LONG-LASTING SURFACE ANTI-MICROBIAL AND METHOD OF APPLICATION

FIELD

This disclosure relates to barrier-forming compositions and methods for treating surfaces to prevent the spread of communicable diseases.

BACKGROUND

There has been a longstanding need for devices, compositions, and other treatments that will effectively prevent communicable diseases. Attempts at solving this problem include wearing masks or respirators and avoiding or quarantining of individuals or animals that are known or expected to be sick or carrying germs. Such approaches are common in certain countries where masks are worn by persons encountering contaminated environments such as public transportation or public gathering places. Various sorts of sanitizers and antimicrobials have also been developed for use on surfaces to prevent the spread of harmful microorganisms from surface contact.

While numerous solutions exist for killing microorganisms once they have contacted a person or animal, the effectiveness of such solutions is dependent on quick recognition of the germ contact and application of the germ-killing composition prior to the microorganism binding to a host cell on mucosal surface or a disrupted area of the skin, whereby it would enter the body and infect the individual. For example, washing with an anti-bacterial soap may be effective for killing bacteria on the hands; however, it is very easy for a person to unwittingly touch a contaminated surface and put their hands near or in their mouth or nose before washing their hands. In addition, by only being “antibacterial,” by definition many germ killing products fail to also inhibit the spread of fungi and viruses. Hand-washing or surface sanitization typically has a very short-lived duration and many microorganisms will quickly begin regrowth or new microorganisms will become deposited on the surface.

Physical devices such as masks are uncomfortable, zinc, vitamin C, and herbal remedies have unproven results, and surface sanitizers and solutions for killing germs that have already contacted the body are often ineffective for prevention of infection since they are intermittent, transitory options that do not provide sustained antimicrobial duration.

Numerous compositions have been developed for killing germs on household surfaces, healthcare facility surfaces, workplace or office surfaces, and other various surfaces that from time to time become contaminated. However, these compositions typically contain highly volatile organic compounds, and although they almost immediately kill microorganisms, they quickly evaporate and/or are washed or wiped away and do not continue to inhibit microorganism growth for more than a few minutes. In addition, it is unlikely that a quick application of such compositions will kill all the microorganisms present on the surface. Thus, the remaining microorganisms will inevitably begin regrowing in a short time.

Compositions have also been developed that mask or destroy odors or act to kill mold or other microorganisms that cause a deterioration in a visual aspect of a surface. However, improvements in a long-lasting, powerful and generally non-toxic solution for such uses is desirable.

SUMMARY

In an embodiment, a method for trapping and neutralizing or killing microorganisms on a surface includes: applying a barrier-forming composition that comprises an antimicrobial agent onto an inanimate surface; forming a barrier layer on the inanimate surface; in the barrier, trapping and killing microorganisms encountered on the surface and encountered from the environment after the applying step is performed. The barrier layer has an antimicrobial cidal or static activity for a duration of at least about one hour.

In an embodiment, a barrier-forming composition for a surface treatment includes: a carbohydrate gum, a humectant, and an antimicrobial agent. The barrier-forming composition meets the following requirements:

about 0.0001%≦C≦about 0.4%;

about 0.07%≦H≦about 70%; and

0.0005%<A

about 0%≦C≦about 0.4%;

about 55%≦H≦about 70%; and

0.0005%<A

wherein all percentages are by weight of the total composition;

wherein C is the carbohydrate gum; H is the humectant; and A is the antimicrobial agent. The barrier composition is active to trap, and neutralize or kill microorganisms.

In an embodiment, a pre-treated, disposable wipe includes a disposable wipe material at least partially saturated with a composition. The composition includes a carbohydrate gum, a humectant, and an antibacterial agent. The barrier-forming composition meets the following requirements:

about 0.0001%≦C≦about 0.4%;

about 0.07%≦H≦about 70%; and

0.0005%<A;

about 0%≦C≦about 0.4%;

about 55%≦H≦about 70%; and

0.0005%<A;

wherein all percentages are by weight of the total composition;

wherein C is the carbohydrate gum; H is the humectant; and A is the antimicrobial agent.

In an embodiment, a method includes: preventing or decreasing odor produced by microorganisms, by applying a barrier-forming composition that comprises an antimicrobial onto an inanimate surface; forming a barrier layer on the inanimate surface that is active to trap and kill or neutralize microorganisms encountered by the barrier layer for a duration of at least about one hour, thereby preventing or decreasing odor generated by microorganisms from escaping the barrier layer.

In an embodiment, a method includes preventing or treating a detraction of a visual appearance of a surface, the detraction being caused by microorganisms that produce a visible growth on the surface or discoloration of the surface, by: applying a barrier-forming composition that comprises an antimicrobial onto the surface; forming a barrier layer on the surface that is active to trap, and kill or neutralize microorganisms encountered by the barrier layer for a duration of at least about one hour thereby preventing or decreasing the visible growth or discoloration on the surface.

In an embodiment, a method for trapping, and neutralizing or killing microorganisms that cause infectious disease, includes: applying a barrier-forming composition that comprises an antimicrobial onto a surface of an apparatus; forming a barrier coating on the surface of the apparatus that is active to trap and kill or neutralize microorganisms encountered by the barrier layer for a duration of at least about one hour thereby preventing or decreasing the risk of infection from the microorganisms.

The articles “a” and “the,” as used herein, mean “one or more” unless the context clearly indicates to the contrary.

The terms “item” and “apparatus” are used synonymously herein.

The term “therapeutic,” as used herein, is meant to also apply to preventative treatment.

The term “or,” as used herein, is not an exclusive or, unless the context clearly indicates to the contrary.

The use of the term “mammal” herein, means a human or animal commonly defined as a mammal.

The use of the term “block” or “blocking” herein, includes blocking passage by trapping.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an illustrated flow chart of a treated and untreated inanimate surface and the results of an encounter with microorganisms.

FIG. 1B is a depiction of a proposed mechanism of antimicrobial activity in an embodiment of the barrier-forming composition.

FIG. 2 is a schematic showing the formation of a barrier on a mucosal surface, as described in Example 2.

FIG. 3 is a graph showing the repair process as a percentage on a wounded epithelial cell sample through cell growth and migration after 6 hours to cover the scratched space in each of Examples 11-15.

FIG. 4 shows microscopic photographs showing epithelial cell growth and migration on both untreated control Example 15 and treated Examples 16 and 17 on a wounded epithelial sample.

FIG. 5 shows photos of magnified cross-sections of the barrier-forming composition-treated and untreated engineered human oral mucosa (EHOM) of Examples 11-15.

FIG. 6 is graph showing an LDH assay of Examples 16-19 and 20-25.

FIG. 7 is a schema showing the method of evaluation of microbial growth in the upper and lower chambers of an EHOM assay, as described in Examples 27-28.

FIG. 8 show photographs of agar media plates showing microbial growth in the upper and lower chambers of an EHOM assay, as described in Examples 27-28.

FIG. 9 shows photographs of magnified cross-sections of the barrier-forming composition-treated and untreated engineered human oral mucosa (EHOM) of Examples 31-32.

FIG. 10 shows photographs of microbial growth on untreated EHOM or EHOM treated with an example barrier-forming composition, followed by infection with C. albicans, as described in Examples 33-40.

FIG. 11 shows photographs of microbial growth on untreated EHOM or EHOM treated with formulations followed by infection with S. mutans, as described in Examples 33-40.

FIG. 12 shows photographs of microbial growth from “flow-through” media (collected from the lower chamber) of EHOM treated with an example barrier-forming composition, as described in Example 33-40.

FIG. 13 presents graphs showing LDH release by EHOM treated with saline (control) or example barrier-forming compositions, followed by infection with (A) C. albicans or (B) S. mutans, as described in Examples 40-47.

FIG. 14 is a graph showing post-antimicrobial effect of barrier-forming compositions against bacteria and fungi, as described in Examples 48-61 and 61-69.

FIG. 15 shows scanning electron micrographs of S. sanguis, C. albicans, and S. mutans, untreated or treated with barrier-forming composition, as described in Examples 71-76.

FIG. 16 presents graphs depicting activity of an example barrier-forming composition against biofilms formed by bacteria and fungi, as described in Examples 77-79.

FIG. 17 is a graph showing activity of an example barrier-forming composition on microbial biofilms after a 1-min exposure, as described in Examples 80-81.

FIG. 18 presents fluorescent microscopy photographs showing the effect of an example barrier-forming composition on cytopathic effects (CPE) of influenza (H1N1)-infected MDCK cells, as described in Examples 85-86.

FIG. 19 presents fluorescent microscopy photographs showing the effect of an example barrier-forming composition on against H1N1 virus, as described in Examples 85-86.

FIG. 20 is a graph showing levels of influenza virus in infected barrier-forming composition treated and -untreated cells, as determined by quantitative PCR, as described in Examples 87-88.

FIG. 21 is a graph showing direct antiviral activity of example barrier-forming compositions prepared with or without preservatives and antimicrobial agent (CPC) against influenza virus, determined using quantitative PCR, as described in Examples 89-91.

FIG. 22 shows the activity of an example barrier-forming composition against H1N1 virus over a 6 hour time period. Panel (A) is a graph showing a percent inhibition in viral growth compared to an untreated control. Panels (B) and (C) are micrographs of (B) untreated and (C) barrier-forming composition treated cells.

FIG. 23 is a graph showing the activity of formulations against HIV, as described in Examples 94-96.

FIG. 24 is a Western blot showing activity of Example 8 against Epstein-Barr Virus (EBV), as described in Example 97.

FIG. 25 is a graph showing LDH levels as an indicator of cellular integrity in untreated (control) EHOM or EHOMs tissues exposed to Examples 5-7, as described in Examples 154-159.

FIG. 26 shows representative photographs of a wounded oral epithelial cell culture treated with Example 3 (5% dilution) for 10 minutes, immediately after the wound (panel A), after about 6 hours (panel D), and after about 24 hours (panel E), as described in Example 160. Panels B and C show an equivalent wound on an untreated control confluent culture of oral epithelial cells after about 6 hours and about 24 hours, respectively.

FIG. 27 are photographs demonstrating the ability of an example barrier-forming composition to coat the oral mucosal surface.

FIG. 28 are photographs showing time-lapse microscopy of bacterial growth after a 1 minute exposure to an example barrier-forming composition, as described in Examples 162-163. Images represent bacterial growth after 20 min, 120 min, or 360 min post-exposure.

FIG. 29 is a graph showing the effect of a single dose of an example barrier-forming composition on oral microbial burden of a healthy individual, as described in Example 164-166. (A)—Microbial load in CFUs, (B) reduction in microbial load (%) compared to baseline.

FIG. 30 is a graph showing the effect of an example barrier-forming composition on levels of oral microbes over a 5-day period in three healthy adults, as described in Examples 167-169.

FIG. 31 is a graph showing the effect of an example barrier-forming composition on microbial burden of the oral cavity after 5-day usage in 31 healthy subjects, as described in Examples 170-198.

FIG. 32 is a graph showing the microbial load in oral samples obtained from three representative study participants, as described in Examples 170-198.

FIG. 33 shows is a schema describing the in vitro filter insert-based model to evaluate penetration of microbes across the barrier formed by example barrier-forming compositions, as described in Examples 199-205.

FIG. 34 is a set of photographs showing growth of MRSA biofilms on the surface of silicone elastomer discs treated with PBS (control, A, C, E) and Example 7 barrier forming composition (B, D, F), as described in Examples 219-224.

FIG. 35 is a set of photographs showing cell monolayers treated with an embodiment of the barrier-forming composition, Example 252, for varying time periods (A), (B), and (C), and a control Example 253 (D).

FIG. 36 is a set of immunofluorescence photographs showing cell monolayers treated with an embodiment of the barrier-forming composition, Example 252, for varying time periods (A), (B), and (C), and a control Example 253 (D).

DETAILED DESCRIPTION

This application discloses a stable composition for treating/cleaning surfaces that provides a long-term sustained microbial static or cidal activity that inhibits or destroys harmful microbial growth that also has a barrier-forming property. In addition, in embodiments, the composition is also safe for human consumption.

The data presented herein is representative of the treatment of a variety of surfaces to the extent it shows a long-lasting antimicrobial barrier-forming composition against a range of microorganisms.

By blocking and/or neutralizing microorganisms that cause infectious disease on a surface, this in turn inhibits or prevents microorganisms from transferring and disseminating into the body and causing infection.

Furthermore, by blocking and/or neutralizing microorganisms on a surface that cause odor, this will in turn inhibit, and/or stop, odor from emitting from the surface.

In addition, by blocking and/or neutralizing microorganisms (such as, for example mold) on a surface that are, or can become, detracting from the visual aesthetic appearance of the surface, this will in turn inhibit, and/or stop, detraction of the visual aesthetic appearance of the surface.

The method and composition incorporates an antimicrobial agent that can inhibit microorganisms, including bacteria, fungi, and viruses, known to cause infections. The method provides a barrier on a surface by forming a barrier film or coating over it and an antimicrobial agent is included that can kill or inhibit microorganisms (bacteria, fungi and viruses). In an embodiment, the barrier-forming composition is also effective against microorganisms that cause odor and detraction of the visual aesthetic appearance of surfaces.

As the data herein shows, even if the mammal touches the surface and some of the barrier composition transfers to their body and comes into contact with human or other mammal mucosa or, for example, surfaces in the oral cavity, nasal cavity, vaginal cavity, throat, the ears and other orifices, the composition, in an embodiment, is safe and non-toxic for such consumption. As the Examples show the product applied directly to mucosal tissue, the surface treatment composition would be expected to be safe even if, for example, a child puts their mouth directly on the treated surface and ingests a portion of the composition. In another example, a utensil or other item that contacts food may be coated with the barrier forming composition and may impart some of the composition to food surfaces that then contact mucosa of a human and become ingested.

A barrier-forming composition that is non-toxic and safe and forms a barrier that inhibits the passage of active pathogenic microbes through to the other side is desirable. Another desirable property is an ability to inhibit microbial growth through static or cidal activity for an extended period of time. Without being bound by theory, the mechanism of action of the barrier-forming composition disclosed herein is based on a synergistic dual-action mechanism, in which germs are trapped in the formed barrier coating, and subsequently killed by the antimicrobial active ingredient. In an embodiment, the barrier-forming composition is not hydrophilic, which, without being bound by theory, is theorized to enhance its sustained effectiveness. For certain applications the composition may have adhesive properties for dry surfaces and/or be hydrophobic.

As shown in the Examples below, the properties of the barrier-forming composition and its effectiveness to prevent a wide variety of communicable diseases were assessed using at least ten different approaches based on: (1) an in vitro anti-microbial susceptibility testing; (2) an in vitro time kill assay; (3) an in vitro biofilm model; (4) an in vitro filter insert-based model, (5) an in vivo-like engineered human oral mucosa (EHOM) model; (6) electron microscopy evaluation; (7) hydrophobicity assay; (8) physico-chemical compatibility assays; (9) cell culture-based model using monolayer of human cell lines; and (10) human clinical trials.

