Title:
METHOD AND COMPOSITION FOR PREVENTION AND TREATMENT OF ORAL FUNGAL INFECTIONS
Kind Code:
A1


Abstract:
A composition of stabilized chlorine dioxide at a concentration range of about 0.0004% to about 0.8% (w/v) having anti fungal properties to prevent oral fungal infections and method of use are disclosed.



Inventors:
Young, Elena J. (Scottsdale, AZ, US)
Ratcliff, James L. (Pueblo West, CO, US)
Application Number:
12/500163
Publication Date:
01/14/2010
Filing Date:
07/09/2009
Assignee:
Micropure, Inc. (Scottsdale, AZ, US)
Primary Class:
International Classes:
A61K33/00; A61P31/04
View Patent Images:



Other References:
Pfaller et al., "Epidemiology of Invasive Candidiasis: a Persistent Public Health Problem." Clinical Microbiology Reviews, Jan. 2007, p. 133-163.
Pfaller et al., "Epidemiology of Invasive Candidiasis: a Persistent Public Health Problem." Clinical Microbiology Reviews, Jan. 2007, p. 133-163; vol. 20, No. 1.
Primary Examiner:
WEBB, WALTER E
Attorney, Agent or Firm:
SNELL & WILMER L.L.P. (Main) (PHOENIX, AZ, US)
Claims:
We claim:

1. A composition inhibiting fungal infection in the oral cavity by inhibiting Candida species including C. albicans, C. dubliniensis, C. glabrata, and C. krusei with a solution of stabilized chlorine dioxide at a concentration in the range of about 0.0004% to about 0.05% (w/v).

2. A composition for the treatment and prevention of fungal infection in the oral cavity by the fungicidal effects on Candida species also including C. albicans, C. dubliniensis, C. glabrata, and C. krusei with a solution of stabilized chlorine dioxide at a concentration in the range of about 0.4% to about 0.8% (w/v).

3. A method for reducing and killing Candida albicans, C. dubliniensis, C. glabrata, and C. krusei by application of a solution of stabilized chlorine dioxide at a concentration in the range of about 0.0004% to about 0.8% (w/v).

4. A method of inhibiting the growth of Candida albicans, C. dubliniensis, C. glabrata, and C. krusei by application of a solution of stabilized chlorine dioxide at a concentration in the range of about 0.0004% to about 0.8% (w/v).

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is related to and claims priority to a provisional application entitled “METHOD AND COMPOSITION FOR PREVENTION AND TREATMENT OF ORAL FUNGAL INFECTIONS” filed Jul. 10, 2008, and assigned Ser. No. 61/079,532.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention relates to the use of stabilized chlorine dioxide in topical oral compositions to prevent oral fungal infections.

2. Description of Related art

Thrush, clinically termed oral candidiasis, is the most common opportunistic fungal infection in humans. Thrush is caused by the imbalance of microorganisms in the oral cavity allowing Candida species (fungus or yeast) to grow out of control causing infection with development of white lesions and potentially spreading to other parts of the body, including the esophagus, lungs, liver, and skin. Four types of oral thrush are recognized: angular cheilitis, denture stomatitis, erythematous candidiasis, and pseudomembranous candidiasis. Thrush may involve several species of Candida resident in the oral ecology, each with its own characteristics and susceptibility to treatments.

Candida species are found in the oral cavity as normal commensal microorganisms and may overgrow when the host response is weakened, such as in immunocompromised individuals. Immunocompromised conditions include HIV/AIDS, nutritional deficiencies, metabolic disorders such as diabetes, malignancies, xerostomia, medication side effects, aging, pregnancy, Sjogrens syndrome, dentures, and smokers.

The amount of Candida colonization in the oral cavity of denture wearers was higher (Abu-Elteen and Abu-Alteen, 1998). Studies that observed oral cavities of immunocompromised patients indicate that patients who wore dentures were associated with increased numbers of yeasts, more specifically Candida species (Willis et al., 1999; Gonclaves et al., 2006).

