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Claims  |
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I claim:
1. A composition for preventing and treating conditions of the epithelium
of the rectal, vaginal, urethral, nasal, ocular, and auditory canal
orifices, brought about by the occurrence of any of fungal and bacterial
infections, leukoplakia, penetration of bacterial toxins, invasion of
antigenic substances and microbiota, living unicellular orctanisms,
vaginitis, endometriosis, Porphyromonas gingivalis, Actinobactillus
actinomycetumcomitans, Pseudomonades, Candida species and leukoplakia
vulvae, said composition comprising a topical preparation selected from
the group consisting of liquid solutions, suspensions, semi-solids,
salves, creams, and suppositories, wherein the topical preparation
contains stabilized chlorine dioxide in a concentration in the range of
0.005%-2.0% and a phosphate compound selected from the group consisting of
disodium hydrogen phosphate, sodium dihydrogen phosphate, trisodium
phosphate, or sodium monofluorophosphate, wherein the concentration of
said phosphate compound is in a range of between about 0.02%-3.0% to
retard escape of chlorine dioxide from the composition at a pH in the
range of 6.0 to 7.4, thereby increasing the shelf life and efficacy of the
composition.
2. A composition for preventing and treating conditions of the epithelium
of the rectal, vaginal, urethral, oral, nasal, ocular, and auditory canal
orifices, brought about by the occurrence of any of fungal and bacterial
infections, leukoplakia, penetration of bacterial toxins, invasion of
antigenic substances and microbiota, living unicellular organisms,
vaginitis, endometriosis, Porphyromonas gingivalis, Actinobactillus
actinomycetumcomitans, Pseudomonades, Candida species and leukoplakia
vulvae, said composition comprising a topical preparation selected from
the group consisting of liquid solutions, suspensions, semi-solids,
salves, creams, and suppositories, wherein the topical preparation
contains stabilized chlorine dioxide in a concentration in the range of
0.005%-2.0% and a phosphate compound comprising trisodium phosphate,
wherein the concentration of said phosphate compound is in a range of
between about 0.02%-3.0% to retard escape of chlorine dioxide from the
composition at a pH in the range of 6.0 to 7.4, thereby increasing the
shelf life and efficacy of the composition.
3. The composition as set forth in claim 1, wherein the phosphate compound
is sodium monofluorophosphate.
4. The composition as set forth in claim 1, wherein the concentration of
stabilized chlorine dioxide is approximately 0.2% and the concentration of
phosphate compound is approximately 1.0%.
5. A composition for preventing and treating conditions of the epithelium
of the rectal, vaginal, urethral, nasal, ocular, and auditory canal
orifices, brought about by the occurence of any of fungal and bacterial
infections, leukoplakia, penetration of bacterial toxins, invasion of
antigenic substances and microbiota, living unicellular organisms,
vaginitis, endometriosis, Porphyromonas gingivalis, Actinobactillus
actinomycetumcomitans, Pseudonionades, Candida species and leukoplakia
vulvae, said composition comprising a topical preparation selected from
the group consisting of liquid solutions, suspensions, semi-solids,
salves, creams, and suppositories, wherein the topical preparation
contains at least 0.1% stabilized chlorine dioxide and at least 0.05% of a
phosphate compound selected from the group consisting of disodium hydrogen
phosphate, sodium dihydrogen phosphate, trisodium phosphate, or sodium
monofluorophosphate to retard escape of chlorine dioxide from the
composition at a pH in the range of 6.0 to 7.4, thereby increasing the
shelf life and efficacy of the composition.
6. A composition for preventing and treating conditions of the epithelium
of the rectal, vaginal, urethral, oral, nasal, ocular, and auditory canal
orifices, brought about by the occurrence of any of fungal and bacterial
infections, leukoplakia, penetration of bacterial toxins, invasion of
antigenic substances and microbiota, living unicellular organisms,
vaginitis, endometriosis, Porphyromonas gingivalis, Actinobactillus
actinomycetumcomitans, Pseudomonades, Candida species and leukoplakia
vulvae, said composition comprising a topical preparation selected from
the group consisting of liquid solutions, suspensions, semi-solids,
salves, creams, and suppositories, wherein the topical preparation
contains at least 0.1% stabilized chlorine dioxide and at least 0.05% of a
phosphate compound comprising trisodium phosphate to retard escape of
chlorine dioxide from the composition at a pH in the range of 6.0 to 7.4,
thereby increasing the shelf life and efficacy of the composition.
7. The composition as set forth in claim 5, wherein the phosphate compound
is sodium monofluorophosphate.
8. A composition for preventing and treating the epithelium of the rectal,
vaginal, urethral, nasal, ocular, and auditory canal orifices by reducing
the number of bacteria including Actinobacillus actinomycetemcomitans and
Porphyromonas gingivalis in said orifices, said composition comprising a
topical preparation selected from the group consisting of liquid
solutions, suspensions, semi-solids, salves, creams, and suppositories,
wherein the topical preparation contains stabilized chlorine dioxide in a
concentration in the range of 0.005%-2.0% and a phosphate compound
selected from the group consisting of disodium hydrogen phosphate, sodium
dihydrogen phosphate, trisodium phosphate, or sodium monofluorophosphate
in a concentration of between 0.02%-3.0% to retard escape of chlorine
dioxide from the composition at a pH in the range of 6.0 to 7.4, thereby
increasing the shelf life and efficacy of the composition.
