WO2012075257A2

WO2012075257A2 - Compositions for oral and nasal hygiene

Info

Publication number: WO2012075257A2
Application number: PCT/US2011/062844
Authority: WO
Inventors: Bing-yan WANG; Tong Zhao; Sebastian Ciancio; Michael Doyle
Original assignee: The University Of Georgia Research Foundation, Inc.
Priority date: 2010-12-01
Filing date: 2011-12-01
Publication date: 2012-06-07
Also published as: WO2012075257A3

Abstract

The present disclosure encompasses pharmaceutically acceptable antimicrobial compositions of an amount of levulinic acid and sodium dodecyl sulfate effective in reducing the microbial population of an internal cranial cavity of a recipient human or animal subject. The disclosure also provides methods for reducing a microbial population of an internal cranial cavity of a recipient human or animal comprising contacting the oral cavity or sinuses of the subject with a composition comprising between about 0.5% to about 5% by weight per volume of levulinic acid and between about 0.05% to about 2% by weight per volume of sodium dodecyl sulfate (SDS) and for a period effective in reducing a microbial population of the oral cavity.

Description

COMPOSITONS FOR ORAL AND NASAL HYGIENE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Serial No. : 61 /418,577, entitled "COMPOSITONS FOR ORAL AND NASAL HYGI ENE" filed on

December 1 , 2010, the entirety of which is hereby incorporated by reference.

TECHNICAL FIELD

The present disclosure is generally related to antimicrobial compositions and methods for the use thereof in reducing microbial populations of cranial cavities, including the mouth and sinus cavities. The compositions and methods can be useful in dental and naso-pharyngeal applications and for enhancing oral hygiene.

BACKGROUND

There is continuing interest in the development of novel antimicrobial treatments such as combinations of natural antimicrobials, including generally recognized as safe (GRAS) chemicals to reduce microbial loads on the tissue surfaces of humans and animals. One especially targeted area for the application of antimicrobial preparations is the oral cavity which is typically populated by heterogenous populations of microbial organisms including bacteria, yeast and viruses. While many of these populations are commensural, they are also frequently the origin of pathological conditions such as dental caries, gingivitis, periodontal disease, and the like. Other cranial cavities such as the nasal passages and the sinuses may also be populated by microbial communities and which can lead to pathological conditions.

There is an ongoing need, therefore, for compositions effective in reducing the microbial load of such internal surface regions of an animal or human head, but which are benign to the recipient, causing low or no irritation to tissues that are ennervated and highly sensitive to damage or inflammation. Such compositions should also be effective against a wide diversity of organisms and have a low propensity for allowing the development of resistant populations. Besides being effective against planktonic populations, i.e. microbial populations of individual cells or suspensions thereof, advatageous antimicrobial compositions should preferably also be able to attack and destroy microbes embedded in biofilms such as those that develop on tooth surfaces, and which can resist mechanical removal.

SUMMARY

The combination of the surfactant sodium dodecyl sulfate (SDS) with levulinic acid produces a synergistic effect in relation to the antimicrobial effectiveness of the individual compounds. Accordingly, this synergy allows the formulation of compositions where the active agents are present at concentrations effective to reduce microbial populations of biological surfaces by a factor between about 10³ and about 10⁷, but low enough to avoid damaging sensitive tissues of a subject treated with such a composition .

One aspect of the present disclosure, therefore, encompasses embodiments of pharmaceutically acceptable antimicrobial compositions that can comprise an amount of levulinic acid and sodium dodecyl sulfate formulated to be effective in reducing the microbial population of an internal cranial cavity of a recipient human or animal subject.

In some embodiments of this aspect of the disclosure, the pharmaceutically acceptable antimicrobial composition can be effective against a microbial population on a surface of the internal cranial cavity of a recipient human or animal subject and is selected from the group consisting of: a nasal mucosal surface, an oral mucosal surface, a cranial sinus mucosal surface, a pharyngeal mucosal surface, and the surface of a tooth.

In some embodiments of this aspect of the disclosure, the pharmaceutically acceptable antimicrobial composition can be effective against a microbial population on an oral surface.

In some embodiments of this aspect of the disclosure, the pharmaceutically acceptable antimicrobial composition can comprise between about 0.5% to about 5% by weight per volume of levulinic acid and between about 0.05% to about 2% by weight per volume of sodium dodecyl sulfate (SDS).

In some embodiments of this aspect of the disclosure, the pharmaceutically acceptable antimicrobial composition can have a pH value between about 3.0 and 5.0.

In some embodiments of this aspect of the disclosure, the pharmaceutically acceptable antimicrobial composition can comprise 0.5% levulinic acid and 0.05% S DS and has a pH value of about 4.2

In some embodiments of this aspect of the disclosure, the pharmaceutically acceptable antimicrobial composition can comprise an amount of levulinic acid and sodium dodecyl sulfate more effective in reducing a microbial population of a cranial internal mucosal surface of a recipient human or animal subject when compared to a composition comprising either sorbitol, propylene glycol, sodium lauryl sulfate, benzoic acid, sodium saccharin, flavor, sucralose, and FD&C green no. 3, or eucalyptol, menthol, methyl salicylate, thymol; an alcohol, benzoic acid, poloxamer 407, and sodium benzoate.

In some embodiments of this aspect of the disclosure, the pharmaceutically acceptable antimicrobial composition can be formulated as a liquid, a gel, a paste, a foam, a cream, or a spray.

In other embodiments of this aspect of the disclosure, the pharmaceutically acceptable antimicrobial composition can be formulated as a liquid.

In some embodiments of this aspect of the disclosure, the pharmaceutically acceptable antimicrobial composition is formulated as a liquid mouth wash. In embodiments of this aspect of the disclosure, the pharmaceutically acceptable antimicrobial composition can further comprise a colorant, a perfume, a flavoring agent, or any combination thereof.

In embodiments of this aspect of the disclosure, the pharmaceutically acceptable antimicrobial composition, when delivered to the oral cavity of the subject human or animal reduces the microbial population of a biofilm thereof.

In embodiments of this aspect of the disclosure, the amount of levulinic acid and sodium dodecyl sulfate is effective in reducing a population of Bacillus subtilis, B. anthracis, B. cereus, E. coli, Salmonella spp. Debaryomyces hansenii Saccharomyces cerevisiae, Candida magnoliae, Zygosaccharomyces bailii, Geot chum candidum, Mucor hiemalis,

Penicillium pubeseus, Penicillium expansum, Paecylomyces variotri, S. mutans, S. gordonii, S. mitis, S. oralis, Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis, or any combination thereof of the cranial internal mucosal surface of a recipient human or animal subject.

Another aspect of the present disclosure encompasses embodiments of methods for reducing a microbial population of an internal cranial cavity of a recipient human or animal subject, the method comprising contacting the internal cranial cavity of a recipient hu man or animal subject with a composition comprising between about 0.5% to about 5% by weight per volume of levulinic acid and between about 0.05% to about 2% by weight per volume of sodium dodecyl sulfate (SDS) and for a period effective in reducing a microbial population of the oral cavity.

In embodiments of this aspect of the disclosure, the composition can comprise about 0.5% levulinic acid and about 0.05% SDS.

In embodiments of this aspect of the disclosure, the composition can be a pharmaceutically acceptable formulation, where the formulation can be a liquid, a gel, a paste, a foam, a cream, or a spray.

I n embodiments of this aspect of the disclosure, the composition can be a pharmaceutically acceptable formulation , and where the formulation is a liquid.

In embodiments of this aspect of the disclosure, the composition can be a pharmaceutically acceptable formulation, and where the formulation is a liquid mouth wash.

In embodiments of this aspect of the disclosure, the pharmaceutically acceptable formulation can further comprise a colorant, a perfume, a flavoring agent, or any combination thereof.

In embodiments of this aspect of the disclosure, the liquid can have a pH value between about 2 and about 7. In embodiments of this aspect of the disclosure, the liquid can have a pH value between about 3.0 and 5.0. In some embodiments of this aspect of the disclosure, the liquid can have a pH value of about 4.2.

In one embodiment of this aspect of the disclosure, the composition can comprise 0.5% levulinic acid and 0.05% SDS and has a pH value of about 4.2.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be more readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.

