Abstract:
The composition and method for the use of stabilized chlorine dioxide as an antimicrobial agent against oral microorganisms for the treatment and prevention of halitosis and prevention of oral diseases, wherein the activation and release of chlorine dioxide from the composition a) occurs rapidly and without a period of induction, b) results from the oxidative reduction and consumption of amino acids and volatile sulfur compound precursors, and c) generates twice the available chlorine dioxide gas as that generated from simply lowering the pH of the composition. The preferred concentrations of stabilized chlorine dioxide in this invention are in the range of 0.005% to 2.0% (w/v) wherein the pH of the composition is initially lowered by a citrate and then stabilized by a peroxy compound.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present application is a continuation of, claims priority to and the benefit of U.S. Ser. No. 12/704,360 filed Feb. 11, 2010 and entitled “Composition and Method for the Generation of Chlorine Dioxide from the Oxidative Consumption of Biomolecules.” The &#39;360 application includes subject matter disclosed in and claims priority to a U.S. Provisional Patent Application No. 61/152,336 filed Feb. 13, 2009 and entitled “Oxidative Consumption of Salivary Biomolecules,” assigned to the present assignee. Both of the aforementioned applications are incorporated herein by reference in their entirety. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates to the oxidative consumption of salivary biomolecules, in particular, it relates to the generation of chlorine dioxide for antibacterial affects in the oral cavity with a stabilized chlorine dioxide composition. 
       BACKGROUND OF INVENTION 
       [0003]    Oral disease refers to a number of generally preventable conditions of the mouth with a variety of causes. Plaque is the most recognizable precursor to oral disease. It is the biofilm that forms on teeth within hours after they are cleaned. The main mineral component of teeth is hydroxyapatite (HAP) and when teeth are cleaned, HAP becomes exposed to the oral environment. Salivary proteins such as mucins, proline-rich proteins, statherins, histatins, and cystatins have a strong affinity for HAP. These proteins quickly bind or adsorb to the exposed HAP of the tooth to form a thin coating called the acquired pellicle. Certain bacteria in the oral cavity selectively adhere to the pellicle, begin to divide, and form colonies. Initially, approximately 80% of the bacteria that colonize pellicle-coated tooth surfaces are facultative, gram-positive, non-motile cocci such as  Streptococcus sanguinis  (formerly  Streptococcus sanguis ). The other 20% include a variety of gram-negative bacteria such as  Veillonella  species. As the colonies grow, the environment changes due to the metabolic activities of these early colonizers and the addition of diverse groups of other bacteria to the biofilm (plaque) mass. An important environmental change in the plaque biofilm is the low-oxygen environment that promotes colonization and growth of anaerobic bacteria. Microorganisms in the biofilm synthesize a slime matrix or glycocalyx from the abundant polysaccharides, glycoproteins, and dietary sugars (e.g., sucrose) present in the oral environment. Eventually, the plaque becomes a characteristic biofilm with a highly structured, matrix-embedded, diverse microbial population in which gene expression is severely altered. The volume and structure of the biofilm created provides protection to the bacteria housed within it, potentially reducing the efficacy of antimicrobials. As a result, disruption of the biofilm of plaque is typically accomplished by mechanical means (e.g., brushing, flossing, professional tooth cleaning). Use of certain anti-plaque and antiseptic agents has been suggested for prevention of biofilms, but these treatments are typically tested in vitro using pure strains of microbes cultured on agar. Such in vitro conditions do not adequately simulate the biofilm environment, which may limit the significance of the test results. 
         [0004]    Within biofilms, continuous metabolic activity of bacteria produces acids that can demineralize tooth enamel and dentin leading to the development of dental caries and progressive tooth decay. This demineralization is irreversible unless there is early intervention by a dental professional who might recommend the inclusion of certain fluoride-containing oral care products in the daily dental routine. If left untouched, demineralization can progress to the inner layers of the tooth, leading to severe pain and increased potential for loss of the tooth. 
         [0005]    If dental plaque is left undisturbed, deeper portions of the plaque biofilm mineralize leading to the formation of calculus. Calculus has two major components, organic material and inorganic material. The organic portion of calculus consists mainly of dead bacteria. The inorganic part of calculus is composed of several minerals derived from calcium and phosphate present in the oral environment. There are two types of calculus, subgingival (below the gum line) and supragingival (above the gum line). Supragingival calculus is highly organized, porous, and visible. Once formed, calculus cannot be removed by conventional brushing and flossing; the intervention of a dental professional is generally required. Calculus retention is problematic for oral health because it harbors biofilm-forming bacteria that can lead to the development of periodontal (gum) infections. 
         [0006]    Halitosis (bad breath) is caused primarily by the presence of volatile sulfur compounds (VSCs) in expired breath. Approximately 90% of foul odors in expired mouth air are due to the presence of the two major VSCs: hydrogen sulfide (H 2 S) and methyl mercaptan (CH 3 SH— also called methanethiol). The sulfur in these VSCs comes from the breakdown by bacteria of sulfur-containing proteins from saliva, plaque, and sloughed epithelial cells. Increased production or build-up of any of the protein sources will lead to higher levels of VSCs in mouth air. 
         [0007]    There are a number of known situations that will lead to increased VSC production. For example, persons who do not perform adequate oral hygiene will have abundant amounts of supragingival and subgingival plaque biofilms on their teeth. This is especially true in difficult-to-clean locations such as interproximal areas between the teeth. In addition, natural teeth that support some dental prostheses are difficult to clean. Finally, the dorsal surface of the tongue is rough, irregular, and harbors large quantities of microorganisms. In general, the microorganisms in chronic intraoral biofilms will produce large quantities of VSCs. Besides being the major contributor to halitosis, VSCs are potent irritants and can aggravate existing inflammation of the gums. High levels of VSCs can kill epithelial cells that may lead to increased permeability and ulceration of the gum tissue. The existence of open wounds coupled with increased gum tissue permeability can promote the entry of bacteria into the bloodstream (i.e., bacteremia). Chronic bacteremia may increase the risk for the development of a numbers of systemic problems such as heart attacks, stroke, and adverse birth outcomes. 
