The use of antimicrobial agents to treat and reduce oral and dental disease is well documented in the professional literature. Among the most efficacious such agents is Clou, a strong oxidizing agent. C102 is well documented as a bactericidal, bacteriostatic, fungicidal, fungistatic, viricidal, and viralstatic agent. It is approved by the EPA under Registration Number 9048-3 for both water purification and food preparation and preservation because of this antimicrobial activity.

C102 is also effective in treating malodor. It achieves this efficacy by two mechanisms of action. First, C102 oxidizes the sulfide bonds of volatile and odoriferous sulfur compounds (specifically hydrogen sulfide and di-methyl mercaptan bonds) that are metabolic byproducts released by certain anaerobic bacteria documented to reside in the oral cavity; and second, its antimicrobial activity lowers the number of such microorganisms that release these volatile sulfur compounds.

However, because of its reactivity, C102 is unstable in an aqueous solution and, as such, cannot be stored at room temperature. Furthermore, since C102 is a gas, it cannot be stored in liquid form at room temperature. Thus, various references to"stabilized"C102 do not refer to gaseous C102, but rather to various chlorous acid- liberating compounds. Unfortunately, chlorous acid, even when buffered, will demineralize tooth enamel and lead to even more significant oral health problems.

One such chlorous acid-liberating compound used is sodium chlorite (NaClO2). References to the use of NaClO2 to generate chlorous acid can be found in the following papers: Chepek CW, Reed OK, Ratcliff PA, Reduction of Bleeding On Probing With Oral Care Products, Compendium 1995, 16 (2): 188-196 ; Bolin V, Ratcliff PA, Germicidal Effect Of Providone Iodide and C102 On Dental Pathogens. <BR> <BR> <P>J. Dent Res. 1987,373. IADR Abstracts; Grootveld M, Silwood C, Lynch E. , Ability of oral heathcare products to alleviate oral malodour. JDent. Res. 1997; 289: 50.

IADR Abstracts.

Compositions for treating oral malodor that employ chlorine-containing compounds are disclosed in U. S. Pat. No. 5,772, 986 to Richter; U. S. Pat. No.

5,738, 840 to Kross; U. S. Pat. No. 4,552, 679 to Schubel, and U. S. Pat. No.

4,808, 389 to Ratcliff. These references disclose various vehicles for introducing the compositions to the oral cavity, including liquid rinses, toothpastes (either with or without suds), lozenges, and sprays, as disclosed in U. S. Pat. No. 4,837, 009 to Ratcliff. The chemical mechanisms for producing compositions containing chlorous acid are varied. Some references, such as Ratcliff'215, describe the generation of chlorous acid at controlled pH levels using phosphate buffers. U. S. Pat. Nos.

4,891, 216 to Kross and 4,902, 498 to Agricola et al. disclose a two part system that generates chlorous acid by mixing a metal chlorite or other chlorous acid-liberating compound with a protic acid at acidic pH levels. U. S. Pat. No. 5,667, 817 to Kross discloses a two-stage system that requires the use of lactic acid and that results in a composition having a very disagreeable taste, making it unsuitable for use in oral healthcare. As a consequence, this product is not commercially available. However, even those products that are commercially available have significant drawbacks due to their complex chemistries, poor shelf life, poor taste, and poor efficacy.

Because chlorous acid will form C102 in aqueous media, there will be some ClO2 generated whenever chlorous acid contacts water. However, no known product is able to consistently provide therapeutic levels of ClO2 capable of reliable and efficacious use, much less to do so in the presence of H202. By employing a single- stage system, known products must control the spontaneous reaction that occurs between the metal chlorite and protic acid to form chlorous acid. For this purpose,

various buffers must be used to regulate the system's pH below the pK., of chlorous acid, resulting in a relatively steady-state generation of chlorous acid. But, for these products to have any useful shelf-life, it is necessary that their steady-state C102 levels be fairly low. Furthermore, because the reaction is unidirectional, not only is the product's shelf-life determined by the amount of metal chlorite initially present in the system and its pH, but the end-user is unable to determine how much chlorous acid is present at any given time, as the amount of chlorous acid in the system decays over time.