Figs. 1 A-1 E illustrate bar graphs demonstrating the efficacy of levulinic acid and

SDS, alone or in combination, to kill spores of Bacillus anthracis Sterne. Spores were exposed to one of six different solutions: A: 3% levulinic acid plus 2% SDS; B: 2% levulinic acid plus 1 % SDS; C: 0.5% levulinic acid plus 0.05% SDS; D: 3% levulinic acid; E: 2% SDS; or F: water (serving as the control) for various lengths of time before testing the spores for viability relative to the control sample. Average plate counts are based on counting three plates; error bars indicate +/- one standard deviation.

Period of exposure: Fig. 1A, 0 min; Fig. 1 B, 10 min; Fig. 1 C, 45 min; Fig. 1 D, 90 min; Fig. 1 E, 180 min.

Figs. 2A-2E illustrate bar graphs demonstrating the efficacy of levulinic acid and SDS, alone or in combination, to kill spores of Bacillus anthracis Sterne. Spores were exposed to one of six different solutions: A: 3% levulinic acid plus 2% SDS; B: 2% levulinic acid plus 1 % SDS; C: 0.5% levulinic acid plus 0.05% SDS; D: 3% levulinic acid; E: 2% SDS; and F: water (serving as the control) for time intervals before testing the spores for viability relative to the control sample. In order to differentiate whether CFU originated from vegetative cells or from spores, at each time point samples were split in two equivalent aiiquots. One aliquot was subjected to heat treatment (65 °C, 30 min) to kill vegetative cells before enumeration of residual heat-resistant spores. The other aliquot was plated at room temperature (RT). Average plate counts are based on counting three plates; error bars indicate +/- one standard deviation.

Period of exposure: Fig. 2A, 0 hr; Fig. 2B, 1 hr; Fig. 2C, 2 hrs; Fig. 2D, 3 hrs; Fig.

2E, 4 hrs.

Figs. 3A-3E represent bar graphs demonstrating the efficacy of levulinic acid and SDS, alone or in combination, to kill spores of Bacillus anthracis Sterne. Spores were exposed to one of six different solutions: A: 3% levulinic acid plus 2% SDS; B: 2% levulinic acid plus 1 % SDS; C: 0.5% levulinic acid plus 0.05% SDS; D: 3% levulinic acid; E: 2% SDS; and F: water (serving as the control) for time intervals before testing the spores for viability relative to the control sample. In order to differentiate whether CFU originated from vegetative cells or from spores, at each time point samples were split in two equivalent aliquots. One aliquot was subjected to heat treatment (65 °C, 30 min) to kill vegetative cells before enumeration of residual heat-resistant spores. The other aliquot was plated at room temperature (RT). Average plate counts are based on counting three plates; error bars indicate +/- one standard deviation.

Period of exposure: Fig. 3A, 0 hr; Fig. 3B, 1 hr; Fig . 3C, 2 hrs; Fig. 3D, 3 hrs; Fig . 3E, 4 hrs.

Fig . 4 is a digital image showing the efficacy of (levulinic acid 0.5% (v/v)) and sodium dodecyl sulfate (SDS) 0.05% (w/v) in reducing the bacterial load in the human oral cavity.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and 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.

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.

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.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and descri be the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed. As will be apparent to those of skill in the art upon reading this disclosure, each of the individ ual 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.

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.

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.

As used herein, the following terms have the meanings ascribed to them unless specified otherwise. In this disclosure, "comprises," "comprising ," "containing" and "having" and the like can have the meaning ascribed to them in U.S . Patent law and can mean " includes," "including ," and the like; "consisting essentially of or "consists essentially" or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein, but which may contain additional structural groups, composition components or method steps (or analogs or derivatives thereof as discussed above). Such additional structural groups, composition components or method steps, etc. , however, do not materially affect the basic and novel characteristic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein. "Consisting essentially of or "consists essentially" or the like, when applied to methods and compositions encompassed by the present disclosure have the meaning ascribed in U .S. Patent law and the term is open-ended , allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.

Period of exposure: Fig. 2A, 0 hr; Fig. 2B, 1 hr; Fig . 2C, 2 hrs; Fig. 2D, 3 hrs; Fig. 2E, 4 hrs. Definitions

In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below.

As used herein the term "microorganism" or "microbe" is intended to include living cellular organisms, both unicellular and multicellular that are less than 5 mm in length, and include but are not limited to bacteria, fungi (yeasts (non-filamentous fungi), molds

(filamentous fungi), archaea, protists; amoebas and spores formed by any of these.

As used herein an "antimicrobial" is a compound that exhibits microbicidal or microbiostatic properties that enables the compound to kill, destroy, inactivate, or neutralize a microorganism; or to prevent or reduce the growth, ability to survive, or propagation of a microorganism.

As used herein the term "acid" refers to any chemical compound that, when dissolved in water, gives a solution with a hydrogen ion activity greater than in pure water, i.e. a pH less than 7.0. An "organic acid" is a carbon containing compound (except for carbonic acid) with acidic properties.

A monoprotic acid is an acid that is able to donate one proton per molecule during ionization.

The term "internal cranial region" as use herein refers to any mucosal or non- mucosal internal surface of the head of a human or animal subject, and in particular of the oral cavity (mouth) such as the cheek, gingival surface, the tongue, and the outer surface of the teeth of the subject, an internal surface of a nasal passage, a sinus internal surface, or of the throat, where any such surface may be colonized or infected by a microbial population either pathologically or non-pathologically.

The term "biofilm" as used herein refers to a protective matrix of excreted polymeric compounds. This matrix protects the cells within it and facilitates communication among them through biochemical signals. Bacteria living in a biofilm usually have significantly different properties from free-floating bacteria of the same species, as the dense and protected environment of the film allows them to cooperate and interact in various ways. One benefit of this environment is increased resistance to detergents and antibiotics, as the dense extracellular matrix and the outer layer of cells protect the interior of the community. In some cases antibiotic resistance can be increased a thousand-fold.

Dental plaque is a biofilm material that adheres to the teeth and consists of bacterial cells (mainly Streptococcus mutans and Streptococcus sanguinis), salivary polymers and bacterial extracellular products. This accumulation of microorganisms subject the teeth and gingival tissues to high concentrations of bacterial metabolites which results in dental disease. As used herein, the term "pharmaceutically acceptable" is intended to encompass any compound that can be safely administered to warm blooded vertebrates including humans. Pharmaceutically acceptable acids and surfactants include acids and surfactants that are classified by the United States Food and Drug Administration (FDA) as being Generally Regarded As Safe (GRAS), and encompass any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans.

The terms "effective amount" or a "therapeutically effective amount" of an antimicrobial composition as used herein refer to concentrations of an active agent that provide desired effects, i.e. , a log order reduction in surface microbial counts of a microbial population.

Description

An antimicrobial composition as provided herein can comprise a pharmaceutically acceptable acid and a pharmaceutically acceptable surfactant. The compositions disclosed herein can reduce a microbial population by a factor greater than 10², including by a factor of 10³ to a factor of 10⁸, using a combination of an acid and surfactant at concentrations that are significantly less effective when used separatedly. The individual active ingredients of the present compositions (i.e., the pharmaceutically acceptable acid and surfactant) are ineffective in reducing microbial cell count by a factor greater than 10², even when the active agents are used separately at 2x or 5x the effective concentration used in the combination. The concentration of the pharmaceutically acceptable acid in the antimicrobial composition is within the range of about 0.03% to about 3%, or about 0.05% to about 2%, or about 0.05% to about 1 %, or about 0.1 % to about 3%, or about 0.3% to about 3%, or about 0.3% to about 2%, or about 0.5% to about 3%, or about 0.5% to about 2%, or about 0.5% to about 1 %, weight per volume in water. In one embodiment the concentration of the pharmaceutically acceptable surfactant in the antimicrobial composition is within the range of about 0.005% to about 1 %, or about 0.01 % to about 1 %, or about 0.05% to about 1 %, or about 0.1 % to about 1 %, or about 0.05% to about 2%, or about 0.5% to about 2% by weight per volume in water.