The oral microbiological environment can be significantly affected by tobacco smoking, specifically having an effect on oral bacteria and fungi, particularly Candida. The impact of smoking on thrush varies in combination to pre-existing conditions (dentures, HIV, and diabetes) (Soysa and Ellepola, 2005). Increasingly, studies show smokers have greater numbers of oral Candida carriage than non-smokers (Abu-Elteen and Abu-Alteen, 1998; Willis et al., 1999). Several studies suggest that smoking has a significant affect on the incidence of thrush in immunocompromised patients. Smoking is an important risk indicator for thrush, especially in HIV infected patients (Chattopadhyay et al., 2005). Conley et al. (1996), Campisis et al. (2002), and Slavinsky et al. (2002) found significant associations between thrush and smoking in HIV infected individuals. Willis et al. (1999) reported that seventy seven percent (77%) of diabetic patients carried Candida species in the mouth. Among these patients, there was a significant increase in the tobacco smokers. Smoking alone or in combination with other factors may be contributory to the development of thrush.

Thrush is the most common and earliest oral manifestation of HIV/AIDS caused mostly by Candida species. HIV/AIDS patients commonly have dry mouth, pain and may develop oral lesions from thrush, which can interfere with oral intake of food and lead to severe malnutrition. HIV related oral manifestations occur in an estimated 30-80% of HIV patients and are often under diagnosed and misdiagnosed. Thrush will develop in up to 90% of all advanced untreated HIV infections, with 60% experiencing at least one episode per year with recurrences. (Samaranayake et al., 1989; McCarthy et al., 1991). Thrush is often the first indicator of progression from HIV to AIDS; this was confirmed in a study by Sharma et al. (2006) who showed that there was a 2.5 time increased risk of progression from HIV to AIDS in patients with thrush. The progression indicates the immunological decline and is manifest by decreased CD4+ T-lymphocyte cell counts, which contribute to the risk of developing both thrush and oral hairy leukoplakia. Chattopadhyay et al. (2005 and 2007) reported a correlation that showed low CD4+ counts and smoking are independent risk factors for thrush and oral hairy leukoplakia.

Cancer treatments, cytoxic chemotherapy and radiotherapy, have short and long term side effects including thrush. The incidence of thrush in cancer patients ranges widely depending on the stage of the cancer, doses of treatments, method of diagnosis and other predisposing factors. Davies et al. (2006) found that 66% of cancer patients carried oral Candida and other yeast species and 30% had thrush. Another study reported 25% of patients receiving radiation for head and neck cancer had high prevalence of Candida colonization in the oral cavity (Redding et al., 2004). There is evidence that thrush can also spread to the esophagus and develop esophageal candidiasis (Samonis et al, 1998). This finding underscores the importance of preventing and reducing the risk of thrush in all immunocompromised patients.

Diabetes mellitus patients have increased susceptibility to certain infections, which can lead to poor metabolic control. Studies have shown that oral candidal infections are more common in diabetic patients than in non-diabetics. Takasawa, et al. (2006) reported a case study of the association of diabetes with thrush. The case involved a 75-year old healthy patient who developed diabetes and candidiasis (oral and esophageal) within a short interval with limited risk factors. The patient was diagnosed with type 2 diabetes accompanied by severe thrush and esophageal candidiasis. The case indicates a relationship between diabetes and oral infection, wherein diabetes may cause oral infections and conversely oral infection may stimulate the development of diabetes (Taylor, 2008).

Candida species have been isolated from oral cavities of diabetic patients. Willis et al., 1999 found 77% of diabetic patients carried oral Candida species. This study also established that a number of contributory factors affect candidal colonization; these include smoking, dentures, type and duration of diabetes and the degree of glycaemic control. Willis et al. also isolated several different species of Candida in combination to the predominate species, Candida albicans. Goncalves et al. (2006) investigated the oral yeast colonization and antifungal susceptibility in diabetic patients, isolating several non-albican species, including C. tropicalis, C. glabrata, C. krusei, C. rugosa, C. guillermondii, and C. parapsilosis. This study tested the resistance of these species to the antifungal treatment fluconazole, and found high levels of resistance by the non-albican species.

Treatment and therapy of thrush varies with each medical condition. Prevalent recommended therapies currently include nystatin, azole antifungal agents and amphotericin B preparations. Initial episodes of oral thrush in healthy children and adults can be treated effectively with topical therapies, including clotrimazole troches, nystatin suspensions or pastilles (Rex et al., 2000); however, individuals with immunocompromised systems will often have recurrent episodes of infections making it difficult to treat with these therapies. A resistance to the therapies may also develop with any regimen. Most patients will initially respond to topical therapies; however, immunocompromised patients will often experience symptomatic relapses sooner.

