immune system of the subject to the desirable responses but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

[0041] As used herein, the terms "treating” and "treatment" can refer generally to obtaining a desired pharmacological and / or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof (e.g., biofilms, dental caries), such as infections and consequences thereof and / or tuning the immune system of the subject to the desirable responses (e.g., to Streptococcus mutans (S. mutans)). The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term "treatment" as used herein can include any treatment of infections in a subject, particularly a human and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease or infection but has not yet been diagnosed as having it; (b) inhibiting the disease or infection, i.e., arresting its development; and (c) relieving the disease or infection i.e., mitigating or ameliorating the disease and / or its symptoms or conditions, (d) and / or tune the immune system of the subject to the desirable responses (e.g., to Streptococcus mutans (S. mutans)). The term "treatment" as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with the disorder and / or those in which the disorder is to be prevented. As used herein, the term "treating", can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and / or condition, and / or tuning the immune system of the subject to the desirable responses (e.g., to Streptococcus mutans (S. mutans)). Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

[0042] As used herein, “therapeutic” can refer to treating, healing, and / or ameliorating a disease, disorder, condition (e.g., biofilms, dental caries), or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect and / or tuning the immune system of the subject to the desirable responses (e.g., to Streptococcus mutans (S. mutans)).

[0043] The term “pharmaceutically acceptable prodrug” or “prodrug” represents those prodrugs of the compounds of the present disclosure which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective fortheir intended use. Prodrugs of the present disclosure can be rapidly transformed in vivo to a parent compound having a structure of a disclosed compound, for example, by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, V. 14 of the A.C.S. Symposium Series, and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press (1987).

[0044] General Discussion

[0045] Dental caries is a multifactorial disease with a complex etiology. The key determinants for the initiation and progression of a carious lesion are biofilm accumulation on the tooth surface and the creation of an acidic (low pH) environment. This problem of loss of pH homeostasis is perpetuated by the ecological shift represented by the increase in proportions of acidogenic and aciduric species, or caries-associated microorganisms (CAMs) at the expense of acid-sensitive commensals that are often active in generating ammonia and / or H2O2. This provides an understanding that a successful therapy should address both the loss of pH homeostasis and ecological imbalance.

[0046] Unlike strategies that target specific caries pathogens or indiscriminately eliminate oral biofilms, embodiments of the present disclosure are based on the most current understanding of the etiology of caries and seeks to reestablish pH homeostasis and modulate the ecology of oral biofilms to promote oral health. N-Acetyl-D-Glucosamine (GIcNAc) (IUPAC name -D-(Acetylamino)-2-deoxy-glucopyranose) can reduce biofilm virulence potential, primarily due to the ability of GIcNAc to modulate bacterial central metabolism, which translates into enhanced pH homeostasis and altered release of antimicrobials such as H2O2(increased) and mutacins (reduced), all in favor of commensals and against colonization and cariogenic traits of CAMs. The present disclosure uses prebiotic GIcNAc, alone without other prebiotics, for an anti-caries therapy by taking advantage of the ability of GIcNAc to modulate physiology of both the pathobionts and the beneficial commensals that make up a significant part of the oral microbiome, without the need for specific probiotic strains or being dependent on the presence of certain enzymatic activities. The present disclosure provides for compositions including GIcNAc, pharmaceutical composition including GIcNAc, methods of treating or preventing oral diseases, and the like. In an aspect, GIcNAc can be used as an agent in a composition to treat and / or prevent oral diseases such as dental caries (or tooth decay), oral candidiasis (or oral thrush), periodontitis, peri-mucositis and peri-implantitis. Each of these diseases are biofilm diseases with a dysbiotic etiology.

[0047] In an aspect, the composition having GIcNAc can include a hydrogel, a powder or tablet, a solution in water or saline, or as a substrate carried by a hydrogel or toothpaste, each of which can be applied directly onto oral or dental surfaces. In an aspect, the composition can be used in conjunction with a delivery apparatus such as a wearable dental device. In an aspect, the composition including the GIcNAc can take the form of a hydrogel microparticle that allows for controlled release (e.g., in response to pH or other specific oral conditions) in targeted microbiomes, such as the dental plaque wherein the environmental pH tends to drop in response to food intake, which both enhances the versatility of the therapy and avoids undesired effects on other microbiomes (such as the Gl tract whose homeostasis is deemed essential to human health). In particular, hydrogel microparticles include the composition are desirable in pediatric, geriatric, and long-term hospitalized patients and individuals with special needs as it does not require daily use, is easy to apply, and minimizes ingestion during and after application.