It is, however, further contemplated that the antimicrobial compositions of the present disclosure may be formulated as a concentrate comprising a reduced amount of a solvent compared to the compositions applied to the animal or human subject. Such concentrates are convenient for storage or transport of the antimicrobial compositions, which may be readily diluted to provide the antimicrobial compositions of the disclosure.

Accordingly, the compositions of the disclsoure can comprise levulinic acid : sodium dodecyl sulfate in weight ratios of: between about 1 .0 : about 66.0 to about 1 .0 : about 0.1 . One advantageous weight ratio of the levulinic acid to the SDS is about 1 .0 : about 4.0; another advantageous ratio is about 1 .0 : about 0.1 . In the latter example, the composition may be combined with a suitable solvent, whereby the antimicrobial composition may have about 0.5% w/v of levulinic acid and about 0.05% w/v SDS .

Previous studies revealed that combinations of different organic acids can be used as anti-bacterial agents based on their killing effects on E. coli 01 57:H7 and Campylobacter (Zhao, ef al. 2006). Levulinic acid is an organic acid that can be produced cost effectively and in high yield from renewable feedstocks (Bozell, et al. 2000, Fang & Hanna, 2002). Its safety for humans has been widely tested and FDA has given it GRAS status for direct addition to food as a flavoring agent or adjunct (21 CFR, 172.515). As disclosed herein , the antimicrobial effect of 1 % by weight levulinic acid alone will not suffice to kill more than 1 log CFU Salmonellalm\ within 30 minutes, and its bactericidal effect was increased only to 3.4 log CFU/ml within 30 minutes when the levulinic acid concentration was increased to 3% by weight (see Tables 1 -3).

Sodium dodecyl sulfate (SDS) also has GRAS status (21 CFR, 172.210) at 0.5% wt of gelatin , as a whipping agent in gelatin used in marshmallows and at 0.0125% in liquid and frozen egg whites. It has been widely studied and is used as a surfactant in household products such as toothpastes, shampoos, shaving foams, and bubble baths. The SDS molecule has a tail of 12 carbon atoms attached to a sulfate group, giving the molecule the amphiphilic properties required of a surfactant. At concentrations as disclosed herein, the use of SDS by itself has very little antimicrobial effect.

The substantial bactericidal effect of a combination of levulinic acid and SDS on E. coli 01 57: H7 and Salmonella was validated in water. A shown in the examples below, the compositions of the disclsoure have antimicrobial activity against a wide variety of microbial organisms that may colonize or infect a cranial cavity, including those bacteria and fungal species that can give rise to pathological conditions of the mouth and the nasal passages. The compositions of the disclsoure are also effective against sporing bodies of such microbial forms, as shown by its activity against the viability of the spores of B. anthracis. Accordingly, the compositions of the disclosure provide a broad spectru m disinfectant advantageous for reducing the microbial population resident on, or advantageously colonizing , a cranial mucosal surface of an animal or human subject. In addition, the bactericidal activity of this combination of chemicals remained effective even in an extreme organic-rich environment such as that containing fecal matter or feathers.

The embodiments of the methods of the present disclosure provide for delivering to an animal or human subject an antimicrobial composition comprising a pharmaceutically acceptable surfactant and a pharmaceutically acceptable organic acid, wherein the concentration of the organic acid is 0.5% by weight/volume or less and the concentration of the su rfactant is 0.05% by weight/volume or less. It is desirable, however, that since the antimicrobial compositions of the disclosure are to be effectively used on sensitive internal tissue surfaces of the subject that the compositions of the disclosure be pharmaceutically acceptable and formulated to provide as little irritation to the subject as possible while retaining antimicrobial efficiacy.

The compositions of the present disclsoure may comprise a maximum concentration of 0.3 to 3% by weight of one or more organic acids selected from the group consisting of lactic acid, acetic acid , and levulinic acid and a maximum concentration of 0.05 to 2% by weight SDS. In one embodiment the composition comprises 0.3 to 3% by weight levulinic acid and 0.05 to 1 % by weight S DS.

The antimicrobial compositions disclosed herein can be used to reduce the population of an undesirable microbe. A reduction of a population of a microbe can be achieved when the populations of the microbe is reduced by at least 2 log .

An antimicrobial composition comprising levulinic acid and a surfactant is provided wherein the composition is effective in reducing resident microbial popu lations of such as an oral cavity. The concentration of said levulinic acid and surfactant are at concentrations that are ineffective in reducing said resident microbial population when used separatedly. The concentration of each of the levulinic acid and surfactant components is at a concentration 0.5x, 0.25x, 0.1 x, or less than 0.1 x, of the concentration required to produce a significant reduction (e.g . , greater than one log reduction within 5 minutes) in a microbial population when the respective component (i.e. , levulinic acid or surfactant) is used separately. In one embodiment the concentration of the levulinic acid in the compositions of the present invention is no more than 3%, 2.5%, 2.0%, 1 .5%, 1 .0%, 0.5% or 0.25% (w/v). I n one embodiment the concentration of the levulinic acid is less than 2.5% (w/v) or less than 2.0% (w/v) and in a further embodiment the concentration of the levulinic acids is about 0.5% (w/v) levulinic acid. These concentrations of levulinic acid in combination with a pharmaceutically acceptable surfactant at concentrations of less than 2% have been found to retain the organoleptic properties of foods, including produce. The concentration of the surfactant in one embodiment of the present compositions is no more than about 0.01 % to about 1 %, or about 0.01 % to about 0.1 % and more typically is about 0.05% (w/v).

A method for the rapid killing of microbial strains, including bacteria, yeasts, and molds, is provided. The method comprises contacting an internal surface of the head of an animal or human subject, and especially, but not only, the mouth of the subject, with a composition comprising a SDS and levulinic acid , wherein the concentration of the orgainic acid is 3.0%, 2.0%, 1 .0% or 0.5% (w/v) or less and the concentration of the surfactant is less than 1 %, 0.5%, 0.1 % or 0.05% (w/v). In these embodiments the composition can comprise a maximum concentration of 0.3 to 3% by weight levulinic acid and a maximum

concentration of 0.05 to 1 % by weight SDS. It is contemplated that the compositions of the present disclosure may further comprise additives such as colorants, flavorings and the like that would be usfeul to improve the acceptance of the compositions to a recipient animal or human, or contribute to the compositions appeal in commercial sale. The compositions may further be formulated by methods well-known in the art for their application to a recipient subject as a wash, such as a mouthwash, a paste or cream or ointment that may be applied to a surface to be treated, a spray for delivery of the composition to such as the internal volume and surfaces of the oral cavity, the nasal passages, sinuses, and the like.

One aspect of the present disclosure, therefore, encompasses embodiments of pharmaceutically acceptable antimicrobial compositions that can comprise an amount of levulinic acid and sodium dodecyl sulfate effective in reducing the microbial population of an internal cranial cavity of a recipient human or animal subject.

In some embodiments of this aspect of the disclosure, the pharmaceutically acceptable antimicrobial composition can be effective against a microbial population on a surface of the internal cranial cavity of a recipient human or animal subject and is selected from the group consisting of. a nasal mucosal surface, an oral mucosal surface, a cranial sinus mucosal surface, a pharyngeal mucosal surface, and the surface of a tooth.

In some embodiments of this aspect of the disclosure, the pharmaceutically acceptable antimicrobial composition can comprise 0.5% levulinic acid and 0.05% SDS and has a pH value of about 4.2

In some embodiments of this aspect of the disclosure, the pharmaceutically acceptable antimicrobial composition can be formulated as a liquid, a gel, a paste, a foam, a cream, or a spray. In other embodiments of this aspect of the disclosure, the pharmaceutically acceptable antimicrobial composition can be formulated as a liquid.

In some embodiments of this aspect of the disclosure, the pharmaceutically acceptable antimicrobial composition is formulated as a liquid mouth wash .

In embodiments of this aspect of the disclosure, the pharmaceutically acceptable antimicrobial composition can further comprise a colorant, a perfume, a flavoring agent, or any combination thereof.