Commercially available, non-chlorous acid-containing products, such as Mentadent (active ingredients: baking soda and H202) and Listerines (active ingredients: thymol, eucalyptol, and methyl salicilate) oral rinses achieve plaque inhibition rates of only 15% and 30%, respectively. These levels are well below the therapeutic and prophylactic benchmark of about 50% plaque inhibition achieved by Peridexs oral rinse (active ingredient: chlorhexidine gluconate), which is available only by prescription. However, even though Peridex is the most-effective, commercially available plaque inhibitor, it has serious drawbacks that limit its applicability. Most significant among these drawbacks is severe staining to hard oral tissues observed even with brief use. In addition to being unsightly, this black staining actually creates an environment for future plaque buildup, necessitating additional follow-up office visits to be removed by abrasion of the tooth surface, which, in turn, increases the teeth's susceptibility to caries.

There is a strong commercial need for a composite formulation that overcomes these problems. First, the ideal oral care composition would be available over-the-counter yet achieve plaque inhibition rates comparable to compositions currently available only by prescription, inhibit gingival inflammation and periodontal inflammation, reduce dental caries, and control oral malodor. Second, the ideal composition should provide equivalent or superior efficacy to known compositions, yet be pleasing to the taste, thereby increasing patient compliance.

Third, the ideal composition should have a superior shelf life due to the chemical stability of the component reactants. Fourth, the composition should be easy to use <BR> <BR> and have a simple chemistry that reacts under normal environmental conditions (i e. ,

at ambient temperature and pressure and without the need for multiple steps, pressurized containers, etc. ). Fifth, the composition once fully constituted should have a pH value that is suitable for oral use and not be harmful to the teeth or oral tissues. Sixth, the ideal composition should have an effervescent quality for increased aeration of the oral tissues to facilitate the reduction of anaerobic bacteria and other microbes. Seventh, the ideal composition should not stain the teeth, provide an environment for future plaque buildup, require additional treatment, or make the teeth more susceptible to caries. Finally, the ideal composition would enable the rapid, reliable, and predictable generation in situ of therapeutic levels of Cl02.

SUMMARY OF THE INVENTION The present invention is directed to a method of treating or reducing the risk of a microbial infection using a composition made by mixing a solution of a water soluble metal chlorite with a solution of hydrogen peroxide and an oxidizing agent (other than hydrogen peroxide). The composition is especially useful as an oral rinse for treating or reducing the risk those microbial infections associated with dental disease, such as gingivitis, dental caries and oral malodor. The method comprises the steps of : a) providing a first solution comprising a water-soluble chlorite compound, said chlorite compound present at a concentration in the range of about 0.1 to 0. 5% by weight, and said first solution having an alkaline pH; b) providing a second solution comprising an oxidizing agent and hydrogen peroxide, said oxidizing agent present at a concentration in the range of about 1.0 to 10% by weight, said hydrogen peroxide present at a concentration in the range of about 0.3 to 1. 5% by weight, and said second solution having a pH in the range of about 1 to 6; c) mixing the first solution and the second solution together to provide an antimicrobial composition, wherein said composition has a pH below about 7; and d) applying the composition of step (c) to the locus of the microbial infection.

DETAILED DESCRIPTION OF THE INVENTION A preferred water soluble chlorite compound for the first solution is NaClO2.

However, other water soluble metal salts of chlorite, including other alkali metal <BR> <BR> salts and group (IT) salts (e. g. , calcium and magnesium salts) can also be used. The water soluble chlorite is present at a concentration in the range of about 0.1 to 0.5% by weight, preferably about 0.25 to 0.32%. This first solution is maintained at an alkaline pH above about 7, preferably around pH 8.