[0048] In an aspect, the composition can be used so that tens of micromolar to low millimolar levels of GIcNAc (e.g. about 10 micromolar to 10 millimolar of GIcNAc) are delivered to the microbial populations residing on the oral surfaces. This approach will induce a metabolic reprogramming in a large group of bacteria that normally ferment carbohydrates into organic acids, and reduce the cariogenic potential of caries-contributing pathobionts, such as Streptococcus mutans by replacing some of the stronger acid (i.e., lactic acid) they produce with a weaker acid (i.e., acetic acid), and enhance the anti-caries activities of the health-promoting commensal bacteria such as the mitis- and sanguinis-g roup streptococci through production of hydrogen peroxide and ammonia, the former of which is a potent antimicrobial and the latter of which neutralizes acids. At the same time, because this GIcNAc-mediated metabolic shift represents a more efficient physiology with production of more energy molecules such as ATP and less lactic acid, the use of GIcNAc compositions should also improve the pH homeostasis and biodiversity of the oral microbiome. In addition, use of compositions including GIcNAc can induce cell death in Candida albicans that is an opportunistic pathogen that can contribute to dental caries and be responsible for oral Candidiasis. These benefits will significantly reduce the risk of developing dental caries, oral candidiasis, periodontitis, peri-mucositis or peri-implantitis by boosting the abundance and health-promoting activities of commensal bacteria against colonization of pathobionts. In particular, through a synergistic interaction with S. mutans, C. albicans is positively associated with development of early childhood caries and root caries that predominantly affects elderly population. By reducing the cariogenicity of both species, use of compositions including GIcNAc should prove effective against both forms of caries that affect our most vulnerable populations.

[0049] In addition, oral Candidiasis is often a result of microbial dysbiosis when commensal species is greatly reduced under conditions such as antibiotic treatment or when the host immunity is compromised. Use of compositions including GIcNAc is expected to both boost the commensal bacteria and reduce the fitness and abundance of the pathogen.

[0050] As described above, the composition including GIcNAc can be used in conjunction with delivery devices. The use of delivery devices can help to increase the retention of composition including GIcNAc in the oral cavity to enhance its efficacy and reduce its leaching into the gastrointestinal track where it may influence the microbial homeostasis. Some of these delivery methods, e.g., toothpaste or mouthwash, are suitable for in-home treatment, whereas others such as varnish or special dental devices are better suited for professional use.

[0051] In an aspect, the present disclosure includes a composition (and pharmaceutical composition) comprising GIcNAc (e.g., in the form of a powder, mouth rinse, toothpaste). In an embodiment, the composition can be in form of a hydrogel that includes the GIcNAc. The hydrogel can be made of materials such as polyethylene glycol) (PEG)-based microparticles or their derivatives PEG-peptide, or chitosan which are known to be highly biocompatible. The composition includes about 1 millimolar to 100 millimolar of GIcNAc.

[0052] In an aspect, the composition (and pharmaceutical composition) can consist essentially of GIcNAc. The phrase “consists essentially of’ means that the composition does not include other prebiotics or active agents that are used to treat the oral disease, but the composition can include inactive compounds (e.g., carriers, binders, flavorings, colorants, polymer that form the hydrogel, inactive compounds found in toothpaste, mouth rinse, and the like).

[0053] In an aspect, the present disclosure includes methods of administering a therapeutically effective amount the composition to the oral cavity of the subject. The composition can be administered by the subject or a caretaker of the subject by disposing the composition (e.g., as a powder, solution, mouth rinse, toothpaste, and the like) into the oral cavity of the subject for a period of time (e.g., 20 seconds to 5 minutes).

[0054] In an aspect, the composition can be disposed on and / or in a device (e.g., dental appliance, orthodontic retainer (removable), denture, aligner, mouthguard, glide) that can deliver the composition over a period of time (e.g., minutes to hours based on the device). For example, the composition can be applied to an orthodontic retainer that is within the oral cavity of the subject and worn while the subject sleeps. In an aspect, the device can have the composition disposed on the device just prior to use or pre-disposed on the device so once the device (having the composition disposed thereon) is taken out of a package, the device can be placed in the oral cavity of the subject.

[0055] Pharmaceutical Formulations and Routes of Administration

[0056] Embodiments of the present disclosure include the composition (e.g., pharmaceutical composition) (e.g., including GIcNAc) as identified herein and can be formulated with one or more pharmaceutically acceptable excipients, diluents, carriers and / or adjuvants. In addition, embodiments of the present disclosure include the composition formulated with one or more pharmaceutically acceptable auxiliary substances. In particular the composition can be formulated with one or more pharmaceutically acceptable excipients, diluents, carriers, and / or adjuvants to provide an embodiment of a composition of the present disclosure.

[0057] A wide variety of pharmaceutically acceptable excipients are known in the art. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7thed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3rded. Amer. Pharmaceutical Assoc.

[0058] The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering compositions, tonicity adjusting compositions, stabilizers, wetting agents and the like, are readily available to the public.

[0059] In an embodiment of the present disclosure, the composition (e.g., including GIcNAc) can be administered to the subject using any means capable of resulting in the desired effect. Thus, the composition can be incorporated into a variety of formulations for therapeutic administration. For example, the composition can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, solutions, injections, inhalants and aerosols.