Oral azoles, nystatin, amphotericin B, and chlorhexidine are several therapies administered orally for the treatment of oral thrush. The azoles include fluconazole, itraconazole, and ketoconazole, which can be capsules/tablets or liquid suspensions taken by mouth and absorbed by the gastrointestinal tract. Oral fluconazole is better tolerated than itraconazole and ketoconazole. Capsule azoles are found to be less effective than the oral suspensions due to variable absorption. Nystatin and amphotericin B are less effective at preventing fungal infections than prophylactic therapies with fluconazole. Most recurring infections are due to prior use of the therapies where the fungi developed resistance to the treatments; individuals with recurring infections must change from one oral treatment to other treatments over time. For instance, thrush infections resistant to fluconazole will respond to oral itraconazole about two-thirds of the time. When the patient is not responding to itraconazole, amphotericin B oral suspension may be effective. A high dose of medication for a short period is recommended to reduce the development of candidal resistance.

Chlorhexidine gluconate (CHX) has antifungal properties, and it is widely used by dental professionals as an antimicrobial oral rinse. While it may be effectively used as a preventive to the development of thrush, it has not been proven effective as a treatment. Objectional taste and teeth staining lead to problematic use of CHX continuously. Worthington et al. (2008) reviewed literature pertaining to the effectiveness of interventions and medications for treating thrush in cancer patients, concluding that drugs absorbed or partially absorbed from the GI tract are more effective than those not absorbed (including nystatin and amphotericin B).

Candida albicans is usually the predominant species in thrush, however other species of Candida have been emerging as significant pathogens in patients. Non-albicans species of Candida have been isolated in combination with C. albicans in cancer and HIV patients. They have been observed to cause more severe immunosuppression, and consequently are more difficult to treat. Cartledge et al. (1999) reported that from 100 non-albicans isolates obtained from HIV patients with thrush, 88 were resistant to fluconazole. There is a need for a treatment with high susceptibility to all types Candida species.

Non-albicans species commonly found in saliva of patients with oral lesions (with or without oral thrush) include C. tropicalis, C. glabrata, C. parapsilosis, C. Krusei, and C. dubliniensis (Oliveria et al., 2007; Coleman et al., 1997). A study by Davies et al. showed 25% of samples taken involved non-albicans species (including C. glabrata, C. dubliniensis, and C. tropicalis) were the predominant organisms and a contributing factor in 19% of samples taken from cancer patients with thrush (2006).

Candida glabrata, formerly known as Torulopsis glabrata, is a significant human pathogen and is the second leading cause of oral thrush (Li et al., 2007). Its association with thrush is unclear as some research suggests that it is only a commensal organism and does not contribute directly to infections. However, it is also observed that its presence with C. albicans in HIV-positive patients present more severe and difficult to treat forms of thrush, requiring higher doses of fluconazole medication. Other treatments for C. glabrata infections include itraconazole and amphotericin B solutions; however much like other treatments for fungal infections, a percentage of C. glabrata treated with these medications become resistant to them. C. glabrata is dose-dependent to fluconazole, and may require higher doses than does Candida albicans in order to be effective. The C. glabrata is the second most frequent species in elderly patients with and without dentures. Lockhart et al. found that patients with dentures had an increase in C. glabrata frequency from 36% to 58% in elderly ages 70-79 yrs and 80 years and older, respectively (1999). Candida glabrata is an increasingly common species found in all cases of thrush infections and is very difficult to treat due to its resistance to commonly used drugs.

Candida dubliniensis has been found mostly in oral cavities of HIV-positive and AIDS patients, especially those that received fluconazole therapy. C. dubliniensis has phenotypic characteristics similar to C. albicans and displays the same antifungal susceptibilities. Research has found that fluconazole can be ineffective for managing diverse infections that include C. albicans and C. dubliniensis species due to their combined development of resistance to the drug (Moran et al., 1997). HIV-positive patients with large doses of medications are more vulnerable to developing resistance. C. dubliniensis has been effectively treated with several common azoles therapies including ketoconazole, itraconazole, and amphotericin B. C. dubliniensis is also susceptible to triazoles, including voriconazole, posaconazole and ravuconazole.

Candida krusei colonization in the oral cavity is increasingly common. Thrush with C. krusei also includes the presence of C. albicans. Itraconazole solutions were proven effective in treating Candida krusei in thrush patients, but C. krusei infections were resistant to both fluconazole and ketoconazole (Cartledge et al., 1999).