9. A composition for preventing and treating the epithelium of the rectal,
vaginal, urethral, nasal, ocular, and auditory canal orifices by reducing
the number of bacteria including Actinobacillus actinomycetemcomitans and
Porphyromonas gingivalis in said orifices, said composition comprising a
topical preparation selected from the group consisting of liquid
solutions, suspensions, semi-solids, salves, creams, and suppositories,
wherein the topical preparation contains at least 0.1% stabilized chlorine
dioxide and at least 0.05% of a phosphate compound selected from the group
consisting of disodium hydrogen phosphate, sodium dihydrogen phosphate,
trisodium phosphate, or sodium monofluorophosphate to retard escape of
chlorine dioxide from the composition at a pH in the range of 6.0 to 7.4,
thereby increasing the shelf life and efficacy of the composition.
10. A composition for preventing and treating the epithelium of the rectal,
vaginal, urethral, nasal, ocular, and auditory canal orifices by reducing
the occurrence of leukoplakia in said orifices, said composition
comprising a topical preparation selected from the group consisting of
liquid solutions, suspensions, semi-solids, salves, creams, and
suppositories, wherein the topical preparation contains stabilized
chlorine dioxide in a concentration in the range of 0.005%-2.0% and a
phosphate compound selected from the group consisting of disodium hydrogen
phosphate, sodium dihydrogen phosphate, trisodium phosphate, or sodium
monofluorophosphate in a concentration of between 0.02%-3.0% to retard
escape of chlorine dioxide from the composition at a pH in the range of
6.0 to 7.4, thereby increasing the shelf life and efficacy of the
composition.
11. A composition for preventing and treating the epithelium of the rectal,
vaginal, urethral, nasal, ocular, and auditory canal orifices by reducing
the occurrence of leukoplakia in said orifices, said composition
comprising a topical preparation selected from the group consisting of
liquid solutions, suspensions, semi-solids, salves, creams, and
suppositories, wherein the topical preparation contains at least 0.1%
stabilized chlorine dioxide and at least 0.05% of a phosphate compound
selected from the group consisting of disodium hydrogen phosphate, sodium
dihydrogen phosphate, trisodium phosphate, or sodium monofluorophosphate
to retard escape of chlorine dioxide from the composition at a pH in the
range of 6.0 to 7.4, thereby increasing the shelf life and efficacy of the
composition.
12. A composition for preventing and treating the epithelium of the rectal,
vaginal, urethral, nasal, ocular, and auditory canal orifices by reducing
the number of yeasts including Candida albicans in said orifices, said
composition comprising a topical preparation selected from the group
consisting of liquid solutions, suspensions, semi-solids, salves, creams,
and suppositories, wherein the topical preparation contains stabilized
chlorine dioxide in a concentration in the range of 0.005%-2.0% and a
phosphate compound selected from the group consisting of disodium hydrogen
phosphate, sodium dihydrogen phosphate, trisodium phosphate, or sodium
monofluorophosphate in a concentration of between 0.02%-3.0% to retard
escape of chlorine dioxide from the composition at a pH in the range of
6.0 to 7.4, thereby increasing the shelf life and efficacy of the
composition.
13. A composition for preventing and treating the epithelium of the rectal,
vaginal, urethral, nasal, ocular, and auditory canal orifices by reducing
the number of yeasts including Candida albicans in said orifices, said
composition comprising a topical preparation selected from the group
consisting of liquid solutions, suspensions, semi-solids, salves, creams,
and suppositories, wherein the topical preparation contains at least 0.1%
stabilized chlorine dioxide and at least 0.05% of a phosphate compound
selected from the group consisting of disodium hydrogen phosphate, sodium
dihydrogen phosphate, trisodium phosphate, or sodium monofluorophosphate
to retard escape of chlorine dioxide from the composition at a pH in the
range of 6.0 to 7.4, thereby increasing the shelf life and efficacy of the
composition. |
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Claims |
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Description |
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BACKGROUND OF THE INVENTION
The present invention is directed to a method and composition for
prevention and treatment of abnormal conditions of the epithelium of
bodily orifices. More particularly, the present invention relates to the
use of activated stabilized chlorine dioxide in conjunction with a
phosphate compound (to provide stability and as a surfactant or nonsudsing
detergent to reduce surface tension on mucosal tissues assisting in the
exposure of the epithelial covering to the activated chlorine dioxide), to
thereby prevent and treat fungal and bacterial infections of the rectal,
vaginal, urethral, oral, nasal, ocular, and auditory canal orifices, and
other abnormal conditions of the epithelium, including leukoplakia.