The method and composition described herein may be particularly useful when a human, or more generally, a mammal, has a disrupted skin or mucosa or has a condition resulting in an immunocompromised state or is otherwise at a greater risk for infection. A disruption may be caused by a wound, scratch, or other opening in the skin or mucosa. The skin and the mucosa of the oral cavity and gastrointestinal (GI) tract serve as an important mechanical barrier that helps to prevent a local or systemic invasion of various microbes and the absorption of microbial products that are normally present in the oral cavity and the lumen of the gut. “Gastrointestinal mucosal injury in experimental models of shock, trauma, and sepsis,” Crit. Care Med. 1991; 19:627-41.). Derangement in the barrier function of the mucosa plays a central role in the pathophysiology of systemic infection. In other words, disruption of the skin or mucosa will lead to infections.

Elimination or reduction of the risk of a breach in the first line of defense is important, and the maintenance of mucosal or skin integrity is important. (Anders Heimdahl, “Prevention and Management of Oral Infections in Cancer Patients” Supportive Care in Cancer, Vol. 7, No. 4, 224-228 (1999).) Thus, having intact skin or mucosa is an important host defense against systemic infection, particularly in immunocompromised patients (e.g. cancer patients). (Shahab A. Khan, John R. Wingard, “Infection and Mucosal Injury,” Cancer Treatment Journal of the National Cancer Institute, Monographs No. 29 (2001). A barrier-forming composition that blocks and kills harmful microorganisms and that does not interfere with healing of a disrupted skin or mucosa is a unique and unexpected solution to the susceptibility of the problems of those with disrupted skin or mucosa, particularly those that also have immunodeficiency.

In an embodiment, a barrier-forming composition may be applied in a method for preventing or inhibiting the transfer of microorganisms from the surrounding environment (including from surfaces and airborne particulates that later become deposited on a treated surface) to a mammal. By prevention, it is not meant that no infection from microorganisms is possible, but that the risk of infection from microorganisms encountered at the time of application and/or subsequent to application of the barrier-forming composition is reduced.

In an embodiment, the barrier-forming composition is applied in an effective amount to an inanimate surface and provides a barrier layer on the surface that traps microorganisms, such as by inhibiting microorganisms from penetrating to the other side of the barrier. An antimicrobial is provided that kills or deactivates (neutralizes) the microorganism's harmful activity. The combined barrier and antimicrobial synergistically act to block, neutralize, and/or kill microorganisms on the inanimate surface or microorganisms that subsequently come into contact with an exposed (top) surface of the barrier, thereby providing a long-lasting antimicrobial that is significantly more powerful than just an antimicrobial alone. The barrier is effective to trap and kill or neutralize microorganisms already present on the treated surface and/or to block and kill or neutralize microorganisms that may be deposited on top of the barrier, i.e., the exposed surface of the barrier layer, after the application of the barrier forming composition is performed. In an embodiment, the barrier layer is active for at least about one hour after application.

This dual action composition and method (barrier plus antimicrobial) is applicable to various surfaces found in a variety of places, including for example, homes, schools, churches, restaurants, daycares, workplaces, vehicles and medical buildings, such as, for example, ceramic, glass, wood, (including, for example, varnished, stained, or waxed wood), linoleum, CORIAN (a composition of acrylic polymer and aluminum trihydrate), Formica, porcelain, metal (including, for example, stainless steel, steel, iron, wrought iron, copper, brass, bronze, silver, gold, platinum, aluminum, and alloys of such metals), ceramic, painted surfaces, carbon fiber, textile materials (including, for example, wool, silk, cotton, hemp, sisal, velvet, aramid, acrylic, olefins, nylon, rayon, and spandex), concrete, stone (including, for example, granite, marble, soapstone, limestone, Jerusalem stone, quartz, travertine, and slate), plastic, tile, carpet and rubber. Types of inanimate surfaces include, for example, countertop, table top, flooring, fixtures, furniture, toilet bowl, toilet seat, toilet flush knob, doorknob, faucet, bathtub, shower, hot tub, sauna, sink, clothing, food preparation equipment, playground equipment, and toys

FIG. 1A is an illustrated flow chart of microbes encountering an untreated inanimate surface (left side) and an inanimate surface with the barrier-forming composition administered on it resulting in a formed barrier layer (right side) that shows a primary efficacy of the barrier-forming composition on an inanimate surface. Instead of protecting a mucosa from infection from microbes, when treating an inanimate surface the barrier layer prevents microorganisms from binding to the surface, colonizing and forming a biofilm. Biofilms are known to be difficult to destroy. The barrier-forming composition thus presents a surprisingly effective solution to providing a sanitized surface in comparison to cleaners that only focus on killing microorganisms that are only already on the surface. While an antimicrobial solution that does not form a barrier will instantly kill some of the microorganisms in a biofilm on a surface, it is practically impossible to kill all microorganisms in a biofilm and the biofilm will soon begin to recolonize. In contrast, the barrier-forming composition prevents biofilms from forming in the first place and also has prolonged activity to destroy already formed biofilms.

In an embodiment, the barrier-forming composition traps and/or kills or neutralizes all harmful microorganisms contacting the barrier-forming composition. In another embodiment, the barrier substantially traps and/or kills or neutralizes enough harmful microorganisms that contact the barrier-forming composition to inhibit or even stop them from causing an infectious disease. In the latter case, if the harmful microorganism's penetration of the barrier is slowed and/or diluted it will enhance the body's own ability to prevent the microorganisms from causing disease or widespread infection. In vitro testing demonstrates that embodiments of the barrier-forming composition prevent all bacteria from reaching the other side of the barrier for long periods, including about two hours or more, about six hours or more, about sixteen hours or more, and about twenty-four hours or more. In vitro testing shows that in viruses exposed to embodiments of the barrier-forming composition, growth may be inhibited for about two or more days (such as influenza), up to about nine days, (such as HIV), after which the viral count is still below the MIC for extended periods, such as about two or three additional days Inhibitory activity against influenza virus was observed for up to 48 hours.

In the event that the treated surface is touched by a mammal, some of the barrier composition may be transferred to the mammal and encounter a disrupted skin or a mucosal surface. Traces of active microorganisms may be present on the transferred barrier composition or may be picked up from elsewhere and come into contact with the transferred barrier composition. In this situation, without being bound to theory, a still-active barrier composition is expected to have additional antimicrobial preventative activity on the disrupted skin or mucosal site. In an embodiment that illustrates a proposed mechanism of the barrier-forming composition in such a case, shown in FIG. 1B, the barrier-forming composition provides anti-viral activity. When a virus comes into contact with a cell, it will bind to receptors on the host cell. Over time, 5 to 6 hours, or so, the virus is internalized by the host cell, the virus multiplies inside the host cell, and it induces cell lysis causing additional virus particles to infect other host cells.

In contrast, in a disrupted skin or mucosal surface, or a mucosal surface that has come into contact with a barrier-forming composition, a protective barrier is on the surface of the host cell. The barrier, may protect the cell and any receptors on the cell, thereby preventing the virus particle from binding to the cell receptors. Thus, infection and lysis is also prevented. Similarly, the disrupted skin or mucosal surface, or a mucosal surface is also protected from bacteria or fungi.

The barrier-forming composition retains the barrier for a long duration, such as a duration of about 2 hours or more, a duration of about 6 hours or more, a duration of about 16 hours or more, a duration of about 16 hours to about 24 hours, or a duration of about 24 hours or more, thereby protecting host cells and preventing infection. The cidal or static antimicrobial activity is also retained for a long duration, such as about 2 hours or more, about 6 hours or more, about 16 hours or more, about 24 hours or more, or about 48 hours or more, thereby killing microorganisms before they can be transferred to mammals and even if transferred to mammals continuing to protect host cells and preventing infection. These durations are applicable for viruses, bacteria, and fungi.

In another embodiment, the barrier-forming composition is applied on an item or apparatus surface prior to the item or apparatus surface encountering a contaminated environment and prior to the apparatus encountering a mucosa of a mammal. The barrier-forming composition provides a barrier on the item or apparatus surface that traps and kills the microorganisms, thereby preventing or inhibiting active microorganisms from passing to the mucosa or causing infection.

Harmful microorganisms are those known to cause infectious disease such as, for example, the treatment and prevention of infectious diseases, such as communicable diseases caused by microorganisms, such as Candida species (e.g. C. albicans, C. glabrata, C. krusei, C. tropicalis), Staphylococcus species (including methicillin-resistant S. aureus, MRSA), Streptococcus species (e.g. S. sanguis, S. oralis, S. mitis, S. salivarius, S. gordonii, S. pneumoniae), Acinetobacter baumannii, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and other microorganisms such as microorganisms that cause upper respiratory infections, and common cold (rhinovirus) and influenza viruses and Pneumonia, P. gingivalis, Y. enterocolitica, Acinetobacter bumanii, Aggregatibacter actinomycetemcomitans, microorganisms that cause odor, microorganisms that can detract from visual appeal of surfaces, Clostridium difficile, Bordetella pertussis, Burkholderia, Aspergillus fumigatus, Penicillium spp, Cladosporium, Klebsiella pneumoniae, Salmonella choleraesuis, Escherichia coli (0157:H7), Trichophyton mentagrophytes, Rhinovirus Type 39, Respiratory Syncytial Virus, Poliovirus Type 1, Rotavirus Wa, Influenza A Virus, Herpes Simplex Virus Types 1 & 2, and Hepatitis A Virus. In an embodiment, the barrier-forming composition and method of treatment and prevention described herein may be useful, for example, for prevention of sexually transmitted diseases such as, for example, infections caused by human immunodeficiency virus (HIV), Herpes simplex, or human papilloma virus (HPV).

The barrier-forming composition has shown effectiveness against microorganisms with a diameter of, for example, about 30 nm or greater, such as about 100 nm (HIV, spherical), about 100 to about 300 nm (influenza, spherical and elongated forms), about 120 nm to about 260 nm (EBV spherical/disk forms), and about 30 nm (rhinovirus, spherical). Thus, the barrier composition should also be effective against other microorganisms with diameters of about 30 nm, or greater than about 30 nm.

The barrier-forming composition has even shown powerful and surprising activity inhibiting biofilms, which can be very difficult to eradicate. In an embodiment, the method comprises administering the barrier-forming composition to a formed biofilm on a surface or inhibiting microorganisms encountered by the barrier coating from forming a biofilm.

In an embodiment, the barrier-forming composition and method of treatment and prevention described herein may be useful, for example, for prevention and/or treatment of odors emanating from microorganisms present on or growing on any of the surfaces mentioned above.

In an embodiment, the barrier-forming composition and method of treatment and prevention described herein may be useful, for example, for the prevention and/or treatment of odors from microorganisms deposited on kitchen or bathroom surfaces, or any other surfaces mentioned herein. For example, the barrier-forming composition may be used to prevent odor from bacteria deposited during food preparation, from spills of food or drink or bodily fluids on carpet or vehicle interiors, or any number of other occurrences where odor causing microorganisms are deposited or grow on surfaces.

Odor causing microorganisms include, for example, bacteria and fungi. Specific examples that may be mentioned, include, Centipeda periodontii, Eikenella corrodens, Enterobacteriaceae, Fusobacterium nucleatum subsp. nucleatum, Fusobacterium nucleatum subsp. polymorphum, Fusobacterium nucleatum subsp. vincentii, Fusobacterium periodonticum, Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella (Bacteroides) melaminogenica, Prevotella intermedia, Bacteroides (Bacteroides) loescheii, Solobacterium moorei, Tannerella forsythia (Bacteroides forsythus), Treponema denticola, such as those disclosed in Scully, C. and J. Greenman (2008). “Halitosis (breath odor).” Periodontol 2000 48: 66-75, which is hereby incorporated by reference. Odor causing microorganisms may also be considered harmful microorganisms for purposes of this application.

In an embodiment, the barrier-forming composition and method of treatment and prevention described herein may be useful, for example, for treatment and prevention of detraction of the visual aesthetic appearance of surfaces. Such detraction of the visual aesthetic appearance of a surface may be caused by microorganisms that produce a visible growth on a surface and/or discoloration of a surface. In an embodiment, the barrier-forming composition and method of treatment and prevention described herein may be useful, for example, for prevention and/or treatment of staining of tile grout by mold, or prevention and/or treatment of basement floor or wall surfaces.

Microorganisms that cause detraction of the visual aesthetic appearance of surfaces include, for example, fungi. Microorganisms that cause detraction of the visual aesthetic appearance of surfaces may also be considered harmful microorganisms for purposes of this application.

The microorganisms may be air-borne microorganisms. In an embodiment, the microorganisms are those that cause communicable diseases. In an embodiment, the microorganisms do not include those that cause allergic reactions or dental problems, such as, for example, cavities (caries), gingivitis, or seasonal allergies. Similarly, in an embodiment, the method of prevention does not solely or additionally prevent dental problems or allergic reactions, such as, for example, cavities (caries), gingivitis, or seasonal allergies.

In another embodiment, however, microorganisms, such as fungi that may generally be classified as allergens, other allergens, and airborne irritants to the mucosa, are blocked by the barrier and the method. It may be especially useful to treat surfaces with the barrier composition if an allergic mammal is expected to be in a location known or expected to produce a high number of allergens or airborne irritants, such as an outdoor environment.

The methods and compositions disclosed herein may be especially applicable for treating surfaces that immunocompromised persons will encounter. In addition, the barrier-forming composition may be useful for prevention and/or treatment of infections by microorganisms that commonly infect wounds on the mucosa or skin.

In an embodiment, the barrier-forming composition and method of treatment and prevention described herein may be useful, for example, for prevention and/or treatment of infections from items that may be contaminated in activity related treatments, such as, for example, ventilator use (which would include medical devices related to the ventilator and contacting the patient). As another example of a contaminated item, treatment and prevention of fungal infections through applications to the body, and or items or surfaces coming into contact with the body, such as shoes, may also be mentioned. In an embodiment the contaminated item may be, for example, a food, a drink, utensils, drink containers and accessories, an item for use by children, a medical apparatus, or a dental apparatus.

In an embodiment, the interior surface of an apparatus protecting the feet, such as a shoe, is treated with an effective amount of the barrier-forming composition to destroy, inhibit or prevent odor from fungal growth. For example, the shoe may be treated proactively by administering the composition prior to a person wearing the shoe. Specifically the shoe may be treated prior to a person exercising or participating in an activity where their feet or shoes may become wet. In another example, the shoe is treated when a person knows that they have a fungal condition on their foot, such as athlete's foot (tinea pedis). In another embodiment, the shoe may be treated if it is producing a foul odor. In each of these embodiments, the barrier-forming composition traps and kills or prevents fungal or other microbial growth, thereby reducing the risk of infection on the human's foot and masking and/or neutralizing the foul odor produced by the microorganisms.

In an embodiment, the surface of a medical device is treated with the barrier-forming composition prior to contacting the human body. The barrier-forming composition provides a coating on the medical device surface that traps and kills microorganisms. Medical devices include, for example, instruments, apparatuses, and other articles of manufacture that are intended to contact or come in close proximity with the human body, such as human tissue, bloodstream, mucosa, open wounds, and internal cavities. Examples of medical devices include ventilators, trachea devices, catheters, central venous catheters, urinary catheters, peritoneal dialysis catheters, contact lenses, total joint replacement prostheses, endotracheal tubes, voice prostheses, penile prostheses, testicular prostheses, prostatic stents, artificial urinary sphincters, breast prostheses, vascular graft, orthopedic devices, prosthetic heart valves, scalpels, scopes, implanted replacement devices, In an embodiment, medical devices also include dental devices.

