Oral care composition

Abstract: A stable mouth wash or dentifrice composition containing stabilized chlorine dioxide and phosphates is disclosed for reducing the motility of and killing microbial pathogens. The preferred concentration ranges are between about 0.005% - 0.5% chlorine dioxide, and between about 0.02% - 3.0% phosphate. The phosphate retards escape of chlorine dioxide in the pH range (6.0 to 7.4) typically found in a mouth.

Description: Background of the Invention 1. Field of the Invention The present invention is directed to a method and composition for prevention and treatment of gingivitis and periodontitis, as well as dental caries. More particularly, the present invention relates to the use of phosphates in conjunction with stabilized chlorine dioxide to provide a mouth rinse or nonsudsing detergent toothpaste having increased stability and shelf life at a pH which maintains the effectiveness of stabilized chlorine dioxide in reducing the motility of and in killing bacterial pathogens. 2. Description of the Prior Art The volatile sulfur compounds, hydrogen sulfide (H 2 S) methylmercaptan (CH 3 SH) and di-methylmercaptan (CH 3 ) 2 S are recognized in the current dental literature as being the major contributors to oral malodor. Numerous researchers using organoleptic, chemical, amperometric, mass spectrometric, gas or liquid chromatographic methods have demonstrated that these volatile sulfur compounds are present in the head space and vapor of putrefied saliva and in individual samples of mouth air. In most persons, hydrogen sulfide and methylmercaptan constitute over 90% of the total volatile sulfur content identified in mouth air. Further, the sulfur compounds increase the ability of bacteria and their toxic byproducts to penetrate the lining epethelial barrier of oral mucosa and penetrate into the underlying connective tissue. (Gaffer and Rizzo papers referenced in "Effect of Hydrogen Sulfide and Methyl Mercaptan on the Permeability of Oral Mucosa, J. Dent Res. 63(7), July 1984, pages 994-997). These malodorous volatile sulfur compounds are generated primarily through the putreficative action of oral microorganisms on sulfur containing amino acids, peptones or proteins found in the mouth. These substrates are readily available in saliva and dental plaque or may be derived from proteinaceous food particles trapped between the teeth, in the gingival crevices or adhering to the mucous membranes and the irregular surface of the tongue as well as exfoliated oral epithelium food debris and the like. Current studies have indicated that mouth odor not only comes from the posterior dorsal surface of the tongue but also from periodontal pockets. People with periodontal involvement have an attendant increase in oral malodor from disintegrated epithelial cells. Starting with a clean tooth surface, plaque formation and resulting ecology occurs in the following steps: 1. Deposition of a coating of glycoproteins from salivary and other oral mucous gland secretions. This is referred to as acquired pellicle. 2. Fastening and colonization of streptococcus organisms to the acquired pellicle primarily by streptococcus sanguis and streptococcus mutans. 3. Conversion of sucrose to glucans (dextran) and fructans by the bacterial enzyme glucosyltransferases. In this plaque mass are imbedded dead cells, cell debris and food debris. High molecular weight polymers of glucose and other sugars, altered salivary glycoproteins, proteases and various chemotactic and inflammatory inducing substances have been detected and partially characterized. 4. Other organisms, primarily gram positive aerobes, become residents in the plaque mass and use the glucans and fructans for nutrition. These are primarily oxygen using organisms and the oxygen source is from the saliva that bathes the plaque mass. 5. With time and the functioning of this ecological system, the oxygen use by the superficial bacteria deprive the lower layers of the plaque matrix of a supply of oxygen. An opportunity is provided for non-oxygen using bacteria (facultative anaerobes) to become established. 6. If left undisturbed, the ecological system now established is self perpetuating. That is, the streptococcus bacteria continue to produce glucans and fructans. Other bacteria product produce toxins that kill cells of the host and the dead cells become other essential nutrients. The superficial bacteria deprive the deeper layers of the plaque mass of oxygen and keep the ecological system going. Thus, both aerobic and anaerobic organisms survive in the plaque mass. 7. The established ecological system attendant the plaque mass produces toxins from the aerobic bacteria that cause gingivitis and toxins from an aerobic bacteria that cause periodontitis. Various substances have been tested for their ability to disrupt plaque or prevent its formation and to treat mouth odor such as antibiotics, chlorhexidines, oxine, and alexidine. Over