Thrush caused by the colonization of Candida tropicalis is rare and is susceptible to any antifungal treatment. However, its presence in thrush of cancer patients receiving chemotherapy can be very pathogenic and may lead to hemotologic infections.

New orally administered, ingested antifungal drugs, including terbafine, azoles, and echnocandins, are currently being tested as treatments of thrush. Studies show these new drugs may be more effective in treating thrush involving non-albicans infections. For instance, Bagg et al. (2005) shows in vitro tests of voriconazole to be effective on fungal oral infections which are resistant to other antifungals including fluconazole and itraconazole. However, this study also showed C. glabrata not to be fully susceptible to voriconazole. Voriconazole must be administered with care due to its significant drug interactions and its contraindication with several other drugs.

There is a limited capacity of current pharmaceutical drugs to prevent and treat Candida infections. Candida species are recognized to become resistant to most fungicidal treatments over time, and different species are more or less resistant to treatment and various medications. In several cases, the resistance to antifungals can be reduced with use at higher doses but such dosing only can be used for a short time (Rose, 2004). Certain individuals suffering from oral thrush (cancer, HIV, and diabetes) require extended treatments that correspond to their medical conditions. Similarly, pregnant women and the elderly may require oral thrush treatments extending over several months and therefore may not be able to use the higher dosages over extended time without untoward consequences. Immunocompromised patients often are diagnosed with underlying conditions that require several medications, complicating treatment with the prospect of negative drug interactions. Given the limitations of antifungals' effectiveness against candidal infections, the higher doses may be useful and appropriate largely for mild cases because most severe cases require longer periods of treatment.

The treatment of thrush becomes particularly difficult when several different Candida species are present and when other existing systemic conditions complicate treatment. Prevention of thrush among populations most at risk is preferable than treatment because it permits immunocompromised patients to maintain their health and diet and may lead to less severe and/or less frequent cases. Therefore, there is a need for a composition for both the prevention and the treatment of thrush, which is safe and effective in inhibiting, reducing and eliminating all oral Candida species involved in infections.

SUMMARY OF THE INVENTION

The present invention relates to a composition containing stabilized chlorine dioxide that may be used for treatment of the mouth either in a solution, for example as a mouthwash, in concentrations below approximately 0.8% (w/v) for the control of disease-causing bacteria, bacterial plaque, and oral malodor. Mint oils or extracts may be added to flavor an oral rinse or oral spray of stabilized chlorine dioxide in such a manner that the flavoring would not interact with stabilized chlorine dioxide or affect the stability of the formulation.

It is therefore a primary object of the present invention to provide a composition of stabilized chlorine dioxide to prevent and treat specific Candida species, including C. dubliniensis, C. glabrata, C. krusei, and C. tropicalis.

Another object of the present invention is to provide a composition of stabilized chlorine dioxide in a concentration equal to or greater than 0.4% (w/v) to prevent and treat fungal infection in the oral cavity.

Still another object of the present invention is to provide a method for prevention and treating fungal infection in the oral cavity.

Yet another object of the present invention is to provide a method for inhibiting the growth of Candida albicans, C. dubliniensis, C. glabrat and C. krusei.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The term chlorine dioxide is widely used in the industry. Those skilled in the art will and do appreciate the various forms or variations thereof, which are available to perform certain intended functions and purposes. Furthermore, U.S. Pat. No. 3,271,242 describes a form of stabilized chlorine dioxide and a method of making the product and a mechanism of action, which is particularly useful in carrying out the present invention.

Masschelein (1979) teaches that the bactericidal properties of chlorine dioxide were well known before its first applicable use in the 1950's. Today, chlorine dioxide is used as a drinking water treatment obtained from sodium chlorite producing a solution free of chlorine. Stabilized chlorine dioxide is an aqueous solution comprising chlorite and stabilizers. When the pH of stabilized chlorine dioxide lowers from a neutral pH, molecular chlorine dioxide releases from the aqueous solution. This mechanism of action of stabilized chlorine dioxide has bactericidal and bacteriostatic effects on the microbial ecology of aerobic, facultative, and anaerobic pathogenic bacteria.