In an embodiment, the medical device is treated with the barrier-forming composition prior to encountering the human body, such as up to about 12 hours prior to encountering the human body, for example, about 5 minutes to about 8 hours, about 12 minutes to about 6 hours, or about 1 hour to about 10 hours, prior to encountering the human body.

In an embodiment, the medical device is treated with the barrier forming composition in a spray formulation by spraying the medical device until a moist coating appears on the surface.

In another embodiment, the medical device is treated by dipping the medical device in the barrier-forming composition and withdrawing it from the composition, which results in a residual coating layer on the medical device.

In an embodiment, the medical device is a catheter or similar device that is inserted into a mammal body canal, such as the throat, the anal, vaginal, or urethral canals. The coating is applied to the medical device prior to encountering the body canal and, as it is inserted, also helps to lubricate the body canal and provide antimicrobial activity along the canal.

In an embodiment, the medical device is an implanted device, such as a replacement hip part. The coating is applied to the medical device prior to encountering the body canal and provides a long-lasting and powerful antimicrobial barrier, thereby guarding against microorganisms that were on the medical device prior to the treatment and also any microorganisms that encounter the medical device after the treatment.

In an embodiment, the medical device that contacts more than one location on the body, such as a catheter, scope or a device used in surgery. Treating such a medical device prior to the first contact with the body may reduce the risk of transferring infectious microorganisms from one area of the body to another.

In an embodiment, the step of applying the barrier-forming composition occurs prior to or during a mammal that is not contaminated encountering the contaminated surface. In an embodiment, the application of the barrier-forming composition occurs in response to the identification of the surface as being contaminated or in response to an observation of a contamination event. For example, the barrier composition may be applied to a surface where a contamination event has occurred, such as when a person has sneezed, coughed, or vomited, or more generally where bodily fluids or matter have been deposited.

In another embodiment, the barrier-forming composition is applied on a mucosa, such as an oral, pharyngeal, or nasal mucosa, in response to encountering an environment that is considered to be contaminated or in response to an observed contamination event. The barrier-forming composition provides a barrier coating on the mucosa surface that traps and kills microorganisms, such as those that are encountered subsequent to the application of the barrier-forming composition, thereby preventing or inhibiting active microorganisms from passing to the mucosa or causing infection.

In another embodiment, the method of treating a surface with the barrier forming composition includes identifying a contaminated surface, wherein the contaminated surface is known or expected to be contaminated with odor causing viral, fungal, or bacterial microorganisms. The step of applying the barrier-forming composition occurs prior to or during an odor emanating from the contaminated surface. For example, the barrier composition may be applied to a surface where food is handled or bodily fluids are deposited, or any of the surfaces listed herein.

In an embodiment, a method includes preventing or decreasing odor produced by microorganisms, by performing the steps of applying a barrier-forming composition that comprises an antimicrobial onto an inanimate surface. The barrier-forming composition then quickly forms a barrier layer on the inanimate surface that is active to trap, and kill or neutralize microorganisms encountered by the barrier layer for a duration of at least about one hour, thereby preventing or decreasing odor generated by microorganisms from escaping the barrier layer. The microorganisms that encounter the barrier layer may be those that were already present on the surface that was treated or those that are disposed on the exposed top surface of the barrier layer after it is applied. In an embodiment, the method includes identifying an area of the inanimate surface that is a source of the odor, and applying the barrier-forming composition to the area.

In another embodiment the method of treating a surface with the barrier forming composition includes identifying a contaminated surface, wherein the contaminated surface is known or expected to be contaminated with viral, fungal, or bacterial microorganisms that cause detraction of the visual aesthetic appearance of surfaces. The step of applying the barrier-forming composition occurs prior to, during, or after a detraction of the visual aesthetic appearance of the contaminated surface occurs. For example, the barrier composition may be applied to bathroom tile grout that show visual signs of mold growth or are expected to come in contact with mold, or on other surfaces mentioned herein that show visual signs of mold growth or are expected to come in contact with mold.

In an embodiment the method includes preventing or treating a detraction of a visual appearance of a surface, where the detraction is caused by microorganisms that produce a visible growth on the surface or discoloration of the surface. The treatment or prevention is facilitated by the steps of applying a barrier-forming composition that comprises an antimicrobial onto the surface; forming a barrier layer on the surface that is active to trap, and kill or neutralize microorganisms encountered by the barrier layer for a duration of at least about one hour, thereby preventing or decreasing the visible growth or discoloration on the surface. The microorganisms that encounter the barrier layer may be those that were already present on the surface that was treated or those that are disposed on the exposed top surface of the barrier layer after it is applied. In an embodiment, the method includes identifying an area of the surface that has a visible microbial growth or discoloration, and applying the barrier-forming composition to the area.

In another embodiment, the method of treating a surface with the barrier forming composition includes treating the surface with the barrier-forming composition proactively, regardless of whether the surface is known or expected to be contaminated with viral, fungal, or bacterial microorganisms. In an embodiment, the administered barrier coating traps and kills microorganisms that encounter the barrier coating after the treating step. As disclosed herein, the barrier coating is effective to kill microorganisms encountered for a long duration after the treatment step, thereby facilitating its effectiveness as a proactive treatment, which stands in contrast to prior art antimicrobial compositions that are not effective for proactive treatment, partly due to their ineffectiveness for long time-periods.

In another embodiment, the method of treating a surface with the barrier forming composition includes treating a food surface with the barrier forming composition. For example, the method of treating a food surface with the barrier forming composition includes identifying a possibly contaminated food surface or a contamination event occurring in the vicinity of the food surface, wherein the surface is known or expected to be contaminated with harmful viral, fungal, or bacterial microorganisms or be exposed to the same.

For example, the food surface may be selected from produce, such as fruits and vegetables, including for example, salad vegetables, lettuce, cabbage, onions, spinach, broccoli, carrots, brussel sprouts, potatoes, nuts, carrots, apples, oranges, bananas, berries. The food surface may include meat, such as, for example, raw meat including, for example, beef, pork, lamb, chicken, and turkey. The food surface may also include processed meats, such as bologna, salami, pepperoni, and frankfurters.

In a particular embodiment, the barrier-forming composition may be applied to a food surface that is deemed to have a heightened risk of e. coli, salmonella, botulism, or listeria. The heightened risk may be based on knowledge of prior infections of a certain type of food surface or known contaminations of food surfaces in a common locality. The heightened risk may be based on a known or expected exposure of the food surface to a contaminant.

In an embodiment, the food surface treated in response to a contamination event, such as a person sneezing or coughing in the vicinity of the food surface. For example in the same room, vehicle, or within about 10 yards of the food surface.

In an embodiment, the food surface may be treated proactively to prevent contamination of microorganisms that may contact the food surface after the treatment, such as for up to 24 hours after treatment, or between about 12 minutes to about 6 hours, or between about 1 hour to about 8 hours post-treatment. In an embodiment, produce that is available to be touched by numerous customers, such as in a supermarket or salad bar, may be treated proactively with the barrier composition in the anticipation of guarding against a contamination event or contaminated environment.

In an embodiment of the method of treatment of food surfaces, the barrier composition is rinsed off prior to consumption. However, in an embodiment, the barrier composition is safe for human consumption in the therapeutically effective amount and is not rinsed off the food surface prior to consumption.

To achieve full effectiveness, the proper amount to apply to a surface is an amount that is enough to coat the targeted surface with enough of the barrier-forming composition to form a barrier layer, for example, this may be determined by whether the surface appears wet or misted. For example, the effective amount for a may be expressed in terms of a volume per square cm, such as, for example, from about 0.5 to about 50 μl/cm², such as, about 5 to about 40 μl/cm², or about 10 to about 25 μl/cm²; or for example, about 0.625 to about 10 μl/cm², such as, about 2.5 to about 5 μl/cm². Other delivery mediums, such as a roll-on or disposable wipes, may have dosages derived from these ranges given the adjustments for concentrations and other factors known to those of skill in the art.

In addition, the average thickness of the film formed on the surface from the barrier-forming composition may range, for example, from about 0.001 to about 0.2 mm, such as about 0.01 mm to about 0.1, or about 0.08 to about 0.15 mm.

In an embodiment, in a continued application method of prevention and/or treatment, the barrier-forming composition may be administered to a surface in a series of doses, such as, for example, about every 1 to 2 days, about every 2 to 4 days, or about every 1 to 2 weeks. This method of prevention and/or treatment can be continued, for example, for 6 to 12 months or for several years. This continued application method may be preferred during flu season or outbreaks of particular illnesses, or simply as part of a routine cleaning schedule.

In an embodiment, methods of applying the barrier-forming composition include, for example, rubbing, mopping, wiping, or spraying the composition onto the surface. The barrier-forming composition may be applied to the surface through many different delivery systems, including, for example: dilutable liquids, gels, lubricants, compositions sprayable by a mechanical action pump, aerosolized spray compositions, or infusion or layering of the barrier-forming composition into or onto products, such as disposable wipes.

In an embodiment, the composition is sprayed onto a surface in an amount sufficient to coat the surface with a mist or thin layer of liquid. One or more sprays may be required to coat the surface depending on the size of the surface. Areas that are considered especially contaminated with microorganisms may be treated more heavily than others. Wiping or rubbing is not required in this embodiment.

A mechanical pump spray or an aerosolized spray device may be used. In the aerosolized embodiment, the barrier-forming composition may be mixed with common propellant agents, such as CO₂, nitrogen, and hydrocarbons. A bag-on-valve embodiment may also be used; however, the composition is stable enough so as not to require a separation of the propellant agent and the composition components.

In an embodiment, the barrier-forming composition is applied by wiping the composition onto the surface from a material that includes the barrier-forming composition. For example, a cloth, a mop, a scrubbing brush, a toilet cleaning brush, or a paper towel, may be at least partially saturated with the barrier-forming composition. The composition may then be wiped or mopped or otherwise applied to the surface from the material. In an embodiment, the composition can be applied by spraying and subsequent wiping of the sprayed composition.

In a particular embodiment, a disposable wipe is pre-treated with the barrier-forming composition. In an embodiment, the wipe can simply be removed from a container and then be rubbed on the desired surface to apply the barrier-forming composition to the surface. The wipe can then be disposed of. Common materials for disposable wipes include, for example, wood pulp, viscose, polyester, cotton, and combinations of these. The wipe can be soaked in and then removed from the barrier forming composition for the pre-treatment step. The wipe should be at least partially saturated with the barrier forming composition. In the application step, the surface should be wetted with the wipe to provide an effective barrier. After wiping the surface, a barrier-forming composition is deposited that forms a thin film barrier on the surface and is active to inhibit microorganisms from passing through the barrier layer.

In an example, the disposable wipe may be used to treat an item, such as a utensil, in order to apply the barrier-forming composition to the utensil.

Without being bound by theory, the same anti-viral barrier mechanism described above and depicted in FIG. 1B is applicable to the anti-bacterial, and anti-fungal activity of the composition and method of prevention described herein.

In an embodiment, the barrier-forming composition comprises a carbohydrate gum (C), a humectant (H), and an antimicrobial agent (A), and the barrier-forming composition meets the following requirements:

about 0.0001%≦C≦about 0.4%;

about 0.07%≦H≦about 70%; and

0.0005%<A

about 0%≦C≦about 0.4%;

about 55%≦H≦about 70%; and

0.0005%<A

All percentages are by weight of the total composition. The ranges in this embodiment reflect the demonstrated effectiveness of the germ killing power of the barrier-forming composition at very low dilutions against many microorganisms reported in MIC experiments in Table V below. After effective application, the barrier layer has antimicrobial cidal or static activity.

In another embodiment the barrier-forming composition meets the following requirements:

about 0.01%≦C≦about 0.4%;

about 4.5%≦H≦about 65%; and

0.0005%<A

about 0%≦C≦about 0.4%;

about 55%≦H≦about 65%; and

0.0005%<A

All percentages are by weight of the total composition.

In another embodiment, the humectant of the barrier-forming composition meets the following requirements: about 0.07%≦H<1%. This low-humectant embodiment reduces the stickiness or adhesiveness of the composition to provide a better tactile sensation to the inanimate surface.

In an embodiment, the barrier-forming composition includes glycerin or one or more similar humectant substances. In an embodiment, the concentration of the humectant may range from about 0.07% to about 10% of the entire composition (by weight), such as about 3% to about 8%, 0.35% to less than 1%, or about 0.1% to less than 0.5%. In another embodiment, the humectant may range from about 2% to about 70% weight percent of the entire composition, such as, for example, about 4.5% to about 65%, about 7% to about 35%, or about 15% to about 45%. Humectants similar to glycerin may be classified generally as polyols. The humectants may be, for example, glycerin, sorbitol, xylitol, propylene glycol, polyethylene glycol, and mixtures thereof. In an embodiment, glycerin may be used at high concentrations such as about 55% to about 65% in the absence of a gum.

In an embodiment, the composition also includes a gum. The gum may be, for example, a polysaccharide, xanthan gum, gum Arabic, or guar gum. Such gums may be generally classified as carbohydrate gums that have an overall negative charge. In another embodiment, the gum may be, for example, xanthan gum, guar gum, gum Arabic, tragacanth, gum karaya, locust bean gum, carob gum, and pectin. These gums may also be generally classified as carbohydrate gums that have an overall negative charge. In an embodiment, the gum may be present in a weight percentage of the total composition ranging from about 0.0001% to about 0.4%, such as about 0.0005 to about 0.25%. In another embodiment, the gum may be present in a weight percentage of the total composition ranging from about 0.01% to about 0.4%, such as for example, about 0.25% to about 0.35%, about 0.05% to about 0.25%, or about 0.4%.

In an embodiment, the barrier composition comprises a humectant, an antimicrobial, and optionally a gum, wherein the gum, if present, is present in an amount of about 0.0001% to about 0.4% by weight of the total barrier-forming composition.

In an embodiment, an antimicrobial agent is present in the composition. For example, the composition may include one or more anti-viral agents, or antifungals or antibacterials or a combination thereof. In addition, the effect of such antimicrobials includes static and/or cidal activity.

The antimicrobial agent may include, but is not limited to cationic antimicrobial agents and pharmaceutically acceptable salts thereof, including, for example, monoquaternary ammonium compounds (QAC, cetrimide, benzalkonium chloride, cetalkonium chloride, cetylpyridinium chloride, myristalkonium chloride, Polycide), biquaternaries and bis-biguanides (Chlorhexidine, Barquat, hibitane), and biguanides, polymeric biguanides, polyhexamethylene biguanides, Vantocil, Cosmocil, diamidines, halogen-releasing agents including chlorine- and iodine-based compounds, silver and antimicrobial compounds of silver, peracetic acid (PAA), silver sulfadiazine, phenols, bisphenols, hydrogen peroxide, hexachloroprene, halophenols, including but not limited to chloroxylenol (4-chloro-3,5-dimethylphenol; p-chloro-m-xylenol).