In embodiments of this aspect of the disclosure, the amount of levulinic acid and sodium dodecyl sulfate is effective in reducing a population of Bacillus subtilis, B. anthracis, B. cereus, E. coli, Salmonella spp. Debaryomyces hansenii Saccharomyces cerevisiae, Candida magnoliae, Zygosaccharomyces bailii, Geotrichum candidum, Mucor hiemalis, Penicillium pubeseus, Penicillium expansum, Paecylomyces variotri, S. mutans, S. gordonii, S. mitis, S. oralis, Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis, or any combination thereof of the cranial internal mucosal surface of a recipient human or animal subject.

Another aspect of the present disclosure encompasses embodiments of methods for reducing a microbial population of an internal cranial cavity of a recipient human or animal subject, the method comprising contacting the internal cranial cavity of a recipient human or animal subject with a composition comprising between about 0.5% to about 5% by weight per volume of levulinic acid and between about 0.05% to about 2% by weight per volume of sodium dodecyl sulfate (SDS) and for a period effective in reducing a microbial population of the oral cavity.

In embodiments of this aspect of the disclosure, the composition can be a pharmaceutically acceptable formulation, where the formulation can be a liquid, a gel , a paste, a foam, a cream, or a spray.

In embodiments of this aspect of the disclosure, the composition can be a pharmaceutically acceptable formulation , and where the formulation is a liquid.

In embodiments of this aspect of the disclosure, the composition can be a pharmaceutically acceptable formulation, and where the formulation is a liquid mouth wash .

In embodiments of this aspect of the disclosure, the pharmaceutically acceptable formulation can further comprise a colorant, a perfume, a flavoring agent, or any combination thereof. In embodiments of this aspect of the disclosure, the liquid can have a pH value between about 2 and about 7.

In embodiments of this aspect of the disclosure, the liquid can have a pH value between about 3.0 and 5.0. In some embodiments of this aspect of the disclosure, the liquid can have a pH value of about 4.2.

The specific examples below are to be construed as merely illustrative, and not limiting of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein , utilize the present disclosure to its fullest extent. All publications recited herein are hereby incorporated by reference in their entirety.

It should be emphasized that the embodiments of the present disclosure, particularly, any "preferred" embodiments, are merely possible examples of the implementations, merely set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiment(s) 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, and the present disclosure and protected by the following claims.

The following 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, efc), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

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. The term "about" can include ± 1 %, ±2%, ±3%, ±4%, ±5%, ±6%, ±7% , ±8% , ±9%, or ±10% , or more of the numerical value(s) being mod ified . EXAMPLES

Example 1

Table 1: Reduction of S. enteritidis in water with organic acids and SDS at 21 °C.

negative by enrichment culture.

Table 2: Reduction of E. coli 0157.Ή7 in water with levulinic acid and SDS at 21 °C

-, negative by enrichment culture

Table 3: Reduction of S. typhimurium DT 104 in water with levulinic acid + SDS at 21 °C

a +, positive by enrichment (minimum detection level is 0.7 log CFU/ml)

b -, negative by enrichment culture Example 2

Efficacy of the Organic Acid/SDS compositions against L. monocytogenes: The efficacy of the antibacterial compositions disclosed herein was tested against Listeria monocytogenes. The results are indicated in Table 4.

Table 4: Reduction of L. monocytogenes by different concentrations of levulinic acid and

SDS individually and in combination at 21 °C.

a The actual time 0 was delayed by 5 to 10 seconds due to time for sample processing. ^b +, Positive by enrichment culture but not by direct plating (minimum detection level is 0.7 log CFU/ml).

c -, Negative by direct plating and enrichment culture.

Example 3

Reduction of microorganisms by different chemical combinations at 21 °C. Different combinations of pharmaceutically acceptable acids in combination with various

pharmaceutically acceptable surfactants were tested for their antibacterial properties.

Microorganisms were contacted with the test compositions using the same assay and procedures as disclosed in Example 1 . The results obtained by contacting microorganisms with different surfactant/acid combinations are indicated in Table 5.

Table 5: Reduction of microorganisms by different chemical combination at 21 °C

a The actual time 0 was delayed by 5 to 10 seconds due to time for sample processing. ^b ND, not determined.

a The actual time 0 may was delayed by 10 to 20 seconds due to time for sample

processing.

b +, Below the minimum detection level by direct plating (<0.7 log CFU/ml), but positive by enrichment culture.

Example 4

Efficacy of compositions to kill spores of Bacillus anthracis Sterne: For all experiments an equal volume of spore suspension of B. anthracis Sterne (34F₂) was added to 25 ml of reagents A, B, C, D, E, and F in 250-ml flasks. The compositions of reagents are A: 3% levulinic acid plus 2% SDS; B: 2% levulinic acid plus 1 % SDS; C: 0.5% levulinic acid plus 0.05% SDS; D: 3% levulinic acid; E: 2% SDS; F: water (control).

Flasks were incubated at 37 °C in a shaker (200 rpm). At each time point 100 μΙ of sample was transferred into 900 μΙ water, vortexed, and 100 μΙ of the dilution spread on Brain Heart Infusion agar plates. Plates were incubated at 37 °C overnight and colonies counted the next morning (approximately 16 hours later).

Experiment A3: 250 μΙ spore suspension (5 x 10⁴ spores) were added to 25 ml of the reagents.

Sampling time points were t = 0 (spores were added and after mixing with the reagent, 100 μΙ of the suspension were removed for enumeration), t = 10 min, t = 45 min, t = 90 min, t = 180 min. Average plate counts were based on counting three plates, as shown in Figs. 1 A- 1 E, for example.

Experiments A4, A5: In experiment A4, 250 μΙ spore suspension (5 x 10⁴ spores) were added to 25 ml of the reagents. In experiment A5, 625 μΙ spore suspension (1 .25 x 10⁵ spores) were added to 25 ml of the reagents. Sampling time points were t = 0, t = 1 h , t = 2h, t = 3h, t = 4h, t = 5h. To differentiate whether CFU originated from vegetative cells or from spores, at each time point samples were split in two equivalent aliquots. One aliquot was subjected to heat treatment (65 °C, 30 min) to kill vegetative cells before enumeration of residual heat-resistant spores. The other aliquot was plated at room temperature (RT). Average plate counts (Figs. 2A-2E and 3A-3E , respectively) are based on counting three plates; error bars indicate +/- one standard deviation.

Experiment A3: At t = 45 min recovery of CFUs from flasks A and B was reduced to 9% ( 1 .7 CFU) and 43% (8 CFU), respectively, as compared to control flask F. At t = 90 min and t = 1 80 min zero colony forming units (CFU) were recovered from flasks A and B. For flasks C and D retrieval decreased over time but did not drop below 6% (reagent C) and 39% (reagent D) at 180 min. Recovery levels from the flask with reagent E did not decrease (Table 6).

Table 6: Experiment A3: CFU % recovery (as compared to control flask F)

Experiments A4, A5: In both experiments CFU recovery from flasks A and B at t = 0 and t = 1 h orig inated from heat-sensitive cells because colony counts were zero for the samples which received heat treatment. No CFU were retrieved from flask A or B for t = 2h , t = 3h, t = 4h (Figs. 2A-2E and 3A-3E). For both reagents C and D % recovery decreased over time but of all compounds tested reagents A and B killed most effectively (Tables 7-10). Reagent E was not more effective than the water control F (Figs. 2A-2E and 3A-3E).

Table 7: Experiment A4 absent heat: CFU % recovery (as compared to control flask F): room temperature

Table 8: Experiment A4 with heat: CFU % recovery (as compared to control flask F): 65° C

Table 9: Experiment A5 absent heat: CFU % recovery (compared to control flask F): room temperature

Table 10: Experiment A5 with heat: CFU % recovery (as compared to control flask F): 65° C

While reagents C and D in a 4-hour time frame had a negative effect on spore survival, neither one of these reagents was as effective in killing spores as reagents A and B. Reagent E was not different from the water control F.

Viable cell counts demonstrated that reagents A and B affected heat sensitivity of spores very quickly at the t = 0 time point suggesting induction of a break in spore dormancy. Chemical disinfectants which are not toxic and able to diminish resistance of spores to killing are potentially of great benefit.