         [0008]    Gingivitis is defined as the presence of gingival inflammation without loss of connective tissue attachment. The precursor to gingivitis is undisturbed dental plaque biofilms. Studies have shown that gingivitis will develop within 10-21 days if all oral hygiene practices are stopped and plaque is allowed to accumulate undisturbed. Clinical signs of gingivitis are redness, swelling (edema), and bleeding gums. 
         [0009]    Periodontitis refers to a group of infections in which the supporting tissues of the teeth such as connective tissue and bone are destroyed by plaque-induced inflammation. The most common form is known as Chronic Periodontitis that affects approximately 20% of the adult U.S. population. Signs of chronic periodontitis include all of those associated with gingivitis (i.e., redness, swelling, bleeding) plus the formation of deep periodontal pockets (increased probing depths), gingival recession, increased tooth mobility, and loss of bone as detected by radiographs. If left untreated, chronic periodontitis can lead to tooth loss. 
         [0010]    Several dozen types of oral bacteria have been implicated as putative periodontal pathogens including gram-negative bacteria such as:  Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Eikenella corrodens, Prevotella intermedia , and  Campylobacter rectus . Gram-positive bacteria of importance include  Streptococcus intermedius, Micromonas micros , and  Eubacterium  species. Spirochetes such as  Treponema denticola  are also important. Low levels of most of these pathogens can be isolated from healthy mouths. These bacteria only become a problem when they are left undisturbed in mature dental plaque biofilms. Finally, chronic periodontitis is a polymicrobial infection with multiple bacteria working together in a biofilm to cause the disease. 
         [0011]    Treatment of both gingivitis and chronic periodontitis is designed to facilitate the frequent removal and disruption of dental plaque biofilms. For gingivitis, effective oral hygiene practices on a daily basis are usually sufficient. This involves thorough removal of plaque from facial and lingual surfaces of the teeth with a toothbrush and good interproximal care with dental floss or other appropriate devices (e.g., toothpicks). Periodic tooth cleaning by an oral health care provider is required to remove mineralized plaque (i.e., calculus). Treatment of chronic periodontitis is more difficult since the disease-causing plaque is usually at subgingival sites and in deep periodontal pockets. Standard interventions usually include oral hygiene instructions followed by thorough subgingival debridement (i.e., scaling and root planing). If the infection persists, surgical intervention may be recommended to reduce the depth of the pockets and to gain access to thoroughly remove the calculus deposits on root surfaces. In some cases, reconstructive surgical procedures are performed in an attempt to regain some of the lost periodontal attachment and supporting bone. Once the infection is under control, the patient is placed on a rigorous maintenance/recall program to reduce the chances of recurrent infection. It is during this maintenance phase of therapy that non-invasive over-the-counter products are especially useful in slowing down the reformation of dental plaque biofilms on tooth surfaces. 
       Chlorine Dioxide 
       [0012]    The use of chlorine dioxide for sanitation was first suggested in 1948 by Eric Woodward to reduce the incidence of unpleasant taste in shrimp. Since then, chlorine dioxide use has spread into a number of other industries. The oxidative power of ClO 2  is used in the manufacture of wood pulp as an agent for the bleaching of cellulose fibers. In water treatment, ClO 2  has become widely used for water sanitation. In this case, it has been shown to be effective at reducing the bacterial content, algae content, and odor associated with wastewater treatment. Additionally, the utilization of ClO 2  for treating drinking water has been effective without adversely affecting its taste. The benefits of ClO 2  over other processes utilizing ozone or bleach for example, are reduced cost, reduced toxicity and reduced production of chlorinated by-products. 
         [0013]    In 1999 the EPA published “Alternative Disinfectants and Oxidants Guidance Manual,” describing disinfectant uses for ClO 2  and containing information on the mechanism of generation, application and standards and regulations governing use of ClO 2  and other disinfectants. Major applications listed by table 4-5, section 4.8.2 in the manual are as follows: primary or secondary disinfectant, taste control, odor control, TTHM/HAA reduction (total trihalomethanes are chlorinated organics, chloroform [CHCl 3 ] and dichlorobromomethane [CHCl 2 Br] for example; haloacetic acids are created when an atom from the halogen group, chlorine, for example, replaces a hydrogen on the acetic acid molecule), Fe and Mn control, color removal, sulfide destruction, phenol destruction and Zebra mussel control [EPA 1999, p. 4-34]. These are accomplished by oxidation of various substances found in water. For example, unpleasant tastes and odors (sulfides, phenols, others) can exist in water due to vegetative decay and algae content. ClO 2  reduces these tastes either by eliminating the source (algae) or oxidizing the causative taste and odor molecules. In the control of iron and manganese, ClO 2  will bring the dissolved ions out of solution to form precipitates, which may be eliminated through filtration and/or sedimentation. Zebra mussel control is important because it helps to maintain the natural ecology of a body of water. Zebra mussels are organisms that will infest a lake or river, strip it of nutrients and create a pseudo-fecal mucous layer on the bottom. The use of ClO 2  for water sanitation and pulp treatment generally involves on-site generation followed by immediate use. 
         [0014]    The term ‘stabilized chlorine dioxide’ on the other hand, refers to the generation and subsequent sequestration of ClO 2 , which allows for its storage and availability for later use. The first reference to stabilized chlorine dioxide in patent was in U.S. Pat. No. 2,482,891 in which ClO 2  is stabilized in a powder for storage. For its application, it is mixed with water to “liberate” the chlorine dioxide. A method and composition for the use of aqueous stabilized chlorine dioxide for antiseptic purposes was noted in U.S. Pat. No. 3,271,242. The 1979 text Chlorine Dioxide, Chemistry and Environmental Impact of Oxychlorine Compounds, describes (aqueous) stabilized chlorine dioxide as follows:
       “The stabilization of chlorine dioxide in aqueous solution was proposed by using perborates and percarbonates. Thus, a stabilized solution of ClO 2  would be obtained at pH 6 to 8 by passing gaseous ClO 2  into an aqueous solution containing 12% Na 2 CO 3 .3H 2 O 2 . Other variants are possible. In reality, it seems that in these methods, the chlorine dioxide is practically completely transformed to chlorite. Dioxide is released upon acidification . . . ” [Masschelein, 1979]       
 