In addition, the antimicrobial may also be or include: antibacterial agents, both cidal and static, and different classes, for example tetracycline, chloramphenicol, fusidic acid, fluoroquinolone, macrolide antibacterial agents, oxazolidinones, quinolone- and naphthyridone-carboxylic acid, citral, trimethoprim and sulfamethoxazole (singly and combined), aminoglycoside, polymyxin, penicillins and their derivatives. In addition, the antimicrobial may also include, for example: antifungal agents in the following classes: azoles, polyenes, echinocandins, and pyrimidines. Combinations of the any of the foregoing antimicrobial agents are also contemplated. Many of the foregoing are cationic species or their pharmaceutically acceptable salts, and in an embodiment, cationic antimicrobials are utilized in the composition. In an embodiment the composition is exclusive agents that release gas fumes, such as, for example, chlorine dioxide, or chlorine dioxide producing reactants.

In an embodiment, the barrier-forming composition does not induce mutations or the development of resistance by microbes. This is because of the mechanism of action against the microorganisms by the barrier and the selected antimicrobial.

The antimicrobial may be present, for example, in an amount ranging from about 0.0005% to 5% by weight of the total composition, such as, for example, about 0.0025% to about 1%, about 0.005 to about 0.006%, or about 0.0006% to about 0.003%. In another embodiment, the antimicrobial may be present, for example, in an amount ranging from about 0.05% to about 0.1% by weight of the total composition, such as, for example, about 0.05% to about 0.06% or about 0.06% to about 0.1%. In an embodiment, the antimicrobial is about 5% or less, or about 3% or less, or about 1.5% or less, such as when the antimicrobial used does not cause solubility problems at higher concentrations.

In embodiments, the composition may further include other components, such as, for example, copovidone and other lubricating agents, parabens such as methyl paraben or propylparaben, scenting agents, preservatives, such as sodium benzoate, buffering agents, such as monosodium and disodium phosphate, sweeteners, hydrogenated castor oil with ethylene oxide, and carboxymethylcellulose. These components may, for example, be included in amounts ranging from about 0.01% to about 5% by weight of the total composition, such as, for example, about 0.1% to about 2%. In another embodiment, the components are included, for example, in amounts of about 0.0001% to about 0.05%. Buffering agents (such as monosodium or disodium phosphate) may also be used.

Purified water and/or alcohol may be used as the diluent component of the composition. In an embodiment, the barrier-forming composition is a free-flowing liquid suitable for spraying. This is in contrast to a paste or toothpaste composition, which is typically not free-flowing and not suitable for spraying. In addition, in an embodiment, the barrier-forming composition is free of abrasives that are commonly used in toothpaste compositions.

Some antimicrobials, including cetylpyridinium chloride, are known to be negatively affected in their antimicrobial properties by additional active components. Thus, in an embodiment, the composition consists essentially of only the gum, the humectant, and the antimicrobial, such as including only preservatives or scenting agents that do not affect the barrier or antimicrobial activity. In an embodiment, the composition is exclusive of agents for acting against the teeth and/or gums, including, for example, abrasives (such as those used in toothpastes) teeth whitening or desensitizing agents. In an embodiment, the composition is exclusive of cellooligosaccharides. In an embodiment, the antimicrobial agent is exclusive of lipids such as fatty acid ethers or esters of polyhydric alcohols or alkoxylated derivatives thereof. In an embodiment, the composition is exclusive of one or more of time-release agents, allergy-relief compounds, azelastine, silicon based oils, essential oils, polyvinyl pyrrolidone, polyvinyl alcohol, and potassium nitrate. In an embodiment, the composition is free of volatile organic compounds, including for example, volatile alcohols. In an embodiment, the composition is free of surfactant or foaming agent. For the avoidance of doubt, none of the above should be construed to mean that all embodiments are exclusive of these compounds.

In an embodiment, a method for making a barrier-forming composition includes mixing and heating the carbohydrate gum, humectant, and antimicrobial agents. In an embodiment, heating is replaced with extended mixing times. Other components may also be mixed in a single or multiple mixing steps. All components of the barrier-forming composition may be mixed at one time to produce a composition with a stable shelf life. This is in contrast to compositions that have active components that must be added separately a short time prior to use. Thus, in an embodiment, the barrier-forming composition is a stable one-part composition that does not require mixing with a second composition to activate it for use.

In general, the dual-action mechanism of providing a barrier from microorganisms and an antimicrobial agent provides a long-lasting effect, characterized by both in vitro, simulated in vivo, and in vivo examples below. In in vivo examples the barrier-forming composition was shown to have antimicrobial effect (cidal or static) for at least 6 hours, while the barrier property was tested in simulated in vivo tests (on artificial human mucosa EHOMs) indicated the barrier itself had a significantly extended duration past 6 hours, such as greater than about 8 hours, about 6 to about 16 hours, and about 24 hours, or more. In addition, in vitro tests indicate the antimicrobial effect had a significantly extended duration past about 2 hours, past about 6 hours, and depending on the microorganism tested, such as greater than about 8 hours, about 6 to about 16 hours, and about 24 hours, or more.

Post antimicrobial effect (PAE) is defined as suppression of microbial growth that persists after limited exposure to an antimicrobial agent. Having a longer PAE is considered advantageous for antimicrobial agents as it allows for persistent inhibition of microbial growth, and may affect dosing regimens as agents with long PAEs may need less frequent administration than those with short PAEs.

In embodiments of the method and composition disclosed herein the PAE of the composition when applied to a mucosa has a PAE that persists for about 6 hours or more, such as about 6 hours to about 16 hours, or about 16 hours to about 24 hours.

In an embodiment, the composition is liquid and is non-foaming.

As mentioned above, in an embodiment, the composition is suitable for spraying, and thus also has a viscosity that is suitable for spraying. In an embodiment, the composition has a viscosity of less than 500 cps such as, for example, about 490 cps to about 10 cps, or about 400 cps to about 15 cps. In another embodiment, the composition has a viscosity of about 16 to about 20 cps, such as, for example, about 17 to about 19 cps.

In an embodiment, the composition is non-toxic to humans, wherein at least a portion of the composition may be ingested and is safe for human consumption.

Without being bound by theory, the barrier-forming composition is not hydrophilic which allows the barrier-forming composition to have a greater affinity to adhere to and cover certain surfaces. Furthermore, in an embodiment, the antimicrobial being embedded in the non-hydrophilic composition will allow for sustained antimicrobial activity on treated surfaces. In an embodiment the barrier-forming composition is amphiphilic or has amphiphilic components.

One measure of hydrophilicity is the Rf (relative front) value, determined by chromatography in water. In an embodiment, the composition has an Rf value in water of 0 to about 0.25, such as about 0.0001 to about 0.15, or about 0.03 to about 0.1.

In an embodiment, the composition has a pH of about 4 to about 8, such as about 5 to about 7, or about 6 to about 7.5. In another embodiment the composition has a pH of greater than 5.5 to about 8, wherein antimicrobials such as cetylpyridinium chloride are most effective.

As the Examples below show, the barrier-forming composition has been shown to trap and block the passage of a wide variety of representative fungi, bacteria and viruses. Because viruses are amongst the smallest infectious microorganisms, and because the barrier-forming composition forms a mechanical barrier blocking viruses, it is expected that the barrier-forming composition would be an effective preventative treatment not only for viruses but also for larger microorganisms, including a wide range of bacteria and fungi.

Several experiments were performed to assess the safety of the composition on mammals and the ability of the spray formulation to form a protective barrier on an Engineered Human Oral Mucosa (EHOM) model. The experimental evidence showed that the composition formed a barrier over tissues, which prevents microorganisms from penetrating into the tissues

EXAMPLES
Example 1
Human Gingival Epithelial Cell and Fibroblast Cultures

Normal human gingival cells (epithelial cells and fibroblasts) were obtained from ScienCell Research Laboratories (Carlsbad, Calif., USA). The fibroblasts were cultured in Dulbecco's modified Eagle's medium (DME, Invitrogen Life Technologies, Burlington, ON, Canada) supplemented with fetal bovine serum (FBS, Gibco, Burlington, ON, Canada) to a final concentration of 10%. The epithelial cells were cultured in Dulbecco's modified Eagle's (DME)-Ham's F12 (3:1) (DMEH) with 5 μg/mL of human transferrin, 2 nM 3,3′,5′ of tri-iodo-L-thyronine.

0.4 μg/mL of hydrocortisone, 10 ng/mL of epidermal growth factor, penicillin and streptomycin, and 10% FBS (final concentration). The medium was changed once a day for epithelial cells and three times a week for fibroblasts. When the cultures reached 90% confluency, the cells were detached from the flasks using a 0.05% trypsin-0.1% ethylenediaminetetra acetic acid (EDTA) solution, washed twice, and resuspended in DMEM (for the fibroblasts) or DMEH-supplemented medium (for the epithelial cells).

Example 2
Engineered Human Oral Mucosa (EHOM) Tissue

The EHOM model was produced by using the gingival fibroblasts and epithelial cells of Example 1 that were used to form a complex three-dimensional spatial cellular organization similar to that found in normal human oral mucosa. The lamina propria was produced by mixing Type I collagen (Gibco-Invitrogen, Burlington, ON, Canada) with gingival fibroblasts, followed by culture in 10% FBS-supplemented medium for four days. The lamina propria was then seeded with gingival epithelial cells to obtain the EHOM. The tissue specimens were grown under submerged conditions until the total surface of the lamina propria was covered with epithelial cells. To produce stratified epithelium, the EHOM was raised to an air-liquid interface for four more days to facilitate the organization of the epithelium into its different strata.

The lamina propria is a thin layer of loose connective tissue that lies beneath the epithelium and together with the epithelium constitutes the mucosa. FIG. 2 shows an illustration of the EHOM mucosal tissue, with an arrow pointing to its location in a schema depicting mucosa covered with the barrier-forming composition.

Examples 3-9

Examples of the barrier-forming compositions were created by adding the ingredients listed below in a 50-mL centrifuge tube, and vortexing to bring to “free-flow” consistency. The constituents of the compositions and their approximate amounts are given in Table I (the values in Table I are percentages by weight of the total composition):

TABLE II

Example 5
Example 6

Example 3
Example 4
(control)
(control)
Example 7
Example 8
Example 9

Glycerin
7
35
35
35
35
7
7

Xanthan Gum
0.01
0.4
0.4
0.4
0.4
0.01
0.01

Cetyl
0.05
0.05

0.1
0.06
0.05

Pyridinium

Chloride

Preservatives
No
No
No
Yes
Yes
Yes
Yes

*Purified water comprised the remaining portion of the composition.

**Preservatives included methylparaben (0.1%), propylparaben (0.1%), sodium benzoate (0.5%)

Based on the results below, the preservatives were found to be superfluous to the barrier formation and antimicrobial activity.

Example 10
Monolayer Wound Repair Assay

Wound repair assays were performed on the epithelial cells and fibroblasts of Example 1. Briefly, gingival epithelial cells (1×10⁴cells) and fibroblasts (1×10³cells) were seeded into wells of 6-well plates and grown in Dulbecco's modified Eagle's (DME)-Ham's F12 (3:1) (DMEH) with 5 μg/mL of human transferrin, 2 nM 3,3′,5′ of tri-iodo-L-thyronine, 0.4 μg/mL of hydrocortisone, 10 ng/mL of epidermal growth factor, penicillin and streptomycin, and 10% FBS (final concentration). Upon confluency, wounds were made in the confluent monolayer of each well using a 200 μL pipette tip.

Examples 11-15

In Examples 11 and 12, the epithelial cell cultures from Example 10 were exposed to diluted barrier-forming compositions of Examples 3 and 4 for about 2 minutes. In Examples 13 and 14, the fibroblast cultures from Example 10 were exposed to diluted barrier-forming compositions of Examples 3 and 4 for about 2 minutes. Prior to exposure, Example 3 was diluted with saline to a 1% concentration and Example 4 was diluted with saline to a 5% concentration. Following exposure, the spray was washed out twice with warm sterile culture medium, then cell cultures were over layered with DME for fibroblasts and DMEH for epithelial cells, and cultured for 6 and 24 hours in a 5% CO₂humid atmosphere at 37° C. Control Example 15, which was an untreated culture from Example 10, was also tested.

Wound repair/cell migration was ascertained using an optical microscope, and digital photographs were taken (Nikon, Coolpix 950). The percentages of wound closure (cell growth/migration) were calculated by comparing relative wound areas before and after exposure to our barrier spray using formula I:

$1. \frac{(initial scratch size) - (size after a specified culture period)}{value of the (initial scratch size) \times 100} .$

FIG. 3 shows a graph of the wound repair data generated from formula I after 6 hours post-treatment. FIG. 4 also shows photographs indicating the wound repair of the epithelial cells cultures treated with the 1% diluted barrier-forming composition and the control Example 15.

Following contact with the barrier-forming compositions, epithelial cells (FIG. 4A) and fibroblasts (data not shown) migrated progressively starting at 6 hours post contact with the barrier-forming compositions of Examples 11 and 12. Epithelial cells were small and cuboidal in shape, in both non-exposed and barrier-forming composition-exposed cultures (FIG. 4A-E). Fibroblasts were elongated cells with small nucleus in both culture conditions (unexposed and cetylpyridinium chloride-based products exposed cells) (data not shown). Cell growth and migration to cover the wounded zone were comparable in both the control Example 15 and the treated Examples 11 and 12 as compared to the original wound (FIG. 4F). Follow up over 24 hours showed that each of Examples 11 and 12 covered the edges of the scratch giving confluent monolayers (FIG. 4D-E).

This experiment was repeated five separate times with similar results. The treated Examples 11 and 12 did not show a major side effect on gingival epithelial cells growth/migration nor on cells differentiation (no cells presenting large cytoplasm and large nucleus).

Examples 16-19
Cytotoxicity Assay

The engineered human oral mucosa (EHOM) model of Example 2 was used to determine whether the composition of Examples 10 and 11 were safe and did not promote tissue damage or cell necrosis. In Example 16 and 17, the epithelium surface (10 cm²) was over layered with a thin layer (300 μl) of the Example 3 barrier-forming composition at a dilution of 5% and the Example 4 composition at a dilution of 1% (both were diluted in serum free culture medium) for time periods of about 2 minutes. The variation in time period was not deemed to have an effect on the results and just reflects the time it took to conduct the procedures. Control Example 18 was a control that was not treated with the barrier-forming composition. The EHOM tissues were then rinsed twice with warm sterile culture medium and incubated in a 5% CO₂humid atmosphere at 37° C. for 24 hours. Following this incubation period, to assess whether the engineered tissue was damaged, each EHOM was macroscopically examined for the presence of holes due to the contact with the barrier spray formulation. Photos were taken of these EHOM to confirm such possibility. Additionally, biopsies were collected from each EHOM and subjected to histological staining using eosin and hematoxylin.

In Examples 16 and 17, EHOM exposed to Examples 3 and 4 in the 1 and 5% dilutions, similar to untreated control Example 18, do not show any macroscopic damage such as holes. (See FIG. 5). The absence of such damage was monitored after 10 minute tissue contact with 5% dilution Example 3 mouthwash (Example 7A) and 1% dilution spray (Example 7B) compositions. This was confirmed by histological analyses showing multilayer organisation of the epithelium on the top of a fibroblast-populated connective tissue. No specific damage was observed in EHOM tissues in Examples 16 or 17. The Example 16 and 17 treated-EHOM structures were comparable to the untreated control Example 18.

The same observation was noted in an Example 19, which was the same as Example 16, except it included an EHOM treated with a 1% dilution (data not shown).