Example 5

Isolates: Bacillus subtilis strain ATCC #82 and B. cereus ATCC# 10987 were obtained from the ATCC, and B. circulans #47-10 and #31028 were from collection at Center for Food Safety. The frozen isolates were grown in brain heart infusion agar (BHA) at 37° C for 24 h. Alicyclobacillus acidocaldarius strain OS-CAJ and SAC (isolated from apple juice concentrate), and N-1 108 (isolated from apple-cranberry juice) were from collection at Center for Food Safety. The isolated were grown in Orange Serum Broth at 43 °C for 72 h and then transferred to potato dextrose agar (PDA) at 43 °C for 48 hr.

Spore production: For β. cereus, B. subtilis, and B. circulans, the isolates were individually grown in 10 ml BHI for 24 hr and then, precipitated, suspended and washed for 3 times by centrifugation at 4,000 x g for 20 min. The final pellet was transferred to 10 ml sporulation medium, containing FeCI₂, 0.0036 mM; MgCI₂, 0.041 mM; MnCI₂, 0.1 mM; NH₄CI, 10 mM; Na₂S0₄, 0.75 mM; KH₂P0₄, 0.5 mM; CaCI₂, 1 mM; NH₄N0₃, 1 .2 mM; D-glucose, 10 mM; and L-glutamic acid, 10 mM, pH 7.1 (Donnellan ei a/., (1964) J. Bact. 87: 332-335) at 30 °C for 5 days with agitation at 200 rpm. The spores were precipitated and suspended in 1 ml sterile H₂0 by centrifugation at 4,000 x g for 20 min. The solution was heated at 65 °C for

I 8 30 min and kept at 4 °C before use. For A. acidoterrestris isolates, the bacterium was individually grown in potato dextrose agar, pH 3.5 at 43 °C for 7 days and bacteria were collected by a plastic loop, suspended in 5 ml sterile H₂0 containing 30 glass beads and vortexed for 2 min at 150 rpm. The solution was heated at 65 °C for 30 min and kept in 5 °C before use.

Spore staining: The Wirtz-Conklin spore stain was used for observation of spore

morphology.

Chemicals: Levulinic acid and sodium dodecyl sulfate were obtained from Sigma-Aldrich (St. Louis, MO).

Water: Deionized, unchlorinated water was filter sterilized through a 0.2 pm regenerated cellulose filter (Corning Inc., Corning, NY) was used for preparing chemical solution.

Inactivation of spores: Each 500 ml flasks containing 199-ml combined chemical solution with a magnetic bar at 200 rpm was individually heated to 62 °C ± 2 °C on a hot plate. A volume of .0 ml of spore suspension was added in the center of the chemical solution under constant mixing condition at 200 rpm.

Enumeration of spores: At pre-determined schedules a sample of 1 .0 ml was removed from the flask and mixed with 9.0 ml 0.1 M phosphate buffer, pH 7.2 and then serial dilution (1 : 10) up to 10 ⁸ CFU/ml was made and 0.1 ml from each diluted tubes was inoculated on the surface of either BHA plates for bacillus species or PDA plates for alicyclobacillus species. The plates were incubated at 37 °C, 48 hr for bacillus and at 43 °C, 72 hr for alicyclobacillus species. The species of colonies randomly picked from the highest dilution plates were confirmed by biochemical assays.

Example 6

Table 11: Counts of B. subtilis (strain) ATCC #82 spores treated by levulinic acid and sodium dodecyl sulfate at 21 "C

Example 7

Table 12: Counts of B. subtilis (strain A TCC #31028) spores treated by levulinic acid and sodium dodecyl sulfate at 21 °C

a, Inoculation of spore is 2.7 x 10^B CFU/ml after heating at 65 °C for 30 min.

Example 8

Table 13: Counts of B. subtilis (strain A TCC #31028) spores treated by levulinic acid and sodium dodecyl sulfate at 21 ° C

Example 9

Table 14: Counts of B. subtilis (strain A TCC #31028) spores treated by levulinic acid and sodium dodecyl sulfate at 21 °C

Example 10

Table 15: Counts of B. subtilis (strain A TCC #31028) spores treated by levulinic acid and sodium dodecyl sulfate at 62 "C

a, Inoculums of B. subtilis ATCC #31028 is 1 .6 x 10⁹ /m (germinated and spores).

Example 11

Table 16: Counts of B. circulans (strain #47-10) spores treated by levulinic acid and sodium dodecyl sulfate at 21 "C

a, Inoculation of spore is 8.8 x 10 CFU/ml after heating at 65 °C for 30 min.

Example 12

Table 17: Counts of B. circulans (strain #47-10) spores treated by levulinic acid and sodium dodecyl sulfate at 21 °C

Inoculation of spore is 8.2 x 10 CFU/ml after heating at 65° C for 30 min. Example13

Table 18: Counts of B. circulans (strain #47-10) spores treated by levulinic acid and sodium dodecyl sulfate at 21 °C

a, Inoculation of spore is 9.8 x 10 CFU/ml after heating at 65 °C for 30 min.

Example 14

Table 19: Counts of B. circulans (strain #47- 10) spores treated by levulinic acid and sodium dodecyl sulfate at 21 °C

a, Inoculation of spore is 9.3 x 10" CFU/ml after heating at 65 °C for 30 min.

Example 15

Table 20: Counts of B. cereus (strain ATCC#10987) spores treated by levulinic acid and sodium dodecyl sulfate at 21 °C

Inoculation of spore is 2.2 x 10 CFU/ml after heating at 65 °C for 30 min Example 16

Table 21: Counts of B. cereus (strain ATCC#10987) spores treated with levulinic acid and sodium dodecyl sulfate at 21 °C

a, Inoculation of spore is 4.0 x 10 CFU/ml after heating at 65 °C for 30 min.

Example 17

Table 22: Counts of B. cereus (strain ATCC#10987) spores treated with levulinic acid and sodium dodecyl sulfate at 21 °C

, Inoculation of spore is 6.1 x 10 CFU/ml after heating at 65 °C for 30 min.

Example 18

Table 23: Counts of B. cereus spores treated with levulinic acid and sodium dodecyl sulfate at 62 °C ± 2 °C

Example 19

Table 24: Counts of Alicyclobacillus acidoterrestris mixture (bacteria + spores) treated by levulinic acid and sodium dodecyl sulfate at 62°+2° C

a, Inoculation of a mixture of 3-strains A. acidoterrestris, including strains #SAC, #OS-CAS, and #N-1 108 is 1 .1 x 10⁹ /ml.

Example 20

Table 25: Counts of Alicyclobacillus acidoterrestns mixture (bacteria + spores) treated by levulinic acid and sodium dodecyl sulfate at 21 °C and 62 °C

a, Inoculation of mixture for isolate SAC is 1.6 x 10 CFU/ml, for OS-CAS is 2.6 x 10 CFU/ml, and for N-1 108 is 6.3 x 10⁷ CFU/ml.

Example 21

Table 26: Counts of Alicyclobacillus acidoterrestns spores (pre-treated for 30 min at 65 °C)

^a, Inoculation of spore (after treated at 65°C for 30 min) for isolate SAC is 8.0 x 10⁷ CFU/ml, for OS-CAS is 7.2 x 10⁷ CFU/ml, and for N-1 108 is 6.3 x 10⁷ CFU/ml.

Example 22

Table 27: Effect of levulinic acids plus Sodium Dodecyl Sulfate (SDS) at different concentrations at 21 °C on various yeast species

G. candidum in 0.5% levulinic acid + 3.0 2.6 2.6 2.4 <0.7 <0.7 <0.7 <0.7 0.05% SDS

G. candidum in 2.0% levulinic acid + 3.3 <0.7 - - - - - - 1 .0% SDS

a Initial inoculation level: Saccharomyces cerevisiae: 7.5 x 10 CFU/ml; Debaryomyces hansenii: 7.4 x 10⁷ CFU/ml; Candida magnoliae: 3.4 x 10⁸ CFU/ml; Zygosaccharomyces bailii: 3.4 x 10⁷ CFU/ml; Geotrichum candidum: 1 .2 x 10⁷ CFU/ml.

b The actual time 0 was delayed by 5 to 10 seconds due to time for sample processing. c Negative by direct plating and enrichment culture.