The oxidizing agent is required to be physiologically acceptable, i. e. , suitable for administration to humans. The oxidizing agent is also required to be suitable for oxidizing the chlorite to Cl02. Suitable oxidizing agents generally have a reduction potential greater than the value of 0.954 volts and include, but are not limited to: (1) inorganic oxidants (e. g. , salts of persulfate, iodate (103-), bromate (BrO3~), permanganate (MnO4~), hypochlorite (OCl-) and the like); (2) organic peroxides (e. g., alkyl and aryl hydroperoxides such as tert-butyl hydroperoxide and benzoyl hydroperoxide, and dialkyl, diaryl, or mixed alkyl aryl peroxides having the required reduction potential); (3) organic peracids (e. g., alkyl and aryl peracids, such as peracetic acid and perbenzoic acid and dialkyl, diaryl, or mixed alkyl aryl peracids having the required reduction potential); and (4) reagents which can be used to <BR> <BR> generate an oxidant in situ (e. g. , reduced species such as tetrahydropterins, reduced flavins, thiols, and the like which can react with molecular oxygen to generate a hydroperoxide species that can then react with chlorite to produce chlorine dioxide). <BR> <BR> <P>Physiologically acceptable metal persulfate salts are preferred, i. e. , metal persulfate salts suitable for administration to humans. Examples include persulfate salts of sodium, potassium, lithium, calcium and the like. Sodium persulfate (Na2S208) is a preferred oxidizing agent.

The second solution comprising the oxidizing agent (preferably sodium persulfate (Na2S208)) and hydrogen peroxide (H202) is maintained at a pH in the range of about 1 to 6. The oxidizing agent is present at a concentration in the range of about 1.0 to 10% by weight, preferably in the range of about 3 to 5%. The term "hydrogen peroxide"as used herein includes hydrogen peroxide itself as well as any

peroxide generator such as urea peroxide, zinc peroxide, calcium peroxide, sodium percarbonate and the like. The hydrogen peroxide is present at a concentration in the range of 0.3 to 1.5% by weight, preferably in the range of about 0.3 to 0.75%.

While this invention is not intended to be limited by any particular theory or mechanism of action, it is believed that the therapeutic effectiveness of the present compositions is due at least in part to the formation of chlorine dioxide (C102).

When the first and second solutions are mixed, ClO2 is formed by the oxidation of the water-soluble chlorite compound as described by the reactions shown below.

Chlorine dioxide is a known antimicrobial agent. Hydrogen peroxide is also believed to be responsible in part for the therapeutic effectiveness of the present compositions, even though it is known that in the presence of H2O2, ClO2 is reduced back to C102 and hydrogen peroxide is consumed.

These reactions are illustrated below when sodium persulfate is used as the oxidizing agent: 2 NaC102 + Na2S2o8 2 ClO2 + 2Na2SO4 ClO2 + H202 ClO2 + 2H++ °2 Applicants have found that when there is an adequate amount of oxidizing agent, as described herein, relatively small amounts of water soluble metal chlorite and hydrogen peroxide may be used together to provide an effective oral rinse.

The amounts of water soluble metal chlorite compound, hydrogen peroxide and oxidizing agent ingredients described above are based on mixing the first and second solutions in approximately equal volumes. These volumes may be varied to adjust for variations in the concentration of the ingredients in the first and second solutions. Accordingly, another embodiment of the invention relates to a method for treating or reducing the risk of a microbial infection comprising the step of applying an antimicrobial composition having a pH below about 7 to the locus or surface of the microbial infection, wherein said composition is prepared by mixing a first . solution with a second solution, the first solution comprising a water soluble metal chlorite and the second solution comprising a suitable oxidizing agent, as described above (e. g. , sodium persulfate), and hydrogen peroxide. The resulting mixture

comprises about 0.05 to 0.25% of the water soluble metal chlorite, about 0.5 to 5% of the oxidizing agent (e. g. , sodium persulfate) and about 0.15 to 0.75% hydrogen peroxide, wherein all quantities are based on the weight of the antimicrobial composition.

The antimicrobial composition obtained after mixing the solutions will have C102 at a concentration in the range of about 1 to 100 ppm, preferably in the range of about 2 to 20 ppm; hydrogen peroxide in the range of about 0.15 to 0. 75%, preferably in the range of about 0. 15 to 0.4% ; oxidizing agent in the range of about 0.5 to 5%, preferably 1.5 to 2.5% ; and C10 ;, in the range of about 0.05 to 0.5%.

Accordingly, another embodiment of this invention relates to a method for treating or reducing the risk of microbial infection comprising the step of applying to the locus of the microbial infection an antimicrobial composition comprising (a) ClO2 at a concentration in the range of about 1 to 100 ppm, preferably in the range of about 2 to 20 ppm; (b) hydrogen peroxide in the range of about 0.15 to 0.75% by weight, preferably in the range of about 0.15 to 0.4% ; (c) oxidizing agent in the range of about 0.5 to 5% by weight, preferably 1.5 to 2.5% ; and (d) C102-in the range of about 0.05 to 0.5% by weight, wherein the pH of the composition is below about 7.