         [0016]    The reference to percarbonates and perborates may be replaced by the term ‘peroxy compounds,’ which would refer to any buffer suitable for maintaining the pH and hence, the stability of the ClO 2  in solution. The buffer is a necessary component, as the ClO 2  is unstable at low pH. Once the solution reaches low pH or encounters an area of low pH, the stabilized ClO 2  is released from solution and available for sanitation and oxidation. 
         [0017]    In oral care products, the use of stabilized ClO 2  has been suggested as an active ingredient by a number of patents: U.S. Pat. Nos. 4,689,215; 4,696,811; 4,786,492; 4,788,053; 4,792,442; 4,793,989; 4,808,389; 4,818,519; 4,837,009; 4,851,213; 4,855,135; 4,886,657; 4,889,714; 4,925,656; 4,975,285; 5,200,171; 5,348,734; 5,489,435; 5,618,550. Additionally, the use of stabilized ClO 2  has been suggested for the degradation of amino acids in U.S. Pat. No. 6,136,348. The premise for these products is that the stabilized chlorine dioxide will remain as such until it encounters the localized reductions in pH. Reduced pH levels can be a result of low pH saliva or oral mucosa, the accumulation of oral disease-causing bacteria or the presence of plaque biofilms on teeth and epithelial cells. Once released, the now active chlorine dioxide is effective at killing bacteria and oxidizing VSCs. Data have shown dramatic reduction in bacteria after exposures as short as 10 seconds, as set forth in U.S. Pat. No. 4,689,215. Additional data show remarkable decrease in VSCs in expired mouth air; the mechanism is believed to be oxidation of VSCs through the cleavage of the sulfide bonds. 
         [0018]    The effectiveness of the chlorine dioxide is likely dependent on the amount of ClO 2  released from stabilized chlorine dioxide when the solution is acidified. The amount of ClO 2  released depends on the initial concentration of the solution, its pH, and the stabilizing buffer or agent used. It could follow that that the efficacy of the chlorine dioxide as an oral care product is dependent on the amount of ClO 2  released from the stabilized chlorine dioxide solution. As a result, it is imperative that accurate, precise measurements are taken so the concentration of stabilized ClO 2  and of the release of ClO 2  from solution can be determined. In addition to the need to quantify the efficacy of the solution, concentrations must be understood to ensure the safety of the product. 
         [0019]    A concern about the stability of stabilized ClO 2  was recited in U.S. Pat. No. 5,738,840 with reference to the inclusion of “other oxychlorine species” which could refer to chloride [Cl − ] or chlorate [ClO 3   − ]. The mechanism of action was questioned and suggested that at pH between 6.2 and 7.0 “any molecular chlorine dioxide which forms by degradation of the chlorite is converted back to chlorite by reaction with the residual stabilizer.” This reverse reaction is unlikely due to the lower pH in the bacteria-laden target areas of the mouth described earlier. U.S. Pat. No. 6,231,830 calls into question the stoichiometry and safety of the formulation presented in U.S. Pat. No. 5,738,840. It is claimed that the formulation described is a ‘chlorinator’ in which “ . . . a build-up of chlorate ion, an unwanted by-product” may occur. 
       PRIOR ART 
       [0020]    Previous inventions contemplate the use of stabilized chloride dioxide as a bactericide for the treatment gingivitis as well as a deodorizing agent for the treatment of oral malodor (Ratcliff, U.S. Pat. No. 4,689,215; Madray, U.S. Pat. No. 6,231,830 B1; Richter, U.S. Pat. No. 5,738,840; Witt, U.S. Pat. No. 6,350,438 B1). There is a large amount of evidence that indicates chlorine dioxide has bactericidal properties and that the chlorine dioxide serves to attack malodorous volatile sulfur compounds in the mouth by splitting of the sulfide bonds (Lynch et al., 1997; Silwood et al., 2001). 
         [0021]    Grootveld et al. (2001) demonstrated that an admixture of oxohalogen oxidants chlorite and chlorine dioxide significantly reduces the number of  Streptococcus mutans  and lactobacilli.  Candida albicans  exhibited a decrease however not statistically significant. The research collected saliva samples from 33 dental patients prior to and following rinsing with the admixture oral rinse and measured the levels of each organism. 
         [0022]    Research completed by Lynch et al. (1997) evaluated the oxidative consumption of salivary biomolecules by an oral rinse preparation containing an admixture of stable free radical species chlorine dioxide with chlorite anions.  1 HNMR spectroscopy was used to obtain multicomponent evaluations of the actions of the oral rinse in the treatment of periodontal diseases and dental caries. Saliva samples were collected from 10 volunteers prior to and following rinsing and analyzed using the  1 HNMR. Results indicated that the oxidative decarboxylation of salivary pyruvate and the oxidative consumption of urate, thiocyanate anion, and amino acids cysteine and methionine. The reductions in biomolecules included, but not limited to the following components: short-chain non-volatile carboxylic acid anions. The study revealed that the oral rinse composition of stable free radical species chlorine dioxide with chlorite anions reduces and removes pathogenic micro-organisms when used as an oral rinse. 
         [0023]    Inventors, Ratcliff and Lynch, U.S. Pat. No. 6,136,348, suggest degradation of amino acids with the use of stabilized chlorine dioxide. The premise for the composition described in the patent is that stabilized chlorine dioxide is chlorine dioxide stabilized as a sodium chlorite at a neutral or alkaline pH. The composition will remain as such until it encounters the localized reductions in pH as in saliva. The formation of chlorine dioxide is a slow process and the effectiveness of the chlorine dioxide is likely dependent on the amount released from the stabilized chlorine dioxide. The patent describes the weak bonds between some amino acids, like cysteine, leading to susceptibility to being destroyed by oxidative consumption. 
         [0024]    While prior art teaches various compositions of stabilized chlorine dioxide relative to oral health, they do not teach a method of stabilized chlorine dioxide to oxidatively consuming salivary biomolecules to produce antimicrobial affects for the reduction of growth and development of oral bacteria and microorganisms concerned with halitosis and oral disease by the generation of chlorine dioxide. 
       SUMMARY OF THE INVENTION 
       [0025]    Stabilized chlorine dioxide has a beneficial effect of tending to prevent a number of factors of oral disease, both by eliminating the bacteria that cause them and also by oxidizing molecules associated with them using a solution in the form of a wash, rinse, soak, paste, gel, aerosol spray, or other suitable delivery system. 
         [0026]    A buffered solution of aqueous sodium chlorite, when in solution at neutral to alkaline pH, is considered stabilized chlorine dioxide because it does not release the chlorine dioxide until it is acidified. It follows that measurement of the concentration of stabilized chlorine dioxide is not, in fact, a measurement of chlorine dioxide (ClO 2 ) contained in solution, but a quantification of the concentration of (aqueous) chlorite (ClO 2 —) in solution. Once acidified, the amount of ClO 2  released is limited by and a direct result of the ClO 2 — concentration. 
         [0027]    For liquids such as mouthwash, the standard unit of measurement when expressing concentration is weight-volume percentage. That is, a certain weight of component, solid, liquid, or dissolved in a solvent, is present in a certain volume of total mouthwash. Preferred concentrations of stabilized chlorine dioxide in this invention are in the range of 0.005% to 2.0% (w/v). 
         [0028]    Halitosis is caused by the presence of volatile sulfur compounds. By which the sulfur compounds are produced from oral bacteria and other microorganisms, including fungi and virus forms, in the oral cavity and when undisturbed or not removed can lead to plaque and development of oral diseases, including gingivitis and periodontitis. Within the diverse ecology of the oral cavity and plaque are complex salivary biomolecules required for microorganisms to function, grow and develop. These salivary biomolecules act as building blocks for reproduction, increasing numbers of microorganisms and volatile sulfur compounds in the oral cavity leading to halitosis. By reducing or eliminating the presence of salivary biomolecules with stabilized chlorine dioxide, the growth and numbers of microorganisms in the oral cavity will be reduced or eliminated and therefore treating and preventing halitosis. 
         [0029]    It is therefore a primary object of the present invention to provide stabilized chlorine dioxide as an antimicrobial agent against the oral microorganisms by generating chlorine dioxide by the oxidative consumption of salivary biomolecules. 
         [0030]    Another object of the present invention is to provide stabilized chlorine dioxide as a halitosis treatment and prevention by the oxidative consumption and inactivation of volatile sulfur compounds and their amino acid precursors to alleviate halitosis. 
         [0031]    Still another object of the present invention is to oxidatively consume and inactivate salivary biomolecules, including pyruvate, methionine, trimethylamine, tyrosine, glycine, creatine, 3-D-hydroxybutyrate, salivary taurine, lactate, and lysine. 
         [0032]    Yet another object of the present invention is to provide stabilized chlorine dioxide composition in a solution or other delivery vehicle such as in the form of a wash, rinse, soak, paste, gel, or aerosol spray to deprive microorganisms of salivary biomolecules as necessary compounds to grow and develop. 
         [0033]    A further object of the present invention is to prevent halitosis with stabilized chlorine dioxide composition by oxidatively consuming salivary biomolecules to eliminate and prevent microorganisms from growing and development in the oral cavity. 
         [0034]    A still further object of the present invention is to treat halitosis with stabilized chlorine dioxide composition by oxidatively consuming salivary biomolecules to eliminate and prevent microorganisms from growth and development in the oral cavity. 
         [0035]    Yet a further object of the present invention is to provide antimicrobial affects of stabilized chlorine dioxide on oral bacterial by producing chlorine dioxide as a product of oxidatively consuming salivary biomolecules. 
         [0036]    These and other objects of the present invention will become apparent to those skilled in the art as the description thereof proceeds. 
     