the years, mouth washes and toothpastes have been supplemented with additives which claim to have beneficial effects against dental diseases. For example, fluoride added to toothpaste has been shown to reduce dental caries but has not been shown to prevent gingivitis and periodontal disease. As described in U.S. Patent No. 4,198,394, the effectiveness of fluoride is reportedly enhanced by adding sodium dihydrogen phosphate to the fluoride containing dentifrice. Phosphate compounds have also been used to stabilize dentifrices, as described in U.S. Patents Nos. 3,622,662 (dental creams) and 3,577,521 (toothpastes). The prior art compositions that have been used and tested have found some acceptance but are generally ineffective in periodontitis, gingivitis, plaque accumulation and mouth malodor. On the other hand, U.S. Patent Nos. 4,689,215 and 4,818,519 describe the use of stabilized chlorine dioxide in aqueous solution for the treatment of the mouth as a deodorizing agent, antiplaque agent, bactericide for treatment of gingivitis and periodontitis and as a bactericidal fungicidal and viralcidal agent in other related applications. Stabilized chlorine dioxide and a method of making it are described in U.S. Patent No. 3,271,242; stabilized chlorine dioxide is sold by Bio-Cide International, P.O. Box 2700, Norman, Oklahoma, 73070 under the name Purogene. Further discussion of stabilized chlorine dioxide may be found in a treatise entitled "Chlorine Dioxide" by W. J. Masschelein and published by the Ann Arbor Science Publishes, Inc., copyright 1979 (note in particular pages 138-140). Various embodiments of chlorine dioxide for various purposes are also reviewed in this treatise. Chlorine dioxide, ClO 2 , functions biochemically in many ways other than as a germicide. These functions include: (1) oxidation of double bonds between two carbon atoms; (2) oxidation of unsaturated fatty acids (lipids) via double bonds between two carbon atoms; (3) acceleration of hydrolysis of carboxalic anhydrides: (4) oxidation of aldehydes to the corresponding carboxylic acids; (5) oxidation of alcohols; (6) oxidation of amines; (7) oxidation of phenols, phenolic derivatives and thiphenolic compounds; (8) moderate oxidation of hydroquinones; (9) oxidation of thiophenols; (10) oxidation of amino acids, proteins and polyamides, primarily by attacking sulphide bonds. These are cystine, methionone and tryosine. Tryptophane also has been shown to be reactive. Keratin, (which makes up the cyto-skeletal structure in epithelial cells cytoplasm), and C1O 2 keratin sulfonic hydrosoluble acids; (11) carbohydrates are altered as the CHO and CH 2 OH radicals to produce carboxylic functions; and (12) Nitrates and sulphides are readily oxidized. Limited attempts have been made to use chlorine dioxide in toothpaste formulations. For example, U.S. Patent No. 4,330,530 discloses the topical application of an aqueous gel comprising sodium chlorite simultaneously with the topical application of another aqueous gel compromising lactic acid. Upon mixture of these two gels, non-stabilized chlorine dioxide is produced within the gel mixture. The two gels cannot be premixed without potential loss of an effective application of the operative element (non-stabilized chlorine dioxide). There is no teaching in that reference of the use of stabilized chlorine dioxide. A relatively new product, Oxyfresh Toothpaste, produced by Oxyfresh USA, Inc. of Spokane, Washington, contains purogene. This formulation uses Sodium Laural Sulphate as a detergent. Inasmuch as C10 2 reacts to sulphates, the C10 2 content is gradually decreased, rendering the formulation inactive with a short shelf life because stabilized chlorine dioxide gas tends to escape from the mixture, the effectiveness and shelf life of the stabilized chlorine dioxide is reportedly accomplished by housing the product in a special aerosol can. Document EP-A-0344701 relates to oral compositions comprising a chlorous acid liberating material. Accordingly, there exists a clear need for a stabilized chlorine dioxide oral health preparation such as a mouth wash and dentifrice such as a toothpaste with an acceptable shelf life which will effectively inhibit the initial pellicle which precedes plaque formation and inhibit or control the formation of bacterial plaque and suppress organisms such as but not limited to (1) "Streptococcus mutans", which is implicated as the major cause of human dental decay; (2) Black Pigmented Bacteriodes, and "Acinobacillus actinomycetumcomitans" which is implied in human periodontitis; and (3) will reduce odor in the mouth through the control of hydrogen and methylmercaptan. Summary of the Invention Briefly, and in accord with one embodiment of the present invention, a composition containing stabilized chlorine dioxide and a phosphate is disclosed as being useful in preventing and treating dental diseases such as gingivitis, periodontitis and dental