U.S. patent application number 12/500163 was filed with the patent office on 2010-01-14 for method and composition for prevention and treatment of oral fungal infections.
This patent application is currently assigned to Micropure, Inc.. Invention is credited to James L. Ratcliff, Elena J. Young.
Application Number | 20100009009 12/500163 |
Document ID | / |
Family ID | 41505373 |
Filed Date | 2010-01-14 |
United States Patent
Application |
20100009009 |
Kind Code |
A1 |
Young; Elena J. ; et
al. |
January 14, 2010 |
METHOD AND COMPOSITION FOR PREVENTION AND TREATMENT OF ORAL FUNGAL
INFECTIONS
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) ; Ratcliff; James L.; (Pueblo
West, CO) |
Correspondence
Address: |
The von HELLENS LAW FIRM, LTD.;C. Robert von Hellens
7330 N 16TH STREET, SUITE C 201
PHOENIX
AZ
85020
US
|
Assignee: |
Micropure, Inc.
Scottsdale
AZ
|
Family ID: |
41505373 |
Appl. No.: |
12/500163 |
Filed: |
July 9, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61079532 |
Jul 10, 2008 |
|
|
|
Current U.S.
Class: |
424/613 |
Current CPC
Class: |
A61P 31/04 20180101;
A61K 9/0053 20130101; A61K 33/20 20130101 |
Class at
Publication: |
424/613 |
International
Class: |
A61K 33/00 20060101
A61K033/00; A61P 31/04 20060101 A61P031/04 |
Claims
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
[0001] 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
[0002] 1. Field of Invention
[0003] The present invention relates to the use of stabilized
chlorine dioxide in topical oral compositions to prevent oral
fungal infections.
[0004] 2. Description of Related art
[0005] 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.
[0006] 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.
[0007] 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).
[0008] 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.
[0009] 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.sup.+ 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.sup.+ counts and smoking are independent risk
factors for thrush and oral hairy leukoplakia.
[0010] 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.
[0011] 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).
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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).
[0016] 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.
[0017] 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).
[0018] 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.
[0019] 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.
[0020] 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).
[0021] 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.
[0022] 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.
[0023] 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.
[0024] 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
[0025] 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.
[0026] 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.
[0027] 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.
[0028] Still another object of the present invention is to provide
a method for prevention and treating fungal infection in the oral
cavity.
[0029] 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
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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).
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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).
[0043] 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.
[0044] 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).
[0045] 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.
[0046] 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).
[0047] 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).
[0048] 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).
[0049] 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.
[0050] In Vitro Evaluation of Stabilized Chlorine Dioxide Oral
Rinse Containing Stabilized Chlorine Dioxide Susceptibility of
Candida Species:
[0051] 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.
[0052] Materials [0053] Four clinical isolates of Candida,
including one each of C. albicans, C. glabrata, C. krusei, and C.
dubliniensis [0054] Stabilized chlorine dioxide oral rinse (0.8%
concentration) [0055] Chlorhexidine gluconate (20% stock solution)
[0056] 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. [0057] Potato Dextrose Agar: Potato dextrose agar 39 g,
Agar 1 g, Distilled water 1L [0058] Cereal (oatmeal) Agar: Heinz
baby oatmeal cerial 100 g, Agar 15 g, Distilled water 1L [0059]
Yeast Nitrogen Base: Yeast Nitrogen Base 6.7 g, Dextrose 5 g,
Distilled water 1L, Filter sterilize (All media stored at
2-8.degree. C.)
[0060] Supplies
[0061] Adjustable volume pipettes, bunsen burner, cell counter,
disposable serological pipettes, eppendorf repipettor,
hemacytometer, 35.degree. 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.
[0062] Experimental Methodology--Susceptibility Testing
[0063] Serial dilutions of the stabilized chlorine dioxide oral
rinse were combined with inoculum (0.5-2.5.times.10.sup.3 colony
forming units (CFU/mL)) in 96-well microdilution trays and
incubated at 35.degree. C. for 24 hours.
[0064] 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).
[0065] 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 .gtoreq.99.9% of Candida cells were reduced
from the starting inoculum count.
[0066] The time-kill assay was performed by adding inocula
(0.5-2.5.times.10.sup.3 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 .mu.l 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.degree. 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.
[0067] Results and Conclusions
[0068] 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).
[0069] 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).
[0070] 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-US-00001 TABLE 1 Minimum inhibitory concentrations (MIC) of
stabilized chlorine dioxide rinse. MIC of stabilized chlorine
Candida Species dioxide rinse C. albicans 0.05% C. dubliniensis
0.05% C. glabrata 0.0004% C. krusei 0.025%
TABLE-US-00002 TABLE 2 Minimum fungicidal concentrations (MFC) of
stabilized chlorine dioxide rinse. MFC of stabilized chlorine
Candida Species dioxide rinse C. albicans 0.40% C. dubliniensis
0.40% C. glabrata 0.40% C. krusei 0.40%
TABLE-US-00003 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 1 Replicate 2 Concentration 30 1 30 1 Candida
Species of rinse seconds minute seconds minute C. albicans 0.40%
240 1020 20 20 0.80% 0 0 0 0 C. dubliniensis 0.40% >2000
>2000 >2000 >2000 0.80% 0 0 0 0 C. glabrata 0.40% >2000
>2000 >2000 >2000 0.80% 0 0 0 0 C. krusei 0.40% >2000
>2000 >2000 >2000 0.80% 0 0 0 0
TABLE-US-00004 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 Species Concentration of CHX Bacteria Count (CFU/mL) C.
albicans 0.015% >2000 0.12% >2000 C. dubliniensis 0.015%
>2000 0.12% >2000 C. glabrata 0.015% >2000 0.12% >2000
C. krusei 0.015% >2000 0.12% >2000
* * * * *