    
     
       BRIEF DESCRIPTION OF FIGURES 
         [0037]      FIG. 1 , (a) and (b), illustrates the expanded 0.80-4.25 ppm regions of the 600.13 Mhz single-pulse  1 H NMR spectra of a human salivary supernatant specimen (pH value 6.78) acquired (a) prior to and (b) subsequent to treatment with oral rinse I according to the procedure outlined in the Materials and Methods section. Abbreviations: A. Acetate-C H   3 ; Ala I and II, alanine-C H   3  and —C H  group proton respectively; Bu I, β-hydroxybutyrate proton γ-CH 3  group protons; Bu II, III and IV, β-hydroxybutyrate β, β′, and α protons respectively (ABX coupling system); iso-But I and II, iso-butyrate-C H   3  and —C H  group protons respectively; n-But I, II and III, n-butyrate γ, β, and α protons respectively; Chol, choline-N +  (C H   3 ) 3 ; Cit, Citrate-AB-CH 2 —CO 2   − ; DMeN, dimethylamine-C H   3 ; Eth I and II, ethanol-C H   3  and —C H   2  group protons respectively; Form, formate- H ; Gly, glycine-C H ; His I and II, histidine ABX system β protons; Lac I and II, lactate-C H   3  and —C H  protons respectively; Leu I, II, III and IV, leucine δ, γ, β, and α protons respectively; MeGu, methylguanidine-CH 3 ; MeN, methylamine-C H   3 ; Meth, methanol-C H   3 ; N—Ac, spectral region for acetamido methyl groups of N-acetyl sugars; Phe I and II, phenylalanine  AB X β protons; Prop I and II, propionate-C H   3  and —C H   2  group protons respectively; Pyr, pyruvate-CH 3 ; Sar I and II, sarcosine-CH 3  and —CH 2  group protons respectively; Suc, succinate-CH 2 ; Tau I and II, Taurine-CH 2 NH 3   +  and —CH 2 SO 3   −  protons respectively; TMeN, trimethylamine-C H   3 , Tyr I and II, tyrosine  AB X β protons; Tyr III, tyrosine AB X  α proton; n-Val I and II, n-valerate δ and γ protons respectively. 
           [0038]      FIG. 2  illustrates a plot of absorbance at 262 nm (A 262 ) versus chlorite concentration for a series of calibration standards in the 1.60-8.00 mM concentration range 
           [0039]      FIG. 3( a )  illustrates a reversed-phase (RP) ion-pair (IP) chromatograms of a 1.00 mM chlorite standard solution. The retention time of ClO 2   −  was 6.90 min. 
           [0040]      FIG. 3( b )  illustrates a reversed-phase (RP) ion-pair (IP) chromatograms of oral rinse I formulation (diluted ¼ with doubly-distilled water prior to analysis). The retention time of ClO 2   −  was 6.90 min. 
           [0041]      FIG. 3( c )  illustrates a reversed-phase (RP) ion-pair (IP) chromatograms of a typical salivary supernatant sample (0.10 ml) pre-treated with 0.50 ml of the above oral rinse I. The retention time of ClO 2   −  was 6.90 min. 
           [0042]      FIG. 4  illustrates a plot of chlorite peak area (μV·s −1 ) obtained from the HPLC analysis versus chlorite concentration for a series of chlorite calibration standards 
       
    
    
     DESCRIPTION OF THE INVENTION 
       [0043]    This invention relates to the discovery through research of the composition for and methodology of generating of chlorine dioxide by a stabilized chlorine dioxide composition through the oxidatively consuming salivary biomolecules in the oral cavity and producing antimicrobial affects on oral bacteria and microorganisms concerned with halitosis and oral disease with the reduction of growth and development. Chlorine dioxide is known to be a strong oxidizer and is capable of oxidizing amino acids. The work of Lynch et al. proves so with the degradation of cysteine and methionine into pyruvate in the presence of an admixture of stable free radical species chlorine dioxide with chlorite anions (1997). This was confirmed with the following evidence of research suggesting oxidative consumptions of salivary biomolecules and interactions of stabilized chlorine dioxide as chlorite with human salivary biomolecules. The oxidative decarboxylation of salivary pyruvate by stabilized chlorine dioxide composition indicates a mechanism of action of the interaction of this invention with salivary biomolecules as an antimicrobial agent. 
         [0044]    The specific mechanism of action of ‘stabilized’ chlorine dioxide (specifically, chlorite anion) on oral organisms and biomolecules has not been fully investigated. The present invention research evidence suggests that stabilized chlorine dioxide oxidatively consumes salivary biomolecules and creates products that may exert bactericidal and bacteriostatic effects on the oral bacterial cells which ultimately gives rise to cell death. These effects can lead to control over the formation of bacterial plaque and the adverse generation of malodorous volatile sulfur compounds, major contributors to oral diseases. 
         [0045]    The purpose of researching the oxidentive consumption of salivary biomolecules this investigation was to determine: (1) the metabolic profile of human saliva and the capacity of salivary biomolecules to react with stabilized chlorine dioxide oral rinse, (2) the amount of chlorine dioxide generated from chlorite when the oral rinse is mixed with saliva, and how much chlorine dioxide is consumed or chlorite remains, and (3) an assay technique for monitoring chlorine dioxide activity in saliva, as well as determining the level of volatile sulfur compounds after being treated with a stabilized chlorine dioxide rinse. The oral rinse compositions included a concentration of 0.1% (w/v) and 0.4% (w/v) stabilized chlorine dioxide. These formulations are designated as oral rinse I and II, respectively. 
         [0046]    This research suggested that the stabilized chlorine dioxide composition has the capacity to clinically alleviate oral malodor by the direct oxidative inactivation of volatile sulfur compounds and their amino acid precursors. These results also reveal a new mechanism of action of stabilized chlorine dioxide (chlorite), specifically its reaction with human salivary biomolecules to produce chlorine dioxide. 
       Materials and Methods 
     Spectrophotometric Determination of Chlorite Concentrations in Oral Rinse Formulations 
       [0047]    For oral rinse I, 1.00 ml aliquots were diluted to a total volume of 3.00 ml with doubly-distilled water and electronic absorption spectra of these solutions were recorded on a Unicam UV-2 spectrophotometer in the 190-400 nm wavelength range. Similarly, 0.20 ml volumes of oral rinse II were diluted to a final volume of 3.00 ml with doubly-distilled water and electronic absorption spectra were also acquired in this manner. Chlorite concentrations were determined via measurement of its absorbance at 262 nm [ε=160 M −1  cm −1 , as determined in this study]. A further series of these oral rinse solutions were pre-treated with the amino acid L-glycine (final concentration 2.00 mM) to remove hypochlorous acid/hypochlorite (HOCl/OCl − ) and chlorine dioxide (ClO 2   • ), the former generating glycine monochloroamine via equation A. 
         [0000]      H 3 N + —CH 2 —CO 2   − +OCl − →Cl—NH—CH 2 —CO 2   − +H 2 O  (A)
 