caries. Stabilized chlorine dioxide is an effective agent for the treatment of the mouth as a deodorizing agent, antiplaque agent, bactericide for treatment of gingivitis and periodontitis and as a bactericidal fungicide and viricidal agent. The addition of activating inhibitor phosphates to the stabilized chlorine dioxide retards the rapid escape of chlorine dioxide gas at the Ph range of 6.0 to 7.4 typical of the mouth. Concentrations of stabilized chlorine dioxide are in the range of between about .005% to 0.5%; concentrations of the phosphate, preferably disodium hydrogen phosphate, sodium dihydrogen phosphate, trisodium phosphate, sodium monofluorophosphate, or pyrophosphates are in the range of between about 0.02% to 3.0%. Description of the Preferred Embodiment Broadly, the present invention contemplates the use of an activating inhibitor in a stabilized chlorine dioxide mixture to make possible the lowering of the Ph of the chlorine dioxide mixture at the time it is used in the mouth. Clinical observations by the inventor have led to the discovery that an activating inhibitor phosphate such as disodium hydrogen phosphate, sodium dihydrogen phosphate, or, preferably, trisodium phosphate, will allow the stabilized chlorine dioxide to remain stable and effective at a lower Ph than has been thought possible. In addition, the phosphate is a detergent which, when used in place of the detergent sodium laurel sulfate, provides a nonsudsing toothpaste. Nonsudsing toothpastes are preferred for use with electric toothbrushes because electric toothbrushes tend to spray sudsing toothpastes about the room during use. Further, people tend to quit brushing when a mouth full of suds accumulates in the mouth; a nonsudsing toothpaste does not cause this psychological event. The first step in the formation of plaque on a clean tooth surface is the formation of acquired pellicle. Studies by others have shown the following to be part of the acquired pellicle formative process. Glycoproteins of salivary and other mucous gland origin are attached to the hydroxyapatite crystals. (Roukima, P.A. and Nieuw Amerongen A.V., Sulphated Glycoproteins in Human Saliva; Saliva and Dental Caries (Sp. Supp. Microbiology Abst.) 1979, pp. 76, Embery, G., The role of anionic glycoconjugates, particularly sulphated glycoproteins in relation to the Oral Cavity, Saliva and Dental Caries. (Supp. Microbiol Abstr.), Information Retrieval 1978, pp. 105-111). Sulphated glycoproteins have a strong affinity to the calcium anion (ibid., pp 105-108). Most major salivary secreted glycoproteins may be bound to certain ester sulphates (ibid). These sulphated glycoproteins have been related to bacterial agglutination or clumping (ibid., pp 108). Clinical observations by the inventor have led to the discovery that the process of acquired pellicle can be inhibited by the use by humans of stabilized chlorine dioxide. Through such observations it has been learned that the chlorine dioxide reacts with the sulphated glycoproteins to inhibit pellicle formation. This process results primarily from, but is not limited to, oxidation of the sulphide bonds. Since acquired pellicle is the first step in plaque formation, this initial inhibition alters the sequence of events to follow. The second step, bacterial adhesion, and subsequent steps are consequently retarded. No disulphate enzymes capable of cleaning the sulphate moieties of glycoproteins are known. Bacterial agglutinigation includes the conversion of sucrose to glucans and fructrans by enzymes known as glycosyltransferases. These enzymes are of bacterial origin. The plaque mass becomes a complex extra cellular (of microorganisms) matrix containing sulphated glucosamineglycans, proteglycans, glycoproteins, sugar, proteins and lipids, which aid in the process of bacterial agglutination (Schluger, S., Yuodelis R. and Page R., Periodontal Disease, Chapter 6, pp. 135 to 166, 1977, Lea and Febiger, Phila, PA. Newbrun E., Polysaccharide Synthesis in Plaque; Microbiol Aspects of Dental Caries, Vol III (Supp. Microbiology Abstr.), 1976, pp 649-664.) These compounds include the presence of sulphur and become unstable in the presence of high oxygen compounds. The oxygen splits the sulphide bonds to form sulphates or SO 2 . Clinical observations by the inventor have led to the conclusion that all of these biochemical compounds are attacked to a greater or lesser extent by stabilized chlorine dioxide. Since these compounds may be used as nutrients for bacteria, the reduction of the compounds will inhibit bacterial growth. More specifically, the stabilized chlorine dioxide oxidizes carbohydrates, chondroitin sulphates, glucosaminglycans, glycoproteins, proteins and lipids. Since these compounds arise as bacterial byproducts and debris from dead and dying cells, are of salivary origin and are the mechanism of agglutination of the plaque mass, their degradation/oxidation retards plaque growth. The initial bacterial residents of the plaque mass are aerobic, oxygen using organisms. The saliva bathing the plaque provides the source of oxygen. As the plaque thickens and the oxygen using bacteria increases in numbers, the deeper layers have a reduced oxygen content. The greater the aerobic population of plaque matrix, the lower the oxygen level in the saliva. This permits the deeper layers of the plaque matrix to develop an anaerobic population of bacteria (Globerman, D. Y., and Kleinberg, I., Intro-Oral PO 2 and it's relation to Bacterial Accumulation on the Oral Tissues: saliva and Dental Caries. (Sp. Supp. Microbiology Abstr.) 