FIG. 5 shows the effect of the barrier-forming composition on EHOM macroscopic shape and structure. Panels A and B show control Example 18. Panels C and D show Example 16 (spray composition) and panels E and F show Example 17 (mouthwash composition).

Examples 20-25

Side effects, if any, of the barrier spray composition on EHOM injury was also assessed by measuring the leakage of intracellular LDH into the culture medium.

In Examples 20-23, EHOM tissues were exposed to (A) 1% or (B) 5% dilutions of Examples 3 and 4, respectively, for 10 minutes, followed by growth in culture medium for 24 hours. Examples 24 and 25 were controls that were not treated. 50 μl of a supernatant of each of Examples 20-25 were then transferred to a sterile 96-well flat-bottom plate. Each well was supplemented with 50 μl of reconstituted substrate mix, and the plate was incubated for 30 min at room temperature in the dark. To estimate LDH levels, aliquots of the culture supernatant were collected and subjected to an LDH cytotoxicity assay (Promega, Madison, Wis., USA), as per the manufacturer's protocol. This assay measures the conversion of L-lactate and NAD to pyruvate and NADH by the released LDH. To stop the reaction, 50 μl of stop solution was added to each well. Next 100 μl of the mixture were transferred to a 96-well flat-bottom plate, and the absorbance was read at 490 nm with an X-Mark microplate spectrophotometer (Bio-Rad, Mississauga, ON, Canada).

In the LDH and wound repair experiments, the following test methods were used. Each experiment was performed at least four times, with experimental values expressed as means±SD. The statistical significance of the differences between the control (absence of barrier spray) and the test (presence of spray) values was determined by one-way ANOVA. Posteriori comparisons were done using Tukey's method. Normality and variance assumptions were verified using the Shapiro-Wilk test and the Brown and Forsythe test, respectively. All of the assumptions were fulfilled. P values were declared significant at ≦0.05. Data were analyzed using the SAS statistical package (version 8.2, SAS Institute Inc., Cary, N.C., USA).

Results presented in FIG. 6 showed no significant difference in LDH levels in barrier-forming composition treated structures as compared to untreated tissues. These observations were made at 6 hours (data not shown) and 24 hours (FIG. 6) post-exposure to the barrier-forming composition treated Examples. FIG. 6(A) shows Control Example 24 and the 1% dilution treated Examples 20 and 21; and FIG. 6(B) shows a Control Example 25, and the 5% dilution treated Examples 22 and 23. This data confirm the non-toxic effect of Examples 3 and 4. Similarly, contact for 2 minutes with Examples 3 and 4 did not show any difference as compared to the controls (data not shown).

Example 26
Determination Whether Barrier-Forming Composition Damages EHOM Structure

EHOMs of Example 2 were treated with Example 4 for about 2 minutes, washed with culture medium then cultured for 24 hours. Tissue was then examined for possible macroscopic tissue damage (presence or not of holes). Tissue damage was also investigated by histological analyses. For this purpose, biopsies were taken from each EHOM. They were fixed with 4% paraformaldehyde solution and then embedded in paraffin. Thin sections (4 μm) were stained with eosin-hematoxilyn. Sections were mounted with a coverslip in 50%-glycerol mounting medium, observed through an optical microscope, and photographed. No damage to the treated EHOMs was ascertained.

Examples 27 and 28
Determination Whether the Barrier-Forming Composition Affects Mechanical Barrier Function of EHOM Against Microbial Passage Through Mucosal Tissue

In Examples 27 and 28, two approaches were used to determine whether the control Examples formed a barrier that blocked the microbial passage through the mucosal tissues and also had an inherent anti-microbial effect. Growth in pass-through chamber and growth on EHOM surface was assessed by evaluating growth in agar media.

In Example 27, EHOMs of Example 2 were put in contact with 1 and 5% dilutions (diluted in serum free culture medium) of Example 4 for 2 minutes. Tissues were then washed twice with serum free culture medium then over layered with 1×10⁶candida microbial cells in a volume of 300 μl. Tissues were then put on air-liquid culture plates and incubated for 24 hours in 5% CO₂humid atmosphere at 37° C. Next, the culture medium underneath the EHOM (ventral chamber) was collected and seeded on Sabouraud agar plate to verify whether or not the microorganisms penetrated through the tissue and reached the culture medium below. A culture was also obtained from the EHOM surface and seeded on Sabouraud agar plate. The process is graphically depicted in FIG. 7.

In Example 28, EHOMs of Example 2 that were treated with 1 and 5% dilutions of the Example 4 composition for 2 minutes were over layered with candida microbial cells for 24 hours were flipped onto Sabouraud dextrose agar plates and left in place for 5 minutes. The EHOMs were then removed and the plates were incubated for 24 hours at 30° C., after which microbial growth was ascertained macroscopically and photographed. Each experiment was repeated 5 independent times with similar results.

FIG. 8 shows the results of the cultures of the EHOM surface (panels C and D) and the culture of the pass-through liquid from the bottom (ventral) chamber (panels A and B). The A and C panels were EHOMs treated with a 1% dilution of Example 4, and the B and D panels were EHOMS treated with a 5% dilution of Example 4. This data indicates that Example 4 composition forms a barrier that prevents passage of microbes through the EHOM tissues but does not have an inherent anti-microbial effect.

Examples 29 and 30

In Examples 29 and 30, Examples 27 and 28 were repeated, except the EHOM were infected with S. mutans. Similar results were obtained that indicated that the barrier-forming compositions formed a barrier preventing the S. mutans microbes from passing through the barrier, but did not have an antimicrobial effect.

Examples 31 and 32
Determination Whether the Barrier-Forming Composition Affects Mechanical Barrier Function of EHOM Against Microbial Invasion

In Example 32, a set of EHOM tissues from Example 2 was treated with the barrier-forming composition of Example 4 and then overlaid with C. albicans. In control Example 31 a control set was not treated with the barrier-forming composition prior to overlayering with C. albicans. Immediately after each contact period, biopsies were taken from each EHOM, fixed with paraformaldehyde solution, and embedded in paraffin. Thin sections (4 μm) were stained with eosin-hematoxylin. Sections were observed using an optical microscope to analyze the invasion/penetration of microbial cells into the tissue. Following microscopic observations, representative photos were taken from each condition and presented. The experiment was repeated three times with similar results. Similar results were also obtained with treatment with Example 3 (data not shown).

FIG. 9 shows the effect of the barrier-forming composition on microbial invasion of EHOM tissues. Panel (A) is a representative photograph of the untreated control Example 31, and panel (B) is a photograph of the treated Example 32. The arrow indicates invading fungal hyphae in the untreated control Example 31.

Examples 33-40

The EHOM model described above was also used to evaluate the ability of Examples 5-7 to form a barrier that: (a) prevents oral bacteria (S. mutans) and fungi (Candida albicans) from penetrating/invading human oral mucosa, and (b) does not cause damage to host cells (cytotoxicity assay).

Examples 33-40 were formulated according to Table III below.

TABLE III

Barrier-forming

composition Pre-

Treatment
Microbe Overlay
FIG. reference

Example 33
None

C. albicans

FIG. 10(A)

Example 34
Example 5

C. albicans

FIG. 10(B)

Example 35
Example 6

C. albicans

FIG. 10(C)

Example 36
Example 7

C. albicans

FIG. 10(D)

Example 37
None

S. mutans

FIG. 11(A)

Example 38
Example 5

S. mutans

FIG. 11(B)

Example 39
Example 6

S. mutans

FIG. 11(C)

Example 40
Example 7

S. mutans

FIG. 11(D)

In Examples 33-40, after pre-treatment and incubation according to the procedures of Examples 27 and 28 (1) the flow-through medium was collected from the lower chamber; and (2) tissues were flipped and placed onto the surface of Sabouraud dextrose agar Petri dishes, and incubated for 24 hours. Collected flow-through media were spread onto agar media plates, and incubated for 24 hours also as described in Examples 27 and 28. Table III also indicates the Figure in which a photo of each Example was taken showing the microbial growth on each flipped Example culture.

FIGS. 10 and 11 show that both Candida and Streptococcus were able to grow on the surface of EHOM treated with the compositions of Examples 5-6. In contrast, as shown in FIG. 12, no microbial growth was observed when the “flow-through” medium collected from the lower chambers of EHOMs of Examples 36 or 40, i.e. those treated with the Example 7 composition. This indicates that treatment of the EHOMs with the Example 7 composition did not cause damage to the surface of the mucosal tissues and organisms were unable to penetrate the treated EHOM. Similar results were obtained with EHOM treated with the compositions of Examples 5 and 6 (data not shown). These data indicate that the combination of glycerine and xanthan gum is capable of forming a protective barrier on mucosal tissues.

Examples 41-47
Tested Formulations are not Toxic and do not Cause Damage to the Cells/Tissues

In Examples 41-47, the EHOM model was used to assess the toxicity of the composition. Examples 41-47 were formulated as stated in Table IV.

TABLE IV

Barrier-forming

composition Pre-

Treatment
Microbe Overlay
FIG. Reference

Example 41
None

C. albicans

FIG. 13(A)

Example 42
Example 5

C. albicans

FIG. 13(A)

Example 43
Example 6

C. albicans

FIG. 13(A)

Example 44
Example 7

C. albicans

FIG. 13(A)

Example 41A
None

S. mutans

FIG. 13(B)

Example 45
Example 5

S. mutans

FIG. 13(B)

Example 46
Example 6

S. mutans

FIG. 13(B)

Example 47
Example 7

S. mutans

FIG. 13(B)

After pre-treatment and incubation according to the procedures of Examples 27 and 28, culture supernatant was collected from the Example 41-48 EHOM tissues and used to measure LDH activity.

As shown in FIG. 13, no significant increase in LDH levels was observed in Examples 41-48 irrespective of whether the formulations contained cetylpyridinium chloride with or without preservatives and infected with either Candida albicans or S. mutans, respectively. These data confirmed the non-toxic effect of the Example barrier-forming compositions and that these formulations maintained the integrity of the host mucosal tissues.

Data are mean±SD and were computed as stated in Example 25 above. No significant difference between untreated and treated tissues was noted.

Taken together, the data indicates that the example compositions represent an effective and a safe barrier that can prevent microorganisms from penetrating and invading human mucosal tissues.

Examples 48-61

Preclinical evaluation of the barrier-forming composition showed that the composition was effective against many bacteria and yeasts. The antimicrobial activities of the Example 7 barrier-forming composition were evaluated against a number of clinical isolates obtained from patients, including S. salivarius, P. gingivalis, S. pyogenes, S. pneumonia, Fusobacterium nucleatum, S. mutans, S. aureus, Y. enterocolitica, S. oralis, S. mitis, C. albicans, C. krusei, C. tropicalis, and C. glabrata. Activity of the Example 7 barrier-forming composition was evaluated by determining its minimum inhibitory concentration (MIC) using reference methods described in the Clinical and Laboratory Standards Institute (CLSI) documents M07-A8, M11-A7, and M27-A3.

A standardized inoculum of several types of aerobic or anaerobic bacteria (1×10⁴cells/ml) was incubated with serially diluted solutions of Example 7 (containing 0.1% CPC, or 1 μg/ml) or 2% chlorhexidine gluconate (CHX, 20 μg/mL) as a comparative example. Cells were allowed to grow in the presence or absence (growth control) of the test agents for 24 hours. The MIC for each agent was defined as the concentration that induced a 100% growth inhibition (compared to no-drug control).

A similar microdilution-based CLSI method (M27-A2) was used to evaluate the activity of Example 7 against albicans and non-albicans Candida species.

TABLE V

Chlorhexidine MIC

Example 7 MIC
(μg/mL

Organism
(μg/mL CPC)
chlorhexidine)

Example 48

S. salivarius

0.98
19.6

Example 49

P. gingivalis

0.98
19.6

Example 50

S. pyogenes

0.98
19.6

Example 51

S. pneumonia

0.98
19.6

Example 52

F. nucleatum

1.95
19.6

Example 53

S. mutans

1.95
19.6

Example 54

S. aureus

3.91
19.6

Example 55

Y. enterocolitica

3.91
19.6

Example 56

S. oralis

500
19.6

Example 57

S. mitis

500
19.6

Example 58

C. albicans

0.25
19.6

Example 59

C. krusei

0.06
19.6

Example 60

C. tropicalis

0.06
19.6

Example 61

C. glabrata

0.125
19.6

The barrier-forming composition was also found to have potent antimicrobial activity against: MRSA, Acinetobacter baumannii, Streptococcus sanguis, S. gordonii, and Aggregatibacter actinomycetemcomitans.

As can be seen in Table V, the Example 7 composition exhibited potent activity against many aerobic and anaerobic bacteria, as well as the fungi.

The MIC of the Example 7 barrier-forming composition against S. oralis and S. mitis was noticeably elevated (500 μg/mL) compared to other organisms. It is interesting to note that S. oralis and S. mitis are normal commensals of the oral cavity. Activity of the commonly used antimicrobial chlorhexidine (2% solution) was also determined by the same method. Table V shows the MIC of the Example 7 barrier-forming composition and chlorhexidine (2% solution) as a comparative example against various microorganisms.

Taken together, these results demonstrate that Example 7 possesses potent activity against pathogenic bacteria and fungi commonly isolated from the oral cavity. This activity was more potent than that observed for chlorhexidine.

A similar activity profile was observed for the barrier-forming compositions of Examples 10 and 11.

Example 62

As a further comparison, published data shows that the tested barrier-forming composition has a better or at least equivalent MIC compared to CPC alone (i.e. not in a composition according to the barrier formulation disclosed herein). See Frank-Albert Pitten and Axel Kramer, “Efficacy of Cetylpyridinium Chloride Used as Oropharyngeal Antiseptic,”Arzneim.-Forsch./Drug Res. 51 (II), pp 588-595 (2001), which is incorporated herein by reference. The data varies based on the microorganism tested, but, for example, CPC (alone) against S. mutans has an MIC of 5.0-6.25 μg/mL, which is much less effective than the 1.95 μg/mL reported in Example 53. This was an unexpected result since CPC has the risk of losing its activity when mixed with other excipient chemicals in a formulation. See Department of Health and Human Services (Food and Drug Administration) (1994) Oral Health Care Drug Products for Over-the-Counter Human Use; Tentative Final Monograph for Oral Antiseptic Drug Products. Proposed Rules (21 CFR Part 356, Docket No. 81N-033A, RIN 0905-AA06). Federal Register 59:6084-124.

Examples 63-69
Duration of Antimicrobial Activity of Barrier-Forming Compositions In Vitro
Determination of Post-Antimicrobial Effect (PAE)

The PAE of Example 8 against several microorganisms was evaluated in Examples 63-68. Control Example 69 was also provided. Several microorganisms were exposed to Example 8 (at a concentration equal to the MIC) for 1 min followed by three washes to remove residual formulation. The treated cells were then spread on agar medium plates, which were incubated at 37° C., and the time taken for the cells to regrow was determined. PAE was expressed as the time (in hours) for which growth inhibition (%) was maintained by the Examples 63-68, compared to the untreated control Example 69.