         [0048]    Results acquired revealed that there were no differences between spectra obtained before and after glycine treatment, indicating that these potentially interfering, further oxohalogen oxidants were absent from the oral rinse formulations examined. 
       Volunteer Recruitment and Collection of Samples 
       [0049]    A series of non-medically-compromised volunteers (n=20) without any form of active periodontal disease or active dental caries were recruited to the study. To avoid interferences arising from the introduction of exogenous agents into the oral environment, volunteers were requested to collect all saliva available, i.e., (‘whole’ saliva expectorated from the mouth) into a plastic universal tube immediately after waking in the morning on a pre-selected day. 
         [0050]    Each volunteer was also requested to refrain completely from oral activities (i.e., eating, drinking, tooth-brushing, oral rinsing, smoking, etc.) during the short period between awakening and sample collection (ca. 5 min.). Each collection tube contained sufficient sodium fluoride (15 μmol.) to ensure that metabolites are not generated or consumed via the actions of micro-organisms or their enzymes present in whole saliva during periods of sample preparation and/or storage. 
         [0051]    Saliva specimens were transported to the laboratory on ice and then centrifuged immediately (3,000 r.p.m for 15 min.) on their arrival to remove cells and debris, and the resulting supernatants were stored at −70° C. for a maximum duration of 18 hr. prior to analysis. The pH values of each supernatant were determined prior to  1 H NMR analysis. 
         [0000]    Spectrophotometric Analysis of Residual (Unreacted) Chlorite Anion (ClO 2   − ) in Oral Rinse/Salivary Supernatant Mixtures 
         [0052]    An ATI Unicam UV-VIS UV-2 spectrophotometer was employed for the determination of residual chlorite in each of the salivary supernatants collected in order to determine its level of consumption by biomolecules therein on equilibration. 
         [0053]    0.09 ml aliquots of each salivary supernatant specimen were treated with 0.450 ml of oral rinse I. This mixture was thoroughly rotamixed and diluted to a final volume of 1.20 ml to yield an absorbance value of approximately 1 at 262 nm. The reference cell contained an equivalent volume of corresponding salivary supernatant diluted to a final volume of 1.20 ml with doubly-distilled H 2 O. Initially, scans were made over the wavelength range of 190-300 nm. 
         [0054]    Since oral rinse II contained exactly four times the concentration of ClO 2   −  [0.4% (w/v)], 0.10 ml aliquots of each salivary supernatant specimen were treated with 0.500 ml of this product, and once thoroughly rotamixed, a 0.135 ml aliquot of this mixture was diluted to a final volume of 1.20 ml with H 2 O. The reference cell contained 22.5 μl of salivary supernatant diluted to 1.20 ml with H 2 O. 
         [0055]    ClO 2   −  has a wavelength of maximum absorbance (λ max ) at 262 nm (ε=160 M −1  cm −1 ) and therefore was readily detectable at the volumes (and hence concentrations of ClO 2   − ) of each oral rinse added. 
         [0056]    Where required, the pH value of samples were adjusted to a value of 1.00 and samples were then equilibrated at ambient temperature for a 24 hr. period (to ensure conversion of each mole of ClO 2   −  remaining to 0.50 of an equivalent of ClO 2   • ) in order to improve the sensitivity of this assay system [ClO 2   •  has a λ max  value in the visible region (360 nm) with ε=1,150 M −1  cm −1 ]. 
         [0000]    HPLC Monitoring of the Interaction of the Oral Rinse Oxohalogen Oxidants with Intact Human Saliva 
         [0057]    The chlorite level remaining in each salivary supernatant sample was also determined using a novel high-performance liquid chromatographic (HPLC) technique employing a reversed-phase C18 column with the ion-pair reagent hexadecyl-trimethylammonium bromide (HTB) present in the mobile phase. The operating system utilised was a Waters Millennium HPLC system, consisting of a Waters 626 Pump, Waters 996 Photodiode Array Detector and a Waters in-line degasser remotely operated using Waters unique Millennium software. 
         [0058]    Samples were prepared via the treatment of 0.10 ml volumes of saliva supernatants with 0.50 ml aliquots of ¼ diluted oral rinses I and II. Once thoroughly rotamixed, 10 μl aliquots of the resulting solutions were injected using a remotely-operated automated auto-sampler with injector onto a reversed-phase C18 ODS Column (4.6×75 mm). A Spherisorb S5-ODS 1 guard column was employed to remove any potential analytical column contaminants. 
         [0059]    The mobile phase was de-gassed using an in-line degasser. The mobile phase consisted of 2% (w/v) borate/gluconate buffer with 2% (v/v) butan-1-ol and 12% (v/v) acetonitrile (final pH 7.2) and operated at a flow rate of 1.10 ml/min. The ion-pair reagent (Hexadecyl-trimethylammonium Bromide) was added at a final concentration of 50.00 mM in order to ensure that ClO 2   −  is readily separated from interfering salivary components. This analyte was identified by comparisons of its peak&#39;s absorption spectrum generated by the photo-diode array detector (λ max.  262 nm) with that of an authentic chlorite standard. 
       Preparation of Human Salivary Supernatant Samples for  1 H NMR Analysis 
       [0060]    Each individual salivary supernatant sample was divided into three equivalent portions (0.60 ml). In total, there were three separate specimen reaction mixtures: 3.0 ml of oral rinses I and II were added to the first and second salivary supernatant samples respectively, whilst the third served as an untreated control in which 3.0 ml of H 2 O was added to the original 0.6 ml volume of salivary supernatant. The samples were then thoroughly rotamixed to ensure a homogenous mixture and then equilibrated at 37° C. for a period of 30 s. 
         [0061]    Samples were prepared by adding 0.05 ml of deuterium oxide ( 2 H 2 O, providing a field frequency lock) and 0.05 ml of a 5.0 mM solution of sodium 3-trimethylsilyl [2,2,3,3- 2 H 4 ] propionate [TSP, chemical shift reference (δ=0.00 ppm) and internal quantitative standard] in  2 H 2 O to a 0.60 ml volume of each sample examined. 
         [0062]    Each sample was then subjected to multicomponent high resolution  1 H NMR analysis in order to identify the nature of salivary biomolecules which react with ClO 2   −  and/or ClO 2   • . i.e., via oxidative consumption or otherwise, together with the products generated from such reaction systems. 
         1 H NMR Measurements 
       [0063]    One-dimensional (1-D)  1 H NMR spectra were acquired on a Bruker AMX-600 spectrometer (ULIRS, Queen Mary, University of London facility, U.K) operating at a frequency of 600.13 MHz and a probe temperature of 298 K. The intense water signal (δ=4.80 ppm) was suppressed by presaturation via gated decoupling during the delay between pulses. 
         [0064]    Pulsing conditions for 1-D spectra acquired on salivary supernatant and oral rinse samples were: 128 free induction decays (FIDS); 16,384 data points; 3-7 μs pulses; 1.0 s pulse repetition rate. Line-broadening functions of 0.30 Hz were routinely utilised for the processing of experimental NMR data. Where present, the methyl group resonances of lactate (δ=1.330 ppm) and alanine (δ=1.481 ppm) served as secondary internal references for the control and oral rinse-treated salivary supernatant samples examined. 
       Results 
       1 H NMR Analysis of Oral Rinse Formulations I and II 
       [0065]      1 H NMR spectra acquired on the oral rinse I formulation contained clear, prominent resonances ascribable to citrate [—C H   2 CO 2   −  protons, δ=2.65 ppm (dd, AB coupling system)] which serves as a buffering agent, with lower intensity signals arising from acetate [—C H   3  group, singlet (s) located at 1.92 ppm] and formate [ − O 2 C— H  singlet (s), δ=8.46 ppm]. Ethanol [—C H   3  and —C H   2 OH group protons, δ=1.21(t) and 3.66(q) respectively] was also detectable at trace levels. 
         [0066]    Spectra acquired on the oral rinse II product also contained resonances ascribable to citrate [—C H   2 CO 2   −  protons, δ=2.65 ppm (dd, AB coupling system)] and lower intensity signals arising from trace levels of acetate [—C H   3  group, singlet (s) located at 1.92 ppm] and formate [ − O 2 C— H  singlet (s), δ=8.46 ppm]. 
         [0000]      1 H NMR Analysis of the Interaction of ClO 2   − -Containing Oral Rinse Formulations with Human Salivary Supernatant Specimens 
         [0067]    600 MHz  1 H NMR spectra were acquired for every salivary supernatant sample examined (i.e., a total of 60, 3 daily specimens collected from each of 20 human volunteers). A typical  1 H NMR spectrum of a human salivary supernatant sample is shown in  FIG. 2( a ) ; that of the same saliva specimen pre-treated with Oral Rinse I is displayed in  FIG. 2( b ) . These  1 H NMR investigations [of the oxidative consumption of salivary biomolecules by oxohalogen oxidants present in Oral Rinses I and II tested (predominantly ClO 2 )] revealed that:
       1. Pyruvate was oxidatively decarboxylated to acetate and CO 2      2. The volatile sulphur compound (VSC) precursor methionine was oxidised to its corresponding sulphoxide   3. A resonance ascribable to malodorous trimethylamine (s, δ=2.91 ppm) was reduced in intensity (a process presumably resulting in its transformation to trimethylamine oxide)   4. Tyrosine was oxidised (presumably to a quinone species)   5. The Glycine α-CH 2  group resonance was reduced in intensity, an observation possibly attributable to its reaction with trace levels of hypochlorite/hypochlorous acid present in the oral rinses (generating mono- and/or dichloroamine species)   6. The concentrations of creatinine and 3-D-hydroxybutyrate were diminished following treatment with each oral rinse, an observation consistent with their oxidative consumption by oxohalogen species present therein.   7. Salivary taurine decreased in concentration post treatment.   8. Lactate-CH 3  and —CH signals were diminished in intensity following treatment.   9. Resonances ascribable to lysine were reduced in intensity post-treatment.       
 