1976 pp. 275-292). Clinical observations by the inventor lead to the discovery that the use of stabilized chlorine dioxide with an activating inhibitor in an oral health preparation such as a mouth wash or toothpaste will raise the level of oxygen in the saliva. The raised level of oxygen within the plaque matrix will inhibit anaerobic bacterial growth. As periodontitis is caused by anaerobic bacteria, the potential for the development of periodontitis is reduced by stabilized chlorine dioxide. The inhibition of acquired pellicle formation, the prevention of bacterial agglutinization and the oxidation of the plaque mass through the use of chlorine dioxide with an activating inhibitor phosphate in a mouth wash or toothpaste are independent of the germicidal capacity of such mouth wash or toothpaste. Furthermore, these factors in combination with the bacteriocidal capacity of chlorine dioxide in the mouth wash or toothpaste renders the mouth wash or toothpaste an effective pellicle and plaque inhibitor. The permeability of sublingual mucous tissue within the mouth is increased substantially by exposure to hydrogen sulfide (H 2 S) and methyl mercaptan (CH 3 SH). (Gaffer and Rizzo papers referenced in "Effect of Hydrogen Sulfide and Methyl Mercaptan on the Permeability of Oral Mucosa, J. Dent Res. 63(7), July 1984, pages 994-997). Accordingly, the toxic bacterial products attendant plaque which produce these compounds have a related effect on tissue permeability. Since chlorine dioxide breaks the disulphide bonds of both these compounds, the use of chlorine dioxide with an activating inhibitor phosphate in a toothpaste would reduce the penetration potential of pathogenic materials. Evidence exists that endotoxin and lipopolysaccharide from gram negative bacteria are the worst of the products to penetrate the tissues. Application of endotoxin to gingiva has caused gingival inflammation. (ibid). Chlorine dioxide used in treatment of plaque acts upon attendant gram negative bacteria. Thereby, the inventor has learned through experimentation and observation that chlorine dioxide with an activating inhibitor phosphate in a mouth wash or toothpaste can be a preventative product leading to oral health. The use and efficacy of this composition is described below. Example I The stability of Chlorine Dioxide at Ph 6.8 in the Presence of Phosphate. Materials: 1. Purogene (Trademark) (2% C10 2 ), Lot #8907.41, 1 gallon, Manufactured by BIO-Cide, International, P.O. Box 2700, Norman, OK 73070. 2. Sodium Phosphate, monobasic, dibasic, and tribasic. Methods: A 10% solution of monobasic sodium phosphate was prepared in distilled water. Ten ml was placed into each of four beakers. One of each of the four beakers received 1, 2.5, 5, and 10ml of chlorine dioxide concentrate (2% C10 2 ), respectively All solutions were diluted to 90ml with distilled water, adjusted to pH 6.8 with 1N NaOH and 1N HC1, diluted to 100ml and placed in screw cap bottles. Solutions containing dibasic and tribasic sodium phosphate and a distilled water blank control were prepared in a similar manner. Chlorine dioxide content and pH was determined for each solution on days 0, 7, 14, 21 and 28 in accordance with Standard Methods for the Examination of Water and Wastewater, 17th edition, 1989. Results and Summary: As shown in Table 1, the content of chlorine dioxide was stable in all sodium phosphate solutions and distilled water control over the 28 day test period. The pH of all samples ranged from 6.1 to 7.6. Example II The Stability of Chlorine Dioxide in Various Toothpastes at pH 5.0, 5.5, and 6.0. Stabilized chlorine dioxide in alkaline solutions is present as sodium chlorite. Acidification of sodium chlorite results in the liberation of chlorine dioxide gas which is very reactive against organic material. Materials: 1. Tartar Control Crest (Trademark) toothpaste, original flavor, lot #9342TK, manufactured by Procter and Gamble, Cincinnati, Ohio, 45202. 2. Aquafresh (Trademark) for Kids Fluoride toothpaste bubblemint flavor, lot M292B, manufactured by Beecham Products, USA, Pittsburgh, Pennsylvania, 15230. 3. Generic toothpaste without a detergent (unlabeled), supplied by Perry Ratcliff, DDS, 7125 East Lincoln Drive, Scottsdale, Arizona 85253. 