As shown in FIG. 14, Example 8 exhibited a PAE ranging between 4 hours to 24 hours, depending on the organism tested (S. aureus, S. pneumonia, S. gordonii, S. sanguis, S. salivarius, and S. mitis). Similar activity of Example 8 was observed against Candida (data not shown). Other Example barrier-forming compositions exhibited similar PAE against microorganisms.

Example 70

Testing of PAE for the Example 7 barrier-forming composition against S. mutans compared to a similar comparative Example with lower CPC content of 0.7% showed that the PAE of Example 7 was 24 hours, while that of Comparative Example 70 was 6 hours. Thus demonstrating that Example 7 exhibits greater prolonged antimicrobial activity than comparative Example 70, and that additional amounts of CPC have more than a simple additive effect on anti-microbial activity.

Examples 71-76

Scanning electron microscopy was also used to show that treatment of S. sanguis, (Example 71), S. oralis, (Example 72), and C. albicans (Example 73) with the composition of Example 3 resulted in destruction of cellular integrity.

In Examples 71-73, cells were grown in the presence of Example 3 for 24 hours. Next, the cells were washed to remove residual formulation, dehydrated by passing through a series of alcohol solutions (10% to 100%, v/v) and processed for SEM analysis. Control Examples 74-76 differed from Examples 71-73 in that they were not grown in the presence of Example 3.

The SEM photos showed that unlike untreated control Examples 74-76, which demonstrated healthy intact cells (FIG. 15 A, C, E), microbes exposed to the Example 3 barrier-forming composition were deformed, collapsed, and exhibited total destruction of cellular integrity with clear evidence of leakage of cytoplasmic material. (FIG. 15, B, D, F).

Examples 77-79

Since biofilms are precursors to certain infectious diseases, in Examples 77-79, experiments were performed to determine whether the barrier-forming compositions can prevent formation of biofilms by bacteria and yeasts. Biofilms were formed using an in vitro model. See Chandra et al. “In vitro Growth and Analysis of Candida Biofilms” Nature Protocols 3(12): 1909-1924 (2008).

In Examples 77-79 a standard biofilm model was employed to determine whether the Example 3 barrier-forming composition exhibits activity against bacterial and fungal biofilms. In Examples 77-79, three different microorganisms (C. albicans, S. oralis, and S. salivarius) were adhered on substrate for 90 minutes to allow biofilms to form to adhesion phase. Next, discs containing the adherent bacteria were incubated for 15, 30 or 60 minutes with 50% concentration of Example 3 (1:1 dilution with appropriate medium). Following incubation, biofilms were scraped, spread on culture media, incubated and colony forming units (CFUs) were determined. Media diluted with phosphate buffered saline (PBS, 1:1) were used as a control. Table VI reports data at 0 (Control), 15, 30, and 60 minutes.

TABLE VI

Effect of Barrier-forming composition on Early Phase Biofilms (log CFU)

Example 77
Example 78
Example 79

Exposure time

C. albicans

S. oralis

S. salivarius

Control
5.44
3.25
3.16

15 min
0
0
0

30 min
0
0
0

60 min
0
0
0

FIG. 16 also reports data on Examples 77-79 as a graph of % inhibition versus growth control. These results showed that Example 3 barrier-forming composition inhibited bacterial and fungal microbes with an MIC of 0.2% against biofilms formed by S. salivarius, S. oralis, or C. albicans.

Examples 80 and 81

In Example 80 we evaluated the effect of 1 minute exposure of C. albicans early phase biofilms to Example 3, and found that even with an exposure for as short a time as 1 minute, it was able to inhibit biofilm formation (FIG. 17). Example 81 was an untreated control sample.

Examples 82-84
Ability of Barrier-Forming Composition to Treat Mature Biofilms

To determine whether the barrier-forming composition can treat biofilms, we evaluated its activity against fully formed mature biofilms. Biofilms were grown to mature phase, and then exposed to Example 7 for 2 or 4 hours, and the resulting CFUs were determined. A composition that causes at least 2-log reduction in microbial CFUs compared to untreated cells is considered to be effective against microbial biofilms.

As shown in Table VII, exposure to Example 7 resulted in complete eradication of biofilms formed by C. albicans and S. oralis, and a 3.4-log reduction in CFUs for biofilms formed by S. salivarius compared to the untreated control (log CFU=3.95 vs. 7.36, respectively).

TABLE VII

Effect of Example 7 on mature biofilms (log CFU)

Exposure
Example 82
Example 83
Example 84

time

C. albicans

S. oralis

S. salivarius

Control
5.60
7.40
7.36

2 h
0
0
4.00

4 h
0
0
3.95

In summary, the results indicate that Example 7 possesses potent activity against biofilms formed by bacteria and fungi.

Examples 85-86
The Barrier-Forming Composition is Also Active Against Viruses

The activity of barrier-forming composition against viruses, including respiratory viruses (influenza virus H1N1, strain 2009/H1N1/infA) and the human immunodeficiency virus (HIV) was determined.

The barrier-forming composition inhibits the infectivity of influenza A

To evaluate the effect of the barrier-forming composition on the infectivity of influenza virus, Madin Darby canine kidney (MDCK) cells were grown to ≧90% confluence at 37° C. prior to infection. MDCK cells are used routinely for assays involving influenza viruses.

In Example 85 cell monolayers were exposed to the Example 7 barrier-forming composition. In control Example 86 the cell layers were exposed to optiMEM (+P/S, +Lglu) tissue culture media for different times: (1) T1: 30 min exposure, (2) T2: 1 h exposure, (3) T3: 2 h exposure. Next, the formulation was removed and the cell monolayers were infected with influenza virus (multiplicity of infection (MOI)=0.1). Cells that were untreated or infected immediately after exposure (TO) were used as baseline controls. Infected cells were then centrifuged, resuspended in 500 μL of growth medium, and incubated at 32.5° C. for 48 hours. Immunofluorescence microscopy (using FITC labeled anti-influenza antibody) was also used to evaluate the effect of the Example 7 barrier-forming composition on the ability of influenza virus to infect mammalian cells.

FIG. 18 shows the effect of Example 7 on cytopathic effects of influenza-infected MDCK cells (Example 85) (panels A and C), and control Example 86 (panels B and D). Images were obtained from: phase contrast (A-B), and immunofluorescence microscopy (C-D). No identifying cytopathic effect (CPE) was observed in formulation-treated cells. Untreated cells displayed typical CPE including focal rounding and degenerative changes.

The data showed that exposure of cell monolayers to Example 7 for 30 minutes, 1 hour, or 2 hours remained confluent and healthy (Example 85). In contrast, in the untreated cells and cells treated immediately prior to infection (TO) (control Example 86) demonstrated substantial cytopathic effect. As seen in FIG. 18 panel C, no fluorescence was observed in the barrier-forming composition treated cells of Example 85, while the untreated cells of Example 86 exhibited fluorescence (FIG. 18 panel D).

Further fluorescence microscopy images corresponding to Examples 85 and 86 are presented in FIG. 19.

Examples 87 and 88
Activity of Barrier-Forming Composition on Viral Load Using Quantitative PCR

FIG. 20 shows levels of influenza virus in infected treated cells (Example 87) and untreated cells (Example 88), as determined by quantitative PCR. In Example 87, cells were treated with Example 7 and in control Example 88 the cells were left untreated. Later the supernatants were collected and analyzed for the presence of virus.

Cell culture supernatants from the same assay as in Examples 87 and 88 were collected and nucleic acid extracted using QIAamp Viral RNA Kit (QIAGEN, Valencia, Calif.). Random hexamer primers (Invitrogen Carlsbad, Calif.) were used to create a cDNA library for each specimen. Reverse transcription reactions were performed with M-MLV RT (Invitrogen, Carlsbad, Calif.) according to the manufacturer's specifications. Quantitative analysis was performed on a StepOne Plus Taqman Real Time PCR (Applied Biosystems, Branchburg, N.J.) using TaqMan Universal PCR Master Mix (Applied Biosystems, Branchburg, N.J.), 2 μl of cDNA sample, and primers/probes targeting the influenza matrix gene. A reference standard was prepared using a cDNA fragment of the H1N1 matrix gene and human RNAse P amplified by conventional RT-PCR, gel purified (QIAquick, Qiagen, Valencia, Calif.), and quantified using a spectrophotometer (Beckman Coulter, Brea, Calif.).

As shown in FIG. 20 and Table IV, the Example 87 cells treated Example 7 for 30 min or 60 min did not have detectable influenza at 48 hours post infection. Moreover, treatment with Example 7 for 2 hours resulted in a 6-fold decrease in viral load, compared to the untreated control or those treated immediately prior to infection (Example 88).

TABLE VIII

Example 87
Example 88 (control)

30 min
0
192000

60 min
0
79800

120 min
23400
143000

Examples 89-91
Barrier-Forming Composition has Direct Antiviral Effect Against Influenza Virus

To determine whether the barrier-forming composition has direct antiviral activity against influenza virus, we infected African Green Monkey Kidney (CV-1) cells (grown in 24-well plates to 90% confluence) with influenza virus that was pre-treated with Example 7. CV-1 cells are routinely used a highly susceptible substrate for diagnosis and study of viruses.

In Examples 89-91, a standardized amount of influenza (0.1 MOI) was pretreated for 5 minutes at room temperature with: (1) Example 7 (to form Example 89), (2) control Example 6, a compound without CPC but with preservatives (to form Example 90), and (3) control Example 5 placebo alone (a compound without CPC and preservatives) (to form Example 91). After the 5 minute incubation virus/drug mix was diluted by an additional equal volume with optiMEM (+P/S, +Lglu) to dilute out the treatment compositions.

In Examples 89-91, CV-1 cells were prepared as described in above. The Example 89-91 treated and untreated viruses were then inoculated onto the cells as described above.

Influenza viral load was determined by real time PCR as described above. The data as shown in FIG. 21 showed significant decrease in viral load for influenza virus pretreated with the Example 7 barrier-forming composition containing the antimicrobial agent CPC (Example 89), compared to those containing only the barrier-forming composition and/or preservative but no CPC (Examples 90 and 91). Pre-treatment of virus with Example 7 exhibited significant decrease in viral copies, compared to formulations with no CPC.

These results demonstrate that the Example 7 barrier-forming composition possesses direct antiviral activity against influenza virus that is not inherent in Examples 5 and 6.

Examples 92 and 93

In Examples 92 and 93, the barrier-forming composition's ability to inhibit the infectivity of influenza A (2009/H1N1/infA) was tested. African Green Monkey Kidney (CV-1) cells were grown in 24-well plates to 90% confluence. Next, the barrier-forming composition, Example 7, was applied to the cells (20% Example 7, 80% OptiMeM, working CPC concentration of 0.02%.) in Example 92. Each time point matched with control Example 93 (No barrier-forming composition applied, 100% OptiMeM). The barrier-forming composition was allowed to dwell on the surface for 30 minutes, and then removed from the ceil monolayer. Cells were thoroughly washed twice with sterile optiMEM (+PfS, +Lglu). Influenza was inoculated at MOi=0.1 at 30 minute intervals from TO through T+6 hours. Following infection, cells were then centrifuged @ 2200 rpm×30 minutes and 500 μl of optiMEM (+P/S, +Lglu, 2 μg/ml trypsin (sigma-Aldrich, St Louis, Mo.)) was applied. Infected cells were grown at 32.5° C. for 96 hours at 5% CO₂. The influenza viral load was determined by real time PCR.

As shown in FIG. 22, pre-treatment of host monolayers with glycerine-xanthan gum formulation results in inhibition of viral infection by up to 84.93% compare to untreated controls. The fact that inhibition of viral infection was observed in host cells despite removal of the barrier-forming composition demonstrates that the barrier-forming composition formed a protective barrier on host cells, which prevented viral invasion for at least 6 hours.

FIG. 1B may be referred to as a possible mechanism accounting for the inhibition of infection.

Examples 94-96
Barrier-Forming Composition Exhibits Activity Against HIV

Examples 94-96 determined whether the barrier-forming composition possessed activity against HIV. Host MT mammalian cells were plated into 96-well round bottom plates at a density of 15,000 cells/well in RPMI/10% FBS/PS. The next day (Day 2), virus was pretreated with control Example 5 (to form Example 94), control Example 6 (to form Example 95), or Example 7 (to form Example 96) for 5 minutes and added to cells. After 24 hours of exposure to formulation, the MT (macaque) mammalian cells were washed 3 times with phosphate buffered saline (PBS) and fresh media was replaced. Supernatant (10 μL) was collected post-treatment on Days 1, 2, 5, 6, 7, and 9, and the viral load was determined by reverse transcriptase (RT) activity. FIG. 23 shows a graph of the viral copies per mL for each of Examples 72-74 over a 9 day span.

The results showed that Example 7 in Example 96 exhibited anti-HIV activity at all time points monitored post-treatment.

The control Example 5 or control Example 6 without CPC and/or preservative in Examples 94 and 95 exhibited only minimal anti-HIV activity.

In summary, our findings demonstrate that the barrier-forming composition Example 7 containing CPC exhibits long-lasting antiviral activity against HIV.

Example 97

Representative organisms viral lesions are important infections in different mucosal tissues. In Example 97 an experiment was performed to determine whether the barrier-forming composition exhibits activity against the common oral Epstein-Barr virus (EBV). Western blotting was used to evaluate the ability of the Example 8 barrier-forming composition to degrade lytic viral protein EAD (indicating inhibition of viral replication).

In Examples 97, EBV-infected gastric epithelial cells were exposed to different dilutions (1:16, 1:32 and 1:64) of Example 8, and the presence of EAD protein was detected using specific antibodies. Presence of cellular β-actin was used as an indicator of epithelial cell integrity. As shown in FIG. 24, 1:64 dilution of Example 8 degraded EAD without affecting cellular actin. These results demonstrate that Example 8 specifically inhibits viral replication, and as such, is an effective anti-viral and useful for prevention of viral infection.

Examples 98-100
Duration of Anti-Microbial Barrier Versus Commercial Mouthwash Product

To determine the duration for which the barrier-forming composition can maintain the antimicrobial activity, bacteria and fungi were exposed to an EHOM of Example 2 that was treated with the barrier-forming composition of Example 7 in a well and an EHOM of Example 2 that was treated with a comparative commercial product in a well for 2 minutes. The bacterial and fungal microbes were overlaid on top of the control untreated EHOM (Example 98) and the treated EHOMs (Example 99 and Comparative Example 100). Next the residual (flow-through) solution was removed from the bottom well (lower chamber of the EHOM model) and spread onto agar medium plates. FIG. 7 depicts this test method for further clarity. These plates were then incubated at 37° C., and the number of microbial cells (colony forming units, CFUs) growing after 24 hours were counted.

In control Example 98 an untreated EHOM was tested. In Example 99 S. mitis bacteria was overlaid on the barrier-forming composition as described above. Example 100 is a comparative example showing the activity of commercially available LISTERINE (containing ethanol (26.9%), menthol, thymol, methyl salicylate, and eucalyptol) against S. mitis bacteria. Table IX shows the results.

TABLE IX

CFUs of S. mitis bacteria in flow through

liquid from EHOM

Time post-
Example 98

Example 100

exposure
(control)
Example 99
(comparative)

2 hours
1150000
5820
780000

4 hours
1400000
5500
800000

6 hours
1600000
6000
840000

Examples 101-103

In Examples 101-103, the same procedure of Examples 98-100 was performed except Candida albicans fungus was tested on the barrier-forming composition as described above. Table X shows the results. Example 103 is comparative, showing the activity of commercially available LISTERINE.