         [0077]    With regard to these  1 H NMR analysis results acquired, the consumption of salivary methionine by chlorite is of much importance to oral hygiene and clinical periodontology since both CH 3 SH and H 2 S are generated from this amino acid via metabolic pathways operational in gram-negative micro-organisms. Hence, data acquired here indicates that the oral rinses examined have the capacity to clinically alleviate oral malodour via the direct oxidative inactivation of VSCs and their amino acid precursors. 
         [0078]    As demonstrated here, the techniques employed are of much value concerning multicomponent assessments of the interactions of chlorite with human salivary biomolecules, and the oxidative decarboxylation of salivary pyruvate by this oxohalogen oxidant serves as an important example of this which may be of some relevance to its mechanisms of action. 
       Spectrophotometric Analysis of Chlorite Calibration Standards 
       [0079]    Prior to spectrophotometric analysis of Oral Rinses I and II, the extinction coefficient of chlorite (ClO 2   − ) was determined at its λ max  value of 262 nm. This was conducted by analysing authentic ClO 2   −  calibration standards (1.60-8.00 mM, Table 1 and  FIG. 2 ). Each measurement was made in triplicate in order to ensure the reproducibility of data acquired. Plots of absorbance at 262 nm (A 262 ) versus chlorite concentration were clearly linear: the extinction coefficient was determined as ε=160 M −1  cm −1 , and the correlation coefficient (r) for the plot shown in Table 1 was 0.9955. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Absorbance values at 262 nm for replicate (n = 3) determinations 
               
               
                 obtained for a series of chlorite calibration standards (1.60-8.00 mM) 
               
             
          
           
               
                   
                 Concentration (mM) 
                 1st 
                 2nd 
                 3rd 
               
               
                   
                   
               
             
          
           
               
                   
                 1.60 
                 0.274 
                 0.274 
                 0.273 
               
               
                   
                 2.40 
                 0.405 
                 0.404 
                 0.403 
               
               
                   
                 3.20 
                 0.509 
                 0.509 
                 0.508 
               
               
                   
                 4.00 
                 0.632 
                 0.63 
                 0.631 
               
               
                   
                 4.80 
                 0.784 
                 0.783 
                 0.783 
               
               
                   
                 5.60 
                 1.032 
                 1.034 
                 1.033 
               
               
                   
                 6.40 
                 1.055 
                 1.056 
                 1.055 
               
               
                   
                 7.20 
                 1.161 
                 1.163 
                 1.161 
               
               
                   
                 8.00 
                 1.253 
                 1.252 
                 1.252 
               
               
                   
                   
               
             
          
         
       
     
         [0080]    Treatment of the water diluent with up to 20% (v/v) ethanol exerted no influence on the final absorbance values obtained, an observation which confirmed that this potential contaminant exerted no influence on the spectrophotometric assay of chlorite performed in this manner (i.e., no reaction between these agents was noted under our experimental conditions). 
       Spectrophotometric Determination of the Consumption of Oral Rinse Chlorite by Human Salivary Supernatant Specimens 
       [0081]    Following the establishment of ClO 2   − &#39;s extinction coefficient (via the acquisition of electronic absorption spectra on a series of its calibration standards), difference spectrophotometric analysis of chlorite in each of the salivary supernatant/oral rinse mixtures was performed in order to determine its level of consumption by biomolecules therein on equilibration. In this manner, the decrease in absorbance at 262 nm observed following equilibration of the oral rinse formulations with human salivary supernatants according to the procedure outlined in methods was employed to estimate the level of oral rinse chlorite (ClO 2   − ) consumption by this biofluid. Table 2(a) gives the concentrations of chlorite consumed (per ml of saliva) for reaction mixtures containing a 5:1 volume ratio of oral rinse:salivary supernatant. 
         [0000]                                                                TABLE 2(a)                   Spectrophotometric determination of the consumption of oral rinse        ClO 2   −  by human salivary supernatant samples (μmol. ClO 2   −         consumed per ml of saliva).             Patient                Code   Oral Rinse I   Oral Rinse II                    J1   0.1640   0.1504   0.1776   0.0944   0.1168   0.1192       J2   0.0200   0.0096   0.0176   0.0360   0.0280   0.0472       J3   0.3040   0.3152   0.3040   0.3552   0.3024   0.3024       BR1   0.0400   0.0760   0.0600   0.0696   0.0584   0.1136       BR2   0.1136   0.0752   0.1008   0.1392   0.1136   0.1808       BR3   0.2968   0.2800   0.3096   0.0976   0.0504   0.0776       G1   0.0008   0.0104   0.0168   0.0584   0.1448   0.1168       G2   0.0168   0.0080   0.0112   0.1528   0.1664   0.1640       G3   0.0392   0.0624   0.0584   0.0864   0.0720   0.0920       U1   0.0200   0.0112   0.0200   0.0528   0.0360   0.0248       U2   0.0168   0.0040   0.0072   0.0168   0.0304   0.0392       U3   0.0168   0.0216   0.0144   0.4504   0.3888   0.4056       M1   0.0696   0.0728   0.0704   0.1000   0.1528   0.1280       M2   0.0072   0.0016   0.0080   0.2024   0.1504   0.1608       M3   0.0000   0.0048   0.0016   0.0192   0.0224   0.0224       L1   0.0064   0.0040   0.0128   0.0248   0.0024   0.0112       L2   0.0104   0.0064   0.0056   0.0416   0.0832   0.0696       L3   0.0296   0.0320   0.0352   0.0664   0.064   0.0608       SB1   0.1408   0.1576   0.1272   0.0720   0.0416   0.0664       SB2   0.1400   0.1496   0.1336   0.2888   0.3584   0.2776       SB3   0.0240   0.0240   0.0264   0.0224   0.0248   0.0336       I1   0.0336   0.0424   0.0296   0.1112   0.1336   0.1528       I2   0.0856   0.0752   0.0544   0.0448   0.0336   0.0248       I3   0.0264   0.0216   0.0240   0.072   0.0976   0.0808       R1   0.0376   0.0536   0.0368   0.0080   0.0224   0.0056       R2   0.0056   0.0016   0.0000   0.1000   0.0752   0.0888       R3   0.0056   0.0104   0.0104   0.0224   0.0112   0.0192       ZK1   0.0088   0.0096   0.0096   0.0664   0.0552   0.0608       ZK2   0.1032   0.1376   0.1248   0.0976   0.0752   0.1136       ZK3   0.0232   0.0192   0.0264   0.1168   0.0808   0.0832       V1   0.2000   0.2128   0.2264   1.4808   1.4888   1.4696       V2   0.0328   0.0408   0.0472   0.2168   0.1552   0.1976       V3   0.0704   0.0680   0.0672   0.7000   0.6528   0.6304       Z1   0.0120   0.0096   0.0128   0.0664   0.0528   0.0552       Z2   0.0240   0.0224   0.0184   0.0056   0.0024   0.0000       Z3   0.0232   0.0184   0.0104   0.0504   0.0472   0.0552       GG1   0.0344   0.0216   0.0328   0.2000   0.1752   0.1944       GG2   0.1400   0.1296   0.1296   0.2392   0.2584   0.2472       GG3   0.0088   0.0136   0.0104   0.0024   0.0136   0.008       N1   0.0056   0.0064   0.0040   0.1976   0.2080   0.2000       N2   0.0112   0.008   0.0048   0.0696   0.0752   0.0528       N3   0.0184   0.0200   0.0120   0.0112   0.0168   0.0056       ED1   0.0792   0.0576   0.0920   0.2752   0.2720   0.2448       ED2   0.3184   0.3352   0.3288   0.3168   0.4640   0.4808       ED3   0.0288   0.0224   0.0160   0.1080   0.1248   0.0608       AB1   0.0112   0.0232   0.0256   0.2112   0.1608   0.1080       AB2   0.0024   0.0032   0.0048   0.0584   0.1000   0.0944       AB3   0.008   0.0104   0.0072   0.0552   0.0696   0.0696       S1   0.2512   0.2736   0.2624   0.964   0.9832   1.0304       S2   0.1952   0.1728   0.1584   0.1472   0.1696   0.1056       S3   0.1176   0.1744   0.1440   0.5448   0.5776   0.5696       DG1   0.1104   0.1144   0.0880   0.0504   0.0392   0.0664       DG2   0.0192   0.0328   0.0208   0.0552   0.1360   0.0080       DG3   0.0088   0.0120   0.0096   0.0808   0.0832   0.0888       SG1   0.0336   0.0456   0.0544   0   0   0.0024       SG2   0.0744   0.0944   0.0656   0.1136   0.1080   0.1080       SG3   0.0144   0.0120   0.0120   0.1504   0.1168   0.1392       P1   0.0128   0.0112   0.0152   0.1112   0.1336   0.1304       P2   0.0176   0.0200   0.0248   0.0472   0.0392   0.0504       P3   0.0600   0.0504   0.0408   0.0640   0.1024   0.1472               Abbreviations: patient codes in the rows refer to volunteers, whilst columns represent oral rinse treatments, with three independent sampling days ‘nested’ within each treatment.            
Multifactorial Analysis-of-Variance of Difference Spectrophotometric Data Involving the Determination of ClO 2   −  Consumption by Salivary Biomolecules
 