Another embodiment of the present invention is the aforementioned composition formed from mixing the two solutions.

For adjusting the pH, any suitable buffer may optionally be used such as a bicarbonate buffer, a citrate buffer or a phosphate buffer. When used, the buffer will typically be present at a concentration in the range of about 0.1 to 1.0% by weight.

Bicarbonate is a preferred buffer system. Any suitable food-grade acid or base may be used to prepare the buffer system or to otherwise adjust the pH. Preferred acids are phosphoric acid and citric acid, optionally supplemented by tannic acid, and a preferred base is sodium bicarbonate.

The composition may contain optional ingredients to improve taste, appearance or mouthfeel in order to enhance its appeal to the consumer. Such optional ingredients include colorants, sweeteners, flavorings and surfactants that are known ingredients in commercially available mouthwash. Examples of colorants include FDC Red 40, FDC Green 3, FDC Brown mixture, FDC Yellow 5, DC Red 19, DC Red 33, DC Yellow 10, and the like, which are typically present in about

0.01 to 0.2 weight percent. Examples of suitable sweeteners include glycerin and sugar alcohols like sorbitol or artificial sweeteners such as aspartam, saccharin or acesulfame. Sweetening agents are generally used at levels of from about 0.005% to about 2% by weight of composition. Examples of flavorants include oils of spearmint, peppermint, wintergreen, sassafras, clove, sage, eucalyptus, cinnamon, lemon, orange, and methyl salicylate. Flavorants are used in a quantity of about 0.1 percent by weight. A surfactant can be added as an optional ingredient in a quantity of about 0.2-2 weight percent, and preferably is selected from orally-compatible nonionic and anionic polymers which are commercially available for oral hygiene applications. Nonionic oral surfactants are illustrated by laurate esters of sorbitol consisting of the monoester condensed with about 15-25 moles of ethylene oxide, such as Tween 20 (ICI Americas). Another suitable type of oral surfactants are the polymers of polyoxyethylene and polyoxypropylene, such as Pluronic F-108 (BASF- Wyandotte). Anionic oral surfactants are illustrated by alkyl sulfonates and sulfates, such as sodium lauryl sulfate or a sulfonate monoglyceride of a Cl0-Cl8 fatty acid.

The present composition may also contain deodorizing agents, anti-foam agents, ethanol or other alcohols, as well as other conventional ingredients.

It is preferred that the present composition be prepared fresh from the two solutions and used at or near the time of preparation. The composition is best used between about 1 to 5 minutes after it is prepared. However, if the composition is left to sit for several hours, its effectiveness will begin to diminish. In the field of oral mouthwashes, suitable containers have been developed for maintaining separate solutions that are to be mixed at the time of use by the consumer. US Patents 5,252, 312,5, 289,950 and 5,392, 947, incorporated herein by reference, describe such dispensing containers for dental moutwash. The containers have at least two discreet compartments so that the contents of the compartments may be dispensed simultaneously.

Accordingly, another embodiment of this invention is directed to a kit or dental mouthwash product comprising two components. The kit or dental mouthwash product can be used in therapy, for example, for treating and/or reducing the risk of microbial infections such as those associated with dental disease. The QS iX tS lfantllrP. ^f a med ment ft, r llRe in treating

or reducing the risk of said microbial infections. The kit or dental mouthwash product comprises: a) first liquid component comprising a water-soluble chlorite compound, said chlorite compound present at a concentration in the range of about 0.1 to 0.5% by weight, and said first component having an alkaline pH; and b) a second liquid component comprising a second solution comprising an oxidizing agent, as described herein (preferably sodium persulfate) and hydrogen peroxide, said oxidizing agent at a concentration in the range of about 1.0 to 10% by weight, said hydrogen peroxide present at a concentration in the range of about 0.3 to 1.5% by weight, and said second component having a pH in the range of about 1 to 6.