[0060] In pharmaceutical dosage forms, the composition may be administered in the form of its pharmaceutically acceptable salts, or a subject active composition may be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting. For oral preparations, the composition can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

[0061] Embodiments of the composition can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[0062] Embodiments of the composition can be utilized in aerosol formulation to be administered via inhalation. Embodiments of the composition can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

[0063] Embodiments of the composition can be formulated in an injectable composition in accordance with the disclosure. Typically, injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the active ingredient (triamino-pyridine derivative and / or the labeled triamino-pyridine derivative) encapsulated in liposome vehicles in accordance with the present disclosure.

[0064] Suitable excipient vehicles for the composition are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Methods of preparing such dosage forms are known, or will be apparent upon consideration of this disclosure, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985. The composition or formulation to be administered will, in any event, contain a quantity of the composition adequate to achieve the desired state in the subject being treated.

[0065] Compositions of the present disclosure can include those that comprise a sustained- release or controlled release matrix (e.g., a hydrogel). In addition, embodiments of the present disclosure can be used in conjunction with other treatments that use sustained- release formulations. As used herein, a sustained-release matrix is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid-based hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids. A sustained-release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxcylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone. Illustrative biodegradable matrices include a polylactide matrix, a polyglycolide matrix, and a polylactide co-glycolide (co-polymers of lactic acid and glycolic acid) matrix.

[0066] Dosages

[0067] Embodiments of the composition (e.g., including GIcNAc) can be administered to a subject in one or more doses. Those of skill will readily appreciate that dose levels can vary as a function of the specific the composition administered, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

[0068] In an embodiment, multiple doses of the composition are administered. The frequency of administration of the composition can vary depending on any of a variety of factors, e.g., severity of the symptoms, and the like. For example, in an embodiment, the composition can be administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).

[0069] The duration of administration of the composition, e.g., the period of time over the composition is administered, can vary, depending on any of a variety of factors, e.g., patient response, etc. For example, the composition in combination or separately, can be administered over a period of time of about one day to one week, about two weeks to four weeks, about one month to two months, about two months to four months, about four months to six months, about six months to eight months, about eight months to 1 year, about 1 year to 2 years, or about 2 years to 4 years, or more.

[0070] Routes of Administration

[0071] Embodiments of the present disclosure provide methods and compositions for the administration of the composition (e.g., including GIcNAc) to a subject (e.g., a human) using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration. Routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical application, intravenous, rectal, nasal, oral, and other enteral and parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the composition and / or the desired effect. A composition can be administered in a single dose or in multiple doses.

[0072] Embodiments of the composition can be administered to a subject using available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes. In general, routes of administration contemplated by the disclosure include, but are not limited to, enteral, parenteral, or inhalational routes.

[0073] Parenteral routes of administration other than inhalation administration include, but are not limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i.e., any route of administration other than through the alimentary canal. Parenteral administration can be conducted to effect systemic or local delivery of the composition. In an embodiment the administration is oral administration.

[0074] Additional features

[0075] Various aspects of the present disclosure are described herein. The following provides aspects of the present disclosure.

[0076] The present disclosure provides for a pharmaceutical composition comprising a therapeutically effective amount of the N-Acetyl-D-Glucosamine (GIcNAc) to treat an oral disease and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is a dentifrice, wherein oral disease is selected from: dental caries, oral candidiasis, periodontitis, peri-mucositis, peri-implantitis, or a combination thereof, and wherein the composition includes about 1 millimolar to 100 millimolar of GIcNAc. The pharmaceutical composition can consist essentially of GIcNAc. The composition can include a hydrogel, wherein the hydrogel is selected from: polyethylene glycol) (PEG), PEG-peptide, chitosan, or a combination thereof. The composition is a sustained release composition. The composition is a powder or a toothpaste.

[0077] The present disclosure provides for a composition comprising N-Acetyl-D- Glucosamine (GIcNAc). The composition can include a hydrogel. The hydrogel is selected from: polyethylene glycol) (PEG), PEG-peptide, chitosan, or a combination thereof. The composition is a sustained release composition. The composition is a dentifrice composition that includes the GIcNAc. The composition includes about 1 millimolar to 100 millimolar of GIcNAc. The composition is a powder. The composition is a toothpaste. The composition is disposed in or on a dental appliance. The composition consists essentially of GIcNAc. The present disclosure provides for a pharmaceutical composition comprising a therapeutically effective amount of the composition, as described above and herein, to treat an oral disease. The pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is a dentifrice. The oral disease is selected from: dental caries, oral candidiasis, periodontitis, peri-mucositis, peri-implantitis, or a combination thereof. The oral disease is dental caries.