Previous inventions contemplate the use of stabilized chlorine dioxide for the prevention and treatment of gingivitis and periodontitis, as well as dental caries (Ratcliff, U.S. Pat. Nos. 5,200,171 and 5,348,734). Theses patents describe the basic composition and use of stabilized chlorine dioxide oral rinse of the present invention. The Ratcliff inventions claim the prevention and treatment of dental diseases by reducing the number of oral microbial pathogens, including yeasts such as Candida albicans, at concentration ranges between about 0.005-0.5% stabilized chlorine dioxide. This prior art does not contemplate the use of stabilized chlorine dioxide for the prevention of oral thrush or in the inhibition of growth of other Candida species.

Prior art compositions that have been used and tested have been accepted to an extent of efficacy in treatments and prevention of abnormal conditions of the epithelium bodily orifices, such as oral nasal, ocular, auditory, rectal, vaginal, and urethral canal orifices (Ratcliff, U.S. Pat. Nos. 5,489,435 and 5,618,550). The claims of the previous invention described the in vitro study of Candida culture exposed to a stabilized chlorine dioxide solution resulting in more than 99% of Candida albicans reduced within 10 seconds.

Several antifungal compounds claim to treat fungal infections of the oral cavity (Francois U.S. Pat. No. 5,707,975 and Lipton U.S. Pat. No. 6,780,838). Francois et al. present an antifungal invention comprising of a cyclodextrin formulation for oral administration to treat fungal infections. Lipton et al. claim an invention with a therapeutically effective amount of one or more selected peptides in combination with a fungicide as a treatment of oral fungal pathologies. However, these inventions do not propose use of stabilized chlorine dioxide as the active ingredient for prevention and treatment of thrush.

There are several well-established advantages to stabilized chlorine dioxide as an antifungal including its broad range of antiseptic abilities, established safety, method of action, ability to be used over time, its low cost (relative to the aforementioned antifungal drugs), and ease of use (Mohammed et al, 2004). The present invention consists of stabilized chlorine dioxide at concentration ranges that exhibit fungistatic and fungicidal properties and may be used for the prevention and treatment of fungal infections and diseases in the oral cavity. Unlike, current treatment for oral fungal infections, the present invention can be used for any length of time without decreasing effectiveness due to fungal resistance, is effective against several major species of Candida found to cause oral thrush, does not have objectional taste, and does not cause teeth staining.

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. For example, preferred concentrations of stabilized chlorine dioxide as used herein may be in the range of 0.0004% to 2% (w/v).

The terms “topical oral care composition” and “oral composition” as used herein are meant to describe a product, which is not intentionally swallowed for purposes of systemic administration of therapeutic agents, but is retained in the oral cavity for a sufficient time to contact substantially all of the dental surfaces and/or oral mucosal tissues for purposes of oral activity.

The present invention focuses on fungicidal properties of stabilized chlorine dioxide. Present evidence shows that the effects of stabilized chlorine dioxide on several Candida species significantly reduce candidal reproduction, both individual species and species in colonial forms. There is no prior art claiming stabilized chlorine dioxide as a preventative or treatment on specific Candida species, including C. dubliniensis, C. glabrata, C. krusei, and C. tropicalis.

Supporting evidence for the antifungal properties of the present invention are observed in Mohammad et al.'s clinical study of investigating the efficacy of chlorine dioxide mouth rinse as a topical antiseptic treating chronic atrophic candidiasis (2004). The study concluded that a 0.8% concentration chlorine dioxide mouth rinse demonstrated management of chronic atrophic candidiasis. Results indicated significant clinical improvement after 10 days. Mohammad et al. shows that the 0.8% chlorine dioxide had a statistically significant effect on improving the oral soft tissues of the thrush as well as reducing the microbial count. The present invention and this study indicate thrush generally, and overgrowth of certain species of Candida specifically, can be prevented safely and effectively with dosages lower than those employed by Mohammad et al.

Wirthlin et al. (2001) supports the present invention's safety and effectiveness of 0.1% stabilized chlorine dioxide oral rinse. Thirty-eight subjects in the clinical study tested 0.1% oral rinse and a placebo for 6 months. Wirthlin et al. observed no clinical overgrowth of Candida species or reported no adverse effects on teeth, restorations, or soft tissues with the use of the oral rinse. Additionally, the oral rinse did not affect taste or stain the teeth of the patients. It was also determined from the study that yeasts, enterics, black-pigmented Porphyromonas, Prevotella, Actinomyces, Fusobacteria or Streptococci species showed no resistance or decreased susceptibility to stabilized chlorine dioxide.