4. Trisodium phosphate, Na 3 PO 4 .12H 2 0. Fisher Scientific, Fair Lawn, NJ. 5. Purogene, (2% Chlorine dioxide), lot #8907:41, manufactured by BIO-CIDE International, Inc., P.O. Box 2700, Normal, Oklahoma 73070. Methods: Crest and Aquafresh Toothpaste: Each toothpaste was divided into three 50g portions and placed in plastic jars with lids. One portion of each toothpaste was adjusted to pH 5.0, 5.5, and 6.0, respectively, with 1N NaOH and 1N HC1. Subsequently 5ml 2% Chlorine dioxide was added to each portion and mixed thoroughly, yielding a chlorine dioxide concentration of approximately 0.15%. Generic Toothpaste: Toothpaste was dispensed into four jars, 50g per jar. Jars were divided into groups of two each. Jars in the first group each received 1.2g Na 3 PO 4 .12H 2 0 (1% Na 3 PO 4 ) while jars in second group each received 2.3g Na 3 PO 4 .12H 2 0 (2% Na 3 PO 4 ). One jar from each group was adjusted to pH5.0 and 6.0, respectively. Subsequently 5ml 2% Chlorine dioxide was added to each portion and mixed thoroughly, yielding a chlorine dioxide concentration of approximately 0.15%. The pH and chlorine dioxide content of all jars was determined. Jars were stored in the dark at @ 20 degrees C and reassayed for pH and chlorine dioxide content on days 7, 14, 21 and 28. RESULTS AND SUMMARY: As shown in Table 2, the pH values of Crest and generic toothpaste with 1% and 2% trisodium phosphate were stable during the twenty-eight day test period. In sharp contrast, the pH values of Aquafresh toothpaste increased from 0.4 to 1.0 units during the 28 day test period. Table 3 shows the percentage of chlorine dioxide remaining in the various toothpastes on days 0, 7, 14, 21, and 28. Chlorine dioxide in Crest toothpaste gradually decreased during the 28 day test period. On day 28, 6% of the original chlorine dioxide was remaining in Crest toothpaste at pH 5.0. In sharp contrast, 54% of the chlorine dioxide was present in Crest toothpaste at pH 6.0, nine fold greater than at pH 5.0 Chlorine dioxide content in Aqua Fresh toothpaste remained stable for the first 21 days (78-100% of C10 2 remaining) at all Ph levels. A sharp drop in chlorine dioxide concentration occurred on day 28; the reason for the decrease is not clear. Chlorine dioxide content in generic toothpastes containing 1% and 2% trisodium phosphate at pH 5.0 gradually decreased during the 28 day test period. On the 28th day, 5 to 27% of the chlorine dioxide remained, respectively. In sharp contrast, generic toothpaste containing 1% and 2% trisodium phosphate at pH 6.0 remained relatively stable during the 28 day test period (80 to 100% chlorine dioxide remaining). This is three to 20 fold greater retention of chlorine dioxide than at pH 5.0. Of all the toothpastes tested, generic toothpaste with either 1% or 2% trisodium phosphate at pH 6.0 appears to be the most compatible with chlorine dioxide. RESULTS SHOWING pH VALUES OF TOOTHPASTES DURING THE TWENTY-EIGHT DAY TEST PERIOD TOOTHPASTE DAY 0 7 14 21 28 Crest 5.0 5.0 5.0 5.0 5.0 5.5 5.5 5.5 5.5 5.5 6.0 6.0 6.0 6.0 6.0 Aqua Fresh 5.0 5.6 5.7 5.9 6.0 5.5 5.7 5.7 5.8 5.9 6.0 6.2 6.3 6.4 6.6 Generic w/ 1% Na 3 PO 4 5.0 5.0 5.0 5.0 5.1 6.0 6.0 6.0 6.0 6.1 Generic w/ 2% Na 3 P0 4 5.0 5.1 5.0 5.1 5.2 6.0 6.0 6.0 6.0 6.1 RESULTS SHOWING THE STABILITY OF CHLORINE DIOXIDE IN TOOTHPASTES AT VARIOUS pH LEVELS DURING TWENTY-EIGHT DAY TEST PERIOD TOOTHPASTE pH PERCENT CHLORINE DIOXIDE REMAINING ON DAYS 0 7 14 21 28 Crest 5.0 100 56 38 19 6 5.5 100 60 47 40 27 6.0 100 85 77 62 54 Aqua Fresh 5.0 100 86 86 93 43 5.5 100 89 89 78 22 6.0 100 100 100 100 17 Generic w/ 1% Na 3 P0 4 5.0 100 55 25 10 5 6.0 100 100 100 93 80 Generic w/ 2% Na 3 P0 4 5.0 100 73 87 33 27 6.0 100 82 82 88 82 Example III The Antimicrobial Effectiveness of Chlorine Dioxide in Phosphate Solution Materials: 1. Purogene (2% chlorine dioxide), lot #8907:41, manufactured by BIO-CIDE International, Inc., P. O. Box 2700, Norman, Oklahoma 73070. 2. Test Organisms: Streptococcus mutans (ATCC #27152), Streptococcus sanguis (ATCC# 10556), and Candida albicans (ATCC# 18804) 3. Saline, 0.9% NaC1. 4. Butterfield's Buffer phosphate dilutent (BFB), pH 7.2. 5. Sterile 15% sodium thiosulfate. 6. Blood agar. 7. Stop watch. 8. Sterile 1N HC1 and 1N NaOH. 9. pH meter. 10. McFarland nephelometer tube No. 1. Density of this tube is equivalent to a bacterial suspension of 3 x 10 8 organisms per ml. 11. N,N-diethyl-p-phenylenediamine (DPD reagent). 12. Phosphate buffer reagent. 13. Sodium dihydrogen phosphate, NaH 2 PO 4 .7H 2 0. (Fisher Scientific, Fair Lawn, NJ. 14. Trisodium phosphate, Na 3 PO 4 .12H 2 0. Albright & Wilson, P.O. Box 80, Oldbury, Narley, West Midlands, B694LN, England. 