Thiols, particularly the volatile sulfur compounds such as hydrogen
sulfide, methylmercaptan and dimethylsulfide, are recognized in the
current literature as being major contributors to the penetration of
bacterial toxins through the epithelial barrier into the underlying basal
lamina and connective tissue. A. Rizzo, Peridontics, 5:233-236 (1967); W.
Ng and J. Tonzetich, J. Dental Research, 63(7):994-997 (1984); M. C.
Solis-Gaffar, T. J. Fischer and A. Gaffar, J. Soc. Cosmetic Chem.,
30:241-247 (1979); I. Kleinberg and G. Westbay, J. Peridontol, 63(9):
768-774 (1992). The penetration of this barrier makes possible the
invasion of antigenic substances such as viral and bacterial toxins and
bacteria into the underlying substrate. Thus, by removing the volatile
sulfur compounds and maintaining the epithelial barrier there is a
reduction in the penetration capacity of antigens and microbiota (A.
Rizzo, Peridontics, 5:233-236 (1967); W. Ng and J. Tonzetich, J. Dental
Research, 63(7): 994-997 (1984); M. C. Solis-Gaffar, T. J. Fischer and A.
Gaffar, J. Soc. Cosmetic Chem., 30:241-247 (1979)) as well as the
destruction of the motility and the death of bacterial and viral forms.
Studies done in the mouth have demonstrated that the penetration of
bacteria takes place in the presence of the volatile sulfur compounds,
resulting in initiation of the inflammatory reaction including initiation
of the complement cascade. I. Kleinberg and G. Westbay, J. Peridontol,
63(9): 768-774 (1992). Initiation of the inflammatory reaction and
development of the complement leads to an eightfold increase in the cell
division or mitosis of epithelial cells in the attachment apparatus of the
gingiva. W. O. Engler, S. P. Ramfjiord and J. J. Hiniker, J.Periodont.,
36:44-56 (1965). Because the epithelia of other orifices, and particularly
vaginal epithelium, are very similar to the gingival epithelium, reactions
similar to those described above for the gingival epithelium occur in all
other parts of the body, as demonstrated by the occurence of vaginitis and
endometriosis of the vagina. Examples of such bacteria which may appear in
any bodily orifice include Porphyromonas (formerly known as Bacteroides)
gingivalis, Actinobacillus actinomycetemcomitans, and Pseudomonades.
The volatile sulfur compounds are generated primarily from the polypeptide
chains of the epithelial cell walls, and from the cell walls, pili,
fimbrae, and flagella of microorganisms, including fungi, that are part of
the normal flora of the organs of the exposed surfaces of the body. The
polypeptide chains are composed of a series of amino acids including
cysteine, cystine, and methionine, each of which contain sulfur side
chains. The death of the microorganisms or the epithelial cells results in
degradation of the polypeptide chains into their amino acid components,
particularly cysteine and methionine, which then become the source of the
sulfur compounds hydrogen sulfide, methylmercaptan and di-methylsulfide
which alter the epithelial barrier, permitting penetration of the barrier
by antigenic substances.
Penetration of the epithelial barrier by volatile sulfur compounds reduces
the capacity of the tissues to protect against bacteria, virus, fungus,
and yeast forms. Tonzetich has shown, using S.sup.35 -labelled
methylmercaptan, the penetration of thiol through the epithelium, plus the
basal lamina, into the underlying connective tissues where it begins
degradation of collagen fibers. W. Ng and J. Tonzetich, J. Dental
Research, 63(7): 994-997 (1984). In addition, it is the nature of many of
the bodily orifices that they are inhabited by both pathogenic and
non-pathogenic organisms. If an antibiotic is used to reduce the organisms
normally present, opportunistic yeast forms and other pathogenic organisms
resistant to the administered antibiotic often invade or multiply at or in
the bodily orifices.
Candida species, particularly Candida albicans, are the yeasts that
primarily affect the mouth and the female vagina. In the mouth, infection
by Candida is called Thrush; in the vagina it is called vaginitis.
With the increase of patients having immunocompromising diseases such as
AIDS, leukemia, diabetes and immunosuppressing diseases such as stress,
alcoholism, etc., a progressively higher percentage of the human
population is susceptible to invasion and growth of bacterial and fungal
Candida organisms. In addition, such patients are susceptible to the
development of conditions of leukoplakia such as oral hairy leukoplakia
and leukoplakia vulvae.
In patients afflicted with diabetes, as well as familial history diabetes,
the neutrophil, which is the first line defense cell against foreign
antigens, has an altered 110 Dalton surface protein which reduces the
capacity of the neutrophil to phagocytizc bacteria by approximately 50%.
R. J. Genco, T. E. Van Dyke, M. J. Levine, R. D. Nelson and M. E. Wilson,
J. Dental Research, 65(12):1379-1391 (1986). As a result of the
development of antibiotics, insulin, and more sophisticated methods of
treating diabetes, early deaths of diabetics from infections have been
prevented, resulting in a several-fold increase in the number of familial
history diabetes in the population. Thus, the increased presence of the
diabetes gene in the gene pool of the human race is rapidly increasing,
resulting in a higher number of humans with an immunocompromised capacity.