Optionally, the kit or dental mouthwash product additionally comprises: c) dispensing container which houses a first compartment with an outlet end containing the first liquid component and a second compartment with an outlet end containing the second liquid component; d) a closure mechanism for closing the compartments over the outlet ends; and e) a closure means for allowing the first and second liquid components to be simultaneously dispensed.

Such containers are especially well-suited to simultaneously dispensing equal quantities of the two liquid components. Optionally, the outer walls of one or both of the compartments may be constructed of a translucent or clear material so that the liquid level within the container may be viewed.

The composition may be used as an oral rinse for treating dental disease.

Such disease includes gingivitis, dental caries and oral malodor. The amount of composition and the frequency of treatment may be varied depending on the type and severity of the disease and on the mode of application. The amount of composition used per treatment may vary from about 0.1 ml to 100 ml depending on

the application. The lower amounts may be sufficient if the composition is to be applied directly, for example, by using a syringe or other means of direct application.

For an oral rinse, the amounts typically vary from a few milliliters to about 100 ml per treatment, preferably from about 25 to 50 ml. The composition may be used as a mouthwash daily or multiple times during the day or in accordance with a treatment regimen that would be prescribed by one skilled in the art of dental care. For treating the dental diseases described herein, treatments will generally be made once to a few times per day, preferably twice per day.

The composition may also be used in dental appliance therapy, especially for treating extra-oral appliances such as removable partial dentures, full dentures, night guards, and orthodontic appliances. The appliance is treated by immersing it in the composition for a suitable period of time, usually about 10 to 15 minutes.

The pH of the resulting composition should be below 7, but above the pKa of chlorous acid. It is known that ClO2 is generated from NaCIO2 at pH values below 7 in the absence of an oxidizing agent such as Na2S208. However, the generation is quite slow (on the order of days and months). In the presence of an oxidizing agent such as Na2S208, under the conditions described above, therapeutic levels of ClO2 are believed to be generated in seconds. Increasing the concentration of the oxidizing by weight will cause a more rapid generation of CIO2. Table 1 shows that for a constant level of NaClO2 the generation of C102 increases with increasing levels of Na2S20g. The levels of ClO2 generated by this reaction are within the levels that are expected to'inhibit the formation of gingival plaque.

Table 1: Generation of C102 in 5 minutes after a 1: 1 mixture of a NaClO2 solution (0.25%) and a solution of Na2S208 (ranging from 1-5%) at pH = 5.

% Na102 % Na2S208 C102ppm 0.25 1 2.5 0.25 2 5.5 0.25 3 10.0 0.25 4 12.5 0.25 5 17.0 Table 2 below shows the effects of H202 on the reaction system. As discussed above, although an oxidant, in this reaction H202 will reduce ClO2 back into Na102. In the above stoichiometry, ClOz is generated even in the presence of H202. While the apparent Cl02 concentration decreases with increasing levels of H202, even at the highest levels of H202 (1.2%), there is sufficient Cl02 to inhibit gingival plaque. For products requiring less H202, either higher levels of C102 can be obtained or the levels of NaClO2 and oxidizing agent may be adjusted, in a manner that would be apparent to one skilled in the art, to yield desired levels of C102.

Table 2: C102 generation 5 minutes after a 1: 1 mixture of a Na102 (0.25%) solution and a solution containing Na2S208 (5%) and H202 (ranging from 0-1.2%) at pH = 5.

% H202 % NaClO2 % NaOs C10, ppm 0 0.25 5 17 0.025 0.25 5 16.5 0.075 0.25 5 15.6 0.15 0.25 5 13.5 0.3 0.25 5 11.3 0.6 0.25 5 9.0 1.2 0.25 5 6.0 In the case where the application also calls for the use of NaHC03, such as in bicarbonate-and peroxide-containing products, the pH of the persulfate/peroxide- containing component should be sufficiently acidic such that upon mixing with the NaClO2/bicarbonate-containing component the resulting pH is less than 7.

Table 3 below shows a formula for the two-component system of the present invention. The ranges given below represent the various conditions that could result

in levels of C102 that inhibit plaque. In general the Cl02 levels will be dependent on the level of NaClO2 in the base and the level of H202and Na2S208 in the activator.