Example 23

Table 28: Effect of levulinic acids plus Sodium Dodecyl Sulfate (SDS) at different concentrations at 21 °C on various mold species

P. variotri in 2.0% SDS 5.6 5.5 5.5 5.4 5.4 5.6 5.6 5.6

P. variotri in 0.5% levulinic acid + 5.2 5.2 4.8 4.5 4.1 4.4 3.8 3.4 0.05% SDS

P. variotri in 2.0% levulinic acid + 4.3 3.8 3.4 3.0 2.7 2.3 1 .5 0.7 1.0% SDS

P. variotri in 3.0% levulinic acid + 4.6 4.4 4.4 3.6 2.9 2.2 <0.7 <0.7 2.0% SDS

a Initial inoculation level: Mucor hiemalis: 3.1 x 10 CFU/ml; Penicillium pubeseus: 6.9 x 10⁷ CFU/ml; Penicillium expansum: 2.9 x 10⁷ CFU/ml; Paecylomyces variotri: 2.7 x 10⁷ CFU/ml. ^b The actual time 0 was delayed by 5 to 10 seconds due to time for sample processing.

The compositions of the present disclosure encompass formulations useful for application to a human or animal subject as a mouthwash. The compositions of the present disclosure comprise, but are not limited to, levulinic acid (including at a concentration of about 0.5% (v/v)) and sodium dodecyl sulfate (SDS) (including at a concentration of about 0.05% (w/v)) in a pharmaceutically acceptable solvent such as, but not limited to, water.

Example 24

Bacterial strains: S. gordonii CH1 , S. gordonii DL1 , S. mutans BM71 , S. mutans GS5. S. mitis, and S. oralis were inoculated on THB agar plates cultured 24-48 hrs at 37 °C in an anaerobic jar. THB medium: 3 g Todd Hewitt Broth (THB) in 100 ml dH₂0, sterilization with autoclave. BM (biofilm) medium (1/4 THBM): 3 g Todd Hewitt Broth (THB) in 400 ml dH₂0 with 40 mg hog gastric mucin (Sigma) (final concentration = 0.01 %), sterilized by autoclave. Procedure:

Day one: Inoculated oral streptococci for agar plates into 1 ml THB and grew overnight to stationary phase.

Day two: Diluted (1 : 100) the overnight cultures of in the BM medium and inoculated 100 μΙ/well in 96-well microtiter plates. Incubated the microtiter plates at 37 °C overnight, in an anaerobic box, with tap water in the water container. Anaerobic bags were not necessary for streptococcal biofilm formation.

Day three: Washed the wells twice using 0.1 % peptone water, not disturbing the biofilms. Added 20 μΙ test reagents or controls to each well. Incubated at 37 °C or room temperature for 0.5, 1 , 5, or 10 mins. Added 100 μΙ/well 0.1 % peptone water to stop the killing.

Sonicated: Output=3, Cycle=30, 3 times/well. Diluted in serial dilutions in sterile PBS or

HANKS on THB or Mitis Salivarius agar plates. Incubated the plates in anaerobic jars at 37 °C for 48 hrs.

Day five: Counted the colonies that survived the killing assay.

Example 25

Formulations of levulinic acid, at concentrations ranging from about 0.0125% to about 3% w/v) and sodium dodecyl sulfate (SDS) at concentrations ranging from about 0.0125% to about 3% w/v) were evaluated individually (as shown in Table 30 (Levulinic acid titration), and Table 31 (sodium dodecyl sulfate titration)) or as a combination (Table 29, titration of combination). Their efficacies in reducing the viability of oral bacteria at 37 °C or at room temperature were examined.

Results showed that a formulation of 0.5% levulinic acid : 0.05% SDS could significantly reduce or eliminate all biofilm oral bacteria tested within 30 seconds of application of the composition and at either 37 °C or room temperature. Organisms against which the compositions were tested included pathogens for dental caries such as S. mutans BM71 and S. mutans GS5, early colonizers such as S. gordonii CH1 , S. gordonii DL1 that provides biological surfaces for further attachment of periodontal pathogens, and oral bacteria such as S. mitis NCTC10712, and S. oralis KS32AR. LISTERI N E. RTM (a market- dominant commercial mouth rinse) was used as a positive control and water was used as the negative control in all the experiments.

Cell counts on the MS plates were graded from "-" to "++++": no survival colony; "±", < 15 colonies; "+", colony numbers of about 1 5 to about 300; "++", colony numbers of about 300 to about 1500; "+++", colonies forming bacterial lawns but with individual colony still distinguishable;" ++++", bacterial lawns, not be able to distinguish individual colony.

Table 29. Titration of Combination of Levulinic Acid and SDS (ratio of 10: 1)

Table 30. Titration of Levulinic Acid

Table 31. Titration of Sodium Dodecyl Sulfate

The combination of levulinic acid plus SDS has been demonstrated to be useful for dental application in a variety of strategies for prevention and/or treatment of oral infectious diseases, and as disinfectants for dental practice.

The antimicrobial effects as tested were stable at least for 6 months at room temperature (20 °C-24 °C). The advantages of the compositions of the present disclosure include: 1 ) high efficacy: it showed evidence of being able to reduce or eliminate populations of oral biofilm bacteria (which can be about 1000 times more resistant to antimicrobial reagents that planktonic bacteria) within 30 sees (a reduction of 1 0⁷ CFU/ml); whereas LISTERINE.RTM, a widely available commercial standard for oral hygiene products only demonstrated 2 log orders of CFU/ml reduction (10⁵ CFU/ml remaining) (as shown in Tables 29 and 34).

This high efficacy of killing biofilm bacteria in a very short time allows the

compositions of the present disclosure to be especially useful in dentistry, since oral bacteria exist mainly as biofilms in the oral cavity; 2) levulinic acid has soothing effects and has been utilized in cosmetic products. Therefore, it will not irritate the soft tissue in the oral cavity, which is especially useful for children and elderly population. In comparison ,

LISTERI NE. RTM contains a high concentration of alcohol that can irritate oral mucosa. The long-term use of a oral hygiene product with alcohol components has also raised concerns of possible oral cancer risk; 3) levulinic acid is produced from waste-grass at low cost;

whereas the mineral oil and alcohol components in LISTERIN E. RTM are more expensive; 4) exhibits a broad range of antimicrobial activity, not only eliminating gram-positive organisms, but also gram-negative organisms. Accordingly, the formulations of the present disclosure are useful in dentistry as a mouth rinse, tooth paste, agents for treatment and/or prevention of common oral bacterial infections such as dental caries and periodontal diseases, disinfective solution for dentures, anti-bacterial solution for WATERPICK.RTM, anti-bacterial solution for dental prophylaxis, and disinfectants for dental instruments and working surfaces; 5) the compositions of the present disclosure are readily soluble in water for incorporation into pastes, gels, varnishes, and local delivery devices; 6) levulinic acid and SDS have been individually designated by the U.S. Food and Drug Administration as generally recognized as safe for direct addition to food as a flavoring substance or adjunct ( see 21 CFR 172.515 for levulinic acid, and 21 CFR 172.822 for SDS); and 7) low risk of provoking other oral conditions such as recurrent aphthous ulcers: the effective SDS concentration of about 0.05% w/v) is less than the concentrations presently utilized in commercial toothpastes (1 -3%). The higher levels of SDS have been considered to be a risk of provoking recurrent aphthous ulcers in the oral cavity.

The organic acid lactic acid has demonstrated the same efficacy as levulinic acid when combined with SDS, as shown in Table 32. However, lactic acid does not possess the soothing effect of levulinic acid.

Toothpaste detergent sodium bicarbonate (NaHC0₃; baking soda) was also tested as a possible substitute for the SDS. The combination of levulinic acid and baking soda did not exhibit any antimicrobial effect, as shown in Table 33.

Table 32. Titration of Combination of Lactic Acid and SDS

Table 33. Titration of Combination of Levulinic Acid and NaHC0₃

Table 34. Titration of LISTERINE.RTM

The results of experiments to determine that elevating pH of the compositions of the present disclosure will affect its killing ability are shown in Table 35. The pH of different bottles of LISTERINE.RTM ranged from 4.12 to 4.23. The compositions of the present disclosure were adjusted accordingly to a higher pH of 4.25 and a range of different of pH from about 4.50 to about 6.0 were tested.