This fact in part explains why some women develop vaginitis whenever they
are treated with antibiotic drugs.
3. Stability of Chlorine Dioxide
Chlorine dioxide is unstable in aqueous solutions at lower pH levels. It is
produced commercially and shipped in an aqueous solution in its hydrolytic
byproduct forms at 8.3 to 9.0 pH. At that range there is complete
retention of the chlorine dioxide hydrolyzed forms within the solution so
that a shelf life of from 1-5 years may be achieved. When the pH of
chlorine dioxide is lowered to 7.2 or below, chlorine dioxide begins to
become activated and, in the gaseous form, it is available for reactivity
with thiols, microorganisms, and organic debris in solution.
At present, there is an inadequate capacity of existing pharmaceutical
drugs to control Candida infections (IADR symposium, March 1993). The
severe diseases may be resistant to the commonly used drugs ketonideozole
and nystatin, etc. Other synthetic drugs which are used systemically may
have limited effects, and infections are resistant to treatment.
Combinations of these drugs systemically and by suppositories may not
always work.
In an in vitro study by the present inventor of Candida culture using the
protocol of a simulated oral environment as stipulated by the Food and
Drug Administration in the Federal Register, Vol. 47, No. 101 (May 25,
1982), wherein calf serum is added to the tryptic soy broth inoculated
with the Candida, one ml. of the Candida culture was withdrawn and plate
counted by standard techniques to determine the baseline content of the
Candida population. Both a solution and a slurry of 1 ml. paste containing
0.1% chlorine dioxide with 0.2% phosphate stabilizer plus 2 ml. of
distilled water was added to the TSB broth with calf serum. Additional
samples were taken at 10, 30 and 60 seconds and again plated to count the
remaining Candida. It was found that at 10 seconds there was a 99+%
reduction of Candida albicans using standard plate count techniques.
In a six month clinical trial by the present inventor, samples were taken
from the gingival crevice of the mouth. After treatment of humans with a
composition comprising 0.10% chlorine dioxide and 0.2% phosphate
stabilizer, the inventor showed by means of standard plate count methods
that during the period from baseline to six months, there was a
statistically significant reduction of Candida albicans. This clinical
trial demonstrates the capacity of a composition comprising 0.1% activated
stabilized chlorine dioxide together with metallic phosphate (the latter
compound acting both to stabilize the chlorine dioxide solution and also
as a surfactant to break the surface tension and allow chlorine dioxide to
effectively interact with the Candida albicans infection) to prevent and
treat the development of a Candida infection.
Further details of the preparation and use of chlorine dioxide/phosphate
compositions can be found in U.S. Pat. No. 5,200,171, issued Apr. 6, 1993
to Ratcliff, which is hereby incorporated by reference.
SUMMARY OF THE INVENTION
Briefly, and in accord with one embodiment of the present invention, a
composition containing stabilized chlorine dioxide and a phosphate is
disclosed as being useful in preventing and treating abnormal conditions
of the epithelium of bodily orifices. Examples of such abnormal conditions
of the epithelium of the rectal, vaginal, urethral, oral, nasal, ocular,
and auditory canal orifices include bacterial and fungal infections, such
as Candida, and leukoplakia. Stabilized chlorine dioxide is an effective
agent for removing thiol compounds for deodorizing the mouth as well as
deodorizing other bodily orifices, such as the vagina. The addition of
activating inhibitor phosphates to the stabilized chlorine dioxide reduces
surface tension and retards the rapid escape of chlorine dioxide gas at
the pH range of 6.5 to 7.0 typical of orifices of the body. Preferred
concentrations of stabilized chlorine dioxide compounds are in the range
of between about 0.005% to 2.0%. The concentration of the phosphate
compound, preferably disodium hydrogen phosphate, sodium dihydrogen
phosphate, trisodium phosphate, and sodium monofluorophosphate, is in the
range of between about 0.02 to 3.0%.
DESCRIPTION OF THE PREFERRED EMBODIMENT
Broadly, the present invention contemplates the use of an activating
inhibitor and surface tension reducing agent, specifically, a phosphate
compound, preferably, disodium hydrogen phosphate, sodium dihydrogen
phosphate, trisodium phosphate, or sodium monofluorophosphate (in
particular, trisodium phosphate, or sodium monofluorophosphate), combined
with a stabilized chlorine dioxide solution, to make possible the lowering
of the pH of the mixture to an optimal value of less than about 7.2 at the
time the mixture is used to prevent and treat abnormal conditions of the
epithelium of bodily orifices, such as those caused by fungal and
bacterial infections of the rectal, vaginal, urethral, oral, nasal,
ocular, and auditory canal orifices, and other abnormal conditions of the
epithelium, including leukoplakia.
The present invention can be used to control the above-described bodily
orifice maladies in humans, and animals which are human companions, such
as dogs, cats, horses, etc., by reducing the presence of fungal and
bacterial infections and leukoplakia in bodily orifices of the human and
animal population, to prevent transference and cross infection from person
to person or animal to person or animal to animal. Thus, the present
invention can be used in both human and veterinary applications.