The level of NaHC03 in the base will determine the level of phosphoric acid (H3PO4) that should be used in order to adjust the pH of the activator. Upon mixing the base and activator, the resulting pH should be below 7. Increasing levels of NaHC03 in the base therefore will require increasing levels of phosphoric acid in the activator to achieve a final pH of less than 7 when the two phases are mixed.

Table 3. Formulation for Two-Component System.

Base (Solution 1) Activator (Solution 2) Component Wt. % Component Wt. % Na102 0.1-0. 5 H202 0.3-1. 5 NaHCO3 up to 1.0 Na2S208 1.0-10. 0 Surfactant 0.01-1. 0 Surfactant 0.01-1. 0 Flavor 0.5-2. 0 Flavor 0.5-2. 0 Ethanol up to 16.0 H3PO4 as needed Water balance Water balance The lower levels of NaClO2 and Na2S208 can be used to generate the desired levels of C102 if the HzOz is eliminated from the formulation. The level of NaHC03 will not effect the generation of C102 where no HzOz is present. However, the pH of the resulting solution should be below 7 if NaHC03 is used in the formula. It may be desirable in some applications to include bicarbonate but not H202.

Example A standardized Ramfjord protocol was followed for all subjects in the study.

Forty three (43) adult subjects, between the ages of 18 and 65, were recruited for a two-cell, 48 hour plaque inhibition study against a water placebo control. The study was conducted in a double blind manner using the formulation described in Table 4 below. The subjects received 50 ml unit doses, two times per day over a 48 hour period. Neither examiner nor subject had knowledge of the test product identity.

Table 4: Clinical formula evaluated in a double-blind, two-cell, 48 hour plaque inhibition study against a water placebo control.

Base Activator Component Wt. % Component Wt % Na102 0.32 H202 0.75 NaHCO3 0.5 Na2S208 5.0 Surfactant 0.5 Surfactant 0.5 Flavor 0.5 Flavor 0.5 Ethanol 10.0 H3PO4 as needed Water balance Water balance On day one the panelists received dental prophylaxis and were instructed to use the assigned rinse. No oral hygiene regimens, besides the rinsing with the test product or placebo, were allowed. At the end of the two-day treatment, panelists received a plaque evaluation of the Ramfjord teeth as well as all other molars, excluding third molars. The plaque were evaluated for supragingival plaque using the Distal Mesial Plaque Index scoring method. The findings are shown in Table 5 below.

Table 5: Clinical results obtained in the double-blind, two-cell, 48 hour plaque inhibition study against a water placebo control.

Tooth Number of Standard Surface Product subjects Mean Deviation P Value Mouth Placebo 22 1.13 0.27 0.0001 Test Rinse 21 0.63 0.12 MO Placebo 22 0.59 0.26 0.0001 Test Rinse 21 0.23 0.12 MM Placebo 22 1.01 0.28 0.0001 Test Rinse 21 0.46 0.22 MG Placebo 22 1.29 0.33 0.0001 Test Rinse 21 0.75 0.20 FM Placebo 22 1.45 0.44 0.0002 Test Rinse 21 0.96 0.31 FD Placebo 22 1.70 0.44 0.0001 Test Rinse 21 1.06 0.20 DG Placebo 22 1.72 0.42 0.0001 Test Rinse 21 1.00 0.22 DM Placebo 22 1.36 0.46 0.0001 Test Rinse 21 0.63 0.32 DO Placebo 22 0. 53 0.37 0.0014 Test Rinse 21 0.21 0.23 R Placebo 22 0.55 0.18 0.0001 Test Rinse 21 0.32 0.14 The mean values shown in Table 5 are mean plaque scores for each tooth surface examined on the Ramfjord teeth. The greater the mean value, the greater the relative plaque accumulation. The data show that the ClO2 group mean plaque scores were significantly lower than the water placebo group mean scores for all individual surfaces and for the total mouth. Therefore it can be concluded that the ClO2 rinse is significantly more efficacious than water in the inhibition of dental plaque on the teeth, over a 48 hour period with four applications.

In addition to use within the oral cavity, it is contemplated that the present invention has applicability for use on the vaginal, anal, nasal, and ocular mucous membrane surfaces, topically, and in vitro, as, e. g., as a contact lens wash or dental apparatus wash.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.