At pH 4.25 there was comparable killing effect as at pH 3.06 within 30 seconds. At pH about 5.0 to about 6.0 there was a similar bactericidal ability as LISTERINE. RTM. Table 35. Efficacy of 0.5% levulinic acid plus 0.05% sodium dodecyl sulfate at Different pH

Example 26

The formulations of the present disclosure were tested in a mouse model and in human volunteers to evaluate safety and efficacy in vivo. As show in Table 36, applications twice a day for 7 days were more effective than LISTERINE.RTM in eliminating S. gordonii DL1 oral biofilm bacteria. 3 mice per experimental group; S. gordonii DL1 was inoculated twice/day for 5 days, followed by the application of the reagents twice/day for 7 days; oral swabs were performed to obtain the numbers of S. gordonii DL1 in the oral cavities the day after the completion of reagent applications.

Table 36. Efficacy of 0.5% levulinic acid plus 0.05% sodium dodecyl sulfate in murine oral cavity

Compositions according to the formulations of the present disclosure (levulinic acid 0.5% (v/v)) and sodium dodecyl sulfate (SDS) 0.05% (w/v)) were applied in the oral cavity of mice twice a day for 14 days. On the day 15, mice were sacrificed, and oral mucosa samples (tips of the tongue and the lower lips) were sent for histological analysis. There was no difference in histology between the (levulinic acid 0.5% (v/v)) and sodium dodecyl sulfate (SDS) 0.05% (w/v) experimental group and water control group.

The efficacy of levulinic acid 0.5% (v/v)) and sodium dodecyl sulfate (SDS) 0.05% (w/v) was tested in human volunteers. Using a split mouth design, a single application of levulinic acid 0.5% (v/v)) and sodium dodecyl sulfate (SDS) 0.05% (w/v) effectively reduced oral supragingival bacteria loads in two human volunteers (Fig. 4).

Example 27

This study evaluated the bactericidal effect of 0.5% levulinic acid plus 0.05% sodium dodecyl sulfate, SDS on oral Gram-negative anaerobic bacteria, including Fusobacterium nucleatum, Prevotella intermedia, and Porphyromonas gingivalis, in pure culture and in biofilms.

Strains: Bacterial isolates (one each) of Fusobacterium nucleatum, Prevotella intermedia, and Porphyromonas gingivalis 381 were used.

Growth conditions: One loop of bacterial suspension from a frozen tube was plated onto tryptic soy agar supplemented with yeast extract, hemin, menadione, and defibrinated sheep blood (TSA H/M medium) and incubated at 37 °C for 48-96 hr in an anaerobic bag

(Environmental Chamber, Bio-Bag Type A, BD BBL). The colonies were transferred onto fresh plates of the same medium and plates were incubated at 37 °C for 48-72 hr under the same conditions as described above. Resulting colonies were transferred into a tube containing tryptic soy broth supplemented with yeast extract, hemin, and menadine

(TSB/H/M), and incubated at 37 °C for 48-96 hr in an anaerobic bag. Two ml of bacterial broth from each tube was transferred into a tube containing 10 ml of fresh TSB/H/M, and tubes were incubated at 37 °C for 24-48 hr in an anaerobic bag.

Inactivation assay for pure cultures: The bacteria in each culture were sedimented by centrifugation at 4,000 x g for 10 min and each pellet was resuspended in 1 .0 ml of sterile water. One-tenth ml of bacterial suspension was treated in 19.9 ml of chemical solution in a 50ml centrifuge tube agitated by hand according to a predetermined sampling schedule. After treatment, 1 .0 ml of treated bacterial suspension was serially (1 :10) diluted in either 0.1 M phosphate buffer or 0.1 % peptone. 0.1 ml from each dilution tube was surface-inoculated onto TSA/H/M plates. Plates were individually placed in an anaerobic bag and sealed, and then incubated at 37 °C for 72-96 hrs.

Biofilm assays: Two-tenths ml of culture medium of each bacterial isolate and 0.2 ml of fresh TSB/HM medium were inoculated into each well of a 24-well plate. The plates were individually placed in an anaerobic bag and sealed, and then incubated at 37 °C for 72-96 h for biofilm formation. The broth from each well was removed and each well was twice washed in 0.1 % peptone to remove cells that were non-specifically bound. 0.2 ml of treatment solution was added to each well containing cells attached in a biofilm, according to a predetermined sampling schedule. The treatment solution was removed and the plate was dried with a sterile paper towel. Five sterile glass beads and 0.3 ml of 0.1 % peptone water were added to each well and vortexed for 1 min at 250 rpm. One-tenth ml of treated bacterial biofilm was either plated directly or serially ( : 10) diluted in 0.1 % peptone water according to the procedure described above.

Results: Pure culture assay results for Fusobacterium nucleatum revealed that treatment with 0.5% levulinic acid plus 0.05% SDS, pH 3.0, and 0.5% levulinic acid plus 0.05% SDS, pH 4.25 (pH adjusted with 1 M carbonate buffer), inactivated 7.5 and 7 log CFU/ml within 30 seconds, respectively (Table 37). Similar results were also obtained with the positive controls (LISTERINE.RTM 1 and USTERINE.RTM 2) within 120 seconds (Table 37). A treatment solution containing either 0.5% levulinic acid or 0.05% SDS did not significantly reduce the bacterial population, as shown in Table 37).

Table 37: Inactivation of Fusobacterium nucleatum in pure culture at 21 "C

inoculated at 1 .1 x 10 CFU/ml.

"LISTERINE.RTM 1 , LISTERINE ZERO.RTM mouthwash contains: water, sorbitol solution, propylene, glycol, sodium lauryl sulfate, benzoic acid, sodium saccharin, flavor, sucralose, FD&C green no. 3.

USTERINE .RTM 2: LISTERINE.RTM antiseptic mouthwash containing: active ingredients, eucalyptol (0.092%), menthol (0.042%), methyl salicylate (0.060%), thymol (0.064%); and inactive ingredients, alcohol (26.9%), benzoic acid, poloxamer 407, sodium benzoate, caramel.

dND: not determined.

For the biofilm assays, results revealed that 0.5% levulinic acid plus 0.05% SDS, pH 3.0, and 0.5% levulinic acid plus 0.05% SDS, pH 4.25, reduced F. nucleatum populations by 3.0 and 3.1 log CFU/cm² within 30 seconds, respectively as shown in Table 2, whereas LISTERINE. RTM 1 and LISTERINE.RTM 2 reduced F. nucleatum populations by about 1 .3 CFU/cm² within 30 seconds.

Table 38: Inactivation of Fusobacterium nucleatum in a biofilm at 21 °C

Results for Prevotella intermedia in pure culture revealed that treatment with 0.5% levulinic acid plus 0.05% SDS, pH at either 3.0 or 4.25, for 30 sees inactivated > 6 log CFU/ml (Table 39) whereas treatment with LISTERINE.RTM 1 and LISTERINE.RTM 2 inactivated 4.9 and 4.6 log CFU/ml, respectively.

Table 39: Inactivation of Prevotella intermedia in pure culture at 21 °C

inoculated at 1 .5 x 10 CFU/ml.

bLISTERINE.RTM 1 , LISTERINE ZERO.RTM mouthwash contained water, sorbitol solution, propylene, glycol, sodium lauryl sulfate, benzoic acid, sodium saccharin, flavor, sucralose, FD&C green no. 3.

^CLISTERINE. RTM 2, LISTERINE.RTM antiseptic mouthwash contained active ingredients, eucalyptol (0.092%), menthol (0.042%), methyl salicylate (0.060%), thymol (0.064%); and inactive ingredients, alcohol (26.9%), benzoic acid, poloxamer 407, sodium benzoate, caramel.

For the biofilm assays, results revealed that 0.5% levulinic acid plus 0.05% SDS at pH 3.0 or 4.25, reduced within 60 seconds 3.5 and 2.7 log P. intermedia counts/cm², respectively (Table 40), whereas LISTERINE.RTM 1 and LISTERINE.RTM 2 reduced P. intermedia counts by 1 .4 and 1 .5 log/cm², respectively (Table 40). Table 40: Inactivation of Prevotella intermedia in a biofilm at 21 °C

Treatment of pure cultures of Porphromonas gingivalis 381 with 0.5% levulinic acid plus 0.05% SDS at either pH 3.0 or 4.25, inactivated greater than 6 log CFU/ml within 30 seconds, as did the LISTERINE.RTM 1 and LISTERINE.RTM 2 (Table 41 ).