Clinical observations and in vitro and in vivo studies by the inventor have
led to the discovery that an activating inhibitor phosphate such as
disodium monohydrogen phosphate, sodium dihydrogen phosphate, or,
preferably, trisodium phosphate, or sodium monofluorophosphate, causes a
reduction in surface tension, as well as stabilizing chlorine dioxide, so
that the chlorine dioxide remains effective at a lower pH than was
previously thought possible. In addition, the phosphate is a detergent
which is used in place of other detergents for lowering surface tension
and allowing the activated chlorine dioxide to become available to the
convoluted surfaces of the body orifices. The preferred concentration
ranges are between about 0.005%-2.0% chlorine dioxide, and between about
0.02%-3.0% phosphate. For most patients, the preferred concentration of
chlorine dioxide will be in the range of between about 0.005-0.5%; in the
case of extremely immunocompromised patients having runaway bacterial or
fungal infections or severe leukoplakia, it is preferred to increase the
concentration of chlorine dioxide up to about 1.0-2.0%.
The permeability of mucus epithelial tissue is increased substantially by
exposure to thiol compounds including hydrogen sulfide (H.sub.2 S) and
methylmercaptan (CH.sub.3 --SH) and dimethylsulfide (CH.sub.3
--S--CH.sub.3). In a Candida infection, there is increased inflammation
and degeneration of epithelial cells, which break down into thiols,
including the above sulfur compounds. A vicious cycle is established,
leading to an environment for the increase of Candida growth. If the
patient is immunocompromised with AIDS, the problem is exacerbated with
ulcerations that could increase the probability of sexually transmitted
disease. Likewise, a non-AIDS patient could be more exposed to sexually
transmitted disease.
The following examples further illustrate various features of the invention
but are intended in no way to limit the scope of the invention which is
defined in the appended claims.
EXAMPLE 1
The stability of Chlorine Dioxide at Ph 6.8 in the Presence of Phosphate.
Materials
1. Purogene (2% ClO.sub.2), Lot #8907.41, 1 gallon, Manufactured by
BIO-Cide, International, P.O. Box 2700, Norman, Okla. 73070.
2. Sodium Phosphate, monobasic, dibasic, and tribasic.
Methods
A 10% solution of monobasic sodium phosphate was prepared in distilled
water. Ten ml was placed into each of four beakers. One of each of the
four beakers received 1, 2.5, 5, and 10 ml of chlorine dioxide concentrate
(2% ClO.sub.2), respectively. All solutions were diluted to 90 ml with
distilled water, adjusted to pH 6.8 with 1N NaOH and 1N HCl, diluted to
100 ml and placed in screw cap bottles.
Solutions containing dibasic and tribasic sodium phosphate and a distilled
water blank control were prepared in a similar manner.
Chlorine dioxide content and pH was determined for each solution on days 0,
7, 14, 21 and 28 in accordance with Standard Methods for the Examination
of Water and Wastewater, 17th edition, 1989.
Results and Summary
As shown in Table 1, the content of chlorine dioxide was stable in all
sodium phosphate solutions and distilled water control over the 28 day
test period. The pH of all samples ranged from 6.1 to 7.6.
TABLE 1
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RESULTS SHOWING THE STABILITY OF CHLORINE DIOXIDE SOLUTION AT
pH 6.8 IN DISTILLED WATER AND 1% SODIUM PHOSPHATE, MONOBASIC,
DIBASIC, AND TRIBASIC
DAY
Theroetical
0 7 14 21 28
SOLUTION % ClO.sub.2
pH
% ClO.sub.2
pH
% ClO.sub.2
pH
% ClO.sub.2
pH
% ClO.sub.2
pH
% ClO.sub.2
__________________________________________________________________________
Distilled Water
0.02 6.8
0.02 6.9
0.02 6.9
0.02 6.5
0.02 6.5
0.02
0.05 6.8
0.05 6.9
0.05 6.9
0.05 7.1
0.05 6.9
0.05
0.1 6.3
0.1 6.9
0.1 7.0
0.1 7.7
0.1 7.6
0.1
0.2 6.8
0.2 6.9
0.2 6.9
0.2 7.2
0.2 7.2
0.2
O/O Na.sub.2 HPO.sub.11
0.02 6.8
0.02 6.1
0.02 6.7
0.02 6.7
0.02 6.8
0.02
(Disodium
0.05 6.8
0.05 6.8
0.05 6.8
0.05 6.8
0.05 6.8
0.05
hydrogen 0.1 6.8
0.1 6.9
0.1 6.9
0.1 6.8
0.1 6.8
0.1
phosphate
0.2 6.8
0.2 6.9
0.2 6.9
0.2 6.9
0.2 6.8
0.2
O/O NaH.sub. 2 PO.sub.4
0.02 6.8
0.02 6.7
0.02 6.8
0.02 6.7
0.02 6.8
0.02
Sodium 0.05 6.8
0.05 6.8
0.05 6.8
0.05 6.8
0.05 6.9
0.05
dihydrogen
0.1 6.8
0.1 6.8
0.1 6.8
0.1 6.9
0.1 6.9
0.1
phosphate)
0.2 6.8
0.2 6.8
0.2 6.8
0.2 6.9
0.2 6.9
0.2
O/O Na.sub.3 PO.sub.4
0.02 6.8
0.02 6.8
0.02 6.4
0.02 6.9
0.02 7.0
0.02
(Trisodium
0.05 6.8
0.05 7.0
0.05 7.1
0.05 6.9
0.05 7.0
0.05
phoshpate)
0.1 6.8
0.1 7.5
0.1 7.5
0.1 7.0
0.1 6.9
0.1
0.2 6.8
0.2 7.0
0.2 7.1
0.2 6.9
0.2 6.9
0.2
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EXAMPLE 2
The stability of Chlorine Dioxide at Ph 6.8 in the Presence of 0.2%
Phosphate
The following is an example of how to test the stability of chlorine
dioxide at pH 6.8 in the presence of 0.2% phosphate.