The present invention concerns oral care compositions including oral washes or rinses, oral gels, toothpaste dentifrices, and oral spray in a solution comprising of stabilized chlorine dioxide. It contemplates the use of stabilized chlorine dioxide as a fungistatic and fungicidal agent against yeast species involved in oral disease such as, but not limited to C. albicans, C. glabrata, C. krusei, and C. dubliniensis. The mechanism for the composition includes the determined inhibitory and fungistatic activity of the stabilized chlorine dioxide compositions against four clinical isolates of Candida.

The present invention consists of a stabilized chlorine dioxide composition, which acts as a fungistatic agent on the aforementioned Candida species at a concentration ranges between 0.0004%-0.05% (w/v) and as a fungicidal agent on the aforementioned Candida species at a concentration ranges between 0.4%-0.8% (w/v).

The present invention proclaims the use of stabilized chlorine dioxide oral rinse, dentifrice, oral spray, or oral gel as a fungistatic treatment on Candida species with a minimum concentration of 0.0004% (w/v). The present invention contemplates the ability of stabilized chlorine dioxide as a fungistatic agent against Candida species involved in thrush. For example, it was shown in the present invention that the re-growth of C. albicans, C. dubliniensis, C. glabrata, and C. krusei were inhibited, showing a fungistatic effect on fungi involved in thrush. There is little or no prior research claiming inhibited growth of Candida species, including C. albicans, C. dubliniensis, C. glabrata, and C. krusei, after exposure to stabilized chlorine dioxide. Present research indicates a stabilized chlorine dioxide composition has fungistatic effects on the Candida species ultimately leading to fungal cell death. This inhibition of cellular metabolism and cell function effectively inhibits or controls the overgrowth and formation candidal infections, the main contributors to human fungal infections.

The present invention has an effect of killing and reducing the number of Candida fungi at concentrations lower than that known in the prior art. The present invention established the fungicidal kinetics of the antimicrobial characteristics of stabilized chlorine dioxide against Candida species at minimum fungicidal concentrations of equal to or greater than 0.4% (w/v). The present invention acts as a fungicide on the following fungi: C. albicans, C. dubliniensis, C. glabrata, and C. krusei. Given the predominance of these Candida species, individually and in naturally occurring colonies, a stabilized chlorine dioxide oral composition is believed to be effective on the majority candidal fungi involved in the oral fungal infection (thrush).

The specific mechanism in which chlorine dioxide inactivates fungi and bacteria is currently postulated and researched. Therefore, it is believed that the present invention's fungistatic properties are due to inhibition of protein synthesis and/or to the inability of the cell to maintain membrane permeability and inhibited metabolic processes. Due to these effects on fungi and bacteria, a stabilized chlorine dioxide oral composition can inhibit plaque production and progression to oral diseases and thrush. This can be accomplished by individuals rinsing their mouths with said composition in a concentration range of 0.0004% to 0.8% (w/v) or brushing teeth and thereby exposing the oral cavity to the active ingredients in comparable concentration, or by using an oral spray in the oral cavity, or by other comparable delivery mechanism. The following mechanisms of action specify the explanations for fungicidal and bacterial kill by chlorine dioxide.

The specific mechanism of action of chlorine dioxide on cells has been debated for a number of years. Early research showed that chlorine dioxide's primary effect was the disruption of protein synthesis, leading to cell death (Benarde et al., 1967). Results from Benarde's studies clearly showed an abrupt inhibition on protein synthesis. Explanations of this occurrence on the cells included possible inhibition of amino acid activation, inactivation of messenger RNA (which prevents translation), and destruction of ribosomes by chlorine dioxide (which causes a loss in cell contents by leakage).

A later study, however, provided an alternate hypothesis to the precise mechanism of action of chlorine dioxide on cells. Roller et al. studied the effects of chlorine dioxide on dehydrogenase enzymes, protein synthesis, and deoxyribonucleic acid of bacteria (Roller et al., 1986). This study found that total dehydrogenase enzymes were inhibited completely within the first 5 seconds of reaction by chlorine dioxide and protein synthesis was partially inhibited. The dosage of chlorine dioxide used was found to be proportional to the extent of inhibition. These studies concluded that the primary effect of chlorine dioxide on cells was occurring in an area in the cell other than the dehydrogenase enzymes, protein-synthesizing complex, or DNA. It was determined that inhibition of protein synthesis of cells, indeed, contributed to cell death. However, Roller et al. concluded that an impairment of the cell's functions is occurring even before protein synthesis. Chlorine dioxide did not cause cell inactivation by altering or impairing the cell's DNA. An explanation or theory of the cell deaths by chlorine dioxide in this study is by a reaction with or oxidation of components related to enzyme activity of the cell (Roller et al, 1986).