Table 41: Inactivation of Porphyromonas gingivalis 381 in pure culture at 21 °C

^bLISTE RINE.RTM 1 , LISTERINE ZERO.RTM mouthwash contained water, sorbitol solution, propylene, glycol, sodium lauryl sulfate, benzoic acid, sodium saccharin, flavor, sucralose, FD&C green no. 3.

USTERINE.RTM 2, LISTERINE.RTM antiseptic mouthwash contained active ingredients, eucalyptol (0.092%), menthol (0.042%), methyl salicylate (0.060%), thymol (0.064%); and inactive ingredients, alcohol (26.9%), benzoic acid, poloxamer 407, sodium benzoate, caramel.

dND: not determined.

Results of the biofilm assays revealed that treatment with 0.5% levulinic acid plus 0.05% SDS, at pH 3.0 and 4.25 reduced P. gingivalis 381 populations by 4.3, and 3.3 log/cm² within 60 seconds, respectively, whereas treatment with LISTER INE.RTM 1 and LISTERINE. RTM 2 reduced P. gingivalis 381 counts by 2.1 and 2.6 log/cm² within 60 seconds, respectively. Table 42 Inactivation of Porphyromonas gingivalis 381 in a biofilm at 21 °C

I n pure culture assay, most (>6.0 log CFU/ml) of the Gram-negative anaerobic bacteria evaluated , including Fusobacterium nucleatum, Prevotella intermedia, and

Porphyromonas gingivalis, were inactivated within 30 seconds at 21 °C by 0.5% levulinic acid plus 0.05% SDS at pH 3.0 and 4.25, whereas LISTERINE . RTM 1 and LISTERI N E.RTM 2 were not as effective for F. nucleatum or P. intermedia. However, LISTERI NE. RTMs 1 and 2 were as effective in killing P. gingivalis in pure culture as the levulinic acid and SDS treatment.

Treatment with 0.5% levulinic acid plus 0.05% SDS at pH 3.0 or 4.25 of

Fusobacterium nucleatum, Prevotella intermedia, and Porphyromonas gingivalis in a biofilm was at least about 0 to about 100 times more effective in reducing cell counts within 30 seconds than either LISTERI NE.RTM 1 (without alcohol) or LISTERI NE. RTM 2 (with alcohol).

Results show that there is a synergistic effect when SDS and levulinic acid are combined, since each component individually only shows a very modest to no antibacterial activity. Antimicrobial activity of the wash of the invention is better than commercially available industry standards, even at slightly higher pH levels, which are indicative that a wash of the compositions of the present disclosure will be far less aggressive toward enamel and dentine than currently available preparations. Activity against biofilm by the wash of the invention is between 10 and 200 times more effective than OTC standard, which leads to better applicability against other oral diseases such as periodontitis and gingivitis.

Claims

What is claimed: 1 . A pharmaceutically acceptable antimicrobial composition comprising an amount of levulinic acid and sodium dodecyl sulfate formulated to be effective in reducing the microbial population of an internal cranial cavity of a recipient human or animal subject.

2. The pharmaceutically acceptable antimicrobial composition of claim 1 , wherein the microbial population is on a surface of the internal cranial cavity of a recipient human or animal subject and is selected from the group consisting of: a nasal mucosal surface, an oral mucosal surface, a cranial sinus mucosal surface, a pharyngeal mucosal surface, and the surface of a tooth. 3. The pharmaceutically acceptable antimicrobial composition of claim 2, wherein the surface of the internal cranial cavity of a recipient human or animal subject is an oral surface.

4. The pharmaceutically acceptable antimicrobial composition of claim 1 , wherein the pharmaceutically acceptable antimicrobial composition comprises between about 0.5% to about 5% by weight per volume of levulinic acid and between about 0.05% to about 2% by weight per volume of sodium dodecyl sulfate (SDS).

5. The pharmaceutically acceptable antimicrobial composition of claim 2, wherein the pharmaceutically acceptable antimicrobial composition has a pH value between about 3.0 and 5.0.

6. The pharmaceutically acceptable antimicrobial composition of claim 4, wherein the pharmaceutically acceptable antimicrobial composition comprises 0.5% levulinic acid and 0.05% SDS and has a pH value of about 4.2.

7. The pharmaceutically acceptable antimicrobial composition of claim 1 , wherein the pharmaceutically acceptable antimicrobial composition comprises an amount of levulinic acid and sodium dodecyl sulfate more effective in reducing a microbial population of a cranial internal mucosal surface of a recipient human or animal subject when compared to a

5 composition comprising either sorbitol, propylene glycol, sodium lauryl sulfate, benzoic acid, sodium saccharin, flavor, sucralose, and FD&C green no. 3, or eucalyptol, menthol, methyl salicylate, thymol; an alcohol, benzoic acid, poloxamer 407, and sodium benzoate.

8. The pharmaceutically acceptable antimicrobial composition of claim 1 , wherein the I 0 pharmaceutically acceptable antimicrobial composition is formulated as a liquid, a gel, a paste, a foam, a cream, or a spray.

9. The pharmaceutically acceptable antimicrobial composition of claim 1 , wherein the pharmaceutically acceptable antimicrobial composition is formulated as a liquid.

1 5

10. The pharmaceutically acceptable antimicrobial composition of claim 1 , wherein the pharmaceutically acceptable antimicrobial composition is formulated as a liquid mouth wash.

1 1 . The pharmaceutically acceptable antimicrobial composition of claim 1 , wherein the 0 pharmaceutically acceptable antimicrobial composition further comprises a colorant, a

perfume, a flavoring agent, or any combination thereof.

12. The pharmaceutically acceptable antimicrobial composition of claim 1 , wherein the pharmaceutically acceptable antimicrobial composition, when delivered to the oral cavity of 5 the subject human or animal reduces the microbial population of a biofilm thereof.

13. The pharmaceutically acceptable antimicrobial composition of claim 1 , wherein the amount of levulinic acid and sodium dodecyl sulfate is effective in reducing a population of Bacillus subtilis, B. anthracis, B. cereus, E. coli, Salmonella spp. Debaryomyces hansenii 0 Saccharomyces cerevisiae, Candida magnoliae, Zygosaccharomyces bailii, Geotrichum candidum, Mucor hiemalis, Penicillium pubeseus, Penicillium expansum, Paecylomyces variotri, S. mutans, S. gordonii, S. mitis, S. oralis, Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis, or any combination thereof of the cranial internal mucosal surface of a recipient human or animal subject.

5

1 . A method of reducing a microbial population of an internal cranial cavity of a recipient human or animal subject, the method comprising contacting the internal cranial cavity of a recipient human or animal subject with a composition comprising between about 0.5% to about 5% by weight per volume of levulinic acid and between about 0.05% to about 2% by weight per volume of sodium dodecyl sulfate (SDS) and for a period effective in reducing a microbial population of the oral cavity.

15. The method of claim 14, wherein the composition comprises about 0.5% levulinic acid and about 0.05% SDS.

16. The method of claim 14, wherein the composition is a pharmaceutically acceptable formulation, and wherein the formulation is a liquid, a gel, a paste, a foam, a cream, or a spray. 17. The method of claim 14, wherein the composition is a pharmaceutically acceptable formulation, and wherein the formulation is a liquid.

18. The method of claim 17, wherein the formulation is a liquid mouth wash. 19. The method of claim 14, wherein the pharmaceutically acceptable formulation comprises a colorant, a perfume, a flavoring agent, or any combination thereof.

20. The method of claim 14, wherein the liquid has a pH value between about 2 and about 7.

21 . The method of claim 14, wherein the liquid has a pH value between about 3.0 and 5.0.

22. The method of claim 14, wherein the liquid has a pH value of about 4.2. 23. The method of claim 14, wherein the composition comprises 0.5% levulinic acid and 0.05% SDS and has a pH value of about 4.2.