[0078] The present disclosure provides for a method for treating an oral disease comprising contacting a biofilm or contacting a cell capable of forming a biofilm with a therapeutically effective amount of the composition or pharmaceutical composition as described above or herein. The oral disease is selected from: dental caries, oral candidiasis, periodontitis, perimucositis, peri-implantitis, or a combination thereof. The oral disease is dental caries. The cell capable of forming a biofilm is Streptococcus mutans.

[0079] EXAMPLES

[0080] Now having described the embodiments of the disclosure, in general, the examples describe some additional embodiments. While embodiments of the present disclosure are described in connection with the example and the corresponding text and figures, there is no intent to limit embodiments of the disclosure to these descriptions. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.

[0081] Example 1

[0082] Dental caries has significantly affected human health since the Stone Age1, and it remains the most prevalent oral disease today. Globally, it is estimated that 3.1 billion people suffer from untreated caries2, particularly those of lower social and economic status3, a severe situation that has barely improved in the last 30 years4. As adults retain their teeth later in life, the prevalence of caries has increased in this population, and the recession of the gums with aging increases susceptibility to root surface caries. In the US, nearly all adults (99.5%) aged 65 years or older have had a cavity, and 1 in 5 have untreated tooth decay5. Thus, there is a need for the development of novel and comprehensive treatment strategies for this preventable disease.

[0083] The diverse microbiota that colonize the teeth form dental plaque, biofilm communities that exist in a dynamic equilibrium with host defenses and are generally compatible with the integrity of tooth tissues6 7. The transition from oral health to most oral microbially driven diseases is characterized by compositional and metabolic changes in oral biofilm communities (dysbiosis)8. In the case of caries, frequent acidification of plaque favors the outgrowth of highly acidogenic and aciduric microorganisms, such as Streptococcus mutans, Lactobacillus spp., Scardovia wiggsiae, and Candida albicans9 10— thus dental caries is a polymicrobial disease. However, the consensus is that S. mutans is the most significant pathobiont due to its high prevalence in cariogenic plaque, contribution to biofilm development, promotion of plaque acidification11 14, and its ability to cause caries in animal models15 16. Cariogenic microorganisms can rapidly ferment dietary carbohydrates, lowering the environmental pH (<5.5) so that significant tooth enamel demineralization occurs6 10 17 18. Acidification periods are followed by alkalinization periods, in which the return of plaque pH to neutrality can foster enamel remineralization17. Dental caries occurs when periods of acidification outweigh those of alkalization. Notably, the microbial composition of healthy tooth surfaces is substantially different from that found during caries activity8 9; with metagenomic studies10 18 20disclosing consistently higher proportions of bacteria associated with oral health, especially alkali- and hydrogen peroxide (H2O2)-producing commensals of the mitis and sanguinis groups of streptococci that are antagonistic to aciduric species such as S. mutans21 22. Thus, approaches that modulate the composition and biochemistry of the oral microbiota to enhance the stability of health- associated microbial communities can be highly effective in preventing and controlling dental caries23.

[0084] The oral ecosystem is a dynamic environment wherein microbe-microbe and microbe-host interactions shape the composition and physiology of the microbial communities24. While the number, complexity, and diversity of these interactions may be incalculable, antagonism and synergism between organisms in the greatest proportions likely have the most profound impact on health and disease24. While relatively abundant members of dental biofilms such as Actinomyces spp., Veillonella spp., and Fusobacterium spp. are undoubtedly crucial in biofilm development and stability, streptococci constitute the bulk (up to -60%) of oral biofilms25and have numerous traits that can significantly influence the biofilm composition, structure, and pathogenic potential towards health or disease.

[0085] Although diverse in many respects, oral streptococci have similar nutritional requirements and ferment carbohydrates as their primary route for ATP generation. Consequently, commensal streptococci such as S. sanguinis and S. gordonii compete directly with S. mutans and other fermentative cariogenic species for nutrients and habitats 26. 27. Not surprisingly, cariogenic and commensal microorganisms have evolved multiple mechanisms to antagonize the growth of one another26. In particular, S. mutans produces multiple bacteriocins (mutacins) that differentially target streptococcal species28 :32and mutacin expression is commonly regulated by secretion and sensing of competence stimulating peptide (CSP) as a quorum-sensing signal28 33. As a defense mechanism, commensal streptococci produce proteases such as the Sgc, originally identified in S. gordonii strain Challis34, which interfere with mutacin production by degrading CSP. Another form of antagonism that confers a major selective advantage to S. mutans is its inherit and adaptive acid tolerance (aciduricity)35 36. Even though S. mutans does not compete well with commensal streptococci at neutral pH, its ability to continue to ferment carbohydrates at pH values of 4 and below is a potent ecological weapon37. Commensal streptococci do not usually grow well below pH values of 5.5. Still, some health-associated oral streptococci produce an alkaline compound, ammonia that moderates biofilm acidification37and secrete H2O2 that inhibits the growth of S. mutans and other caries-associated microorganisms (CAMs)26 38 40. Thus, providing prebiotics that favor the production of alkali and H2O2can give a competitive advantage to commensal streptococci over CAMs.