TABLE X

CFUs of Candida albicans in flow through

liquid from EHOM

Time post-
Example 101

Example 103

exposure
(control)
Example 102
(comparative)

2 hours
1150000
12000
124000

4 hours
2900000
12000
252000

6 hours
3900000
13000
350000

The data further showed that Example 7 barrier-forming composition maintained activity for up to and including 24 hours. Taken together, these results showed that unlike LISTERINE, the Example 7 barrier-forming composition continued to maintain an intact barrier on EHOM tissues for up to and including 24 hours.

Examples 104-153

To identify further examples of concentrations of glycerin and xanthan gum that can form a barrier effective in preventing the passage of microorganisms, different concentrations of the gum xanthan gum and humectant glycerin were tested (5%-95% glycerin; 0.005%-0.5% xanthan gum) singly and in combination using an in vitro filter insert-based barrier model. FIG. 33 shows the general test method used for Examples 104-153.

Filter inserts of 3 μm and 8 μm diameter pore size were used for testing the passage of bacteria (Streptococcus salivarius) and fungi (Candida albicans), respectively. Glycerin or xanthan gum or their combinations (100 μL aliquots) were overlaid on the surface of the filter to form a barrier. The filter had a diameter of 24 mm. Thus, the film had a thickness of approximately 0.01 mm on the filter, mimicking a value in the range of thicknesses of the composition film when applied in a therapeutically effective amount to the mouth. Next, 5×10⁴cells of either bacteria or fungi were applied on top of the formed barrier in the filter inserts. Next, we placed these filter inserts on the surface of agar medium (Brain Heart Infusion (BHI) medium for bacteria, Sabouraud Dextrose (SD) medium for fungi) in 6-well plates. The plates along with the filter inserts were incubated overnight for 24 hours at 37° C.

The plates were monitored for the presence of bacterial or fungal growth CFUs (colony forming units) in the agar medium as well as in the filter insert. Microbial growth in the filter insert only, but not in the agar medium, demonstrated that an effective barrier was formed on the filter, which prevented passage of microorganisms. Conversely, growth in the agar medium around the filter insert suggested that the tested agents failed to form an effective barrier, allowing the organisms to go through the filter.

The results reported in Table XI showed that glycerin was able to form a barrier at concentrations greater than or equal to 55%, when tested alone. In contrast, xanthan gum alone did not form a barrier at any of the concentrations tested (ranging from 0.005% to 0.4%). However, it was observed that when combined with 0.01% xanthan gum, a barrier was formed at glycerin concentrations 7%, 45%, 55%, and 65%. Furthermore, combination of 0.4% xanthan gum with glycerin concentrations of 7%, 15%, 25%, 35%, 45%, 55%, and 65% also formed a barrier. Therefore, specific combinations of glycerin and xanthan gum were identified that can form a barrier that prevents passage of microorganisms in an in vitro filter insert-based model.

TABLE XI

Examples
Example
Examples
Examples
Examples
Examples
Examples

104-112
113
114-121
122-129
130-137
138-145
146-153

Xanthan Gum (%)

0
0.005
0.01
0.05
0.1
0.2
0.4

Glycerine (%)
0
No
No
No
No
No
No
No

5
No

No
No
No
Yes
No

7
No

Yes

Yes

15
No

No
No
No
Yes
Yes

25
No

No
No
No
Yes
Yes

35
No

No
No
No
Yes
Yes

45
No

Yes
No
No
Yes
Yes

55
Yes

Yes
No
No
Yes
Yes

65
Yes

Yes
No
No
Yes
Yes

‘No’: no barrier formed;

‘Yes’: barrier formed

Microbial cells retained by the compositions of Examples 104-153 formed on filter inserts were trapped by the barrier, and were viable, thus demonstrating that the formed barrier does not have an inherent antimicrobial property without an antimicrobial agent. In other words, the microbes retained in the barrier were still active and could pose a threat to infection; for example, if they are freed from the barrier by abrasion or after the barrier loses its integrity.

It should be noted that an effective barrier may be formed at lower concentrations of glycerine and/or xanthan gum when an effective antimicrobial is added. This is because the antimicrobial and barrier act in tandem to stop and/or kill the harmful microbes.

Examples 154-157

In addition to the Examples above on EHOM samples, further testing was performed to further demonstrate the barrier-forming composition (a) does not damage the host tissues, and (b) is able to prevent microbial invasion into the human mucosal tissues. These criteria were tested for two representative combinations (glycerine:xanthan gum; 7%:0.01% and 35%:0.4%), selected based on the in vitro results of Examples 104-153, because they successfully formed a barrier. Formulations containing these two combinations were tested using the EHOMs of Example 2 that mimic the mucosal lining.

The EHOMs were treated with the various formulations of Examples 3 and 4 in either 1% (Examples 154 and 155) or 5% (Examples 156 and 157) dilutions in normal saline [0.9%]) for about 2 minutes to form Examples 154-157. Each EHOM was covered with 300 μL of one of the tested formulations and left under the sterile hood for 10 minutes at room temperature (25° C.). At the end of the contact period, tissues were washed twice with culture medium to remove the formulations.

Example 158

After treatment with Examples 3 and 4, the EHOMs were then examined for possible macroscopic tissue damage (presence of holes). Tissue damage was also investigated by histological analyses. For this purpose, biopsies were taken from each EHOM and fixed with 4% paraformaldehyde solution and then embedded in paraffin. Thin sections (4 μm) were stained with eosin-hematoxilyn and mounted with a coverslip in 50%-glycerine mounting medium, observed through an optical microscope, and photographed. The treated EHOMs and a control untreated EHOM were examined for macroscopic tissue damage and structural changes. There was no visible damage (holes) in the untreated or treated tissues at a magnification of ×500 in five different EHOMs tested.

Example 159
Evaluation of Barrier-Forming Composition Causing Damage to Host Cells (Cytotoxicity Assay)

The Example 156 and 157 EHOMs that were treated with the 5% dilution of Examples 3-7 of the barrier-forming composition were then over-layered with 1×10⁶cells of either C. albicans or S. mutans in a volume of 100 μl. The EHOMs were then placed on air-liquid culture plates and incubated for 24 hours in 5% CO₂humid atmosphere at 37° C. Following this incubation period, aliquots of the culture supernatant were collected and subjected to a Lactate dehydrogenase (LDH) cytotoxicity assay (Promega, Madison, Wis., USA), as per the manufacturer instructions. 50 μl of each supernatant were transferred to a sterile 96-well flat-bottom plate. Each well was supplemented with 50 μl of reconstituted substrate mix, and the plate was incubated for 30 minutes at room temperature in the dark. To stop the reaction, 50 μl of stop solution was added to each well. Next 100 μl of the mixture was transferred to a 96-well flat-bottom plate, and the absorbance was read at 490 nm with an X-Mark microplate spectrophotometer (Bio-Rad, Mississauga, ON, Canada). LDH was assessed using LDH cytotoxicity assay. Data are means±SD. No significant difference between EHOMs treated with the Example composition and an untreated, uninfected EHOM control was noted. FIG. 25 graphically shows the results of these tests on an untreated control example, and those treated with Examples 5, 6, and 7. Similar results were obtained for the EHOMs treated with Examples 3 and 4. The tested Examples all maintained the integrity of the mucosa.

Example 160
Effect of Barrier-Forming Composition Formulations on Gingival Cell Growth/Migration

Wound repair assays were performed. Briefly, oral (gingival) epithelial cells (1×10⁴) and fibroblasts (1×10³) were seeded into wells of 6-well plates and grown in appropriate culture medium. Upon confluency, wounds were made in the confluent monolayer of each well using a 200 μL pipette tip. Cultures were then exposed to 1 and 5% by weight dilutions of Examples 3 and 4 for about 2 minutes. Following exposure, the formulations were washed out twice with warm sterile culture medium, and then cell cultures were over layered with DEM for fibroblasts and with DEM for fibroblasts and DMEH for epithelial cells, and cultured for 6 and 24 hours in a 5% CO₂humid atmosphere at 37° C. Wound repair/cell migration was ascertained using optical microscope, and digital photographs were taken (Nikon, Coolpix 950). FIG. 26 shows representative photographs of a wounded oral epithelial cell culture treated with Example 3 (5% dilution) for 10 minutes, immediately after the wound (panel A), after about 6 hours (panel D), and after about 24 hours (panel E). Panels B and C show an equivalent wound on an untreated control confluent culture of oral epithelial cells after about 6 hours and about 24 hours, respectively. Similar results were seen for the cell culture treated with Example 4.

The percentage of wound closure (cell growth/migration) was calculated by comparing relative wound areas before and after exposure to the formulations using formula I stated above.

The epithelial cells were small and cuboidal in shape in both treated and untreated cultures. Similar results were observed for scratch wound model using fibroblasts (data not shown). Taken together, this data indicate that example compositions are not toxic and do not negatively impact cell growth/migration and wound healing.

Example 161
Glycerine-Xanthan Gum Formulations Form a Coating on the Human Oral Mucosa

To determine whether glycerine-xanthan gum formulation can form a coating on the human oral mucosa, we spiked the Example 7 formulation with Gentian Violet (GV) as a marker dye. The spiked product (750 μL) was sprayed onto the oral cavity of human volunteers. Post-application, the oral cavity was inspected for staining, and the images were captured using a digital camera. As shown in FIG. 27, the formulation stained both cheeks and the dorsal/ventral surface of the tongue.

Examples 162 and 163
Exposure of Microbes to Barrier-Forming Composition Inhibits Cell Growth
Time-Lapse Microscopy

To determine the inhibitory activity and duration for which barrier-forming compositions exhibit activity against microbes, time-lapse analysis was performed on cells exposed to the barrier-forming composition, compared to untreated bacteria and fungi.

In Example 162, S. mutans microbial cells were exposed to Example 7 for one minute, washed to remove any residual agent, and allowed to grow in a petri-dish containing fresh growth medium. Growth of organisms at 37° C. was monitored for a 6 hour period, and photomicrographs were taken every 20 minutes over the 6 hour incubation period using a camera connected to the microscope.

In control Example 163 the same procedure was followed with untreated cells.

As shown in FIG. 28, in contrast to the untreated bacteria, where cells reached confluence by 6 hours, microbes treated with the Example 7 barrier-forming composition failed to regrow during the same time period post-exposure. Similarly, exposure of Candida cells to the Example 7 barrier-forming composition completely inhibited growth during the incubation period (data not shown).

These results further confirmed that the barrier-forming composition possesses prolonged antimicrobial activity.

Examples 164-166
In vivo Study
Barrier-Forming Composition (Example 7) Lowers the Oral Microbial Load in Humans: Short- and Long-Term Activity

Short-Term Activity

The duration of activity of Example 7 was determined in healthy individuals by evaluating the effect of a single application on microbial burden of the oral cavity. In Examples 164-166, three healthy individuals (over 18 years of age, healthy mouth) were enrolled with informed consent, and asked to apply a single application of the composition of Example 7 on their cheeks. A single application was defined as three sprays of 0.25 ml each in volume. Next, swabs were collected from these individuals at baseline (pre-treatment), 1 hour, 2 hours, and 6 hours post-treatment. Swabs were cultured on agar media plates specific for aerobic or anaerobic organisms, incubated for 24-28 hours at 37° C., and the number of CFUs were counted. Effect of Example 7 on microbial burden was determined (CFUs), and percentage inhibition was calculated for each post-exposure time point relative to the baseline (0 minutes) CFUs.

The results showed that application of Example 7 led to consistent reduction in microbial load for up to 6 hours (See FIG. 29A, which shows CFUs of a representative tested individual. Treatment with the barrier-forming composition resulted in 69% to 96% reduction of the microbial burden in the oral cavity (See FIG. 29B, which shows a representative individual's reduction in microbial load.)

Examples 167-169
Long-Term Activity

The activity of the barrier-forming composition over a 5-day period against oral microbes was evaluated. In Examples 167-169, three healthy individuals were enrolled, and asked to apply a single dosage (three sprays 0.75 mLs total) of Example 7 three times daily (approximately 9 AM, noon, and 3 PM) for a 5-day period (representing a typical 5-day work-week). Swabs were collected from these individuals at baseline (before application on day 1) and at the end of the day on each day during the 5-day period. Collected swabs were cultured on agar media plates, incubated for 24-28 hours at 37° C. and at 5% CO₂humidity, and the number of CFUs were counted.

The effect of the Example 7 barrier-forming composition on microbial burden was determined (as median CFUs for the three subjects), and percentage inhibition was calculated for each post-exposure time point relative to the baseline (0 min) CFUs. FIG. 30 shows these results in a graph of CFUs versus time (FIG. 30A) and reduction in microbial load versus time (FIG. 30B). Examples 167-169 demonstrate that application of Example 7 over 5 days led to consistent reduction in microbial load over the 5-day test period (FIG. 30A). Treatment with the Example 7 barrier-forming composition resulted in 65%-88% reduction of the median microbial burden in the oral cavity of the study participants (FIG. 30B).

Examples 170-198

In a clinical study, twenty-nine healthy individuals were enrolled after informed consent. Baseline information was recorded (age in years, gender, ethnicity, and date of enrolment). Oral examination of the mouth was undertaken, and the inside of the mouth (cheek) was swabbed with a sterile culture swab. Baseline oral swab samples were cultured to determine bacterial load prior to study. In Examples 170-198, each of the twenty-nine participants were given a spray bottle containing the barrier-forming composition of Example 7 and instructed to spray the inside of their mouth for a total volume of 0.75 ml, then swish for 30 seconds and swallow. Two groups of approximately equal number of participants were tested. One group used the example barrier-forming composition every two hours, three times a day, for five days (a typical work week). The other group used the example barrier-forming composition every two hours, four times a day, for five days (a typical work week). No substantial difference was noted in the two groups. Swabs were collected on days 1, 2, 3, and 5 at the end of the day (8 hours after the first administration of the barrier-forming composition) and cultured on media specific for aerobic and anaerobic bacteria. Data were presented as number of microbes: total, aerobic and anaerobic. FIG. 31 shows a graph of total microbial load and breaks down the total into aerobic and anaerobic counts from just prior to treatment and on day 5 of treatment. FIG. 32 shows graphs of microbial load over the 5 day period in oral samples obtained from three representative study participants.

Overall, the in vivo testing showed that the barrier-forming composition exhibits antimicrobial activity against oral microbes, as measured by reduction in the levels of these organisms, over both short- and long-term duration.

The data showed that treatment with the barrier-forming composition over a 5-day period resulted in reduction in the oral microbial load, for total microbes, aerobic and anaerobic organisms.

Example 199-205
Identification of Additional Humectants for Forming a Barrier to Prevent Microbial Penetration

In Example 199 an in vitro filter insert-based model (see FIG. 33) was used to test different humectants at different concentrations.

Six compositions were prepared according to Table XII based on the mixing procedures used for Examples 3-8.

TABLE XII

Ex.

Ex.