         [0082]    Statistical analysis of data acquired regarding the difference spectrophotometric determination of ClO 2   −  consumption by salivary biomolecules [i.e., multifactorial analysis-of-variance (ANOVA)] revealed highly significant differences between (1) the ClO 2   −  content of each oral rinse investigated (p&lt;&lt;0.001), (2) volunteers (p&lt;0.01) and (3) ‘days nested within volunteers’ (p&lt;0.001). Indeed, estimates of the overall mean consumption of ClO 2   −  determined for a reaction mixture containing a 5:1 (v/v) ratio of oral rinse:human salivary supernatant were 6.334×10 −2  and 1.626×10 −1  μmol. ClO 2   −  per ml of salivary supernatant for Oral Rinses I and II respectively. The ‘between replicates’ mean square value was only 1.266×10 −4 , indicating a high level of reproducibility on repeat (triplicate) determinations conducted on each sample tested. The full ANOVA table is shown in Table 2(b). 
         [0000]                                                                                TABLE 2(b)                   Multifactorial analysis-of-variance (ANOVA) table for data acquired       from the study involving the difference spectrophotometric        determination of ClO 2   −  consumption by salivary biomolecules.             Source of                                Variation   d.f   SS   MS   F   p   EMS                    (1) Between ClO 2   −     1   1.3839   1.3839   64.37   &lt;&lt;0.001           concentrations                               (Fixed Effect)                               (2) Between   19   6.5421   0.3443   2.54   &lt;0.01   σ 2  + 6σ o   2  + 18σ 2   v         Volunteers                               (Random Effect)                               (3) Between   40   5.4146   0.1354   6.30   &lt;0.001   σ 2  + 6σ o   2         Sampling                               Days within                               Volunteers                               (Random Effect)                               (4) Error   295   6.3504   0.0215           σ 2          (Residual)                               (5) Between   4   5.065 × 10 −4     1.266 × 10 −4                     Replicates                               Total   359   19.6915                               Abbreviations: d.f., degrees-of freedom;       SS, sum of squares values;       MS, mean square values;       F, F variance ratio statistic;       EMS, expected mean square.            
Development of a Novel HPLC Method for Monitoring Oral Rinse Chlorite Consumption and its Oxidative Interaction with Salivary Biomolecules
 
         [0083]    In this section, the development of an HPLC method for the determination of ClO 2   •  in human saliva specimens (i.e., prior and subsequent to its treatment with the oral rinse formulations) is described. 
         [0084]    The chlorite level remaining in each salivary supernatant sample was determined using a high-performance liquid chromatographic (HPLC) technique employing a reversed-phase C18 column with the ion-pair reagent hexadecyl-trimethylammonium bromide (HTB) present in the mobile phase. 
         [0085]    Experiments involving alteration of the ion pair reagent concentration from 5.00 to 50.00 mM showed that a concentration of 50.00 mM gave rise to a good resolution of ClO 2   −  from salivary components in all samples investigated. Identification of the ClO 2   −  peak was based on its retention time (6.9 min) and the diode-array spectrum of its HPLC peak (λ max  262 nm). Injection of authentic sodium chlorite calibration standards (1.00-10.00 mM) demonstrated a clear linear relationship between peak intensity and concentration. Typical chromatograms of a 1.00 mM chlorite standard solution, the oral rinse I formulation (diluted ¼ with doubly-distilled water prior to analysis) and a typical salivary supernatant sample (0.10 ml) pre-treated with 0.50 ml of the above oral rinse (I) are shown in  FIGS. 3( a ), ( b ) and ( c )  respectively. The retention time of ClO 2   −  was 6.90 min. 
         [0086]    Plots of chlorite peak area (Table 3) versus its concentration were clearly linear ( FIG. 4 ). 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Area under chlorite peak (μV/sec.) values obtained 
               
               
                 via HPLC analysis of known chlorite calibration standards 
               
             
          
           
               
                   
                 Concentration (mM) 
                 Mean value 
                 uV/sec 
               
               
                   
                   
               
             
          
           
               
                   
                 0.80 
                 70833 
                 69102 
                 69879 
               
               
                   
                 1.60 
                 151673 
                 151878 
                 153334 
               
               
                   
                 2.40 
                 208530 
                 209419 
                 210975 
               
               
                   
                 3.20 
                 259823 
                 258413 
                 259662 
               
               
                   
                 4.00 
                 326322 
                 326592 
                 326771 
               
               
                   
                 4.80 
                 394229 
                 394023 
                 394386 
               
               
                   
                 5.60 
                 514239 
                 510086 
                 513058 
               
               
                   
                 6.40 
                 535511 
                 535418 
                 530565 
               
               
                   
                 7.20 
                 586871 
                 592830 
                 585209 
               
               
                   
                 8.00 
                 628810 
                 628254 
                 622356 
               
               
                   
                   
               
             
          
         
       
     
       CONCLUSIONS 
       [0087]    Results acquired on the consumption of (relatively) simple amino acids such as glycine, alanine and taurine by the oral rinse tested here (predominantly containing ClO 2   −  as an oxidant) are explicable by previous investigations conducted on the kinetics and mechanisms of the reactions of such biomolecules with oxyhalogen oxidants (including ClO 2   − ) as outlined below. 
         [0088]    Of much relevance to the substantial extent of salivary taurine consumption by the oral rinses investigated in the studies are experiments reported by Chinake and Simoyi (1997) on the oxidation of this β-amino acid by ClO 2   −  (at neutral to acidic pH values, i.e., those which are relevant to the oral environment). Indeed, the stoichiometry of this reaction system was found to involve the consumption of 3 molar equivalents of ClO 2   −  per mole of taurine to generate 1 of taurine&#39;s N-monochloroamine [Cl(H)NCH 2 CH 2 SO 3 H] and 2 of ClO 2   •  (the production of N-monochlorotaurine is rapid when expressed relative to that of ClO 2   •  accumulation); at the lower pH values investigated, N-monochlorotaurine disassociated to taurine and N-dichlorotaurine. An important characteristic of this reaction system involves a significant induction period in which both HOCl and the reactive intermediate H(OH)NCH 2 CH 2 SO 3 H are produced, a process leading to the formation of N-chlorotaurine and ClO 2   •  autocatalytically. As expected for redox reactions involving ClO 2   − , this autocatalysis is mediated by a Cl 2 O 2  intermediate species, and interestingly, taurine&#39;s C—S bond is not cleaved, despite the availability of the powerful oxidant HOCl. 
         [0089]    Hence, these previously reported studies clearly explain the substantial  1 H NMR-detectable reductions in salivary taurine observed on treatment of human salivary supernatant specimens with the tested oral rinse ClO 2   − . They also indicate that the oral rinse-induced oxidative consumption of a range of α-amino acids present in this biofluid also detected in this investigation, specifically free (non-protein-incorporated) alanine, arginine, aspartate, cysteine, glutamate, glutamine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, tyrosine and valine, also proceed via this mechanism. 
         [0090]    However, since many N α -monochloroamines generated in this manner are unstable at physiological temperature (37° C.) (Hazen et. al. (1998)), and decompose to corresponding aldehydes (equation 1), and hence further investigations focused on the detection and quantification of such species corresponding to the side-chains of α-amino acids (e.g., formaldehyde from salivary glycine, acetaldehyde from alanine, etc.) are required in order to demonstrate this. 
         [0000]      Cl(H)N—CHR—CO 2   − +H 2 O→RCHO+NH 3 +CO 2 +Cl −   (1)
 