[0086] A well-known synergistic relationship in caries occurs between S. mutans and the yeast pathobiont C. albicans that are commonly co-isolated from children with severe caries41'44, which is supported by animal studies that demonstrate that co-infection with S. mutans and C. albicans elicit more extensive caries than infection with only one of them45. Several reasons could help to explain how C. albicans and S. mutans contribute synergistically to caries severity: C. albicans can utilize lactic acid excreted by S. mutans and other lactic acid bacteria as an energy source, it is highly acidogenic and aciduric, and it can reduce the oxygen tension of oral biofilms to favor often oxygen-sensitive pathobionts46. In turn, glucosyltransferase B secreted by S. mutans can bind to the surface mannans of C. albicans, allowing the yeast to synthesize adhesive glucan polysaccharides in the presence of sucrose and further enhance the formation of a cariogenic biofilm matrix47 48. Of interest, the oral commensal Streptococcus parasanguinis has been shown to disrupt S. mutans-C. albicans synergism by dispersing their biofilms in vitro49. Therefore, while the synergism among CAMs enhances cariogenicity, it can also be targeted by beneficial commensals, especially if proper conditions are provided (i.e., prebiotics).

[0087] Arginine is a prebiotic used worldwide in some commercially available oral care products to prevent and arrest caries. It works by inducing ammonia production via the arginine deiminase (AD) pathway present in many beneficial commensal streptococci, resulting in increased environmental pH and lower abundance of CAMs even in the presence of highly cariogenic sucrose50’52. Clinical studies have also demonstrated that commensal streptococci with high AD activities correlates with better dental health50 53 54. Considering the enormous complexity of oral microbial interactions and the dependence of arginine on the presence of AD for exerting its benefit, these earlier examples of success in modulating biofilm activity and ecology are especially encouraging as they reaffirmed the central role of pH homeostasis in the widely accepted “ecological plaque hypothesis”55 56. An example of less explored prebiotics in oral health is the amino sugar N- acetylglucosamine (GIcNAc), widely consumed as a food supplement that may benefit joint health57. GIcNAc is ubiquitous in the biosphere as a major component of the chitin exoskeleton of arthropods, fungal cell walls, and bacterial cell envelopes. It is also one of several sugar moieties decorating the mucins and other glycoproteins present in significant quantities in saliva and oral epithelium58 59. Consumed by many commensal bacteria60, GIcNAc is a significant carbon and nitrogen source for oral microbes, contributing to the maintenance of a stable, healthy biofilm. Most microorganisms degrade GIcNAc via a two- step process: deacetylation of GlcNAc-6-P followed by deamination of GlcN-6-P, generating ammonia, acetate, and fructose-6-P (Fig. 1). As such, even in the absence of arginine, GIcNAc catabolism reduces lactic acid production and moderates the environmental pH61. We recently showed that GIcNAc further enhances the competitive advantage of commensal streptococci over S. mutans by stimulating H2O2production and alkali generation via AD60, resulting in suppression of S. mutans growth and establishment62 63. Moreover in C. albicans, GIcNAc has been shown to impact fungal metabolism and yeast-hypha transition, reduce surface attachment, and induce cell death64 65. Thus, GIcNAc has great potential in remodeling the biofilm metabolism and ecology towards health.

[0088] Clinical and laboratory studies strongly support that nutrient composition, availability, and metabolic interactions profoundly influence the balance between healthy and dysbiotic states. Our recent advancement in the effects and application of GIcNAc in caries-relevant model systems (see the preliminary data) demonstrated the feasibility of using GIcNAc as a prebiotic to modulate biofilm homeostasis and virulence potential to prevent and control dental caries. Our goal is to develop and validate a carrier system, in the form of a hydrogel and a mouth rinse, to efficiently deliver GIcNAc to oral biofilms in a controlled manner that can be further developed into a novel and low-cost therapy for enhancing oral health and preventing and controlling dental caries.