199
Ex. 200
Ex. 201
Ex. 202
Ex. 203
Ex. 204
205

Xanthan
0.4
0.4
0.4
0.4
0.4
0.4

Gum

Glycerin
4.5
4.5
4.5

4.5

Sorbitol

4.5
4.5
4.5

4.5

Xylitol

4.5
4.5
4.5

4.5

Next, 100 μL of Examples 199-205 were placed into filter inserts (pore size 0.8 μm diameter, that allows both bacteria and fungi to pass through) and allowed to form a layer. Next, organisms were overlaid on the layer formed by the test solutions. The filter inserts containing the layer of test solutions and microorganisms were then placed on the surface of agar media plates and incubated for 24 hours at 37° C. After the incubation period, the agar media plates were evaluated for growth on filter insert and in the agar media. Growth on filter insert but no growth in agar media indicated that the test solution formed a barrier, which prevented the microbes from passing through. In contrast, microbial growth in the filter insert as well as the agar media indicated that no such barrier was formed.

The results showed that each of the xanthan gum-based solutions containing the tested humectants (singly or in combination) formed intact barriers on the filter insert that prevented the passage of microorganisms into underlying agar medium.

Example 206-213
Determination of the Solubility Limits of Xanthan Gum

To determine the solubility of xanthan gum, it was mixed at different concentrations in water and the solubility observed by monitoring the presence or absence of clumps and free flow of the mixture. Table XIII reports the results and concentrations.

TABLE XIII

Xanthan Gum

Example
Concentration
Solubility

206
0.40%
free flowing viscous solution

207
0.45%
some clumps, viscous solution

208
0.5%
more clumps, viscous solution

209
0.6%
clumps, more viscous than above

210
0.7%
clumps, more viscous than above

211
0.8%
Extensive clumps, highly viscous solution,

no free flow

212
0.9%
Extensive clumps, highly viscous solution,

no free flow

213
1.00%
Extensive clumps, highly viscous jelly,

no free flow

We found that when mixed at 0.4%, xanthan gum formed a free-flowing solution (Table XIII). In contrast, mixtures containing 0.45% or 0.5% xanthan gum formed a viscous fluid but contained small clumps. The extent of clumps increased with increasing concentration of xanthan gum (0.6% and 0.7%). At concentrations ≧0.8%, xanthan gum mixture contained extensive clumps, with a jelly-like consistency and no free flow.

Example 214
Comparison of Cationic CPC in Barrier-Forming Composition with Neutral Antimicrobial Agent in Barrier-Forming Composition

In Example 214, the formulation of Example 7 was made, except the neutral agent Citral was used instead of CPC. The antimicrobial activity of formulations containing CPC (0.1%) or Citral (0.5%) against Streptococcus was ascertained. The assay described above in Examples 48-61 was used to perform these studies.

The results showed that the formulation containing citral exhibited antimicrobial activity (MIC=12.5%). However, activity of formulation containing citral was significantly less potent than that containing CPC (MIC=0.098%).

Example 215
Physico-Chemical Testing of Hydrophobicity and Comparison

In Example 215 thin layer chromatography analysis was used to compare the hydrophobicity of Example 7 with a hydrophobic composition. The hydrophobic composition was comprised of the components in Table XIV.

TABLE XIV

Wt %

Glycerin
7

Sorbitol
5

Poloxamer 338
1

PEG 60 Hydrogenated castor oil
1

VP/VA copolymer
0.75

Sodium benzoate
0.5

Cellulose Gum
0.2

CPC
0.05

Methyl Paraben
0.05

Propyl paraben
0.05

Sodium Saccharin
0.05

Xanthan Gum
0.01

Disodium Phosphate
0.006

Flavoring and coloring agents
0.121

*the remainder of the composition was purified water

10 μL of Example 7 and the hydrophobic composition were deposited on pre-made TLC plates (at a distance of 2 cm from the bottom edge). The spots were air-dried for 5 minutes, and the plates were placed in a TLC chromatography jar containing water as a solvent. The TLC system was allowed to run until the solvent front reached the top edge of the plate. Plates were removed and the solvent and sample fronts were marked. The Relative Front (Rf) values were calculated for the two samples using the formula II:

Rf=Distance travelled by spot/Distance travelled by solvent front II.

The results showed that the Rf value for the hydrophobic composition and Example 7 were 0.33 and 0, respectively, indicating that the hydrophobic composition was highly miscible in water. In contrast, Example 7 did not exhibit any mobility in the aqueous solvent, demonstrating that this formulation is hydrophobic or not hydrophilic.

Example 216

A barrier-forming composition was made by mixing the components according to Table XV below in water to form a solution. A eucalyptol component was also included in an amount of 5× per the Homeopathic Pharmacopeia, but also did not affect the test results, other than demonstrating that the composition still works with this component added into it. All percentages are by weight.

TABLE XV

Antimicrobial
Humectant
Gum

(CPC)
(Glycerin)
(Xanthan Gum)

Example 216
0.01%
35%
0.4%

Examples 217-219

The barrier-forming composition was also shown to have effectiveness in killing allergy causing molds. MIC tests were performed on a polystyrene plastic surface.

In Example 217 the barrier-forming composition of Example 216 was tested to determine its MIC against Stachybotrys MRL 9740. The Example 7 composition had an MIC of 0.06 micrograms/ml.

In Example 218 the barrier-forming composition of Example 216 was tested to determine its MIC against Aspergillus fumigatus 18748. The Example 7 composition had an MIC of 0.49 micrograms/ml.

In Example 219 the barrier-forming composition of Example 216 was tested to determine its MIC against Cladosporium. The Example 7 composition had an MIC of 0.39 micrograms/ml.

Because Stachybotrys and Aspergillus fumigatus are mold-causing organisms, these examples further support the embodiment wherein the barrier-forming composition is applied to surfaces to prevent or treat mold growth or discoloration.

Examples 219-224

In Examples 219-224 the effect of barrier-forming composition on MRSA biofilm formation on a silicone elastomer disc surface was evaluated.

In Examples 219-221, three silicone elastomer discs with a 1 cm diameter were pre-sprayed with 0.25 mL with the Example 7 barrier-forming composition for 60 min and incubated at 37° C. In Examples 222-224 a control example was performed by treating a silicone elastomer disc with an equivalent amount of a phosphate-buffered saline (PBS) for 60 minutes and incubated at 37° C.

The Example 219-224 pretreated discs were each submerged in 4 mL MRSA suspension (1×10⁷cells/mL), and incubated at 37° C. for 90 min (“Adhesion Phase”). Next, the discs with adherent cells were removed and transferred to wells containing 4 mL of Brain Heart Infusion (BHI). The wells were incubated at 37° C. on a rocker for 24 hours. Biofilm formation on the discs was evaluated by quantitative culturing on BHI agar plates. Scanned images of the wells were recorded using a scanner.

As shown in Table XVI, pre-treatment with Example 7 barrier-forming composition prevented formation of biofilms on the disc surface. FIG. 34 shows images of colony burden in biofilms formed by MRSA on the PBS treated (A, C, E) and Example 7 treated (B, D, F) discs.

TABLE XVI

Treatment
Example
MRSA CFUs/mL

Example 7
219
0

barrier-
220
0

forming
221
0

composition

PBS
222
1.58 × 10⁸

223
1.72 × 10⁸

224
1.53 × 10⁸

Examples 225-246

The Example 7 barrier-forming composition was tested to determine its efficacy against several strains of Bordetella pertussis. In test Examples 225-235, agar-based assays were constructed in which Example 7 barrier-forming composition was incorporated in Regan-Lowe Charcoal agar BBL #297883 plates as a 64 microgram/mL dilution in water. Control Examples 236-246 were agar plates containing no Example 7 barrier-forming composition. In each of Examples 225-246 5×10⁴cells (50 uL) of Bordetella pertussis were spotted on the test surface and plates were incubated at 37 degrees C. for 24 hours. As shown in Table XVII, confluent growth was observed in control Examples 236 to 246, while no growth was observed in test Examples 236-246. The designation 4+ means luxurious growth.

TABLE XVII

Bordetella

Microbial

Example

pertussis Strain #
Growth

225
J11E
None

226
J11F
None

227
J14B
None

228
J14C
None

229
J14D
None

230
J14G
None

231
J32B
None

232
J32C
None

233
J32D
None

234
J36E
None

235
J36F
None

236
J11E
4+

237
J11F
4+

238
J14B
4+

239
J14C
4+

240
J14D
4+

241
J14G
4+

242
J32B
4+

243
J32C
4+

244
J32D
4+

245
J36E
4+

246
J36F
4+

Examples 247-252

The antiviral activity of the barrier-forming composition, Example 7 (in various diluted concentrations) was evaluated against the ATCC VR-1200 strain of rhinovirus.

Human Hepatoma (HUH-7) Cells were prepared in 24-well plates with Dulbecco's Modified Eagle Medium (DMEM) with 10% heat inactivated fetal calf serum and supplemented with L-glutamine (Lglu) and penicillin/streptomycin (P/S) (unless specified, all reagents produced by Gibco, N.Y., USA). All culture cells were grown to 90-100% confluence at 37° at 5% CO₂and then washed with OptiMEM+P/S+Lglu once prior to infection.

In Examples 247-251, the Example 7 composition was applied to cell monolayers at varying concentrations (5%, 10%, 15%, 20%, 50% diluted in 400 microliter optiMEM (+P/S, +Lglu)) for a working CPC concentration of 0.005%, 0.01%, 0.015%, 0.02% and 0.05% respectively, and was allowed to dwell for 1 hour prior to inoculation. In control Example 252 400 microliter optiMEM (+P/S, +Lglu) was applied to the cells and allowed to dwell for 1 hour prior to inoculation.

The cell monolayers were then removed from the Example 7 dilutions or control optiMEM and rhinovirus was applied at a multiplicity of infection (MOI) of 0.1. Cells were incubated with virus at 32.5° C. for 1 hour. After which the inoculum was removed and 500 μl OptiMEM+P/S+Lglu was placed on the cells. Cells were then grown at 32.5° C. at 5% CO2. After 5 days incubation, cell culture supernatants were collected for rhinovirus viral load quantification.

Rhinovirus viral titer of the Example 247-251 cell culture supernatants were measured by real time PCR. In comparison to Control Example 252 significantly decreased rhinovirus viral load was demonstrated in Example 251, which was a 50% concentration of Example 7. See Table XVIII below.

TABLE XVIII

Example
Wt. % Example 7
Amount
Viral load/mL

247
5%
303354.64
12141854.69

248
10%
5628.209
2251283.75

249
15%
92717.83
37087131.25

250
20%
8776.60
3510638.67

251
50%
0
0

252
0 (control)
95307.36
38122943.75

Examples 253 and 254

A test Example 253 was formulated with a 50% Example 7 diluted suspension (0.05 CPC concentration) in 500 microliter optiMEM (+P/S, +Lglu). A control Example 254 was formulated as a control solution with no Example 7 (500 microliter optiMEM (+P/S, +Lglu)). Examples 253 and 254 were applied the cells disclosed in Examples 246-252, but at defined intervals: T−1 hour, T−30 min, and T−0 (Immediate) prior to infection.

The cell monolayers were then removed from the Example 253 suspension and the Example 254 control solution. The rhinovirus viral particles were applied to the treated cell monolayers at a multiplicity of infection (MOI) of 0.1. Cells were incubated with virus at 32.5° C. for 1 hour. After which the inoculum was removed and 500 μl OptiMEM+P/S+Lglu was placed on the cells. Cells were then grown at 32.5° C. at 5% CO₂for 5 days. The cells treated with Example 253 and 254 were viewed daily for the presence of cytopathic effect. After 5 days incubation, cell culture supernatant was gathered for immunofluorescence and rhinovirus viral load quantification.

FIG. 35 discloses photos of cells treated with test Example 253 at FIG. 35(a) T−1 hr, FIG. 35(c) T−30 min and FIG. 35(b) T 0 (immediate). None of these photos demonstrated any cytopathic effect and healthy cells overgrew the plate. However, as shown in FIG. 35(d) the Example 254 untreated control cells demonstrated focal rounding, detachment and cell death. Cytopathic effect determination included the development of focal rounding, cell size enlargement or reduction, syncytial formation, development of multinucleated giant cells, and detachment.

Immunofluorescence was determined as follows: Virus infected cell monolayers and uninfected control were washed with sterile PBS. The cells were trypsinized, spotted upon wells on slides and fixed with acetone. The slides were tested by DFA employing FITC labeled monoclonal antibodies. An indirect immunofluorescence assay was performed using Light Diagnostics Pan-Enterovirus Detection Kit (Millipore). This detection kit is well described for having cross reactivity with rhinovirus infected cells. All antibody dwell steps occurred for 1 hour at 37° C. Following a final wash, cells were evaluated at a wavelength of 488 nm for the presence of fluorescence.

FIG. 36 discloses immunofluorescence photos of cells pretreated treated with test Example 253 at FIG. 36(d) T−1 hr, FIG. 36(b) T−30 min and FIG. 36(c) T−0 (immediate). The cells treated with Example 253 for 1 hour and 30 minutes displayed no immunofluorescence. The cells treated with Example 253 for T−0 (immediate) demonstrated scant fluorescence. However, the untreated control Example 254 showed substantial immunofluorescence suggesting profound viral infection (FIG. 36(a)).

Viral load for the samples was quantified as follows: Cell culture supernatants were collected and stored at −80° C. Nucleic acid was extracted using QIAamp Viral RNA Kit (QIAGEN, Valencia, Calif.). Random hexamer primers (Invitrogen Carlsbad, Calif.) were used to create a cDNA library for each specimen. Reverse transcription reactions were performed with M-MLV RT (Invitrogen, Carlsbad, Calif.) according to the manufacturer's specifications. Quantitative analysis was performed on a StepOne Plus Taqman Real Time PCR (Applied Biosystems, Branchburg, N.J.) using TaqMan Universal PCR Master Mix (Applied Biosystems, Branchburg, N.J.), 2 microliter of cDNA sample, and primers/probes targeting the rhinovirus polyprotein gene. A reference standard was prepared using an amplicon amplified by conventional RT-PCR, gel purified (QIAquick, Qiagen, Valencia, Calif.), and quantified using a spectrophotometer (Beckman Coulter, Brea, Calif.). The results are shown in Table XIX.

TABLE XIX

Amount
Viral load/mL

Example 253:
0
0

1 hour pretreatment

Example 253:
0
0

30 minute pretreatment

Example 253:
0
0

Immediate pretreatment

Example 254 (control)
331025.2
1.32 × 10⁸

No rhinovirus amplification was apparent at T−1 hour, T−30 min, or T−0 (immediate) timepoints at 5 day post infection. Untreated (control) cells demonstrated substantial amplification (>10⁸copies/ml) suggesting viral infection.

Number	Date	Country
61477147	Apr 2011	US
61477147	Apr 2011	US
61477147	Apr 2011	US
61725375	Nov 2012	US

	Number	Date	Country
Parent	13448926	Apr 2012	US
Child	13734363		US
Parent	13448957	Apr 2012	US
Child	13448926		US
Parent	PCT/US12/33921	Apr 2012	US
Child	13448957		US

LONG-LASTING SURFACE ANTI-MICROBIAL AND METHOD OF APPLICATION

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

US Classifications

International Classifications

Abstract

Description

Claims

CROSS-REFERENCE TO RELATED APPLICATIONS

Provisional Applications (4)

Continuation in Parts (3)