15. Sodium monofluorophosphate, Na 2 FPO 3 , Ref No. OB 12837, manufactured by Albright and Wilson, P.O. Box 80, Oldbury, Narley, West Midlands, B694LN, England. DPD reagent and phosphate buffer reagent were prepared in accord with Standard Methods for the Examination of Water and Wastewater, 17th Edition, p. 9-54 (1989). Methods: 1. Test Solutions: A ten percent sodium dihydrogen phosphate solution was prepared in distilled water. Ten m1 was placed into each of five beakers. One of each of the five beakers received 0, 1, 2.5, 5, and 10ml of chlorine dioxide concentrate (2% C10 2 ), respectively. All solutions were diluted to 90ml with distilled water, adjusted to pH 6.0 with 1N NaOH and 1N HC1, diluted to 100ml and placed in screw cap bottles. Solutions containing 0 ppm chlorine dioxide were filter sterilized prior to use. Solutions containing trisodium phosphate and sodium monofluorophosphate were prepared in a similar manner. II. Test Suspensions: Suspensions of each organism were prepared in Butterfield's buffer from 48 hour agar cultures and turbidity adjusted to a McFarland Tube #1. Subsequently 0.1ml of this suspension was diluted in 50ml of saline. The diluted microorganism suspensions were now ready for use. III. Test Procedure: 1. Test: One ml of test suspension was aliquoted into each of five sterile 16 x 125mm screw cap tubes. Each of the five tubes received 4ml of a solution containing either 0, 200, 500, 1000, or 2000ppm chlorine dioxide in 1% sodium dihydrogen phosphate. Each tube was shaken for ten seconds and immediately inactivated with 0.25ml 15% sodium thiosulfate. Solutions containing 1% trisodium phosphate and 1% sodium monofluorophosphate were handled in a similar manner. 2. Controls: One ml of test suspension was dispensed into two sterile 16 x 125mm screw cap tubes. Each tube received 4ml 2000ppm chlorine dioxide in 1% sodium dihydrogen phosphate. The first tube received 0.25ml sodium thiosulfate, while the second tube received none. Subsequently each tube was tested for residual chlorine dioxide by adding 0.3ml phosphate buffer reagent and 0.3m1 DPD reagent to each tube. Neutralized tubes were colorless, while nonneutralized tubes were pink. Solutions of trisodium phosphate and sodium monofluorophosphate containing 2,000ppm chlorine dioxide were handled in a similar manner. One m1 test suspension of each organism was treated with 4m1 Butterfield's buffer and 0.25 m1 10% sodium thiosulfate as a negative control. After inactivation with sodium thiosulfate all tubes were plate counted. Sterility tests on all reagents were run parallel to experiments by plate counted method. The plate counted method and sterility tests were conducted in accord with Standard Methods for the Examination of Water and Wastewater, 17th Edition, p. 9-54 (1989). RESULTS AND SUMMARY: As shown in Tables 4, 5 and 6, 99-100% of the organisms were killed when challenged with 2,000ppm (0.2%) chlorine dioxide in either 1% sodium dihydrogen phosphate or trisodium phosphate. In sharp contrast, only fifty percent of S.mutans were killed when challenged with 2,000ppm chlorine dioxide in sodium monofluorophosphate. S.sanguis and C.albicans were 99-100% killed when challenged with 1,000ppm (0.1%) chlorine dioxide in either 1% sodium dihydrogen phosphate or trisodium phosphate. In sharp contrast, S.mutans was killed 24 and 76%, respectively, when similarly challenged. 1,000ppm chlorine dioxide in 1% sodium monofluorophosphate yielded a 12% kill when used against S. mutans. Chlorine dioxide concentrations of 200ppm (0.02%) and 500ppm (0.05%) in the presence of phosphates demonstrated marginal bacteriocidal activity against S.mutans, S.sanguis, and C.albicans (6-66% kill). RESULTS SHOWING THE BACTERIOCIDAL ACTIVITY OF CHLORINE DIOXIDE IN PHOSPHATE SOLUTIONS AT pH 6.0 AGAINST STREPTOCOCCUS MUTANS. CLO 2 (PPM) PHOSPHATE SOLUTION Negative Control NaH 2 HP0 4 Na 3 P0 4 Na 2 FPO 3 0 17,000 16,000 (6) 18,000 (0) 14,000 (18) 200 ND 15,000 (12) 16,000 (6) 14,000 (18) 500 ND 15,000 (12) 15,000 (12) 14,000 (18) 1000 ND 13,000 (24) 4,100 (76) 15,000 (12) 2000 ND 2 (99) 120 (99) 8,500 (50) ND = Not Done RESULTS SHOWING THE BACTERIOCIDAL ACTIVITY OF CHLORINE DIOXIDE IN PHOSPHATE SOLUTIONS AT pH 6.0 AGAINST STREPTOCOCCUS SANGUIS CLO 2 (PPM) PHOSPHATE SOLUTION Negative Control 1% NaH 2 HP0 4 1% Na 3 P0 4 0 29,000 22,000 (24) 25,000 (14) 200 ND 22,000 (24) 19,000 (34) 500 ND 16,000 (45) 11,000 (62) 1000 ND 360 (99) 0 (100) 2000 ND 0 (100) 0 (100) ND = Not Done RESULTS SHOWING THE BACTERIOCIDAL ACTIVITY OF CHLORINE DIOXIDE IN PHOSPHATE SOLUTIONS AT pH 6.0 AGAINST CANDIDA ALBICANS CLO 2 (PPM) PHOSPHATE SOLUTION Negative Control 1% NaH 2 HP0 4 1% Na 3 P0 4 0 95,000 64,000 (33) 55,000 (42) 200 ND 58,000 (39) 64,000 (33) 500 ND 47,000 (51) 32,000 (66) 1000 ND 250 (99) 0 (100) 2000 ND 17 (99) 5 (99) ND = Not Done Example IV The stability of Chlorine Dioxide at Ph 6.8 in the Presence of 0.02% Phosphate. The following is an example of how to test the stability of chlorine dioxide at Ph 6.8 in the presence of 0.02% phosphate. Materials: 1. Purogene (2% C10 2 ), Lot #8907.41, 1 gallon, Manufactured by BIO-Cide, International, P.O. Box 2700, Norman, OK 73070. 