[0089] Dental caries is a multifactorial disease with a complex etiology. Still, the key determinants for the initiation and progression of a carious lesion are biofilm accumulation on the tooth surface and the creation of an acidic (low pH) environment. This problem of loss of pH homeostasis is perpetuated by the ecological shift represented by the increase in proportions of acidogenic and aciduric species (CAMs) at the expense of acid-sensitive commensals that are often active in generating ammonia and / or H2O2. This reality determines that any successful therapy must deal with both loss of pH homeostasis and ecological imbalance. Unlike strategies that target specific caries pathogens or indiscriminately eliminate oral biofilms, our approach is conceptually innovative because it is founded on the most current understanding of the etiology of caries and seeks to reestablish pH homeostasis and modulate the ecology of oral biofilms to promote oral health. This example is built on groundbreaking studies that provided a proof of principle for the ability of GIcNAc to reduce biofilm virulence potential, primarily due to the ability of GIcNAc to modulate bacterial central metabolism, which translates into enhanced pH homeostasis and altered release of antimicrobials such as H2O2(increased) and mutacins (reduced), all in favor of commensals and against colonization and cariogenic traits of CAMs (see60 63 66). GIcNAc, a naturally occurring and safe carbohydrate, allows us to address fundamental principles of the disease while creating opportunities for rapid translation of the findings to diminish the burden of dental caries using ecologically based tactics. Importantly, our strategy of using the prebiotic GIcNAc alone represents a novel approach to developing an anti-caries therapy by taking advantage of the ability of GIcNAc to modulate physiology of both the pathobionts and the beneficial commensals that make up a significant part of the oral microbiome, without the need for specific probiotic strains or being dependent on the presence of certain enzymatic activities, e.g., AD which tends to be variable in different populations and subjected to influence by environmental factors67 69.

[0090] Embodiments of the present disclosure use a hydrogel microparticle system that allows for controlled release (e.g., in response to pH or other specific oral conditions) of GIcNAc in targeted microbiomes, which both enhances the versatility of the therapy and avoids undesired effects on other microbiomes. Hydrogel applications are desirable in pediatric, geriatric, and long-term hospitalized patients and individuals with special needs as it does not require daily use, is easy to apply, and minimizes ingestion during and after application. We will also prepare less costly GIcNAc mouth rinse formulations and compare their effectiveness to the hydrogel. According to the American Dental Association, mouth rinse formulations do not require application by a professional and can be used daily as part of oral hygiene in 6+ years old persons.

[0091] This example is built upon extensive preliminary data and recent developments in two fronts of dental research: i) clinical and in vitro findings by the Abranches, Garcia, and Roesch groups that support and expand findings from others that synergistic interactions between S. mutans and C. albicans are strongly associated with recalcitrant infections in early childhood caries (ECC) patients that did not respond to clinical interventions74, and ii) molecular genetics and ecological studies by the Zeng group60 63 66 75on metabolism of amino sugars that uncovered the capacity of GIcNAc in reprograming bacterial central metabolism, acid production, and gene regulation, which altered bacterial interactions and biofilm biodiversity in favor of oral health. Also, Dr. Andrew’s group developed a safe and reliable microparticle system for delivering bioactives suitable for application in GIcNAc- based treatment. Given the enormous inter- and intra-taxa heterogeneity present across health-associated streptococci, using prebiotics to boost the competitive advantage of endogenous commensals against CAMs can effectively prevent and control caries.

[0092] We and others showed that S. mutans and C. albicans synergize to promote the assembly of highly cariogenic biofilms45 47 48 76 78, resulting in the most aggressive forms of caries in children41 44 74. To date, most studies on S. mutans-C. albicans synergism have centered on the overproduction of adhesive glucans when these two species are cocultivated, resulting in increased biomass and microbial recruitment to the growing biofilms 47, 79, so, si Recently, the Abranches group showed that the catalase-positive C. albicans protects S. mutans (catalase negative) against the deleterious effect of H2O2, either produced by the beneficial commensals such as Streptococcus A12 or added exogenously, in a catalase-dependent but glucan matrix-independent manner, expanding the synergistic repertoire of these two CAMs82. Preliminary results from the Abranches group while studying root caries in the elderly population (>65 yrs) also suggested a strong association between root caries and the presence of C. albicans and S. mutans (data not shown). These developments thus strongly support targeting S. mutans-C. albicans synergism as the most prominent and virulent CAMs in our model systems.

[0093] Dr. Zeng’s completed R03 DE024782 program generated novel and fundamental knowledge on the physiological and genetic aspects of amino sugar metabolism by S. mutans and related streptococci (Figs. 1 and 2)66 75. As detailed below, we uncovered that amino sugar metabolism (especially GIcNAc) by the mitis and sanguinis groups of streptococci profoundly impacted central metabolism61and gene regulation60, enhancing the competitive fitness of these commensals against S. mutans60 62. Pertinent to this application, the Zeng group also developed and validated an ex vivo biofilm model to study microbial interactions within complex biofilm communities62 63 83and showed that GIcNAc inhibited S. mutans62and boosted biodiversity in complex biofilms84.