Berg et al. (1986) studied the effect of chlorine dioxide on membrane functions of Escherichia coli, finding that the permeability control was impaired, leading to cell death. This study also showed that the inactivation by the chlorine dioxide did not cause a significant loss of intracellular macromolecules existing inside the cell to the surroundings. However, the membrane damage led to the loss of intracellular potassium destroying the trans-membrane ionic gradient; this is understood in the research to result in lethal inhibition of the metabolic processes and cell death. Thus, the permeability barrier of the cell was determined to be important to the sensitivity to chlorine dioxide and growth characteristics of the cell (Berg et al., 1986).

The present research evidence suggests that stabilized chlorine dioxide causes fungistatic and fungicidal effects, as well as bactericidal and bacteriostatic effects, on the fungal and bacterial cells, which ultimately lead to cell death. The current knowledge relative to the mechanism of action of chlorine dioxide on cell morphology indicates that Candida species would not be able to develop resistance to the method of action.

In Vitro Evaluation of Stabilized Chlorine Dioxide Oral Rinse Containing Stabilized Chlorine Dioxide Susceptibility of Candida Species:

To test the fungistatic and antifungal properties of stabilized chlorine dioxide oral rinse against several Candida species, as measured by minimum inhibitory concentration (MIC), minimum fungicidal concentrations (MFC), and time-kill colony counts after exposure, the following experiments were performed.

Materials

    • Four clinical isolates of Candida, including one each of C. albicans, C. glabrata, C. krusei, and C. dubliniensis
    • Stabilized chlorine dioxide oral rinse (0.8% concentration)
    • Chlorhexidine gluconate (20% stock solution)
    • RPMI 1640: Buffered with MOPS [3-(N-morpholino) propanesulfonic acid], with glutamine, without bicarbonate, pH=7.0. Prepared according to manufacturer's specifications and filter sterilize.
    • Potato Dextrose Agar: Potato dextrose agar 39 g, Agar 1 g, Distilled water 1L
    • Cereal (oatmeal) Agar: Heinz baby oatmeal cerial 100 g, Agar 15 g, Distilled water 1L
    • Yeast Nitrogen Base: Yeast Nitrogen Base 6.7 g, Dextrose 5 g, Distilled water 1L, Filter sterilize (All media stored at 2-8° C.)

Supplies

Adjustable volume pipettes, bunsen burner, cell counter, disposable serological pipettes, eppendorf repipettor, hemacytometer, 35° C. incubator, inoculation loop, microscope, microtiter plates, multichannel pipettor, pipette tips, sterile conical tubes (15 ml), sterile saline (0.85%), sterile water, sterile cotton swab, vortex mixer, weighing scale.

Experimental Methodology—Susceptibility Testing

Serial dilutions of the stabilized chlorine dioxide oral rinse were combined with inoculum (0.5-2.5×103 colony forming units (CFU/mL)) in 96-well microdilution trays and incubated at 35° C. for 24 hours.

Solutions of specified concentrations (concentration range up to 0.8% (w/v)) in the minimum inhibitory concentration (MIC) were tested according to the standard method described in NCCLS M27-A document. The plates were removed from incubation after 24 hours. The MIC was recorded as the lowest concentration to inhibit 50% of fungal growth as compared to the growth control (no drug exposure).

Minimum fungicidal concentration (MFC) testing was determined according to modifications suggested by Canton et al. (2003). Contents of each clear well from the MIC assay were sub-cultured onto potato dextrose agar. In order to avoid antifungal carryover, the aliquots were allowed to soak into the agar and were streaked for isolation once dry, removing the cells from the drug source. The MFC was measured as the lowest concentration at which ≧99.9% of Candida cells were reduced from the starting inoculum count.