2. Sodium Phosphate, monobasic, dibasic, and tribasic. Methods: A 0.2% solution of monobasic sodium phosphate is prepared in distilled water. Ten ml is placed into each of four beakers. One of each of the four beakers receives 1, 2.5, 5, and 10ml of chlorine dioxide concentrate (2% C10 2 ), respectively. All solutions are diluted to 90ml with distilled water, adjusted to pH 6.8 with 1N NaOH and 1N HC1, diluted to 100ml and placed in screw cap bottles. Solutions containing dibasic and tribasic sodium phosphate and a distilled water blank control are prepared in a similar manner. Chlorine dioxide content and pH is determined for each solution on days 0, 7, 14, 21 and 28 in accordance with Standard Methods for the Examination of Water and Wastewater, 17th edition, 1989, in order to determine the stability of chlorine dioxide over time. Example V The Stability of Chlorine Dioxide in Various Toothpastes having a 0.02% and 0.05% Na 3 PO 4 at pH 5.0, 5.5, and 6.0. The following is an example of how to test the stability of chlorine dioxide in various toothpastes having either 0.02% or 0.05% Na 3 PO 4 . Stabilized chlorine dioxide in alkaline solutions is present as sodium chlorite. Acidification of sodium chlorite results in the liberation of chlorine dioxide gas which is very reactive against organic material. Materials: 1. Tartar Control Crest toothpaste, original flavor, lot #9342TK, manufactured by Procter and Gamble, Cincinnati, Ohio, 45202. 2. Aquafresh for Kids Fluoride toothpaste bubblemint flavor, lot M292B, manufactured by Beecham Products, USA, Pittsburgh, Pennsylvania, 15230. 3. Generic toothpaste without a detergent (unlabeled), supplied by Perry Ratcliff, DDS, 7125 East Lincoln Drive, Scottsdale, Arizona 85253. 4. Trisodium phosphate, Na 3 PO 4 .12H 2 0. Fisher Scientific, Fair Lawn, NJ. 5. Purogene, (2% Chlorine dioxide), lot #8907:41, manufactured by BIO-CIDE International, Inc., P.O. Box 2700, Normal, Oklahoma 73070. Methods: Crest and Aquafresh Toothpaste: Each toothpaste is divided into three 50g portions and placed in plastic jars with lids. One portion of each toothpaste is adjusted to pH 5.0, 5.5, and 6.0, respectively, with 1N NaOH and 1N HC1. Subsequently 5ml 2% Chlorine dioxide is added to each portion and mixed thoroughly to yield a chlorine dioxide concentration of approximately 0.15%. Generic Toothpaste: Toothpaste is dispensed into four jars, 50g per jar. The jars are divided into groups of two each. Jars in the first group each receive 0.24 Na 3 PO 4 .12H 2 0 (0.02 Na 3 PO 4 ) while jars in second group each receive 0.6g Na 3 PO 4 .12H 2 0 (0.05% Na 3 PO 4 ). One jar from each group is adjusted to pH5.0 and 6.0, respectively. Subsequently a sufficient amount of 2% Chlorine dioxide is added to each portion and mixed thoroughly to yield a chlorine dioxide concentration of approximately 0.15%. The pH and chlorine dioxide content of all jars is determined. The jars are stored in the dark at @ 20 degrees C and reassayed for pH and chlorine dioxide content on days 7, 14, 21 and 28, in order to determine the stability of the various toothpastes. Example VI The Antimicrobial Effectiveness of Chlorine Dioxide in 0.02% Phosphate Solution The following is an example of how to test the antimicrobial effectiveness of chlorine dioxide in 0.02% phosphate solution. Materials: 1. Purogene (2% chlorine dioxide), lot #8907:41, manufactured by BIO-CIDE International, Inc., P. O. Box 2700, Norman, Oklahoma 73070. 2. Test Organisms: Streptococcus mutans (ATCC #27152), Streptococcus sanguis (ATCC# 10556), and Candida albicans (ATCC# 18804) 3. Saline, 0.9% NaC1. 4. Butterfield's Buffer phosphate dilutent (BFB), pH 7.2. 5. Sterile 15% sodium thiosulfate. 6. Blood agar. 7. Stop watch. 8. Sterile 1N HC1 and 1N NaOH. 9. pH meter. 10. McFarland nephelometer tube No. 1. Density of this tube is equivalent to a bacterial suspension of 3 x 10 8 organisms per ml. 11. N,N-diethyl-p-phenylenediamine (DPD reagent). 12. Phosphate buffer reagent. 13. Sodium dihydrogen phosphate, NaH 2 PO 4 .7H 2 0. (Fisher Scientific, Fair Lawn, NJ. 14. Trisodium phosphate, Na 3 PO 4 .12H 2 0. Albright & Wilson, P.O. Box 80, Oldbury, Narley, West Midlands, B694LN, England. 15. Sodium monofluorophosphate, Na 2 FPO 3 , Ref No. OB 12837, manufactured by Albright and Wilson, P.O. Box 80, Oldbury, Narley, West Midlands, B694LN, England. DPD reagent and phosphate buffer reagent is prepared in accord with standard Methods for the Examination of Water and Wastewater, 17th Edition, p. 9-54 (1989). Methods: 1. Test Solutions: A 0.2 percent sodium dihydrogen phosphate solution is prepared in distilled water. Ten m1 is placed into each of five beakers. One of each of the five beakers receives 0, 1, 2.5, 5, and 10ml of chlorine dioxide concentrate (2% C10 2 ), respectively. All solutions are diluted to 90ml with distilled water, adjusted to pH 6.0 with 1N NaOH and 1N HC1, diluted to 100ml and placed in screw cap bottles. Solutions containing 0 ppm chlorine dioxide are filter sterilized prior to use. Solutions containing