Materials
1. Purogene (2% ClO.sub.2), Lot #8907.41, 1 gallon, Manufactured by
BIO-Cide, International, P.O. Box 2700, Norman, Okla. 73070.
2. Sodium Phosphate, monobasic, dibasic, and tribasic.
Methods
A 0.2% solution of monobasic sodium phosphate is prepared in distilled
water. Ten ml is placed into each of four beakers. One of each of the four
beakers receives 1, 2.5, 5, and 10 ml of chlorine dioxide concentrate (2%
ClO.sub.2), respectively. All solutions were diluted to 90 ml with
distilled water, adjusted to pH 6.8 with 1N NaOH and 1N HCl, diluted to
100 ml and placed in screw cap bottles.
Solutions containing dibasic and tribasic sodium phosphate and a distilled
water blank control are prepared in a similar manner.
Chlorine dioxide content and pH is determined for each solution on days 0,
7, 14, 21 and 28 in accordance with Standard Methods for the Examination
of Water and Wastewater, 17th edition, 1989, in order to determine the
stability of chlorine dioxide over time.
EXAMPLE 3
The Effectiveness of Chlorine Dioxide in Phosphate Mixture Against Candida
albicans
Materials
1. Purogene (2% chlorine dioxide), lot #8907:41, manufactured by BIO-CIDE
International, Inc., P.O. Box 2700, Norman, Okla. 73070.
2. Test Organism: Candida albicans (ATCC#18804)
3. Saline, 0.9% NaCl.
4. Butterfield's Buffer phosphate dilutent (BFB), pH 7.2.
5. Sterile 15% sodium thiosulfate.
6. Blood agar.
7. Stop watch.
8. Sterile 1N HCl and 1N NaOH.
9. pH meter.
10. McFarland nephelometer tube No. 1. Density of this tube is equivalent
to a bacterial suspension of 3.times.10.sup.8 organisms per ml.
11. N,N-diethyl-p-phenylenediamine (DPD reagent).
12. Phosphate buffer reagent.
13. Sodium dihydrogen phosphate, NaH.sub.2 PO.sub.4.7H.sub.2 0.
14. Trisodium phosphate, Na.sub.3 PO.sub.4.12H.sub.2 0.
15. Sodium monofluorophosphate, Na.sub.2 FPO.sub.3, Ref No. OB 12837,
manufactured by Albright and Wilson, P.O. Box 80, Oldbury, Narley, West
Midlands, B694LN, England.
DPD reagent and phosphate buffer reagent were prepared in accord with
Standard Methods for the Examination of Water and Wastewater, 17th
Edition, p. 9-54 (1989).
Methods
1. Test Solutions
A ten percent sodium dihydrogen phosphate solution was prepared in
distilled water. Ten ml was placed into each of five beakers. One of each
of the five beakers received 0, 1, 2.5, 5, and 10 ml of chlorine dioxide
concentrate (2% ClO.sub.2), respectively. All solutions were diluted to 90
ml with distilled water, adjusted to pH 6.0 with 1N NaOH and 1N HCl,
diluted to 100 ml and placed in screw cap bottles. Solutions containing 0
ppm chlorine dioxide were filter sterilized prior to use.
Solutions containing trisodium phosphate and sodium monofluorophosphate
were prepared in a similar manner.
II. Test Suspensions
Suspensions of the Candida albicans organism were prepared in Butterfield's
buffer from 48 hour agar cultures and turbidity adjusted to a McFarland
Tube #1. Subsequently 0.1 ml of this suspension was diluted in 50 ml of
saline. The diluted microorganism suspensions were now ready for use.
III. Test Procedure
1. Test
One ml of test suspension was aliquoted into each of five sterile
16.times.125 mm screw cap tubes. Each of the five tubes received 4 ml of a
solution containing either 0, 200, 500, 1000, or 2000 ppm chlorine dioxide
in 1% sodium dihydrogen phosphate. Each tube was shaken for ten seconds
and immediately inactivated with 0.25 ml 15% sodium thiosulfate. Solutions
containing 1% trisodium phosphate and 1% sodium monofluorophosphate were
handled in a similar manner.