[0094] We compared the transcriptomes of planktonic cultures of S. mutans UA159 and S. gordonii DL1 grown on glucose to those grown on GIcNAc61. A total of 45 and 30 genes were differentially expressed during growth on GIcNAc in S. mutans and S. gordonii, respectively. Most of these genes were involved in energy metabolism, followed by genes required for interbacterial competition. In S. mutans, growth on GIcNAc increased the expression of genes responsible for pyruvate oxidation; and reduced the expression of most bacteriocin genes and lactate dehydrogenase (Jdh), the lactic acid-producing enzyme. For S. gordonii, GIcNAc also affected pyruvate metabolism, increasing the expression of genes required for pyruvate oxidation, production of H2O2; and the arginine deiminase system61. Thus, GIcNAc induced a metabolic reprogramming in both bacteria that shunted pyruvate from lactate production into oxidation, which not only was bioenergetically favorable but for S. gordonii also meant increased production of H2O2and ammonia. Our findings further validated that central metabolism and its regulation are crucial to maintaining bacterial homeostasis in dental biofilms21’85 87.

[0095] To demonstrate these physiological shifts, acid production by both bacteria was measured when catabolizing GIcNAc. As shown in Figs. 2A and B, both S. mutans UA159 and S. gordonii DL1 produced significantly less lactic acid when growing on GIcNAc compared to glucose, yet higher levels of acetic acid were produced, indicative of a redirection of pyruvate away from LDH. Meanwhile, several commensal streptococci, including S. gordonii produced elevated levels of H2O2when growing on GIcNAc (Fig. 2C). Importantly, catabolism of GIcNAc by S. mutans (and similarly in commensals) resulted in a drastically flattened glycolytic pH drop curve compared to glucose, with the resting pH of the culture nearing 5.0 instead of 3.5, which is typical of glucose (Fig. 2D). This effect is likely the combined result of a reduction in strong acid production and the additional release of ammonia through the deamination of GIcNAc. This outcome also distinguishes GIcNAc from arginine, as the latter depends on AD-positive commensals for its benefit. As a result of these physiological changes in pH homeostasis and antagonistic activity by oral streptococci, growth on GIcNAc significantly benefited commensal species, including S. gordonii, S. sanguinis, S. cristatus and S. oralis60 62, and decreased the overall fitness of S. mutans (see Fig. 3A for example).

[0096] An ex vivo microcosm biofilm model88was adapted to study complex microbial interactions using pooled untreated saliva as inoculum and a semi-synthetic biofilm medium (BM) containing glucose, sucrose, or GIcNAc. This cell-containing saliva (CCS) biofilm model was subjected to taxonomic analysis, revealing 35-37 major taxa known to inhabit the supragingival plaque84. S. salivarius was predominant in most sugars except when GIcNAc was present. CCS growth in GIcNAc resulted in a significant increase in biodiversity with an increased abundance of mitis and sanguinis groups of streptococci (S. mitis, S. oralis, S. cristatus, and S. sanguinis). In support of the metabolic reprogramming discussed above, targeted metabolomic analyses revealed a systematic change in a panel of organic acids produced by the biofilm fed with GIcNAc (relative to glucose), including a decrease in lactate production63. To further validate our ex vivo model, we tested the ability of the CCS biofilms spiked with S. mutans (106 / ml) to demineralize the enamel of human tooth samples for 30 days, with daily media refreshment63. Scanning electron microscopy revealed drastically less enamel damage (Fig. 3B) when the biofilms were grown in GIcNAc compared to glucose63. We monitored the survival of S. mutans in these CCS biofilms and observed that GIcNAc prevented its implantation in the growing biofilm62. It is worth noting that for most of these biofilm assays (Fig. 3&5) conducted using BM-based media, 2 mM sucrose was included in all conditions to simulate the Western diet.

[0097] We observed that C. albicans clinical and lab strains grew poorly on GIcNAc relative to glucose (data not shown), a finding supported by a recent study showing that GIcNAc triggers cell death in C. albicans64. Notably, other research showed that carbohydrates can alter Candida physiology and Candida-S. mutans interactions65 89 90. To test whether these effects of GIcNAc on microbial homeostasis observed so far can be observed in vivo, we conducted a pilot rat caries experiment using S. mutans UA159 and C. albicans SC5314, in the presence or absence of GIcNAc. As shown in Fig. 4, co-infection with S. mutans and C. albicans resulted in successful colonization by both microbes and the development of significant caries lesions at the end of the 7-week experiment as visualized by Nano-CT (Fig. 4A, arrows). While co-infection with UA159 and SC5314 increased caries severity (dashed arrow) and colonization by S. mutans in relation to mono-infection with UA159, treatment of the co-infection group with GIcNAc significantly reduced the occurrence and severity of carious lesions and lowered abundance of both S. mutans and C. albicans (Fig. 4). In parallel, we modified the CCS biofilm model by deriving it from dental plaque samples collected from caries-active patients (IRB201600851) that naturally contained S. mutans and C. albicans and tested whether treatment with GIcNAc can revert dysbiosis. Consistent with the animal experiment, the use of GIcNAc significantly reduced C. albicans abundance, as indicated by CFU counting on selective agar plates (data not shown). Nanopore sequencing91analysis on the same biofilms confirmed the reduced abundance of C. albicans (Fig. 5C), and S. mutans (Fig. 5D), when GIcNAc was the supporting sugar relative to glucose or sucrose. Importantly, plaque biofilms grown with GIcNAc differed significantly in microbial composition from those grown on glucose and sucrose (Fig. 5A and 5B). These studies validate our findings regarding the beneficial effects of GIcNAc on microbial homeostasis by modulating the abundance of health-associated streptococci and CAMs.