trisodium phosphate and sodium monofluorophosphate are prepared in a similar manner. II. Test Suspensions: Suspensions of each organism are prepared in Butterfield's buffer from 48 hour agar cultures and turbidity adjusted to a McFarland Tube #1. Subsequently 0.1ml of this suspension is diluted in 50ml of saline. The diluted microorganism suspensions are then ready for use. III. Test Procedure: 1. Test: One ml of test suspension is aliquoted into each of five sterile 16 x 125mm screw cap tubes. Each of the five tubes receives 4ml of a solution containing either 0, 200, 500, 1000, or 2000ppm chlorine dioxide in 1% sodium dihydrogen phosphate. Each tube is shaken for ten seconds and immediately inactivated with 0.25ml 15% sodium thiosulfate. Solutions containing 1% trisodium phosphate and 1% sodium monofluorophosphate are handled in a similar manner. 2. Controls: One ml of test suspension is dispensed into two sterile 16 x 125mm screw cap tubes. Each tube receives 4ml 2000ppm chlorine dioxide in 0.02% sodium dihydrogen phosphate. The first tube receives 0.25ml sodium thiosulfate, while the second tube receives none. Subsequently each tube is tested for residual chlorine dioxide by adding 0.3ml phosphate buffer reagent and 0.3m1 DPD reagent to each tube. Neutralized tubes are colorless, while nonneutralized tubes are pink. Solutions of trisodium phosphate and sodium monofluorophosphate containing 2,000ppm chlorine dioxide are handled in a similar manner. One m1 test suspension of each organism is treated with 4m1 Butterfield's buffer and 0.25 m1 10% sodium thiosulfate as a negative control. After inactivation with sodium thiosulfate all tubes are plate counted. Sterility tests on all reagents are run parallel to experiments by plate counted method. The plate counted method and sterility tests are conducted in accord with Standard Methods for the Examination of Water and Wastewater, 17th Edition, p. 9-54 (1989), in order to determine the antimicrobial effectiveness of chlorine dioxide in 0.02% phosphate solution. It will be seen from the foregoing that the mixture of stabilized chlorine dioxide and phosphates as part of a mouth wash or toothpaste has an improved shelf life, and is effective as a bactericide superior to other compositions used today. Both stabilized chlorine dioxide, and phosphates, have been used for many years in other areas and extensive study in animals and in man have demonstrated the compounds' low toxicity and safety. The invention is defined by the claims.

Claims: A composition in the form of a dentifrice or mouthwash for preventing and treating dental disease by reducing the number of microorganisms in the mouth, said composition comprising: (i) stabilized chlorine dioxide in a concentration in the range of 0.005% - 0.5% by volume based on the total volume of the composition; and (ii) a stabiliser comprising a phosphate compound in a concentration in the range of 0.02% - 3.0% by volume to retard escape of chlorine dioxide from the composition at a pH of from 6.0 to 7.4 thereby increasing the shelf life and efficacy of the composition. A composition as claimed in claim 1, containing at least 0.1% by volume of stabilised chlorine dioxide and at least 0.05% by volume of the phosphate. The composition as set forth in claim 2, wherein the concentration of stabilized chlorine dioxide is approximately 0.2% by volume and the concentration of phosphate is approximately 1.0% by volume. A composition as claimed in claim 1, wherein the phosphate is mono-, or di-basic sodium phosphate, or pyrophosphate. The composition as set forth in any of claims 1 to 4, wherein the phosphate is disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium monofluorophosphate or pyrophosphate. The composition as set forth in claim 5, wherein the phosphate is sodium dihydrogen phosphate. The composition as set forth in any of claims 1 to 3, wherein the phosphate is trisodium phosphate. A composition as claimed in any preceding claim, wherein the disease is caused by Streptococcus mutans , S. sanguis or Candida albicans . Use of a phosphate compound at a concentration in the range of from 0.02 to 3.0% by volume as a stabiliser to retard escape of chlorine dioxide from a mouthwash or dentifrice composition at a pH of from 6.0 to 7.4, said composition containing 0.005 to 0.5% by volume stabilised chlorine dioxide. Use as claimed in claim 9, wherein the phosphate is sodium monofluorophosphate or pyrophosphate, disodium hydrogen phosphate or sodium dihydrogen phosphate. Use as claimed in claim 9, wherein the phosphate is trisodium phosphate. Use of a composition as defined in any one of claims 1 to 7 in the manufacture of a dentifrice or mouthwash for treating dental disease by reducing the number of microorganisms in the mouth.

Inventors: RATCLIFF PERRY A, RATCLIFF, PERRY A.
Applicants: MICROPURE INC, MICROPURE, INC.
Application year: 1993
Main classification: A61K7
IPC codes: A61Q11, A61K8