2. Controls
One ml of test suspension was dispensed into two sterile 16.times.125 mm
screw cap tubes. Each tube received 4 ml 2000 ppm chlorine dioxide in 1%
sodium dihydrogen phosphate. The first tube received 0.25 ml sodium
thiosulfate, while the second tube received none. Subsequently each tube
was tested for residual chlorine dioxide by adding 0.3 ml phosphate buffer
reagent and 0.3 ml DPD reagent to each tube. Neutralized tubes were
colorless, while nonneutralized tubes were pink. Solutions of trisodium
phosphate and sodium monofluorophosphate containing 2,000 ppm chlorine
dioxide were handled in a similar manner.
One ml test suspension of the Candida albacans organism was treated with 4
ml Butterfield's buffer and 0.25 ml 10% sodium thiosulfate as a negative
control.
After inactivation with sodium thiosulfate all tubes were plate counted.
Sterility tests on all reagents were run parallel to experiments by plate
counted method. The plate counted method and sterility tests were
conducted in accord with Standard Methods for the Examination of Water and
Wastewater, 17th Edition, p. 9-54 (1989).
Results and Summary
As shown in Table 2, 99-100% of the Candida albicans organisms were killed
when challenged with 1,000 ppm (0.1%) -2,000 ppm (0.2%) chlorine dioxide
in either 1% sodium dihydrogen phosphate or trisodium phosphate. Chlorine
dioxide concentrations of 200 (0.02%) and 500 ppm (0.05%) in the presence
of phosphates demonstrated marginal bacteriocidal activity against
C.albicans (39-51% kill).
TABLE 2
______________________________________
RESULTS SHOWING THE BACTERIOCIDAL
ACTIVITY OF CHLORINE DIOXIDE IN
PHOSPHATE SOLUTIONS AT pH 6.0
AGAINST CANDIDA ALBICANS
PHOSPHATE SOLUTION
CLO.sub.2
Negative
(PPM) Control* 1% NaH.sub.2 HPO.sub.4
1% Na.sub.3 PO.sub.4
______________________________________
0 95,000** 64,000 (33)***
55,000 (42)
200 ND 58,000 (39) 64,000 (33)
500 ND 47,000 (51) 32,000 (66)
1000 ND 250 (99) 0 (100)
2000 ND 17 (99) 5 (99)
______________________________________
*Buttertield's buffer
**Organisms/ml
***Percent Kill
ND Not Done
EXAMPLE 4
THE EFFECTIVENESS OF CHLORINE DIOXIDE IN PHOSPHATE MIXTURE AGAINST CANDIDA
ALBICANS IN THE PRESENCE AND ABSENCE OF SERUM
Materials
1. Purogene, Lot #8907:41, 1 gallon (contains 2% ClO.sub.2), manufactured
by BIO-CIDE International, Inc., P.O. Box 2700, Norman, Okla. 73070.
2. Test Organism: Candida albicans (ATCC #18804) obtained from American
Type Culture Collection, (ATCC) 12301 Parklawn Drive, Rockville, Md.
20852.
3. 15% Sodium thiosulfate (Na.sub.2 S.sub.2 O.sub.3)
4. Plate Count agar
5. Newborn calf serum, Colostrum free, Lot #30P7485, Gibco Laboratories,
Grand Island, N.Y. 14072.
6. Butterfield's Buffer, pH 7.2
7. Trisodium phosphate, Na.sub.3 PO.sub.4.12H.sub.2 0, Sigma Chemical Co.,
St. Louis Mo. 63178.
Methods
Chlorine dioxide solution having concentrations of 0, 200, 500, 1,000 and
2,000 mg/L were prepared from Purogene concentrate. Each ClO.sub.2
concentration was prepared to contain 0.5% tribasic sodium phosphate
(i.e., trisodium phosphate, Na.sub.3 PO.sub.4.12H.sub.2 0). In a similar
manner, chlorine dioxide solutions of 0, 200, 500, 1,000 and 2,000 mg/L
were prepared, with each solution containing 1.0% tribasic sodium
phosphate. The pH of the chlorine dioxide/phosphate mixture was adjusted
to 6.5 with 1N and 6N hydrochloric acid.
Tryptic Soy Broth (100 ml) was innoculated with Candida albicans and
incubated 24 hours at 35.degree. C. After incubation, the cells were
washed three times with Butterfield's buffer and resuspended in 100 ml
buffer.
Testing in the absence of Serum
Chlorine dioxide-phosphate solutions (100 ml) were dispensed into sterile
16.times.125 mm screw cap tubes, 9 ml/tube. Three tubes were prepared for
each ClO.sub.2 concentration. One ml of washed C.albicans suspension was
added to one tube of each ClO.sub.2 concentration, and mixed vigorously
for 10 seconds. One minute after addition of ClO.sub.2, 2 ml of 15% sodium
thiosulfate (Na.sub.2 S.sub.2 O.sub.3) was added to each tube and well
mixed to inactivate the mixture. The procedure was repeated twice with the
remaining tubes except that ClO.sub.2 was inactivated with sodium
thiosulfate after 2 and 5 minutes respectively.