         [0091]    Interestingly, it is well known that aldehydes act as potent microbicidal agents, and hence those derived from the above processes may also exert this activity in the oral environment. Indeed, a 2.0% (w/v) solution of this agent is frequently employed as a disinfectant (Follente et. al.). 
         [0092]    Similarly, the oxidative consumption of γ-aminobutyrate (GABA) noted here is likely to proceed via a similar mechanism. However, the amino acids cysteine, methionine and tyrosine, each with redox-active side-chains can, of course, also be oxidatively modified by ClO 2   −  (and also ClO 2   •  and HOCl/OCl −  produced via its reaction with these and/or further α-amino acids, together with GABA and particularly taurine) to cysteine sulphonate (and cysteine), methionine sulphoxide (equation 2) and a tyrosine-derived quinone species respectively. 
         [0000]      H 3 N + CH(CH 2 CH 2 SCH 3 )CO 2   − +ClO 2   − →H 3 N + CH(CH 2 CH 2 SOCH 3 )CO 2   − +OCl −   (2)
 
         [0093]    With regard to the oxidative consumption of salivary α-keto acid anions, particularly pyruvate and α-ketoglutarte, by ClO 2   −  present in the tested oral rinses, which was also observed in our investigations, it has been previously noted that an intense green Cl 2 /OCl −  colouration is generated on reaction of ClO 2   −  with pyruvate (equation 3) [Lynch et. al. 1997]. Hence, such reaction systems clearly generate HOCl/OCl −  which can, of course, subsequently produce N-monochloro- and -dichloroamines from free or, in selected cases, protein-incorporated amino acids, the former decomposing to corresponding aldehydes under physiological conditions. 
         [0000]      CH 3 COCO 2   − +ClO 2   − →CH 3 CO 2   − +CO 2 +OCl −   (3)
 
         [0094]    Therefore, it should be noted that the production of reactive HOCl/OCl −  during an induction period observed during the reaction of ClO 2   −  with the β-amino acid taurine (Chinake and Simoyi (1997)) (and also presumably the salivary α-amino acids and γ-aminobutyrate consumed on reaction with tested oral rinse ClO 2   − ) will also serve to further reduce the amino acid concentrations of human saliva. Indeed, even if this mechanistic process only proceeds in the reactions of selected free amino acids with ClO 2   −  (or those located at the N-termini of salivary proteins), the HOCl/OCl −  generated will, of course, be available to react with a much wider range of such HOCl/OCl −  ‘scavenger’ species in a (relatively) unselective manner to form N α -monochloro- and dichloroamines, together with N ε -monochloro- and -dichloroamines in lysine residues (either free or protein-incorporated). As noted above, specific aldehydes arising from the decomposition of their parent amino acid N α -monochloroamine precursors will serve as valuable indicators of the activity of HOCl/OCl −  arising from these reaction systems (RCHO, where R represents an amino acid side-chain moiety). 
         [0095]    Aldehydes produced from the interaction of HOCl/OCl −  with salivary α-amino acids and the decomposition of the primary N α -monochloroamine products can also react with ClO 2   − , and the oxidation of formaldehyde (HCHO) by this oxyhalogen oxidant was critically examined by Chinake et. al. (1998) in both mildly acidic and alkaline media. This reaction gave rise to CO 2  and ClO 2   •  as products, the latter in virtually quantitative yield, and was autocatalytic with respect to hypochlorous acid/hypochlorite (HOCl/OCl − ). Indeed, the primary phase of the process generated HOCl which facilitated (catalysed) the production of ClO 2   •  and the additional oxidation of formic acid/formate (HCO 2 H/HCO 2   − ); ClO 2   •  rapidly accumulated in view of its (relative) lack of reactivity towards both HCHO and HCO 2 H/HCO 2   − . Although with excess HCHO the stoichiometry of this process was determined to be 3ClO 2   − +HCHO→HCO 2 H+2ClO 2 (aq.)   • +Cl − +2H 2 O, when large excesses of ClO 2   −  were present [as, of course, expected in the case of 5:1 (v/v) mixtures of tested oral rinses:human salivary supernatant], the stoichiometric profile involved in the consumption of 6 molar equivalents of ClO 2   −  per mole of HCHO to generate 4 of ClO 2   • , 2 of Cl −  and 1 of CO 2 . 
         [0096]    With regard to the oral rinse-mediated decrease in the intensities of salivary cysteine resonances observed here (and also in previously-conducted chemical model studies (Lynch et. al., 1997), Darkwa et. al. (2003) investigated the oxidative consumption of N-acetylcysteine by ClO 2   − , and found that the final product generated from this reaction system was N-acetylsulphonate and that the process had a stoichiometry of 3ClO 2   − +2RSH→3Cl − +2RSO 3 H; as expected, there was no evidence for the production of N-chloroamine derivatives. This oxidation proceeds via a mechanism involving a stepwise S-oxygenation process involving the consecutive generation of sulphenic and sulphonic acid adducts. Intriguingly, a notable characteristic of the reaction is the rapid, immediate formation of chlorine dioxide (ClO 2   • ) without a monitorable induction period since oxidation of the thiol by this oxyhalogen free radical species is sufficiently slow for it to accumulate without such a time lag which, in general, represents a characteristic of the oxidation of organosulphur compounds by ClO 2   − . A full description of the ‘global’ dynamics of this system involves 8 reactions in a truncated mechanism. 
         [0097]    In conclusion, evidence provided in our investigations clearly demonstrate that the generation of ClO 2   •  from ClO 2   −  in the oral environment is not entirely dependent on entry of the latter into acidotic environments therein (equations 4 and 5, the pK a  value of the ClO 2   − /HClO 2  system being 2.31 (Lynch et al. 1997)). Although the mean pH value of this biofluid is ca. 7 when unchallenged with oral stimuli (i.e., ‘resting’), the consumption of relatively large volumes of beverages of lower pH value (ca. pH 4) can clearly exert a significant influence on this parameter. However, it should also be noted that the pH value of primary root caries lesions can approach a limit of 4.5, and therefore this represents an environment in which there are expected to be marked elevations in the level of HClO 2  generated (i.e., from 0.0020% at pH 7.00 to 0.64% of total available oxyhalogen oxidant at pH 4.50), although it should be noted that, in view of the pK a  value of the ClO 2   − /HClO 2  couple, this value still remains very low when expressed relative the total amount of oxyhalogen oxidant available (the remainder being ClO 2   −  in the absence of alternative means of producing ClO 2   • , or HOCl/OCl − , from the interaction of ClO 2   −  with α-, β- and γ-amino acids available). Of course, from the stoichiometry of equation 5, 2 molar equivalents of ClO 2   •  are generated per 4 of HClO 2 , and hence the above figures for HClO 2  generation represent double that of the total ClO 2   •  producible (i.e., maximum percentages of 0.0010 and 0.32% of total oxyhalogen oxidant at pH values of 7.00 and 4.50 respectively). Clearly, the rate of ClO 2   •  generation from HClO 2  should also be considered in view of the short oral rinse-salivary supernatant equilibration time involved in our studies. 
         [0000]      ClO 2   − +H + →HClO 2 (pK a =2.31)  (4)
 
         [0000]      4HClO 2 →2ClO 2   • +ClO 3   − +Cl − +H 2 O  (5)