[0098] Hydrogel nano- and micro-particles are attractive for drug delivery due to their ability to protect the drug from degradation, deliver it specifically to the target tissue, and subsequently release the drug in a controlled manner92 94. Drugs can be encapsulated within the hydrogel matrix, and release of the drug from the microparticles is typically achieved by the hydrogel’s swelling and degradation, either through a natural process or in response to a specific stimulus such as pH, temperature, or hydrogel-degrading enzymes. As a proof of principle, a hydrophobic compound (dexamethasone) and a hydrophilic compound (methylene blue) were each encapsulated in a biocompatible PEG-peptide microparticle and successfully released by treatment with an enzyme solution that targeted the peptide in a dose-dependent manner95. When these microparticles were used to treat two healthy human cell lines, lung fibroblasts IMR-90 and lung carcinoma cells A549, no cytotoxicity was detected by measuring LDH activities and live / dead staining (Fig. 6). Here, we will primarily utilize the pH-dependent mechanism for GIcNAc release from these microparticles, and the safety will be further validated using relevant human oral cell lines. Thus, we are well-positioned to test the prebiotic GIcNAc as an effective and ecological approach to prevent and control dental caries.

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[0215] It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of “about 0.1% to about 5%” should be interpreted to include not only the explicitly recited concentration of about 0.1 wt% to about 5 wt%, but also include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range. In an embodiment, the term “about” can include traditional rounding according to significant figures of the numerical value. In addition, the phrase “about ‘x’ to ‘y’” includes “about ‘x’ to about ‘y’”.

[0216] It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations, and are set forth only for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiments of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure.

Claims

CLAIMS1. A pharmaceutical composition comprising a therapeutically effective amount of the N- Acetyl-D-Glucosamine (GIcNAc) to treat an oral disease and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is a dentifrice, wherein oral disease is selected from: dental caries, oral candidiasis, periodontitis, peri-mucositis, peri-implantitis, or a combination thereof, and wherein the composition includes about 1 millimolar to 100 millimolar of GIcNAc.

2. The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition consists essentially of GIcNAc.

3. The pharmaceutical composition of claim 1 , wherein the composition includes a hydrogel, wherein the hydrogel is selected from: polyethylene glycol) (PEG), PEG-peptide, chitosan, or a combination thereof.

4. The pharmaceutical composition of claims 1 to 3, wherein the composition is a sustained release composition.

5. The pharmaceutical composition of claim 4, wherein the composition is a powder or a toothpaste.

7. A composition comprising N-Acetyl-D-Glucosamine (GIcNAc).

8. The composition of claim 7, wherein the composition includes a hydrogel.

9. The composition of claim 2, wherein the hydrogel is selected from: polyethylene glycol) (PEG), PEG-peptide, chitosan, or a combination thereof.

10. The composition of claims 7 to 9, wherein the composition is a sustained release composition.11 . The composition of claims 7 to 10, wherein the composition is a dentifrice composition that includes the GIcNAc.

12. The composition of claims 7 to 11 , wherein the composition includes about 1 millimolar to 100 millimolar of GIcNAc.

13. The composition of claims 7 to 12, wherein the composition is a powder.

14. The composition of claims 7 to 12, wherein the composition is a toothpaste.

15. The composition of claims 7 to 14, wherein the composition is disposed in a dental appliance.

16. The composition of claims 7 to 15, wherein the composition consists essentially of GIcNAc.

17. A pharmaceutical composition comprising a therapeutically effective amount of the composition of any one of claims 7-16 to treat an oral disease.

18. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

19. The pharmaceutical composition of claim 18, wherein the pharmaceutically acceptable carrier is a dentifrice.

20. The pharmaceutical composition of claim 17, wherein the oral disease is selected from: dental caries, oral candidiasis, periodontitis, peri-mucositis, peri-implantitis, or a combination thereof.21 . The pharmaceutical composition of claim 17, wherein the oral disease is dental caries.

22. A method for treating an oral disease comprising contacting a biofilm or contacting a cell capable of forming a biofilm with a therapeutically effective amount of the composition or pharmaceutical composition of any one of claims 1-16.

23. The method of claim 22, wherein the oral disease is selected from: dental caries, oral candidiasis, periodontitis, peri-mucositis, peri-implantitis, or a combination thereof.

24. The method of claim 22, wherein the oral disease is dental caries.

25. The method of claim 22, where the cell capable of forming a biofilm is Streptococcus mutans.

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