The time-kill assay was performed by adding inocula (0.5-2.5×103 CFU/mL) of Candida albicans, C. dubliniensis, C. glabrata, and C. krusei to serial dilutions of concentrations ranging from 0.1-0.8% of stabilized chlorine dioxide oral rinse for 30 second and 1-minute exposure times. Following exposure, 100 μl aliquots were diluted 50% with 0.85% saline and plated onto potato dextrose agar plates. The aliquots were allowed to dry and then were streaked to remove the yeast from the compound. The plates were incubated at 35° C. for 24 hours. Colony counts were taken and were compared to initial inoculum. The same test was done treating the four Candida species with chlorhexidine gluconate at concentrations ranging from 0.015-0.12%. Chlorhexidine gluconate at concentration 0.12% is commonly prescribed to patients with oral disease.

All tests were performed in duplicate.

Results and Conclusions

The stabilized chlorine dioxide oral rinse showed strong inhibition against all strains of Candida species tested. The MIC range was 0.0004-0.05% (w/v) concentration (Table 1). The concentration at which C. albicans and C. dubliniensis were inhibited by stabilized chlorine dioxide oral rinse was 0.05%. C. krusei and C. glabrata both have lower concentrations of 0.025% and 0.0004%, respectively. The MFC range for all species was found to be greater than or equal to 0.4% concentration (Table 2).

Time-kill at 30 seconds and 1-minute exposures were also determined from this study. It has been determined that stabilized chlorine dioxide oral rinse is very effective in killing Candida species completely within 30 seconds of exposure at a concentration of 0.8% stabilized chlorine dioxide (Table 3). A 0.4% concentration solution also showed reduction of the count of Candida albicans after 30 seconds as shown in Replicate 1 and Replicate 2. This suggests that stabilized chlorine dioxide oral rinses at higher concentrations have a fungicidal effect within 1 minute of exposure at concentrations between 0.4% and 0.8%. Chlorhexidine gluconate is commonly prescribed at a concentration of 0.12% for the treatment of oral diseases and was used as a positive control. The chlorhexidine gluconate concentrations tested did not reduce the colony count of any of the Candida species within one minute of exposure (Table 4).

The in vitro test results of stabilized chlorine dioxide against Candida species shows fungistatic and fungicidal properties at the suggested concentrations. The present invention relates to use of stabilized chlorine dioxide as a pharmaceutically acceptable topical oral care product, including washes, rinses, soaks, pastes, gels, and aerosol sprays. The compositions of the present invention may be used to prevent or treat fungal infections and diseases, such as candidiasis or thrush. The present invention may also be used as a substitute or adjunct therapy to current treatments for oral fungal infections to promote overall oral health, especially for immunocompromised individuals.

TABLE 1
Minimum inhibitory concentrations (MIC) of
stabilized chlorine dioxide rinse.
MIC of stabilized chlorine
Candida Speciesdioxide rinse
C. albicans0.05%
C. dubliniensis0.05%
C. glabrata0.0004%
C. krusei0.025% 

TABLE 2
Minimum fungicidal concentrations (MFC) of
stabilized chlorine dioxide rinse.
MFC of stabilized chlorine
Candida Speciesdioxide rinse
C. albicans0.40%
C. dubliniensis0.40%
C. glabrata0.40%
C. krusei0.40%

TABLE 3
Two replications of Time-kill after 30 seconds and 1 minute of
stabilized chlorine dioxide oral rinse exposure against four
Candida species (after 24 hours incubation). Growth
control for each species was >2000 CFU/mL.
Bacteria Count (CFU/mL)
Replicate 1Replicate 2
Concentration301301
Candida Speciesof rinsesecondsminutesecondsminute
C. albicans0.40%24010202020
0.80%0000
C. dubliniensis0.40%>2000>2000>2000>2000
0.80%0000
C. glabrata0.40%>2000>2000>2000>2000
0.80%0000
C. krusei0.40%>2000>2000>2000>2000
0.80%0000

TABLE 4
Time kill after 1-minute exposure of chlorhexidine gluconate
against Candida species (after 24 hours incubation).
Growth control for each species was >2000 CFU/mL.
Candida SpeciesConcentration of CHXBacteria Count (CFU/mL)
C. albicans0.015%>2000
 0.12%>2000
C. dubliniensis0.015%>2000
 0.12%>2000
C. glabrata0.015%>2000
 0.12%>2000
C. krusei0.015%>2000
 0.12%>2000