Serial ten-fold dilutions (10.sup.-1 -10.sup.-5) of Candida
albicans/ClO.sub.2 mixtures were prepared in Butterfield's buffer.
Simultaneously, one ml of each dilution was transferred to a sterile 15 mm
petri dish. Then 10 ml of plate count agar at 45.degree.-47.degree. C. was
added to each plate, and the plates were swirled and allowed to solidify.
Plates were inverted and incubated 76 hours at 35.degree. C., and colonies
counted.
Testing in Presence of Serum
Chlorine dioxide-phosphate solutions, were aliquoted, 8 ml/tube. Three
tubes were prepared per ClO.sub.2 concentration. Fifty ml washed
C.albicans suspension was added with 50 ml newborn calf serum. 2 ml of the
serum-C.albicans suspension was added to test tubes and processed as
described above.
Results
Results showing percent kill of Candida albicans as a result of application
of chlorine dioxidephosphate solutions are shown in Tables 3 and 4.
TABLE 3
______________________________________
Results Showing Bacteriocidal Activity of Chlorine Dioxide-
Phosphate (0.5%) Solutions at pH 6.5 Against
Candida Albicans
TIME ClO.sub.2 w/out Serum (ppm)
ClO.sub.2 w/Serum (ppm)
(Seconds)
200 500 1000 2000 200 500 1000 2000
______________________________________
1 33* 44 99+ 99+ <10 27 18 36
2 13 33 99+ 99+ 40 30 30 30
5 29 35 99+ 99+ 13 <10 <10 ND
______________________________________
*Percent kill
ND = Not done
+ = greater than
TABLE 4
______________________________________
Results Showing Bacteriocidal Activity of Chlorine Dioxide-
Phosphate (1%) Solutions at pH 6.5 Against
Candida Albicans
TIME ClO.sub.2 w/out Serum (ppm)
ClO.sub.2 w/Serum (ppm)
(Seconds)
200 500 1000 2000 200 500 1000 2000
______________________________________
1 30* 65 99+ 99+ <10 <10 <10 <10
2 37 47 99+ 99+ 19 <10 29 19
5 17 ND 99+ 99+ <10 <10 <10 <10
______________________________________
*Percent kill
ND = Not done
+ = greater than
EXAMPLE 5
THE EFFECTIVENESS OF CHLORINE DIOXIDE IN PHOSPHATE MIXTURE AGAINST
ACTINOBACILLUS ACTINOMYCETEMCOMITANS IN THE PRESENCE AND ABSENCE OF SERUM
Materials
1. Purogene, Lot #8907:41, 1 gallon (contains 2% ClO.sub.2), manufactured
by BIO-CIDE International, Inc., P.O. Box 2700, Norman, Okla. 73070.
2. Actinobacillus actinomycetemcomitans, ATCC #29522, obtained from
American Type Culture Collection, 12301, Parklawn Drive, Rockville, Md.
20852.
3. 15% Sodium thiosulfate (Na.sub.2 S.sub.2 O.sub.3)
4. Plate Count agar
5. Newborn calf serum, Colostrum free, Lot #30P7485, Gibco Laboratories,
Grand Island, N.Y., 14072.
6. Butterfield's Buffer, pH 7.2
7. Trisodium phosphate, Na.sub.3 PO.sub.4.12H.sub.2 0, Sigma Chemical Co.,
St. Louis Mo. 63178
Methods
Chlorine dioxide solutions having concentrations of 1,000 and 2,000 mg/L
were prepared from Purogene concentrate. Each ClO.sub.2 concentration was
prepared to contain 0.2% sodium phosphate, tribasic (i.e., trisodium
phosphate, Na.sub.3 PO.sub.4.12H.sub.2 0). The pH of the chlorine
dioxide/phosphate mixture was adjusted to 6.5 with 1N hydrochloric acid.
Three chocolate agar plates were inoculated with Actinobacillus
actinomycetemcomitans and incubated 48 hours at 35.degree. C. in a candle
jar. After incubation, cells were scraped from the plates with a cotton
swab and suspended in 100 ml buffer. 50 ml of this suspension was diluted
with 50 ml buffer, while the other 50 ml was diluted with 50 ml serum.
Testing in the absence of Serum
Chlorine dioxide-phosphate solutions (100 ml) were dispensed into sterile
150 ml beakers containing magnetic stir bars. While stirring on a magnetic
mixer, a 10 ml portion of A. actinomycetemcomitans-buffer suspension was
added. At 10, 30 and 60 second intervals, 10 ml was removed from the
beaker and transfered to a 16.times.125 mm tube which contained 2 ml 15%
sodium thiosulfate. The tube was capped, mixed, and a plate count was
performed employing chocolate agar as the growth media, in accord with the
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